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WO2016008051A1 - Contactine-1 (cntn1) pour utilisation dans des procédés de diagnostic et de traitement du cancer de la prostate - Google Patents

Contactine-1 (cntn1) pour utilisation dans des procédés de diagnostic et de traitement du cancer de la prostate Download PDF

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WO2016008051A1
WO2016008051A1 PCT/CA2015/050663 CA2015050663W WO2016008051A1 WO 2016008051 A1 WO2016008051 A1 WO 2016008051A1 CA 2015050663 W CA2015050663 W CA 2015050663W WO 2016008051 A1 WO2016008051 A1 WO 2016008051A1
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cntn1
cells
level
prostate cancer
expression level
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Damu Tang
Judy YAN
Diane OJO
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McMaster University
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McMaster University
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Definitions

  • CONTACTIN-1 (CNTN1) FOR USE IN METHODS OF DIAGNOSIS AND
  • the present invention generally relates to diagnostic and therapeutic methods for prostate cancers, and more particularly, to the use of the cell surface neural cell adhesion factor, contactin 1 (CNTN1), in such methods.
  • CNTN1 cell surface neural cell adhesion factor
  • PC Prostate cancer
  • PSA prostate specific antigen
  • CSCs cancer stem cells
  • Tumors consist of heterogeneous cell populations, in which a minor group of cells (CSCs) are the unit of evolution selection.
  • Prostate stem cells PSC have been identified in both humans and mice. Akin to orchiectomy leading to robust prostate involution, androgen- removal results in marked PC regression.
  • PCSCs Prostate Cancer Stem Cells
  • CNTN1 contactin 1
  • a method of determining prostate cancer, or progression, recurrence or metastasis of prostate cancer, in a male mammal comprises the steps of determining in a biological sample obtained from the mammal the expression level of CNTN1 in the sample, comparing the CNTN1 expression level to a control level, and determining that the mammal has prostate cancer when the expression level of CNTN1 in the sample is greater than the control level.
  • a method of treating prostate cancer in a mammal comprising administering to the mammal a CNTN1 inhibitor.
  • composition comprising a CNTN1 inhibitor and a pharmaceutically acceptable carrier is provided.
  • a method of determining tamoxifen resistance in breast cancer comprising determining the level of CNTN1 in breast cancer cells, comparing the CNTN1 level to a control level, and determining that the mammal has tamoxifen resistant breast cancer when the level of CNTN1 in the sample is greater than the control level.
  • Figure 1 shows CNTN1 expression promotes prostate cancer cell invasion.
  • C) cell lysates were collected from the indicated cell lines and examined for protein expression by western blot for CNTN1 and actin. Experiments were repeated at least three times; representative image from a single repeat is shown. M: monolayer cells.
  • D) whole DU145 spheres were prepared onto slides by cytospin and stained by immunofiuoresence for CNTN1 expression. Similar images were obtained from multiple spheres; a typical image is included.
  • PC3 10% FBS
  • PC3 SF serum free PCSC conditions
  • CNTN1 mRNA in PC3 SF is shown as a fold change to PC3 cells (mean ⁇ s.d.). Experiments were repeated two times. F) western blot analysis of CNTN1 in DU145 EV, CNTN1 , parental (Par) cells from which spheres were derived, and sphere cells. G) DU145 EV and DU145 CNTN1 cells were examined for their ability to pass through a control and matrigel membrane. Experiments were repeated three times. Typical images from one experiment are shown (left panel). Cell invasion was quantified (right panel). *:p ⁇ 0.05 by a 2-tailed student t- test.
  • FIG. 2 shows Ectopic CNTN1 does not affect DU145 cell proliferation. 1000 cells of DU145 EV and CNTN1 cells were seeded in triplicate in 96 well plates, followed by determining cell proliferation daily using WST-1 for 5 days. Experiments were repeated 3 times.
  • FIG. 3 shows the characterization of the impact of CNTN1 on LNCaP C4-2 cell biology.
  • LNCaP C4-2 EV and CNTN1 cells were serum starved over night, stimulated with 10% FBS for the indicated timepoints, and examined by western blot for serine 473-phosphorylated AKT (pAKT) and AKT. Experiments were repeated twice; typical images from a single repeat are shown.
  • FIG. 4 shows CNTN1 affects AKT activation and E-cadherin expression.
  • DU145 EV and CNTN1 cells were serum starved over night followed by the addition of media with 10% FBS for the indicated timepoints.
  • AKT activation was examined by western blot for serine 473-phosphorylated AKT (pAKT). Experiments were repeated twice; typical images from a single repeat are shown.
  • B) DU145 sphere cells were infected with shCTRL (control shRNA) and shCNTN1 , followed by western blot examination for the indicated proteins (inset). Experiments were repeated three times; the levels of pAKT were standardized to total AKT and quantified (means ⁇ s.d.). *:p ⁇ 0.05 by a 2-tailed student t-test.
  • Figure S shows CNTN1 promotes xenograft tumor formation.
  • the indicated sphere cells (A) and monolayer cells (B) were subcutaneously implanted into NOD/SCID mice at 10 4 sphere cells or 10 6 monolayer cells per mouse. Four or five mice were used per line.
  • Xenograft tumors were monitored weekly; