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WO2014071218A2 - Biomarkers for breast cancer and methods of using same - Google Patents

Biomarkers for breast cancer and methods of using same Download PDF

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WO2014071218A2
WO2014071218A2 PCT/US2013/068117 US2013068117W WO2014071218A2 WO 2014071218 A2 WO2014071218 A2 WO 2014071218A2 US 2013068117 W US2013068117 W US 2013068117W WO 2014071218 A2 WO2014071218 A2 WO 2014071218A2
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ron
ctd
gene
expression level
msp
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WO2014071218A3 (en
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Stephanie CUNHA
Alana Lee WELM
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University of Utah Research Foundation Inc
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • C12Q2531/00—Reactions of nucleic acids characterised by
    • C12Q2531/10—Reactions of nucleic acids characterised by the purpose being amplify/increase the copy number of target nucleic acid
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    • C12Q2565/00—Nucleic acid analysis characterised by mode or means of detection
    • C12Q2565/50—Detection characterised by immobilisation to a surface
    • C12Q2565/501—Detection characterised by immobilisation to a surface being an array of oligonucleotides
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • Metastasis is the main cause of death in cancer patients, and there are currently no therapies that specifically prevent the metastatic process or cure metastatic disease. Metastasis is a multistep, dynamic process that remains highly enigmatic, and a better understanding of mechanisms that facilitate metastasis continues to be required for development of new treatments. Transcriptional profiling has clearly demonstrated that there are sets of genes, or signatures, for which expression in primary tumors correlates with metastasis and/or poor survival, although the mechanisms orchestrating many of these gene expression programs have not been defined. For example, a 70-gene signature has been identified that is associated with metastasis, which is now used clinically as the MAMMAPRINT® test (AGendia, Irvina CA).
  • this disclosure provides methods of treating breast cancer in a subject.
  • the methods may include determining in a sample of the breast cancer an expression level of one or more genes selected from CSGALNACT1, SIGLEC6, SHC4, ELTD1 , CGA, MST1R, KRT8P41 , BTF3P4, RP11-478C6.4, EPHA3, LXN, ENSG00000252044, ENSG00000242932, ENSG00000241115, AP001347.6, LHFPL2, CTD-2330K9.2, RASD1, ENSG00000242693, AC093388.3, AOC1, CPE, PCDH8, DCHS2, DPP4, ENSG00000242777, GUSBP5, ENSG00000244466, ENSG00000252331 , RP11- 664D7.4, RP11-206M11.7, ENSG00000244580, EYA4, CTA-714B7.5, ENSG00000252318, ABC
  • this disclosure provides methods of assessing the likelihood that breast cancer in a subject will metastasize or has metastasized.
  • the methods may include determining in a sample of the breast cancer an expression level of one or more genes selected from CSGALNACT1 , SIGLEC6, SHC4, ELTD1 , CGA, MST1 R, KRT8P41 , BTF3P4, RP1 1-478C6.4, EPHA3, LXN, ENSG00000252044, ENSG00000242932, ENSG00000241 1 15, AP001347.6, LHFPL2, CTD-2330K9.2, RASD1 , ENSG00000242693, AC093388.3, AOC1 , CPE, PCDH8, DCHS2, DPP4, ENSG00000242777, GUSBP5, ENSG00000244466, ENSG00000252331 , RP1 1-664D7.4, RP1 1-206M1 1.7, ENSG00000244580
  • the disclosure provides methods of determining whether a subject having breast cancer would benefit from treatment with at least one of a RON inhibitor and a PI3K kinase inhibitor.
  • the methods may include assessing the likelihood that the breast cancer will metastasize or has metastasized; and determining whether the subject would benefit from treatment with at least one of a RON inhibitor and a PI3K kinase inhibitor based on the likelihood that the breast cancer will metastasize or has metastasized.
  • the expression level of at least one gene selected from CSGALNACT1 , SIGLEC6, SHC4, ABCA1 , PLD1 , RNF144A, SLC44A4, SLC2A13, and AXIN2 is different relative to the control value.
  • control value associated with each gene is determined by determining the expression level of that gene in one or more control samples, and calculating an average expression level of that gene in the one or more control samples, wherein each control sample is obtained from healthy tissue of the same or a different subject.
  • the expression of the gene is increased or decreased relative to the expression level of a control by an amount of at least about 2-fold. In some embodiments, the expression of at least one gene is increased at least about 2-fold relative to the control value associated with the gene. In some embodiments, the expression of at least one gene is decreased at least about 2-fold relative to the control value associated with the gene.
  • the disclosure provides methods of monitoring a molecular response of a subject to treatment of breast cancer with at least one of a RON inhibitor and a PI3K inhibitor.
  • the methods may include determining in a first sample of the breast cancer an expression level of one or more genes selected from CSGALNACT1 , SIGLEC6, SHC4, ELTD1 , CGA, MST1 R, KRT8P41 , BTF3P4, RP1 1-478C6.4, EPHA3, LXN, ENSG00000252044, ENSG00000242932, ENSG00000241 1 15, AP001347.6, LHFPL2, CTD-2330K9.2, RASD1 , ENSG00000242693, AC093388.3, AOC1 , CPE, PCDH8, DCHS2, DPP4, ENSG00000242777, GUSBP5, ENSG00000244466, ENSG00000252331 , RP1 1- 664D7.4, RP1
  • determining the expression level of a first gene comprises measuring the expression level of an RNA transcript of the first gene, or an expression product thereof. In some embodiments, measuring the expression level of the RNA transcript of the first gene, or the expression product thereof, includes using at least one of a PCR-based method, a Northern blot method, a microarray method, and an immunohistochemical method.
  • kits may include one or more primers, each primer adapted to amplify an RNA transcript of an independent one of the genes selected from CSGALNACT1 , SIGLEC6, SHC4, ELTD1 , CGA, MST1 R, KRT8P41 , BTF3P4, RP1 1-478C6.4, EPHA3, LXN, ENSG00000252044, ENSG00000242932, ENSG00000241 1 15, AP001347.6, LHFPL2, CTD-2330K9.2, RASD1 , ENSG00000242693, AC093388.3, AOC1 , CPE, PCDH8, DCHS2, DPP4, ENSG00000242777, GUSBP5, ENSG00000244466, ENSG00000252331 , RP1 1- 664D7.4, RP1 1-206M1 1.7, ENSG00000244580, EYA4, CTA-714B7.5, ENSG00000252318, ABCA1 , P
  • the at least one primer is adapted to amplify an RNA transcript of a gene selected from CSGALNACT1 , SIGLEC6, SHC4, ABCA1 , PLD1 , RNF144A, SLC44A4, SLC2A13, and AXIN2.
  • a first probe comprises an antibody to an expression product.
  • a first probe comprises an oligonucleotide complementary to an RNA transcript.
  • kits may include one or more probes, each probe adapted to specifically bind to an RNA transcript, or an expression product thereof, of an independent one of the genes selected from CSGALNACT1 , SIGLEC6, SHC4, ELTD1 , CGA, MST1 R, KRT8P41 , BTF3P4, RP1 1-478C6.4, EPHA3, LXN, ENSG00000252044, ENSG00000242932, ENSG00000241 1 15, AP001347.6, LHFPL2, CTD-2330K9.2, RASD1 , ENSG00000242693, AC093388.3, AOC1 , CPE, PCDH8, DCHS2, DPP4, ENSG00000242777, GUSBP5, ENSG00000244466, ENSG00000252331 , RP1 1-664D7.4, RP1 1-206M11.7, ENSG00000244580, EYA4, CTA-714B7.5, ENSG00000252318,
  • the at least one probe is adapted to bind to an RNA transcript, or an expression product thereof, of a gene selected from CSGALNACT1 , SIGLEC6, SHC4, ABCA1 , PLD1 , RNF144A, SLC44A4, SLC2A13, and AXIN2.
  • a first probe comprises an antibody to an expression product.
  • a first probe comprises an oligonucleotide complementary to an RNA transcript.
  • FIG. 1 RON/MSP signaling promotes widespread metastasis of human breast cancer in vivo
  • A Immunoblot analysis of RON levels (top panel) in immortalized mammary epithelial cells (MCF-10A; blue), non-aggressive breast cancer cell lines (MCF7 and T47D; green), aggressive breast cancer cell lines (MDA-MB-453, HCC1 143, HCC1806; orange) and MCF7 and T47D cells engineered to overexpress RON/MSP (red).
  • MDF-10A immortalized mammary epithelial cells
  • MDF7 and T47D green
  • aggressive breast cancer cell lines MDA-MB-453, HCC1 143, HCC1806; orange
  • MCF7 and T47D cells engineered to overexpress RON/MSP red.
  • the ⁇ -actin (ACTB) loading control is shown in the bottom panel.
  • B The effect of RON/MSP on spontaneous lung, bone, liver and brain metastasis of orthotopic M
  • FIG. 1 Shown are representative bioluminescent images of primary tumors, lung, bone, liver and brain metastasis from single mice.
  • FIG. 1 RON/MSP signaling upregulates MBD4 and drives aberrant DNA methylation
  • A Circular representation of genome-wide aberrant DNA methylation caused by RON/MSP expression. Average methylation levels for all of the CGs in 10-Mbp-wide windows are shown in the blue-green tracks (the outermost track shows MCF7 cells; the middle track shows MCF7-RON/MSP cells). The innermost track indicates the differentially methylated regions (DMRs) between MCF7 and MCF7-RON/MSP cells (FDR>20, differential methylation>0.25).
  • DMRs differentially methylated regions
  • Top panel Distribution of differentially methylated CpGs on CpG islands (CpGi) shelves (>2 to 4 kb from island edge) and on CpGi shores (0-2 kb from island edge); within and outside CpGi.
  • Bottom panel Distribution of differentially methylated CpGs across regions of other significance.
  • TSS 200 within the region 1 -200 bp upstream of the TSS; TSS 1500, within the region 201 -1500 bp upstream of the transcription start site (TSS); UTR, untranslated region; HyperMe, hypermethylated; HypoMe, hypomethylated.
  • FIG. 3 Knockdown of MBD4 blocks RON/MSP-mediated breast cancer metastasis.
  • A Boxplot showing RON and MBD4 mRNA expression in normal breast tissues (white) and breast tumors (dark grey) from The Cancer Genome Atlas (TCGA) data set.
  • B Western blot for RON and MBD4 proteins in normal human breast tissues from reduction mammoplasties and human primary breast tumor specimens; the ⁇ -actin (ACTB) loading control is also shown.
  • FIG. 4 Knockdown of MBD4 reverses DNA aberrant methylation and expression of RON/MSP regulated genes.
  • A Representation of smoothed methylation values from bisulfite sequencing data for MCF7 (black), MCF7-RON/MSP (blue) and MCF7- RON/MSP-shMBD4 cells (purple) in a representative region of chromosome 2. The hypomethylation block in MCF7-RON/MSP that becomes hypermethylated by knocking- down MBD4 is indicated by a red bar.
  • (B) Example of DNA methylation levels in promoter regions in MCF7 (black), MCF7-RON/MSP (blue) and MCF7-RON/MSP-shMBD4 cells (purple). The hypomethylated block in MCF7-RON/MSP cells that becomes re-methylated by knocking down MBD4 is indicated by a red bar.
  • (C) Example of DNA methylation levels in a promoter region that becomes hypermethylated in shMBD4 cells, independent of RON/MSP expression (red bar) Bisulfite sequencing data for MCF7 (black), MCF7-RON/MSP (blue) and MCF7-RON/MSP-shMBD4 cells (purple) are shown.
  • Figure 5 Specific subclasses of human breast tumors possess the RON/MBD4 epigenetic signature, which correlates with poor prognosis (A) Circular representation of the RON/MBD4 epigenetic signature.
  • the outside track represents the 192 genes regulated by RON/MSP for which expression was reversed following knockdown of MBD4 (blue: genes downregulated by RON/MSP and then reversed by knocking down MBD4 with shRNA; orange: genes upregulated by RON/MSP and then reversed by knocking down MBD4).
  • In larger characters are the genes that are annotated in the METABRIC dataset.
  • the inner track represents the statistically significant DMRs at these regions (red: hypermethylated DMRs in MCF7-RON/MSP and then reversed by knocking down MBD4; green: hypomethylated DMRs in MCF7-RON/MSP and then reversed by knocking down MBD4).
  • B Enrichment pattern of the gene set comprising the RON/MBD4 epigenetic signature (rows) across 997 breast tumors (columns). Red and green indicated significantly over or underexpressed gene, respectively. Are represented the 582 patients having enrichment or underrepresentation of the gene set. Blue bars (right) indicate individual tumor annotations for breast cancer subtype.
  • the table (right) shows the association of the RON/MBD4 epigenetic signature with ER, HER2 and PR status, as well as intrinsic breast cancer subtype, grade and death (we assigned a p value according to the hypergeometric distribution)
  • C Kaplan-Meier analysis of overall survival in 997 breast cancer patients from the METABRIC discovery dataset (Curtis et al., 2012). Survival curve of individuals with tumors showing an enrichment of the 1 16 annotated genes from the RON/MBD4 epigenetic signature is shown in red; all other patients are shown in blue (No signature). The p value indicates a statistically significant survival difference between these two groups of patients. The survival hazard ratio was calculated using Univariate Cox's regression analysis. [0023] Figure 6. MBD4 is regulated by RON/MSP signaling through PI3K pathway.
  • FIG. 7 Treatment with a new RON inhibitor, OSI-296, drastically decreases breast cancer metastasis in human patient-derived breast tumor grafts.
  • FIG. 8 (A) Tumor growth curves following orthotopic transplantation of MCF7 or T47D cells (blue) or MCF7-RON/MSP or T47-RON/MSP (red) into cleared inguinal mammary fat pads of 3-week old NOD/SCID mice. (B) Representative lung sections from mice carrying MCF7-RON/MSP or T47D-RON/MSP tumors, stained with H&E or immunostained with an antibody specific for human cytokeratin to positively identify metastasis. (C) Scatter plot showing differential expression obtained using microarray (x axis) and RNA-seq (y axis) between MCF7 and MCF7-RON/MSP.
  • Points represent genes, located based on estimates of differential expression using RNA-Seq and Microarray.
  • Red points represent genes for which FDR ⁇ 0.1 and fold change>2.
  • Green points represent genes differentially expressed identified by RNA-seq only.
  • Purple points represent genes differentially expressed identified by microarray only. The line corresponds to perfect agreement between the technologies.
  • Figure 9 (A) Real-time quantitative RT-PCR for various DNMT mRNA expression levels, normalized to ⁇ -actin mRNA expression, in MCF7 and MCF7-RON/MSP cells. (B) DNA methylation activity in MCF7 and MCF7-RON/MSP cells, as assessed by EpiQuick DNA methyltransferase assay.
  • FIG. 10 Figure 10.
  • A Tumor growth following orthotopic implantation of MCF7 (dark blue), MCF 7- RON/MSP (red), MCF7-RON/MSP-shScr (green), MCF7-RON/MSP-shMBD4 (yellow) and MCF7-RON/MSP-shMBD4R cells (light blue) in MCF7 (left) and T47D (right) models.
  • B Metastasis frequencies for MCF7, MCF7-RON/MSP, MCF7-RON/MSP-shScr, and MCF7-RON/MSP-shMBD2 tumors.
  • C Representative images showing lack of effect of sh-MBD2 on spontaneous lung, bone, liver and brain metastasis of RON/MSP-expressing MCF7 tumors.
  • FIG. 11 (A) Western blots showing expression of EGFR, p-EGFR, MBD4, p- AKT, p-ERK, AKT, ERK and ⁇ -actin in MDA-MB-231 cells treated with EGF (15 nM) for 1 hour. (B) The expression of MET, MBD4 and ACTB in MCF7 cell line infected with retroviruses carrying cDNA of MET. (C) MCF7 and MCF7-RON/MSP cells were treated with or without alkaline phosphatase for 30 min.
  • the lysates were subjected to (a) conventional SDS-PAGE on 8% (wt/vol) polyacrylamide gel or (b) SDS-PAGE on 6% (wt/vol) polyacrylamide gel containing 20 ⁇ Mn2+-Phos-tag, followed by immunoblotting with the anti-MBD4 antibody.
  • a) conventional SDS-PAGE on 8% (wt/vol) polyacrylamide gel or (b) SDS-PAGE on 6% (wt/vol) polyacrylamide gel containing 20 ⁇ Mn2+-Phos-tag followed by immunoblotting with the anti-MBD4 antibody.
  • alkaline phosphatase shifts in the mobility of MBD4 were suppressed.
  • RON also known as macrophage stimulating 1 -receptor (MST1 R)
  • MST1 R macrophage stimulating 1 -receptor
  • MSP macrophage-stimulating protein
  • HGFL hepatocyte growth factor-like protein
  • MST1 macrophage stimulating 1
  • Pro-MSP is cleaved by membrane-bound proteases such as membrane type serine protease 1 (MT- SP1 ; also known as matriptase) or hepatocyte growth factor activator, leading to activation of MSP at the cell surface. Binding of MSP to its receptor, RON, activates RON and leads to cellular growth, motility, and invasion. While RON overexpression may be a feature of cancers, the molecular mechanisms by which RON induces tumorgenesis and metastasis are still unclear.
  • MT- SP1 membrane type serine protease 1
  • matriptase hepatocyte growth factor activator
  • RON/MSP enhances metastasis of breast cancer xenografts, and that metastasis is enhanced by driving DNA demethylation at key target gene promoters.
  • RON/MSP-initiated aberrant DNA methylation is the result of PI3K- dependent upregulation of MBD4, which is a component of the DNA demethylase complex.
  • knockdown of MBD4 in RON/MSP-expressing breast cancer cells reverses the DNA methylation and blocks metastasis.
  • the inventors have further discovered a set of genes that are specifically regulated by RON/MSP through MBD4- directed aberrant DNA methylation, and that the MBD4/RON signature is enriched in basal- like (triple negative) breast cancers and associated with poor prognosis.
  • inhibition of Ron signaling with a small molecule kinase inhibitor blocks metastasis of patient-derived tumor grafts, indicating that RON inhibitors can be used as novel anti-metastatic agents.
  • the gene signature may include at least one of the genes shown in Table 1 .
  • the expression of these genes was found to be deregulated when RON/MSP is overexpressed, as detailed in the Examples.
  • the expression was reversed (i.e. , the gene overexpression was decreased) when expression of MBD4 was down regulated, demonstrating these genes are downstream of RON/MSP and MBD4.
  • Some of the genes (corresponding to SEQ ID NOs 1-56) further demonstrated aberrant expression upon administration of a RON inhibitor.
  • SEQ ID NOs. 1-56 correspond to genes for which the expression in a xenograft tumor became reversed after treatment with a RON inhibitor, wherein SEQ ID NOs 1-47 correspond to the polynucleotide sequences and variants and SEQ ID NOs 48-56 in parentheses correspond to the polypeptide sequences.
  • the gene signature includes at least one of the genes corresponding to SEQ ID NOs 1-56. In some embodiments, the gene signature includes at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 of the genes listed in Table 1. In some embodiments, the gene signature includes at least 5 of the genes listed in Table 1. [0033] In some embodiments, provided are methods of assessing the likelihood that breast cancer in a subject will metastasize or has metastasized.
  • the method may include determining in a sample of the breast cancer an expression level of one or more genes, comparing the expression level of each gene to a control value associated with that same gene, and assessing the likelihood that the breast cancer will metastasize or has metastasized based on the relative difference between the expression level and the control value associated with each gene.
  • An increase or decrease (as detailed below) in the expression level relative to the control value of at least one gene may correlate with an increased likelihood that the breast cancer in the subject will metastasize or has metastasized.
  • the gene may be selected from CSGALNACT1 , SIGLEC6, SHC4, ELTD1 , CGA, MST1 R, KRT8P41 , BTF3P4, RP1 1-478C6.4, EPHA3, LXN, ENSG00000252044, ENSG00000242932, ENSG00000241 1 15, AP001347.6, LHFPL2, CTD-2330K9.2, RASD1 , ENSG00000242693, AC093388.3, AOC1 , CPE, PCDH8, DCHS2, DPP4, ENSG00000242777, GUSBP5, ENSG00000244466, ENSG00000252331 , RP1 1-664D7.4, RP1 1-206M11.7, ENSG00000244580, EYA4, CTA-714B7.5, ENSG00000252318, ABCA1 , PLD1 , RNF144A, SLC44A4, SLC2A13, AXIN
  • the gene may be selected from CSGALNACT1 , SIGLEC6, SHC4 , ELTD1 , CGA, MST1 R, KRT8P41 , BTF3P4, RP1 1-478C6.4, EPHA3, LXN, ENSG00000252044, ENSG00000242932, ENSG00000241 1 15, AP001347.6, LHFPL2, CTD-2330K9.2, RASD1 , ENSG00000242693, AC093388.3, AOC1 , CPE, PCDH8, DCHS2, DPP4, ENSG00000242777, GUSBP5, ENSG00000244466, ENSG00000252331 , RP1 1- 664D7.4, RP1 1-206M1 1.7, ENSG00000244580, EYA4, CTA-714B7.5, and ENSG00000252318.
  • An increase in the expression level of at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 of these genes relative to the control value may correlate with an increased likelihood that the breast cancer in the subject will metastasize or has metastasized.
  • An increase in the expression level of at least 1 of the genes corresponding to SEQ ID NOs 1-22 and 48-50 relative to the control value may correlate with an increased likelihood that the breast cancer in the subject will metastasize or has metastasized.
  • the gene may be selected from CSGALNACT1 , SIGLEC6, and SHC4 (corresponding to SEQ ID NOs 1-22 and 48-50).
  • the gene may be selected from ABCA1 , PLD1 , RNF144A, SLC44A4, SLC2A13, AXIN2, PGR, XXbac-BPG254F23.6, AMY2B, LIMCH1 , RP1 1- 356J5.12, ID2, MFAP2, LPHN2, RP1 1-594N15.2, RP1 1-575A19.2, SPINK5, HLA-DQB1- AS1 , RN7SL151 P, ATP8A1 , FAM189A2, ZNF423, CTD-2001 C12.1 , ELOVL2, TESC, KCNMB4, STOX2, PLEKHM3, MT-TL1 , KLK1 1 , NPNT, ENSG00000234543, SHC2, BTNL9, ARMC4, OLFML2A, AMOT, RP1 1-556114.1 , SLFN5, PKIA, CTC-339F2.2, ADAM23,
  • a decrease in the expression level of at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 of these genes relative to the control value may correlate with an increased likelihood that the breast cancer in the subject will metastasize or has metastasized.
  • a decrease in the expression level of at 1 of the genes corresponding to SEQ ID NOs 23-47 and 51-56 relative to the control value may correlate with an increased likelihood that the breast cancer in the subject will metastasize or has metastasized.
  • the gene may be selected from ABCA1 , PLD1 , RNF144A, SLC44A4, SLC2A13, AXIN2 (corresponding to SEQ ID NOs 23-47 and 51-56).
  • the gene may be selected from CSGALNACT1 , SIGLEC6, SHC4, ABCA1 , PLD1 , RNF144A, SLC44A4, SLC2A13, AXIN2 (corresponding to SEQ ID NOs 1-56).
  • a difference (decrease or increase) in the expression level of at least one of the genes corresponding to SEQ ID NOs 1-56 relative to the control value may correlate with an increased likelihood that the breast cancer in the subject will metastasize or has metastasized.
  • the expression level of the gene may be increased relative to the expression level of a control by an amount of at least about 0.5-fold, at least about 1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5- fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, at least about 10-fold, at least about 1 1-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold
  • the expression of the gene may be increased relative to the expression level of a control by an amount of about 0.5-fold to about 500-fold, about 1-fold to about 50-fold, or about 2-fold to about 10-fold. In some embodiments, the expression of the gene is increased relative to the expression level of a control by an amount of at least about 2-fold.
  • the expression level of the gene may be decreased relative to the expression level of a control by an amount of at least about 0.5-fold, at least about 1 -fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5- fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, at least about 10-fold, at least about 1 1 -fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 4.5
  • the expression of the gene may be decreased relative to the expression level of a control by an amount of about 0.5-fold to about 500-fold, about 1 -fold to about 50-fold, or about 2-fold to about 10-fold. In some embodiments, the expression of the gene is decreased relative to the expression level of a control by an amount of at least about 2-fold.
  • a subject can be an animal, a vertebrate animal, a mammal, a rodent (e.g. a guinea pig, a hamster, a rat, a mouse), murine (e.g. a mouse), canine (e.g. a dog), feline (e.g. a cat), equine (e.g. a horse), a primate, simian (e.g. a monkey or ape), a monkey (e.g. marmoset, baboon), an ape (e.g. gorilla, chimpanzee, orangutan, gibbon), or a human.
  • the subject is a mammal.
  • the mammal is a human.
  • sample or “biological sample” relates to any material that is taken from its native or natural state, so as to facilitate any desirable manipulation or further processing and/or modification.
  • a sample or a biological sample can comprise a cell, a tissue, a fluid (e.g., a biological fluid), a protein (e.g., antibody, enzyme, soluble protein, insoluble protein), a polynucleotide (e.g., RNA, DNA), a membrane preparation, and the like, that can optionally be further isolated and/or purified from its native or natural state.
  • a “biological fluid” refers to any a fluid originating from a biological organism.
  • Exemplary biological fluids include, but are not limited to, blood, serum, and plasma.
  • a biological fluid may be in its natural state or in a modified state by the addition of components such as reagents, or removal of one or more natural constituents (e.g., blood plasma).
  • a sample can be from any tissue or fluid from an organism.
  • the sample comprises tissue from the breast, milk ducts, breast lobules, digestive tract, lung, liver, kidney, brain, lip, mouth, esophagus, urinary bladder, prostate, vagina, and/or cervix.
  • the sample is from a tissue that is part of, or associated with, the breast of the organism.
  • the sample may be tissue from a neoplasm.
  • a neoplasm may include cancer.
  • the sample may be cancerous tissue or from a tumor.
  • the sample may comprise tissue surrounding cancerous tissue or a tumor.
  • the sample may comprise tissue surrounding or around the perimeter of cancerous tissue or a tumor that was surgically excised.
  • the cancer may comprise breast cancer.
  • the methods described herein can include any suitable method for evaluating gene expression. Determining expression of at least one gene may include, for example, detection of an RNA transcript or portion thereof, and/or an expression product such as a protein or portion thereof. Expression of a gene may be detected using any suitable method known in the art, including but not limited to, detection and/or binding with antibodies, detection and/or binding with antibodies tethered to or associated with an imaging agent, real time RT-PCR, Northern analysis, magnetic particles (e.g., microparticles or nanoparticles), Western analysis, expression reporter plasmids, immunofluorescence, immunohistochemistry, detection based on an activity of an expression product of the gene such as an activity of a protein, any method or system involving flow cytometry, and any suitable array scanner technology.
  • any suitable method known in the art including but not limited to, detection and/or binding with antibodies, detection and/or binding with antibodies tethered to or associated with an imaging agent, real time RT-PCR, Northern analysis, magnetic particles (e.
  • the expression level of a protein may be evaluated by immunofluorescence by visualizing cells stained with a fluorescently-labeled protein-specific antibody, Western blot analysis of protein expression, and RT-PCR of protein transcripts.
  • the antibody or fragment thereof may suitably recognize a particular intracellular protein, protein isoform, or protein configuration.
  • determining the expression level of at least one gene comprises measuring the expression level of an RNA transcript of the at least one gene, or an expression product thereof.
  • an "imaging agent” or “reporter” is any compound or composition that enhances visualization or detection of a target. Any type of detectable imaging agent or reporter may be used in the methods disclosed herein for the detection of an expression product. Exemplary imaging agents and reporters may include, but are not limited to, compounds and compositions comprising magnetic beads, fluorophores, radionuclides, and nuclear stains (e.g., DAPI), and further comprising a targeting moiety for specifically targeting or binding to the target expression product.
  • DAPI nuclear stains
  • an imaging agent may include a compound that comprises an unstable isotope (i.e., a radionuclide), such as an alpha- or beta-emitter, or a fluorescent moiety, such as Cy-5, Alexa 647, Alexa 555, Alexa 488, fluorescein, rhodamine, and the like.
  • suitable radioactive moieties may include labeled polynucleotides and/or polypeptides coupled to the targeting moiety.
  • the imaging agent may comprise a radionuclide such as, for example, a radionuclide that emits low-energy electrons (e.g., those that emit photons with energies as low as 20 keV).
  • Such nuclides can irradiate the cell to which they are delivered without irradiating surrounding cells or tissues.
  • Non-limiting examples of radionuclides that are can be delivered to cells may include, but are not limited to, 137 Cs, 103 Pd, 111 ln, 125 l, 211 At, 212 Bi and 213 Bi, among others known in the art.
  • Further imaging agents may include paramagnetic species for use in MRI imaging, echogenic entities for use in ultrasound imaging, fluorescent entities for use in fluorescence imaging (including quantum dots), and light-active entities for use in optical imaging.
  • a suitable species for MRI imaging is a gadolinium complex of diethylenetriamine pentacetic acid (DTPA).
  • determining the expression level of at least one gene includes measuring the expression level of an RNA transcript of the at least one gene, or an expression product thereof. In some embodiments, measuring the expression level of the RNA transcript of the at least one gene, or the expression product thereof, includes using at least one of a PCR-based method, a Northern blot method, a microarray method, and an immunohistochemical method.
  • the expression level of a gene in a sample may be compared to a control value associated with that same gene.
  • a control may include comparison to the level of expression in a control cell, such as a non-cancerous cell or other normal cell, or comparison to a cancer sample that does not express RON from a different patient or from historical controls.
  • the control may be from a non-cancerous or normal cell from the same subject, or it may be from a different subject.
  • a control may include an average range of the level of expression from a population of normal cells.
  • control value associated with each gene may be determined by determining the expression level of that gene in one or more control samples, and calculating an average expression level of that gene in the one or more control samples, wherein each control sample is obtained from normal or healthy tissue of the same or a different subject.
  • the method may include determining in a sample of the cancer an expression level of one or more genes selected from the genes listed in Table 1 , comparing the expression level of each gene to a control value associated with that same gene, and administering at least one of a RON inhibitor and a PI3K kinase inhibitor to the subject when the expression level of at least one or five of the genes corresponding to CSGALNACT1 , SIGLEC6, SHC4, ELTD1 , CGA, MST1 R, KRT8P41 , BTF3P4, RP1 1-478C6.4, EPHA3, LXN, ENSG00000252044, ENSG00000242932, ENSG00000241 1 15, AP001347.6, LHFPL2, CTD-2330K9.2, RASD1 , ENSG00000242693, AC093388.3, AOC1 , CPE, PCDH8, DCHS2, DPP4, ENSG00000242777, GUSBP5, ENSG00000244466,
  • the method may include administering at least one of a RON inhibitor and a PI3K kinase inhibitor to the subject when the expression level of at least one or five of the genes corresponding to ABCA1 , PLD1 , RNF144A, SLC44A4, SLC2A13, AXIN2, PGR, XXbac- BPG254F23.6, AMY2B, LIMCH1 , RP1 1-356J5.12, ID2, MFAP2, LPHN2, RP1 1-594N15.2, RP1 1-575A19.2, SPINK5, HLA-DQB1-AS1 , RN7SL151 P, ATP8A1 , FAM189A2, ZNF423, CTD-2001 C12.1 , ELOVL2, TESC, KCNMB4, STOX2, PLEKHM3, MT-TL1 , KLK1 1 , NPNT, ENSG00000234543, SHC2, BTNL9, ARMC4, OLF
  • Alternative therapies include therapies other than administration of a RON inhibitor or a PI3K kinase inhibitor.
  • Alternative therapies may include, but are not limited to, radiation therapy, a surgical procedure, anti-cancer drugs, or any combination thereof.
  • methods of determining whether a subject having breast cancer would benefit from treatment with at least one of a RON inhibitor and a PI3K kinase inhibitor may include assessing the likelihood that the breast cancer will metastasize or has metastasized according to the method detailed above, and determining whether the subject would benefit from treatment with at least one of a RON inhibitor and a PI3K kinase inhibitor based on the likelihood that the breast cancer will metastasize or has metastasized.
  • kits for monitoring a molecular response of a subject to treatment of breast cancer with at least one of a RON inhibitor and a PI3K inhibitor may include determining in a first sample of the breast cancer an expression level of one or more genes, administering to the subject a therapeutic amount of a medicament comprising at least one of a RON inhibitor and a PI3K inhibitor, determining in a second sample of the breast cancer an expression level of each of the one or more genes, wherein the second sample is collected after administration of the medicament, and comparing for each gene the expression level in the first sample to the expression level in the second sample.
  • Inhibitors encompass agents that inhibit the activity of or reduce the amount of RON, MSP, MBD4, PI3K, or other members of the RON/MBD4 pathway.
  • the amount or the activity of the protein may be reduced or inhibited using a variety of techniques known in the art.
  • an inhibitor may indirectly or directly bind and inhibit the activity of the protein, including binding activity or catalytic activity.
  • An inhibitor may inhibit the ability of the protein to interact with cellular and extracellular components.
  • An inhibitor may prevent or reduce expression of the protein.
  • a therapeutic composition adapted to reduce the amount or the activity of the protein may comprise a small molecule inhibitor of the protein itself or of a binding partner, an antibody specific for the protein, or a siRNA.
  • the therapeutic composition may comprise a siRNA adapted to reduce the expression of the protein.
  • RON inhibitors may include, but are not limited to, OSI-296, BMS- 77607, and any other compounds that inhibit the activity of RON.
  • PI3K inhibitors may include, but are not limited to, LY294002, BEZ-235, BKM-120, and any other compounds that inhibit the activity of PI3K.
  • compositions may be administered to a subject by any convenient route of administration, whether systemically/ peripherally or at the site of desired action, including but not limited to, oral (e.g. by ingestion); topical (including e.g. transdermal, intranasal, ocular, buccal, and sublingual); pulmonary (e.g. by inhalation or insufflation therapy using, e.g. an aerosol, e.g.
  • oral e.g. by ingestion
  • topical including e.g. transdermal, intranasal, ocular, buccal, and sublingual
  • pulmonary e.g. by inhalation or insufflation therapy using, e.g. an aerosol, e.g.
  • vaginal parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal; by implant of a depot, for example, subcutaneously or intramuscularly.
  • treatment refers generally to treatment and therapy, whether of a human or an animal (e.g. in veterinary applications), in which a desired therapeutic effect is achieved.
  • treatment may ameliorate the condition or may inhibit the progress of the condition (e.g., reduce the rate of progress or halt the rate of progress).
  • kits for assessing the likelihood that breast cancer in a subject will metastasize or has metastasized are provided. In some embodiments, provided are kits for determining whether the subject would benefit from treatment with at least one of a RON inhibitor and a PI3K kinase inhibitor.
  • the kit may include one or more primers.
  • Each primer may be adapted to amplify an RNA transcript of an independent one of a genes selected from CSGALNACT1 , SIGLEC6, SHC4, ELTD1 , CGA, MST1 R, KRT8P41 , BTF3P4, RP1 1-478C6.4, EPHA3, LXN, ENSG00000252044, ENSG00000242932, ENSG00000241 1 15, AP001347.6, LHFPL2, CTD-2330K9.2, RASD1 , ENSG00000242693, AC093388.3, AOC1 , CPE, PCDH8, DCHS2, DPP4, ENSG00000242777, GUSBP5, ENSG00000244466, ENSG00000252331 , RP1 1-664D7.4, RP1 1-206M1 1.7, ENSG00000244580, EYA4, CTA-714B7.5, ENSG00000252318, ABCA1 , PLD1 , RNF
  • kits for assessing the likelihood that breast cancer in a subject will metastasize or has metastasized are provided. In some embodiments, provided are kits for determining whether the subject would benefit from treatment with at least one of a RON inhibitor and a PI3K kinase inhibitor.
  • the kit may include one or more probes.
  • Each probe may be adapted to specifically bind to an RNA transcript, or an expression product thereof, of an independent one of the genes selected from CSGALNACT1 , SIGLEC6, SHC4, ELTD1 , CGA, MST1 R, KRT8P41 , BTF3P4, RP1 1-478C6.4, EPHA3, LXN, ENSG00000252044, ENSG00000242932, ENSG00000241 1 15, AP001347.6, LHFPL2, CTD-2330K9.2, RASD1 , ENSG00000242693, AC093388.3, AOC1 , CPE, PCDH8, DCHS2, DPP4, ENSG00000242777, GUSBP5, ENSG00000244466, ENSG00000252331 , RP1 1- 664D7.4, RP1 1-206M1 1.7, ENSG00000244580, EYA4, CTA-714B7.5, ENSG00000252318, ABCA1 , P
  • a probe may comprise an antibody to an expression product.
  • a probe may comprise an oligonucleotide complementary to an RNA transcript.
  • a probe may comprise an "imaging agent” or “reporter” as detailed above.
  • the method may include administering at least one of a RON inhibitor and a PI3K kinase inhibitor.
  • the inhibitor may be formulated into a composition. Standard pharmaceutical formulation techniques are used, such as those disclosed in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. (1990).
  • MCF-7 cells were cultured in Dulbecco's modified Eagle's medium (DME/F-12, Thermo Scientific) supplemented with 1 % penicillin/streptomycin (Thermo Scientific), 10 ⁇ g/mL insulin (Gibco), and 10% heat inactivated fetal bovine serum (Thermo Scientific) at 37°C in 5% C02.
  • T47D cells were cultured in the same conditions in RPMI1640 (RPMI1640, Thermo Scientific) supplemented with 1 % penicillin/streptomycin (Thermo Scientific) and 10% heat inactivated fetal bovine serum.
  • RON and MSP were stably expressed in MCF7 cells by retroviral infection (Liu et al., 201 1 ).
  • MCF7-RON/MSP cells were maintained in MCF7 medium supplemented with 0.2 ⁇ g/mL puromycin and 50 ⁇ g/mL hygromycin.
  • MCF7 and T47D cell lines were infected with lentiviral constructs that direct the synthesis of shRNA (based on PLKO.1 but with a neo resistant cassette substituted for the puromycin resistant cassette) and selected by the addition of 1 mg/mL G418 48 hours later.
  • Bioluminescence images were obtained with a 15 cm field of view, binning (resolution) factor of 8, 1/f stop, open filter, and an imaging time of 8 sec.
  • a metastatic event as any detectable luciferase signal above background and validated metastasis histologically.
  • female NOD-SCID mice were each orthotopically transplanted with a single primary breast cancer tumor fragment from patient lines HCI-003 and HCI-01 1. Mice bearing these established xenograft tumors (2-4 mm in diameter) received vehicle (40% Trappsol) or OSI-296 (200 mg/kg) by oral gavage every other day until a tumor diameter of 1.2-1.5 cm was reached. Mice were killed and analyzed for metastasis histologically.
  • Genomic DNA for MeDIP and bisulfite modification was isolated using the DNeasy Blood Kit (Qiagen).
  • Total RNA for gene expression analysis by microarray and quantitative RT-PCR was isolated with the RNeasy Kit (Qiagen) and tested for integrity on RNA 6000 NanoChips using an Agilent 2100 Bioanalyzer.
  • RNA polymerase and dye-labeled nucleotides are combined with the reaction mixture to simultaneously amplify the target material through the generation of cRNA and incorporate cyanine 3-CTP.
  • Fluorescently labeled, cRNA molecules are purified from the reaction mixture using the Qiagen RNeasy mini kit. The concentration of the purified samples is determined using a NanoDrop ND-1000 spectrophotometer. Fluorescently labeled cRNA samples (825 ng each) were fragmented and combined with Agilent Hi-RPM Hybridization Buffer. Microarray hybridizations were performed using Agilent SureHyb Hybridization chambers.
  • Hybridization chambers were loaded onto a rotisserie in an Agilent Hybridization oven and were incubated at 65°C for 17 hours with a rotational speed of 10 rpm. Following incubation, the microarray slide was washed for 1 minute each in Gene Expression Wash Buffer 1 (6X SSPE, 0.005% N-lauroylsarcosine; room temperature) and Gene Expression Wash Buffer (0.06X SSPE, 0.005% N-lauroylsarcosine; 31 °C) for 1 minute each. Microarray slides were briefly dipped in a solution of acetonitrile and dried. Microarray slides were scanned in an Agilent Technologies G2505C Microarray Scanner at 5 ⁇ resolution.
  • the scanner performs detection of Cyanine-3 signal on the hybridized slide.
  • TIF files generated from the scanned microarray image are loaded into Agilent Feature Extraction Software version 10.5.
  • the software automatically positions a grid and finds the centroid positions of each feature on the microarray. This information is used to perform calculations that include feature intensities, background measurements and statistical analyses.
  • Data generated by the software is recorded as a tab-delimited text file.
  • the text data were analyzed using the AgilentFilter and Genesifter software. Differential gene expression was evaluated using the t- test (p ⁇ 0.01 ) and Benjamini and Hochberg correction. The threshold was set at 2-fold change for both upregulated and downregulated genes. Specific differentially expressed genes were confirmed by RT-qPCR.
  • RNA (1 ⁇ g) was primed with oligo (dT) primers and converted into cDNA with RevertAid First Strand cDNA Synthesis Kit (Fermentas).
  • SYBR green-based real time PCR was performed with a Roche LightCycler system, and the reaction mix contained 1 X SYBR green master mix (SABiosciences) and 0.5 ⁇ each of the forward and reverse primers in a volume of 25 ⁇ _.
  • PCR cycling consisted of 95 ° C for 10 min, then 30 cycles of 95 ° C for 10 sec, 60 ° C for 30 sec, 72 ° C for 10 sec, followed by a melting-curve analysis. All PCR primers used are available on request.
  • Genomic DNA (approximately 50 ng to 2 ⁇ g) was sheared to an average size range of 275 bp in a volume of 52.5 ⁇ using a Covaris S2 Focused-ultrasonicator with the following settings: Intensity 5.0; Duty cycle 10%; 25 cycles per Burst 200; Treatment Time 60 seconds. Sheared DNA was converted to blunt-ended fragments with 5'-phosphates and 3'-hydroxyl groups using a combination of enzymes that perform fill-in reactions and exhibit exonuclease activity.
  • Size selection of the blunt-ended DNA was accomplished using bead-based methodologies.
  • An A-base was added to the blunt ends as a means to prepare the fragments for adapter ligation and block concatamer formation during the ligation step.
  • Adapters containing a T-base overhang were ligated to the A-tailed DNA fragments.
  • Adapter-ligated molecules were purified by bead based methodologies and were bisulfite converted using the Qiagen EpiTect Bisulfite Kit (cat# 59104).
  • Bisulfite treated DNA was PCR amplified for 12 cycles using Agilent Pfu Turbo Cx (Agilent cat# 600410) to enrich those fragments that have adapter molecules ligated to both ends.
  • the concentration of the amplified library was measured using the Invitrogen Qubit dsDNA HS Assay (Q32851 ) and an aliquot of the library was resolved on an Agilent 2200 Tape Station using a D1 K (cat# 5067-5361 and 5067-5362) or a High Sensitivity D1 K (cat# 5067-5363 and 5067-5364) assay to define the size range.
  • Libraries were adjusted to a concentration of approximately 10 nM and quantitative PCR was performed using the KapaBiosystems Kapa Library Quant Kit (cat# KK4824) to quantify adapter-ligated library molecules. The concentration was further adjusted following qPCR to prepare the library for lllumina sequence analysis.
  • SAM alignments were generated from lllumina Fastq files aligned to the human hg19 genome using Novocraft's novoalign aligner (http://www.novocrafi.com) in bisulfite mode with the following parameters: -r Random -t 240 -h 120 -b 2 -p bisulfite.
  • An in silico chrLambda sequence was used to align the fully methylated lambda sequence that was spiked into the samples in order to measure the bisulfite conversion efficiency and displayed very low non-conversion/ sequencing error rates of 0.00348 (MCF7) and 0.00245 (MCF7-RON/MSP). These rates were used for scoring individual cytosines for significant methylation with a binomial test.
  • DMRs Differentially- methylated regions
  • NovoalignBisulfiteParser http://useq.sourceforge.net/cmdLnMenus.html#Novoalig nBisulfiteParser was used to parse the text based novoalignments into four binary "PointData" sets containing the number of observed converted Cs (Ts - non methylCs) and non-converted Cs (methylCs) at each reference C sequenced in the genome for both the plus and minus strands.
  • BisStat http://useq.sourceforge.net/cmdLnMenus.html#BisStat) application was used to calculate per base fraction methylation scores for bases with five or more reads from both strands and generate tracks for visualization in IGB. BisStat also calculated fraction methylation for 500bp windows containing a minimum of 5CpGs.
  • Differentially expressed genes (p ⁇ 0.05) were functionally annotated and classified by using the functional annotation tool database for annotation, visualization, and integrated discovery (DAVID), which provides integrated annotation and analysis of genome- scale datasets derived from high-throughput technologies.
  • DAVID integrated discovery
  • EXAMPLE 2 RON/MSP signaling promotes widespread metastasis of human breast cancer in vivo.
  • MCF7- RON/MSP and T47D-RON/MSP RON and MSP cDNAs
  • Overexpressing cells had comparable RON levels with the metastatic breast cancer cell lines.
  • the effects of RON/MSP gain-of-function on tumor growth and/or metastasis of MCF7 and T47D tumors were assessed in immune- compromised mice by orthotopic implantation into the cleared inguinal mammary fat pads of 3-week old female NOD/SCID mice.
  • FIG. 8A Shown in Figure 8A are tumor growth curves following orthotopic transplantation of MCF7 or T47D cells (blue) or MCF7-RON/MSP or T47-RON/MSP (red) into cleared inguinal mammary fat pads of 3-week old NOD/SCID mice.
  • mice were euthanized for analysis of metastasis when the tumors reached the same size endpoint for each group (after 25 and 16 weeks for orthotopically implanted MCF7 and MCF7-RON/MSP, respectively, and after 25 weeks for both T47D and T47D-RON/MSP; Figure 8A).
  • RON/MSP expression was sufficient to induce high rates of spontaneous lung, bone, liver and brain metastasis from MCF7-RON/MSP and T47D-RON/MSP tumors, compared with little or no metastasis from the parental cell xenografts ( Figures 1 B and C).
  • RNA sequencing RNA-seq
  • microarray gene expression profiling of MCF7 and MCF7-RON/MSP cells. Sequencing was performed with 3 biological replicates for each cell line. Sequencing reads were aligned to the hg19 human genome with all known and theoretical splice junctions using Novocraft's novoalign aligner. From individual samples, we obtained 24 million reads that mapped to the human genome. The microarray was performed with four biological replicates for each cell line and the data were median normalized.
  • RNA-seq For comparative analysis between RNA-seq and microarray, we focused only on those reads which aligned to annotated regions of the human genome, as microarray probes are designed primarily against annotated regions. Differentially expressed genes, based on a false discovery rate threshold of 1 % and a fold change of 2, were highly correlated between the two methods (Figure 8C). Shown in Figure 8C is a scatter plot showing differential expression obtained using microarray (x axis) and RNA-seq (y axis) between MCF7 and MCF7-RON/MSP. Points represent genes, located based on estimates of differential expression using RNA-Seq and Microarray. Red points represent genes for which FDR ⁇ 0.1 and fold change>2.
  • Green points represent genes differentially expressed identified by RNA-seq only.
  • Purple points represent genes differentially expressed identified by microarray only. The line corresponds to perfect agreement between the technologies.
  • the GO term analysis was not used in our subsequent analysis, but indicated that RON/MSP signaling induces expression of genes whose functions are consistent with biological pathways relevant to metastasis. However, the data did not immediately implicate a clear mechanistic pathway to explain how RON/MSP drives metastasis.
  • DEGs Differentially expressed genes identified from both RNA-sequencing and microarray.
  • EXAMPLE 3 RON/MSP signaling upregulates the thymine glycosylase MBD4 and drives DNA methylation reprogramming.
  • FIG. 2A Shown in Figure 2A is a circular representation of genome-wide aberrant DNA methylation caused by RON/MSP expression. Average methylation levels for all of the CGs in 10-Mbp-wide windows are shown in the blue-green tracks (the outermost track shows MCF7 cells; the middle track shows MCF7- RON/MSP cells). The innermost track indicates the differentially methylated regions (DMRs) between MCF7 and MCF7-RON/MSP cells (FDR>20, differential methylation>0.25).
  • DMRs differentially methylated regions
  • DMRs occurred in both gene-body and intergenic regions, both inside and outside CpG islands (Figure 2B). Shown in Figure 2B is (top panel) a distribution of differentially methylated CpGs on CpG islands (CpGi) shelves (>2 to 4 kb from island edge) and on CpGi shores (0-2 kb from island edge); within and outside CpGi; u, upstream of CpGi; d, downstream of CpGi. In the bottom panel is a distribution of differentially methylated CpGs across regions of other significance.
  • TSS 200 within the region 1-200 bp upstream of the TSS; TSS 1500, within the region 201-1500 bp upstream of the transcription start site (TSS); UTR, untranslated region; HyperMe, hypermethylated; HypoMe, hypomethylated. DMRs regulated by RON/MSP were often intergenic, and there was a significant enrichment of hypomethylated versus hypermethylated regions (Figure 2B-C). Shown in Figure 2C is an example of smoothed methylation levels from bisulfite sequencing data for MCF7 (black) and MCF7-RON/MSP (blue) on chromosome 14. A hypomethylation block is indicated by a red bar.
  • TDG thymine DNA glycosylase
  • RON and MBD4 are coordinately expressed in actual human breast tumors.
  • TCGA Cancer Genome Atlas
  • RON and MBD4 were both upregulated in breast tumors compared to the normal tissue ( Figure 3A, a boxplot showing RON and MBD4 mRNA expression in normal breast tissues (white) and breast tumors (dark grey) from The Cancer Genome Atlas (TCGA) data set).
  • Figure 3A a boxplot showing RON and MBD4 mRNA expression in normal breast tissues (white) and breast tumors (dark grey) from The Cancer Genome Atlas (TCGA) data set.
  • RON and MBD4 protein expression in 3 breast reduction tissues and 13 human primary breast tumors by western analysis. RON and MBD4 were highly expressed in all 7 poorly differentiated carcinomas.
  • EXAMPLE 4 Knockdown of MBD4 blocks MSP/RON-mediated breast cancer metastasis.
  • MBD4 knockdown was validated by Western blotting (Figure 3C).
  • Shown in Figure 10A is tumor growth following orthotopic implantation of MCF7 (dark blue), MCF7-RON/MSP (red), MCF7-RON/MSP-shScr (green), MCF7-RON/MSP-shMBD4 (yellow) and MCF7- RON/MSP-shMBD4R cells (light blue) in MCF7 (left) and T47D (right) models.
  • spontaneous lung, liver, bone and brain metastasis was significantly inhibited in animals carrying MCF7-RON/MSP-shMBD4 and T47D-RON/MSP-shMBD4 tumors relative to the control tumors expressing RON/MSP ( Figure 3D and 3E).
  • the catalytic mutant shMBD4R D560A
  • metastasis frequencies of RON/MSP-shMBD4 cells were not significantly different from the original parental cell lines, approaching 0%.
  • the RON/MSP-shMBD2 and the RON/MSP- shScr control tumors showed a similar frequency of metastasis as the RON/MSP tumors ( Figure 3D, 3E, 10B, and 10C).
  • FIG. 10B Shown in Figure 10B are metastasis frequencies for MCF7, MCF7-RON/MSP, MCF7-RON/MSP-shScr, and MCF7-RON/MSP-shMBD2 tumors
  • Figure 10C are representative images showing lack of effect of sh-MBD2 on spontaneous lung, bone, liver and brain metastasis of RON/MSP-expressing MCF7 tumors.
  • MBD4 rescue constructs (MBD4R-3UTR and MBD4R-CDS) that were resistant to their respective shRNAs.
  • MCF7 and T47D cells expressing RON/MSP-shMBD4R (D560A) into mammary fad pads of NOD/SCID mice and monitored metastasis.
  • the mutation prevented the rescue of the metastasis phenotype observed with the wild type MBD4 rescue constructs, suggesting that MBD4 is driving metastasis through its glycosylase activity ( Figure 3D and 3E).
  • MBD4 rescue constructs suggesting that MBD4 is driving metastasis through its glycosylase activity
  • EXAMPLE 5 Knockdown of MBD4 reverses abnormal DNA methylation patterns and reverses expression of RON/MSP-regulated genes.
  • MBD4 knockdown reversed the methylation status of a specific collection of loci in MCF7-RON/MSP cells. These regions of aberrant DNA methylation were reprogrammed following the knockdown of MBD4, to levels that were comparable with parental MCF7 cells ( Figure 4A and 4B).
  • Shown in Figure 4A is a representation of smoothed methylation values from bisulfite sequencing data for MCF7 (black), MCF7-RON/MSP (blue) and MCF7- RON/MSP-shMBD4 cells (purple) in a representative region of chromosome 2.
  • the hypomethylation block in MCF7-RON/MSP that becomes hypermethylated by knocking- down MBD4 is indicated by a red bar.
  • Shown in Figure 4B is an example of DNA methylation levels in promoter regions in MCF7 (black), MCF7-RON/MSP (blue) and MCF7- RON/MSP-shMBD4 cells (purple).
  • the hypomethylated block in MCF7-RON/MSP cells that becomes re-methylated by knocking down MBD4 is indicated by a red bar. It is important to note that knocking down MBD4 also caused changes in methylation of some loci that were independent of RON/MSP (Figure 4C). Shown in Figure 4C is an example of DNA methylation levels in a promoter region that becomes hypermethylated in shMBD4 cells, independent of RON/MSP expression (red bar) Bisulfite sequencing data for MCF7 (black), MCF7-RON/MSP (blue) and MCF7-RON/MSP-shMBD4 cells (purple) are shown. In all panels, the orange bars indicate CpG islands and the black bars show the genes.
  • the outside track represents the 192 genes regulated by RON/MSP for which expression was reversed following knockdown of MBD4 (blue: genes downregulated by RON/MSP and then reversed by knocking down MBD4 with shRNA; orange: genes upregulated by RON/MSP and then reversed by knocking down MBD4).
  • the inner track represents the statistically significant DMRs at these regions (red: hypermethylated DMRs in MCF7-RON/MSP and then reversed by knocking down MBD4; green: hypomethylated DMRs in MCF7-RON/MSP and then reversed by knocking down MBD4).
  • EXAMPLE 6 Specific subclasses of human breast tumors possess the RON/MBD4 epigenetic signature, which correlates with poor prognosis
  • RON/MBD4 epigenetic signature was present in human breast tumors.
  • a gene module map (Segal et al., Nature Genetics 2004, 36, 1090-1098) to examine the expression of 1 16 genes of the 192 above-mentioned genes in 997 primary human breast cancers from the Metabric discovery cohort (the remainder of the genes were not annotated in the Metabric dataset, Figure 5A genes in large characters).
  • the RON/MBD4 epigenetic metastasis signature presence of the signature was defined as a minimum of 5 genes differentially expressed from the RON/MBD4 signature, i.e., a minimum of 5 genes shown in Table 1 ) was present approximately in 25% of the breast cancers (Figure 5B).
  • Shown in Figure 5B is an enrichment pattern of the gene set comprising the RON/MBD4 epigenetic signature (rows) across 997 breast tumors (columns). Red and green indicated significantly over or underexpressed gene, respectively. Are represented the 582 patients having enrichment or underrepresentation of the gene set. Blue bars (right) indicate individual tumor annotations for breast cancer subtype. The table (right) shows the association of the RON/MBD4 epigenetic signature with ER, HER2, and PR status, as well as intrinsic breast cancer subtype, grade and death (we assigned a p value according to the hypergeometric distribution).
  • the survival hazard ratio was calculated using Univariate Cox's regression analysis. These data strongly suggest that the RON/MBD4 pathway exists in a fairly large subset of human breast cancers (25%), and that detection of the novel RON/MBD4 epigenetic pathway could significantly contribute to risk assessment in breast cancer patients. Thus, an important corollary to this work was to ask how RON regulates MBD4 and whether the presence of a RON/MBD4 epigenetic signature would predict response to anti-Ron therapy.
  • EXAMPLE 7 MBD4 is upregulated by RON/MSP through PI3K signaling
  • RON was knocked down using shRNA via lentiviral infection in both the RON-positive DU4475 breast cancer cell line and in primary culture from a RON-positive patient-derived breast tumor graft. Scrambled shRNA were used as control. RON knockdown, although not complete, caused a reduction in MBD4 expression in both models ( Figure 6B).
  • HCI-014 patient derived breast tumor graft
  • RON is known to transduce a variety of signals that regulate different downstream pathways, most notably the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways.
  • MAPK mitogen-activated protein kinase
  • PI3K phosphatidylinositol 3-kinase
  • MBD4 phosphorylation was not altered as a result of RON/MSP pathway activation ( Figure 11 B, showing the expression of MET, MBD4 and ACTB in MCF7 cell line infected with retroviruses carrying cDNA of MET) suggesting that MBD4 is not a direct target of RON kinase or kinases in the PI3K pathway.
  • Figure 11C Shown in Figure 11C are MCF7 and MCF7- RON/MSP cells were treated with or without alkaline phosphatase for 30 min.
  • the lysates were subjected to (a) conventional SDS-PAGE on 8% (wt/vol) polyacrylamide gel or (b) SDS-PAGE on 6% (wt/vol) polyacrylamide gel containing 20 ⁇ Mn2+-Phos-tag, followed by immunoblotting with the anti-MBD4 antibody.
  • a) conventional SDS-PAGE on 8% (wt/vol) polyacrylamide gel or (b) SDS-PAGE on 6% (wt/vol) polyacrylamide gel containing 20 ⁇ Mn2+-Phos-tag followed by immunoblotting with the anti-MBD4 antibody.
  • alkaline phosphatase shifts in the mobility of MBD4 were suppressed.
  • EXAMPLE 8 Treatment with a RON kinase inhibitor prevents metastasis of primary patient-derived tumor grafts.
  • Ron kinase activity can be blocked by small molecule kinase inhibitors.
  • small molecule kinase inhibitors To determine if the RON/MBD4 pathway can be blocked in patient tumors using RON kinase inhibitors, and if there is a resulting effect on metastasis, we utilized four independent experimental systems that are highly relevant to bona fide breast tumors. First, we chose two patient-derived tumor grafts that survive primary culture conditions and treated them with the RON/MET dual inhibitor, OSI-296 in vitro. Inhibition of RON (verified by reduction of phosphorylated RON protein) caused downregulation of MBD4 ( Figure 7A).
  • FIG. 7A Shown in Figure 7A are Western blots with anti-p-RON, MBD4 and ⁇ -actin antibodies on protein lysates from primary cultures of the patient-derived tumor graft HCI-007, treated without (-) or with 1 ⁇ of the RON inhibitor OSI-296 over the indicated times.
  • the tumor grafts were implanted orthotopically into cleared mammary fat pads of NOD/SCID mice and allowed to grow to 100 mm 3 before starting treatment with OSI-296.
  • Shown in Figure 7C are sections of axillary lymph nodes isolated from mice carrying orthotopic (inguinal fat pad) patient-derived breast tumor grafts HCI-003 and HCI- 01 1 following treatment with trappsol or OSI-296. The sections were stained with H&E (left panels) or were immunostained with antibodies specific for human cytokeratin (right panels) to detect human tumor cells.
  • Shown in Figure 7D are sections of lungs isolated from mice carrying orthotopic patient-derived breast tumor grafts HCI-003 or HCI-01 1 following treatment with trappsol or OSI-296. The sections were stained with H&E (left) or were immunostained with antibodies specific for human cytokeratin (CK; right) to identify human tumor cells.
  • FIG. 7F Shown in Figure 7F is Kaplan-Meier analysis of overall survival in 997 breast cancer patients from the METABRIC discovery dataset (left) and in 977 breast cancer patients from the compendium dataset (right). Survival curve of individuals with tumors showing an enrichment of the 9 RON/MBD4 epigenetic signature genes that were deregulated in tumors from mice treated with OSI-296 compared to the trappsol control is shown in red; all other patients are shown in blue (No signature). The p value indicates a statistically significant survival difference between these two groups of patients. The survival hazard ratio was calculated using Univariate Cox's regression analysis. To validate our findings using additional, independent data, we generated a meta- collection of gene expression data from an additional 977 patients from five independently published studies of breast cancer.
  • FIG. 7G Shown in Figure 7G are univariate and multivariate cox regression analyses for overall survival in the 997 patients from the METABRIC dataset, examining ER, PR, status, basal-like subtype, and the MBD4/RON signature as variables (HR, hazard ratio). While ER- tumors have a poor prognosis, there are no targeted therapy options due to lack of a defined pathway driving these tumors. Together, these data highlight the promising potential for (A) identifying breast cancer patients that might benefit from a RON inhibitor; (B) identifying a set of biomarkers for RON inhibition, and (C) blocking RON to inhibit breast cancer metastasis.
  • Polypeptide of SEQ ID NO: 1 homo sapiens, 53 AA

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Abstract

Provided are methods of treating breast cancer. Further provided are methods and kits for assessing the likelihood that breast cancer in a subject will metastasize or has metastasized. Further provided are methods and kits for determining whether a subject having breast cancer would benefit from treatment with at least one of a RON inhibitor and a PI3K kinase inhibitor. Further provided are methods of monitoring a molecular response of a subject to treatment of breast cancer with at least one of a RON inhibitor and a PI3K inhibitor.

Description

BIOMARKERS FOR BREAST CANCER AND METHODS OF USING SAME
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent Application No. 61/721 ,977, filed November 2, 2012, and U.S. Provisional Patent Application No. 61/851 ,796, filed March 13, 2013, each of which is incorporated herein by reference in its entirety.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made in part with government support under grant DOD 08-1 - 0109 awarded by the United States Department of Defense Breast Cancer Research Program and in part with foundation support under grant KG081251 from Susan G. Komen for the Cure. The government has certain rights in the invention.
INTRODUCTION
[0003] Metastasis is the main cause of death in cancer patients, and there are currently no therapies that specifically prevent the metastatic process or cure metastatic disease. Metastasis is a multistep, dynamic process that remains highly enigmatic, and a better understanding of mechanisms that facilitate metastasis continues to be required for development of new treatments. Transcriptional profiling has clearly demonstrated that there are sets of genes, or signatures, for which expression in primary tumors correlates with metastasis and/or poor survival, although the mechanisms orchestrating many of these gene expression programs have not been defined. For example, a 70-gene signature has been identified that is associated with metastasis, which is now used clinically as the MAMMAPRINT® test (AGendia, Irvina CA). Moreover, some specific gene sets have been found to be associated with metastasis to lung, bones, or the brain. A variety of experimental systems have revealed many potentially critical mediators of metastasis that convey pro- metastatic features upon tumor cells; however, it is likely that multiple, coordinated events contribute to metastasis. The astonishing heterogeneity of tumors also suggests that tumor cells may achieve metastasis through many independent pathways, so it is not likely that targeting a single gene or pathway would be beneficial for a large number of patients. On the other hand, if there are coordinated transcriptional programs that drive a significant proportion of metastasis, and if such programs can be identified in tumors and targeted for therapy, it could advance the field considerably. SUMMARY
[0004] In some aspects, this disclosure provides methods of treating breast cancer in a subject. The methods may include determining in a sample of the breast cancer an expression level of one or more genes selected from CSGALNACT1, SIGLEC6, SHC4, ELTD1 , CGA, MST1R, KRT8P41 , BTF3P4, RP11-478C6.4, EPHA3, LXN, ENSG00000252044, ENSG00000242932, ENSG00000241115, AP001347.6, LHFPL2, CTD-2330K9.2, RASD1, ENSG00000242693, AC093388.3, AOC1, CPE, PCDH8, DCHS2, DPP4, ENSG00000242777, GUSBP5, ENSG00000244466, ENSG00000252331 , RP11- 664D7.4, RP11-206M11.7, ENSG00000244580, EYA4, CTA-714B7.5, ENSG00000252318, ABCA1, PLD1, RNF144A, SLC44A4, SLC2A13, AXIN2, PGR, XXbac-BPG254F23.6, AMY2B, LIMCH1, RP11-356J5.12, ID2, MFAP2, LPHN2, RP11-594N15.2, RP11-575A19.2, SPINK5, HLA-DQB1-AS1, RN7SL151P, ATP8A1, FAM189A2, ZNF423, CTD-2001C12.1 , ELOVL2, TESC, KCNMB4, STOX2, PLEKHM3, MT-TL1, KLK11, NPNT, ENSG00000234543, SHC2, BTNL9, ARMC4, OLFML2A, AMOT, RP11-556114.1 , SLFN5, PKIA, CTC-339F2.2, ADAM23, NPSR1-AS1, NREP, AC009410.1, IMPG1, C12orf40, AC097500.2, AC004069.2, CDH26, CCDC33, PCDP1, ALCAM, LRRN1, WWC3-AS1, MT- TY, MXRA8, RASL11A, COL3A1, PDE1A, COL21A1, NTM, GNG4, RAMP3, AC026188.1, ELOVL2-AS1, SMAD9, ZBTB20, GNG12-AS1, EFNA5, RP11-713M6.2, AC078883.3, PAMR1, BMPER, NYAP2, RP4-813D12.3, HMCN1, FAT4, CYP2C8, PLLP, NXPH1, CTD- 2260A17.2, MUC5AC, PP13439, SOCS2-AS1, CCM2L, ZNF827, ADAMTSL3, SDC2, RBM11, PRICKLE2-AS3, RNF180, DOCK10, FAM198A, TMEM86A, LGALS3BP, KLK8, AC012307.2, AC004053.1, CTD-3179P9.1 , NOD2, RP11-556114.2, C3, ZNF396, ZNF792, ENSG00000244288, CHRM3, AGBL4, KLF7, AP004372.1, DKK2, NPAS3, SMARCA1, DOK6, SULF1, RORC, YPEL1, LRP1B, RP4-710M3.2, DCLK1, ENSG00000235732, NIPSNAP3B, SEPP1, LY6D, ENSG00000244428, AKR1C3, GJA1, CXXC4, FSTL5, CYBRD1, SERPINA1, AC004540.5, RP11-600K15.1 , ASTL, C20orf197, KCNS3, CTC- 448D22.1, TMEM45ARP11-86516.2, SNCG, CCDC152, MURC, TMCC2, FBN2, FREM1, ENSG00000235471, ENSG00000170233, THRB, AC004947.2, C1orf168, RN7SKP3, THBS2, TREHAC007365.3, ATOH8, SEMA3D, and GPC6; comparing the expression level of each gene to a control value associated with that same gene; and administering at least one of a RON inhibitor and a PI3K kinase inhibitor to the subject when the expression level of at least one of CSGALNACT1, SIGLEC6, SHC4, ELTD1, CGA, MST1R, KRT8P41, BTF3P4, RP11-478C6.4, EPHA3, LXN, ENSG00000252044, ENSG00000242932, ENSG00000241115, AP001347.6, LHFPL2, CTD-2330K9.2, RASD1, ENSG00000242693, AC093388.3, AOC1, CPE, PCDH8, DCHS2, DPP4, ENSG00000242777, GUSBP5, ENSG00000244466, ENSG00000252331 , RP11-664D7.4, RP11-206M11.7, ENSG00000244580, EYA4, CTA-714B7.5, and ENSG00000252318 is increased at least about 2-fold relative to the control, or administering an alternative therapy to the subject when the expression level of the gene is increased less than about 2-fold or decreased relative to the control; or administering at least one of a RON inhibitor and a PI3K kinase inhibitor to the subject when the expression level of at least one of ABCA1 , PLD1 , RNF144A, SLC44A4, SLC2A13, AXIN2, PGR, XXbac-BPG254F23.6, AMY2B, LIMCH1 , RP1 1- 356J5.12, ID2, MFAP2, LPHN2, RP1 1-594N15.2, RP1 1-575A19.2, SPINK5, HLA-DQB1- AS1 , RN7SL151 P, ATP8A1 , FAM189A2, ZNF423, CTD-2001 C12.1 , ELOVL2, TESC, KCNMB4, STOX2, PLEKHM3, MT-TL1 , KLK1 1 , NPNT, ENSG00000234543, SHC2, BTNL9, ARMC4, OLFML2A, AMOT, RP1 1-556114.1 , SLFN5, PKIA, CTC-339F2.2, ADAM23, NPSR1-AS1 , NREP, AC009410.1 , IMPG1 , C12orf40, AC097500.2, AC004069.2, CDH26, CCDC33, PCDP1 , ALCAM, LRRN1 , WWC3-AS1 , MT-TY, MXRA8, RASL1 1A, COL3A1 , PDE1A, COL21A1 , NTM, GNG4, RAMP3, AC026188.1 , ELOVL2-AS1 , SMAD9, ZBTB20, GNG12-AS1 , EFNA5, RP1 1-713M6.2, AC078883.3, PAMR1 , BMPER, NYAP2, RP4- 813D12.3, HMCN1 , FAT4, CYP2C8, PLLP, NXPH1 , CTD-2260A17.2, MUC5AC, PP13439, SOCS2-AS1 , CCM2L, ZNF827, ADAMTSL3, SDC2, RBM1 1 , PRICKLE2-AS3, RNF180, DOCK10, FAM198A, TMEM86A, LGALS3BP, KLK8, AC012307.2, AC004053.1 , CTD- 3179P9.1 , NOD2, RP1 1-556114.2, C3, ZNF396, ZNF792, ENSG00000244288, CHRM3, AGBL4, KLF7, AP004372.1 , DKK2, NPAS3, SMARCA1 , DOK6, SULF1 , RORC, YPEL1 , LRP1 B, RP4-710M3.2, DCLK1 , ENSG00000235732, NIPSNAP3B, SEPP1 , LY6D, ENSG00000244428, AKR1 C3, GJA1 , CXXC4, FSTL5, CYBRD1 , SERPINA1 , AC004540.5, RP1 1-600K15.1 , ASTL, C20orf197, KCNS3, CTC-448D22.1 , TMEM45A, RP1 1-86516.2, SNCG, CCDC152, MURC, TMCC2, FBN2, FREM1 , ENSG00000235471 , ENSG00000170233, THRB, AC004947.2, C1orf168, RN7SKP3, THBS2, TREH, AC007365.3, ATOH8, SEMA3D, and GPC6 is decreased at least about 2-fold relative to the control, or administering an alternative therapy to the subject when the expression level of the gene is decreased less than about 2-fold or increased relative to the control. In some embodiments, the alternative therapy is selected from the group consisting of radiation therapy, a surgical procedure, anti-cancer drugs, or any combination thereof.
[0005] In some aspects, this disclosure provides methods of assessing the likelihood that breast cancer in a subject will metastasize or has metastasized. The methods may include determining in a sample of the breast cancer an expression level of one or more genes selected from CSGALNACT1 , SIGLEC6, SHC4, ELTD1 , CGA, MST1 R, KRT8P41 , BTF3P4, RP1 1-478C6.4, EPHA3, LXN, ENSG00000252044, ENSG00000242932, ENSG00000241 1 15, AP001347.6, LHFPL2, CTD-2330K9.2, RASD1 , ENSG00000242693, AC093388.3, AOC1 , CPE, PCDH8, DCHS2, DPP4, ENSG00000242777, GUSBP5, ENSG00000244466, ENSG00000252331 , RP1 1-664D7.4, RP1 1-206M1 1.7, ENSG00000244580, EYA4, CTA-714B7.5, ENSG00000252318, ABCA1 , PLD1 , RNF144A, SLC44A4, SLC2A13, ΑΧΙΝ2, PGR, XXbac-BPG254F23.6, ΑΜΥ2Β, LIMCH1 , RP1 1- 356J5.12, ID2, MFAP2, LPHN2, RP1 1-594Ν15.2, RP1 1-575Α19.2, SPINK5, HLA-DQB1- AS1 , RN7SL151 P, ΑΤΡ8Α1 , FAM189A2, ZNF423, CTD-2001 C12.1 , ELOVL2, TESC, KCNMB4, STOX2, PLEKHM3, MT-TL1 , KLK1 1 , ΝΡΝΤ, ENSG00000234543, SHC2, BTNL9, ARMC4, OLFML2A, ΑΜΟΤ, RP1 1-556114.1 , SLFN5, ΡΚΙΑ, CTC-339F2.2, ADAM23, NPSR1-AS1 , NREP, AC009410.1 , IMPG1 , C12orf40, AC097500.2, AC004069.2, CDH26, CCDC33, PCDP1 , ALCAM, LRRN1 , WWC3-AS1 , ΜΤ-ΤΥ, MXRA8, RASL1 1A, COL3A1 , PDE1A, COL21A1 , ΝΤΜ, GNG4, RAMP3, AC026188.1 , ELOVL2-AS1 , SMAD9, ΖΒΤΒ20, GNG12-AS1 , EFNA5, RP1 1-713Μ6.2, AC078883.3, PAMR1 , BMPER, ΝΥΑΡ2, RP4- 813D12.3, HMCN1 , FAT4, CYP2C8, PLLP, ΝΧΡΗ1 , CTD-2260A17.2, MUC5AC, ΡΡ13439, SOCS2-AS1 , CCM2L, ZNF827, ADAMTSL3, SDC2, RBM1 1 , PRICKLE2-AS3, RNF180, DOCK10, FAM198A, ΤΜΕΜ86Α, LGALS3BP, KLK8, AC012307.2, AC004053.1 , CTD- 3179Ρ9.1 , NOD2, RP1 1-556114.2, C3, ZNF396, ZNF792, ENSG00000244288, CHRM3, AGBL4, KLF7, ΑΡ004372.1 , DKK2, NPAS3, SMARCA1 , DOK6, SULF1 , RORC, YPEL1 , LRP1 B, RP4-710M3.2, DCLK1 , ENSG00000235732, NIPSNAP3B, SEPP1 , LY6D, ENSG00000244428, AKR1 C3, GJA1 , CXXC4, FSTL5, CYBRD1 , SERPINA1 , AC004540.5, RP1 1-600K15.1 , ASTL, C20orf197, KCNS3, CTC-448D22.1 , ΤΜΕΜ45Α, RP1 1-86516.2, SNCG, CCDC152, MURC, TMCC2, FBN2, FREM1 , ENSG00000235471 , ENSG00000170233, THRB, AC004947.2, C1orf168, RN7SKP3, THBS2, TREH, AC007365.3, ΑΤΟΗ8, SEMA3D, and GPC6; comparing the expression level of each gene to a control value associated with that same gene; and assessing the likelihood that the breast cancer will metastasize or has metastasized based on the relative difference between the expression level and the control value associated with each gene, wherein an increase in the expression level relative to the control value of at least one gene selected from CSGALNACT1 , SIGLEC6, SHC4, ELTD1 , CGA, MST1 R, KRT8P41 , BTF3P4, RP1 1- 478C6.4, EPHA3, LXN, ENSG00000252044, ENSG00000242932, ENSG00000241 1 15, AP001347.6, LHFPL2, CTD-2330K9.2, RASD1 , ENSG00000242693, AC093388.3, AOC1 , CPE, PCDH8, DCHS2, DPP4, ENSG00000242777, GUSBP5, ENSG00000244466, ENSG00000252331 , RP1 1-664D7.4, RP1 1-206M1 1.7, ENSG00000244580, EYA4, CTA- 714B7.5, and ENSG00000252318 correlates with an increased likelihood that the breast cancer in the subject will metastasize or has metastasized, or wherein a decrease in the expression level relative to the control value of at least one gene selected from ABCA1 , PLD1 , RNF144A, SLC44A4, SLC2A13, AXIN2, PGR, XXbac-BPG254F23.6, AMY2B, LIMCH1 , RP1 1-356J5.12, ID2, MFAP2, LPHN2, RP11-594N15.2, RP1 1-575A19.2, SPINK5, HLA-DQB1-AS1 , RN7SL151 P, ATP8A1 , FAM189A2, ZNF423, CTD-2001 C12.1 , ELOVL2, TESC, KCNMB4, STOX2, PLEKHM3, MT-TL1 , KLK1 1 , NPNT, ENSG00000234543, SHC2, BTNL9, ARMC4, OLFML2A, AMOT, RP1 1-556114.1 , SLFN5, PKIA, CTC-339F2.2, ADAM23, NPSR1-AS1 , NREP, AC009410.1 , IMPG1 , C12orf40, AC097500.2, AC004069.2, CDH26, CCDC33, PCDP1 , ALCAM, LRRN1 , WWC3-AS1 , MT-TY, MXRA8, RASL1 1A, COL3A1 , PDE1A, COL21A1 , NTM, GNG4, RAMP3, AC026188.1 , ELOVL2-AS1 , SMAD9, ZBTB20, GNG12-AS1 , EFNA5, RP1 1-713M6.2, AC078883.3, PAMR1 , BMPER, NYAP2, RP4- 813D12.3, HMCN1 , FAT4, CYP2C8, PLLP, NXPH1 , CTD-2260A17.2, MUC5AC, PP13439, SOCS2-AS1 , CCM2L, ZNF827, ADAMTSL3, SDC2, RBM1 1 , PRICKLE2-AS3, RNF180, DOCK10, FAM198A, TMEM86A, LGALS3BP, KLK8, AC012307.2, AC004053.1 , CTD- 3179P9.1 , NOD2, RP1 1-556114.2, C3, ZNF396, ZNF792, ENSG00000244288, CHRM3, AGBL4, KLF7, AP004372.1 , DKK2, NPAS3, SMARCA1 , DOK6, SULF1 , RORC, YPEL1 , LRP1 B, RP4-710M3.2, DCLK1 , ENSG00000235732, NIPSNAP3B, SEPP1 , LY6D, ENSG00000244428, AKR1 C3, GJA1 , CXXC4, FSTL5, CYBRD1 , SERPINA1 , AC004540.5, RP1 1-600K15.1 , ASTL, C20orf197, KCNS3, CTC-448D22.1 , TMEM45A, RP1 1-86516.2, SNCG, CCDC152, MURC, TMCC2, FBN2, FREM1 , ENSG00000235471 , ENSG00000170233, THRB, AC004947.2, C1orf168, RN7SKP3, THBS2, TREH, AC007365.3, ATOH8, SEMA3D, and GPC6 correlates with an increased likelihood that the breast cancer in the subject will metastasize or has metastasized.
[0006] In other aspects, the disclosure provides methods of determining whether a subject having breast cancer would benefit from treatment with at least one of a RON inhibitor and a PI3K kinase inhibitor. The methods may include assessing the likelihood that the breast cancer will metastasize or has metastasized; and determining whether the subject would benefit from treatment with at least one of a RON inhibitor and a PI3K kinase inhibitor based on the likelihood that the breast cancer will metastasize or has metastasized.
[0007] In some embodiments, the expression level of at least five genes selected from CSGALNACT1 , SIGLEC6, SHC4, ELTD1 , CGA, MST1 R, KRT8P41 , BTF3P4, RP1 1- 478C6.4, EPHA3, LXN, ENSG00000252044, ENSG00000242932, ENSG00000241 1 15, AP001347.6, LHFPL2, CTD-2330K9.2, RASD1 , ENSG00000242693, AC093388.3, AOC1 , CPE, PCDH8, DCHS2, DPP4, ENSG00000242777, GUSBP5, ENSG00000244466, ENSG00000252331 , RP1 1-664D7.4, RP1 1-206M1 1.7, ENSG00000244580, EYA4, CTA- 714B7.5, and ENSG00000252318 is increased relative to the control value. In some embodiments, the expression level of at least one gene selected from CSGALNACT1 , SIGLEC6, and SHC4 is increased relative to the control value.
[0008] In some embodiments, the expression level of at least five genes selected from ABCA1 , PLD1 , RNF144A, SLC44A4, SLC2A13, AXIN2, PGR, XXbac-BPG254F23.6, AMY2B, LIMCH1, RP11-356J5.12, ID2, MFAP2, LPHN2, RP11-594N15.2, RP11-575A19.2, SPINK5, HLA-DQB1-AS1 , RN7SL151P, ATP8A1 , FAM189A2, ZNF423, CTD-2001C12.1 , ELOVL2, TESC, KCNMB4, STOX2, PLEKHM3, MT-TL1 , KLK11, ΝΡΝΤ, ENSG00000234543, SHC2, BTNL9, ARMC4, OLFML2A, AMOT, RP11-556114.1 , SLFN5, ΡΚΙΑ, CTC-339F2.2, ADAM23, NPSR1-AS1 , NREP, AC009410.1, IMPG1 , C12orf40, AC097500.2, AC004069.2, CDH26, CCDC33, PCDP1 , ALCAM, LRRN1 , WWC3-AS1 , ΜΤ- ΤΥ, MXRA8, RASL11A, COL3A1, PDE1A, COL21A1, ΝΤΜ, GNG4, RAMP3, AC026188.1, ELOVL2-AS1 , SMAD9, ΖΒΤΒ20, GNG12-AS1 , EFNA5, RP11-713Μ6.2, AC078883.3, PAMR1 , BMPER, ΝΥΑΡ2, RP4-813D12.3, HMCN1 , FAT4, CYP2C8, PLLP, ΝΧΡΗ1 , CTD- 2260Α17.2, MUC5AC, ΡΡ13439, SOCS2-AS1 , CCM2L, ZNF827, ADAMTSL3, SDC2, RBM11, PRICKLE2-AS3, RNF180, DOCK10, FAM198A, ΤΜΕΜ86Α, LGALS3BP, KLK8, AC012307.2, AC004053.1, CTD-3179P9.1 , NOD2, RP11-556114.2, C3, ZNF396, ZNF792, ENSG00000244288, CHRM3, AGBL4, KLF7, ΑΡ004372.1, DKK2, NPAS3, SMARCA1, DOK6, SULF1, RORC, YPEL1, LRP1B, RP4-710M3.2, DCLK1, ENSG00000235732, NIPSNAP3B, SEPP1, LY6D, ENSG00000244428, AKR1C3, GJA1, CXXC4, FSTL5, CYBRD1, SERPINA1, AC004540.5, RP11-600Κ15.1 , ASTL, C20orf197, KCNS3, CTC- 448D22.1, TMEM45ARP11-86516.2, SNCG, CCDC152, MURC, TMCC2, FBN2, FREM1, ENSG00000235471, ENSG00000170233, THRB, AC004947.2, C1orf168, RN7SKP3, THBS2, TREHAC007365.3, ΑΤΟΗ8, SEMA3D, and GPC6 is decreased relative to the control value. In some embodiments, the expression level of at least one gene selected from ABCA1, PLD1, RNF144A, SLC44A4, SLC2A13, and AXIN2 is decreased relative to the control value.
[0009] In some embodiments, the expression level of at least five genes selected from CSGALNACT1, SIGLEC6, SHC4, ELTD1, CGA, MST1R, KRT8P41, BTF3P4, RP11- 478C6.4, EPHA3, LXN, ENSG00000252044, ENSG00000242932, ENSG00000241115, AP001347.6, LHFPL2, CTD-2330K9.2, RASD1, ENSG00000242693, AC093388.3, AOC1, CPE, PCDH8, DCHS2, DPP4, ENSG00000242777, GUSBP5, ENSG00000244466, ENSG00000252331, RP11-664D7.4, RP11-206M11.7, ENSG00000244580, EYA4, CTA- 714B7.5, ENSG00000252318, ABCA1, PLD1, RNF144A, SLC44A4, SLC2A13, AXIN2, PGR, XXbac-BPG254F23.6, AMY2B, LIMCH1, RP11-356J5.12, ID2, MFAP2, LPHN2, RP11-594N15.2, RP11-575A19.2, SPINK5, HLA-DQB1-AS1 , RN7SL151P, ATP8A1, FAM189A2, ZNF423, CTD-2001C12.1 , ELOVL2, TESC, KCNMB4, STOX2, PLEKHM3, MT- TL1, KLK11, NPNT, ENSG00000234543, SHC2, BTNL9, ARMC4, OLFML2A, AMOT, RP11- 556114.1, SLFN5, PKIA, CTC-339F2.2, ADAM23, NPSR1-AS1, NREP, AC009410.1, IMPG1, C12orf40, AC097500.2, AC004069.2, CDH26, CCDC33, PCDP1, ALCAM, LRRN1, WWC3-AS1, MT-TY, MXRA8, RASL11A, COL3A1, PDE1A, COL21A1, NTM, GNG4, RAMP3, AC026188.1 , ELOVL2-AS1 , SMAD9, ZBTB20, GNG12-AS1 , EFNA5, RP1 1- 713Μ6.2, AC078883.3, PAMR1 , BMPER, ΝΥΑΡ2, RP4-813D12.3, HMCN1 , FAT4, CYP2C8, PLLP, ΝΧΡΗ1 , CTD-2260A17.2, MUC5AC, ΡΡ13439, SOCS2-AS1 , CCM2L, ZNF827, ADAMTSL3, SDC2, RBM1 1 , PRICKLE2-AS3, RNF180, DOCK10, FAM198A, ΤΜΕΜ86Α, LGALS3BP, KLK8, AC012307.2, AC004053.1 , CTD-3179P9.1 , NOD2, RP1 1-556114.2, C3, ZNF396, ZNF792, ENSG00000244288, CHRM3, AGBL4, KLF7, ΑΡ004372.1 , DKK2, NPAS3, SMARCA1 , DOK6, SULF1 , RORC, YPEL1 , LRP1 B, RP4-710M3.2, DCLK1 , ENSG00000235732, NIPSNAP3B, SEPP1 , LY6D, ENSG00000244428, AKR1 C3, GJA1 , CXXC4, FSTL5, CYBRD1 , SERPINA1 , AC004540.5, RP1 1-600Κ15.1 , ASTL, C20orf197, KCNS3, CTC-448D22.1 , ΤΜΕΜ45Α RP1 1-86516.2, SNCG, CCDC152, MURC, TMCC2, FBN2, FREM1 , ENSG00000235471 , ENSG00000170233, THRB, AC004947.2, C1orf168, RN7SKP3, THBS2, TREH, AC007365.3, ΑΤΟΗ8, SEMA3D, and GPC6 is different relative to the control value. In some embodiments, the expression level of at least one gene selected from CSGALNACT1 , SIGLEC6, SHC4, ABCA1 , PLD1 , RNF144A, SLC44A4, SLC2A13, and AXIN2 is different relative to the control value.
[0010] In some embodiments, the control value associated with each gene is determined by determining the expression level of that gene in one or more control samples, and calculating an average expression level of that gene in the one or more control samples, wherein each control sample is obtained from healthy tissue of the same or a different subject.
[0011] In some embodiments, the expression of the gene is increased or decreased relative to the expression level of a control by an amount of at least about 2-fold. In some embodiments, the expression of at least one gene is increased at least about 2-fold relative to the control value associated with the gene. In some embodiments, the expression of at least one gene is decreased at least about 2-fold relative to the control value associated with the gene.
[0012] In other aspects, the disclosure provides methods of monitoring a molecular response of a subject to treatment of breast cancer with at least one of a RON inhibitor and a PI3K inhibitor. The methods may include determining in a first sample of the breast cancer an expression level of one or more genes selected from CSGALNACT1 , SIGLEC6, SHC4, ELTD1 , CGA, MST1 R, KRT8P41 , BTF3P4, RP1 1-478C6.4, EPHA3, LXN, ENSG00000252044, ENSG00000242932, ENSG00000241 1 15, AP001347.6, LHFPL2, CTD-2330K9.2, RASD1 , ENSG00000242693, AC093388.3, AOC1 , CPE, PCDH8, DCHS2, DPP4, ENSG00000242777, GUSBP5, ENSG00000244466, ENSG00000252331 , RP1 1- 664D7.4, RP1 1-206M1 1.7, ENSG00000244580, EYA4, CTA-714B7.5, ENSG00000252318, ABCA1 , PLD1 , RNF144A, SLC44A4, SLC2A13, ΑΧΙΝ2, PGR, XXbac-BPG254F23.6, AMY2B, LIMCH1 , RP1 1-356J5.12, ID2, MFAP2, LPHN2, RP1 1-594N15.2, RP1 1-575A19.2, SPINK5, HLA-DQB1-AS1 , RN7SL151 P, ATP8A1 , FAM189A2, ZNF423, CTD-2001 C12.1 , ELOVL2, TESC, KCNMB4, STOX2, PLEKHM3, MT-TL1 , KLK1 1 , NPNT, ENSG00000234543, SHC2, BTNL9, ARMC4, OLFML2A, AMOT, RP1 1-556114.1 , SLFN5, PKIA, CTC-339F2.2, ADAM23, NPSR1-AS1 , NREP, AC009410.1 , IMPG1 , C12orf40, AC097500.2, AC004069.2, CDH26, CCDC33, PCDP1 , ALCAM, LRRN1 , WWC3-AS1 , MT- TY, MXRA8, RASL1 1A, COL3A1 , PDE1A, COL21A1 , NTM, GNG4, RAMP3, AC026188.1 , ELOVL2-AS1 , SMAD9, ZBTB20, GNG12-AS1 , EFNA5, RP1 1-713M6.2, AC078883.3, PAMR1 , BMPER, NYAP2, RP4-813D12.3, HMCN1 , FAT4, CYP2C8, PLLP, NXPH1 , CTD- 2260A17.2, MUC5AC, PP13439, SOCS2-AS1 , CCM2L, ZNF827, ADAMTSL3, SDC2, RBM1 1 , PRICKLE2-AS3, RNF180, DOCK10, FAM198A, TMEM86A, LGALS3BP, KLK8, AC012307.2, AC004053.1 , CTD-3179P9.1 , NOD2, RP1 1-556114.2, C3, ZNF396, ZNF792, ENSG00000244288, CHRM3, AGBL4, KLF7, AP004372.1 , DKK2, NPAS3, SMARCA1 , DOK6, SULF1 , RORC, YPEL1 , LRP1 B, RP4-710M3.2, DCLK1 , ENSG00000235732, NIPSNAP3B, SEPP1 , LY6D, ENSG00000244428, AKR1 C3, GJA1 , CXXC4, FSTL5, CYBRD1 , SERPINA1 , AC004540.5, RP1 1-600K15.1 , ASTL, C20orf197, KCNS3, CTC- 448D22.1 , TMEM45A RP11-86516.2, SNCG, CCDC152, MURC, TMCC2, FBN2, FREM1 , ENSG00000235471 , ENSG00000170233, THRB, AC004947.2, C1orf168, RN7SKP3, THBS2, TREHAC007365.3, ATOH8, SEMA3D, and GPC6; administering to the subject a therapeutic amount of a medicament comprising at least one of a RON inhibitor and a PI3K inhibitor; determining in a second sample of the breast cancer an expression level of each of the one or more genes, wherein the second sample is collected after administration of the medicament; and comparing for each gene the expression level in the first sample to the expression level in the second sample. In some embodiments, the one or more genes are selected from CSGALNACT1 , SIGLEC6, SHC4, ABCA1 , PLD1 , RNF144A, SLC44A4, SLC2A13, and AXIN2.
[0013] In some embodiments, determining the expression level of a first gene comprises measuring the expression level of an RNA transcript of the first gene, or an expression product thereof. In some embodiments, measuring the expression level of the RNA transcript of the first gene, or the expression product thereof, includes using at least one of a PCR-based method, a Northern blot method, a microarray method, and an immunohistochemical method.
[0014] In other aspects, the disclosure provides kits for assessing the likelihood that breast cancer in a subject will metastasize or has metastasized, or for determining whether the subject would benefit from treatment with at least one of a RON inhibitor and a PI3K kinase inhibitor. The kits may include one or more primers, each primer adapted to amplify an RNA transcript of an independent one of the genes selected from CSGALNACT1 , SIGLEC6, SHC4, ELTD1 , CGA, MST1 R, KRT8P41 , BTF3P4, RP1 1-478C6.4, EPHA3, LXN, ENSG00000252044, ENSG00000242932, ENSG00000241 1 15, AP001347.6, LHFPL2, CTD-2330K9.2, RASD1 , ENSG00000242693, AC093388.3, AOC1 , CPE, PCDH8, DCHS2, DPP4, ENSG00000242777, GUSBP5, ENSG00000244466, ENSG00000252331 , RP1 1- 664D7.4, RP1 1-206M1 1.7, ENSG00000244580, EYA4, CTA-714B7.5, ENSG00000252318, ABCA1 , PLD1 , RNF144A, SLC44A4, SLC2A13, AXIN2, PGR, XXbac-BPG254F23.6, AMY2B, LIMCH1 , RP1 1-356J5.12, ID2, MFAP2, LPHN2, RP1 1-594N15.2, RP1 1-575A19.2, SPINK5, HLA-DQB1-AS1 , RN7SL151 P, ATP8A1 , FAM189A2, ZNF423, CTD-2001 C12.1 , ELOVL2, TESC, KCNMB4, STOX2, PLEKHM3, MT-TL1 , KLK1 1 , NPNT, ENSG00000234543, SHC2, BTNL9, ARMC4, OLFML2A, AMOT, RP1 1-556114.1 , SLFN5, PKIA, CTC-339F2.2, ADAM23, NPSR1-AS1 , NREP, AC009410.1 , IMPG1 , C12orf40, AC097500.2, AC004069.2, CDH26, CCDC33, PCDP1 , ALCAM, LRRN1 , WWC3-AS1 , MT- TY, MXRA8, RASL1 1A, COL3A1 , PDE1A, COL21A1 , NTM, GNG4, RAMP3, AC026188.1 , ELOVL2-AS1 , SMAD9, ZBTB20, GNG12-AS1 , EFNA5, RP1 1-713M6.2, AC078883.3, PAMR1 , BMPER, NYAP2, RP4-813D12.3, HMCN1 , FAT4, CYP2C8, PLLP, NXPH1 , CTD- 2260A17.2, MUC5AC, PP13439, SOCS2-AS1 , CCM2L, ZNF827, ADAMTSL3, SDC2, RBM1 1 , PRICKLE2-AS3, RNF180, DOCK10, FAM198A, TMEM86A, LGALS3BP, KLK8, AC012307.2, AC004053.1 , CTD-3179P9.1 , NOD2, RP1 1-556114.2, C3, ZNF396, ZNF792, ENSG00000244288, CHRM3, AGBL4, KLF7, AP004372.1 , DKK2, NPAS3, SMARCA1 , DOK6, SULF1 , RORC, YPEL1 , LRP1 B, RP4-710M3.2, DCLK1 , ENSG00000235732, NIPSNAP3B, SEPP1 , LY6D, ENSG00000244428, AKR1 C3, GJA1 , CXXC4, FSTL5, CYBRD1 , SERPINA1 , AC004540.5, RP1 1-600K15.1 , ASTL, C20orf197, KCNS3, CTC- 448D22.1 , TMEM45A RP11-86516.2, SNCG, CCDC152, MURC, TMCC2, FBN2, FREM1 , ENSG00000235471 , ENSG00000170233, THRB, AC004947.2, C1orf168, RN7SKP3, THBS2, TREHAC007365.3, ATOH8, SEMA3D, and GPC6, and instructions for use. In some embodiments, the at least one primer is adapted to amplify an RNA transcript of a gene selected from CSGALNACT1 , SIGLEC6, SHC4, ABCA1 , PLD1 , RNF144A, SLC44A4, SLC2A13, and AXIN2. In some embodiments, a first probe comprises an antibody to an expression product. In some embodiments, a first probe comprises an oligonucleotide complementary to an RNA transcript.
[0015] In other aspects, the disclosure provides kits for assessing the likelihood that breast cancer in a subject will metastasize or has metastasized, or for determining whether the subject would benefit from treatment with at least one of a RON inhibitor and a PI3K kinase inhibitor. The kits may include one or more probes, each probe adapted to specifically bind to an RNA transcript, or an expression product thereof, of an independent one of the genes selected from CSGALNACT1 , SIGLEC6, SHC4, ELTD1 , CGA, MST1 R, KRT8P41 , BTF3P4, RP1 1-478C6.4, EPHA3, LXN, ENSG00000252044, ENSG00000242932, ENSG00000241 1 15, AP001347.6, LHFPL2, CTD-2330K9.2, RASD1 , ENSG00000242693, AC093388.3, AOC1 , CPE, PCDH8, DCHS2, DPP4, ENSG00000242777, GUSBP5, ENSG00000244466, ENSG00000252331 , RP1 1-664D7.4, RP1 1-206M11.7, ENSG00000244580, EYA4, CTA-714B7.5, ENSG00000252318, ABCA1 , PLD1 , RNF144A, SLC44A4, SLC2A13, AXIN2, PGR, XXbac-BPG254F23.6, AMY2B, LIMCH1 , RP1 1-356J5.12, ID2, MFAP2, LPHN2, RP11-594N15.2, RP1 1-575A19.2, SPINK5, HLA-DQB1-AS1 , RN7SL151 P, ATP8A1 , FAM189A2, ZNF423, CTD-2001 C12.1 , ELOVL2, TESC, KCNMB4, STOX2, PLEKHM3, MT-TL1 , KLK1 1 , NPNT, ENSG00000234543, SHC2, BTNL9, ARMC4, OLFML2A, AMOT, RP1 1-556114.1 , SLFN5, PKIA, CTC-339F2.2, ADAM23, NPSR1-AS1 , NREP, AC009410.1 , IMPG1 , C12orf40, AC097500.2, AC004069.2, CDH26, CCDC33, PCDP1 , ALCAM, LRRN1 , WWC3-AS1 , MT-TY, MXRA8, RASL1 1A, COL3A1 , PDE1A, COL21A1 , NTM, GNG4, RAMP3, AC026188.1 , ELOVL2-AS1 , SMAD9, ZBTB20, GNG12-AS1 , EFNA5, RP1 1-713M6.2, AC078883.3, PAMR1 , BMPER, NYAP2, RP4- 813D12.3, HMCN1 , FAT4, CYP2C8, PLLP, NXPH1 , CTD-2260A17.2, MUC5AC, PP13439, SOCS2-AS1 , CCM2L, ZNF827, ADAMTSL3, SDC2, RBM1 1 , PRICKLE2-AS3, RNF180, DOCK10, FAM198A, TMEM86A, LGALS3BP, KLK8, AC012307.2, AC004053.1 , CTD- 3179P9.1 , NOD2, RP1 1-556114.2, C3, ZNF396, ZNF792, ENSG00000244288, CHRM3, AGBL4, KLF7, AP004372.1 , DKK2, NPAS3, SMARCA1 , DOK6, SULF1 , RORC, YPEL1 , LRP1 B, RP4-710M3.2, DCLK1 , ENSG00000235732, NIPSNAP3B, SEPP1 , LY6D, ENSG00000244428, AKR1 C3, GJA1 , CXXC4, FSTL5, CYBRD1 , SERPINA1 , AC004540.5, RP1 1-600K15.1 , ASTL, C20orf197, KCNS3, CTC-448D22.1 , TMEM45A, RP1 1-86516.2, SNCG, CCDC152, MURC, TMCC2, FBN2, FREM1 , ENSG00000235471 , ENSG00000170233, THRB, AC004947.2, C1orf168, RN7SKP3, THBS2, TREH, AC007365.3, ATOH8, SEMA3D, and GPC6, and instructions for use. In some embodiments, the at least one probe is adapted to bind to an RNA transcript, or an expression product thereof, of a gene selected from CSGALNACT1 , SIGLEC6, SHC4, ABCA1 , PLD1 , RNF144A, SLC44A4, SLC2A13, and AXIN2. In some embodiments, a first probe comprises an antibody to an expression product. In some embodiments, a first probe comprises an oligonucleotide complementary to an RNA transcript.
[0016] The disclosure provides for other aspects and embodiments that will be apparent in light of the following detailed description and accompanying Figures. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
[0018] Figure 1. RON/MSP signaling promotes widespread metastasis of human breast cancer in vivo (A) Immunoblot analysis of RON levels (top panel) in immortalized mammary epithelial cells (MCF-10A; blue), non-aggressive breast cancer cell lines (MCF7 and T47D; green), aggressive breast cancer cell lines (MDA-MB-453, HCC1 143, HCC1806; orange) and MCF7 and T47D cells engineered to overexpress RON/MSP (red). The β-actin (ACTB) loading control is shown in the bottom panel. (B) The effect of RON/MSP on spontaneous lung, bone, liver and brain metastasis of orthotopic MCF7 and T47D tumors. Shown are representative bioluminescent images of primary tumors, lung, bone, liver and brain metastasis from single mice. (C) Metastasis frequencies for MCF7 (n=15), MCF7- RON/MSP (n=20), T47D (n=16) and T47D-RON/MSP (n=14) tumors following orthotopic injection into mammary fat pad of NOD/SCID mice. All RON/MSP tumors were harvested when size-matched to parental tumors.
[0019] Figure 2. RON/MSP signaling upregulates MBD4 and drives aberrant DNA methylation (A) Circular representation of genome-wide aberrant DNA methylation caused by RON/MSP expression. Average methylation levels for all of the CGs in 10-Mbp-wide windows are shown in the blue-green tracks (the outermost track shows MCF7 cells; the middle track shows MCF7-RON/MSP cells). The innermost track indicates the differentially methylated regions (DMRs) between MCF7 and MCF7-RON/MSP cells (FDR>20, differential methylation>0.25). This diagram for visualization of genome-wide DNA methylation was generated using the Circos software (Krzywinski et al., 2009). (B) Top panel: Distribution of differentially methylated CpGs on CpG islands (CpGi) shelves (>2 to 4 kb from island edge) and on CpGi shores (0-2 kb from island edge); within and outside CpGi. u, upstream of CpGi; d, downstream of CpGi. Bottom panel: Distribution of differentially methylated CpGs across regions of other significance. TSS 200, within the region 1 -200 bp upstream of the TSS; TSS 1500, within the region 201 -1500 bp upstream of the transcription start site (TSS); UTR, untranslated region; HyperMe, hypermethylated; HypoMe, hypomethylated. (C) Example of smoothed methylation levels from bisulfite sequencing data for MCF7 (black) and MCF7-RON/MSP (blue) on chromosome 14. A hypomethylation block is indicated by a red bar. (D) Representative methylation patterns for MCF7 (black) and MCF7-RON/MSP cells (blue), showing both hypomethylation (Top panel) and hypermethylation (Bottom panel) due to RON/MSP expression. (E) Real-time quantitative RT-PCR data showing MBD4 and TDG mRNA expression levels, normalized to β-actin mRNA levels, in MCF7, MCF7- RON/MSP, T47D and T47D-RON/MSP cells (*p<0.001 ). (F) Western blot with anti-MBD4 antibodies showing upregulation of MBD4 protein expression in MCF7-RON/MSP and T47D- RON/MSP cells compared to parental MCF7 and T47D cells (fold change = 5 for MCF7 and fold change=3 for T47D, p<0.001 , as quantified by ImageJ).
[0020] Figure 3. Knockdown of MBD4 blocks RON/MSP-mediated breast cancer metastasis. (A) Boxplot showing RON and MBD4 mRNA expression in normal breast tissues (white) and breast tumors (dark grey) from The Cancer Genome Atlas (TCGA) data set. (B) Western blot for RON and MBD4 proteins in normal human breast tissues from reduction mammoplasties and human primary breast tumor specimens; the β-actin (ACTB) loading control is also shown. (C) Successful knockdown of MBD4 expression, shown by Western blot on protein lysates from MCF7-RON/MSP-shMBD4 cells (shRNA directed to the 3'UTR or CDS) and T47D-RON/MSP-shMBD4 cells compared with controls (MCF7- RON/MSP, T47D-RON/MSP, and shRNA scramble control, shScrb). MBD4 re-expression following introduction of rescue cDNA into shMBD4-expressing cells (shMBD4R_3UTR and shMBD4R_CDS) is also shown for both cell lines, β-actin (ACTB) levels are shown as a loading control. (D) Top panel: Metastasis frequencies for MCF7 parental tumors (n=15), MCF7-RON/MSP tumors (n=20), or tumors arising from MCF7 cells infected with RON/MSP and either shScrb (n=10), sh-MBD4-3UTR (n=19), or shMBD4-CDS (n=9). The effects of rescue constructs (shMBD4R_3UTR (n=7), shMBD4R_CDS (n=9) or the catalytic mutant shMBD4R (D560A) (n=9)) are shown at the bottom. Bottom panel: Metastasis frequencies for T47D parental tumors (n=16), T47D-RON/MSP tumors (n=14), or tumors arising from T47D cells infected with RON/MSP and either shScrb (n=7), sh-MBD4-3UTR (n=12), or shMBD4-CDS (n=10). The effects of rescue constructs (shMBD4R_3UTR (n=8), shMBD4R_CDS (n=9) or the catalytic mutant shMBD4R (D560A) (n=8)) are shown at the bottom. (E) Representative bioluminescent images of lung, bone and liver metastasis from single mice illustrate the effects of shScrb, sh-MBD4, rescued MBD4 and rescued catalytic mutant on spontaneous lung, bone, liver and brain metastasis of various orthotopic MCF7 tumors.
[0021] Figure 4. Knockdown of MBD4 reverses DNA aberrant methylation and expression of RON/MSP regulated genes. (A) Representation of smoothed methylation values from bisulfite sequencing data for MCF7 (black), MCF7-RON/MSP (blue) and MCF7- RON/MSP-shMBD4 cells (purple) in a representative region of chromosome 2. The hypomethylation block in MCF7-RON/MSP that becomes hypermethylated by knocking- down MBD4 is indicated by a red bar. (B) Example of DNA methylation levels in promoter regions in MCF7 (black), MCF7-RON/MSP (blue) and MCF7-RON/MSP-shMBD4 cells (purple). The hypomethylated block in MCF7-RON/MSP cells that becomes re-methylated by knocking down MBD4 is indicated by a red bar. (C) Example of DNA methylation levels in a promoter region that becomes hypermethylated in shMBD4 cells, independent of RON/MSP expression (red bar) Bisulfite sequencing data for MCF7 (black), MCF7-RON/MSP (blue) and MCF7-RON/MSP-shMBD4 cells (purple) are shown. In all panels, the orange bars indicate CpG islands and the black bars show the genes. (D) Diagrams representing the proportion of genes for which expression was reversed by knocking down MBD4 (outside shifted circle). In blue are represented the genes that are downregulated in MCF7-RON/MSP versus MCF7 (the outside shifted track represents the genes that become upregulated in MCF7-RON/MSP-shMBD4). In orange are represented the genes that are upregulated in MCF7-RON/MSP versus MCF7 (outside track represents the genes that become downregulated in MCF7-RON/MSP-shMBD4).
[0022] Figure 5. Specific subclasses of human breast tumors possess the RON/MBD4 epigenetic signature, which correlates with poor prognosis (A) Circular representation of the RON/MBD4 epigenetic signature. The outside track represents the 192 genes regulated by RON/MSP for which expression was reversed following knockdown of MBD4 (blue: genes downregulated by RON/MSP and then reversed by knocking down MBD4 with shRNA; orange: genes upregulated by RON/MSP and then reversed by knocking down MBD4). In larger characters are the genes that are annotated in the METABRIC dataset. The inner track represents the statistically significant DMRs at these regions (red: hypermethylated DMRs in MCF7-RON/MSP and then reversed by knocking down MBD4; green: hypomethylated DMRs in MCF7-RON/MSP and then reversed by knocking down MBD4). (B) Enrichment pattern of the gene set comprising the RON/MBD4 epigenetic signature (rows) across 997 breast tumors (columns). Red and green indicated significantly over or underexpressed gene, respectively. Are represented the 582 patients having enrichment or underrepresentation of the gene set. Blue bars (right) indicate individual tumor annotations for breast cancer subtype. The table (right) shows the association of the RON/MBD4 epigenetic signature with ER, HER2 and PR status, as well as intrinsic breast cancer subtype, grade and death (we assigned a p value according to the hypergeometric distribution) (C) Kaplan-Meier analysis of overall survival in 997 breast cancer patients from the METABRIC discovery dataset (Curtis et al., 2012). Survival curve of individuals with tumors showing an enrichment of the 1 16 annotated genes from the RON/MBD4 epigenetic signature is shown in red; all other patients are shown in blue (No signature). The p value indicates a statistically significant survival difference between these two groups of patients. The survival hazard ratio was calculated using Univariate Cox's regression analysis. [0023] Figure 6. MBD4 is regulated by RON/MSP signaling through PI3K pathway.
(A) Western blot with anti-RON, anti-MBD4 and anti- β-actin antibodies on protein lysates from the MCF7-TRE-RON/MSCV-tTA cell line treated with or without (+/-) doxycycline for the indicated times (duplicate samples). (B) Western blot showing expression of endogenous RON and MBD4 proteins in the DU-4475 breast cancer cell line and in a patient derived breast tumor graft (HCI-014) before and after infection with lentiviruses carrying RON shRNA (fold change = 3, p<0.02, and fold change = 5, p<0.01 , respectively, as quantified by ImageJ). (C) Top: Western blot with anti-MBD4, anti-p-PRAS and anti- β-actin antibodies in MCF7 and MCF7-RON/MSP cells treated with DMSO or BKM-120 (50 nM, 100 nM, or 500 nM for the time indicated; fold change = 12, p<0.001 , as quantified by ImageJ). Bottom: Western blot with anti-MBD4, anti-p-ERK and anti- β-actin antibodies in MCF7 and MCF7- RON/MSP cells treated with DMSO or U0126 (5 μΜ, 10 μΜ, or 50 μΜ for the time indicated). (D) Western blot showing the expression of MBD4 and p-PRAS in MCF7 and MCF7- RON/MSP cells infected with lentiviruses carrying PI3K-p1 10 shRNA or scrambled shRNA control (fold change = 2, p<0.01 , as quantified by ImageJ).
[0024] Figure 7. Treatment with a new RON inhibitor, OSI-296, drastically decreases breast cancer metastasis in human patient-derived breast tumor grafts. (A)
Western blots with anti-p-RON, MBD4 and β-actin antibodies on protein lysates from primary cultures of the patient-derived tumor graft HCI-007, treated without (-) or with 1 μΜ of the RON inhibitor OSI-296 over the indicated times. (B) Metastasis frequencies for orthotopically-implanted patient-derived tumor grafts HCI-003 and HCI-01 1 following treatment with OSI-296 (n=3 and n=9, for HCI-003 and HCI-01 1 , respectively) or trappsol (n=5 for both HCI-003 and HCI-01 1 ) vehicle control (p value determined by Fisher's exact test). (C) Sections of axillary lymph nodes isolated from mice carrying orthotopic (inguinal fat pad) patient-derived breast tumor grafts HCI-003 and HCI-01 1 following treatment with trappsol or OSI-296. The sections were stained with H&E (left panels) or were immunostained with antibodies specific for human cytokeratin (right panels) to detect human tumor cells. (D) Sections of lungs isolated from mice carrying orthotopic patient-derived breast tumor grafts HCI-003 or HCI-01 1 following treatment with trappsol or OSI-296. The sections were stained with H&E (left) or were immunostained with antibodies specific for human cytokeratin (CK; right) to identify human tumor cells. (E) Table representing the 9 genes of the RON/MBD4 epigenetic signature that become deregulated in tumors from mice treated with OSI296 compared to the trappsol control. (F) Kaplan-Meier analysis of overall survival in 997 breast cancer patients from the METABRIC discovery dataset (left) and in 977 breast cancer patients from the compendium dataset (right). Survival curve of individuals with tumors showing an enrichment of the 9 RON/MBD4 epigenetic signature genes that were deregulated in tumors from mice treated with OSI-296 compared to the trappsol control is shown in red; all other patients are shown in blue (No signature). The p value indicates a statistically significant survival difference between these two groups of patients. The survival hazard ratio was calculated using Univariate Cox's regression analysis. (G) Univariate and multivariate cox regression analyses for overall survival in the 997 patients from the METABRIC dataset, examining ER, PR, status, basal-like subtype, and the MBD4/RON signature as variables. HR, hazard ratio.
[0025] Figure 8. (A) Tumor growth curves following orthotopic transplantation of MCF7 or T47D cells (blue) or MCF7-RON/MSP or T47-RON/MSP (red) into cleared inguinal mammary fat pads of 3-week old NOD/SCID mice. (B) Representative lung sections from mice carrying MCF7-RON/MSP or T47D-RON/MSP tumors, stained with H&E or immunostained with an antibody specific for human cytokeratin to positively identify metastasis. (C) Scatter plot showing differential expression obtained using microarray (x axis) and RNA-seq (y axis) between MCF7 and MCF7-RON/MSP. Points represent genes, located based on estimates of differential expression using RNA-Seq and Microarray. Red points represent genes for which FDR<0.1 and fold change>2. Green points represent genes differentially expressed identified by RNA-seq only. Purple points represent genes differentially expressed identified by microarray only. The line corresponds to perfect agreement between the technologies.
[0026] Figure 9. (A) Real-time quantitative RT-PCR for various DNMT mRNA expression levels, normalized to β-actin mRNA expression, in MCF7 and MCF7-RON/MSP cells. (B) DNA methylation activity in MCF7 and MCF7-RON/MSP cells, as assessed by EpiQuick DNA methyltransferase assay.
[0027] Figure 10. (A) Tumor growth following orthotopic implantation of MCF7 (dark blue), MCF 7- RON/MSP (red), MCF7-RON/MSP-shScr (green), MCF7-RON/MSP-shMBD4 (yellow) and MCF7-RON/MSP-shMBD4R cells (light blue) in MCF7 (left) and T47D (right) models. (B) Metastasis frequencies for MCF7, MCF7-RON/MSP, MCF7-RON/MSP-shScr, and MCF7-RON/MSP-shMBD2 tumors. (C) Representative images showing lack of effect of sh-MBD2 on spontaneous lung, bone, liver and brain metastasis of RON/MSP-expressing MCF7 tumors.
[0028] Figure 11. (A) Western blots showing expression of EGFR, p-EGFR, MBD4, p- AKT, p-ERK, AKT, ERK and β-actin in MDA-MB-231 cells treated with EGF (15 nM) for 1 hour. (B) The expression of MET, MBD4 and ACTB in MCF7 cell line infected with retroviruses carrying cDNA of MET. (C) MCF7 and MCF7-RON/MSP cells were treated with or without alkaline phosphatase for 30 min. The lysates were subjected to (a) conventional SDS-PAGE on 8% (wt/vol) polyacrylamide gel or (b) SDS-PAGE on 6% (wt/vol) polyacrylamide gel containing 20 μΜ Mn2+-Phos-tag, followed by immunoblotting with the anti-MBD4 antibody. By treatment with alkaline phosphatase, shifts in the mobility of MBD4 were suppressed.
DETAILED DESCRIPTION
[0029] RON, also known as macrophage stimulating 1 -receptor (MST1 R), is a member of the Met family of receptor tyrosine kinases. The biological activity of RON is mediated by binding of its extracellular ligand, macrophage-stimulating protein (MSP), also known as hepatocyte growth factor-like protein (HGFL) and macrophage stimulating 1 (MST1 ). MSP is primarily produced by hepatocytes as a single chain inactive precursor, pro-MSP. Pro-MSP is cleaved by membrane-bound proteases such as membrane type serine protease 1 (MT- SP1 ; also known as matriptase) or hepatocyte growth factor activator, leading to activation of MSP at the cell surface. Binding of MSP to its receptor, RON, activates RON and leads to cellular growth, motility, and invasion. While RON overexpression may be a feature of cancers, the molecular mechanisms by which RON induces tumorgenesis and metastasis are still unclear.
[0030] The inventors have discovered that RON/MSP enhances metastasis of breast cancer xenografts, and that metastasis is enhanced by driving DNA demethylation at key target gene promoters. RON/MSP-initiated aberrant DNA methylation is the result of PI3K- dependent upregulation of MBD4, which is a component of the DNA demethylase complex. As detailed in the Examples, knockdown of MBD4 in RON/MSP-expressing breast cancer cells reverses the DNA methylation and blocks metastasis. The inventors have further discovered a set of genes that are specifically regulated by RON/MSP through MBD4- directed aberrant DNA methylation, and that the MBD4/RON signature is enriched in basal- like (triple negative) breast cancers and associated with poor prognosis. As further described in the Examples, inhibition of Ron signaling with a small molecule kinase inhibitor blocks metastasis of patient-derived tumor grafts, indicating that RON inhibitors can be used as novel anti-metastatic agents.
[0031 ] The gene signature may include at least one of the genes shown in Table 1 . The expression of these genes was found to be deregulated when RON/MSP is overexpressed, as detailed in the Examples. The expression was reversed (i.e. , the gene overexpression was decreased) when expression of MBD4 was down regulated, demonstrating these genes are downstream of RON/MSP and MBD4. Some of the genes (corresponding to SEQ ID NOs 1-56) further demonstrated aberrant expression upon administration of a RON inhibitor.
Table 1. Genes downstream of RON/MBD4. Shown in the right column is the fold change in expression of the gene relative to basal levels that was observed when RON/MSP was overexpressed. SEQ ID NOs. 1-56 correspond to genes for which the expression in a xenograft tumor became reversed after treatment with a RON inhibitor, wherein SEQ ID NOs 1-47 correspond to the polynucleotide sequences and variants and SEQ ID NOs 48-56 in parentheses correspond to the polypeptide sequences.
Figure imgf000018_0001
CPE ENSG00000109472 chr4 166282345 166419472 1817.29 2.42
PCDH8 ENSG00000136099 chr13 53418108 53422775 38.65 2.33
DCHS2 ENSG00000197410 chr4 155155526 155412930 31 .09 2.33
DPP4 ENSG00000197635 chr2 162848750 162931052 27.42 2.31
ENSG00000242777 ENSG00000242777 chr6 144692019 144692175 24.04 2.31
GUSBP5 ENSG00000236296 chr4 144480624 144570458 44.99 2.31
ENSG00000244466 ENSG00000244466 chr17 55868618 55868752 3057.09 2.27
ENSG00000252331 ENSG00000252331 chi 1 77597678 77597752 24.35 2.23
RP1 1 -664D7.4 ENSG00000248801 chr8 69215702 69243727 49.35 2.12
RP1 1 -206M1 1.7 ENSG00000244468 chr3 149002568 149051440 161.75 2.08
ENSG00000244580 ENSG00000244580 chr6 133593942 133594166 3057.09 2.06
EYA4 ENSG000001 12319 chr6 133561735 133853258 2384.70 2.06
CTA-714B7.5 ENSG00000233080 chr22 35515816 35627049 68.59 2.03
ENSG00000252318 ENSG00000252318 chr2 133038646 133038738 3057.09 2.03
PGR ENSG00000082175 chrl 1 100900354 101001255 -1629.09 -2.00
XXbac- BPG254F23.6 ENSG00000241287 chr6 32627662 32630227 -72.52 -2.01
AMY2B ENSG00000240038 chii 104096436 104122156 -27.1 1 -2.02
LIMCH1 ENSG00000064042 chr4 41361623 41702061 -128.86 -2.03
RP1 1 -356J5.12 ENSG00000250303 chr11 1 12141471 1 12233257 -145.29 -2.03
ID2 ENSG000001 15738 chr2 8818974 8824583 -67.75 -2.03
MFAP2 ENSG000001 17122 chrl 17300996 17307330 -30.51 -2.03
LPHN2 ENSG000001 17114 chii 81771844 82458107 -101 .24 -2.04
RP1 1 -594N15.2 ENSG00000254266 chr8 79338337 79470738 -140.96 -2.04
RP1 1 -575A19.2 ENSG00000238246 chr10 20359733 20380713 -41.54 -2.05
SPINK5 ENSG00000133710 chr5 147405245 147516852 -62.76 -2.05
HLA-DQB1 -AS1 ENSG00000223534 chr6 32627656 32628506 -32.98 -2.07
RN7SL151 P ENSG00000244230 chr9 21699310 21699523 -30.84 -2.08
ATP8A1 ENSG00000124406 chr4 42410389 42659122 -68.88 -2.09
FAM189A2 ENSG00000135063 chr9 71939487 72007371 -98.88 -2.09
ZNF423 ENSG00000102935 chr16 49524521 49860918 -35.91 -2.09
CTD-2001 C12.1 ENSG00000247877 chr5 99785457 99870966 -37.65 -2.09
ELOVL2 ENSG00000197977 chr6 10980991 1 1044547 -873.57 -2.1 1
TESC ENSG00000088992 chr12 1 17476727 1 17537284 -36.88 -2.12
KCNMB4 ENSG00000135643 chr12 70760055 70828072 -182.83 -2.13
STOX2 ENSG00000173320 chr4 184774583 184938894 -78.35 -2.15
PLEKHM3 ENSG00000178385 chr2 208693026 208890284 -104.18 -2.16
MT-TL1 ENSG00000209082 chrM 3229 3304 -62.85 -2.16
KLK1 1 ENSG00000167757 chr19 51525488 51531290 -21 .29 -2.17
NPNT ENSG00000168743 chr4 106815931 106925184 -1489.66 -2.17
ENSG00000234543 ENSG00000234543 chr9 96301209 96322860 -28.09 -2.19
SHC2 ENSG00000129946 chr19 416586 460996 -56.24 -2.19
BTNL9 ENSG00000165810 chr5 180467224 180488523 -24.74 -2.20
ARMC4 ENSG00000169126 chr10 280641 14 28287977 -22.94 -2.21
0LFML2A ENSG00000185585 chr9 127539436 127577164 -36.22 -2.21 AMOT ENSG00000126016 chrX 112017730 1 12084043 -63.12 -2.21
RP1 1 -556114.1 ENSG00000248373 chr4 105828513 106041 157 -90.29 -2.21
SLFN5 ENSG00000166750 chr17 33570085 33594739 -271 .08 -2.23
PKIA ENSG00000171033 chr8 79428373 79517502 -261 .59 -2.26
CTC-339F2.2 ENSG00000250015 chr5 1 15638050 1 15639831 -28.82 -2.26
ADAM23 ENSG000001 14948 chr2 207308262 207485851 -25.30 -2.26
NPSR1-AS1 ENSG00000197085 chr7 34386123 34911 194 -30.00 -2.29
NREP ENSG00000134986 chr5 1 10998317 1 1 1333161 -206.47 -2.30
AC009410.1 ENSG00000232023 chr2 229347976 2294761 1 1 -35.98 -2.33
IMPG1 ENSG000001 12706 chr6 76630831 76782395 -32.43 -2.36
C12orf40 ENSG00000180116 chr12 40019968 40302102 -233.24 -2.36
AC097500.2 ENSG00000237877 chr2 186897871 186947960 -44.97 -2.36
AC004069.2 ENSG00000251259 chr4 106058436 106061776 -29.20 -2.37
CDH26 ENSG00000124215 chr20 58533470 58609066 -274.87 -2.37
CCDC33 ENSG00000140481 chr15 74509612 7462881 1 -30.71 -2.38
PCDP1 ENSG00000163075 chr2 120302007 120419827 -49.28 -2.39
ALCAM ENSG00000170017 chr3 105085752 105295744 -516.75 -2.40
LRRN1 ENSG00000175928 chr3 3841120 3889387 -39.68 -2.41
WWC3-AS1 ENSG00000225076 chrX 9992881 10006694 -22.73 -2.41
MT-TY ENSG00000210144 chrM 5825 5891 -549.75 -2.41
MXRA8 ENSG00000162576 chrl 1288068 1297157 -41 .20 -2.41
RASL1 1A ENSG00000122035 chr13 27844463 27847827 -292.75 -2.42
COL3A1 ENSG00000168542 chr2 189839045 189877472 -33.43 -2.42
PDE1A ENSG000001 15252 chr2 183004762 183387919 -22.49 -2.46
COL21A1 ENSG00000124749 chr6 55921387 56258892 -29.98 -2.48
NTM ENSG00000182667 chr11 131240372 132206716 -91 .66 -2.48
GNG4 ENSG00000168243 chrl 235710986 235814054 -31 .50 -2.48
RAMP3 ENSG00000122679 chr7 45197389 45225901 -517.51 -2.49
AC026188.1 ENSG00000223727 chr3 3292370 3668980 -48.12 -2.50
ELOVL2-AS1 ENSG00000230314 chr6 1 1043992 1 1078459 -20.24 -2.51
SMAD9 ENSG00000120693 chr13 37418967 37494902 -24.99 -2.51
ZBTB20 ENSG00000181722 chr3 114056940 1 148661 18 -32.77 -2.52
GNG12-AS1 ENSG00000232284 chrl 68297985 68668670 -86.20 -2.53
EFNA5 ENSG00000184349 chr5 106712589 107006596 -2591 .01 -2.53
RP1 1 -713M6.2 ENSG00000251081 chr4 108179856 108223027 -1 10.95 -2.54
AC078883.3 ENSG00000232788 chr2 173328989 173330750 -62.58 -2.55
PAMR1 ENSG00000149090 chr11 35453369 35551848 -56.91 -2.55
BMPER ENSG00000164619 chr7 33944522 34195484 -44.68 -2.55
NYAP2 ENSG00000144460 chr2 226265363 226518734 -29.58 -2.56
RP4-813D12.3 ENSG00000226308 chr20 55841852 55858054 -137.00 -2.56
HMCN1 ENSG00000143341 chrl 185703682 186160085 -1835.60 -2.59
FAT4 ENSG00000196159 chr4 126237553 126414087 -35.59 -2.60
CYP2C8 ENSG00000138115 chr10 96796529 96829254 -34.03 -2.60
PLLP ENSG00000102934 chr16 57290008 57318599 -30.93 -2.60 NXPH1 ENSG00000122584 chr7 8473584 8792593 -73.07 -2.62
CTD-2260A17.2 ENSG00000247121 chr5 96149730 96271513 -20.38 -2.64
MUC5AC ENSG00000215182 chi 1 1 151579 1222364 -32.29 -2.65
PP13439 ENSG00000183657 chr3 171509579 171527714 -29.25 -2.66
SOCS2-AS1 ENSG00000246985 chr12 93936239 93965544 -57.63 -2.68
CCM2L ENSG00000101331 chr20 30598244 30619984 -60.54 -2.70
ZNF827 ENSG00000151612 chr4 146678778 146859787 -190.88 -2.70
ADAMTSL3 ENSG00000156218 chr15 84322837 84708594 -66.25 -2.71
SDC2 ENSG00000169439 chr8 97505578 97624000 -150.35 -2.73
RBM1 1 ENSG00000185272 chr21 15588450 15600693 -72.44 -2.74
PRICKLE2-AS3 ENSG00000226017 chr3 64173219 64186641 -40.90 -2.75
RNF180 ENSG00000164197 chr5 63461670 63668696 -148.61 -2.76
DOCK10 ENSG00000135905 chr2 225629806 225907162 -329.83 -2.77
FAM198A ENSG00000144649 chr3 43020758 43101703 -36.39 -2.78
TMEM86A ENSG00000151 1 17 chr11 18714668 18726332 -72.12 -2.81
LGALS3BP ENSG00000108679 chr17 76967335 76976061 -100.84 -2.84
KLK8 ENSG00000129455 chr19 51499263 51504958 -21 .09 -2.92
AC012307.2 ENSG00000228873 chr2 96811340 96812751 -77.99 -2.93
AC004053.1 ENSG00000245384 chr4 105412121 105618749 -80.99 -2.94
CTD-3179P9.1 ENSG00000249797 chr5 1 17260702 1 17601730 -266.83 -2.95
NOD2 ENSG00000167207 chr16 50727513 50766987 -37.71 -2.96
RP1 1 -556114.2 ENSG00000248242 chr4 105575030 105887950 -73.53 -3.00
C3 ENSG00000125730 chr19 6677845 6730573 -239.06 -3.04
ZNF396 ENSG00000186496 chr18 32946660 32957301 -28.92 -3.07
ZNF792 ENSG00000180884 chr19 35447257 35454953 -28.92 -3.07
ENSG00000244288 ENSG00000244288 chr18 77772846 77773065 -2767.1 1 -3.09
CHRM3 ENSG00000133019 chrl 239549864 240078750 -52.71 -3.13
AGBL4 ENSG00000186094 chi 48998526 50489585 -179.28 -3.17
KLF7 ENSG000001 18263 chr2 207938860 208031991 -75.20 -3.17
AP004372.1 ENSG000002381 17 chr11 131747573 131767002 -28.85 -3.20
DKK2 ENSG0000015501 1 chr4 107842958 108204963 -47.46 -3.27
NPAS3 ENSG00000151322 chr14 33408448 34273382 -27.35 -3.27
SMARCA1 ENSG00000102038 chrX 128580479 128657477 -39.41 -3.30
DOK6 ENSG00000206052 chr18 67068283 67516323 -20.22 -3.34
SULF1 ENSG00000137573 chr8 70378858 70573150 -1533.76 -3.42
RORC ENSG00000143365 chrl 151778546 151804348 -36.62 -3.43
YPEL1 ENSG00000100027 chr22 22051832 22090123 -92.62 -3.43
LRP1 B ENSG00000168702 chr2 140988991 142889270 -2591 .01 -3.56
RP4-710M3.2 ENSG00000255480 chrl 1 30447098 30450815 -20.42 -3.62
DCLK1 ENSG00000133083 chr13 36343121 36705467 -161 .53 -3.75
ENSG00000235732 ENSG00000235732 chrl 228544747 228546097 -50.24 -3.82
NIPSNAP3B ENSG00000165028 chr9 107526437 107539738 -35.97 -3.92
SEPP1 ENSG00000250722 chr5 42799981 42887494 -71 .47 -3.97
LY6D ENSG00000167656 chr8 143866295 143868008 -81 .14 -4.00 ENSG00000244428 ENSG00000244428 chrY 3005050 3005141 -1281 .00 -4.04
AKR1 C3 ENSG00000196139 chr10 5077548 5149878 -1 15.66 -4.16
GJA1 ENSG00000152661 chr6 121756790 121770873 -386.12 -4.16
CXXC4 ENSG00000168772 chr4 105389468 105416058 -125.07 -4.19
FSTL5 ENSG00000168843 chr4 162305048 163085187 -26.96 -4.20
CYBRD1 ENSG00000071967 chr2 172378756 172414643 -46.39 -4.21
SERPINA1 ENSG00000197249 chr14 94843083 94857029 -122.09 -4.22
AC004540.5 ENSG00000214870 chr7 26438212 26538594 -23.95 -4.37
RP1 1 -600K15.1 ENSG00000253658 chr8 69824037 70016425 -54.16 -4.37
ASTL ENSG00000188886 chr2 96789588 96804175 -102.05 -4.65
C20orf197 ENSG00000176659 chr20 58630979 58648008 -61 .38 -4.68
KCNS3 ENSG00000170745 chr2 180591 13 18542882 -628.11 -4.72
CTC-448D22.1 ENSG00000249426 chr5 1 18096168 1 18121 154 -63.23 -4.76
TMEM45A ENSG00000181458 chr3 100211462 100296288 -23.29 -4.84
RP1 1 -86516.2 ENSG00000254337 chr8 69760976 69764998 -440.01 -5.24
SNCG ENSG00000173267 chr10 88718374 88723017 -27.03 -5.28
CCDC152 ENSG00000198865 chr5 42756902 42802462 -71 .35 -5.33
MURC ENSG00000170681 chr9 103340360 103350188 -28.91 -5.50
TMCC2 ENSG00000133069 chrl 205197303 205242471 -28.91 -5.50
FBN2 ENSG00000138829 chr5 127593600 127994878 -70.61 -5.64
FREM1 ENSG00000164946 chr9 14734663 14910993 -26.42 -6.24
ENSG00000235471 ENSG00000235471 chrl 168048780 168054469 -83.20 -6.31
ENSG00000170233 ENSG00000170233 chr17 5402758 5404465 -28.24 -6.54
THRB ENSG00000151090 chr3 24158650 24536773 -132.23 -6.65
AC004947.2 ENSG00000233760 chr7 26591440 26596819 -32.50 -7.14
C1 orf168 ENSG00000187889 chi 57184476 57285369 -91.23 -7.23
RN7SKP36 ENSG00000200867 chr4 71 13814 71 14126 -21 .67 -7.33
THBS2 ENSG00000186340 chr6 169615874 169654139 -22.98 1 1 .16
TREH ENSG000001 18094 chr11 1 18528025 1 18550399 -22.98 1 1 .16
AC007365.3 ENSG00000226756 chr2 64871745 64873832 -27.89 12.85
AT0H8 ENSG00000168874 chr2 85978466 86015189 -60.02 15.13
SEMA3D ENSG00000153993 chr7 84624868 84816171 -1721.43 15.42
GPC6 ENSG00000183098 chr13 93879094 95055812 -668.77 32.90
[0032] In some embodiments, the gene signature includes at least one of the genes corresponding to SEQ ID NOs 1-56. In some embodiments, the gene signature includes at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 of the genes listed in Table 1. In some embodiments, the gene signature includes at least 5 of the genes listed in Table 1. [0033] In some embodiments, provided are methods of assessing the likelihood that breast cancer in a subject will metastasize or has metastasized. The method may include determining in a sample of the breast cancer an expression level of one or more genes, comparing the expression level of each gene to a control value associated with that same gene, and assessing the likelihood that the breast cancer will metastasize or has metastasized based on the relative difference between the expression level and the control value associated with each gene. An increase or decrease (as detailed below) in the expression level relative to the control value of at least one gene may correlate with an increased likelihood that the breast cancer in the subject will metastasize or has metastasized.
[0034] The gene may be selected from CSGALNACT1 , SIGLEC6, SHC4, ELTD1 , CGA, MST1 R, KRT8P41 , BTF3P4, RP1 1-478C6.4, EPHA3, LXN, ENSG00000252044, ENSG00000242932, ENSG00000241 1 15, AP001347.6, LHFPL2, CTD-2330K9.2, RASD1 , ENSG00000242693, AC093388.3, AOC1 , CPE, PCDH8, DCHS2, DPP4, ENSG00000242777, GUSBP5, ENSG00000244466, ENSG00000252331 , RP1 1-664D7.4, RP1 1-206M11.7, ENSG00000244580, EYA4, CTA-714B7.5, ENSG00000252318, ABCA1 , PLD1 , RNF144A, SLC44A4, SLC2A13, AXIN2, PGR, XXbac-BPG254F23.6, AMY2B, LIMCH1 , RP1 1-356J5.12, ID2, MFAP2, LPHN2, RP11-594N15.2, RP1 1-575A19.2, SPINK5, HLA-DQB1-AS1 , RN7SL151 P, ATP8A1 , FAM189A2, ZNF423, CTD-2001 C12.1 , ELOVL2, TESC, KCNMB4, STOX2, PLEKHM3, MT-TL1 , KLK1 1 , NPNT, ENSG00000234543, SHC2, BTNL9, ARMC4, OLFML2A, AMOT, RP1 1-556114.1 , SLFN5, PKIA, CTC-339F2.2, ADAM23, NPSR1-AS1 , NREP, AC009410.1 , IMPG1 , C12orf40, AC097500.2, AC004069.2, CDH26, CCDC33, PCDP1 , ALCAM, LRRN1 , WWC3-AS1 , MT-TY, MXRA8, RASL1 1A, COL3A1 , PDE1A, COL21A1 , NTM, GNG4, RAMP3, AC026188.1 , ELOVL2-AS1 , SMAD9, ZBTB20, GNG12-AS1 , EFNA5, RP1 1-713M6.2, AC078883.3, PAMR1 , BMPER, NYAP2, RP4- 813D12.3, HMCN1 , FAT4, CYP2C8, PLLP, NXPH1 , CTD-2260A17.2, MUC5AC, PP13439, SOCS2-AS1 , CCM2L, ZNF827, ADAMTSL3, SDC2, RBM1 1 , PRICKLE2-AS3, RNF180, DOCK10, FAM198A, TMEM86A, LGALS3BP, KLK8, AC012307.2, AC004053.1 , CTD- 3179P9.1 , NOD2, RP1 1-556114.2, C3, ZNF396, ZNF792, ENSG00000244288, CHRM3, AGBL4, KLF7, AP004372.1 , DKK2, NPAS3, SMARCA1 , DOK6, SULF1 , RORC, YPEL1 , LRP1 B, RP4-710M3.2, DCLK1 , ENSG00000235732, NIPSNAP3B, SEPP1 , LY6D, ENSG00000244428, AKR1 C3, GJA1 , CXXC4, FSTL5, CYBRD1 , SERPINA1 , AC004540.5, RP1 1-600K15.1 , ASTL, C20orf197, KCNS3, CTC-448D22.1 , TMEM45A, RP1 1-86516.2, SNCG, CCDC152, MURC, TMCC2, FBN2, FREM1 , ENSG00000235471 , ENSG00000170233, THRB, AC004947.2, C1orf168, RN7SKP3, THBS2, TREH, AC007365.3, ATOH8, SEMA3D, and GPC6. In some embodiments, the gene is selected from at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 of the genes listed in Table 1 and listed above.
[0035] In some embodiments, the gene may be selected from CSGALNACT1 , SIGLEC6, SHC4 , ELTD1 , CGA, MST1 R, KRT8P41 , BTF3P4, RP1 1-478C6.4, EPHA3, LXN, ENSG00000252044, ENSG00000242932, ENSG00000241 1 15, AP001347.6, LHFPL2, CTD-2330K9.2, RASD1 , ENSG00000242693, AC093388.3, AOC1 , CPE, PCDH8, DCHS2, DPP4, ENSG00000242777, GUSBP5, ENSG00000244466, ENSG00000252331 , RP1 1- 664D7.4, RP1 1-206M1 1.7, ENSG00000244580, EYA4, CTA-714B7.5, and ENSG00000252318. An increase in the expression level of at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 of these genes relative to the control value may correlate with an increased likelihood that the breast cancer in the subject will metastasize or has metastasized. An increase in the expression level of at least 1 of the genes corresponding to SEQ ID NOs 1-22 and 48-50 relative to the control value may correlate with an increased likelihood that the breast cancer in the subject will metastasize or has metastasized.
[0036] In some embodiments, the gene may be selected from CSGALNACT1 , SIGLEC6, and SHC4 (corresponding to SEQ ID NOs 1-22 and 48-50).
[0037] In some embodiments, the gene may be selected from ABCA1 , PLD1 , RNF144A, SLC44A4, SLC2A13, AXIN2, PGR, XXbac-BPG254F23.6, AMY2B, LIMCH1 , RP1 1- 356J5.12, ID2, MFAP2, LPHN2, RP1 1-594N15.2, RP1 1-575A19.2, SPINK5, HLA-DQB1- AS1 , RN7SL151 P, ATP8A1 , FAM189A2, ZNF423, CTD-2001 C12.1 , ELOVL2, TESC, KCNMB4, STOX2, PLEKHM3, MT-TL1 , KLK1 1 , NPNT, ENSG00000234543, SHC2, BTNL9, ARMC4, OLFML2A, AMOT, RP1 1-556114.1 , SLFN5, PKIA, CTC-339F2.2, ADAM23, NPSR1-AS1 , NREP, AC009410.1 , IMPG1 , C12orf40, AC097500.2, AC004069.2, CDH26, CCDC33, PCDP1 , ALCAM, LRRN1 , WWC3-AS1 , MT-TY, MXRA8, RASL1 1A, COL3A1 , PDE1A, COL21A1 , NTM, GNG4, RAMP3, AC026188.1 , ELOVL2-AS1 , SMAD9, ZBTB20, GNG12-AS1 , EFNA5, RP1 1-713M6.2, AC078883.3, PAMR1 , BMPER, NYAP2, RP4- 813D12.3, HMCN1 , FAT4, CYP2C8, PLLP, NXPH1 , CTD-2260A17.2, MUC5AC, PP13439, SOCS2-AS1 , CCM2L, ZNF827, ADAMTSL3, SDC2, RBM1 1 , PRICKLE2-AS3, RNF180, DOCK10, FAM198A, TMEM86A, LGALS3BP, KLK8, AC012307.2, AC004053.1 , CTD- 3179P9.1 , NOD2, RP1 1-556114.2, C3, ZNF396, ZNF792, ENSG00000244288, CHRM3, AGBL4, KLF7, AP004372.1 , DKK2, NPAS3, SMARCA1 , DOK6, SULF1 , RORC, YPEL1 , LRP1 B, RP4-710M3.2, DCLK1 , ENSG00000235732, NIPSNAP3B, SEPP1 , LY6D, ENSG00000244428, AKR1 C3, GJA1 , CXXC4, FSTL5, CYBRD1 , SERPINA1 , AC004540.5, RP1 1-600K15.1 , ASTL, C20orf197, KCNS3, CTC-448D22.1 , TMEM45A, RP1 1-86516.2, SNCG, CCDC152, MURC, TMCC2, FBN2, FREM1 , ENSG00000235471 , ENSG00000170233, THRB, AC004947.2, C1orf168, RN7SKP3, THBS2, TREH, AC007365.3, ATOH8, SEMA3D, and GPC6. A decrease in the expression level of at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 of these genes relative to the control value may correlate with an increased likelihood that the breast cancer in the subject will metastasize or has metastasized. A decrease in the expression level of at 1 of the genes corresponding to SEQ ID NOs 23-47 and 51-56 relative to the control value may correlate with an increased likelihood that the breast cancer in the subject will metastasize or has metastasized.
[0038] In some embodiments, the gene may be selected from ABCA1 , PLD1 , RNF144A, SLC44A4, SLC2A13, AXIN2 (corresponding to SEQ ID NOs 23-47 and 51-56).
[0039] In some embodiments, the gene may be selected from CSGALNACT1 , SIGLEC6, SHC4, ABCA1 , PLD1 , RNF144A, SLC44A4, SLC2A13, AXIN2 (corresponding to SEQ ID NOs 1-56). A difference (decrease or increase) in the expression level of at least one of the genes corresponding to SEQ ID NOs 1-56 relative to the control value may correlate with an increased likelihood that the breast cancer in the subject will metastasize or has metastasized.
[0040] The expression level of the gene may be increased relative to the expression level of a control by an amount of at least about 0.5-fold, at least about 1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5- fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, at least about 10-fold, at least about 1 1-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, at least about 50-fold, at least about 55-fold, at least about 60-fold, at least about 65-fold, at least about 70-fold, at least about 75-fold, at least about 80-fold, at least about 85-fold, at least about 90-fold, at least about 95-fold, at least about 100-fold, at least about 150-fold, at least about 200-fold, at least about 250-fold, at least about 300-fold, at least about 350-fold, at least about 400-fold, at least about 450-fold, at least about 500-fold, or at least about 550- fold. In some embodiments, the expression of the gene may be increased relative to the expression level of a control by an amount of about 0.5-fold to about 500-fold, about 1-fold to about 50-fold, or about 2-fold to about 10-fold. In some embodiments, the expression of the gene is increased relative to the expression level of a control by an amount of at least about 2-fold.
[0041] The expression level of the gene may be decreased relative to the expression level of a control by an amount of at least about 0.5-fold, at least about 1 -fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5- fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, at least about 10-fold, at least about 1 1 -fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, at least about 50-fold, at least about 55-fold, at least about 60-fold, at least about 65-fold, at least about 70-fold, at least about 75-fold, at least about 80-fold, at least about 85-fold, at least about 90-fold, at least about 95-fold, at least about 100-fold, at least about 150-fold, at least about 200-fold, at least about 250-fold, at least about 300-fold, at least about 350-fold, at least about 400-fold, at least about 450-fold, at least about 500-fold, or at least about 550- fold. The expression of the gene may be decreased relative to the expression level of a control by an amount of about 0.5-fold to about 500-fold, about 1 -fold to about 50-fold, or about 2-fold to about 10-fold. In some embodiments, the expression of the gene is decreased relative to the expression level of a control by an amount of at least about 2-fold.
[0042] A subject can be an animal, a vertebrate animal, a mammal, a rodent (e.g. a guinea pig, a hamster, a rat, a mouse), murine (e.g. a mouse), canine (e.g. a dog), feline (e.g. a cat), equine (e.g. a horse), a primate, simian (e.g. a monkey or ape), a monkey (e.g. marmoset, baboon), an ape (e.g. gorilla, chimpanzee, orangutan, gibbon), or a human. In some embodiments, the subject is a mammal. In further embodiments, the mammal is a human.
[0043] As used herein, the term "sample" or "biological sample" relates to any material that is taken from its native or natural state, so as to facilitate any desirable manipulation or further processing and/or modification. A sample or a biological sample can comprise a cell, a tissue, a fluid (e.g., a biological fluid), a protein (e.g., antibody, enzyme, soluble protein, insoluble protein), a polynucleotide (e.g., RNA, DNA), a membrane preparation, and the like, that can optionally be further isolated and/or purified from its native or natural state. A "biological fluid" refers to any a fluid originating from a biological organism. Exemplary biological fluids include, but are not limited to, blood, serum, and plasma. A biological fluid may be in its natural state or in a modified state by the addition of components such as reagents, or removal of one or more natural constituents (e.g., blood plasma). A sample can be from any tissue or fluid from an organism. In some embodiments, the sample comprises tissue from the breast, milk ducts, breast lobules, digestive tract, lung, liver, kidney, brain, lip, mouth, esophagus, urinary bladder, prostate, vagina, and/or cervix. In some embodiments the sample is from a tissue that is part of, or associated with, the breast of the organism. In some embodiments, the sample may be tissue from a neoplasm. A neoplasm may include cancer. In some embodiments, the sample may be cancerous tissue or from a tumor. In some embodiments, the sample may comprise tissue surrounding cancerous tissue or a tumor. In some embodiments, the sample may comprise tissue surrounding or around the perimeter of cancerous tissue or a tumor that was surgically excised. The cancer may comprise breast cancer.
[0044] The methods described herein can include any suitable method for evaluating gene expression. Determining expression of at least one gene may include, for example, detection of an RNA transcript or portion thereof, and/or an expression product such as a protein or portion thereof. Expression of a gene may be detected using any suitable method known in the art, including but not limited to, detection and/or binding with antibodies, detection and/or binding with antibodies tethered to or associated with an imaging agent, real time RT-PCR, Northern analysis, magnetic particles (e.g., microparticles or nanoparticles), Western analysis, expression reporter plasmids, immunofluorescence, immunohistochemistry, detection based on an activity of an expression product of the gene such as an activity of a protein, any method or system involving flow cytometry, and any suitable array scanner technology. For example, the expression level of a protein may be evaluated by immunofluorescence by visualizing cells stained with a fluorescently-labeled protein-specific antibody, Western blot analysis of protein expression, and RT-PCR of protein transcripts. The antibody or fragment thereof may suitably recognize a particular intracellular protein, protein isoform, or protein configuration. In some embodiments, determining the expression level of at least one gene comprises measuring the expression level of an RNA transcript of the at least one gene, or an expression product thereof.
[0045] As used herein, an "imaging agent" or "reporter" is any compound or composition that enhances visualization or detection of a target. Any type of detectable imaging agent or reporter may be used in the methods disclosed herein for the detection of an expression product. Exemplary imaging agents and reporters may include, but are not limited to, compounds and compositions comprising magnetic beads, fluorophores, radionuclides, and nuclear stains (e.g., DAPI), and further comprising a targeting moiety for specifically targeting or binding to the target expression product. For example, an imaging agent may include a compound that comprises an unstable isotope (i.e., a radionuclide), such as an alpha- or beta-emitter, or a fluorescent moiety, such as Cy-5, Alexa 647, Alexa 555, Alexa 488, fluorescein, rhodamine, and the like. In some embodiments, suitable radioactive moieties may include labeled polynucleotides and/or polypeptides coupled to the targeting moiety. In some embodiments, the imaging agent may comprise a radionuclide such as, for example, a radionuclide that emits low-energy electrons (e.g., those that emit photons with energies as low as 20 keV). Such nuclides can irradiate the cell to which they are delivered without irradiating surrounding cells or tissues. Non-limiting examples of radionuclides that are can be delivered to cells may include, but are not limited to, 137Cs, 103Pd, 111ln, 125l, 211At, 212Bi and 213Bi, among others known in the art. Further imaging agents may include paramagnetic species for use in MRI imaging, echogenic entities for use in ultrasound imaging, fluorescent entities for use in fluorescence imaging (including quantum dots), and light-active entities for use in optical imaging. A suitable species for MRI imaging is a gadolinium complex of diethylenetriamine pentacetic acid (DTPA). For positron emission tomography (PET), 18F or 11C may be delivered. Other non-limiting examples of reporter molecules are discussed throughout the disclosure. In some embodiments, determining the expression level of at least one gene includes measuring the expression level of an RNA transcript of the at least one gene, or an expression product thereof. In some embodiments, measuring the expression level of the RNA transcript of the at least one gene, or the expression product thereof, includes using at least one of a PCR-based method, a Northern blot method, a microarray method, and an immunohistochemical method.
[0046] The expression level of a gene in a sample may be compared to a control value associated with that same gene. A control may include comparison to the level of expression in a control cell, such as a non-cancerous cell or other normal cell, or comparison to a cancer sample that does not express RON from a different patient or from historical controls. The control may be from a non-cancerous or normal cell from the same subject, or it may be from a different subject. Alternatively, a control may include an average range of the level of expression from a population of normal cells. Those skilled in the art will appreciate that a variety of controls may be used. In some embodiments, the control value associated with each gene may be determined by determining the expression level of that gene in one or more control samples, and calculating an average expression level of that gene in the one or more control samples, wherein each control sample is obtained from normal or healthy tissue of the same or a different subject. [0047] In some embodiments, provided are methods of treating cancer in a subject. The method may include determining in a sample of the cancer an expression level of one or more genes selected from the genes listed in Table 1 , comparing the expression level of each gene to a control value associated with that same gene, and administering at least one of a RON inhibitor and a PI3K kinase inhibitor to the subject when the expression level of at least one or five of the genes corresponding to CSGALNACT1 , SIGLEC6, SHC4, ELTD1 , CGA, MST1 R, KRT8P41 , BTF3P4, RP1 1-478C6.4, EPHA3, LXN, ENSG00000252044, ENSG00000242932, ENSG00000241 1 15, AP001347.6, LHFPL2, CTD-2330K9.2, RASD1 , ENSG00000242693, AC093388.3, AOC1 , CPE, PCDH8, DCHS2, DPP4, ENSG00000242777, GUSBP5, ENSG00000244466, ENSG00000252331 , RP1 1-664D7.4, RP1 1-206M1 1.7, ENSG00000244580, EYA4, CTA-714B7.5, and ENSG00000252318 (or at least one of CSGALNACT1 , SIGLEC6, and SHC4) is increased at least about 2-fold relative to the control, or administering an alternative therapy to the subject when the expression level of at least one or five of the genes corresponding to CSGALNACT1 , SIGLEC6, SHC4, ELTD1 , CGA, MST1 R, KRT8P41 , BTF3P4, RP1 1-478C6.4, EPHA3, LXN, ENSG00000252044, ENSG00000242932, ENSG00000241 1 15, AP001347.6, LHFPL2, CTD-2330K9.2, RASD1 , ENSG00000242693, AC093388.3, AOC1 , CPE, PCDH8, DCHS2, DPP4, ENSG00000242777, GUSBP5, ENSG00000244466, ENSG00000252331 , RP1 1- 664D7.4, RP1 1-206M1 1.7, ENSG00000244580, EYA4, CTA-714B7.5, and ENSG00000252318 (or at least one of CSGALNACT1 , SIGLEC6, and SHC4) is increased less than about 2-fold or decreased relative to the control.
[0048] The method may include administering at least one of a RON inhibitor and a PI3K kinase inhibitor to the subject when the expression level of at least one or five of the genes corresponding to ABCA1 , PLD1 , RNF144A, SLC44A4, SLC2A13, AXIN2, PGR, XXbac- BPG254F23.6, AMY2B, LIMCH1 , RP1 1-356J5.12, ID2, MFAP2, LPHN2, RP1 1-594N15.2, RP1 1-575A19.2, SPINK5, HLA-DQB1-AS1 , RN7SL151 P, ATP8A1 , FAM189A2, ZNF423, CTD-2001 C12.1 , ELOVL2, TESC, KCNMB4, STOX2, PLEKHM3, MT-TL1 , KLK1 1 , NPNT, ENSG00000234543, SHC2, BTNL9, ARMC4, OLFML2A, AMOT, RP1 1-556114.1 , SLFN5, PKIA, CTC-339F2.2, ADAM23, NPSR1-AS1 , NREP, AC009410.1 , IMPG1 , C12orf40, AC097500.2, AC004069.2, CDH26, CCDC33, PCDP1 , ALCAM, LRRN1 , WWC3-AS1 , MT- TY, MXRA8, RASL1 1A, COL3A1 , PDE1A, COL21A1 , NTM, GNG4, RAMP3, AC026188.1 , ELOVL2-AS1 , SMAD9, ZBTB20, GNG12-AS1 , EFNA5, RP1 1-713M6.2, AC078883.3, PAMR1 , BMPER, NYAP2, RP4-813D12.3, HMCN1 , FAT4, CYP2C8, PLLP, NXPH1 , CTD- 2260A17.2, MUC5AC, PP13439, SOCS2-AS1 , CCM2L, ZNF827, ADAMTSL3, SDC2, RBM1 1 , PRICKLE2-AS3, RNF180, DOCK10, FAM198A, TMEM86A, LGALS3BP, KLK8, AC012307.2, AC004053.1 , CTD-3179P9.1 , NOD2, RP1 1-556114.2, C3, ZNF396, ZNF792, ENSG00000244288, CHRM3, AGBL4, KLF7, AP004372.1 , DKK2, NPAS3, SMARCA1 , DOK6, SULF1 , RORC, YPEL1 , LRP1 B, RP4-710M3.2, DCLK1 , ENSG00000235732, NIPSNAP3B, SEPP1 , LY6D, ENSG00000244428, AKR1 C3, GJA1 , CXXC4, FSTL5, CYBRD1 , SERPINA1 , AC004540.5, RP1 1-600Κ15.1 , ASTL, C20orf197, KCNS3, CTC- 448D22.1 , TMEM45A RP11-86516.2, SNCG, CCDC152, MURC, TMCC2, FBN2, FREM1 , ENSG00000235471 , ENSG00000170233, THRB, AC004947.2, C1orf168, RN7SKP3, THBS2, TREHAC007365.3, ΑΤΟΗ8, SEMA3D, and GPC6 (or at least one of ABCA1 , PLD1 , RNF144A, SLC44A4, SLC2A13, and AXIN2) is decreased at least about 2-fold relative to the control, or administering an alternative therapy to the subject when the expression level of at least one or five of the genes corresponding to ABCA1 , PLD1 , RNF144A, SLC44A4, SLC2A13, AXIN2, PGR, XXbac-BPG254F23.6, AMY2B, LIMCH1 , RP1 1-356J5.12, ID2, MFAP2, LPHN2, RP1 1-594N15.2, RP1 1-575A19.2, SPINK5, HLA-DQB1-AS1 , RN7SL151 P, ATP8A1 , FAM189A2, ZNF423, CTD-2001 C12.1 , ELOVL2, TESC, KCNMB4, STOX2, PLEKHM3, MT-TL1 , KLK1 1 , NPNT, ENSG00000234543, SHC2, BTNL9, ARMC4, OLFML2A, AMOT, RP1 1-556114.1 , SLFN5, PKIA, CTC-339F2.2, ADAM23, NPSR1-AS1 , NREP, AC009410.1 , IMPG1 , C12orf40, AC097500.2, AC004069.2, CDH26, CCDC33, PCDP1 , ALCAM, LRRN1 , WWC3-AS1 , MT-TY, MXRA8, RASL1 1A, COL3A1 , PDE1A, COL21A1 , NTM, GNG4, RAMP3, AC026188.1 , ELOVL2-AS1 , SMAD9, ZBTB20, GNG12- AS1 , EFNA5, RP1 1-713M6.2, AC078883.3, PAMR1 , BMPER, NYAP2, RP4-813D12.3, HMCN1 , FAT4, CYP2C8, PLLP, NXPH1 , CTD-2260A17.2, MUC5AC, PP13439, SOCS2- AS1 , CCM2L, ZNF827, ADAMTSL3, SDC2, RBM11 , PRICKLE2-AS3, RNF180, DOCK10, FAM198A, TMEM86A, LGALS3BP, KLK8, AC012307.2, AC004053.1 , CTD-3179P9.1 , NOD2, RP1 1-556114.2, C3, ZNF396, ZNF792, ENSG00000244288, CHRM3, AGBL4, KLF7, AP004372.1 , DKK2, NPAS3, SMARCA1 , DOK6, SULF1 , RORC, YPEL1 , LRP1 B, RP4- 710M3.2, DCLK1 , ENSG00000235732, NIPSNAP3B, SEPP1 , LY6D, ENSG00000244428, AKR1 C3, GJA1 , CXXC4, FSTL5, CYBRD1 , SERPINA1 , AC004540.5, RP1 1-600K15.1 , ASTL, C20orf197, KCNS3, CTC-448D22.1 , TMEM45A, RP1 1-86516.2, SNCG, CCDC152, MURC, TMCC2, FBN2, FREM1 , ENSG00000235471 , ENSG00000170233, THRB, AC004947.2, C1orf168, RN7SKP3, THBS2, TREH, AC007365.3, ATOH8, SEMA3D, and GPC6 (or at least one of ABCA1 , PLD1 , RNF144A, SLC44A4, SLC2A13, and AXIN2) is decreased less than about 2-fold or increased relative to the control. In some embodiments, the cancer is breast cancer.
[0049] Alternative therapies include therapies other than administration of a RON inhibitor or a PI3K kinase inhibitor. Alternative therapies may include, but are not limited to, radiation therapy, a surgical procedure, anti-cancer drugs, or any combination thereof. [0050] In some embodiments, provided are methods of determining whether a subject having breast cancer would benefit from treatment with at least one of a RON inhibitor and a PI3K kinase inhibitor. The method may include assessing the likelihood that the breast cancer will metastasize or has metastasized according to the method detailed above, and determining whether the subject would benefit from treatment with at least one of a RON inhibitor and a PI3K kinase inhibitor based on the likelihood that the breast cancer will metastasize or has metastasized.
[0051] In some embodiments, provided are methods of monitoring a molecular response of a subject to treatment of breast cancer with at least one of a RON inhibitor and a PI3K inhibitor. The method may include determining in a first sample of the breast cancer an expression level of one or more genes, administering to the subject a therapeutic amount of a medicament comprising at least one of a RON inhibitor and a PI3K inhibitor, determining in a second sample of the breast cancer an expression level of each of the one or more genes, wherein the second sample is collected after administration of the medicament, and comparing for each gene the expression level in the first sample to the expression level in the second sample.
[0052] Inhibitors encompass agents that inhibit the activity of or reduce the amount of RON, MSP, MBD4, PI3K, or other members of the RON/MBD4 pathway. The amount or the activity of the protein may be reduced or inhibited using a variety of techniques known in the art. For example, an inhibitor may indirectly or directly bind and inhibit the activity of the protein, including binding activity or catalytic activity. An inhibitor may inhibit the ability of the protein to interact with cellular and extracellular components. An inhibitor may prevent or reduce expression of the protein. For example, a therapeutic composition adapted to reduce the amount or the activity of the protein may comprise a small molecule inhibitor of the protein itself or of a binding partner, an antibody specific for the protein, or a siRNA. In some embodiments, the therapeutic composition may comprise a siRNA adapted to reduce the expression of the protein. RON inhibitors may include, but are not limited to, OSI-296, BMS- 77607, and any other compounds that inhibit the activity of RON. PI3K inhibitors may include, but are not limited to, LY294002, BEZ-235, BKM-120, and any other compounds that inhibit the activity of PI3K.
[0053] Compositions may be administered to a subject by any convenient route of administration, whether systemically/ peripherally or at the site of desired action, including but not limited to, oral (e.g. by ingestion); topical (including e.g. transdermal, intranasal, ocular, buccal, and sublingual); pulmonary (e.g. by inhalation or insufflation therapy using, e.g. an aerosol, e.g. through mouth or nose); rectal; vaginal; parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal; by implant of a depot, for example, subcutaneously or intramuscularly.
[0054] The term "treatment", as used herein in the context of treating a condition, pertains generally to treatment and therapy, whether of a human or an animal (e.g. in veterinary applications), in which a desired therapeutic effect is achieved. For example, treatment may ameliorate the condition or may inhibit the progress of the condition (e.g., reduce the rate of progress or halt the rate of progress).
[0055] In some embodiments, provided are kits for assessing the likelihood that breast cancer in a subject will metastasize or has metastasized. In some embodiments, provided are kits for determining whether the subject would benefit from treatment with at least one of a RON inhibitor and a PI3K kinase inhibitor. The kit may include one or more primers. Each primer may be adapted to amplify an RNA transcript of an independent one of a genes selected from CSGALNACT1 , SIGLEC6, SHC4, ELTD1 , CGA, MST1 R, KRT8P41 , BTF3P4, RP1 1-478C6.4, EPHA3, LXN, ENSG00000252044, ENSG00000242932, ENSG00000241 1 15, AP001347.6, LHFPL2, CTD-2330K9.2, RASD1 , ENSG00000242693, AC093388.3, AOC1 , CPE, PCDH8, DCHS2, DPP4, ENSG00000242777, GUSBP5, ENSG00000244466, ENSG00000252331 , RP1 1-664D7.4, RP1 1-206M1 1.7, ENSG00000244580, EYA4, CTA-714B7.5, ENSG00000252318, ABCA1 , PLD1 , RNF144A, SLC44A4, SLC2A13, AXIN2, PGR, XXbac-BPG254F23.6, AMY2B, LIMCH1 , RP1 1- 356J5.12, ID2, MFAP2, LPHN2, RP1 1-594N15.2, RP1 1-575A19.2, SPINK5, HLA-DQB1- AS1 , RN7SL151 P, ATP8A1 , FAM189A2, ZNF423, CTD-2001 C12.1 , ELOVL2, TESC, KCNMB4, STOX2, PLEKHM3, MT-TL1 , KLK1 1 , NPNT, ENSG00000234543, SHC2, BTNL9, ARMC4, OLFML2A, AMOT, RP1 1-556114.1 , SLFN5, PKIA, CTC-339F2.2, ADAM23, NPSR1-AS1 , NREP, AC009410.1 , IMPG1 , C12orf40, AC097500.2, AC004069.2, CDH26, CCDC33, PCDP1 , ALCAM, LRRN1 , WWC3-AS1 , MT-TY, MXRA8, RASL1 1A, COL3A1 , PDE1A, COL21A1 , NTM, GNG4, RAMP3, AC026188.1 , ELOVL2-AS1 , SMAD9, ZBTB20, GNG12-AS1 , EFNA5, RP1 1-713M6.2, AC078883.3, PAMR1 , BMPER, NYAP2, RP4- 813D12.3, HMCN1 , FAT4, CYP2C8, PLLP, NXPH1 , CTD-2260A17.2, MUC5AC, PP13439, SOCS2-AS1 , CCM2L, ZNF827, ADAMTSL3, SDC2, RBM1 1 , PRICKLE2-AS3, RNF180, DOCK10, FAM198A, TMEM86A, LGALS3BP, KLK8, AC012307.2, AC004053.1 , CTD- 3179P9.1 , NOD2, RP1 1-556114.2, C3, ZNF396, ZNF792, ENSG00000244288, CHRM3, AGBL4, KLF7, AP004372.1 , DKK2, NPAS3, SMARCA1 , DOK6, SULF1 , RORC, YPEL1 , LRP1 B, RP4-710M3.2, DCLK1 , ENSG00000235732, NIPSNAP3B, SEPP1 , LY6D, ENSG00000244428, AKR1 C3, GJA1 , CXXC4, FSTL5, CYBRD1 , SERPINA1 , AC004540.5, RP1 1-600K15.1 , ASTL, C20orf197, KCNS3, CTC-448D22.1 , ΤΜΕΜ45Α, RP1 1-86516.2, SNCG, CCDC152, MURC, TMCC2, FBN2, FREM1 , ENSG00000235471 , ENSG00000170233, THRB, AC004947.2, C1orf168, RN7SKP3, THBS2, TREH, AC007365.3, ΑΤΟΗ8, SEMA3D, and GPC6 (listed in Table 1 ). The kit may further include instructions for use.
[0056] In some embodiments, provided are kits for assessing the likelihood that breast cancer in a subject will metastasize or has metastasized. In some embodiments, provided are kits for determining whether the subject would benefit from treatment with at least one of a RON inhibitor and a PI3K kinase inhibitor. The kit may include one or more probes. Each probe may be adapted to specifically bind to an RNA transcript, or an expression product thereof, of an independent one of the genes selected from CSGALNACT1 , SIGLEC6, SHC4, ELTD1 , CGA, MST1 R, KRT8P41 , BTF3P4, RP1 1-478C6.4, EPHA3, LXN, ENSG00000252044, ENSG00000242932, ENSG00000241 1 15, AP001347.6, LHFPL2, CTD-2330K9.2, RASD1 , ENSG00000242693, AC093388.3, AOC1 , CPE, PCDH8, DCHS2, DPP4, ENSG00000242777, GUSBP5, ENSG00000244466, ENSG00000252331 , RP1 1- 664D7.4, RP1 1-206M1 1.7, ENSG00000244580, EYA4, CTA-714B7.5, ENSG00000252318, ABCA1 , PLD1 , RNF144A, SLC44A4, SLC2A13, AXIN2, PGR, XXbac-BPG254F23.6, AMY2B, LIMCH1 , RP1 1-356J5.12, ID2, MFAP2, LPHN2, RP1 1-594N15.2, RP1 1-575A19.2, SPINK5, HLA-DQB1-AS1 , RN7SL151 P, ATP8A1 , FAM189A2, ZNF423, CTD-2001 C12.1 , ELOVL2, TESC, KCNMB4, STOX2, PLEKHM3, MT-TL1 , KLK1 1 , NPNT, ENSG00000234543, SHC2, BTNL9, ARMC4, OLFML2A, AMOT, RP1 1-556114.1 , SLFN5, PKIA, CTC-339F2.2, ADAM23, NPSR1-AS1 , NREP, AC009410.1 , IMPG1 , C12orf40, AC097500.2, AC004069.2, CDH26, CCDC33, PCDP1 , ALCAM, LRRN1 , WWC3-AS1 , MT- TY, MXRA8, RASL1 1A, COL3A1 , PDE1A, COL21A1 , NTM, GNG4, RAMP3, AC026188.1 , ELOVL2-AS1 , SMAD9, ZBTB20, GNG12-AS1 , EFNA5, RP1 1-713M6.2, AC078883.3, PAMR1 , BMPER, NYAP2, RP4-813D12.3, HMCN1 , FAT4, CYP2C8, PLLP, NXPH1 , CTD- 2260A17.2, MUC5AC, PP13439, SOCS2-AS1 , CCM2L, ZNF827, ADAMTSL3, SDC2, RBM1 1 , PRICKLE2-AS3, RNF180, DOCK10, FAM198A, TMEM86A, LGALS3BP, KLK8, AC012307.2, AC004053.1 , CTD-3179P9.1 , NOD2, RP1 1-556114.2, C3, ZNF396, ZNF792, ENSG00000244288, CHRM3, AGBL4, KLF7, AP004372.1 , DKK2, NPAS3, SMARCA1 , DOK6, SULF1 , RORC, YPEL1 , LRP1 B, RP4-710M3.2, DCLK1 , ENSG00000235732, NIPSNAP3B, SEPP1 , LY6D, ENSG00000244428, AKR1 C3, GJA1 , CXXC4, FSTL5, CYBRD1 , SERPINA1 , AC004540.5, RP1 1-600K15.1 , ASTL, C20orf197, KCNS3, CTC- 448D22.1 , TMEM45A RP11-86516.2, SNCG, CCDC152, MURC, TMCC2, FBN2, FREM1 , ENSG00000235471 , ENSG00000170233, THRB, AC004947.2, C1orf168, RN7SKP3, THBS2, TREHAC007365.3, ATOH8, SEMA3D, and GPC6 (listed in Table 1 ). The kit may further include instructions for use.
[0057] A probe may comprise an antibody to an expression product. A probe may comprise an oligonucleotide complementary to an RNA transcript. A probe may comprise an "imaging agent" or "reporter" as detailed above.
[0058] In other aspects, provided are methods of inhibiting breast cancer metastasis. In other aspects, provided are methods of treating breast cancer. The method may include administering at least one of a RON inhibitor and a PI3K kinase inhibitor. The inhibitor may be formulated into a composition. Standard pharmaceutical formulation techniques are used, such as those disclosed in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. (1990).
[0059] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including but not limited to") unless otherwise noted. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to illustrate aspects and embodiments of the disclosure and does not limit the scope of the claims.
EXAMPLES
EXAMPLE 1 : Experimental Procedures
[0060] Cell culture
[0061] MCF-7 cells were cultured in Dulbecco's modified Eagle's medium (DME/F-12, Thermo Scientific) supplemented with 1 % penicillin/streptomycin (Thermo Scientific), 10 μg/mL insulin (Gibco), and 10% heat inactivated fetal bovine serum (Thermo Scientific) at 37°C in 5% C02. T47D cells were cultured in the same conditions in RPMI1640 (RPMI1640, Thermo Scientific) supplemented with 1 % penicillin/streptomycin (Thermo Scientific) and 10% heat inactivated fetal bovine serum. RON and MSP were stably expressed in MCF7 cells by retroviral infection (Liu et al., 201 1 ). MCF7-RON/MSP cells were maintained in MCF7 medium supplemented with 0.2 μg/mL puromycin and 50 μg/mL hygromycin. MCF7 and T47D cell lines were infected with lentiviral constructs that direct the synthesis of shRNA (based on PLKO.1 but with a neo resistant cassette substituted for the puromycin resistant cassette) and selected by the addition of 1 mg/mL G418 48 hours later.
[0062] In vivo metastasis
[0063] All animal procedures were reviewed and approved by the University of Utah Institutional Animal Care and Use Committee. Breast cancer cells (2 X 106 cells in 25 μΙ_ matrigel) were implanted into the cleared right inguinal mammary fat pads of 3 week-old female NOD/SCID mice as previously described. An intrascapular estrogen pellet was subcutaneously implanted into the mice at the same time. For bioluminescence imaging, mice were anesthetized and given 150 mg/kg of D-luciferin in PBS by i.p. injection. Five minutes after injection, mice were killed and the organs extracted and imaged ex vivo to detect metastasis foci. Bioluminescence was imaged with a charge-coupled device camera (MS; Xenogen). Bioluminescence images were obtained with a 15 cm field of view, binning (resolution) factor of 8, 1/f stop, open filter, and an imaging time of 8 sec. We defined a metastatic event as any detectable luciferase signal above background and validated metastasis histologically. For drug treatment studies, female NOD-SCID mice were each orthotopically transplanted with a single primary breast cancer tumor fragment from patient lines HCI-003 and HCI-01 1. Mice bearing these established xenograft tumors (2-4 mm in diameter) received vehicle (40% Trappsol) or OSI-296 (200 mg/kg) by oral gavage every other day until a tumor diameter of 1.2-1.5 cm was reached. Mice were killed and analyzed for metastasis histologically.
[0064] DNA and RNA isolation procedures
[0065] Genomic DNA for MeDIP and bisulfite modification was isolated using the DNeasy Blood Kit (Qiagen). Total RNA for gene expression analysis by microarray and quantitative RT-PCR was isolated with the RNeasy Kit (Qiagen) and tested for integrity on RNA 6000 NanoChips using an Agilent 2100 Bioanalyzer.
[0066] DNA methyltransferase (Dnmt) activity assay
[0067] We quantified the total Dnmt activity levels using a colorimetric assay with the EpiQuik DNA Methyltransferase Activity Assay kit (Epigentek Group). In this assay, Dnmt transfers a methyl group from S-adenosylmethionine (AdoMet) to a cytosine in the DNA substrate. Methylated DNA was probed using an anti-5-methylcytosine antibody. The levels of methylated DNA, which were proportional to the enzymatic activity, were then colorimetrically quantified and expressed as absorbance units 450 nm h~1 mg protein-1. [0068] Expression array hybridization and data analysis
[0069] Triplicate biological replicates of hybridization were performed for each cell line. The Agilent Quick Amp Labeling Kit is used to generate fluorescently labeled cRNA for one- color microarray hybridizations. Agilent RNA spike-in controls are combined with input total RNA samples (50 to 1000 ng). The polyadenylated fraction of the total RNA sample is primed with oligo dT/T7 RNA polymerase promoter oligonucleotide sequences and cDNA synthesis is accomplished through the addition of MMLV-RT. Following cDNA synthesis, T7 RNA polymerase and dye-labeled nucleotides are combined with the reaction mixture to simultaneously amplify the target material through the generation of cRNA and incorporate cyanine 3-CTP. Fluorescently labeled, cRNA molecules are purified from the reaction mixture using the Qiagen RNeasy mini kit. The concentration of the purified samples is determined using a NanoDrop ND-1000 spectrophotometer. Fluorescently labeled cRNA samples (825 ng each) were fragmented and combined with Agilent Hi-RPM Hybridization Buffer. Microarray hybridizations were performed using Agilent SureHyb Hybridization chambers. Hybridization chambers were loaded onto a rotisserie in an Agilent Hybridization oven and were incubated at 65°C for 17 hours with a rotational speed of 10 rpm. Following incubation, the microarray slide was washed for 1 minute each in Gene Expression Wash Buffer 1 (6X SSPE, 0.005% N-lauroylsarcosine; room temperature) and Gene Expression Wash Buffer (0.06X SSPE, 0.005% N-lauroylsarcosine; 31 °C) for 1 minute each. Microarray slides were briefly dipped in a solution of acetonitrile and dried. Microarray slides were scanned in an Agilent Technologies G2505C Microarray Scanner at 5 μιη resolution. The scanner performs detection of Cyanine-3 signal on the hybridized slide. TIF files generated from the scanned microarray image are loaded into Agilent Feature Extraction Software version 10.5. The software automatically positions a grid and finds the centroid positions of each feature on the microarray. This information is used to perform calculations that include feature intensities, background measurements and statistical analyses. Data generated by the software is recorded as a tab-delimited text file. The text data were analyzed using the AgilentFilter and Genesifter software. Differential gene expression was evaluated using the t- test (p<0.01 ) and Benjamini and Hochberg correction. The threshold was set at 2-fold change for both upregulated and downregulated genes. Specific differentially expressed genes were confirmed by RT-qPCR.
[0070] Quantitative real-time RT-PCR
[0071] Total RNA (1 μg) was primed with oligo (dT) primers and converted into cDNA with RevertAid First Strand cDNA Synthesis Kit (Fermentas). SYBR green-based real time PCR was performed with a Roche LightCycler system, and the reaction mix contained 1 X SYBR green master mix (SABiosciences) and 0.5 μΜ each of the forward and reverse primers in a volume of 25 μΙ_. PCR cycling consisted of 95°C for 10 min, then 30 cycles of 95°C for 10 sec, 60°C for 30 sec, 72°C for 10 sec, followed by a melting-curve analysis. All PCR primers used are available on request.
[0072] RNA-sequencing
[0073] Alignments were generated from lllumina Fastq files to the hg19 human genome with all known and theorical splice junctions using Novocraft's novoalign aligner. NovoalignParser (http://useq.sourceforge.net cmdLnMenus. htm l#NovoalignParser) application was used to parse the alignment files into binary point data. DefineRegionScanSeqs
(http://useq.sourceforge.net/cmdLri lylenus.html#DefinedRegionScanSeqs) application was used to define differentially expressed genes.
[0074] Whole-Genome Bisulfite Sequencing (WGBS)
[0075] Library construction was performed using the lllumina TruSeq. DNA Sample Preparation Kit (cat# FC-121-2001 , FC-121-2002) using the following methods: Genomic DNA (approximately 50 ng to 2 μg) was sheared to an average size range of 275 bp in a volume of 52.5 μί using a Covaris S2 Focused-ultrasonicator with the following settings: Intensity 5.0; Duty cycle 10%; 25 cycles per Burst 200; Treatment Time 60 seconds. Sheared DNA was converted to blunt-ended fragments with 5'-phosphates and 3'-hydroxyl groups using a combination of enzymes that perform fill-in reactions and exhibit exonuclease activity. Size selection of the blunt-ended DNA (200-450 bp average) was accomplished using bead-based methodologies. An A-base was added to the blunt ends as a means to prepare the fragments for adapter ligation and block concatamer formation during the ligation step. Adapters containing a T-base overhang were ligated to the A-tailed DNA fragments. Adapter-ligated molecules were purified by bead based methodologies and were bisulfite converted using the Qiagen EpiTect Bisulfite Kit (cat# 59104). Bisulfite treated DNA was PCR amplified for 12 cycles using Agilent Pfu Turbo Cx (Agilent cat# 600410) to enrich those fragments that have adapter molecules ligated to both ends. The concentration of the amplified library was measured using the Invitrogen Qubit dsDNA HS Assay (Q32851 ) and an aliquot of the library was resolved on an Agilent 2200 Tape Station using a D1 K (cat# 5067-5361 and 5067-5362) or a High Sensitivity D1 K (cat# 5067-5363 and 5067-5364) assay to define the size range. Libraries were adjusted to a concentration of approximately 10 nM and quantitative PCR was performed using the KapaBiosystems Kapa Library Quant Kit (cat# KK4824) to quantify adapter-ligated library molecules. The concentration was further adjusted following qPCR to prepare the library for lllumina sequence analysis.
[0076] Computational analytical method for WGBS
[0077] The software packages used in this analysis are open source and available from the USeq (http://useq.sourceforge.net/usaqeBisSeq.html) project website (Nix, D.A., Di Sera, T.L., Dalley, B.K., Milash, B.A., Cundick, R.M., Quinn, K.S., and Courdy, S.J. BMC bioinformatics 2010, 11, 455). SAM alignments were generated from lllumina Fastq files aligned to the human hg19 genome using Novocraft's novoalign aligner (http://www.novocrafi.com) in bisulfite mode with the following parameters: -r Random -t 240 -h 120 -b 2 -p bisulfite. An in silico chrLambda sequence was used to align the fully methylated lambda sequence that was spiked into the samples in order to measure the bisulfite conversion efficiency and displayed very low non-conversion/ sequencing error rates of 0.00348 (MCF7) and 0.00245 (MCF7-RON/MSP). These rates were used for scoring individual cytosines for significant methylation with a binomial test. A chrPhiX sequence was added to estimate the read quality and a chrAdapter sequence containing all permutations of the adapter sequences was used to remove these artifacts from the data. Differentially- methylated regions (DMRs) were determined by comparing methylation levels in each cell line using a sliding window of 150 base pairs. Overlapping windows passing two thresholds, a Benjamini-Hochberg corrected a chi-square test of independence FDR of < 0.01 , and fold difference in methylation of > 2, were merged into continuous DMRs and assigned the best window scores (See http://usea.sourceforqe.net/ usaqeBisSea.html for details). Next, DMRs were used to score mean fraction methylation in every sample.
[0078] NovoalignBisulfiteParser(http://useq.sourceforge.net/cmdLnMenus.html#Novoalig nBisulfiteParser) was used to parse the text based novoalignments into four binary "PointData" sets containing the number of observed converted Cs (Ts - non methylCs) and non-converted Cs (methylCs) at each reference C sequenced in the genome for both the plus and minus strands.
[0079] The PoinData was then parsed into mCG context using the ParsePointDataContexts
(http://useq.sourceforge.net/cmdLnMenus.html#ParsePointDataContexts) application.
[0080] BisStat (http://useq.sourceforge.net/cmdLnMenus.html#BisStat) application was used to calculate per base fraction methylation scores for bases with five or more reads from both strands and generate tracks for visualization in IGB. BisStat also calculated fraction methylation for 500bp windows containing a minimum of 5CpGs.
[0081] Mean fraction methylation at each DMR was scored using the ScoreMethylatedRegions(http://useq. sourceforge.net/cmdLnMenus. htm l#ScoreMethylatedR egions) application. Only regions with a minimum of 5Cs and a minimum read coverage of 5 in all samples were used in further analysis.
[0082] Differentially methylated regions were identified using the BisSeq (http://useq.sourceforge.net/cmdLnMenus.html#BisSeq) application. Overlapping 150bp windows that met or exceeded an FDR of 0.01 and a 2-fold difference were merged and included in a spread sheet output of differentially methylated regions. This list was further filtered in excel for including regions with sample having > 0.25 fraction methylation difference.
[0083] Human annotation and genomic sequence (Feb. 2009, GRCh37/hg19) were obtained from the UCSC Genome Bioinformatic website.
[0084] Pathway and functional analyses of differentially methylated and differentially expressed genes
[0085] Differentially expressed genes (p < 0.05) were functionally annotated and classified by using the functional annotation tool database for annotation, visualization, and integrated discovery (DAVID), which provides integrated annotation and analysis of genome- scale datasets derived from high-throughput technologies.
[0086] Human breast tumor gene expression data pre-processing
[0087] We imported expression data from the referenced studies. Raw data was downloaded from the NCBI GEO website or from websites indicated in the original publications and processed as previously described (Segal, E., et al. Nature genetics 2004, 36, 1090-1098). Briefly, we first log2 transformed the expression values and then calculated the mean expression level for each gene across all samples in a given dataset. These mean values were subtracted from all data points, such that expression was represented relative to the mean of each gene, negative values representing below-mean expression and vice versa. To construct the breast cancer compendium, we first normalized each of the five- included studies independently, and then concatenated these sets. In cases where the same individuals were included in more than one of the eight studies comprising the compendium, such redundancy was eliminated so that each individual was included only once in the compendium. Specifically, individuals of the Uppsala cohort analyzed in the Miller set were eliminated from the Desmedt set.
[0088] Analysis of gene-set enrichment patterns
[0089] To identify gene-set enrichment patterns we used methods described previously (Segal, E., et al. Nature genetics 2004, 36, 1090-1098), applied using the Genomica software. For each sample, we first scored the genes whose expression was at least twofold above or below the mean expression level. We then assessed the fraction of over- or under- expressed genes that belong to each tested gene set, calculating a P value according to the hypergeometric distribution. This was repeated for every sample, using a threshold of P < 0.05 for significant enrichment. To compare enrichment patterns across sample groups, we included clinical annotations for each individual sample (such as tumor grade, size and ER status) derived from the original publications. For all samples showing enrichment for the signature (minimum of 5 genes from the RON/MBD4 signature and at least 1 gene from the 9 genes that were deregulated in tumors from mice treated with OSI-296), we calculated the fraction of samples that possessed each annotation, and assigned a P value according to the hypergeometric distribution. We used a more stringent threshold, P < 0.01 , for this calculation. Heat maps showing gene-set enrichments in individual samples include only those samples enriched for the signature.
[0090] Survival analysis
[0091] All survival data were extracted from the original publication (Curtis, C, et al. Nature 2012, 486, 346-352). P values were calculated using the log rank test comparing the group of individuals with tumors showing the RON/MBD4 signature to all other individuals. Univariate and multivariate analyses with Cox proportional-hazards regression were done on the individual clinical variables. Statistical analyses used WinSTAT.
EXAMPLE 2: RON/MSP signaling promotes widespread metastasis of human breast cancer in vivo.
[0092] We examined RON expression in 6 breast epithelial cell lines, including immortalized normal mammary epithelial cells, MCF-10A; 2 non-metastatic cancer cell lines, MCF7 and T47D; 3 metastatic cancer cell lines, MDA-MB-453, HCC1 143, and HCC1806; and MCF7 and T47D cells engineered to overexpress RON/MSP (Figure 1A). RON was overexpressed in metastatic cancer cell lines compared to normal and non-metastatic cancer cell lines. To determine whether RON/MSP drives metastasis, we engineered the non-metastatic MCF7 and T47D breast cancer cell lines to stably express firefly luciferase, and infected them with retroviruses carrying RON and MSP cDNAs (referred to as MCF7- RON/MSP and T47D-RON/MSP; Figure 1A). Overexpressing cells had comparable RON levels with the metastatic breast cancer cell lines. The effects of RON/MSP gain-of-function on tumor growth and/or metastasis of MCF7 and T47D tumors were assessed in immune- compromised mice by orthotopic implantation into the cleared inguinal mammary fat pads of 3-week old female NOD/SCID mice. The growth of tumors was monitored weekly, and tumor metastasis was measured by bioluminescence imaging and histology upon necropsy. The MCF7-RON/MSP cells formed tumors faster compared to parental MCF7 cells, but this was not the case in the T47D model (Figure 8A). Shown in Figure 8A are tumor growth curves following orthotopic transplantation of MCF7 or T47D cells (blue) or MCF7-RON/MSP or T47-RON/MSP (red) into cleared inguinal mammary fat pads of 3-week old NOD/SCID mice. To avoid confounding effects of tumor size on metastasis, the mice were euthanized for analysis of metastasis when the tumors reached the same size endpoint for each group (after 25 and 16 weeks for orthotopically implanted MCF7 and MCF7-RON/MSP, respectively, and after 25 weeks for both T47D and T47D-RON/MSP; Figure 8A). RON/MSP expression was sufficient to induce high rates of spontaneous lung, bone, liver and brain metastasis from MCF7-RON/MSP and T47D-RON/MSP tumors, compared with little or no metastasis from the parental cell xenografts (Figures 1 B and C). Shown in Figure 1 B are representative bioluminescent images of primary tumors, lung, bone, liver and brain metastasis from single mice. Shown in Figure 1C are metastasis frequencies for MCF7 (n=15), MCF7-RON/MSP (n=20), T47D (n=16) and T47D-RON/MSP (n=14) tumors following orthotopic injection into mammary fat pad of NOD/SCID mice. All RON/MSP tumors were harvested when size-matched to parental tumors. The metastases were verified by histological analysis and immunostaining for human cytokeratin proteins (Figure 8B, showing representative lung sections from mice carrying MCF7-RON/MSP or T47D- RON/MSP tumors, stained with H&E or immunostained with an antibody specific for human cytokeratin to positively identify metastasis). These results indicated that RON/MSP expression was sufficient to facilitate the metastatic spread of MCF7 and T47D breast cancers in vivo to sites that are highly relevant for breast cancer metastasis. These results are consistent with our previous findings, which showed a significant correlation between RON/MSP expression and metastasis in patients with breast cancer.
[0093] To determine the molecular mechanisms by which RON/MSP drives breast cancer metastasis, we performed RNA sequencing (RNA-seq) and microarray gene expression profiling of MCF7 and MCF7-RON/MSP cells. Sequencing was performed with 3 biological replicates for each cell line. Sequencing reads were aligned to the hg19 human genome with all known and theoretical splice junctions using Novocraft's novoalign aligner. From individual samples, we obtained 24 million reads that mapped to the human genome. The microarray was performed with four biological replicates for each cell line and the data were median normalized. For comparative analysis between RNA-seq and microarray, we focused only on those reads which aligned to annotated regions of the human genome, as microarray probes are designed primarily against annotated regions. Differentially expressed genes, based on a false discovery rate threshold of 1 % and a fold change of 2, were highly correlated between the two methods (Figure 8C). Shown in Figure 8C is a scatter plot showing differential expression obtained using microarray (x axis) and RNA-seq (y axis) between MCF7 and MCF7-RON/MSP. Points represent genes, located based on estimates of differential expression using RNA-Seq and Microarray. Red points represent genes for which FDR<0.1 and fold change>2. Green points represent genes differentially expressed identified by RNA-seq only. Purple points represent genes differentially expressed identified by microarray only. The line corresponds to perfect agreement between the technologies. We identified 220 significant differentially expressed genes in common with both platforms (Table 2). We used the DAVID bio informatics database to identify the gene ontology (GO) terms for the differentially expressed genes from RNA-seq and microarray and found significant enrichment for genes involved in plasma membrane, developmental processes, receptor activity and cell signaling/communication. The GO term analysis was not used in our subsequent analysis, but indicated that RON/MSP signaling induces expression of genes whose functions are consistent with biological pathways relevant to metastasis. However, the data did not immediately implicate a clear mechanistic pathway to explain how RON/MSP drives metastasis.
Table 2. Differentially expressed genes (DEGs) identified from both RNA-sequencing and microarray.
Figure imgf000042_0001
DI02 ENSG0000021 1448 2.47 3.52
DPF3 ENSG00000205683 3.27 2.54
EGR1 ENSG00000120738 2.53 3.31
EHD2 ENSG00000024422 2.12 2.02
ELTD1 ENSG00000162618 8.38 50.63
EPHA3 ENSG00000044524 2.31 4.06
FAM126A ENSG00000122591 3 2.07
FOS ENSG00000170345 2.04 2.01
FSTL4 ENSG00000053108 2.28 3.12
GPR133 ENSG000001 1 1452 3.02 3.30
GRAMD2 ENSG00000175318 3.46 3.23
HMGCLL1 ENSG00000146151 2.15 2.24
HOXB3 ENSG00000120093 4.23 4.70
HRH1 ENSG00000196639 3.16 1.91
IFI27L2 ENSG000001 19632 2.22 2.32
INHA ENSG00000123999 3.1 1 3.22
IRS2 ENSG00000185950 3.04 2.89
KIAA1 199 ENSG00000103888 2.04 2.04
LHFPL2 ENSG00000145685 2.44 3.15
LXN ENSG00000079257 3.32 3.61
MAOB ENSG00000069535 2.52 2.02
MST1 R ENSG00000164078 22.79 8.23
NMNAT2 ENSG00000157064 2.58 2.55
NR3C1 ENSG000001 13580 2.96 2.36
NR4A3 ENSG000001 19508 3.71 3.68
NRXN3 ENSG00000021645 2.43 3.36
P2RY6 ENSG00000171631 2.28 3.01
PYGM ENSG00000068976 2.1 2.65
RASD1 ENSG00000108551 3.09 2.92
RASGRF1 ENSG00000058335 2.44 3.38
RIMS2 ENSG00000176406 3.37 3.13
RNF43 ENSG00000108375 2.59 2.85
SHC4 ENSG00000185634 3.69 3.78
SIGLEC6 ENSG00000105492 3.67 15.18
STC1 ENSG00000159167 2.42 2.56
TM4SF1 ENSG00000169908 2.58 2.16
TRPM6 ENSG000001 19121 3.02 3.67
UNC13A ENSG00000130477 3.17 3.10
UPK3A ENSG00000100373 2.09 2.34
UPP1 ENSG00000183696 2.8 2.28
A2M ENSG00000175899 -7.24 -3.65
ABCA1 ENSG00000165029 -2.84 -2.18
ABCA4 ENSG00000198691 -2.46 -3.03
ABCC13 ENSG00000243064 -4.23 -2.59 ADAM23 ENSG000001 14948 -2.31 -2.26
ADAMTSL3 ENSG00000156218 -2.99 -2.71
ADD3 ENSG00000148700 -3.33 -2.72
AGR3 ENSG00000173467 -8.14 -7.27
AKR1 C3 ENSG00000196139 -3.37 -4.16
AMOT ENSG00000126016 -2.63 -1.99
ARHGAP24 ENSG00000138639 -3.17 -2.12
ARMC4 ENSG00000169126 -3.1 -1.1 1
ASCL1 ENSG00000139352 -19.32 -20.14
ATOH8 ENSG00000168874 -2.55 -4.09
BIRC3 ENSG00000023445 -2.5 -2.47
BMF ENSG00000104081 -2.41 -1.81
BST2 ENSG00000130303 -1.15 -3.04
BTG3 ENSG00000154640 -3.01 -2.83
C15orf48 ENSG00000166920 -2.53 -2.51
C1orf168 ENSG00000187889 -7.97 -6.64
C1orf64 ENSG00000183888 -4.32 -2.55
C21orf88 ENSG00000184809 -2.98 -2.15
C2orf54 ENSG00000172478 -4.21 -4.01
C3 ENSG00000125730 -2.1 1 -3.04
CA8 ENSG00000178538 -2.03 -3.66
CAPS ENSG00000105519 -3.09 -2.46
CCDC85A ENSG00000055813 -2.44 -2.43
CCL2 ENSG00000108691 -2.29 -6.81
CD36 ENSG00000135218 -3 -6.29
CDH26 ENSG00000124215 -2.33 -2.17
CEACAM6 ENSG00000086548 -2.96 -2.14
CFB ENSG00000243649 -17.09 -4.15
CFL2 ENSG00000165410 -2.57 -2.45
CHRD ENSG00000090539 -2.06 -2.51
CNTNAP2 ENSG00000174469 -2.27 -2.35
COL12A1 ENSG000001 1 1799 -3.67 -2.44
COL3A1 ENSG00000168542 -2.36 -2.26
CPS1 ENSG00000021826 -3.02 -2.41
CPXM2 ENSG00000121898 -2.9 -2.99
CXXC4 ENSG00000168772 -3.19 -2.67
CYP4X1 ENSG00000186377 -5.99 -17.08
DCLK1 ENSG00000133083 -2.77 -3.17
DOCK10 ENSG00000135905 -3.8 -2.31
EFNA5 ENSG00000184349 -2.1 1 -2.49
ELOVL2 ENSG00000197977 -2.39 -2.1 1
ENTPD8 ENSG00000188833 -2.04 -1.91
EPHA7 ENSG00000135333 -1.98 -2.01
FAM149A ENSG00000109794 -2.4 -3.65 FAM49A ENSG00000197872 -3 -4.92
FAM5C ENSG00000162670 -8.8 -8.51
FGF13 ENSG00000129682 -4.91 -4.10
FHOD3 ENSG00000134775 -3.44 -2.97
FSCN1 ENSG00000075618 -2.48 -2.01
FZD8 ENSG00000177283 -2.96 -2.47
GJA1 ENSG00000152661 -3.64 -4.16
GJA3 ENSG00000121743 -2.26 -2.14
GJB2 ENSG00000165474 -3.21 -2.94
GLI1 ENSG000001 1 1087 -2.72 -5.50
GLRA3 ENSG00000145451 -3.58 -4.03
GPC6 ENSG00000183098 -52.07 -19.25
GPR161 ENSG00000143147 -6.31 -3.99
GPR162 ENSG00000250510 -2.26 -3.68
GUCY1A2 ENSG00000152402 -2.97 -5.81
HCK ENSG00000101336 -3.14 -4.77
HDAC9 ENSG00000048052 -3.25 -2.36
HMCN1 ENSG00000143341 -3.04 -2.59
HOXD8 ENSG00000175879 -2.5 -2.11
ID4 ENSG00000172201 -4.7 -4.50
IL20RA ENSG00000016402 -2.78 -2.68
IL32 ENSG00000008517 -5.2 -6.72
ITGA6 ENSG00000091409 -2.31 -2.07
ITGB8 ENSG00000105855 -2.69 -2.17
JAKMIP2 ENSG00000176049 -3.43 -3.47
KCNK2 ENSG00000082482 -5.51 -4.27
KCNS3 ENSG00000170745 -4.98 -4.72
KIAA2022 ENSG00000050030 -2.67 -2.10
KLF7 ENSG000001 18263 -2.64 -2.69
KLHDC7B ENSG00000130487 -2.01 -2.42
KLK1 1 ENSG00000167757 -4.04 -2.17
KLK8 ENSG00000129455 -2.52 -2.92
KRT2 ENSG00000172867 -3.64 -14.54
LAMB1 ENSG00000091136 -3.27 -2.46
LAMP3 ENSG00000078081 -2.16 -3.45
LCN2 ENSG00000148346 -4.56 -3.91
LIMCH1 ENSG00000064042 -2.55 -1.83
LRFN5 ENSG00000165379 -4.31 -3.21
LRG1 ENSG00000171236 -3.1 1 -2.96
LRRC4C ENSG00000148948 -3.1 -3.73
LTB ENSG00000227507 -8.9 -5.69
LY6D ENSG00000167656 -2.81 -4.00
MARCKS ENSG00000155130 -2.54 -2.59
MARK1 ENSG000001 16141 -6.37 -6.39 MICALCL ENSG00000133808 -4.28 -3.05
MIDI ENSG00000101871 -3.83 -2.75
MMP16 ENSG00000156103 -2.81 -2.05
MSMB ENSG00000138294 -2.96 -2.82
MYCN ENSG00000134323 -3.81 -2.56
NCAM2 ENSG00000154654 -3.31 -2.43
NKAIN1 ENSG00000084628 -3.92 -3.13
NOD2 ENSG00000167207 -2.06 -1.89
NPNT ENSG00000168743 -2.4 -2.27
NUPR1 ENSG00000176046 -2.72 -2.66
OLFML2A ENSG00000185585 -2.3 -1.81
OLFML3 ENSG000001 16774 -2.74 -2.45
PAH ENSG00000171759 -4.28 -3.99
PARP8 ENSG00000151883 -2.48 -1.67
PCDH10 ENSG00000138650 -7.01 -4.75
PCDH17 ENSG000001 18946 -2.55 -3.76
PCDH7 ENSG00000169851 -1.22 -4.46
PCDH9 ENSG00000184226 -4.35 -3.64
PDLIM3 ENSG00000154553 -2.67 -2.00
PGR ENSG00000082175 -2.44 -1.80
PHACTR3 ENSG00000087495 -5.1 -5.65
PKIA ENSG00000171033 -2.02 -1.72
PLD1 ENSG00000075651 -1.91 -2.62
PRDM5 ENSG00000138738 -2.23 -1.77
PRSS1 ENSG00000204983 -4.03 -5.06
PRUNE2 ENSG00000106772 -3.34 -3.24
PVRL3 ENSG00000177707 -3.97 -3.22
RAMP3 ENSG00000122679 -2.09 -2.31
RASGRP1 ENSG00000172575 -2.22 -2.01
RASL1 1A ENSG00000122035 -2.02 -2.42
RBM1 1 ENSG00000185272 -2.25 -2.08
RBM20 ENSG00000203867 -3.49 -5.56
RBP7 ENSG00000162444 -2.92 -2.77
RCAN3 ENSG000001 17602 -2.44 -2.01
RCN1 ENSG00000049449 -4 -2.34
RGMA ENSG00000182175 -2.5 -2.63
RLN2 ENSG00000107014 -2.34 -2.00
RORC ENSG00000143365 -3.9 -2.38
S100A7 ENSG00000143556 -10.47 -8.32
S100A7A ENSG00000184330 -8 -10.31
S100A8 ENSG00000143546 -15.7 -14.12
S100A9 ENSG00000163220 -6.45 -5.67
SCIN ENSG00000006747 -4.84 -4.42
SEMA3D ENSG00000153993 -16.63 -14.89 SEMA5A ENSG000001 12902 -4.91 -2.90
SEPP1 ENSG00000250722 -4.03 -3.97
SERPINA1 ENSG00000197249 -2.48 -2.77
SLC14A1 ENSG00000141469 -2.41 -2.14
SLC44A4 ENSG00000204385 -2.16 -3.27
SLC6A14 ENSG00000087916 -21.24 -19.51
SLFN5 ENSG00000166750 -2.28 -2.23
SLITRK4 ENSG00000179542 -5.68 -5.00
SLITRK6 ENSG00000184564 -2.94 -2.43
STON1 ENSG00000243244 -3.05 -2.37
STOX2 ENSG00000173320 -2.64 -1.57
STRA6 ENSG00000137868 -4.41 -4.67
SULF1 ENSG00000137573 -3.82 -3.42
SYCP1 ENSG00000198765 -4.16 -1 1.16
SYCP2 ENSG00000196074 -3.48 -2.65
TFF3 ENSG00000160180 -2.12 -2.06
TGFB2 ENSG00000092969 -3.14 -2.30
TGM2 ENSG00000198959 -2.79 -2.28
THRB ENSG00000151090 -8.31 -11.00
TLR2 ENSG00000137462 -4.14 -3.54
TMEM158 ENSG00000249992 -2.41 -2.13
TMSB15A ENSG00000158164 -2.45 -6.38
TNS4 ENSG00000131746 -2.33 -2.55
TOX3 ENSG00000103460 -2.74 -3.43
TSPAN5 ENSG00000168785 -2.87 -2.10
WNT4 ENSG00000162552 -2.39 -2.87
ZDHHC22 ENSG00000177108 -2.29 -2.69
ZNF469 ENSG00000225614 -2.75 -2.14
ZNF704 ENSG00000164684 -2.4 -2.15
ZNF827 ENSG00000151612 -2.2 -1.91
ZNF835 ENSG00000127903 -2.57 -16.96
EXAMPLE 3: RON/MSP signaling upregulates the thymine glycosylase MBD4 and drives DNA methylation reprogramming.
[0094] Regulation of DNA methylation is a major mechanism involved in cell differentiation and neoplastic transformation. The epigenetic modifications contributing to metastasis, however, are much less understood. In order to determine whether alterations in DNA methylation contributed to the selective regulation of RON/MSP-regulated genes, we performed whole-genome bisulfite sequencing on MCF7 and MCF7-RON/MSP cells. We generated 295 million (MCF7) and 297 million (MCF7-RON/MSP) raw reads resulting in 25.9 GB (MCF7) and 26.1 GB (MCF7-RON/MSP) of raw paired-end sequence data. Of these, 236 million (MCF7) and 232 million (MCF7-RON/MSP) (80 and 78% respectively) reads were successfully aligned using Novocraft's (http://www.novocraft.com) novoalign in bisulfite mode to the hg19 human reference sequence. Differentially-methylated regions (DMRs) were determined by comparing methylation levels in each cell line using a sliding window of 150 base pairs. Overlapping windows passing two thresholds, a Benjamini- Hochberg corrected a chi-square test of independence FDR of < 0.01 , and fold difference in methylation of > 2, were merged into continuous DMRs and assigned the best window scores. Only regions with a methylation difference of > 0.25 were included in further analysis, resulting in 1232 DMRs (Figure 2A). Shown in Figure 2A is a circular representation of genome-wide aberrant DNA methylation caused by RON/MSP expression. Average methylation levels for all of the CGs in 10-Mbp-wide windows are shown in the blue-green tracks (the outermost track shows MCF7 cells; the middle track shows MCF7- RON/MSP cells). The innermost track indicates the differentially methylated regions (DMRs) between MCF7 and MCF7-RON/MSP cells (FDR>20, differential methylation>0.25). This diagram for visualization of genome-wide DNA methylation was generated using the Circos software. DMRs occurred in both gene-body and intergenic regions, both inside and outside CpG islands (Figure 2B). Shown in Figure 2B is (top panel) a distribution of differentially methylated CpGs on CpG islands (CpGi) shelves (>2 to 4 kb from island edge) and on CpGi shores (0-2 kb from island edge); within and outside CpGi; u, upstream of CpGi; d, downstream of CpGi. In the bottom panel is a distribution of differentially methylated CpGs across regions of other significance. TSS 200, within the region 1-200 bp upstream of the TSS; TSS 1500, within the region 201-1500 bp upstream of the transcription start site (TSS); UTR, untranslated region; HyperMe, hypermethylated; HypoMe, hypomethylated. DMRs regulated by RON/MSP were often intergenic, and there was a significant enrichment of hypomethylated versus hypermethylated regions (Figure 2B-C). Shown in Figure 2C is an example of smoothed methylation levels from bisulfite sequencing data for MCF7 (black) and MCF7-RON/MSP (blue) on chromosome 14. A hypomethylation block is indicated by a red bar.
[0095] Next, we wanted to determine to what degree the gene expression differences among cell lines were affected by DNA methylation changes. Examples are shown in Figure 2D (hypomethylation, top panel; hypermethylation, bottom panel). These observations indicated that RON/MSP expression in MCF7 cells induced massive epigenomic remodeling, which also affected expression of particular genes. These data suggested a potential role for altered DNA methylation in the mechanism of RON/MSP-mediated metastasis. [0096] To assess if the aberrant DNA methylation in RON/MSP cells was due to differential expression levels or activities of DNA methyltransferases (Dnmts), we examined mRNA levels of DNMT3A, DNMT3B and DNMT1 in MCF7 and MCF7-RON/MSP cells. We found no statistically significant differences (Figure 9A, showing results for real-time quantitative RT-PCR for various DNMT mRNA expression levels, normalized to β-actin mRNA expression, in MCF7 and MCF7-RON/MSP cells). We also measured Dnmt activity in these cells and found no significant difference between parental cells and those expressing RON/MSP (Figure 9B, showing DNA methylation activity in MCF7 and MCF7-RON/MSP cells, as assessed by EpiQuick DNA methyltransferase assay).
[0097] The increased representation of hypomethylated versus hypermethylated DMRs in RON/MSP-expressing cells led us to consider DNA demethylation as a potential effect of RON/MSP. A mechanism for active DNA demethylation in zebrafish has been discovered that involves the cooperative actions of proteins from the cytidine deaminase family (AID and Apobecs), a G:T mismatch-specific glycosylase (MBD4) and a DNA repair protein (Gadd45). We examined components of this DNA demethylase complex in MCF7-RON/MSP and T47D-RON/MSP cells. We noted robust upregulation of the thymine DNA glycosylase MBD4 in cells expressing RON/MSP (Figure 2E). In contrast, another thymine DNA glycosylase (TDG), which is implicated in a different mechanism of active DNA demethylation, was not significantly regulated by RON/MSP (Figure 2E). Upregulation of MBD4 protein by RON/MSP was validated by Western analysis of protein extracts from MCF7 and T47D cell lines (Figure 2F, showing upregulation of MBD4 protein expression in MCF7-RON/MSP and T47D-RON/MSP cells compared to parental MCF7 and T47D cells; fold change = 5 for MCF7 and fold change=3 for T47D, p<0.001 , as quantified by ImageJ).
[0098] To determine whether RON and MBD4 are coordinately expressed in actual human breast tumors, we analyzed RON and MBD4 RNA expression between breast cancer and tumor-adjacent normal samples in the Cancer Genome Atlas (TCGA) data set. RON and MBD4 were both upregulated in breast tumors compared to the normal tissue (Figure 3A, a boxplot showing RON and MBD4 mRNA expression in normal breast tissues (white) and breast tumors (dark grey) from The Cancer Genome Atlas (TCGA) data set). We also examined RON and MBD4 protein expression in 3 breast reduction tissues and 13 human primary breast tumors by western analysis. RON and MBD4 were highly expressed in all 7 poorly differentiated carcinomas. Lower RON and MBD4 levels were also found in well- differentiated tumor samples, and no MBD4 was detected in control samples taken from breast reduction tissue (Figure 3B, a Western blot for RON and MBD4 proteins in normal human breast tissues from reduction mammoplasties and human primary breast tumor specimens; the β-actin (ACTB) loading control is also shown). RON was barely detectable in breast reduction samples. Together with our data showing that MBD4 is expressed downstream of RON/MSP in breast cancer cell lines, these data suggested that RON/MSP might promote aberrant DNA methylation through the upregulation of MBD4. However, the necessity of MBD4 or DNA methylation for epigenetic regulation of RON/MSP downstream effectors, and any role in metastasis, had never been described.
EXAMPLE 4: Knockdown of MBD4 blocks MSP/RON-mediated breast cancer metastasis.
[0099] To investigate the possible role of MBD4 downstream of RON/MSP in breast cancer metastasis in vivo, MCF7-RON/MSP and T47D-RON/MSP cells were infected with lentiviruses carrying two different MBD4-specific shRNAs directed to either the 3'UTR or the coding region (shMBD4_3UTR and shMBD4_CDS, respectively) or, as controls, an MBD2- specific shRNA (shMBD2) or a scrambled shRNA (shScr). MBD4 knockdown was validated by Western blotting (Figure 3C). The effect of MBD4 knockdown on spontaneous lung, bone, liver and brain metastasis was assessed for both cell lines in NOD/SCID mice. The growth of tumors was monitored weekly; RON/MSP tumors expressing shMBD4 and shScr from both cell lines grew similarly to the tumors derived from RON/MSP cells; therefore, MBD4 loss of function had no effect on primary tumor growth (Figure 10A). Shown in Figure 10A is tumor growth following orthotopic implantation of MCF7 (dark blue), MCF7-RON/MSP (red), MCF7-RON/MSP-shScr (green), MCF7-RON/MSP-shMBD4 (yellow) and MCF7- RON/MSP-shMBD4R cells (light blue) in MCF7 (left) and T47D (right) models. However, spontaneous lung, liver, bone and brain metastasis was significantly inhibited in animals carrying MCF7-RON/MSP-shMBD4 and T47D-RON/MSP-shMBD4 tumors relative to the control tumors expressing RON/MSP (Figure 3D and 3E). Shown in Figure 3D are (top panel) metastasis frequencies for MCF7 parental tumors (n=15), MCF7-RON/MSP tumors (n=20), or tumors arising from MCF7 cells infected with RON/MSP and either shScrb (n=10), sh-MBD4-3UTR (n=19), or shMBD4-CDS (n=9). The effects of rescue constructs (shMBD4R_3UTR (n=7), shMBD4R_CDS (n=9) or the catalytic mutant shMBD4R (D560A) (n=9)) are shown at the bottom. In the bottom panel, shown are metastasis frequencies for T47D parental tumors (n=16), T47D-RON/MSP tumors (n=14), or tumors arising from T47D cells infected with RON/MSP and either shScrb (n=7), sh-MBD4-3UTR (n=12), or shMBD4- CDS (n=10). The effects of rescue constructs (shMBD4R_3UTR (n=8), shMBD4R_CDS (n=9) or the catalytic mutant shMBD4R (D560A) (n=8)) are shown at the bottom. In fact, metastasis frequencies of RON/MSP-shMBD4 cells were not significantly different from the original parental cell lines, approaching 0%. The RON/MSP-shMBD2 and the RON/MSP- shScr control tumors showed a similar frequency of metastasis as the RON/MSP tumors (Figure 3D, 3E, 10B, and 10C). Shown in Figure 10B are metastasis frequencies for MCF7, MCF7-RON/MSP, MCF7-RON/MSP-shScr, and MCF7-RON/MSP-shMBD2 tumors, and shown in Figure 10C are representative images showing lack of effect of sh-MBD2 on spontaneous lung, bone, liver and brain metastasis of RON/MSP-expressing MCF7 tumors. To rule out off-target effects of the MBD4 shRNAs, we generated MBD4 rescue constructs (MBD4R-3UTR and MBD4R-CDS) that were resistant to their respective shRNAs. We then conducted rescue experiments in MCF7-RON/MSP-shMBD4 and T47D-RON/MSP-shMBD4 cells where re-expression of MBD4 was at similar levels to that found in RON/MSP cells (Figure 3C). Expression of MBD4R reversed the inhibition of RON/MSP-mediated metastasis by shMBD4 (Figure 3D and 3E). Shown in Figure 3E are representative bioluminescent images of lung, bone and liver metastasis from single mice illustrate the effects of shScrb, sh-MBD4, rescued MBD4 and rescued catalytic mutant on spontaneous lung, bone, liver and brain metastasis of various orthotopic MCF7 tumors. These data confirm that RON/MSP drives metastasis through upregulation of MBD4, at least in these two models.
[00100] To determine whether MBD4 requires its glycosylase activity to drive metastasis, we engineered an MBD4 rescue cDNA containing a mutation of the catalytic residue, D560A. This mutant has previously been shown to be catalytically dead. We transplanted MCF7 and T47D cells expressing RON/MSP-shMBD4R (D560A) into mammary fad pads of NOD/SCID mice and monitored metastasis. The mutation prevented the rescue of the metastasis phenotype observed with the wild type MBD4 rescue constructs, suggesting that MBD4 is driving metastasis through its glycosylase activity (Figure 3D and 3E). We next sought to determine if MBD4 was also required for reprogramming of DNA methylation downstream of RON/MSP.
EXAMPLE 5: Knockdown of MBD4 reverses abnormal DNA methylation patterns and reverses expression of RON/MSP-regulated genes.
[00101] To investigate the requirement for MBD4 in aberrant DNA methylation driven by RON/MSP, we analyzed WGBS data from MCF7-RON/MSP-shMBD4 compared to MCF7- RON/MSP. MBD4 knockdown reversed the methylation status of a specific collection of loci in MCF7-RON/MSP cells. These regions of aberrant DNA methylation were reprogrammed following the knockdown of MBD4, to levels that were comparable with parental MCF7 cells (Figure 4A and 4B). Shown in Figure 4A is a representation of smoothed methylation values from bisulfite sequencing data for MCF7 (black), MCF7-RON/MSP (blue) and MCF7- RON/MSP-shMBD4 cells (purple) in a representative region of chromosome 2. The hypomethylation block in MCF7-RON/MSP that becomes hypermethylated by knocking- down MBD4 is indicated by a red bar. Shown in Figure 4B is an example of DNA methylation levels in promoter regions in MCF7 (black), MCF7-RON/MSP (blue) and MCF7- RON/MSP-shMBD4 cells (purple). The hypomethylated block in MCF7-RON/MSP cells that becomes re-methylated by knocking down MBD4 is indicated by a red bar. It is important to note that knocking down MBD4 also caused changes in methylation of some loci that were independent of RON/MSP (Figure 4C). Shown in Figure 4C is an example of DNA methylation levels in a promoter region that becomes hypermethylated in shMBD4 cells, independent of RON/MSP expression (red bar) Bisulfite sequencing data for MCF7 (black), MCF7-RON/MSP (blue) and MCF7-RON/MSP-shMBD4 cells (purple) are shown. In all panels, the orange bars indicate CpG islands and the black bars show the genes.
[00102] To further understand the role of MBD4-mediated aberrant DNA methylation in gene expression downstream of RON/MSP, we also performed RNA-sequencing and microarray gene expression profiling of MCF7-RON/MSP-shMBD4 cells. We found that a statistically significant 25% of genes that were regulated by RON/MSP in MCF7 cells became reversed by MBD4 knockdown (Figure 4D). Shown in Figure 4D are diagrams representing the proportion of genes for which expression was reversed by knocking down MBD4 (outside shifted circle). In blue are represented the genes that are downregulated in MCF7-RON/MSP versus MCF7 (the outside shifted track represents the genes that become upregulated in MCF7-RON/MSP-shMBD4). In orange are represented the genes that are upregulated in MCF7-RON/MSP versus MCF7 (outside track represents the genes that become downregulated in MCF7-RON/MSP-shMBD4). Reversal of this gene expression pattern correlated with lack of metastasis upon MBD4 knockdown. We performed a GO-term analysis on these genes and discovered significant enrichment for genes involved in extracellular regions, developmental processes and cell signaling/communication. It is important to note that there were other gene expression changes induced by RON/MSP that were independent of MBD4 and vice-versa. Thus, as expected, RON signaling does contribute other changes to cancer cells. Likewise, knockdown of MBD4 also affected expression of genes other than those regulated by RON/MSP, suggesting that MBD4 can also regulate expression of genes outside of the RON/MSP pathway. We focused on the gene set that was both RON/MSP-dependent and MBD4-dependent.
[00103] Using our cell line data, we identified all of the genes that were regulated by RON/MSP in MCF7 cells that were also reversed by MBD4 knockdown. We identified 192 genes (Figure 5A), of which 64 were also differentially methylated. We refer to these 192 genes as the "RON/MBD4 epigenetic signature" (Figure 5A). Shown in Figure 5A is a Circular representation of the RON/MBD4 epigenetic signature. The outside track represents the 192 genes regulated by RON/MSP for which expression was reversed following knockdown of MBD4 (blue: genes downregulated by RON/MSP and then reversed by knocking down MBD4 with shRNA; orange: genes upregulated by RON/MSP and then reversed by knocking down MBD4). In larger characters are the genes that are annotated in the METABRIC dataset. The inner track represents the statistically significant DMRs at these regions (red: hypermethylated DMRs in MCF7-RON/MSP and then reversed by knocking down MBD4; green: hypomethylated DMRs in MCF7-RON/MSP and then reversed by knocking down MBD4).
EXAMPLE 6: Specific subclasses of human breast tumors possess the RON/MBD4 epigenetic signature, which correlates with poor prognosis
[00104] While experimentation on human cancer cell lines is essential for dissection of mechanistic pathways in cancer biology, it is an extrapolation from bona fide tumors. Therefore, we asked whether the RON/MBD4 epigenetic reprogramming pathway that we identified and functionally characterized in cell line models exists in human breast tumors and, if so, whether it is associated with survival outcome. To first determine whether the RON/MBD4 epigenetic signature is present in human breast tumors, we used a gene module map (Segal et al., Nature Genetics 2004, 36, 1090-1098) to examine the expression of 1 16 genes of the 192 above-mentioned genes in 997 primary human breast cancers from the Metabric discovery cohort (the remainder of the genes were not annotated in the Metabric dataset, Figure 5A genes in large characters). The RON/MBD4 epigenetic metastasis signature (presence of the signature was defined as a minimum of 5 genes differentially expressed from the RON/MBD4 signature, i.e., a minimum of 5 genes shown in Table 1 ) was present approximately in 25% of the breast cancers (Figure 5B).
[00105] Using the published clinical annotations for each tumor (tumor grade, expression of estrogen and progesterone receptors (ER/PR)), we found that the RON/MBD4 epigenetic signature was significantly associated with grade-3, ER-, and PR- tumors (Figure 5B). Several "intrinsic subtypes" of breast cancer have been previously defined on the basis of more comprehensive gene expression profiles: normal-like, luminal type A, luminal type B, HER2-like and basal-like. Indeed, the RON/MBD4 epigenetic signature was significantly enriched in basal-like tumors (Figure 5B). Shown in Figure 5B is an enrichment pattern of the gene set comprising the RON/MBD4 epigenetic signature (rows) across 997 breast tumors (columns). Red and green indicated significantly over or underexpressed gene, respectively. Are represented the 582 patients having enrichment or underrepresentation of the gene set. Blue bars (right) indicate individual tumor annotations for breast cancer subtype. The table (right) shows the association of the RON/MBD4 epigenetic signature with ER, HER2, and PR status, as well as intrinsic breast cancer subtype, grade and death (we assigned a p value according to the hypergeometric distribution).
[00106] We also performed Kaplan-Meier analysis examining overall survival for individuals with breast cancer across the 997-patient METABRIC dataset, using 1 16-gene RON/MBD4 epigenetic signature. The expression of these genes was significantly associated with poor survival (p<0.05, HR=1.30, Figure 5C). Shown in Figure 5C is a Kaplan-Meier analysis of overall survival in 997 breast cancer patients from the METABRIC discovery dataset (Curtis et al., 2012). Survival curve of individuals with tumors showing an enrichment of the 1 16 annotated genes from the RON/MBD4 epigenetic signature is shown in red; all other patients are shown in blue (No signature). The p value indicates a statistically significant survival difference between these two groups of patients. The survival hazard ratio was calculated using Univariate Cox's regression analysis. These data strongly suggest that the RON/MBD4 pathway exists in a fairly large subset of human breast cancers (25%), and that detection of the novel RON/MBD4 epigenetic pathway could significantly contribute to risk assessment in breast cancer patients. Thus, an important corollary to this work was to ask how RON regulates MBD4 and whether the presence of a RON/MBD4 epigenetic signature would predict response to anti-Ron therapy.
EXAMPLE 7: MBD4 is upregulated by RON/MSP through PI3K signaling
[00107] In order to further characterize MBD4 regulation by RON, we engineered an MCF7-TRE-RON/MSCV-tTA cell line, where RON expression is under the control of the tetracycline (Tet) response element (TRE) and is repressed by binding of doxycycline (dox) to the Tet transactivator protein (tTA). As expected, in the absence of dox the cells expressed RON, which began to be repressed 4 hours after the addition of dox to the culture medium and was maximally repressed 48 hours after adding dox (Figure 6A). Consistent with data in Figures 2E and 2F showing that RON regulates MBD4, we found that repression of RON also causes downregulation of MBD4 (Figure 6A). To determine whether repression of endogenous RON causes downregulation of MBD4 in other model systems, RON was knocked down using shRNA via lentiviral infection in both the RON-positive DU4475 breast cancer cell line and in primary culture from a RON-positive patient-derived breast tumor graft. Scrambled shRNA were used as control. RON knockdown, although not complete, caused a reduction in MBD4 expression in both models (Figure 6B). Shown in Figure 6B is a Western blot showing expression of endogenous RON and MBD4 proteins in the DU-4475 breast cancer cell line and in a patient derived breast tumor graft (HCI-014) before and after infection with lentiviruses carrying RON shRNA (fold change = 3, p<0.02, and fold change = 5, p<0.01 , respectively, as quantified by ImageJ). Thus, we concluded that, in all five different breast cancer models we examined, RON upregulates MBD4, which can be reversed with RON downregulation. Thus the pathway has potential to be reversed.
[00108] RON is known to transduce a variety of signals that regulate different downstream pathways, most notably the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways. To elucidate which signaling pathway(s) are required for regulation of MBD4 by RON, we treated the cell lines with varying doses of U0126 (a MEK/ERK inhibitor), or BKM-120 (a PI3K inhibitor) for 0.5 hour or 1 hour, followed by protein extraction and MBD4 Western blotting. Examination of activated (phosphorylated) Erk and PRAS proteins in each experiment verified that the drugs were effective in inhibiting their respective targets (Figure 6C). Treatment with BKM-120, but not U0126, blocked RON/MSP-mediated MBD4 upregulation (Figure 6C). Shown in Figure 6C is (top) a Western blot with anti-MBD4, anti-p-PRAS and anti- β-actin antibodies in MCF7 and MCF7- RON/MSP cells treated with DMSO or BKM-120 (50 nM, 100 nM, or 500 nM for the time indicated; fold change = 12, p<0.001 , as quantified by ImageJ). In the bottom is a Western blot with anti-MBD4, anti-p-ERK and anti- β-actin antibodies in MCF7 and MCF7-RON/MSP cells treated with DMSO or U0126 (5 μΜ, 10 μΜ, or 50 μΜ for the time indicated). To further validate that MBD4 upregulation by RON/MSP requires PI3K/Akt signaling, the PI3K p1 10 alpha catalytic subunit was knocked down in MCF7-RON/MSP cells by shRNA via lentiviral infection. Knockdown of PI3K activity was determined using PRAS as a surrogate measure (Figure 6D). This experiment showed that PI3K knockdown led to a reduction in MBD4 expression (Figure 6D), similar to the results with BKM-120 (Figure 6C), demonstrating that RON/MSP regulates MBD4 through activation of the PI3K pathway. Shown in Figure 6D is a Western blot showing the expression of MBD4 and p-PRAS in MCF7 and MCF7-RON/MSP cells infected with lentiviruses carrying PI3K-p1 10 shRNA or scrambled shRNA control (fold change = 2, p<0.01 , as quantified by ImageJ). To determine whether any activator of PI3K signaling could increase MBD4 expression, we treated EGFR-positive MDA-MB-231 cells with EGF (15 nM) for 30 min, which was sufficient to activate PI3K as determined by phosphorylation of AKT (Figure 11 A, Western blots showing expression of EGFR, p-EGFR, MBD4, p-AKT, p-ERK, AKT, ERK and β-actin in MDA-MB-231 cells treated with EGF (15 nM) for 1 hour). We observed no upregulation of MBD4, indicating that MBD4 is not universally regulated by activation of the PI3K pathway (Figure 11 A). It is also important to note that MBD4 phosphorylation was not altered as a result of RON/MSP pathway activation (Figure 11 B, showing the expression of MET, MBD4 and ACTB in MCF7 cell line infected with retroviruses carrying cDNA of MET) suggesting that MBD4 is not a direct target of RON kinase or kinases in the PI3K pathway. Shown in Figure 11C are MCF7 and MCF7- RON/MSP cells were treated with or without alkaline phosphatase for 30 min. The lysates were subjected to (a) conventional SDS-PAGE on 8% (wt/vol) polyacrylamide gel or (b) SDS-PAGE on 6% (wt/vol) polyacrylamide gel containing 20 μΜ Mn2+-Phos-tag, followed by immunoblotting with the anti-MBD4 antibody. By treatment with alkaline phosphatase, shifts in the mobility of MBD4 were suppressed.
EXAMPLE 8: Treatment with a RON kinase inhibitor prevents metastasis of primary patient-derived tumor grafts.
[00109] Ron kinase activity, and it resulting signaling, can be blocked by small molecule kinase inhibitors. To determine if the RON/MBD4 pathway can be blocked in patient tumors using RON kinase inhibitors, and if there is a resulting effect on metastasis, we utilized four independent experimental systems that are highly relevant to bona fide breast tumors. First, we chose two patient-derived tumor grafts that survive primary culture conditions and treated them with the RON/MET dual inhibitor, OSI-296 in vitro. Inhibition of RON (verified by reduction of phosphorylated RON protein) caused downregulation of MBD4 (Figure 7A). Shown in Figure 7A are Western blots with anti-p-RON, MBD4 and β-actin antibodies on protein lysates from primary cultures of the patient-derived tumor graft HCI-007, treated without (-) or with 1 μΜ of the RON inhibitor OSI-296 over the indicated times. Next, we chose two human tumor graft lines for in vivo studies based on their RON/MBD4 signature (not shown) and on their relatively fast growth and robust metastasis, which allows for assessment of tumor growth and metastasis within a timeline that is practical for drug treatment. The tumor grafts were implanted orthotopically into cleared mammary fat pads of NOD/SCID mice and allowed to grow to 100 mm3 before starting treatment with OSI-296. Although there was no significant effect on tumor growth (data not shown), inhibition of RON in the in vivo setting caused complete blockade of lymph node (Figure 7B and 7C) and lung (Figure 7D) metastasis in these two models. Shown in Figure 7B are Metastasis frequencies for orthotopically-implanted patient-derived tumor grafts HCI-003 and HCI-01 1 following treatment with OSI-296 (n=3 and n=9, for HCI-003 and HCI-01 1 , respectively) or trappsol (n=5 for both HCI-003 and HCI-01 1 ) vehicle control (p value determined by Fisher's exact test). Shown in Figure 7C are sections of axillary lymph nodes isolated from mice carrying orthotopic (inguinal fat pad) patient-derived breast tumor grafts HCI-003 and HCI- 01 1 following treatment with trappsol or OSI-296. The sections were stained with H&E (left panels) or were immunostained with antibodies specific for human cytokeratin (right panels) to detect human tumor cells. Shown in Figure 7D are sections of lungs isolated from mice carrying orthotopic patient-derived breast tumor grafts HCI-003 or HCI-01 1 following treatment with trappsol or OSI-296. The sections were stained with H&E (left) or were immunostained with antibodies specific for human cytokeratin (CK; right) to identify human tumor cells. We then analyzed gene expression in tumors from mice treated with the vehicle (Trappsol) or OSI-296. We found that 9 genes from the RON/MBD4 epigenetic signature were deregulated following treatment with the RON inhibitor (Figure 7E). Shown in Figure 7E is a table representing the 9 genes of the RON/MBD4 epigenetic signature that become deregulated in tumors from mice treated with OSI296 compared to the trappsol control. These data indicated that short term inhibition of RON kinase with OSI-296 could not only block metastasis, but also reverse expression of some genes within the RON/MBD4 epigenetic metastasis signature.
[00110] To assess if expression of these 9 RON/MBD4-dependent genes were associated with patient outcome, we performed Kaplan-Meier analysis examining overall survival for individuals with breast cancer across the 997-patient METABRIC dataset. The expression of these genes, which are a subset of the RON/MBD4 epigenetic signature that can be reversed with a RON inhibitor, was significantly associated with poor survival (presence of the signature was defined as the presence of at least 1 gene from the 9 genes that were deregulated in tumors from mice treated with OSI-296, i.e., SEQ ID NOs 1 -47 and 48-56) (p= 0.04, HR=1.55, Figure 7F). Shown in Figure 7F is Kaplan-Meier analysis of overall survival in 997 breast cancer patients from the METABRIC discovery dataset (left) and in 977 breast cancer patients from the compendium dataset (right). Survival curve of individuals with tumors showing an enrichment of the 9 RON/MBD4 epigenetic signature genes that were deregulated in tumors from mice treated with OSI-296 compared to the trappsol control is shown in red; all other patients are shown in blue (No signature). The p value indicates a statistically significant survival difference between these two groups of patients. The survival hazard ratio was calculated using Univariate Cox's regression analysis. To validate our findings using additional, independent data, we generated a meta- collection of gene expression data from an additional 977 patients from five independently published studies of breast cancer. The method we utilized to normalize data across the different datasets was previously described and is described in detail in the Methods section. Using these data, we confirmed that the 9-gene RON/MBD4 epigenetic signature was significantly associated with poor survival (p= 0.008, HR=1.55, Figure 7F). However, multivariate analysis of the 9-gene RON/MBD4 epigenetic signature revealed that it was not an independent prognosis factor for survival in this dataset, and may give similar risk as ER- status does (Figure 7G). Shown in Figure 7G are univariate and multivariate cox regression analyses for overall survival in the 997 patients from the METABRIC dataset, examining ER, PR, status, basal-like subtype, and the MBD4/RON signature as variables (HR, hazard ratio). While ER- tumors have a poor prognosis, there are no targeted therapy options due to lack of a defined pathway driving these tumors. Together, these data highlight the promising potential for (A) identifying breast cancer patients that might benefit from a RON inhibitor; (B) identifying a set of biomarkers for RON inhibition, and (C) blocking RON to inhibit breast cancer metastasis.
SEQUENCE LISTING
CSGALNACT1. ENSG00000147408
SEQ ID NO: 1 = variant a = ENSG00000147408|ENST00000520003
Polynucleotide, homo sapiens, 161 bp
ATGATGATGGTTCGCCGGGGGCTGCTTGCGTGGATTTCCCGGGTGGTGGTTTTGCTGGTGCTCCTCTGCTGTGCT ATCTCTGTCCTGTACATGTTGGCCTGCACCCCAAAAGGTGACGAGGAGCAGCTGGCACTGCCCAGGGCCAACAGC CCCACGGGGAA
SEQ ID NO: 2 = variant b = ENSG00000147408|ENST00000523262
Polynucleotide, homo sapiens, 312 bp
ATGATGATGGTTCGCCGGGGGCTGCTTGCGTGGATTTCCCGGGTGGTGGTTTTGCTGGTGCTCCTCTGCTGTGCT ATCTCTGTCCTGTACATGTTGGCCTGCACCCCAAAAGGTGACGAGGAGCAGCTGGCACTGCCCAGGGCCAACAGC CCCACGGGGAAGGAGGGGTACCAGGCCGTCCTTCAGGAGTGGGAGGAGCAGCACCGCAACTACGTGAGCAGCCTG AAGCGGCAGATCGCACAGCTCAAGGAGGAGCTGCAGGAGAGGAGTGAGCAGCTCAGGAATGGGCAGTACCAAGCC AGCGATGCTGCT
SEQ ID NO: 3 = variant c = ENSG00000147408|ENST00000397998
Polynucleotide, homo sapiens, 894 bp
ATGATGATGGTTCGCCGGGGGCTGCTTGCGTGGATTTCCCGGGTGGTGGTTTTGCTGGTGCTCCTCTGCTGTGCT ATCTCTGTCCTGTACATGTTGGCCTGCACCCCAAAAGGTGACGAGGAGCAGCTGGCACTGCCCAGGGCCAACAGC CCCACGGGGAAGGAGGGGTACCAGGCCGTCCTTCAGGAGTGGGAGGAGCAGCACCGCAACTACGTGAGCAGCCTG AAGCGGCAGATCGCACAGCTCAAGGAGGAGCTGCAGGAGAGGAGTGAGCAGCTCAGGAATGGGCAGTACCAAGCC AGCGATGCTGCTGGCCTGGGTCTGGACAGGAGCCCCCCAGAGAAAACCCAGGCCGACCTCCTGGCCTTCCTGCAC TCGCAGGTGGACAAGGCAGAGGTGAATGCTGGCGTCAAGCTGGCCACAGAGTATGCAGCAGTGCCTTTCGATAGC TTTACTCTACAGAAGGTGTACCAGCTGGAGACTGGCCTTACCCGCCACCCCGAGGAGAAGCCTGTGAGGAAGGAC AAGCGGGATGAGTTGGTGGAAGCCATTGAATCAGCCTTGGAGACCCTGAACAGTCCTGCAGAGAACAGCCCCAAT CACCGTCCTTACACGGCCTCTGATTTCATAGAAGGGATCTACCGAACAGAAAGGGACAAAGGGACATTGTATGAG CTCACCTTCAAAGGGGACCACAAACACGAATTCAAACGGCTCATCTTATTTCGACCATTCGGCCCCATCATGAAA GTGAAAAATGAAAAGCTCAACATGGCCAACACGCTTATCAATGTTATCGTGCCTCTAGCAAAAAGGGTGGACAAG TTCCGGCAGTTCATGCAGAATTTCAGGCCTGCTGATGAAGTTTTTAGATGTGTGCCTTTAAGCCCTTGA
SEQ ID NO: 4 = variant d = ENSG00000147408|ENST00000332246
Polynucleotide, homo sapiens, 1599 bp
ATGATGATGGTTCGCCGGGGGCTGCTTGCGTGGATTTCCCGGGTGGTGGTTTTGCTGGTGCTCCTCTGCTGTGCT ATCTCTGTCCTGTACATGTTGGCCTGCACCCCAAAAGGTGACGAGGAGCAGCTGGCACTGCCCAGGGCCAACAGC CCCACGGGGAAGGAGGGGTACCAGGCCGTCCTTCAGGAGTGGGAGGAGCAGCACCGCAACTACGTGAGCAGCCTG AAGCGGCAGATCGCACAGCTCAAGGAGGAGCTGCAGGAGAGGAGTGAGCAGCTCAGGAATGGGCAGTACCAAGCC AGCGATGCTGCTGGCCTGGGTCTGGACAGGAGCCCCCCAGAGAAAACCCAGGCCGACCTCCTGGCCTTCCTGCAC TCGCAGGTGGACAAGGCAGAGGTGAATGCTGGCGTCAAGCTGGCCACAGAGTATGCAGCAGTGCCTTTCGATAGC TTTACTCTACAGAAGGTGTACCAGCTGGAGACTGGCCTTACCCGCCACCCCGAGGAGAAGCCTGTGAGGAAGGAC AAGCGGGATGAGTTGGTGGAAGCCATTGAATCAGCCTTGGAGACCCTGAACAGTCCTGCAGAGAACAGCCCCAAT CACCGTCCTTACACGGCCTCTGATTTCATAGAAGGGATCTACCGAACAGAAAGGGACAAAGGGACATTGTATGAG CTCACCTTCAAAGGGGACCACAAACACGAATTCAAACGGCTCATCTTATTTCGACCATTCGGCCCCATCATGAAA GTGAAAAATGAAAAGCTCAACATGGCCAACACGCTTATCAATGTTATCGTGCCTCTAGCAAAAAGGGTGGACAAG TTCCGGCAGTTCATGCAGAATTTCAGGGAGATGTGCATTGAGCAGGATGGGAGAGTCCATCTCACTGTTGTTTAC TTTGGGAAAGAAGAAATAAATGAAGTCAAAGGAATACTTGAAAACACTTCCAAAGCTGCCAACTTCAGGAACTTT ACCTTCATCCAGCTGAATGGAGAATTTTCTCGGGGAAAGGGACTTGATGTTGGAGCCCGCTTCTGGAAGGGAAGC AACGTCCTTCTCTTTTTCTGTGATGTGGACATCTACTTCACATCTGAATTCCTCAATACGTGTAGGCTGAATACA CAGCCAGGGAAGAAGGTATTTTATCCAGTTCTTTTCAGTCAGTACAATCCTGGCATAATATACGGCCACCATGAT GCAGTCCCTCCCTTGGAACAGCAGCTGGTCATAAAGAAGGAAACTGGATTTTGGAGAGACTTTGGATTTGGGATG ACGTGTCAGTATCGGTCAGACTTCATCAATATAGGTGGGTTTGATCTGGACATCAAAGGCTGGGGCGGAGAGGAT GTGCACCTTTATCGCAAGTATCTCCACAGCAACCTCATAGTGGTACGGACGCCTGTGCGAGGACTCTTCCACCTC TGGCATGAGAAGCGCTGCATGGACGAGCTGACCCCCGAGCAGTACAAGATGTGCATGCAGTCCAAGGCCATGAAC GAGGCATCCCACGGCCAGCTGGGCATGCTGGTGTTCAGGCACGAGATAGAGGCTCACCTTCGCAAACAGAAACAG AAGACAAGTAGCAAAAAAACATGA SEQ ID NO: 5 = variant e = ENSG00000147408|ENST00000524213
Polynucleotide, homo sapiens, 43 bp
ATGATGATGGTTCGCCGGGGGCTGCTTGCGTGGATTTCCCGGG
SEQ ID NO: 6 = variant f = ENSG00000147408|ENST0000031 1540
Polynucleotide, homo sapiens, 1599 bp
ATGATGATGGTTCGCCGGGGGCTGCTTGCGTGGATTTCCCGGGTGGTGGTTTTGCTGGTGCTCCTCTGCTGTGCT ATCTCTGTCCTGTACATGTTGGCCTGCACCCCAAAAGGTGACGAGGAGCAGCTGGCACTGCCCAGGGCCAACAGC CCCACGGGGAAGGAGGGGTACCAGGCCGTCCTTCAGGAGTGGGAGGAGCAGCACCGCAACTACGTGAGCAGCCTG AAGCGGCAGATCGCACAGCTCAAGGAGGAGCTGCAGGAGAGGAGTGAGCAGCTCAGGAATGGGCAGTACCAAGCC AGCGATGCTGCTGGCCTGGGTCTGGACAGGAGCCCCCCAGAGAAAACCCAGGCCGACCTCCTGGCCTTCCTGCAC TCGCAGGTGGACAAGGCAGAGGTGAATGCTGGCGTCAAGCTGGCCACAGAGTATGCAGCAGTGCCTTTCGATAGC TTTACTCTACAGAAGGTGTACCAGCTGGAGACTGGCCTTACCCGCCACCCCGAGGAGAAGCCTGTGAGGAAGGAC AAGCGGGATGAGTTGGTGGAAGCCATTGAATCAGCCTTGGAGACCCTGAACAGTCCTGCAGAGAACAGCCCCAAT CACCGTCCTTACACGGCCTCTGATTTCATAGAAGGGATCTACCGAACAGAAAGGGACAAAGGGACATTGTATGAG CTCACCTTCAAAGGGGACCACAAACACGAATTCAAACGGCTCATCTTATTTCGACCATTCGGCCCCATCATGAAA GTGAAAAATGAAAAGCTCAACATGGCCAACACGCTTATCAATGTTATCGTGCCTCTAGCAAAAAGGGTGGACAAG TTCCGGCAGTTCATGCAGAATTTCAGGGAGATGTGCATTGAGCAGGATGGGAGAGTCCATCTCACTGTTGTTTAC TTTGGGAAAGAAGAAATAAATGAAGTCAAAGGAATACTTGAAAACACTTCCAAAGCTGCCAACTTCAGGAACTTT ACCTTCATCCAGCTGAATGGAGAATTTTCTCGGGGAAAGGGACTTGATGTTGGAGCCCGCTTCTGGAAGGGAAGC AACGTCCTTCTCTTTTTCTGTGATGTGGACATCTACTTCACATCTGAATTCCTCAATACGTGTAGGCTGAATACA CAGCCAGGGAAGAAGGTATTTTATCCAGTTCTTTTCAGTCAGTACAATCCTGGCATAATATACGGCCACCATGAT GCAGTCCCTCCCTTGGAACAGCAGCTGGTCATAAAGAAGGAAACTGGATTTTGGAGAGACTTTGGATTTGGGATG ACGTGTCAGTATCGGTCAGACTTCATCAATATAGGTGGGTTTGATCTGGACATCAAAGGCTGGGGCGGAGAGGAT GTGCACCTTTATCGCAAGTATCTCCACAGCAACCTCATAGTGGTACGGACGCCTGTGCGAGGACTCTTCCACCTC TGGCATGAGAAGCGCTGCATGGACGAGCTGACCCCCGAGCAGTACAAGATGTGCATGCAGTCCAAGGCCATGAAC GAGGCATCCCACGGCCAGCTGGGCATGCTGGTGTTCAGGCACGAGATAGAGGCTCACCTTCGCAAACAGAAACAG AAGACAAGTAGCAAAAAAACATGA
SEQ ID NO: 7 = variant g = ENSG00000147408|ENST00000522854
Polynucleotide, homo sapiens, 1599 bp
ATGATGATGGTTCGCCGGGGGCTGCTTGCGTGGATTTCCCGGGTGGTGGTTTTGCTGGTGCTCCTCTGCTGTGCT ATCTCTGTCCTGTACATGTTGGCCTGCACCCCAAAAGGTGACGAGGAGCAGCTGGCACTGCCCAGGGCCAACAGC CCCACGGGGAAGGAGGGGTACCAGGCCGTCCTTCAGGAGTGGGAGGAGCAGCACCGCAACTACGTGAGCAGCCTG AAGCGGCAGATCGCACAGCTCAAGGAGGAGCTGCAGGAGAGGAGTGAGCAGCTCAGGAATGGGCAGTACCAAGCC AGCGATGCTGCTGGCCTGGGTCTGGACAGGAGCCCCCCAGAGAAAACCCAGGCCGACCTCCTGGCCTTCCTGCAC TCGCAGGTGGACAAGGCAGAGGTGAATGCTGGCGTCAAGCTGGCCACAGAGTATGCAGCAGTGCCTTTCGATAGC TTTACTCTACAGAAGGTGTACCAGCTGGAGACTGGCCTTACCCGCCACCCCGAGGAGAAGCCTGTGAGGAAGGAC AAGCGGGATGAGTTGGTGGAAGCCATTGAATCAGCCTTGGAGACCCTGAACAGTCCTGCAGAGAACAGCCCCAAT CACCGTCCTTACACGGCCTCTGATTTCATAGAAGGGATCTACCGAACAGAAAGGGACAAAGGGACATTGTATGAG CTCACCTTCAAAGGGGACCACAAACACGAATTCAAACGGCTCATCTTATTTCGACCATTCGGCCCCATCATGAAA GTGAAAAATGAAAAGCTCAACATGGCCAACACGCTTATCAATGTTATCGTGCCTCTAGCAAAAAGGGTGGACAAG TTCCGGCAGTTCATGCAGAATTTCAGGGAGATGTGCATTGAGCAGGATGGGAGAGTCCATCTCACTGTTGTTTAC TTTGGGAAAGAAGAAATAAATGAAGTCAAAGGAATACTTGAAAACACTTCCAAAGCTGCCAACTTCAGGAACTTT ACCTTCATCCAGCTGAATGGAGAATTTTCTCGGGGAAAGGGACTTGATGTTGGAGCCCGCTTCTGGAAGGGAAGC AACGTCCTTCTCTTTTTCTGTGATGTGGACATCTACTTCACATCTGAATTCCTCAATACGTGTAGGCTGAATACA CAGCCAGGGAAGAAGGTATTTTATCCAGTTCTTTTCAGTCAGTACAATCCTGGCATAATATACGGCCACCATGAT GCAGTCCCTCCCTTGGAACAGCAGCTGGTCATAAAGAAGGAAACTGGATTTTGGAGAGACTTTGGATTTGGGATG ACGTGTCAGTATCGGTCAGACTTCATCAATATAGGTGGGTTTGATCTGGACATCAAAGGCTGGGGCGGAGAGGAT GTGCACCTTTATCGCAAGTATCTCCACAGCAACCTCATAGTGGTACGGACGCCTGTGCGAGGACTCTTCCACCTC TGGCATGAGAAGCGCTGCATGGACGAGCTGACCCCCGAGCAGTACAAGATGTGCATGCAGTCCAAGGCCATGAAC GAGGCATCCCACGGCCAGCTGGGCATGCTGGTGTTCAGGCACGAGATAGAGGCTCACCTTCGCAAACAGAAACAG AAGACAAGTAGCAAAAAAACATGA
SEQ ID NO: 8 = variant h = ENSG00000147408|ENST00000517494
Polynucleotide, homo sapiens, 290 bp
ATGATGATGGTTCGCCGGGGGCTGCTTGCGTGGATTTCCCGGGTGGTGGTTTTGCTGGTGCTCCTCTGCTGTGCT ATCTCTGTCCTGTACATGTTGGCCTGCACCCCAAAAGGTGACGAGGAGCAGCTGGCACTGCCCAGGGCCAACAGC CCCACGGGGAAGGAGGGGTACCAGGCCGTCCTTCAGGAGTGGGAGGAGCAGCACCGCAACTACGTGAGCAGCCTG AAGCGGCAGATCGCACAGCTCAAGGAGGAGCTGCAGGAGAGGAGTGAGCAGCTCAGGAATGGGCA SEQ ID NO: 9 = variant i = ENSG00000147408|ENST00000519222
Polynucleotide, homo sapiens, 894 bp
ATGATGATGGTTCGCCGGGGGCTGCTTGCGTGGATTTCCCGGGTGGTGGTTTTGCTGGTGCTCCTCTGCTGTGCT ATCTCTGTCCTGTACATGTTGGCCTGCACCCCAAAAGGTGACGAGGAGCAGCTGGCACTGCCCAGGGCCAACAGC CCCACGGGGAAGGAGGGGTACCAGGCCGTCCTTCAGGAGTGGGAGGAGCAGCACCGCAACTACGTGAGCAGCCTG AAGCGGCAGATCGCACAGCTCAAGGAGGAGCTGCAGGAGAGGAGTGAGCAGCTCAGGAATGGGCAGTACCAAGCC AGCGATGCTGCTGGCCTGGGTCTGGACAGGAGCCCCCCAGAGAAAACCCAGGCCGACCTCCTGGCCTTCCTGCAC TCGCAGGTGGACAAGGCAGAGGTGAATGCTGGCGTCAAGCTGGCCACAGAGTATGCAGCAGTGCCTTTCGATAGC TTTACTCTACAGAAGGTGTACCAGCTGGAGACTGGCCTTACCCGCCACCCCGAGGAGAAGCCTGTGAGGAAGGAC AAGCGGGATGAGTTGGTGGAAGCCATTGAATCAGCCTTGGAGACCCTGAACAGTCCTGCAGAGAACAGCCCCAAT CACCGTCCTTACACGGCCTCTGATTTCATAGAAGGGATCTACCGAACAGAAAGGGACAAAGGGACATTGTATGAG CTCACCTTCAAAGGGGACCACAAACACGAATTCAAACGGCTCATCTTATTTCGACCATTCGGCCCCATCATGAAA GTGAAAAATGAAAAGCTCAACATGGCCAACACGCTTATCAATGTTATCGTGCCTCTAGCAAAAAGGGTGGACAAG TTCCGGCAGTTCATGCAGAATTTCAGGCCTGCTGATGAAGTTTTTAGATGTGTGCCTTTAAGCCCTTGA
SEQ ID NO: 10 = variant j = ENSG00000147408|ENST00000454498
Polynucleotide, homo sapiens, 1599 bp
ATGATGATGGTTCGCCGGGGGCTGCTTGCGTGGATTTCCCGGGTGGTGGTTTTGCTGGTGCTCCTCTGCTGTGCT ATCTCTGTCCTGTACATGTTGGCCTGCACCCCAAAAGGTGACGAGGAGCAGCTGGCACTGCCCAGGGCCAACAGC CCCACGGGGAAGGAGGGGTACCAGGCCGTCCTTCAGGAGTGGGAGGAGCAGCACCGCAACTACGTGAGCAGCCTG AAGCGGCAGATCGCACAGCTCAAGGAGGAGCTGCAGGAGAGGAGTGAGCAGCTCAGGAATGGGCAGTACCAAGCC AGCGATGCTGCTGGCCTGGGTCTGGACAGGAGCCCCCCAGAGAAAACCCAGGCCGACCTCCTGGCCTTCCTGCAC TCGCAGGTGGACAAGGCAGAGGTGAATGCTGGCGTCAAGCTGGCCACAGAGTATGCAGCAGTGCCTTTCGATAGC TTTACTCTACAGAAGGTGTACCAGCTGGAGACTGGCCTTACCCGCCACCCCGAGGAGAAGCCTGTGAGGAAGGAC AAGCGGGATGAGTTGGTGGAAGCCATTGAATCAGCCTTGGAGACCCTGAACAGTCCTGCAGAGAACAGCCCCAAT CACCGTCCTTACACGGCCTCTGATTTCATAGAAGGGATCTACCGAACAGAAAGGGACAAAGGGACATTGTATGAG CTCACCTTCAAAGGGGACCACAAACACGAATTCAAACGGCTCATCTTATTTCGACCATTCGGCCCCATCATGAAA GTGAAAAATGAAAAGCTCAACATGGCCAACACGCTTATCAATGTTATCGTGCCTCTAGCAAAAAGGGTGGACAAG TTCCGGCAGTTCATGCAGAATTTCAGGGAGATGTGCATTGAGCAGGATGGGAGAGTCCATCTCACTGTTGTTTAC TTTGGGAAAGAAGAAATAAATGAAGTCAAAGGAATACTTGAAAACACTTCCAAAGCTGCCAACTTCAGGAACTTT ACCTTCATCCAGCTGAATGGAGAATTTTCTCGGGGAAAGGGACTTGATGTTGGAGCCCGCTTCTGGAAGGGAAGC AACGTCCTTCTCTTTTTCTGTGATGTGGACATCTACTTCACATCTGAATTCCTCAATACGTGTAGGCTGAATACA CAGCCAGGGAAGAAGGTATTTTATCCAGTTCTTTTCAGTCAGTACAATCCTGGCATAATATACGGCCACCATGAT GCAGTCCCTCCCTTGGAACAGCAGCTGGTCATAAAGAAGGAAACTGGATTTTGGAGAGACTTTGGATTTGGGATG ACGTGTCAGTATCGGTCAGACTTCATCAATATAGGTGGGTTTGATCTGGACATCAAAGGCTGGGGCGGAGAGGAT GTGCACCTTTATCGCAAGTATCTCCACAGCAACCTCATAGTGGTACGGACGCCTGTGCGAGGACTCTTCCACCTC TGGCATGAGAAGCGCTGCATGGACGAGCTGACCCCCGAGCAGTACAAGATGTGCATGCAGTCCAAGGCCATGAAC GAGGCATCCCACGGCCAGCTGGGCATGCTGGTGTTCAGGCACGAGATAGAGGCTCACCTTCGCAAACAGAAACAG AAGACAAGTAGCAAAAAAACATGA
SEQ ID NO: 1 1 = variant k = ENSG00000147408|ENST00000544602
Polynucleotide, homo sapiens, 1599 bp
ATGATGATGGTTCGCCGGGGGCTGCTTGCGTGGATTTCCCGGGTGGTGGTTTTGCTGGTGCTCCTCTGCTGTGCT ATCTCTGTCCTGTACATGTTGGCCTGCACCCCAAAAGGTGACGAGGAGCAGCTGGCACTGCCCAGGGCCAACAGC CCCACGGGGAAGGAGGGGTACCAGGCCGTCCTTCAGGAGTGGGAGGAGCAGCACCGCAACTACGTGAGCAGCCTG AAGCGGCAGATCGCACAGCTCAAGGAGGAGCTGCAGGAGAGGAGTGAGCAGCTCAGGAATGGGCAGTACCAAGCC AGCGATGCTGCTGGCCTGGGTCTGGACAGGAGCCCCCCAGAGAAAACCCAGGCCGACCTCCTGGCCTTCCTGCAC TCGCAGGTGGACAAGGCAGAGGTGAATGCTGGCGTCAAGCTGGCCACAGAGTATGCAGCAGTGCCTTTCGATAGC TTTACTCTACAGAAGGTGTACCAGCTGGAGACTGGCCTTACCCGCCACCCCGAGGAGAAGCCTGTGAGGAAGGAC AAGCGGGATGAGTTGGTGGAAGCCATTGAATCAGCCTTGGAGACCCTGAACAGTCCTGCAGAGAACAGCCCCAAT CACCGTCCTTACACGGCCTCTGATTTCATAGAAGGGATCTACCGAACAGAAAGGGACAAAGGGACATTGTATGAG CTCACCTTCAAAGGGGACCACAAACACGAATTCAAACGGCTCATCTTATTTCGACCATTCGGCCCCATCATGAAA GTGAAAAATGAAAAGCTCAACATGGCCAACACGCTTATCAATGTTATCGTGCCTCTAGCAAAAAGGGTGGACAAG TTCCGGCAGTTCATGCAGAATTTCAGGGAGATGTGCATTGAGCAGGATGGGAGAGTCCATCTCACTGTTGTTTAC TTTGGGAAAGAAGAAATAAATGAAGTCAAAGGAATACTTGAAAACACTTCCAAAGCTGCCAACTTCAGGAACTTT ACCTTCATCCAGCTGAATGGAGAATTTTCTCGGGGAAAGGGACTTGATGTTGGAGCCCGCTTCTGGAAGGGAAGC AACGTCCTTCTCTTTTTCTGTGATGTGGACATCTACTTCACATCTGAATTCCTCAATACGTGTAGGCTGAATACA CAGCCAGGGAAGAAGGTATTTTATCCAGTTCTTTTCAGTCAGTACAATCCTGGCATAATATACGGCCACCATGAT GCAGTCCCTCCCTTGGAACAGCAGCTGGTCATAAAGAAGGAAACTGGATTTTGGAGAGACTTTGGATTTGGGATG ACGTGTCAGTATCGGTCAGACTTCATCAATATAGGTGGGTTTGATCTGGACATCAAAGGCTGGGGCGGAGAGGAT GTGCACCTTTATCGCAAGTATCTCCACAGCAACCTCATAGTGGTACGGACGCCTGTGCGAGGACTCTTCCACCTC TGGCATGAGAAGCGCTGCATGGACGAGCTGACCCCCGAGCAGTACAAGATGTGCATGCAGTCCAAGGCCATGAAC GAGGCATCCCACGGCCAGCTGGGCATGCTGGTGTTCAGGCACGAGATAGAGGCTCACCTTCGCAAACAGAAACAG AAGACAAGTAGCAAAAAAACATGA
SEQ ID NO: 48
Polypeptide of SEQ ID NO: 1 , homo sapiens, 53 AA
MMMVRRGLLAWISRWVLLVLLCCAISVLYMLACTPKGDEEQLALPRANSPTG
SIGLEC6. ENSG00000105492
SEQ ID NO: 12 = variant a = ENSG00000105492|ENST00000436458
Polynucleotide, homo sapiens, 1206 bp
ATGCAGGGAGCCCAGGAAGCCTCCGCCTCAGAGATGCTACCGCTGCTGCTGCCCCTGCTGTGGGCAGCCTCGTAC TATGGTTATGGCTACTGGTTCCTGGAAGGGGCTGATGTTCCAGTGGCCACAAACGACCCAGACGAAGAAGTGCAG GAGGAGACCCGGGGCCGATTCCACCTCCTCTGGGATCCCAGAAGGAAGAACTGCTCCCTGAGCATCAGAGATGCC CGGAGGAGGGACAATGCTGCATACTTCTTTCGGTTGAAGTCCAAATGGATGAAATACGGTTATACATCTTCCAAG CTCTCTGTGCGTGTGATGGCCCTGACCCACAGGCCCAACATCTCCATCCCAGGGACCCTGGAGTCTGGCCATCCC AGCAATCTGACCTGCTCTGTGCCCTGGGTCTGTGAGCAGGGGACGCCCCCCATCTTCTCCTGGATGTCAGCTGCC CCCACCTCCCTGGGCCCCAGGACCACCCAGTCCTCGGTGCTCACAATCACCCCACGGCCCCAGGACCACAGCACC AACCTCACCTGTCAGGTGACGTTCCCTGGAGCCGGTGTGACCATGGAGAGAACCATCCAGCTCAATGTCTCCTCC TTCAAAATCCTGCAAAACACCTCGTCCCTCCCTGTCCTGGAGGGCCAGGCTCTGCGGCTGCTCTGTGATGCTGAC GGCAACCCCCCTGCACACCTGAGCTGGTTCCAGGGCTTCCCCGCCCTGAACGCCACCCCCATCTCCAATACCGGG GTCCTGGAGCTGCCTCAAGTAGGGTCTGCAGAAGAAGGAGATTTCACCTGCCGTGCTCAGCATCCTCTGGGCTCC CTGCAAATCTCTCTGAGTCTCTTTGTGCATTGGAAACCAGAAGGCAGGGCTGGTGGTGTCCTGGGAGCAGTCTGG GGAGCTAGCATCACAACCCTGGTTTTCCTCTGTGTTTGCTTCATCTTCAGAGTGAAGACTAGAAGGAAGAAAGCA GCCCAGCCAGTGCAAAACACGGATGATGTGAACCCCGTCATGGTCTCAGGCTCCAGGGGTCATCAGCACCAGTTC CAGACAGGCATAGTTTCAGACCACCCTGCTGAGGCTGGCCCCATCTCAGAAGATGAGCAGGAGCTCCACTACGCT GTCCTACACTTCCACAAGGTGCAACCTCAGGAACCAAAGGTCACCGACACTGAGTACTCAGAAATCAAGATACAC AAGTGA
SEQ ID NO: 13 = variant b = ENSG00000105492|ENST00000391797
Polynucleotide, homo sapiens, 1029 bp
ATGCAGGGAGCCCAGGAAGCCTCCGCCTCAGAGATGCTACCGCTGCTGCTGCCCCTGCTGTGGGCAGGGGCCCTG GCTCAGGAGCGGAGATTCCAGCTGGAGGGGCCAGAGTCACTGACGGTGCAGGAGGGTCTGTGCGTCCTCGTACCC TGCAGATTGCCCACTACCCTTCCAGCCTCGTACTATGGTTATGGCTACTGGTTCCTGGAAGGGGCTGATGTTCCA GTGGCCACAAACGACCCAGACGAAGAAGTGCAGGAGGAGACCCGGGGCCGATTCCACCTCCTCTGGGATCCCAGA AGGAAGAACTGCTCCCTGAGCATCAGAGATGCCCGGAGGAGGGACAATGCTGCATACTTCTTTCGGTTGAAGTCC AAATGGATGAAATACGGTTATACATCTTCCAAGCTCTCTGTGCGTGTGATGGGGACCCTGGAGTCTGGCCATCCC AGCAATCTGACCTGCTCTGTGCCCTGGGTCTGTGAGCAGGGGACGCCCCCCATCTTCTCCTGGATGTCAGCTGCC CCCACCTCCCTGGGCCCCAGGACCACCCAGTCCTCGGTGCTCACAATCACCCCACGGCCCCAGGACCACAGCACC AACCTCACCTGTCAGGTGACGTTCCCTGGAGCCGGTGTGACCATGGAGAGAACCATCCAGCTCAATGTCTCCTAT GCTCCACAGAAAGTGGCCATCAGCATCTTCCAAGGAAACAGCGCAGCCTTCAAAATCCTGCAAAACACCTCGTCC CTCCCTGTCCTGGAGGGCCAGGCTCTGCGGCTGCTCTGTGATGCTGACGGCAACCCCCCTGCACACCTGAGCTGG TTCCAGGGCTTCCCCGCCCTGAACGCCACCCCCATCTCCAATACCGGGGTCCTGGAGCTGCCTCAAGTAGGGTCT GCAGAAGAAGGAGATTTCACCTGCCGTGCTCAGCATCCTCTGGGCTCCCTGCAAATCTCTCTGAGTCTCTTTGTG CATTGGTCATCAGCACCAGTTCCAGACAGGCATAGTTTCAGACCACCCTGCTGA
SEQ ID NO: 14 = variant c = ENSG00000105492|ENST00000359982
Polynucleotide, homo sapiens, 1 170 bp
ATGCAGGGAGCCCAGGAAGCCTCCGCCTCAGAGATGCTACCGCTGCTGCTGCCCCTGCTGTGGGCAGGGGCCCTG GCTCAGGAGCGGAGATTCCAGCTGGAGGGGCCAGAGTCACTGACGGTGCAGGAGGGTCTGTGCGTCCTCGTACCC TGCAGATTGCCCACTACCCTTCCAGCCTCGTACTATGGTTATGGCTACTGGTTCCTGGAAGGGGCTGATGTTCCA GTGGCCACAAACGACCCAGACGAAGAAGTGCAGGAGGAGACCCGGGGCCGATTCCACCTCCTCTGGGATCCCAGA AGGAAGAACTGCTCCCTGAGCATCAGAGATGCCCGGAGGAGGGACAATGCTGCATACTTCTTTCGGTTGAAGTCC AAATGGATGAAATACGGTTATACATCTTCCAAGCTCTCTGTGCGTGTGATGGCCCTGACCCACAGGCCCAACATC TCCATCCCAGGGACCCTGGAGTCTGGCCATCCCAGCAATCTGACCTGCTCTGTGCCCTGGGTCTGTGAGCAGGGG ACGCCCCCCATCTTCTCCTGGATGTCAGCTGCCCCCACCTCCCTGGGCCCCAGGACCACCCAGTCCTCGGTGCTC ACAATCACCCCACGGCCCCAGGACCACAGCACCAACCTCACCTGTCAGGTGACGTTCCCTGGAGCCGGTGTGACC ATGGAGAGAACCATCCAGCTCAATGTCTCCTGGATGTTGAGGCGGCCACCTCTTTCCACCCCAGATGCTCCACAG AAAGTGGCCATCAGCATCTTCCAAGGAAACAGCGCAGCCTTCAAAATCCTGCAAAACACCTCGTCCCTCCCTGTC CTGGAGGGCCAGGCTCTGCGGCTGCTCTGTGATGCTGACGGCAACCCCCCTGCACACCTGAGCTGGTTCCAGGGC TTCCCCGCCCTGAACGCCACCCCCATCTCCAATACCGGGGTCCTGGAGCTGCCTCAAGTAGGGTCTGCAGAAGAA GGAGATTTCACCTGCCGTGCTCAGCATCCTCTGGGCTCCCTGCAAATCTCTCTGAGTCTCTTTGTGCATTGGAAA CCAGAAGGCAGGGCTGGTGGTGTCCTGGGAGCAGTCTGGGGAGCTAGCATCACAACCCTGGTTTTCCTCTGTGTT TGCTTCATCTTCAGGGTCATCAGCACCAGTTCCAGACAGGCATAG
SEQ ID NO: 15 = variant d = ENSG00000105492|ENST00000425629
Polynucleotide, homo sapiens, 1362 bp
ATGCAGGGAGCCCAGGAAGCCTCCGCCTCAGAGATGCTACCGCTGCTGCTGCCCCTGCTGTGGGCAGGGGCCCTG GCTCAGGAGCGGAGATTCCAGCTGGAGGGGCCAGAGTCACTGACGGTGCAGGAGGGTCTGTGCGTCCTCGTACCC TGCAGATTGCCCACTACCCTTCCAGCCTCGTACTATGGTTATGGCTACTGGTTCCTGGAAGGGGCTGATGTTCCA GTGGCCACAAACGACCCAGACGAAGAAGTGCAGGAGGAGACCCGGGGCCGATTCCACCTCCTCTGGGATCCCAGA AGGAAGAACTGCTCCCTGAGCATCAGAGATGCCCGGAGGAGGGACAATGCTGCATACTTCTTTCGGTTGAAGTCC AAATGGATGAAATACGGTTATACATCTTCCAAGCTCTCTGTGCGTGTGATGGCCCTGACCCACAGGCCCAACATC TCCATCCCAGGGACCCTGGAGTCTGGCCATCCCAGCAATCTGACCTGCTCTGTGCCCTGGGTCTGTGAGCAGGGG ACGCCCCCCATCTTCTCCTGGATGTCAGCTGCCCCCACCTCCCTGGGCCCCAGGACCACCCAGTCCTCGGTGCTC ACAATCACCCCACGGCCCCAGGACCACAGCACCAACCTCACCTGTCAGGTGACGTTCCCTGGAGCCGGTGTGACC ATGGAGAGAACCATCCAGCTCAATGTCTCCTATGCTCCACAGAAAGTGGCCATCAGCATCTTCCAAGGAAACAGC GCAGCCTTCAAAATCCTGCAAAACACCTCGTCCCTCCCTGTCCTGGAGGGCCAGGCTCTGCGGCTGCTCTGTGAT GCTGACGGCAACCCCCCTGCACACCTGAGCTGGTTCCAGGGCTTCCCCGCCCTGAACGCCACCCCCATCTCCAAT ACCGGGGTCCTGGAGCTGCCTCAAGTAGGGTCTGCAGAAGAAGGAGATTTCACCTGCCGTGCTCAGCATCCTCTG GGCTCCCTGCAAATCTCTCTGAGTCTCTTTGTGCATTGGAAACCAGAAGGCAGGGCTGGTGGTGTCCTGGGAGCA GTCTGGGGAGCTAGCATCACAACCCTGGTTTTCCTCTGTGTTTGCTTCATCTTCAGAGTGAAGACTAGAAGGAAG AAAGCAGCCCAGCCAGTGCAAAACACGGATGATGTGAACCCCGTCATGGTCTCAGGCTCCAGGGGTCATCAGCAC CAGTTCCAGACAGGCATAGTTTCAGACCACCCTGCTGAGGCTGGCCCCATCTCAGAAGATGAGCAGGAGCTCCAC TACGCTGTCCTACACTTCCACAAGGTGCAACCTCAGGAACCAAAGGTCACCGACACTGAGTACTCAGAAATCAAG ATACACAAGTGA
SEQ ID NO: 16 = variant e = ENSG00000105492|ENST00000343300
Polynucleotide, homo sapiens, 1062 bp
ATGCAGGGAGCCCAGGAAGCCTCCGCCTCAGAGATGCTACCGCTGCTGCTGCCCCTGCTGTGGGCAGGGGCCCTG GCTCAGGAGCGGAGATTCCAGCTGGAGGGGCCAGAGTCACTGACGGTGCAGGAGGGTCTGTGCGTCCTCGTACCC TGCAGATTGCCCACTACCCTTCCAGCCTCGTACTATGGTTATGGCTACTGGTTCCTGGAAGGGGCTGATGTTCCA GTGGCCACAAACGACCCAGACGAAGAAGTGCAGGAGGAGACCCGGGGCCGATTCCACCTCCTCTGGGATCCCAGA AGGAAGAACTGCTCCCTGAGCATCAGAGATGCCCGGAGGAGGGACAATGCTGCATACTTCTTTCGGTTGAAGTCC AAATGGATGAAATACGGTTATACATCTTCCAAGCTCTCTGTGCGTGTGATGGCCCTGACCCACAGGCCCAACATC TCCATCCCAGGGACCCTGGAGTCTGGCCATCCCAGCAATCTGACCTGCTCTGTGCCCTGGGTCTGTGAGCAGGGG ACGCCCCCCATCTTCTCCTGGATGTCAGCTGCCCCCACCTCCCTGGGCCCCAGGACCACCCAGTCCTCGGTGCTC ACAATCACCCCACGGCCCCAGGACCACAGCACCAACCTCACCTGTCAGGTGACGTTCCCTGGAGCCGGTGTGACC ATGGAGAGAACCATCCAGCTCAATGTCTCCTATGCTCCACAGAAAGTGGCCATCAGCATCTTCCAAGGAAACAGC GCAGCCTTCAAAATCCTGCAAAACACCTCGTCCCTCCCTGTCCTGGAGGGCCAGGCTCTGCGGCTGCTCTGTGAT GCTGACGGCAACCCCCCTGCACACCTGAGCTGGTTCCAGGGCTTCCCCGCCCTGAACGCCACCCCCATCTCCAAT ACCGGGGTCCTGGAGCTGCCTCAAGTAGGGTCTGCAGAAGAAGGAGATTTCACCTGCCGTGCTCAGCATCCTCTG GGCTCCCTGCAAATCTCTCTGAGTCTCTTTGTGCATTGGTCATCAGCACCAGTTCCAGACAGGCATAGTTTCAGA CCACCCTGCTGA
SEQ ID NO: 17 = variant f = ENSG00000105492|ENST00000346477
Polynucleotide, homo sapiens, 1314 bp
ATGCAGGGAGCCCAGGAAGCCTCCGCCTCAGAGATGCTACCGCTGCTGCTGCCCCTGCTGTGGGCAGGGGCCCTG GCTCAGGAGCGGAGATTCCAGCTGGAGGGGCCAGAGTCACTGACGGTGCAGGAGGGTCTGTGCGTCCTCGTACCC TGCAGATTGCCCACTACCCTTCCAGCCTCGTACTATGGTTATGGCTACTGGTTCCTGGAAGGGGCTGATGTTCCA GTGGCCACAAACGACCCAGACGAAGAAGTGCAGGAGGAGACCCGGGGCCGATTCCACCTCCTCTGGGATCCCAGA AGGAAGAACTGCTCCCTGAGCATCAGAGATGCCCGGAGGAGGGACAATGCTGCATACTTCTTTCGGTTGAAGTCC AAATGGATGAAATACGGTTATACATCTTCCAAGCTCTCTGTGCGTGTGATGGCCCTGACCCACAGGCCCAACATC TCCATCCCAGGGACCCTGGAGTCTGGCCATCCCAGCAATCTGACCTGCTCTGTGCCCTGGGTCTGTGAGCAGGGG ACGCCCCCCATCTTCTCCTGGATGTCAGCTGCCCCCACCTCCCTGGGCCCCAGGACCACCCAGTCCTCGGTGCTC ACAATCACCCCACGGCCCCAGGACCACAGCACCAACCTCACCTGTCAGGTGACGTTCCCTGGAGCCGGTGTGACC ATGGAGAGAACCATCCAGCTCAATGTCTCCTCCTTCAAAATCCTGCAAAACACCTCGTCCCTCCCTGTCCTGGAG GGCCAGGCTCTGCGGCTGCTCTGTGATGCTGACGGCAACCCCCCTGCACACCTGAGCTGGTTCCAGGGCTTCCCC GCCCTGAACGCCACCCCCATCTCCAATACCGGGGTCCTGGAGCTGCCTCAAGTAGGGTCTGCAGAAGAAGGAGAT TTCACCTGCCGTGCTCAGCATCCTCTGGGCTCCCTGCAAATCTCTCTGAGTCTCTTTGTGCATTGGAAACCAGAA GGCAGGGCTGGTGGTGTCCTGGGAGCAGTCTGGGGAGCTAGCATCACAACCCTGGTTTTCCTCTGTGTTTGCTTC ATCTTCAGAGTGAAGACTAGAAGGAAGAAAGCAGCCCAGCCAGTGCAAAACACGGATGATGTGAACCCCGTCATG GTCTCAGGCTCCAGGGGTCATCAGCACCAGTTCCAGACAGGCATAGTTTCAGACCACCCTGCTGAGGCTGGCCCC ATCTCAGAAGATGAGCAGGAGCTCCACTACGCTGTCCTACACTTCCACAAGGTGCAACCTCAGGAACCAAAGGTC ACCGACACTGAGTACTCAGAAATCAAGATACACAAGTGA
SEQ ID NO: 49
Polypeptide of SEQ ID NO: 12, homo sapiens, 401 AA
MQGAQEASASEMLPLLLPLLWAASYYGYGYWFLEGADVPVATNDPDEEVQEETRGRFHLLWDPRRKNCSLSIRDA RRRDNAAYFFRLKSKWMKYGYTSSKLSVRVMALTHRPNISI PGTLESGHPSNLTCSVPWVCEQGTPPIFSWMSAA PTSLGPRTTQSSVLTITPRPQDHSTNLTCQVTFPGAGVTMERTIQLNVSSFKILQNTSSLPVLEGQALRLLCDAD GNPPAHLSWFQGFPALNATPISNTGVLELPQVGSAEEGDFTCRAQHPLGSLQISLSLFVHWKPEGRAGGVLGAVW GAS ITTLVFLCVCFIFRVKTRRKKAAQPVQNTDDVNPVMVSGSRGHQHQFQTGIVSDHPAEAGPISEDEQELHYA VLHFHKVQPQEPKVTDTEYSEIKIHK*
SHC4. ENSG00000185634
SEQ ID NO: 18 = variant a = ENSG00000185634|ENST00000332408
Polynucleotide, homo sapiens, 1893 bp
ATGCGAGAACGCGGCCAGGACAGCCTGGCAGGACTCGTGCTGTATGTAGGACTCTTCGGGCACCCCGGGATGCTG CACAGGGCCAAGTACAGCCGCTTTCGGAACGAGTCGATCACGTCCTTGGACGAAGGTAGCTCCGGAGGCTCGGTC GGGAACAAGGGCTCGCCGCAGCCTCCCCACCCCGCCCTGGCACCTCACCTGCCGACTGAAGATGCCACCTTGCCG TCGCAGGAGAGCCCCACCCCACTGTGCACCTTGATCCCCCGCATGGCAAGCATGAAGCTGGCCAACCCGGCCACT TTGCTGAGTCTGAAAAACTTTTGCCTGGGTACCAAAGAGGTGCCTCGGCTGAAGCTCCAGGAAAGCCGGGACCCA GGTTCCAGCGGCCCCTCTTCCCCAGAAACCAGTTTAAGTAGGTCCGGGACTGCACCTCCACCGCAGCAGGACCTG GTGGGACACAGGGCAACCGCCCTAACCCCTGATTCGTGCCCGCTTCCTGGCCCTGGGGAGCCAACACTTAGGAGC AGGCAGGACAGGCACTTTCTACAGCACCTGTTGGGGATGGGCATGAACTACTGTGTGAGGTACATGGGCTGTGTT GAAGTGCTGCAATCAATGAGATCACTGGATTTTGGAATGAGAACCCAAGTTACAAGGGAAGCAATAAGTCGCCTG TGTGAAGCTGTCCCCGGGGCAAATGGAGCCATTAAAAAGCGAAAGCCTCCAGTTAAGTTCCTATCAACAGTCCTT GGCAAAAGTAATCTTCAGTTTTCAGGAATGAATATAAAACTGACCATCTCAACATGCAGTCTCACATTGATGAAT CTTGACAACCAACAGATTATTGCAAATCATCATATGCAGTCTATTTCATTTGCCTCTGGAGGGGATCCTGATACT ACAGACTATGTTGCCTACGTAGCTAAAGATCCAGTTAATCAACGAGCCTGTCACATATTGGAATGCCACAATGGA ATGGCCCAAGACGTCATAAGTACCATAGGGCAGGCTTTTGAACTCCGGTTTAAACAGTACTTGAAAAATCCTTCT TTGAATACTTCTTGTGAAAGTGAGGAGGTGCATATTGATAGCCATGCCGAGGAGAGAGAAGATCATGAATATTAC AATGAAATTCCAGGGAAGCAGCCACCAGTAGGTGGTGTTTCAGATATGCGGATCAAAGTTCAAGCCACGGAACAA ATGGCTTACTGCCCCATACAGTGTGAAAAGTTGTGCTATTTGCCTGGAAACTCCAAGTGCAGCAGTGTATATGAG AACTGTTTAGAACAAAGCAGGGCAATAGGTAATGTCCATCCAAGAGGGGTGCAGTCCCAGCGAGATACCTCATTA TTGAAGCACACGTGCCGAGTGGATCTCTTTGATGACCCCTGCTACATTAATACACAGGCTCTTCAAAGTACACCT GGCTCTGCTGGAAATCAAAGGTCAGCCCAACCACTGGGGAGCCCATGGCACTGCGGAAAGGCACCAGAAACTGTT CAGCCGGGTGCCACAGCCCAGCCTGCCAGCTCACATTCTTTGCCACACATTAAGCAGCAGCTGTGGAGCGAAGAA TGCTATCATGGCAAGCTGAGCAGGAAGGCGGCAGAGAGCCTCTTGGTAAAGGATGGGGACTTTTTGGTTCGAGAG AGTGCAACATCCCCTGGCCAATATGTGCTGAGTGGACTACAGGGAGGCCAAGCAAAACATCTTCTCCTGGTGGAT CCTGAAGGCAAGGTGAGGACCAAGGATCATGTATTTGATAATGTCGGCCACCTTATCAGATACCATATGGATAAC AGTTTGCCAATCATCTCCTCTGGAAGCGAAGTAAGCCTTAAACAACCAGTGAGAAAAGATAATAATCCAGCACTT TTGCATTCCAACAAATGA
SEQ ID NO: 19 = variant b = ENSG00000185634|ENST00000396535
Polynucleotide, homo sapiens, 1 164 bp
ATGCTTCCTGCCCTCGAACATTGGATTCCCAAGTTCTTCAGTTTCAGAACTCGGACTGGCTCTCCTCTCTCCTTA GCCTGCAGACAGCCTATTGTAGGACCTTGTGATCATATTATTGCAAATCATCATATGCAGTCTATTTCATTTGCC TCTGGAGGGGATCCTGATACTACAGACTATGTTGCCTACGTAGCTAAAGATCCAGTTAATCAACGAGCCTGTCAC ATATTGGAATGCCACAATGGAATGGCCCAAGACGTCATAAGTACCATAGGGCAGGCTTTTGAACTCCGGTTTAAA CAGTACTTGAAAAATCCTTCTTTGAATACTTCTTGTGAAAGTGAGGAGGTGCATATTGATAGCCATGCCGAGGAG AGAGAAGATCATGAATATTACAATGAAATTCCAGGGAAGCAGCCACCAGTAGGTGGTGTTTCAGATATGCGGATC AAAGTTCAAGCCACGGAACAAATGGCTTACTGCCCCATACAGTGTGAAAAGTTGTGCTATTTGCCTGGAAACTCC AAGTGCAGCAGTGTATATGAGAACTGTTTAGAACAAAGCAGGGCAATAGGTAATGTCCATCCAAGAGGGGTGCAG TCCCAGCGAGATACCTCATTATTGAAGCACACGTGCCGAGTGGATCTCTTTGATGACCCCTGCTACATTAATACA CAGGCTCTTCAAAGTACACCTGGCTCTGCTGGAAATCAAAGGTCAGCCCAACCACTGGGGAGCCCATGGCACTGC GGAAAGGCACCAGAAACTGTTCAGCCGGGTGCCACAGCCCAGCCTGCCAGCTCACATTCTTTGCCACACATTAAG CAGCAGCTGTGGAGCGAAGAATGCTATCATGGCAAGCTGAGCAGGAAGGCGGCAGAGAGCCTCTTGGTAAAGGAT GGGGACTTTTTGGTTCGAGAGAGTGCAACATCCCCTGGCCAATATGTGCTGAGTGGACTACAGGGAGGCCAAGCA AAACATCTTCTCCTGGTGGATCCTGAAGGCAAGGTGAGGACCAAGGATCATGTATTTGATAATGTCGGCCACCTT ATCAGATACCATATGGATAACAGTTTGCCAATCATCTCCTCTGGAAGCGAAGTAAGCCTTAAACAACCAGTGAGA AAAGATAATAATCCAGCACTTTTGCATTCCAACAAATGA
SEQ ID NO: 20 = variant c = ENSG00000185634|ENST00000557797
Polynucleotide, homo sapiens, 435 bp
ATGCAGTCTATTTCATTTGCCTCTGGAGGGGATCCTGATACTACAGACTATGTTGCCTACGTAGCTAAAGATCCA GTTAATCAACGAGCCTGTCACATATTGGAATGCCACAATGGAATGGCCCAAGACGTCATAAGTACCATAGGGCAG GCTTTTGAACTCCGGTTTAAACAGTACTTGAAAAATCCTTCTTTGAATACTTCTTGTGAAAGTGAGGAGGTGCAT ATTGATAGCCATGCCGAGGAGAGAGAAGATCATGAATATTACAATGAAATTCCAGGGAAGCAGCCACCAGTAGGT GGTGTTTCAGATATGCGGATCAAAGTTCAAGCCACGGAACAAATGGCTTACTGCCCCATACAGTGTGAAAAGTTG TGCTATTTGGTAATGTCCATCCAAGAGGGGTGCAGTCCCAGCGAGATACCTCATTATTGA
SEQ ID NO: 21 = variant d = ENSG00000185634|ENST00000537958
Polynucleotide, homo sapiens, 1035 bp
ATGCAGTCTATTTCATTTGCCTCTGGAGGGGATCCTGATACTACAGACTATGTTGCCTACGTAGCTAAAGATCCA GTTAATCAACGAGCCTGTCACATATTGGAATGCCACAATGGAATGGCCCAAGACGTCATAAGTACCATAGGGCAG GCTTTTGAACTCCGGTTTAAACAGTACTTGAAAAATCCTTCTTTGAATACTTCTTGTGAAAGTGAGGAGGTGCAT ATTGATAGCCATGCCGAGGAGAGAGAAGATCATGAATATTACAATGAAATTCCAGGGAAGCAGCCACCAGTAGGT GGTGTTTCAGATATGCGGATCAAAGTTCAAGCCACGGAACAAATGGCTTACTGCCCCATACAGTGTGAAAAGTTG TGCTATTTGCCTGGAAACTCCAAGTGCAGCAGTGTATATGAGAACTGTTTAGAACAAAGCAGGGCAATAGGTAAT GTCCATCCAAGAGGGGTGCAGTCCCAGCGAGATACCTCATTATTGAAGCACACGTGCCGAGTGGATCTCTTTGAT GACCCCTGCTACATTAATACACAGGCTCTTCAAAGTACACCTGGCTCTGCTGGAAATCAAAGGTCAGCCCAACCA CTGGGGAGCCCATGGCACTGCGGAAAGGCACCAGAAACTGTTCAGCCGGGTGCCACAGCCCAGCCTGCCAGCTCA CATTCTTTGCCACACATTAAGCAGCAGCTGTGGAGCGAAGAATGCTATCATGGCAAGCTGAGCAGGAAGGCGGCA GAGAGCCTCTTGGTAAAGGATGGGGACTTTTTGGTTCGAGAGAGTGCAACATCCCCTGGCCAATATGTGCTGAGT GGACTACAGGGAGGCCAAGCAAAACATCTTCTCCTGGTGGATCCTGAAGGCAAGGTGAGGACCAAGGATCATGTA TTTGATAATGTCGGCCACCTTATCAGATACCATATGGATAACAGTTTGCCAATCATCTCCTCTGGAAGCGAAGTA AGCCTTAAACAACCAGTGAGAAAAGATAATAATCCAGCACTTTTGCATTCCAACAAATGA
SEQ ID NO: 22 = variant e = ENSG00000185634|ENST00000558220
Polynucleotide, homo sapiens, 384 bp
ATGCAGTCTATTTCATTTGCCTCTGGAGGGGATCCTGATACTACAGACTATGTTGCCTACGTAGCTAAAGATCCA GTTAATCAACGAGCCTGTCACATATTGGAATGCCACAATGGAATGGCCCAAGACGTCATAAGTACCATAGGGCAG GCTTTTGAACTCCGGTTTAAACAGTACTTGAAAAATCCTTCTTTGAATACTTCTTGTGAAAGTGAGGAGGTGCAT ATTGATAGCCATGCCGAGGAGAGAGAAGATCATGAATATTACAATGAAATTCCAGGGAAGCAGCCACCAGTAGGT GGTGTTTCAGATATGCGGATCAAAGTTCAAGCCACGGAACAAATGGCTTACTGCCCCATACAGTGTGAAAAGTTG TGCTATTTG
SEQ ID NO: 50
Polypeptide of SEQ ID NO: 18, homo sapiens, 630 AA
MRERGQDSLAGLVLYVGLFGHPGMLHRAKYSRFRNES ITSLDEGSSGGSVGNKGSPQPPHPALAPHLPTEDATLP SQESPTPLCTLI PRMASMKLANPATLLSLKNFCLGTKEVPRLKLQESRDPGSSGPSSPETSLSRSGTAPPPQQDL VGHRATALTPDSCPLPGPGEPTLRSRQDRHFLQHLLGMGMNYCVRYMGCVEVLQSMRSLDFGMRTQVTREAISRL CEAVPGANGAIKKRKPPVKFLSTVLGKSNLQFSGMNIKLTISTCSLTLMNLDNQQI IANHHMQSISFASGGDPDT TDYVAYVAKDPVNQRACHILECHNGMAQDVISTIGQAFELRFKQYLKNPSLNTSCESEEVHIDSHAEEREDHEYY NEI PGKQPPVGGVSDMRIKVQATEQMAYCPIQCEKLCYLPGNSKCSSVYENCLEQSRAIGNVHPRGVQSQRDTSL LKHTCRVDLFDDPCYINTQALQSTPGSAGNQRSAQPLGSPWHCGKAPETVQPGATAQPASSHSLPHIKQQLWSEE CYHGKLSRKAAESLLVKDGDFLVRESATSPGQYVLSGLQGGQAKHLLLVDPEGKVRTKDHVFDNVGHLIRYHMDN SLPI ISSGSEVSLKQPVRKDNNPALLHSNK
ABCA1. ENSG00000165029
SEQ ID NO: 23 = variant a = ENSG00000165029|ENST00000374736
Polynucleotide, homo sapiens, 7190 bp
ATGGCTTGTTGGCCTCAGCTGAGGTTGCTGCTGTGGAAGAACCTCACTTTCAGAAGAAGACAAACATGTCAGCTG CTGCTGGAAGTGGCCTGGCCTCTATTTATCTTCCTGATCCTGATCTCTGTTCGGCTGAGCTACCCACCCTATGAA CAACATGAATGCCATTTTCCAAATAAAGCCATGCCCTCTGCAGGAACACTTCCTTGGGTTCAGGGGATTATCTGT AATGCCAACAACCCCTGTTTCCGTTACCCGACTCCTGGGGAGGCTCCCGGAGTTGTTGGAAACTTTAACAAATCC ATTGTGGCTCGCCTGTTCTCAGATGCTCGGAGGCTTCTTTTATACAGCCAGAAAGACACCAGCATGAAGGACATG CGCAAAGTTCTGAGAACATTACAGCAGATCAAGAAATCCAGCTCAAACTTGAAGCTTCAAGATTTCCTGGTGGAC AATGAAACCTTCTCTGGGTTCCTGTATCACAACCTCTCTCTCCCAAAGTCTACTGTGGACAAGATGCTGAGGGCT GATGTCATTCTCCACAAGGTATTTTTGCAAGGCTACCAGTTACATTTGACAAGTCTGTGCAATGGATCAAAATCA GAAGAGATGATTCAACTTGGTGACCAAGAAGTTTCTGAGCTTTGTGGCCTACCAAGGGAGAAACTGGCTGCAGCA GAGCGAGTACTTCGTTCCAACATGGACATCCTGAAGCCAATCCTGAGAACACTAAACTCTACATCTCCCTTCCCG AGCAAGGAGCTGGCTGAAGCCACAAAAACATTGCTGCATAGTCTTGGGACTCTGGCCCAGGAGCTGTTCAGCATG AGAAGCTGGAGTGACATGCGACAGGAGGTGATGTTTCTGACCAATGTGAACAGCTCCAGCTCCTCCACCCAAATC TACCAGGCTGTGTCTCGTATTGTCTGCGGGCATCCCGAGGGAGGGGGGCTGAAGATCAAGTCTCTCAACTGGTAT GAGGACAACAAC TACAAAGCCCTCTTTGGAGGCAATGGCACTGAGGAAGATGCTGAAACCTTCTATGACAACTCT ACAACTCCTTACTGCAATGATTTGATGAAGAATTTGGAGTCTAGTCCTCTTTCCCGCATTATCTGGAAAGCTCTG AAGCCGCTGCTCGTTGGGAAGATCCTGTATACACCTGACACTCCAGCCACAAGGCAGGTCATGGCTGAGGTGAAC AAGACCTTCCAGGAACTGGCTGTGTTCCATGATCTGGAAGGCATGTGGGAGGAACTCAGCCCCAAGATCTGGACC TTCATGGAGAACAGCCAAGAAATGGACCTTGTCCGGATGCTGTTGGACAGCAGGGACAATGACCACTTTTGGGAA CAGCAGTTGGATGGCTTAGATTGGACAGCCCAAGACATCGTGGCGTTTTTGGCCAAGCACCCAGAGGATGTCCAG TCCAGTAATGGTTCTGTGTACACCTGGAGAGAAGCTTTCAACGAGAC TAACCAGGCAATCCGGACCATATCTCGC TTCATGGAGTGTGTCAACCTGAACAAGCTAGAACCCATAGCAACAGAAGTCTGGCTCATCAACAAGTCCATGGAG CTGCTGGATGAGAGGAAGTTCTGGGCTGGTATTGTGTTCACTGGAATTACTCCAGGCAGCATTGAGCTGCCCCAT CATGTCAAGTACAAGATCCGAATGGACATTGACAATGTGGAGAGGACAAATAAAATCAAGGATGGGTACTGGGAC CCTGGTCCTCGAGCTGACCCCTTTGAGGACATGCGGTACGTCTGGGGGGGCTTCGCCTACTTGCAGGATGTGGTG GAGCAGGCAATCATCAGGGTGCTGACGGGCACCGAGAAGAAAACTGGTGTCTATATGCAACAGATGCCCTATCCC TGTTACGTTGATGACATCTTTCTGCGGGTGATGAGCCGGTCAATGCCCCTCTTCATGACGCTGGCCTGGATTTAC TCAGTGGCTGTGATCATCAAGGGCATCGTGTATGAGAAGGAGGCACGGCTGAAAGAGACCATGCGGATCATGGGC CTGGACAACAGCATCCTCTGGTTTAGCTGGTTCATTAGTAGCCTCATTCCTCTTCTTGTGAGCGCTGGCCTGCTA GTGGTCATCCTGAAGTTAGGAAACCTGCTGCCCTACAGTGATCCCAGCGTGGTGTTTGTCTTCCTGTCCGTGTTT GCTGTGGTGACAATCCTGCAGTGCTTCCTGATTAGCACACTCTTCTCCAGAGCCAACCTGGCAGCAGCCTGTGGG GGCATCATCTACTTCACGCTGTACCTGCCCTACGTCCTGTGTGTGGCATGGCAGGACTACGTGGGCTTCACACTC AAGATCTTCGCTAGCCTGCTGTCTCCTGTGGCTTTTGGGTTTGGCTGTGAGTACTTTGCCCTTTTTGAGGAGCAG GGCATTGGAGTGCAGTGGGACAACCTGTTTGAGAGTCCTGTGGAGGAAGATGGCTTCAATCTCACCACTTCGGTC TCCATGATGCTGTTTGACACCTTCCTCTATGGGGTGATGACCTGGTACATTGAGGCTGTCTTTCCAGGCCAGTAC GGAATTCCCAGGCCCTGGTATTTTCCTTGCACCAAGTCCTACTGGTTTGGCGAGGAAAGTGATGAGAAGAGCCAC CCTGGTTCCAACCAGAAGAGAATATCAGAAATCTGCATGGAGGAGGAACCCACCCACTTGAAGCTGGGCGTGTCC ATTCAGAACCTGGTAAAAGTCTACCGAGATGGGATGAAGGTGGCTGTCGATGGCCTGGCACTGAATTTTTATGAG GGCCAGATCACCTCCTTCCTGGGCCACAATGGAGCGGGGAAGACGACCACCATGTCAATCCTGACCGGGTTGTTC CCCCCGACCTCGGGCACCGCCTACATCCTGGGAAAAGACATTCGCTCTGAGATGAGCACCATCCGGCAGAACCTG GGGGTCTGTCCCCAGCATAACGTGCTGTTTGACATGCTGACTGTCGAAGAACACATCTGGTTCTATGCCCGCTTG AAAGGGCTCTCTGAGAAGCACGTGAAGGCGGAGATGGAGCAGATGGCCCTGGATGTTGGTTTGCCATCAAGCAAG CTGAAAAGCAAAACAAGCCAGCTGTCAGGTGGAATGCAGAGAAAGCTATCTGTGGCCTTGGCCTTTGTCGGGGGA TCTAAGGTTGTCATTCTGGATGAACCCACAGCTGGTGTGGACCCTTACTCCCGCAGGGGAATATGGGAGCTGCTG CTGAAATACCGACAAGGCCGCACCATTATTCTCTCTACACACCACATGGATGAAGCGGACGTCCTGGGGGACAGG ATTGCCATCATCTCCCATGGGAAGCTGTGCTGTGTGGGCTCCTCCCTGTTTCTGAAGAACCAGCTGGGAACAGGC TAC TACCTGACCTTGGTCAAGAAAGATGTGGAATCCTCCCTCAGTTCCTGCAGAAACAGTAGTAGCACTGTGTCA TACCTGAAAAAGGAGGACAGTGTTTCTCAGAGCAGTTCTGATGCTGGCCTGGGCAGCGACCATGAGAGTGACACG CTGACCATCGATGTCTCTGCTATCTCCAACCTCATCAGGAAGCATGTGTCTGAAGCCCGGCTGGTGGAAGACATA GGGCATGAGCTGACCTATGTGCTGCCATATGAAGCTGCTAAGGAGGGAGCCTTTGTGGAACTCTTTCATGAGATT GATGACCGGCTCTCAGACCTGGGCATTTCTAGTTATGGCATCTCAGAGACGACCCTGGAAGAAATATTCCTCAAG GTGGCCGAAGAGAGTGGGGTGGATGCTGAGACCTCAGATGGTACCTTGCCAGCAAGACGAAACAGGCGGGCCTTC GGGGACAAGCAGAGCTGTCTTCGCCCGTTCACTGAAGATGATGCTGCTGATCCAAATGATTCTGACATAGACCCA GAATCCAGAGAGACAGACTTGCTCAGTGGGATGGATGGCAAAGGGTCCTACCAGGTGAAAGGCTGGAAACTTACA CAGCAACAGTTTGTGGCCCTTTTGTGGAAGAGACTGCTAATTGCCAGACGGAGTCGGAAAGGATTTTTTGCTCAG ATTGTCTTGCCAGCTGTGTTTGTCTGCATTGCCCTTGTGTTCAGCCTGATCGTGCCACCCTTTGGCAAGTACCCC AGCCTGGAACTTCAGCCCTGGATGTACAACGAACAGTACACATTTGTCAGCAATGATGCTCCTGAGGACACGGGA ACCCTGGAACTCTTAAACGCCCTCACCAAAGACCCTGGCTTCGGGACCCGCTGTATGGAAGGAAACCCAATCCCA GACACGCCCTGCCAGGCAGGGGAGGAAGAGTGGACCACTGCCCCAGTTCCCCAGACCATCATGGACCTCTTCCAG AATGGGAACTGGACAATGCAGAACCCTTCACCTGCATGCCAGTGTAGCAGCGACAAAATCAAGAAGATGCTGCCT GTGTGTCCCCCAGGGGCAGGGGGGCTGCCTCCTCCACAAAGAAAACAAAACACTGCAGATATCCTTCAGGACCTG ACAGGAAGAAACATTTCGGATTATCTGGTGAAGACGTATGTGCAGATCATAGCCAAAAGCTTAAAGAACAAGATC TGGGTGAATGAGTTTAGGTATGGCGGCTTTTCCCTGGGTGTCAGTAATACTCAAGCACTTCCTCCGAGTCAAGAA GTTAATGATGCCATCAAACAAATGAAGAAACACCTAAAGCTGGCCAAGGACAGTTCTGCAGATCGATTTCTCAAC AGCTTGGGAAGATTTATGACAGGACTGGACACCAAAAATAATGTCAAGGTGTGGTTCAATAACAAGGGCTGGCAT GCAATCAGCTCTTTCCTGAATGTCATCAACAATGCCATTCTCCGGGCCAACCTGCAAAAGGGAGAGAACCCTAGC CATTATGGAATTACTGCTTTCAATCATCCCCTGAATCTCACCAAGCAGCAGCTCTCAGAGGTGGCTCTGATGACC ACATCAGTGGATGTCCTTGTGTCCATCTGTGTCATCTTTGCAATGTCCTTCGTCCCAGCCAGCTTTGTCGTATTC CTGATCCAGGAGCGGGTCAGCAAAGCAAAACACCTGCAGTTCATCAGTGGAGTGAAGCCTGTCATCTACTGGCTC TCTAATTTTGTCTGGGATATGTGCAATTACGTTGTCCCTGCCACACTGGTCATTATCATCTTCATCTGCTTCCAG CAGAAGTCCTATGTGTCCTCCACCAATCTGCCTGTGCTAGCCCTTCTACTTTTGCTGTATGGGTGGTCAATCACA CCTCTCATGTACCCAGCCTCCTTTGTGTTCAAGATCCCCAGCACAGCCTATGTGGTGCTCACCAGCGTGAACCTC TTCATTGGCATTAATGGCAGCGTGGCCACCTTTGTGCTGGAGCTGTTCACCGACAATAAGCTGAATAATATCAAT GATATCCTGAAGTCCGTGTTCTTGATCTTCCCACATTTTTGCCTGGGACGAGGGCTCATCGACATGGTGAAAAAC CAGGCAATGGCTGATGCCCTGGAAAGGTTTGGGGAGAATCGCTTTGTGTCACCATTATCTTGGGACTTGGTGGGA CGAAACCTCTTCGCCATGGCCGTGGAAGGGGTGGTGTTCTTCCTCATTACTGTTCTGATCCAGTACAGATTCTTC ATCAGGCCCAGACCTGTAAATGCAAAGCTATCTCCTCTGAATGATGAAGATGAAGATGTGAGGCGGGAAAGACAG AGAATTCTTGATGGTGGAGGCCAGAATGACATCTTAGAAATCAAGGAGTTGACGAAGATATATAGAAGGAAGCGG AAGCCTGCTGTTGACAGGATTTGCGTGGGCATTCCTCCTGGTGAGTGCTTTGGGCTCCTGGGAGTTAATGGGGCT GGAAAATCATCAACTTTCAAGATGTTAACAGGAGATACCACTGTTACCAGAGGAGATGCTTTCCTTAACAAAAAT AGTATCTTATCAAACATCCATGAAGTACATCAGAACATGGGCTACTGCCCTCAGTTTGATGCCATCACAGAGCTG TTGACTGGGAGAGAACACGTGGAGTTCTTTGCCCTTTTGAGAGGAGTCCCAGAGAAAGAAGTTGGCAAGGTTGGT GAGTGGGCGATTCGGAAACTGGGCCTCGTGAAGTATGGAGAAAAATATGCTGGTAACTATAGTGGAGGCAACAAA CGCAAGCTCTCTACAGCCATGGCTTTGATCGGCGGGCCTCCTGTGGTGTTTCTGGATGAACCCACCACAGGCATG GATCCCAAAGCCCGGCGGTTCTTGTGGAATTGTGCCCTAAGTGTTGTCAAGGAGGGGAGATCAGTAGTGCTTACA TCTCATAGTATGGAAGAATGTGAAGCTCTTTGCACTAGGATGGCAATCATGGTCAATGGAAGGTTCAGGTGCCTT GGCAGTGTCCAGCATCTAAAAAATAGGTTTGGAGATGGTTATACAATAGTTGTACGAATAGCAGGGTCCAACCCG GACCTGAAGCCTGTCCAGGATTTCTTTGGACTTGCATTTCCTGGAAGTGTTCTAAAAGAGAAACACCGGAACATG CTACAATACCAGCTTCCATCTTCATTATCTTCTCTGGCCAGGATATTCAGCATCCTCTCCCAGAGCAAAAAGCGA CTCCACATAGAAGACTACTCTGTTTCTCAGACAACACTTGACCAAGTATTTGTGAACTTTGCCAAGGACCAAAGT GATGATGACCACTTAAAAGACCTCTCATTACACAAAAACCAGACAGTAGTGGACGTTGCAGTTCTCACATCTTTT CTACAGGATGAGAAAGTGAAAGAAAGCTATGTATGA>ENSGO00001650291 ENSTO 0000374733ATGCCCT CTGCAGGAACACTTCCTTGGGTTCAGGGGATTATCTGTAATGCCAACAACCCCTGTTTCCGTTACCCGACTCCTG GGGAGGCTCCCGGAGTTGTTGGAAACTTTAACAAATCCATTGTGGCTCGCCTGTTCTCAGATGCTCGGAGGCTTC TTTTATACAGCCAGAAAGACACCAGCATGAAGGACATGCGCAAAGTTCTGAGAACATTACAGCAGATCAAGAAAT CCAGCTCAAACTTGAAGCTTCAAGATTTCCTGGTGGACAATGAAACCTTCTCTGGGTTCCTGTATCACAACCTCT CTCTCCCAAAGTCTACTGTGGACAAGATGCTGAGGGCTGATGTCATTCTCCACAAGGTAAGCTGA
SEQ ID NO: 24 = variant b = ENSG00000165029|ENST00000423487
Polynucleotide, homo sapiens, 1092 bp
ATGGCTTGTTGGCCTCAGCTGAGGTTGCTGCTGTGGAAGAACCTCACTTTCAGAAGAAGACAAACATGTCAGCTG CTGCTGGAAGTGGCCTGGCCTCTATTTATCTTCCTGATCCTGATCTCTGTTCGGCTGAGCTACCCACCCTATGAA CAACATGAATGCCATTTTCCAAATAAAGCCATGCCCTCTGCAGGAACACTTCCTTGGGTTCAGGGGATTATCTGT AATGCCAACAACCCCTGTTTCCGTTACCCGACTCCTGGGGAGGCTCCCGGAGTTGTTGGAAACTTTAACAAATCC ATTGTGGCTCGCCTGTTCTCAGATGCTCGGAGGCTTCTTTTATACAGCCAGAAAGACACCAGCATGAAGGACATG CGCAAAGTTCTGAGAACATTACAGCAGATCAAGAAATCCAGCTCAAACTTGAAGCTTCAAGATTTCCTGGTGGAC AATGAAACCTTCTCTGGGTTCCTGTATCACAACCTCTCTCTCCCAAAGTCTACTGTGGACAAGATGCTGAGGGCT GATGTCATTCTCCACAAGGTATTTTTGCAAGGCTACCAGTTACATTTGACAAGTCTGTGCAATGGATCAAAATCA GAAGAGATGATTCAACTTGGTGACCAAGAAGTTTCTGAGCTTTGTGGCCTACCAAGGGAGAAACTGGCTGCAGCA GAGCGAGTACTTCGTTCCAACATGGACATCCTGAAGCCAATCCTGATGGATGTGACATGTGATGACATTGCACAT GGGCAGTTAACTGTGCCAAGAAGTGCAGCAGTAGCAGCAACCGGAGATGCAAAGCCCAACATGATGGGGAGAGAA ACTCTTCTTTCAATATGTGCTTCTGTACCAAAAGTGGAATTTCACGAGAGACATATTTTGAAACATTTCTCCTTT TGTGTGTGCGTGAGTGTTTCCCTGTTTCCAGCCAAGGGTATTGTGAGTTTCTCCTGGGCCTCCTTCAGAATCTGG GTGCTCTGGAAAGCAGTGTTTTGGCAACATGGGGAAAGTATGGCAGTGTGGGAGGGTCAGCTGGGTCTGGGTTTG AATATTGCATTTGAATATTTTACCAGCATTGATGTCGGATAA
SEQ ID NO: 51
Polypeptide of SEQ ID NO: 23, homo sapiens, 2261 AA
MACWPQLRLLLWKNLTFRRRQTCQLLLEVAWPLFIFLILISVRLSYPPYEQHECHFPNKAMPSAGTLPWVQGIIC NANNPCFRYPTPGEAPGVVGNFNKSIVARLFSDARRLLLYSQKDTSMKDMRKVLRTLQQIKKSSSNLKLQDFLVD NETFSGFLYHNLSLPKSTVDKMLRADVILHKVFLQGYQLHLTSLCNGSKSEEMIQLGDQEVSELCGLPREKLAAA ERVLRSNMDILKPILRTLNSTSPFPSKELAEATKTLLHSLGTLAQELFSMRSWSDMRQEVMFLTNVNSSSSSTQI YQAVSRIVCGHPEGGGLKIKSLNWYEDNNYKALFGGNGTEEDAETFYDNSTTPYCNDLMKNLESSPLSRI IWKAL KPLLVGKILYTPDTPATRQVMAEVNKTFQELAVFHDLEGMWEELSPKIWTFMENSQEMDLVRMLLDSRDNDHFWE QQLDGLDWTAQDIVAFLAKHPEDVQSSNGSVYTWREAFNETNQAIRTISRFMECVNLNKLEPIATEVWLINKSME LLDERKFWAGIVFTGITPGS IELPHHVKYKIRMDIDNVERTNKIKDGYWDPGPRADPFEDMRYVWGGFAYLQDVV EQAI IRVLTGTEKKTGVYMQQMPYPCYVDDIFLRVMSRSMPLFMTLAWIYSVAVI IKGIVYEKEARLKETMRIMG LDNSILWFSWFISSLIPLLVSAGLLVVILKLGNLLPYSDPSWFVFLSVFAWTILQCFLISTLFSRANLAAACG GIIYFTLYLPYVLCVAWQDYVGFTLKIFASLLSPVAFGFGCEYFALFEEQGIGVQWDNLFESPVEEDGFNLTTSV SMMLFDTFLYGVMTWYIEAVFPGQYGIPRPWYFPCTKSYWFGEESDEKSHPGSNQKRISEICMEEEPTHLKLGVS IQNLVKVYRDGMKVAVDGLALNFYEGQITSFLGHNGAGKTTTMSILTGLFPPTSGTAYILGKDIRSEMSTIRQNL GVCPQHNVLFDMLTVEEHIWFYARLKGLSEKHVKAEMEQMALDVGLPSSKLKSKTSQLSGGMQRKLSVALAFVGG SKVVILDEPTAGVDPYSRRGIWELLLKYRQGRTI ILSTHHMDEADVLGDRIAI ISHGKLCCVGSSLFLKNQLGTG YYLTLVKKDVESSLSSCRNSSSTVSYLKKEDSVSQSSSDAGLGSDHESDTLTIDVSAISNLIRKHVSEARLVEDI GHELTYVLPYEAAKEGAFVELFHEIDDRLSDLGISSYGISETTLEEIFLKVAEESGVDAETSDGTLPARRNRRAF GDKQSCLRPFTEDDAADPNDSDIDPESRETDLLSGMDGKGSYQVKGWKLTQQQFVALLWKRLLIARRSRKGFFAQ IVLPAVFVCIALVFSLIVPPFGKYPSLELQPWMYNEQYTFVSNDAPEDTGTLELLNALTKDPGFGTRCMEGNPIP DTPCQAGEEEWTTAPVPQTIMDLFQNGNWTMQNPSPACQCSSDKIKKMLPVCPPGAGGLPPPQRKQNTADILQDL TGRNISDYLVKTYVQI IAKSLKNKIWVNEFRYGGFSLGVSNTQALPPSQEVNDAIKQMKKHLKLAKDSSADRFLN SLGRFMTGLDTKNNVKVWFNNKGWHAISSFLNVINNAILRANLQKGENPSHYGITAFNHPLNLTKQQLSEVALMT TSVDVLVS ICVIFAMSFVPASFWFLIQERVSKAKHLQFISGVKPVIYWLSNFVWDMCNYWPATLVI I IFICFQ QKSYVSSTNLPVLALLLLLYGWSITPLMYPASFVFKI PSTAYVVLTSVNLFIGINGSVATFVLELFTDNKLNNIN DILKSVFLIFPHFCLGRGLIDMVKNQAMADALERFGENRFVSPLSWDLVGRNLFAMAVEGWFFLITVLIQYRFF IRPRPVNAKLSPLNDEDEDVRRERQRILDGGGQNDILEIKELTKIYRRKRKPAVDRICVGIPPGECFGLLGVNGA GKSSTFKMLTGDTTVTRGDAFLNKNS ILSNIHEVHQNMGYCPQFDAITELLTGREHVEFFALLRGVPEKEVGKVG EWAIRKLGLVKYGEKYAGNYSGGNKRKLSTAMALIGGPPWFLDEPTTGMDPKARRFLWNCALSVVKEGRSVVLT SHSMEECEALCTRMAIMVNGRFRCLGSVQHLKNRFGDGYTIWRIAGSNPDLKPVQDFFGLAFPGSVLKEKHRNM LQYQLPSSLSSLARIFSILSQSKKRLHIEDYSVSQTTLDQVFVNFAKDQSDDDHLKDLSLHKNQTVVDVAVLTSF LQDEKVKESYV
PLD1. ENSG00000075651
SEQ ID NO: 25 = variant a = ENSG00000075651 |ENST00000440204
Polynucleotide, homo sapiens, 174 bp
ATGTCACTGAAAAACGAGCCACGGGTAAATACCTCTGCACTGCAGAAAATTGCTGCTGACATGAGTAATATCATA GAAAATCTGGACACGCGGGAACTCCACTTTGAGGGAGAGGAGGTAGACTACGACGTGTCTCCCAGCGATCCCAAG ATACAAGAAGGTACCAAGTATTAA
SEQ ID NO: 26 = variant b = ENSG00000075651 |ENST00000418087
Polynucleotide, homo sapiens, 425 bp
ATGTCACTGAAAAACGAGCCACGGGTAAATACCTCTGCACTGCAGAAAATTGCTGCTGACATGAGTAATATCATA GAAAATCTGGACACGCGGGAACTCCACTTTGAGGGAGAGGAGGTAGACTACGACGTGTCTCCCAGCGATCCCAAG ATACAAGAAGTGTATATCCCTTTCTCTGCTATTTATAACACTCAAGGATTTAAGGAGCCTAATATACAGACGTAT CTCTCCGGCTGTCCAATAAAAGCACAAGTTCTGGAAGTGGAACGCTTCACATCTACAACAAGGGTACCAAGTATT AATCTTTACACTATTGAATTAACACATGGGGAATTTAAATGGCAAGTTAAGAGGAAATTCAAGCATTTTCAAGAA TTTCACAGAGAGCTGCTCAAGTACAAAGCCTTTATCCGCATCCCCATTCC
SEQ ID NO: 27 = variant c = ENSG00000075651 |ENST00000446289
Polynucleotide, homo sapiens, 705 bp
NTCCATGCTAACGTACAGTTGCTCCGCTCTGCTGCTGATTGGTCTGCTGGTATAAAGTACCATGAAGAGTCCATC CACGCCGCTTACGTCCATGTGATAGAGAACAGCAGGCACTATATCTATATCGAAAACCAGTTTTTCATAAGCTGT GCTGATGACAAAGTTGTGTTCAACAAGATAGGCGATGCCATTGCCCAGAGGATCCTGAAAGCTCACAGGGAAAAC CAGAAATACCGGGTATATGTCGTGATACCACTTCTGCCAGGGTTCGAAGGAGACATTTCAACCGGCGGAGGAAAT GCTCTACAGGCAATCATGCACTTCAACTACAGAACCATGTGCAGAGGAGAAAATTCCATCCTTGGACAGTTAAAA GCAGAGCTTGGTAATCAGTGGATAAATTACATATCATTCTGTGGTCTTAGAACACATGCAGAGCTCGAAGGAAAC CTAGTAACTGAGCTTATCTATGTCCACAGCAAGTTGTTAATTGCTGATGATAACACTGTTATTATTGGCTCTGCC AACATAAATGACCGCAGCATGCTGGGAAAGCGTGACAGTGAAATGGCTGTCATTGTGCAAGATACAGAGACTGTT CCTTCAGTAATGGATGGAAAAGAGTACCAAGCTGGCCGGTTTGCCCGAGGACTTCGGCTACAGTGCTTTAGGTCT AAAATGACTCCAGGTGTCGAAGATCCCTGA
SEQ ID NO: 28 = variant d = ENSG00000075651 |ENST00000356327
Polynucleotide, homo sapiens, 31 1 1 bp
ATGTCACTGAAAAACGAGCCACGGGTAAATACCTCTGCACTGCAGAAAATTGCTGCTGACATGAGTAATATCATA GAAAATCTGGACACGCGGGAACTCCACTTTGAGGGAGAGGAGGTAGACTACGACGTGTCTCCCAGCGATCCCAAG ATACAAGAAGTGTATATCCCTTTCTCTGCTATTTATAACACTCAAGGATTTAAGGAGCCTAATATACAGACGTAT CTCTCCGGCTGTCCAATAAAAGCACAAGTTCTGGAAGTGGAACGCTTCACATCTACAACAAGGGTACCAAGTATT AATCTTTACACTATTGAATTAACACATGGGGAATTTAAATGGCAAGTTAAGAGGAAATTCAAGCATTTTCAAGAA TTTCACAGAGAGCTGCTCAAGTACAAAGCCTTTATCCGCATCCCCATTCCCACTAGAAGACACACGTTTAGGAGG CAAAACGTCAGAGAGGAGCCTCGAGAGATGCCCAGTTTGCCCCGTTCATCTGAAAACATGATAAGAGAAGAACAA TTCCTTGGTAGAAGAAAACAACTGGAAGATTACTTGACAAAGATACTAAAAATGCCCATGTATAGAAACTATCAT GCCACAACAGAGTTTCTTGATATAAGCCAGCTGTCTTTCATCCATGATTTGGGACCAAAGGGCATAGAAGGTATG ATAATGAAAAGATCTGGAGGACACAGAATACCAGGCTTGAATTGCTGTGGTCAGGGAAGAGCCTGCTACAGATGG TCAAAAAGATGGTTAATAGTGAAAGATTCCTTTTTATTGTATATGAAACCAGACAGCGGTGCCATTGCCTTCGTC CTGCTGGTAGACAAAGAATTCAAAATTAAGGTGGGGAAGAAGGAGACAGAAACGAAATATGGAATCCGAATTGAT AATCTTTCAAGGACACTTATTTTAAAATGCAACAGCTATAGACATGCTCGGTGGTGGGGAGGGGCTATAGAAGAA TTCATCCAGAAACATGGCACCAACTTTCTCAAAGATCATCGATTTGGGTCATATGCTGCTATCCAAGAGAATGCT TTAGCTAAATGGTATGTTAATGCCAAAGGATATTTTGAAGATGTGGCAAATGCAATGGAAGAGGCAAATGAAGAG ATTTTTATCACAGACTGGTGGCTGAGTCCAGAAATCTTCCTGAAACGCCCAGTGGTTGAGGGAAATCGTTGGAGG TTGGACTGCATTCTTAAACGAAAAGCACAACAAGGAGTGAGGATCTTCATAATGCTCTACAAAGAGGTGGAACTC GCTCTTGGCATCAATAGTGAATACACCAAGAGGACTTTGATGCGTCTACATCCCAACATAAAGGTGATGAGACAC CCGGATCATGTGTCATCCACCGTCTATTTGTGGGCTCACCATGAGAAGCTTGTCATCATTGACCAATCGGTGGCC TTTGTGGGAGGGATTGACCTGGCCTATGGAAGGTGGGACGACAATGAGCACAGACTCACAGACGTGGGCAGTGTG AAGCGGGTCACTTCAGGACCGTCTCTGGGTTCCCTCCCACCTGCCGCAATGGAGTCTATGGAATCCTTAAGACTC AAAGATAAAAATGAGCCTGTTCAAAACCTACCCATCCAGAAGAGTATTGATGATGTGGATTCAAAACTGAAAGGA ATAGGAAAGCCAAGAAAGTTCTCCAAATTTAGTCTCTACAAGCAGCTCCACAGGCACCACCTGCACGACGCAGAT AGCATCAGCAGCATTGACAGCACCTCCAATACCGGGTCCATCCGTAGTTTACAGACAGGTGTGGGAGAGCTGCAT GGGGAAACCAGATTCTGGCATGGAAAGGACTACTGCAATTTCGTCTTCAAAGACTGGGTTCAACTTGATAAACCT TTTGCTGATTTCATTGACAGGTACTCCACGCCCCGGATGCCCTGGCATGACATTGCCTCTGCAGTCCACGGGAAG GCGGCTCGTGATGTGGCACGTCACTTCATCCAGCGCTGGAACTTCACAAAAATTATGAAATCAAAATATCGGTCC CTTTCTTATCCTTTTCTGCTTCCAAAGTCTCAAACAACAGCCCATGAGTTGAGATATCAAGTGCCTGGGTCTGTC CATGCTAACGTACAGTTGCTCCGCTCTGCTGCTGATTGGTCTGCTGGTATAAAGTACCATGAAGAGTCCATCCAC GCCGCTTACGTCCATGTGATAGAGAACAGCAGGCACTATATCTATATCGAAAACCAGTTTTTCATAAGCTGTGCT GATGACAAAGTTGTGTTCAACAAGATAGGCGATGCCATTGCCCAGAGGATCCTGAAAGCTCACAGGGAAAACCAG AAATACCGGGTATATGTCGTGATACCACTTCTGCCAGGGTTCGAAGGAGACATTTCAACCGGCGGAGGAAATGCT CTACAGGCAATCATGCACTTCAACTACAGAACCATGTGCAGAGGAGAAAATTCCATCCTTGGACAGTTAAAAGCA GAGCTTGGTAATCAGTGGATAAATTACATATCATTCTGTGGTCTTAGAACACATGCAGAGCTCGAAGGAAACCTA GTAACTGAGCTTATCTATGTCCACAGCAAGTTGTTAATTGCTGATGATAACACTGTTATTATTGGCTCTGCCAAC ATAAATGACCGCAGCATGCTGGGAAAGCGTGACAGTGAAATGGCTGTCATTGTGCAAGATACAGAGACTGTTCCT TCAGTAATGGATGGAAAAGAGTACCAAGCTGGCCGGTTTGCCCGAGGACTTCGGCTACAGTGCTTTAGGGTTGTC CTTGGCTATCTTGATGACCCAAGTGAGGACATTCAGGATCCAGTGAGTGACAAATTCTTCAAGGAGGTGTGGGTT TCAACAGCAGCTCGAAATGCTACAATTTATGACAAGGTTTTCCGGTGCCTTCCCAATGATGAAGTACACAATTTA ATTCAGCTGAGAGACTTTATAAACAAGCCCGTATTAGCTAAGGAAGATCCCATTCGAGCTGAGGAGGAACTGAAG AAGATCCGTGGATTTTTGGTGCAATTCCCCTTTTATTTCTTGTCTGAAGAAAGCCTACTGCCTTCTGTTGGGACC AAAGAGGCCATAGTGCCCATGGAGGTTTGGACTTAA
SEQ ID NO: 29 = variant e = ENSG00000075651 |ENST00000342215
Polynucleotide, homo sapiens, 1794 bp
ATGTCACTGAAAAACGAGCCACGGGTAAATACCTCTGCACTGCAGAAAATTGCTGCTGACATGAGTAATATCATA GAAAATCTGGACACGCGGGAACTCCACTTTGAGGGAGAGGAGGTAGACTACGACGTGTCTCCCAGCGATCCCAAG ATACAAGAAGTGTATATCCCTTTCTCTGCTATTTATAACACTCAAGGATTTAAGGAGCCTAATATACAGACGTAT CTCTCCGGCTGTCCAATAAAAGCACAAGTTCTGGAAGTGGAACGCTTCACATCTACAACAAGGGTACCAAGTATT AATCTTTACACTATTGAATTAACACATGGGGAATTTAAATGGCAAGTTAAGAGGAAATTCAAGCATTTTCAAGAA TTTCACAGAGAGCTGCTCAAGTACAAAGCCTTTATCCGCATCCCCATTCCCACTAGAAGACACACGTTTAGGAGG CAAAACGTCAGAGAGGAGCCTCGAGAGATGCCCAGTTTGCCCCGTTCATCTGAAAACATGATAAGAGAAGAACAA TTCCTTGGTAGAAGAAAACAACTGGAAGATTACTTGACAAAGATACTAAAAATGCCCATGTATAGAAACTATCAT GCCACAACAGAGTTTCTTGATATAAGCCAGCTGTCTTTCATCCATGATTTGGGACCAAAGGGCATAGAAGGTATG ATAATGAAAAGATCTGGAGGACACAGAATACCAGGCTTGAATTGCTGTGGTCAGGGAAGAGCCTGCTACAGATGG TCAAAAAGATGGTTAATAGTGAAAGATTCCTTTTTATTGTATATGAAACCAGACAGCGGTGCCATTGCCTTCGTC CTGCTGGTAGACAAAGAATTCAAAATTAAGGTGGGGAAGAAGGAGACAGAAACGAAATATGGAATCCGAATTGAT AATCTTTCAAGGACACTTATTTTAAAATGCAACAGCTATAGACATGCTCGGTGGTGGGGAGGGGCTATAGAAGAA TTCATCCAGAAACATGGCACCAACTTTCTCAAAGATCATCGATTTGGGTCATATGCTGCTATCCAAGAGAATGCT TTAGCTAAATGGTATGTTAATGCCAAAGGATATTTTGAAGATGTGGCAAATGCAATGGAAGAGGCAAATGAAGAG ATTTTTATCACAGACTGGTGGCTGAGTCCAGAAATCTTCCTGAAACGCCCAGTGGTTGAGGGAAATCGTTGGAGG TTGGACTGCATTCTTAAACGAAAAGCACAACAAGGAGTGAGGATCTTCATAATGCTCTACAAAGAGGTGGAACTC GCTCTTGGCATCAATAGTGAATACACCAAGAGGACTTTGATGCGTCTACATCCCAACATAAAGGTGATGAGACAC CCGGATCATGTGTCATCCACCGTCTATTTGTGGGCTCACCATGAGAAGCTTGTCATCATTGACCAATCGGTGGCC TTTGTGGGAGGGATTGACCTGGCCTATGGAAGGTGGGACGACAATGAGCACAGACTCACAGACGTGGGCAGTGTG AAGCGGGTCACTTCAGGACCGTCTCTGGGTTCCCTCCCAATACCGGGTCCATCCGTAGTTTACAGACAGGTGTGG GAGAGCTGCATGGGGAAACCAGATTCTGGCATGGAAAGGACTACTGCAATTTCGTCTTCAAAGACTGGGTTCAAC TTGATAAACCTTTTGCTGATTTCATTGACAGGTACTCCACGCCCCGGATGCCCTGGCATGACATTGCCTCTGCAG TCCACGGGAAGGCGGCTCGTGATGTGGCACGTCACTTCATCCAGCGCTGGAACTTCACAAAAATTATGA
SEQ ID NO: 30 = variant f = ENSG00000075651 |ENST00000351298
Polynucleotide, homo sapiens, 3225 bp
ATGTCACTGAAAAACGAGCCACGGGTAAATACCTCTGCACTGCAGAAAATTGCTGCTGACATGAGTAATATCATA GAAAATCTGGACACGCGGGAACTCCACTTTGAGGGAGAGGAGGTAGACTACGACGTGTCTCCCAGCGATCCCAAG ATACAAGAAGTGTATATCCCTTTCTCTGCTATTTATAACACTCAAGGATTTAAGGAGCCTAATATACAGACGTAT CTCTCCGGCTGTCCAATAAAAGCACAAGTTCTGGAAGTGGAACGCTTCACATCTACAACAAGGGTACCAAGTATT AATCTTTACACTATTGAATTAACACATGGGGAATTTAAATGGCAAGTTAAGAGGAAATTCAAGCATTTTCAAGAA TTTCACAGAGAGCTGCTCAAGTACAAAGCCTTTATCCGCATCCCCATTCCCACTAGAAGACACACGTTTAGGAGG CAAAACGTCAGAGAGGAGCCTCGAGAGATGCCCAGTTTGCCCCGTTCATCTGAAAACATGATAAGAGAAGAACAA TTCCTTGGTAGAAGAAAACAACTGGAAGATTACTTGACAAAGATACTAAAAATGCCCATGTATAGAAACTATCAT GCCACAACAGAGTTTCTTGATATAAGCCAGCTGTCTTTCATCCATGATTTGGGACCAAAGGGCATAGAAGGTATG ATAATGAAAAGATCTGGAGGACACAGAATACCAGGCTTGAATTGCTGTGGTCAGGGAAGAGCCTGCTACAGATGG TCAAAAAGATGGTTAATAGTGAAAGATTCCTTTTTATTGTATATGAAACCAGACAGCGGTGCCATTGCCTTCGTC CTGCTGGTAGACAAAGAATTCAAAATTAAGGTGGGGAAGAAGGAGACAGAAACGAAATATGGAATCCGAATTGAT AATCTTTCAAGGACACTTATTTTAAAATGCAACAGCTATAGACATGCTCGGTGGTGGGGAGGGGCTATAGAAGAA TTCATCCAGAAACATGGCACCAACTTTCTCAAAGATCATCGATTTGGGTCATATGCTGCTATCCAAGAGAATGCT TTAGCTAAATGGTATGTTAATGCCAAAGGATATTTTGAAGATGTGGCAAATGCAATGGAAGAGGCAAATGAAGAG ATTTTTATCACAGACTGGTGGCTGAGTCCAGAAATCTTCCTGAAACGCCCAGTGGTTGAGGGAAATCGTTGGAGG TTGGACTGCATTCTTAAACGAAAAGCACAACAAGGAGTGAGGATCTTCATAATGCTCTACAAAGAGGTGGAACTC GCTCTTGGCATCAATAGTGAATACACCAAGAGGACTTTGATGCGTCTACATCCCAACATAAAGGTGATGAGACAC CCGGATCATGTGTCATCCACCGTCTATTTGTGGGCTCACCATGAGAAGCTTGTCATCATTGACCAATCGGTGGCC TTTGTGGGAGGGATTGACCTGGCCTATGGAAGGTGGGACGACAATGAGCACAGACTCACAGACGTGGGCAGTGTG AAGCGGGTCACTTCAGGACCGTCTCTGGGTTCCCTCCCACCTGCCGCAATGGAGTCTATGGAATCCTTAAGACTC AAAGATAAAAATGAGCCTGTTCAAAACCTACCCATCCAGAAGAGTATTGATGATGTGGATTCAAAACTGAAAGGA ATAGGAAAGCCAAGAAAGTTCTCCAAATTTAGTCTCTACAAGCAGCTCCACAGGCACCACCTGCACGACGCAGAT AGCATCAGCAGCATTGACAGCACCTCCAGTTATTTTAATCACTATAGAAGTCATCACAATTTAATCCATGGTTTA AAACCCCACTTCAAACTCTTTCACCCGTCCAGTGAGTCTGAGCAAGGACTCACTAGACCTCATGCTGATACCGGG TCCATCCGTAGTTTACAGACAGGTGTGGGAGAGCTGCATGGGGAAACCAGATTCTGGCATGGAAAGGACTACTGC AATTTCGTCTTCAAAGACTGGGTTCAACTTGATAAACCTTTTGCTGATTTCATTGACAGGTACTCCACGCCCCGG ATGCCCTGGCATGACATTGCCTCTGCAGTCCACGGGAAGGCGGCTCGTGATGTGGCACGTCACTTCATCCAGCGC TGGAACTTCACAAAAATTATGAAATCAAAATATCGGTCCCTTTCTTATCCTTTTCTGCTTCCAAAGTCTCAAACA ACAGCCCATGAGTTGAGATATCAAGTGCCTGGGTCTGTCCATGCTAACGTACAGTTGCTCCGCTCTGCTGCTGAT TGGTCTGCTGGTATAAAGTACCATGAAGAGTCCATCCACGCCGCTTACGTCCATGTGATAGAGAACAGCAGGCAC TATATCTATATCGAAAACCAGTTTTTCATAAGCTGTGCTGATGACAAAGTTGTGTTCAACAAGATAGGCGATGCC ATTGCCCAGAGGATCCTGAAAGCTCACAGGGAAAACCAGAAATACCGGGTATATGTCGTGATACCACTTCTGCCA GGGTTCGAAGGAGACATTTCAACCGGCGGAGGAAATGCTCTACAGGCAATCATGCACTTCAACTACAGAACCATG TGCAGAGGAGAAAATTCCATCCTTGGACAGTTAAAAGCAGAGCTTGGTAATCAGTGGATAAATTACATATCATTC TGTGGTCTTAGAACACATGCAGAGCTCGAAGGAAACCTAGTAACTGAGCTTATCTATGTCCACAGCAAGTTGTTA ATTGCTGATGATAACACTGTTATTATTGGCTCTGCCAACATAAATGACCGCAGCATGCTGGGAAAGCGTGACAGT GAAATGGCTGTCATTGTGCAAGATACAGAGACTGTTCCTTCAGTAATGGATGGAAAAGAGTACCAAGCTGGCCGG TTTGCCCGAGGACTTCGGCTACAGTGCTTTAGGGTTGTCCTTGGCTATCTTGATGACCCAAGTGAGGACATTCAG GATCCAGTGAGTGACAAATTCTTCAAGGAGGTGTGGGTTTCAACAGCAGCTCGAAATGCTACAATTTATGACAAG GTTTTCCGGTGCCTTCCCAATGATGAAGTACACAATTTAATTCAGCTGAGAGACTTTATAAACAAGCCCGTATTA GCTAAGGAAGATCCCATTCGAGCTGAGGAGGAACTGAAGAAGATCCGTGGATTTTTGGTGCAATTCCCCTTTTAT TTCTTGTCTGAAGAAAGCCTACTGCCTTCTGTTGGGACCAAAGAGGCCATAGTGCCCATGGAGGTTTGGACTTAA
SEQ ID NO: 31 = variant g = ENSG00000075651 |ENST00000340989
Polynucleotide, homo sapiens, 2916 bp
ATGTCACTGAAAAACGAGCCACGGGTAAATACCTCTGCACTGCAGAAAATTGCTGCTGACATGAGTAATATCATA GAAAATCTGGACACGCGGGAACTCCACTTTGAGGGAGAGGAGGTAGACTACGACGTGTCTCCCAGCGATCCCAAG ATACAAGAAGTGTATATCCCTTTCTCTGCTATTTATAACACTCAAGGATTTAAGGAGCCTAATATACAGACGTAT CTCTCCGGCTGTCCAATAAAAGCACAAGTTCTGGAAGTGGAACGCTTCACATCTACAACAAGGGTACCAAGTATT AATCTTTACACTATTGAATTAACACATGGGGAATTTAAATGGCAAGTTAAGAGGAAATTCAAGCATTTTCAAGAA TTTCACAGAGAGCTGCTCAAGTACAAAGCCTTTATCCGCATCCCCATTCCCACTAGAAGACACACGTTTAGGAGG CAAAACGTCAGAGAGGAGCCTCGAGAGATGCCCAGTTTGCCCCGTTCATCTGAAAACATGATAAGAGAAGAACAA TTCCTTGGTAGAAGAAAACAACTGGAAGATTACTTGACAAAGATACTAAAAATGCCCATGTATAGAAACTATCAT GCCACAACAGAGTTTCTTGATATAAGCCAGCTGTCTTTCATCCATGATTTGGGACCAAAGGGCATAGAAGGTATG ATAATGAAAAGATCTGGAGGACACAGAATACCAGGCTTGAATTGCTGTGGTCAGGGAAGAGCCTGCTACAGATGG TCAAAAAGATGGTTAATAGTGAAAGATTCCTTTTTATTGTATATGAAACCAGACAGCGGTGCCATTGCCTTCGTC CTGCTGGTAGACAAAGAATTCAAAATTAAGGTGGGGAAGAAGGAGACAGAAACGAAATATGGAATCCGAATTGAT AATCTTTCAAGGACACTTATTTTAAAATGCAACAGCTATAGACATGCTCGGTGGTGGGGAGGGGCTATAGAAGAA TTCATCCAGAAACATGGCACCAACTTTCTCAAAGATCATCGATTTGGGTCATATGCTGCTATCCAAGAGAATGCT TTAGCTAAATGGTATGTTAATGCCAAAGGATATTTTGAAGATGTGGCAAATGCAATGGAAGAGGCAAATGAAGAG ATTTTTATCACAGACTGGTGGCTGAGTCCAGAAATCTTCCTGAAACGCCCAGTGGTTGAGGGAAATCGTTGGAGG TTGGACTGCATTCTTAAACGAAAAGCACAACAAGGAGTGAGGATCTTCATAATGCTCTACAAAGAGGTGGAACTC GCTCTTGGCATCAATAGTGAATACACCAAGAGGACTTTGATGCGTCTACATCCCAACATAAAGGTGATGAGACAC CCGGATCATGTGTCATCCACCGTCTATTTGTGGGCTCACCATGAGAAGCTTGTCATCATTGACCAATCGGTGGCC TTTGTGGGAGGGATTGACCTGGCCTATGGAAGGTGGGACGACAATGAGCACAGACTCACAGACGTGGGCAGTGTG AAGCGGGTCACTTCAGGACCGTCTCTGGGTTCCCTCCCACCTGCCGCAATGGAGTCTATGGAATCCTTAAGACTC AAAGATAAAAATGAGCCTGTTCAAAACCTACCCATCCAGAAGAGTATTGATGATGTGGATTCAAAACTGAAAGGA ATAGGAAAGCCAAGAAAGTTCTCCAAATTTAGTCTCTACAAGCAGCTCCACAGGCACCACCTGCACGACGCAGAT AGCATCAGCAGCATTGACAGCACCTCCAGTTATTTTAATCACTATAGAAGTCATCACAATTTAATCCATGGTTTA AAACCCCACTTCAAACTCTTTCACCCGTCCAGTGAGTCTGAGCAAGGACTCACTAGACCTCATGCTGATACCGGG TCCATCCGTAGTTTACAGACAGGTGTGGGAGAGCTGCATGGGGAAACCAGATTCTGGCATGGAAAGGACTACTGC AATTTCGTCTTCAAAGACTGGGTTCAACTTGATAAACCTTTTGCTGATTTCATTGACAGGTACTCCACGCCCCGG ATGCCCTGGCATGACATTGCCTCTGCAGTCCACGGGAAGGCGGCTCGTGATGTGGCACGTCACTTCATCCAGCGC TGGAACTTCACAAAAATTATGAAATCAAAATATCGGTCCCTTTCTTATCCTTTTCTGCTTCCAAAGTCTCAAACA ACAGCCCATGAGTTGAGATATCAAGTGCCTGGGTCTGTCCATGCTAACGTACAGTTGCTCCGCTCTGCTGCTGAT TGGTCTGCTGGTATAAAGTACCATGAAGAGTCCATCCACGCCGCTTACGTCCATGTGATAGAGAACAGCAGGCAC TATATCTATATCGAAAACCAGTTTTTCATAAGCTGTGCTGATGACAAAGTTGTGTTCAACAAGATAGGCGATGCC ATTGCCCAGAGGATCCTGAAAGCTCACAGGGAAAACCAGAAATACCGGGTATATGTCGTGATACCACTTCTGCCA GGGTTCGAAGGAGACATTTCAACCGGCGGAGGAAATGCTCTACAGGCAATCATGCACTTCAACTACAGAACCATG TGCAGAGGAGAAAATTCCATCCTTGGACAGTTAAAAGCAGAGCTTGGTAATCAGTGGATAAATTACATATCATTC TGTGGTCTTAGAACACATGCAGAGCTCGAAGGAAACCTAGTAACTGAGCTTATCTATGTCCACAGCAAGTTGTTA ATTGCTGATGATAACACTGTTATTATTGGCTCTGCCAACATAAATGACCGCAGCATGCTGGGAAAGCGTGACAGT GAAATGGCTGTCATTGTGCAAGATACAGAGACTGTTCCTTCAGTAATGGATGGAAAAGAGTACCAAGCTGGCCGG TTTGCCCGAGGACTTCGGCTACAGTGCTTTAGGTCTAAAATGACTCCAGGTGTCGAAGATCCCTGA
SEQ ID NO: 52
Polypeptide of SEQ ID NO: 25, homo sapiens, 57 AA
MSLKNEPRVNTSALQKIAADMSNI IENLDTRELHFEGEEVDYDVSPSDPKIQEGTKY
RNF144A. ENSG00000151692
SEQ ID NO: 32 = variant a = ENSG00000151692|ENST00000432850
Polynucleotide, homo sapiens, 795 bp
NGGTACCGGCCCACCTGGGACCTGGCCCTCGACCCGCTGGTGTCTTGCAAGCTCTGTCTTGGGGAGTACCCAGTG GAGCAGATGACAACCATAGCCCAGTGCCAATGCATCTTCTGTACTCTGTGCCTGAAACAGTATGTTGAGCTCTTG ATCAAAGAAGGATTAGAAACTGCAATTAGCTGCCCAGATGCTGCCTGCCCTAAACAGGGCCACCTACAGGAGAAC GAGATTGAGTGCATGGTTGCAGCTGAAATTATGCAAAGATATAAAAAGCTACAATTTGAAAGAGAGGTGCTGTTT GATCCCTGTCGGACTTGGTGCCCGGCGTCCACCTGCCAAGCTGTGTGTCAGCTCCAGGACGTGGGGCTGCAGACC CCCCAGCCAGTGCAGTGCAAAGCCTGCCGTATGGAATTCTGCTCCACCTGCAAAGCCAGCTGGCACCCTGGCCAG GGCTGCCCGGAGACCATGCCGATCACCTTCCTCCCCGGGGAGACCAGTGCTGCTTTCAAAATGGAAGAAGATGAC GCGCCCATCAAGCGCTGCCCCAAGTGCAAAGTCTACATCGAGCGAGACGAAGGCTGCGCGCAGATGATGTGCAAG AACTGCAAGCACGCCTTCTGCTGGTACTGCCTGGAGTCTCTGGACGATGATTTCCTTCTGATACACTACGATAAG GGACCCTGCCGGAACAAGCTGGGCCACTCCCGGGCATCTGTGATCTGGCATCGGACACAGGGTGATGCCATCTTA GAGAAATGCATTCTCCATAATACTGAATCTGAGTGTCATCTGTAA
SEQ ID NO: 33 = variant b = ENSG00000151692|ENST00000320892
Polynucleotide, homo sapiens, 879 bp
ATGACCACAACAAGGTACCGGCCCACCTGGGACCTGGCCCTCGACCCGCTGGTGTCTTGCAAGCTCTGTCTTGGG GAGTACCCAGTGGAGCAGATGACAACCATAGCCCAGTGCCAATGCATCTTCTGTACTCTGTGCCTGAAACAGTAT GTTGAGCTCTTGATCAAAGAAGGATTAGAAACTGCAATTAGCTGCCCAGATGCTGCCTGCCCTAAACAGGGCCAC CTACAGGAGAACGAGATTGAGTGCATGGTTGCAGCTGAAATTATGCAAAGATATAAAAAGCTACAATTTGAAAGA GAGGTGCTGTTTGATCCCTGTCGGACTTGGTGCCCGGCGTCCACCTGCCAAGCTGTGTGTCAGCTCCAGGACGTG GGGCTGCAGACCCCCCAGCCAGTGCAGTGCAAAGCCTGCCGTATGGAATTCTGCTCCACCTGCAAAGCCAGCTGG CACCCTGGCCAGGGCTGCCCGGAGACCATGCCGATCACCTTCCTCCCCGGGGAGACCAGTGCTGCTTTCAAAATG GAAGAAGATGACGCGCCCATCAAGCGCTGCCCCAAGTGCAAAGTCTACATCGAGCGAGACGAAGGCTGCGCGCAG ATGATGTGCAAGAACTGCAAGCACGCCTTCTGCTGGTACTGCCTGGAGTCTCTGGACGATGATTTCCTTCTGATA CACTACGATAAGGGACCCTGCCGGAACAAGCTGGGCCACTCCCGGGCATCTGTGATCTGGCATCGGACACAGGTT GTGGGCATTTTTGCAGGATTTGGGCTGCTGCTCTTGGTGGCCTCACCTTTCCTACTCCTGGCCACTCCCTTTGTA CTTTGCTGCAAGTGCAAGTGCAGTAAAGGTGACGACGACCCGTTACCCACCTAG
SEQ ID NO: 34 = variant c = ENSG00000151692|ENST00000416587
Polynucleotide, homo sapiens, 165 bp
ATGACCACAACAAGGTACCGGCCCACCTGGGACCTGGCCCTCGACCCGCTGGTGTCTTGCAAGCTCTGTCTTGGG GAGTACCCAGTGGAGCAGATGACAACCATAGCCCAGTGCCAATGCATCTTCTGTACTCTGTGCCTGAAACAGTAT GTTGAGCTCTTGATC
SEQ ID NO: 35 = variant d = ENSG00000151692|ENST00000433456
Polynucleotide, homo sapiens, 165 bp
ATGACCACAACAAGGTACCGGCCCACCTGGGACCTGGCCCTCGACCCGCTGGTGTCTTGCAAGCTCTGTCTTGGG GAGTACCCAGTGGAGCAGATGACAACCATAGCCCAGTGCCAATGCATCTTCTGTACTCTGTGCCTGAAACAGTAT GTTGAGCTCTTGATC
SEQ ID NO: 53
Polypeptide of SEQ ID NO: 32, homo sapiens, 264 AA
XYRPTWDLALDPLVSCKLCLGEYPVEQMTTIAQCQCIFCTLCLKQYVELLIKEGLETAISCPDAACPKQGHLQEN EIECMVAAEIMQRYKKLQFEREVLFDPCRTWCPASTCQAVCQLQDVGLQTPQPVQCKACRMEFCSTCKASWHPGQ GCPETMPITFLPGETSAAFKMEEDDAPIKRCPKCKVYIERDEGCAQMMCKNCKHAFCWYCLESLDDDFLLIHYDK GPCRNKLGHSRASVIWHRTQGDAILEKCILHNTESECHL
SLC44A4. ENSG00000204385
SEQ ID NO: 36 = variant a = ENSG00000204385|ENST00000229729
Polynucleotide, homo sapiens, 2133 bp
ATGGGGGGAAAGCAGCGGGACGAGGATGACGAGGCCTACGGGAAGCCAGTCAAATACGACCCCTCCTTTCGAGGC CCCATCAAGAACAGAAGCTGCACAGATGTCATCTGCTGCGTCCTCTTCCTGCTCTTCATTCTAGGTTACATCGTG GTGGGGATTGTGGCCTGGTTGTATGGAGACCCCCGGCAAGTCCTCTACCCCAGGAACTCTACTGGGGCCTACTGT GGCATGGGGGAGAACAAAGATAAGCCGTATCTCCTGTACTTCAACATCTTCAGCTGCATCCTGTCCAGCAACATC ATCTCAGTTGCTGAGAACGGCCTACAGTGCCCCACACCCCAGGTGTGTGTGTCCTCCTGCCCGGAGGACCCATGG ACTGTGGGAAAAAACGAGTTCTCACAGACTGTTGGGGAAGTCTTCTATACAAAAAACAGGAACTTTTGTCTGCCA GGGGTACCCTGGAATATGACGGTGATCACAAGCCTGCAACAGGAACTCTGCCCCAGTTTCCTCCTCCCCTCTGCT CCAGCTCTGGGGCGCTGCTTTCCATGGACCAACGTTACTCCACCGGCGCTCCCAGGGATCACCAATGACACCACC ATACAGCAGGGGATCAGCGGTCTTATTGACAGCCTCAATGCCCGAGACATCAGTGTTAAGATCTTTGAAGATTTT GCCCAGTCCTGGTATTGGATTCTTGTTGCCCTGGGGGTGGCTCTGGTCTTGAGCCTACTGTTTATCTTGCTTCTG CGCCTGGTGGCTGGGCCCCTGGTGCTGGTGCTGATCCTGGGAGTGCTGGGCGTGCTGGCATACGGCATCTACTAC TGCTGGGAGGAGTACCGAGTGCTGCGGGACAAGGGCGCCTCCATCTCCCAGCTGGGTTTCACCACCAACCTCAGT GCCTACCAGAGCGTGCAGGAGACCTGGCTGGCCGCCCTGATCGTGTTGGCGGTGCTTGAAGCCATCCTGCTGCTG ATGCTCATCTTCCTGCGGCAGCGGATTCGTATTGCCATCGCCCTCCTGAAGGAGGCCAGCAAGGCTGTGGGACAG ATGATGTCTACCATGTTCTACCCACTGGTCACCTTTGTCCTCCTCCTCATCTGCATTGCCTACTGGGCCATGACT GCTCTGTACCTGGCTACATCGGGGCAACCCCAGTATGTGCTCTGGGCATCCAACATCAGCTCCCCCGGCTGTGAG AAAGTGCCAATAAATACATCATGCAACCCCACGGCCCACCTTGTGAACTCCTCGTGCCCAGGGCTGATGTGCGTC TTCCAGGGCTACTCATCCAAAGGCCTAATCCAACGTTCTGTCTTCAATCTGCAAATCTATGGGGTCCTGGGGCTC TTCTGGACCCTTAACTGGGTACTGGCCCTGGGCCAATGCGTCCTCGCTGGAGCCTTTGCCTCCTTCTACTGGGCC TTCCACAAGCCCCAGGACATCCCTACCTTCCCCTTAATCTCTGCCTTCATCCGCACACTCCGTTACCACACTGGG TCATTGGCATTTGGAGCCCTCATCCTGACCCTTGTGCAGATAGCCCGGGTCATCTTGGAGTATATTGACCACAAG CTCAGAGGAGTGCAGAACCCTGTAGCCCGCTGCATCATGTGCTGTTTCAAGTGCTGCCTCTGGTGTCTGGAAAAA TTTATCAAGTTCCTAAACCGCAATGCATACATCATGATCGCCATCTACGGGAAGAATTTCTGTGTCTCAGCCAAA AATGCGTTCATGCTACTCATGCGAAACATTGTCAGGGTGGTCGTCCTGGACAAAGTCACAGACCTGCTGCTGTTC TTTGGGAAGCTGCTGGTGGTCGGAGGCGTGGGGGTCCTGTCCTTCTTTTTTTTCTCCGGTCGCATCCCGGGGCTG GGTAAAGACTTTAAGAGCCCCCACCTCAACTATTACTGGCTGCCCATCATGACCTCCATCCTGGGGGCCTATGTC ATCGCCAGCGGCTTCTTCAGCGTTTTCGGCATGTGTGTGGACACGCTCTTCCTCTGCTTCCTGGAAGACCTGGAG CGGAACAACGGCTCCCTGGACCGGCCCTACTACATGTCCAAGAGCCTTCTAAAGATTCTGGGCAAGAAGAACGAG GCGCCCCCGGACAACAAGAAGAGGAAGAAGTGA SEQ ID NO: 37 = variant b = ENSG00000204385|ENST00000414427
Polynucleotide, homo sapiens, 1234 bp
NAGCGGGACGAGGATGACGAGGCCTACGGGAAGCCAGTCAAATACGACCCCTCCTTTCGAGGCCCCATCAAGAAC AGAAGCTGCACAGATGTCATCTGCTGCGTCCTCTTCCTGCTCTTCATTCTAGGTTACATCGTGGTGGGGATTGTG GCCTGGTTGTATGGAGACCCCCGGCAAGTCCTCTACCCCAGGAACTCTACTGGGGCCTACTGTGGCATGGGGGAG AACAAAGATAAGCCGTATCTCCTGTACTTCAACATCTTCAGCTGCATCCTGTCCAGCAACATCATCTCAGTTGCT GAGAACGGCCTACAGTGCCCCACACCCCAGGTGTGTGTGTCCTCCTGCCCGGAGGACCCATGGACTGTGGGAAAA AACGAGTTCTCACAGACTGTTGGGGAAGTCTTCTATACAAAAAACAGGAACTTTTGTCTGCCAGGGGTACCCTGG AATATGACGGTGATCACAAGCCTGCAACAGGAACTCTGCCCCAGTTTCCTCCTCCCCTCTGCTCCAGCTCTGGGG CGCTGCTTTCCATGGACCAACGTTACTCCACCGGCGCTCCCAGGGATCACCAATGACACCACCATACAGCAGGGG ATCAGCGGTCTTATTGACAGCCTCAATGCCCGAGACATCAGTGTTAAGATCTTTGAAGATTTTGCCCAGTCCTGG TATTGGATTCTTGTGGCTGTGGGACAGATGATGTCTACCATGTTCTACCCACTGGTCACCTTTGTCCTCCTCCTC ATCTGCATTGCCTACTGGGCCATGACTGCTCTGTACCTGGCTACATCGGGGCAACCCCAGTATGTGCTCTGGGCA TCCAACATCAGCTCCCCCGGCTGTGAGAAAGTGCCAATAAATACATCATGCAACCCCACGGCCCACCTTGTGAAC TCCTCGTGCCCAGGGCTGATGTGCGTCTTCCAGGGCTACTCATCCAAAGGCCTAATCCAACGTTCTGTCTTCAAT CTGCAAATCTATGGGGTCCTGGGGCTCTTCTGGACCCTTAACTGGGTACTGGCCCTGGGCCAATGCGTCCTCGCT GGAGCCTTTGCCTCCTTCTACTGGGCCTTCCACAAGCCCCAGGACATCCCTACCTTCCCCTTAATCTCTGCCTTC ATCCGCACACTCCGTTACCACACTGGGTCATTGGCATTTGGAGCCCTCATCCTGACCCTTGTGCAGATAGCCCGG GTCATCTTGGAGTATATTGACCACAAGCTCAGAG
SEQ ID NO: 38 = variant c = ENSG00000204385|ENST00000375562
Polynucleotide, homo sapiens, 2007 bp
ATGGGGGGAAAGCAGCGGGACGAGGATGACGAGGCCTACGGGAAGCCAGTCAAATACGACCCCTCCTTTCGAGGC CCCATCAAGAACAGAAGCTGCACAGATGTCATCTGCTGCGTCCTCTTCCTGCTCTTCATTCTAGGTTACATCGTG GTGGGGATTGTGGCCTGGTTGTATGGAGACCCCCGGCAAGTCCTCTACCCCAGGAACTCTACTGGGGCCTACTGT GGCATGGGGGAGAACAAAGATAAGCCGTATCTCCTGTACTTCAACATCTTCAGCTGCATCCTGTCCAGCAACATC ATCTCAGTTGCTGAGAACGGCCTACAGTGCCCCACACCCCAGACGGTGATCACAAGCCTGCAACAGGAACTCTGC CCCAGTTTCCTCCTCCCCTCTGCTCCAGCTCTGGGGCGCTGCTTTCCATGGACCAACGTTACTCCACCGGCGCTC CCAGGGATCACCAATGACACCACCATACAGCAGGGGATCAGCGGTCTTATTGACAGCCTCAATGCCCGAGACATC AGTGTTAAGATCTTTGAAGATTTTGCCCAGTCCTGGTATTGGATTCTTGTTGCCCTGGGGGTGGCTCTGGTCTTG AGCCTACTGTTTATCTTGCTTCTGCGCCTGGTGGCTGGGCCCCTGGTGCTGGTGCTGATCCTGGGAGTGCTGGGC GTGCTGGCATACGGCATCTACTACTGCTGGGAGGAGTACCGAGTGCTGCGGGACAAGGGCGCCTCCATCTCCCAG CTGGGTTTCACCACCAACCTCAGTGCCTACCAGAGCGTGCAGGAGACCTGGCTGGCCGCCCTGATCGTGTTGGCG GTGCTTGAAGCCATCCTGCTGCTGATGCTCATCTTCCTGCGGCAGCGGATTCGTATTGCCATCGCCCTCCTGAAG GAGGCCAGCAAGGCTGTGGGACAGATGATGTCTACCATGTTCTACCCACTGGTCACCTTTGTCCTCCTCCTCATC TGCATTGCCTACTGGGCCATGACTGCTCTGTACCTGGCTACATCGGGGCAACCCCAGTATGTGCTCTGGGCATCC AACATCAGCTCCCCCGGCTGTGAGAAAGTGCCAATAAATACATCATGCAACCCCACGGCCCACCTTGTGAACTCC TCGTGCCCAGGGCTGATGTGCGTCTTCCAGGGCTACTCATCCAAAGGCCTAATCCAACGTTCTGTCTTCAATCTG CAAATCTATGGGGTCCTGGGGCTCTTCTGGACCCTTAACTGGGTACTGGCCCTGGGCCAATGCGTCCTCGCTGGA GCCTTTGCCTCCTTCTACTGGGCCTTCCACAAGCCCCAGGACATCCCTACCTTCCCCTTAATCTCTGCCTTCATC CGCACACTCCGTTACCACACTGGGTCATTGGCATTTGGAGCCCTCATCCTGACCCTTGTGCAGATAGCCCGGGTC ATCTTGGAGTATATTGACCACAAGCTCAGAGGAGTGCAGAACCCTGTAGCCCGCTGCATCATGTGCTGTTTCAAG TGCTGCCTCTGGTGTCTGGAAAAATTTATCAAGTTCCTAAACCGCAATGCATACATCATGATCGCCATCTACGGG AAGAATTTCTGTGTCTCAGCCAAAAATGCGTTCATGCTACTCATGCGAAACATTGTCAGGGTGGTCGTCCTGGAC AAAGTCACAGACCTGCTGCTGTTCTTTGGGAAGCTGCTGGTGGTCGGAGGCGTGGGGGTCCTGTCCTTCTTTTTT TTCTCCGGTCGCATCCCGGGGCTGGGTAAAGACTTTAAGAGCCCCCACCTCAACTATTACTGGCTGCCCATCATG ACCTCCATCCTGGGGGCCTATGTCATCGCCAGCGGCTTCTTCAGCGTTTTCGGCATGTGTGTGGACACGCTCTTC CTCTGCTTCCTGGAAGACCTGGAGCGGAACAACGGCTCCCTGGACCGGCCCTACTACATGTCCAAGAGCCTTCTA AAGATTCTGGGCAAGAAGAACGAGGCGCCCCCGGACAACAAGAAGAGGAAGAAGTGA
SEQ ID NO: 39 = variant d = ENSG00000204385|ENST00000544672
Polynucleotide, homo sapiens, 1905 bp
ATGGGGGAGAACAAAGATAAGCCGTATCTCCTGTACTTCAACATCTTCAGCTGCATCCTGTCCAGCAACATCATC TCAGTTGCTGAGAACGGCCTACAGTGCCCCACACCCCAGGTGTGTGTGTCCTCCTGCCCGGAGGACCCATGGACT GTGGGAAAAAACGAGTTCTCACAGACTGTTGGGGAAGTCTTCTATACAAAAAACAGGAACTTTTGTCTGCCAGGG GTACCCTGGAATATGACGGTGATCACAAGCCTGCAACAGGAACTCTGCCCCAGTTTCCTCCTCCCCTCTGCTCCA GCTCTGGGGCGCTGCTTTCCATGGACCAACGTTACTCCACCGGCGCTCCCAGGGATCACCAATGACACCACCATA CAGCAGGGGATCAGCGGTCTTATTGACAGCCTCAATGCCCGAGACATCAGTGTTAAGATCTTTGAAGATTTTGCC CAGTCCTGGTATTGGATTCTTGTTGCCCTGGGGGTGGCTCTGGTCTTGAGCCTACTGTTTATCTTGCTTCTGCGC CTGGTGGCTGGGCCCCTGGTGCTGGTGCTGATCCTGGGAGTGCTGGGCGTGCTGGCATACGGCATCTACTACTGC TGGGAGGAGTACCGAGTGCTGCGGGACAAGGGCGCCTCCATCTCCCAGCTGGGTTTCACCACCAACCTCAGTGCC TACCAGAGCGTGCAGGAGACCTGGCTGGCCGCCCTGATCGTGTTGGCGGTGCTTGAAGCCATCCTGCTGCTGATG CTCATCTTCCTGCGGCAGCGGATTCGTATTGCCATCGCCCTCCTGAAGGAGGCCAGCAAGGCTGTGGGACAGATG ATGTCTACCATGTTCTACCCACTGGTCACCTTTGTCCTCCTCCTCATCTGCATTGCCTACTGGGCCATGACTGCT CTGTACCTGGCTACATCGGGGCAACCCCAGTATGTGCTCTGGGCATCCAACATCAGCTCCCCCGGCTGTGAGAAA GTGCCAATAAATACATCATGCAACCCCACGGCCCACCTTGTGAACTCCTCGTGCCCAGGGCTGATGTGCGTCTTC CAGGGCTACTCATCCAAAGGCCTAATCCAACGTTCTGTCTTCAATCTGCAAATCTATGGGGTCCTGGGGCTCTTC TGGACCCTTAACTGGGTACTGGCCCTGGGCCAATGCGTCCTCGCTGGAGCCTTTGCCTCCTTCTACTGGGCCTTC CACAAGCCCCAGGACATCCCTACCTTCCCCTTAATCTCTGCCTTCATCCGCACACTCCGTTACCACACTGGGTCA TTGGCATTTGGAGCCCTCATCCTGACCCTTGTGCAGATAGCCCGGGTCATCTTGGAGTATATTGACCACAAGCTC AGAGGAGTGCAGAACCCTGTAGCCCGCTGCATCATGTGCTGTTTCAAGTGCTGCCTCTGGTGTCTGGAAAAATTT ATCAAGTTCCTAAACCGCAATGCATACATCATGATCGCCATCTACGGGAAGAATTTCTGTGTCTCAGCCAAAAAT GCGTTCATGCTACTCATGCGAAACATTGTCAGGGTGGTCGTCCTGGACAAAGTCACAGACCTGCTGCTGTTCTTT GGGAAGCTGCTGGTGGTCGGAGGCGTGGGGGTCCTGTCCTTCTTTTTTTTCTCCGGTCGCATCCCGGGGCTGGGT AAAGACTTTAAGAGCCCCCACCTCAACTATTACTGGCTGCCCATCATGACCTCCATCCTGGGGGCCTATGTCATC GCCAGCGGCTTCTTCAGCGTTTTCGGCATGTGTGTGGACACGCTCTTCCTCTGCTTCCTGGAAGACCTGGAGCGG AACAACGGCTCCCTGGACCGGCCCTACTACATGTCCAAGAGCCTTCTAAAGATTCTGGGCAAGAAGAACGAGGCG CCCCCGGACAACAAGAAGAGGAAGAAGTGA
SEQ ID NO: 54
Polypeptide of SEQ ID NO: 36, homo sapiens, 710 bp
MGGKQRDEDDEAYGKPVKYDPSFRGPIKNRSCTDVICCVLFLLFILGYIWGIVAWLYGDPRQVLYPRNSTGAYC GMGENKDKPYLLYFNIFSCILSSNI ISVAENGLQCPTPQVCVSSCPEDPWTVGKNEFSQTVGEVFYTKNRNFCLP GVPWNMTVITSLQQELCPSFLLPSAPALGRCFPWTNVTPPALPGITNDTTIQQGISGLIDSLNARDISVKIFEDF AQSWYWILVALGVALVLSLLFILLLRLVAGPLVLVLILGVLGVLAYGIYYCWEEYRVLRDKGASISQLGFTTNLS AYQSVQETWLAALIVLAVLEAILLLMLIFLRQRIRIAIALLKEASKAVGQMMSTMFYPLVTFVLLLICIAYWAMT ALYLATSGQPQYVLWASNISSPGCEKVPINTSCNPTAHLVNSSCPGLMCVFQGYSSKGLIQRSVFNLQIYGVLGL FWTLNWVLALGQCVLAGAFASFYWAFHKPQDIPTFPLISAFIRTLRYHTGSLAFGALILTLVQIARVILEYIDHK LRGVQNPVARCIMCCFKCCLWCLEKFIKFLNRNAYIMIAIYGKNFCVSAKNAFMLLMRNIVRVWLDKVTDLLLF FGKLLVVGGVGVLSFFFFSGRI PGLGKDFKSPHLNYYWLPIMTSILGAYVIASGFFSVFGMCVDTLFLCFLEDLE RNNGSLDRPYYMSKSLLKILGKKNEAPPDNKKRKK
SLC2A13. ENSG00000151229
SEQ ID NO: 40 = variant a = ENSG00000151229|ENST00000280871
Polynucleotide, homo sapiens, 1947 bp
ATGTCCCGCAAGGCAAGCGAGAATGTGGAGTACACGCTGCGGAGCCTGAGCAGCCTGATGGGCGAGCGGCGCAGG AAGCAGCCGGAGCCGGACGCGGCGAGCGCGGCCGGGGAGTGCAGCCTCCTGGCTGCCGCCGAATCGAGCACCAGC CTGCAGAGCGCGGGCGCGGGCGGCGGCGGCGTCGGGGACCTGGAGCGCGCGGCGCGGCGGCAGTTCCAGCAGGAC GAGACCCCCGCCTTCGTGTACGTGGTGGCCGTCTTCTCCGCGCTGGGCGGCTTCCTGTTTGGCTATGACACCGGG GTGGTGTCAGGGGCCATGCTGCTGCTCAAGCGGCAGCTCAGTCTGGACGCGCTGTGGCAGGAGCTGCTGGTGTCC AGCACGGTGGGGGCGGCTGCCGTCTCGGCGCTGGCCGGAGGCGCCCTCAACGGCGTCTTCGGCCGCCGCGCTGCC ATCCTCCTGGCCAGTGCCCTCTTCACCGCCGGCTCCGCGGTGCTGGCTGCGGCCAACAACAAGGAGACACTGCTC GCCGGCCGCCTGGTCGTGGGACTCGGCATCGGCATTGCTTCTATGACAGTGCCAGTGTACATTGCGGAGGTCTCA CCACCCAATTTAAGAGGCCGATTAGTCACCATTAATACCCTCTTCATCACAGGAGGGCAGTTCTTTGCAAGTGTT GTTGATGGAGCCTTCAGTTATCTCCAGAAGGATGGATGGAGGTACATGTTGGGACTTGCAGCAGTTCCGGCGGTT ATACAGTTTTTTGGCTTTCTCTTTTTGCCTGAAAGCCCTCGATGGCTTATTCAGAAAGGACAGACTCAGAAGGCC CGTAGAATTTTATCTCAGATGCGTGGTAACCAGACCATTGATGAGGAATATGATAGCATCAAAAACAACATTGAA GAGGAGGAAAAAGAGGTTGGCTCAGCTGGACCTGTGATCTGCAGAATGCTGAGTTATCCCCCAACTCGCCGAGCT TTAATTGTGGGTTGTGGCCTACAAATGTTCCAGCAGCTCTCAGGCATTAACACCATCATGTACTACAGTGCAACC ATTCTGCAGATGTCTGGTGTTGAAGATGATAGACTTGCAATATGGCTGGCTTCAGTTACAGCCTTCACAAATTTC ATTTTCACACTTGTGGGAGTCTGGCTTGTTGAGAAGGTGGGCCGCAGAAAGCTTACCTTTGGTAGTTTAGCAGGT ACCACCGTAGCACTCATTATTCTTGCCTTGGGATTTGTGCTATCAGCCCAAGTTTCCCCACGCATCACTTTTAAG CCAATAGCTCCGTCAGGTCAGAACGCCACTTGCACAAGATACAGTTACTGTAATGAATGTATGTTGGATCCAGAC TGCGGTTTCTGCTACAAGATGAACAAATCAACTGTCATTGACTCCTCCTGTGTTCCAGTTAATAAAGCATCTACA AATGAGGCAGCCTGGGGCAGGTGTGAAAATGAAACCAAGTTCAAAACAGAAGATATATTTTGGGCTTACAATTTC TGCCCTACTCCATACTCCTGGACTGCACTTCTGGGCCTTATTTTATATCTTGTCTTCTTTGCACCTGGAATGGGA CCAATGCCTTGGACTGTGAATTCTGAAATATATCCCCTTTGGGCAAGAAGTACAGGAAATGCATGTTCATCTGGA ATAAACTGGATTTTCAATGTCCTGGTTTCACTAACATTTTTACACACAGCAGAGTATCTTACATACTATGGAGCT TTCTTCCTCTATGCTGGATTTGCTGCTGTGGGACTCCTTTTCATCTATGGCTGTCTTCCTGAGACCAAAGGCAAA AAATTAGAGGAAATTGAATCACTCTTTGACAACAGGCTATGTACATGTGGCACTTCAGATTCTGATGAAGGGAGA TATATTGAATATATTCGGGTAAAGGGAAGTAACTATCATCTTTCTGACAATGATGCTTCTGATGTGGAATAA
SEQ ID NO: 41 = variant b = ENSG00000151229|ENST00000380858
Polynucleotide, homo sapiens, 1074 bp
ATGTCCCGCAAGGCAAGCGAGAATGTGGAGTACACGCTGCGGAGCCTGAGCAGCCTGATGGGCGAGCGGCGCAGG AAGCAGCCGGAGCCGGACGCGGCGAGCGCGGCCGGGGAGTGCAGCCTCCTGGCTGCCGCCGAATCGAGCACCAGC CTGCAGAGCGCGGGCGCGGGCGGCGGCGGCGTCGGGGACCTGGAGCGCGCGGCGCGGCGGCAGTTCCAGCAGGAC GAGACCCCCGCCTTCGTGTACGTGGTGGCCGTCTTCTCCGCGCTGGGCGGCTTCCTGTTTGGCTATGACACCGGG GTGGTGTCAGGGGCCATGCTGCTGCTCAAGCGGCAGCTCAGTCTGGACGCGCTGTGGCAGGAGCTGCTGGTGTCC AGCACGGTGGGGGCGGCTGCCGTCTCGGCGCTGGCCGGAGGCGCCCTCAACGGCGTCTTCGGCCGCCGCGCTGCC ATCCTCCTGGCCAGTGCCCTCTTCACCGCCGGCTCCGCGGTGCTGGCTGCGGCCAACAACAAGGAGACACTGCTC GCCGGCCGCCTGGTCGTGGGACTCGGCATCGGCATTGCTTCTATGACAGTGCCAGTGTACATTGCGGAGGTCTCA CCACCCAATTTAAGAGGCCGATTAGTCACCATTAATACCCTCTTCATCACAGGAGGGCAGTTCTTTGCAAGTGTT GTTGATGGAGCCTTCAGTTATCTCCAGAAGGATGGATGGAGGTACATGTTGGGACTTGCAGCAGTTCCGGCGGTT ATACAGTTTTTTGGCTTTCTCTTTTTGCCTGAAAGCCCTCGATGGCTTATTCAGAAAGGACAGACTCAGAAGGCC CGTAGAATTTTATCTCAGATGCGTGGTAACCAGACCATTGATGAGGAATATGATAGCATCAAAAACAACATTGAA GAGGAGGAAAAAGAGGTTGGCTCAGCTGGACCTGTGATCTGCAGAATGCTGAGTTATCCCCCAACTCGCCGAGCT TTAATTGTGGGTTGTGGCCTACAAATGTTCCAGCAGCTCTCAGGCATTAACACCATCATGTATGTATTTCTTTCT GGCTTTTTACTGAAAAGATTGTGA
SEQ ID NO: 55
Polypeptide of SEQ ID NO: 40, homo sapiens, 648 AA
MSRKASENVEYTLRSLSSLMGERRRKQPEPDAASAAGECSLLAAAESSTSLQSAGAGGGGVGDLERAARRQFQQD ETPAFVYVVAVFSALGGFLFGYDTGVVSGAMLLLKRQLSLDALWQELLVSSTVGAAAVSALAGGALNGVFGRRAA ILLASALFTAGSAVLAAANNKETLLAGRLVVGLGIGIASMTVPVYIAEVSPPNLRGRLVTINTLFITGGQFFASV VDGAFSYLQKDGWRYMLGLAAVPAVIQFFGFLFLPESPRWLIQKGQTQKARRILSQMRGNQTIDEEYDSIKNNIE EEEKEVGSAGPVICRMLSYPPTRRALIVGCGLQMFQQLSGINTIMYYSATILQMSGVEDDRLAIWLASVTAFTNF IFTLVGVWLVEKVGRRKLTFGSLAGTTVALI ILALGFVLSAQVSPRITFKPIAPSGQNATCTRYSYCNECMLDPD CGFCYKMNKSTVIDSSCVPVNKASTNEAAWGRCENETKFKTEDIFWAYNFCPTPYSWTALLGLILYLVFFAPGMG PMPWTVNSEIYPLWARSTGNACSSGINWIFNVLVSLTFLHTAEYLTYYGAFFLYAGFAAVGLLFIYGCLPETKGK KLEEIESLFDNRLCTCGTSDSDEGRYIEYIRVKGSNYHLSDNDASDVE
AXIN2. ENSG00000168646
SEQ ID NO: 42 = variant a = ENSG00000168646|ENST00000544103
Polynucleotide, homo sapiens, 452 bp
ATGAGTAGCGCTATGTTGGTGACTTGCCTCCCGGACCCCAGCAGCAGCTTCCGTGAGGATGCCCCGCGGCCCCCA GTGCCAGGGGAAGAAGGGGAGACCCCACCGTGTCAGCCAGGGGTGGGCAAGGGCCAGGTCACCAAACCCATGCCT GTCTCTTCCAACACCAGGCGGAACGAAGATGGGTTGGGGGAGCCGGAGGGGCGGGCATCTCCGGATTCCCCTCTG ACCCGGTGGACCAAGTCCTTACACTCCTTATTGGGCGATCAAGACGGTGCTTACCTGTTCCGAACTTTCCTGGAG AGGGAGAAATGCGTGGATACCTTAGACTTCTGGTTTGCCTGCAATGGATTCAGGCAGATGAACCTGAAGGATACC AAAACTTTACGAGTAGCCAAAGCGATCTACAAAAGGTACATTGAGAACAACAGCATTGTCTCCAAGCAGCTGAAG CC
SEQ ID NO: 43 = variant b = ENSG00000168646|ENST00000577278
Polynucleotide, homo sapiens, 815 bp
ATGAGTAGCGCTATGTTGGTGACTTGCCTCCCGGACCCCAGCAGCAGCTTCCGTGAGGATGCCCCGCGGCCCCCA GTGCCAGGGGAAGAAGGGGAGACCCCACCGTGTCAGCCAGGGGTGGGCAAGGGCCAGGTCACCAAACCCATGCCT GTCTCTTCCAACACCAGGCGGAACGAAGATGGGTTGGGGGAGCCGGAGGGGCGGGCATCTCCGGATTCCCCTCTG ACCCGGTGGACCAAGTCCTTACACTCCTTATTGGGCGATCAAGACGGTGCTTACCTGTTCCGAACTTTCCTGGAG AGGGAGAAATGCGTGGATACCTTAGACTTCTGGTTTGCCTGCAATGGATTCAGGCAGATGAACCTGAAGGATACC AAAACTTTACGAGTAGCCAAAGCGATCTACAAAAGGTACATTGAGAACAACAGCATTGTCTCCAAGCAGCTGAAG CCTGCCACCAAGACCTACATAAGAGATGGCATCAAGAAGCAGCAGATTGATTCCATCATGTTTGACCAGGCGCAG ACCGAGATCCAGTCGGTGATGGAGGAAAATGCCTACCAGATGTTTTTGACTTCTGATATATACCTCGAATATGTG AGGAGTGGGGGAGAAAACACAGCTTACATGAGTAATGGGGGACTCGGGAGCCTAAAGGTCGTGTGTGGCTATCTC CCCACCTTGAATGAAGAAGAGGAGTGGACTTGTGCCGACTTCAAGTGCAAACTTTCGCCAACCGTGGTTGGCTTG TCCAGCAAAACTCTGAGGGCCACGGCGAGTGTGAGGTCCACGGAAACTGTTGACAGTGGATACAG SEQ ID NO: 44 = variant c = ENSG00000168646|ENST00000307078
Polynucleotide, homo sapiens, 2532 bp
ATGAGTAGCGCTATGTTGGTGACTTGCCTCCCGGACCCCAGCAGCAGCTTCCGTGAGGATGCCCCGCGGCCCCCA GTGCCAGGGGAAGAAGGGGAGACCCCACCGTGTCAGCCAGGGGTGGGCAAGGGCCAGGTCACCAAACCCATGCCT GTCTCTTCCAACACCAGGCGGAACGAAGATGGGTTGGGGGAGCCGGAGGGGCGGGCATCTCCGGATTCCCCTCTG ACCCGGTGGACCAAGTCCTTACACTCCTTATTGGGCGATCAAGACGGTGCTTACCTGTTCCGAACTTTCCTGGAG AGGGAGAAATGCGTGGATACCTTAGACTTCTGGTTTGCCTGCAATGGATTCAGGCAGATGAACCTGAAGGATACC AAAACTTTACGAGTAGCCAAAGCGATCTACAAAAGGTACATTGAGAACAACAGCATTGTCTCCAAGCAGCTGAAG CCTGCCACCAAGACCTACATAAGAGATGGCATCAAGAAGCAGCAGATTGATTCCATCATGTTTGACCAGGCGCAG ACCGAGATCCAGTCGGTGATGGAGGAAAATGCCTACCAGATGTTTTTGACTTCTGATATATACCTCGAATATGTG AGGAGTGGGGGAGAAAACACAGCTTACATGAGTAATGGGGGACTCGGGAGCCTAAAGGTCGTGTGTGGCTATCTC CCCACCTTGAATGAAGAAGAGGAGTGGACTTGTGCCGACTTCAAGTGCAAACTTTCGCCAACCGTGGTTGGCTTG TCCAGCAAAACTCTGAGGGCCACGGCGAGTGTGAGGTCCACGGAAACTGTTGACAGTGGATACAGGTCCTTCAAG AGGAGCGATCCTGTTAATCCTTATCACATAGGTTCTGGCTATGTCTTTGCACCAGCCACCAGCGCCAACGACAGT GAGATATCCAGTGATGCGCTGACGGATGATTCCATGTCCATGACGGACAGCAGTGTAGATGGAATTCCTCCTTAT CGTGTGGGCAGTAAGAAACAGCTCCAGAGAGAAATGCATCGCAGTGTGAAGGCCAATGGCCAAGTGTCTCTACCT CATTTCCCGAGAACCCACCGCCTGCCCAAGGAGATGACCCCCGTGGAACCCGCCACCTTTGCAGCTGAGCTGATC TCGAGGCTGGAAAAGCTGAAGCTGGAGTTGGAGAGCCGCCACAGCCTGGAGGAGCGCCTGCAGCAGATCCGAGAG GATGAAGAGAGAGAGGGCTCCGAGCTCACACTCAATTCGCGGGAGGGGGCGCCCACGCAGCACCCCCTCTCCCTA CTGCCCTCCGGCAGCTACGAGGAAGACCCGCAGACGATACTGGACGATCACCTGTCCAGGGTCCTCAAGACCCCT GGCTGCCAGTCTCCAGGCGTAGGCCGCTATAGCCCCCGCTCCCGCTCCCCGGACCACCACCACCACCACCATTCG CAGTACCACTCCCTGCTCCCGCCCGGTGGCAAGCTGCCTCCCGCGGCCGCCTCGCCGGGCGCCTGCCCCCTCCTC GGGGGCAAAGGCTTTGTGACCAAGCAGACGACGAAGCATGTCCACCACCACTACATCCACCACCATGCCGTCCCC AAGACCAAGGAGGAGATCGAGGCGGAGGCCACGCAGCGGGTGCACTGCTTCTGCCCTGGGGGCAGCGAGTATTAC TGCTACTCGAAATGCAAAAGCCACTCCAAGGCTCCGGAAACCATGCCCAGCGAGCAGTTTGGCGGCAGCAGAGGC AGTACCTTGCCCAAACGCAATGGGAAAGGCACGGAGCCGGGCCTGGCCCTGCCCGCCAGGGAAGGAGGGGCCCCC GGCGGAGCTGGGGCCCTGCAGCTTCCCCGGGAGGAAGGAGACAGGTCGCAGGATGTCTGGCAGTGGATGCTGGAG AGTGAGCGGCAGAGCAAGCCCAAGCCCCATAGTGCCCAAAGCACAAAAAAGGCCTACCCCTTGGAGTCTGCCCGC TCGTCTCCAGGCGAACGAGCCAGCCGGCACCATCTGTGGGGGGGCAACAGCGGGCACCCCCGCACCACCCCCCGT GCCCACCTGTTCACCCAGGACCCTGCGATGCCTCCCCTGACCCCACCCAACACGCTGGCTCAGCTGGAGGAGGCC TGTCGCAGGCTAGCTGAGGTGTCGAAGCCCCCAAAGCAGCGGTGCTGTGTGGCCAGTCAGCAGAGGGACAGGAAT CATTCGGCCACTGTTCAGACGGGAGCCACACCCTTCTCCAATCCAAGCCTGGCTCCAGAAGATCACAAAGAGCCA AAGAAACTGGCAGGTGTCCACGCGCTCCAGGCCAGTGAGTTGGTTGTCACTTACTTTTTCTGTGGGGAAGAAATT CCATACCGGAGGATGCTGAAGGCTCAGAGCTTGACCCTGGGCCACTTTAAAGAGCAGCTCAGCAAAAAGGGAAAT TATAGGTATTACTTCAAAAAAGCAAGCGATGAGTTTGCCTGTGGAGCGGTGTTTGAGGAGATCTGGGAGGATGAG ACGGTGCTCCCGATGTATGAAGGCCGGATTCTGGGCAAAGTGGAGCGGATCGATTGA
SEQ ID NO: 45 = variant d = ENSG00000168646|ENST00000375702
Polynucleotide, homo sapiens, 2337 bp
ATGAGTAGCGCTATGTTGGTGACTTGCCTCCCGGACCCCAGCAGCAGCTTCCGTGAGGATGCCCCGCGGCCCCCA GTGCCAGGGGAAGAAGGGGAGACCCCACCGTGTCAGCCAGGGGTGGGCAAGGGCCAGGTCACCAAACCCATGCCT GTCTCTTCCAACACCAGGCGGAACGAAGATGGGTTGGGGGAGCCGGAGGGGCGGGCATCTCCGGATTCCCCTCTG ACCCGGTGGACCAAGTCCTTACACTCCTTATTGGGCGATCAAGACGGTGCTTACCTGTTCCGAACTTTCCTGGAG AGGGAGAAATGCGTGGATACCTTAGACTTCTGGTTTGCCTGCAATGGATTCAGGCAGATGAACCTGAAGGATACC AAAACTTTACGAGTAGCCAAAGCGATCTACAAAAGGTACATTGAGAACAACAGCATTGTCTCCAAGCAGCTGAAG CCTGCCACCAAGACCTACATAAGAGATGGCATCAAGAAGCAGCAGATTGATTCCATCATGTTTGACCAGGCGCAG ACCGAGATCCAGTCGGTGATGGAGGAAAATGCCTACCAGATGTTTTTGACTTCTGATATATACCTCGAATATGTG AGGAGTGGGGGAGAAAACACAGCTTACATGAGTAATGGGGGACTCGGGAGCCTAAAGGTCGTGTGTGGCTATCTC CCCACCTTGAATGAAGAAGAGGAGTGGACTTGTGCCGACTTCAAGTGCAAACTTTCGCCAACCGTGGTTGGCTTG TCCAGCAAAACTCTGAGGGCCACGGCGAGTGTGAGGTCCACGGAAACTGTTGACAGTGGATACAGGTCCTTCAAG AGGAGCGATCCTGTTAATCCTTATCACATAGGTTCTGGCTATGTCTTTGCACCAGCCACCAGCGCCAACGACAGT GAGATATCCAGTGATGCGCTGACGGATGATTCCATGTCCATGACGGACAGCAGTGTAGATGGAATTCCTCCTTAT CGTGTGGGCAGTAAGAAACAGCTCCAGAGAGAAATGCATCGCAGTGTGAAGGCCAATGGCCAAGTGTCTCTACCT CATTTCCCGAGAACCCACCGCCTGCCCAAGGAGATGACCCCCGTGGAACCCGCCACCTTTGCAGCTGAGCTGATC TCGAGGCTGGAAAAGCTGAAGCTGGAGTTGGAGAGCCGCCACAGCCTGGAGGAGCGCCTGCAGCAGATCCGAGAG GATGAAGAGAGAGAGGGCTCCGAGCTCACACTCAATTCGCGGGAGGGGGCGCCCACGCAGCACCCCCTCTCCCTA CTGCCCTCCGGCAGCTACGAGGAAGACCCGCAGACGATACTGGACGATCACCTGTCCAGGGTCCTCAAGACCCCT GGCTGCCAGTCTCCAGGCGTAGGCCGCTATAGCCCCCGCTCCCGCTCCCCGGACCACCACCACCACCACCATTCG CAGTACCACTCCCTGCTCCCGCCCGGTGGCAAGCTGCCTCCCGCGGCCGCCTCGCCGGGCGCCTGCCCCCTCCTC GGGGGCAAAGGCTTTGTGACCAAGCAGACGACGAAGCATGTCCACCACCACTACATCCACCACCATGCCGTCCCC AAGACCAAGGAGGAGATCGAGGCGGAGGCCACGCAGCGGGTGCACTGCTTCTGCCCTGGGGGCAGCGAGTATTAC TGCTACTCGAAATGCAAAAGCCACTCCAAGGCTCCGGAAACCATGCCCAGCGAGCAGTTTGGTGCCCAAAGCACA AAAAAGGCCTACCCCTTGGAGTCTGCCCGCTCGTCTCCAGGCGAACGAGCCAGCCGGCACCATCTGTGGGGGGGC AACAGCGGGCACCCCCGCACCACCCCCCGTGCCCACCTGTTCACCCAGGACCCTGCGATGCCTCCCCTGACCCCA CCCAACACGCTGGCTCAGCTGGAGGAGGCCTGTCGCAGGCTAGCTGAGGTGTCGAAGCCCCCAAAGCAGCGGTGC TGTGTGGCCAGTCAGCAGAGGGACAGGAATCATTCGGCCACTGTTCAGACGGGAGCCACACCCTTCTCCAATCCA AGCCTGGCTCCAGAAGATCACAAAGAGCCAAAGAAACTGGCAGGTGTCCACGCGCTCCAGGCCAGTGAGTTGGTT GTCACTTACTTTTTCTGTGGGGAAGAAATTCCATACCGGAGGATGCTGAAGGCTCAGAGCTTGACCCTGGGCCAC TTTAAAGAGCAGCTCAGCAAAAAGGGAAATTATAGGTATTACTTCAAAAAAGCAAGCGATGAGTTTGCCTGTGGA GCGGTGTTTGAGGAGATCTGGGAGGATGAGACGGTGCTCCCGATGTATGAAGGCCGGATTCTGGGCAAAGTGGAG CGGATCGATTGA
SEQ ID NO: 46 = variant e = ENSG00000168646|ENST00000580513
Polynucleotide, homo sapiens, 577 bp
ATGAGTAGCGCTATGTTGGTGACTTGCCTCCCGGACCCCAGCAGCAGCTTCCGTGAGGATGCCCCGCGGCCCCCA GTGCCAGGGGAAGAAGGGGAGACCCCACCGTGTCAGCCAGGGGTGGGCAAGGGCCAGGTCACCAAACCCATGCCT GTCTCTTCCAACACCAGGCGGAACGAAGATGGGTTGGGGGAGCCGGAGGGGCGGGCATCTCCGGATTCCCCTCTG ACCCGGTGGACCAAGTCCTTACACTCCTTATTGGGCGATCAAGACGGTGCTTACCTGTTCCGAACTTTCCTGGAG AGGGAGAAATGCGTGGATACCTTAGACTTCTGGTTTGCCTGCAATGGATTCAGGCAGATGAACCTGAAGGATACC AAAACTTTACGAGTAGCCAAAGCGATCTACAAAAGGTACATTGAGAACAACAGCATTGTCTCCAAGCAGCTGAAG CCTGCCACCAAGACCTACATAAGAGATGGCATCAAGAAGCAGCAGATTGATTCCATCATGTTTGACCAGGCGCAG ACCGAGATCCAGTCGGTGATGGAGGAAAATGCCTACCAGATGTTTTTGACTT
SEQ ID NO: 47 = variant f = ENSG00000168646|ENST00000585045
Polynucleotide, homo sapiens, 575 bp
ATGAGTAGCGCTATGTTGGTGACTTGCCTCCCGGACCCCAGCAGCAGCTTCCGTGAGGATGCCCCGCGGCCCCCA GTGCCAGGGGAAGAAGGGGAGACCCCACCGTGTCAGCCAGGGGTGGGCAAGGGCCAGGTCACCAAACCCATGCCT GTCTCTTCCAACACCAGGCGGAACGAAGATGGGTTGGGGGAGCCGGAGGGGCGGGCATCTCCGGATTCCCCTCTG ACCCGGTGGACCAAGTCCTTACACTCCTTATTGGGCGATCAAGACGGTGCTTACCTGTTCCGAACTTTCCTGGAG AGGGAGAAATGCGTGGATACCTTAGACTTCTGGTTTGCCTGCAATGGATTCAGGCAGATGAACCTGAAGGATACC AAAACTTTACGAGTAGCCAAAGCGATCTACAAAAGGTACATTGAGAACAACAGCATTGTCTCCAAGCAGCTGAAG CCTGCCACCAAGACCTACATAAGAGATGGCATCAAGAAGCAGCAGATTGATTCCATCATGTTTGACCAGGCGCAG ACCGAGATCCAGTCGGTGATGGAGGAAAATGCCTACCAGATGTTTTTGAC
SEQ ID NO: 56
Polypeptide of SEQ ID NO: 42, homo sapiens, 151 AA
MSSAMLVTCLPDPSSSFREDAPRPPVPGEEGETPPCQPGVGKGQVTKPMPVSSNTRRNEDGLGEPEGRASPDSPL TRWTKSLHSLLGDQDGAYLFRTFLEREKCVDTLDFWFACNGFRQMNLKDTKTLRVAKAIYKRYIENNS IVSKQLK P

Claims

CLAIMS We claim:
1. A method of treating breast cancer in a subject, the method comprising:
determining in a sample of the breast cancer an expression level of one or more genes selected from CSGALNACT1 , SIGLEC6, SHC4, ELTD1 , CGA, MST1 R, KRT8P41 , BTF3P4, RP1 1-478C6.4, EPHA3, LXN, ENSG00000252044, ENSG00000242932,
ENSG000002411 15, AP001347.6, LHFPL2, CTD-2330K9.2, RASD1 , ENSG00000242693, AC093388.3, AOC1 , CPE, PCDH8, DCHS2, DPP4, ENSG00000242777, GUSBP5, ENSG00000244466, ENSG00000252331 , RP1 1-664D7.4, RP1 1-206M1 1.7,
ENSG00000244580, EYA4, CTA-714B7.5, ENSG00000252318, ABCA1 , PLD1 , RNF144A, SLC44A4, SLC2A13, AXIN2, PGR, XXbac-BPG254F23.6, AMY2B, LIMCH1 , RP1 1- 356J5.12, ID2, MFAP2, LPHN2, RP1 1-594N15.2, RP1 1-575A19.2, SPINK5, HLA-DQB1- AS1 , RN7SL151 P, ATP8A1 , FAM189A2, ZNF423, CTD-2001 C12.1 , ELOVL2, TESC, KCNMB4, STOX2, PLEKHM3, MT-TL1 , KLK1 1 , NPNT, ENSG00000234543, SHC2, BTNL9, ARMC4, OLFML2A, AMOT, RP1 1-556114.1 , SLFN5, PKIA, CTC-339F2.2, ADAM23, NPSR1-AS1 , NREP, AC009410.1 , IMPG1 , C12orf40, AC097500.2, AC004069.2, CDH26, CCDC33, PCDP1 , ALCAM, LRRN1 , WWC3-AS1 , MT-TY, MXRA8, RASL1 1A, COL3A1 , PDE1A, COL21A1 , NTM, GNG4, RAMP3, AC026188.1 , ELOVL2-AS1 , SMAD9, ZBTB20, GNG12-AS1 , EFNA5, RP1 1-713M6.2, AC078883.3, PAMR1 , BMPER, NYAP2, RP4- 813D12.3, HMCN1 , FAT4, CYP2C8, PLLP, NXPH1 , CTD-2260A17.2, MUC5AC, PP13439, SOCS2-AS1 , CCM2L, ZNF827, ADAMTSL3, SDC2, RBM1 1 , PRICKLE2-AS3, RNF180, DOCK10, FAM198A, TMEM86A, LGALS3BP, KLK8, AC012307.2, AC004053.1 , CTD- 3179P9.1 , NOD2, RP1 1-556114.2, C3, ZNF396, ZNF792, ENSG00000244288, CHRM3, AGBL4, KLF7, AP004372.1 , DKK2, NPAS3, SMARCA1 , DOK6, SULF1 , RORC, YPEL1 , LRP1 B, RP4-710M3.2, DCLK1 , ENSG00000235732, NIPSNAP3B, SEPP1 , LY6D,
ENSG00000244428, AKR1 C3, GJA1 , CXXC4, FSTL5, CYBRD1 , SERPINA1 , AC004540.5, RP1 1 -600K15.1 , ASTL, C20orf197, KCNS3, CTC-448D22.1 , TMEM45A, RP1 1-86516.2, SNCG, CCDC152, MURC, TMCC2, FBN2, FREM1 , ENSG00000235471 ,
ENSG00000170233, THRB, AC004947.2, C1orf168, RN7SKP3, THBS2, TREH,
AC007365.3, ATOH8, SEMA3D, and GPC6;
comparing the expression level of each gene to a control value associated with that same gene; and
administering at least one of a RON inhibitor and a PI3K kinase inhibitor to the subject when the expression level of at least one of CSGALNACT1 , SIGLEC6, SHC4, ELTD1 , CGA, MST1 R, KRT8P41 , BTF3P4, RP1 1-478C6.4, EPHA3, LXN, ENSG00000252044, ENSG00000242932, ENSG00000241 1 15, AP001347.6, LHFPL2, CTD-2330K9.2, RASD1 , ENSG00000242693, AC093388.3, AOC1 , CPE, PCDH8, DCHS2, DPP4, ENSG00000242777, GUSBP5, ENSG00000244466, ENSG00000252331 , RP1 1- 664D7.4, RP1 1-206M1 1.7, ENSG00000244580, EYA4, CTA-714B7.5, and ENSG00000252318 is increased at least about 2-fold relative to the control, or administering an alternative therapy to the subject when the expression level of the gene is increased less than about 2-fold or decreased relative to the control; or
administering at least one of a RON inhibitor and a PI3K kinase inhibitor to the subject when the expression level of at least one of ABCA1 , PLD1 , RNF144A, SLC44A4, SLC2A13, AXIN2, PGR, XXbac-BPG254F23.6, AMY2B, LIMCH1 , RP1 1-356J5.12, ID2, MFAP2, LPHN2, RP1 1-594N15.2, RP1 1-575A19.2, SPINK5, HLA-DQB1-AS1 , RN7SL151 P, ATP8A1 , FAM189A2, ZNF423, CTD-2001 C12.1 , ELOVL2, TESC, KCNMB4, STOX2, PLEKHM3, MT-TL1 , KLK1 1 , NPNT, ENSG00000234543, SHC2, BTNL9, ARMC4, OLFML2A, AMOT, RP1 1-556114.1 , SLFN5, PKIA, CTC-339F2.2, ADAM23, NPSR1-AS1 , NREP, AC009410.1 , IMPG1 , C12orf40, AC097500.2, AC004069.2, CDH26, CCDC33, PCDP1 , ALCAM, LRRN1 , WWC3-AS1 , MT-TY, MXRA8, RASL1 1A, COL3A1 , PDE1A, COL21A1 , NTM, GNG4, RAMP3, AC026188.1 , ELOVL2-AS1 , SMAD9, ZBTB20, GNG12- AS1 , EFNA5, RP1 1-713M6.2, AC078883.3, PAMR1 , BMPER, NYAP2, RP4-813D12.3, HMCN1 , FAT4, CYP2C8, PLLP, NXPH1 , CTD-2260A17.2, MUC5AC, PP13439, SOCS2- AS1 , CCM2L, ZNF827, ADAMTSL3, SDC2, RBM11 , PRICKLE2-AS3, RNF180, DOCK10, FAM198A, TMEM86A, LGALS3BP, KLK8, AC012307.2, AC004053.1 , CTD-3179P9.1 , NOD2, RP1 1-556114.2, C3, ZNF396, ZNF792, ENSG00000244288, CHRM3, AGBL4, KLF7, AP004372.1 , DKK2, NPAS3, SMARCA1 , DOK6, SULF1 , RORC, YPEL1 , LRP1 B, RP4- 710M3.2, DCLK1 , ENSG00000235732, NIPSNAP3B, SEPP1 , LY6D, ENSG00000244428, AKR1 C3, GJA1 , CXXC4, FSTL5, CYBRD1 , SERPINA1 , AC004540.5, RP1 1-600K15.1 , ASTL, C20orf197, KCNS3, CTC-448D22.1 , TMEM45A, RP1 1-86516.2, SNCG, CCDC152, MURC, TMCC2, FBN2, FREM1 , ENSG00000235471 , ENSG00000170233, THRB, AC004947.2, C1orf168, RN7SKP3, THBS2, TREH, AC007365.3, ATOH8, SEMA3D, and GPC6 is decreased at least about 2-fold relative to the control, or administering an alternative therapy to the subject when the expression level of the gene is decreased less than about 2-fold or increased relative to the control.
2. The method of claim 1 , wherein the alternative therapy is selected from the group consisting of radiation therapy, a surgical procedure, anti-cancer drugs, or any combination thereof.
3. A method of assessing the likelihood that breast cancer in a subject will metastasize or has metastasized, the method comprising:
determining in a sample of the breast cancer an expression level of one or more genes selected from CSGALNACT1, SIGLEC6, SHC4, ELTD1, CGA, MST1R, KRT8P41, BTF3P4, RP11-478C6.4, EPHA3, LXN, ENSG00000252044, ENSG00000242932, ENSG00000241115, AP001347.6, LHFPL2, CTD-2330K9.2, RASD1, ENSG00000242693, AC093388.3, AOC1, CPE, PCDH8, DCHS2, DPP4, ENSG00000242777, GUSBP5, ENSG00000244466, ENSG00000252331 , RP11-664D7.4, RP11-206M11.7, ENSG00000244580, EYA4, CTA-714B7.5, ENSG00000252318, ABCA1, PLD1, RNF144A, SLC44A4, SLC2A13, AXIN2, PGR, XXbac-BPG254F23.6, AMY2B, LIMCH1, RP11- 356J5.12, ID2, MFAP2, LPHN2, RP11-594N15.2, RP11-575A19.2, SPINK5, HLA-DQB1- AS1, RN7SL151P, ATP8A1, FAM189A2, ZNF423, CTD-2001C12.1 , ELOVL2, TESC, KCNMB4, STOX2, PLEKHM3, MT-TL1, KLK11, NPNT, ENSG00000234543, SHC2, BTNL9, ARMC4, OLFML2A, AMOT, RP11-556114.1 , SLFN5, PKIA, CTC-339F2.2, ADAM23, NPSR1-AS1, NREP, AC009410.1, IMPG1, C12orf40, AC097500.2, AC004069.2, CDH26, CCDC33, PCDP1, ALCAM, LRRN1, WWC3-AS1, MT-TY, MXRA8, RASL11A, COL3A1, PDE1A, COL21A1, NTM, GNG4, RAMP3, AC026188.1, ELOVL2-AS1, SMAD9, ZBTB20, GNG12-AS1, EFNA5, RP11-713M6.2, AC078883.3, PAMR1, BMPER, NYAP2, RP4- 813D12.3, HMCN1, FAT4, CYP2C8, PLLP, NXPH1, CTD-2260A17.2, MUC5AC, PP13439, SOCS2-AS1, CCM2L, ZNF827, ADAMTSL3, SDC2, RBM11, PRICKLE2-AS3, RNF180, DOCK10, FAM198A, TMEM86A, LGALS3BP, KLK8, AC012307.2, AC004053.1, CTD- 3179P9.1, NOD2, RP11-556114.2, C3, ZNF396, ZNF792, ENSG00000244288, CHRM3, AGBL4, KLF7, AP004372.1, DKK2, NPAS3, SMARCA1, DOK6, SULF1, RORC, YPEL1, LRP1B, RP4-710M3.2, DCLK1, ENSG00000235732, NIPSNAP3B, SEPP1, LY6D, ENSG00000244428, AKR1C3, GJA1, CXXC4, FSTL5, CYBRD1, SERPINA1, AC004540.5, RP11-600K15.1, ASTL, C20orf197, KCNS3, CTC-448D22.1 , TMEM45A, RP11-86516.2, SNCG, CCDC152, MURC, TMCC2, FBN2, FREM1, ENSG00000235471 , ENSG00000170233, THRB, AC004947.2, C1orf168, RN7SKP3, THBS2, TREH, AC007365.3, ATOH8, SEMA3D, and GPC6;
comparing the expression level of each gene to a control value associated with that same gene; and
assessing the likelihood that the breast cancer will metastasize or has metastasized based on the relative difference between the expression level and the control value associated with each gene, wherein an increase in the expression level relative to the control value of at least one gene selected from CSGALNACT1, SIGLEC6, SHC4, ELTD1, CGA, MST1R, KRT8P41, BTF3P4, RP11-478C6.4, EPHA3, LXN, ENSG00000252044, ENSG00000242932, ENSG00000241115, AP001347.6, LHFPL2, CTD-2330K9.2, RASD1, ENSG00000242693, AC093388.3, AOC1 , CPE, PCDH8, DCHS2, DPP4, ENSG00000242777, GUSBP5, ENSG00000244466, ENSG00000252331 , RP1 1-664D7.4, RP1 1-206M11.7, ENSG00000244580, EYA4, CTA-714B7.5, and ENSG00000252318 correlates with an increased likelihood that the breast cancer in the subject will metastasize or has metastasized, or wherein a decrease in the expression level relative to the control value of at least one gene selected from ABCA1 , PLD1 , RNF144A, SLC44A4, SLC2A13, AXIN2, PGR, XXbac-BPG254F23.6, AMY2B, LIMCH1 , RP1 1-356J5.12, ID2, MFAP2, LPHN2, RP1 1-594N15.2, RP1 1-575A19.2, SPINK5, HLA-DQB1-AS1 , RN7SL151 P, ATP8A1 , FAM189A2, ZNF423, CTD-2001 C12.1 , ELOVL2, TESC, KCNMB4, STOX2, PLEKHM3, MT-TL1 , KLK1 1 , NPNT, ENSG00000234543, SHC2, BTNL9, ARMC4, OLFML2A, AMOT, RP1 1-556114.1 , SLFN5, PKIA, CTC-339F2.2, ADAM23, NPSR1-AS1 , NREP, AC009410.1 , IMPG1 , C12orf40, AC097500.2, AC004069.2, CDH26, CCDC33, PCDP1 , ALCAM, LRRN1 , WWC3-AS1 , MT-TY, MXRA8, RASL1 1A, COL3A1 , PDE1A, COL21A1 , NTM, GNG4, RAMP3, AC026188.1 , ELOVL2-AS1 , SMAD9, ZBTB20, GNG12- AS1 , EFNA5, RP1 1-713M6.2, AC078883.3, PAMR1 , BMPER, NYAP2, RP4-813D12.3, HMCN1 , FAT4, CYP2C8, PLLP, NXPH1 , CTD-2260A17.2, MUC5AC, PP13439, SOCS2- AS1 , CCM2L, ZNF827, ADAMTSL3, SDC2, RBM11 , PRICKLE2-AS3, RNF180, DOCK10, FAM198A, TMEM86A, LGALS3BP, KLK8, AC012307.2, AC004053.1 , CTD-3179P9.1 , NOD2, RP1 1-556114.2, C3, ZNF396, ZNF792, ENSG00000244288, CHRM3, AGBL4, KLF7, AP004372.1 , DKK2, NPAS3, SMARCA1 , DOK6, SULF1 , RORC, YPEL1 , LRP1 B, RP4- 710M3.2, DCLK1 , ENSG00000235732, NIPSNAP3B, SEPP1 , LY6D, ENSG00000244428, AKR1 C3, GJA1 , CXXC4, FSTL5, CYBRD1 , SERPINA1 , AC004540.5, RP1 1-600K15.1 , ASTL, C20orf197, KCNS3, CTC-448D22.1 , TMEM45A, RP1 1-86516.2, SNCG, CCDC152, MURC, TMCC2, FBN2, FREM1 , ENSG00000235471 , ENSG00000170233, THRB, AC004947.2, C1orf168, RN7SKP3, THBS2, TREH, AC007365.3, ATOH8, SEMA3D, and GPC6 correlates with an increased likelihood that the breast cancer in the subject will metastasize or has metastasized.
4. A method of determining whether a subject having breast cancer would benefit from treatment with at least one of a RON inhibitor and a PI3K kinase inhibitor, the method comprising:
assessing the likelihood that the breast cancer will metastasize or has metastasized according to the method of claim 3; and
determining whether the subject would benefit from treatment with at least one of a RON inhibitor and a PI3K kinase inhibitor based on the likelihood that the breast cancer will metastasize or has metastasized.
5. The method of any one of claims 1-4, wherein the expression level of at least five genes selected from CSGALNACT1, SIGLEC6, SHC4, ELTD1, CGA, MST1R, KRT8P41, BTF3P4, RP11-478C6.4, EPHA3, LXN, ENSG00000252044, ENSG00000242932, ENSG00000241115, AP001347.6, LHFPL2, CTD-2330K9.2, RASD1, ENSG00000242693, AC093388.3, AOC1, CPE, PCDH8, DCHS2, DPP4, ENSG00000242777, GUSBP5, ENSG00000244466, ENSG00000252331 , RP11-664D7.4, RP11-206M11.7, ENSG00000244580, EYA4, CTA-714B7.5, and ENSG00000252318 is increased relative to the control value.
6. The method of any one of claims 1-4, wherein the expression level of at least one gene selected from CSGALNACT1, SIGLEC6, and SHC4 is increased relative to the control value.
7. The method of any one of claims 1-4, wherein the expression level of at least five genes selected from ABCA1, PLD1, RNF144A, SLC44A4, SLC2A13, AXIN2, PGR, XXbac- BPG254F23.6, AMY2B, LIMCH1, RP11-356J5.12, ID2, MFAP2, LPHN2, RP11-594N15.2, RP11-575A19.2, SPINK5, HLA-DQB1-AS1 , RN7SL151P, ATP8A1, FAM189A2, ZNF423, CTD-2001C12.1, ELOVL2, TESC, KCNMB4, STOX2, PLEKHM3, MT-TL1 , KLK11 , NPNT, ENSG00000234543, SHC2, BTNL9, ARMC4, OLFML2A, AMOT, RP11-556114.1 , SLFN5, PKIA, CTC-339F2.2, ADAM23, NPSR1-AS1, NREP, AC009410.1, IMPG1, C12orf40, AC097500.2, AC004069.2, CDH26, CCDC33, PCDP1, ALCAM, LRRN1, WWC3-AS1, MT- TY, MXRA8, RASL11A, COL3A1, PDE1A, COL21A1, NTM, GNG4, RAMP3, AC026188.1, ELOVL2-AS1, SMAD9, ZBTB20, GNG12-AS1, EFNA5, RP11-713M6.2, AC078883.3, PAMR1, BMPER, NYAP2, RP4-813D12.3, HMCN1, FAT4, CYP2C8, PLLP, NXPH1, CTD- 2260A17.2, MUC5AC, PP13439, SOCS2-AS1, CCM2L, ZNF827, ADAMTSL3, SDC2, RBM11, PRICKLE2-AS3, RNF180, DOCK10, FAM198A, TMEM86A, LGALS3BP, KLK8, AC012307.2, AC004053.1, CTD-3179P9.1 , NOD2, RP11-556114.2, C3, ZNF396, ZNF792, ENSG00000244288, CHRM3, AGBL4, KLF7, AP004372.1, DKK2, NPAS3, SMARCA1, DOK6, SULF1, RORC, YPEL1, LRP1B, RP4-710M3.2, DCLK1, ENSG00000235732, NIPSNAP3B, SEPP1, LY6D, ENSG00000244428, AKR1C3, GJA1, CXXC4, FSTL5, CYBRD1, SERPINA1, AC004540.5, RP11-600K15.1 , ASTL, C20orf197, KCNS3, CTC- 448D22.1, TMEM45ARP11-86516.2, SNCG, CCDC152, MURC, TMCC2, FBN2, FREM1, ENSG00000235471, ENSG00000170233, THRB, AC004947.2, C1orf168, RN7SKP3, THBS2, TREHAC007365.3, ATOH8, SEMA3D, and GPC6 is decreased relative to the control value.
8. The method of any one of claims 1-4, wherein the expression level of at least one gene selected from ABCA1, PLD1, RNF144A, SLC44A4, SLC2A13, and AXIN2 is decreased relative to the control value.
9. The method of any one of claims 1-4, wherein the expression level of at least five genes selected from CSGALNACT1, SIGLEC6, SHC4, ELTD1, CGA, MST1R, KRT8P41, BTF3P4, RP11-478C6.4, EPHA3, LXN, ENSG00000252044, ENSG00000242932, ENSG00000241115, AP001347.6, LHFPL2, CTD-2330K9.2, RASD1, ENSG00000242693, AC093388.3, AOC1, CPE, PCDH8, DCHS2, DPP4, ENSG00000242777, GUSBP5, ENSG00000244466, ENSG00000252331 , RP11-664D7.4, RP11-206M11.7, ENSG00000244580, EYA4, CTA-714B7.5, ENSG00000252318, ABCA1, PLD1, RNF144A, SLC44A4, SLC2A13, AXIN2, PGR, XXbac-BPG254F23.6, AMY2B, LIMCH1, RP11- 356J5.12, ID2, MFAP2, LPHN2, RP11-594N15.2, RP11-575A19.2, SPINK5, HLA-DQB1- AS1, RN7SL151P, ATP8A1, FAM189A2, ZNF423, CTD-2001C12.1 , ELOVL2, TESC, KCNMB4, STOX2, PLEKHM3, MT-TL1 , KLK11, NPNT, ENSG00000234543, SHC2, BTNL9, ARMC4, OLFML2A, AMOT, RP11-556114.1 , SLFN5, PKIA, CTC-339F2.2, ADAM23, NPSR1-AS1, NREP, AC009410.1, IMPG1, C12orf40, AC097500.2, AC004069.2, CDH26, CCDC33, PCDP1, ALCAM, LRRN1, WWC3-AS1, MT-TY, MXRA8, RASL11A, COL3A1, PDE1A, COL21A1, NTM, GNG4, RAMP3, AC026188.1, ELOVL2-AS1, SMAD9, ZBTB20, GNG12-AS1, EFNA5, RP11-713M6.2, AC078883.3, PAMR1, BMPER, NYAP2, RP4- 813D12.3, HMCN1, FAT4, CYP2C8, PLLP, NXPH1, CTD-2260A17.2, MUC5AC, PP13439, SOCS2-AS1, CCM2L, ZNF827, ADAMTSL3, SDC2, RBM11, PRICKLE2-AS3, RNF180, DOCK10, FAM198A, TMEM86A, LGALS3BP, KLK8, AC012307.2, AC004053.1, CTD- 3179P9.1, NOD2, RP11-556114.2, C3, ZNF396, ZNF792, ENSG00000244288, CHRM3, AGBL4, KLF7, AP004372.1, DKK2, NPAS3, SMARCA1, DOK6, SULF1, RORC, YPEL1, LRP1B, RP4-710M3.2, DCLK1, ENSG00000235732, NIPSNAP3B, SEPP1, LY6D, ENSG00000244428, AKR1C3, GJA1, CXXC4, FSTL5, CYBRD1, SERPINA1, AC004540.5, RP11-600K15.1, ASTL, C20orf197, KCNS3, CTC-448D22.1 , TMEM45A, RP11-86516.2, SNCG, CCDC152, MURC, TMCC2, FBN2, FREM1, ENSG00000235471 , ENSG00000170233, THRB, AC004947.2, C1orf168, RN7SKP3, THBS2, TREH, AC007365.3, ATOH8, SEMA3D, and GPC6 is different relative to the control value.
10. The method of any one of claims 1-4, wherein the expression level of at least one gene selected from CSGALNACT1, SIGLEC6, SHC4, ABCA1, PLD1, RNF144A, SLC44A4, SLC2A13, and AXIN2 is different relative to the control value.
11. The method of any one of claims 1-10, wherein the control value associated with each gene is determined by determining the expression level of that gene in one or more control samples, and calculating an average expression level of that gene in the one or more control samples, wherein each control sample is obtained from healthy tissue of the same or a different subject.
12. The method of any one of claims 1-10, wherein the expression of the gene is increased or decreased relative to the expression level of a control by an amount of at least about 2-fold.
13. A method of monitoring a molecular response of a subject to treatment of breast cancer with at least one of a RON inhibitor and a PI3K inhibitor, the method comprising: determining in a first sample of the breast cancer an expression level of one or more genes selected from CSGALNACT1 , SIGLEC6, SHC4, ELTD1 , CGA, MST1 R, KRT8P41 , BTF3P4, RP1 1-478C6.4, EPHA3, LXN, ENSG00000252044, ENSG00000242932, ENSG00000241 1 15, AP001347.6, LHFPL2, CTD-2330K9.2, RASD1 , ENSG00000242693, AC093388.3, AOC1 , CPE, PCDH8, DCHS2, DPP4, ENSG00000242777, GUSBP5, ENSG00000244466, ENSG00000252331 , RP1 1-664D7.4, RP1 1-206M11.7, ENSG00000244580, EYA4, CTA-714B7.5, ENSG00000252318, ABCA1 , PLD1 , RNF144A, SLC44A4, SLC2A13, AXIN2, PGR, XXbac-BPG254F23.6, AMY2B, LIMCH1 , RP1 1- 356J5.12, ID2, MFAP2, LPHN2, RP1 1-594N15.2, RP1 1-575A19.2, SPINK5, HLA-DQB1- AS1 , RN7SL151 P, ATP8A1 , FAM189A2, ZNF423, CTD-2001 C12.1 , ELOVL2, TESC, KCNMB4, STOX2, PLEKHM3, MT-TL1 , KLK1 1 , NPNT, ENSG00000234543, SHC2, BTNL9, ARMC4, OLFML2A, AMOT, RP1 1-556114.1 , SLFN5, PKIA, CTC-339F2.2, ADAM23, NPSR1-AS1 , NREP, AC009410.1 , IMPG1 , C12orf40, AC097500.2, AC004069.2, CDH26, CCDC33, PCDP1 , ALCAM, LRRN1 , WWC3-AS1 , MT-TY, MXRA8, RASL1 1A, COL3A1 , PDE1A, COL21A1 , NTM, GNG4, RAMP3, AC026188.1 , ELOVL2-AS1 , SMAD9, ZBTB20, GNG12-AS1 , EFNA5, RP1 1-713M6.2, AC078883.3, PAMR1 , BMPER, NYAP2, RP4- 813D12.3, HMCN1 , FAT4, CYP2C8, PLLP, NXPH1 , CTD-2260A17.2, MUC5AC, PP13439, SOCS2-AS1 , CCM2L, ZNF827, ADAMTSL3, SDC2, RBM1 1 , PRICKLE2-AS3, RNF180, DOCK10, FAM198A, TMEM86A, LGALS3BP, KLK8, AC012307.2, AC004053.1 , CTD- 3179P9.1 , NOD2, RP1 1-556114.2, C3, ZNF396, ZNF792, ENSG00000244288, CHRM3, AGBL4, KLF7, AP004372.1 , DKK2, NPAS3, SMARCA1 , DOK6, SULF1 , RORC, YPEL1 , LRP1 B, RP4-710M3.2, DCLK1 , ENSG00000235732, NIPSNAP3B, SEPP1 , LY6D, ENSG00000244428, AKR1 C3, GJA1 , CXXC4, FSTL5, CYBRD1 , SERPINA1 , AC004540.5, RP1 1-600K15.1 , ASTL, C20orf197, KCNS3, CTC-448D22.1 , TMEM45A, RP1 1-86516.2, SNCG, CCDC152, MURC, TMCC2, FBN2, FREM1 , ENSG00000235471 , ENSG00000170233, THRB, AC004947.2, C1orf168, RN7SKP3, THBS2, TREH, AC007365.3, ATOH8, SEMA3D, and GPC6;
administering to the subject a therapeutic amount of a medicament comprising at least one of a RON inhibitor and a PI3K inhibitor;
determining in a second sample of the breast cancer an expression level of each of the one or more genes, wherein the second sample is collected after administration of the medicament; and
comparing for each gene the expression level in the first sample to the expression level in the second sample.
14. The method of any one of claims 1-13, wherein the one or more genes are selected from CSGALNACT1 , SIGLEC6, SHC4, ABCA1 , PLD1 , RNF144A, SLC44A4, SLC2A13, and AXIN2.
15. The method of any one of claims 1-14, wherein the expression of at least one gene is increased at least about 2-fold relative to the control value associated with the gene.
16. The method of any one of claims 1-14, wherein the expression of at least one gene is decreased at least about 2-fold relative to the control value associated with the gene.
17. The method of any of claims 1-16, wherein determining the expression level of a first gene comprises measuring the expression level of an RNA transcript of the first gene, or an expression product thereof.
18. The method of claim 17, wherein measuring the expression level of the RNA transcript of the first gene, or the expression product thereof, includes using at least one of a PCR-based method, a Northern blot method, a microarray method, and an immunohistochemical method.
19. A kit for assessing the likelihood that breast cancer in a subject will metastasize or has metastasized, or for determining whether the subject would benefit from treatment with at least one of a RON inhibitor and a PI3K kinase inhibitor, the kit comprising one or more primers, each primer adapted to amplify an RNA transcript of an independent one of the genes selected from CSGALNACT1 , SIGLEC6, SHC4, ELTD1 , CGA, MST1 R, KRT8P41 , BTF3P4, RP1 1-478C6.4, EPHA3, LXN, ENSG00000252044, ENSG00000242932, ENSG00000241 1 15, AP001347.6, LHFPL2, CTD-2330K9.2, RASD1 , ENSG00000242693, AC093388.3, AOC1 , CPE, PCDH8, DCHS2, DPP4, ENSG00000242777, GUSBP5, ENSG00000244466, ENSG00000252331 , RP1 1-664D7.4, RP1 1-206M1 1.7, ENSG00000244580, EYA4, CTA-714B7.5, ENSG00000252318, ABCA1 , PLD1 , RNF144A, SLC44A4, SLC2A13, ΑΧΙΝ2, PGR, XXbac-BPG254F23.6, ΑΜΥ2Β, LIMCH1 , RP1 1- 356J5.12, ID2, MFAP2, LPHN2, RP1 1-594Ν15.2, RP1 1-575Α19.2, SPINK5, HLA-DQB1- AS1 , RN7SL151 P, ΑΤΡ8Α1 , FAM189A2, ZNF423, CTD-2001 C12.1 , ELOVL2, TESC, KCNMB4, STOX2, PLEKHM3, MT-TL1 , KLK1 1 , ΝΡΝΤ, ENSG00000234543, SHC2, BTNL9, ARMC4, OLFML2A, ΑΜΟΤ, RP1 1-556114.1 , SLFN5, ΡΚΙΑ, CTC-339F2.2, ADAM23, NPSR1-AS1 , NREP, AC009410.1 , IMPG1 , C12orf40, AC097500.2, AC004069.2, CDH26, CCDC33, PCDP1 , ALCAM, LRRN1 , WWC3-AS1 , ΜΤ-ΤΥ, MXRA8, RASL1 1A, COL3A1 , PDE1A, COL21A1 , ΝΤΜ, GNG4, RAMP3, AC026188.1 , ELOVL2-AS1 , SMAD9, ΖΒΤΒ20, GNG12-AS1 , EFNA5, RP1 1-713Μ6.2, AC078883.3, PAMR1 , BMPER, ΝΥΑΡ2, RP4- 813D12.3, HMCN1 , FAT4, CYP2C8, PLLP, ΝΧΡΗ1 , CTD-2260A17.2, MUC5AC, ΡΡ13439, SOCS2-AS1 , CCM2L, ZNF827, ADAMTSL3, SDC2, RBM1 1 , PRICKLE2-AS3, RNF180, DOCK10, FAM198A, ΤΜΕΜ86Α, LGALS3BP, KLK8, AC012307.2, AC004053.1 , CTD- 3179Ρ9.1 , NOD2, RP1 1-556114.2, C3, ZNF396, ZNF792, ENSG00000244288, CHRM3, AGBL4, KLF7, ΑΡ004372.1 , DKK2, NPAS3, SMARCA1 , DOK6, SULF1 , RORC, YPEL1 , LRP1 B, RP4-710M3.2, DCLK1 , ENSG00000235732, NIPSNAP3B, SEPP1 , LY6D, ENSG00000244428, AKR1 C3, GJA1 , CXXC4, FSTL5, CYBRD1 , SERPINA1 , AC004540.5, RP1 1-600K15.1 , ASTL, C20orf197, KCNS3, CTC-448D22.1 , ΤΜΕΜ45Α, RP1 1-86516.2, SNCG, CCDC152, MURC, TMCC2, FBN2, FREM1 , ENSG00000235471 , ENSG00000170233, THRB, AC004947.2, C1orf168, RN7SKP3, THBS2, TREH, AC007365.3, ΑΤΟΗ8, SEMA3D, and GPC6, and instructions for use.
20. The kit of claim 19, wherein the at least one primer is adapted to amplify an RNA transcript of a gene selected from CSGALNACT1 , SIGLEC6, SHC4, ABCA1 , PLD1 , RNF144A, SLC44A4, SLC2A13, and AXIN2.
21. A kit for assessing the likelihood that breast cancer in a subject will metastasize or has metastasized, or for determining whether the subject would benefit from treatment with at least one of a RON inhibitor and a PI3K kinase inhibitor, the kit comprising one or more probes, each probe adapted to specifically bind to an RNA transcript, or an expression product thereof, of an independent one of the genes selected from CSGALNACT1 , SIGLEC6, SHC4, ELTD1 , CGA, MST1 R, KRT8P41 , BTF3P4, RP1 1-478C6.4, EPHA3, LXN, ENSG00000252044, ENSG00000242932, ENSG00000241 1 15, AP001347.6, LHFPL2, CTD-2330K9.2, RASD1 , ENSG00000242693, AC093388.3, AOC1 , CPE, PCDH8, DCHS2, DPP4, ENSG00000242777, GUSBP5, ENSG00000244466, ENSG00000252331 , RP1 1- 664D7.4, RP1 1-206M1 1.7, ENSG00000244580, EYA4, CTA-714B7.5, ENSG00000252318, ABCA1 , PLD1 , RNF144A, SLC44A4, SLC2A13, ΑΧΙΝ2, PGR, XXbac-BPG254F23.6, AMY2B, LIMCH1, RP11-356J5.12, ID2, MFAP2, LPHN2, RP11-594N15.2, RP11-575A19.2, SPINK5, HLA-DQB1-AS1, RN7SL151P, ATP8A1, FAM189A2, ZNF423, CTD-2001C12.1 , ELOVL2, TESC, KCNMB4, STOX2, PLEKHM3, MT-TL1, KLK11, NPNT, ENSG00000234543, SHC2, BTNL9, ARMC4, OLFML2A, AMOT, RP11-556114.1 , SLFN5, PKIA, CTC-339F2.2, ADAM23, NPSR1-AS1, NREP, AC009410.1, IMPG1, C12orf40, AC097500.2, AC004069.2, CDH26, CCDC33, PCDP1, ALCAM, LRRN1, WWC3-AS1, MT- TY, MXRA8, RASL11A, COL3A1, PDE1A, COL21A1, NTM, GNG4, RAMP3, AC026188.1, ELOVL2-AS1, SMAD9, ZBTB20, GNG12-AS1, EFNA5, RP11-713M6.2, AC078883.3, PAMR1, BMPER, NYAP2, RP4-813D12.3, HMCN1, FAT4, CYP2C8, PLLP, NXPH1, CTD- 2260A17.2, MUC5AC, PP13439, SOCS2-AS1, CCM2L, ZNF827, ADAMTSL3, SDC2, RBM11, PRICKLE2-AS3, RNF180, DOCK10, FAM198A, TMEM86A, LGALS3BP, KLK8, AC012307.2, AC004053.1, CTD-3179P9.1 , NOD2, RP11-556114.2, C3, ZNF396, ZNF792, ENSG00000244288, CHRM3, AGBL4, KLF7, AP004372.1, DKK2, NPAS3, SMARCA1, DOK6, SULF1, RORC, YPEL1, LRP1B, RP4-710M3.2, DCLK1, ENSG00000235732, NIPSNAP3B, SEPP1, LY6D, ENSG00000244428, AKR1C3, GJA1, CXXC4, FSTL5, CYBRD1, SERPINA1, AC004540.5, RP11-600K15.1 , ASTL, C20orf197, KCNS3, CTC- 448D22.1, TMEM45ARP11-86516.2, SNCG, CCDC152, MURC, TMCC2, FBN2, FREM1, ENSG00000235471, ENSG00000170233, THRB, AC004947.2, C1orf168, RN7SKP3, THBS2, TREHAC007365.3, ATOH8, SEMA3D, and GPC6, and instructions for use.
22. The kit of claim 21, wherein the at least one probe is adapted to bind to an RNA transcript, or an expression product thereof, of a gene selected from CSGALNACT1, SIGLEC6, SHC4, ABCA1, PLD1, RNF144A, SLC44A4, SLC2A13, and AXIN2.
23. The kit of either one of claims 19 or 21, wherein a first probe comprises an antibody to an expression product.
24. The kit of either one of claims 19 or 21, wherein a first probe comprises an oligonucleotide complementary to an RNA transcript.
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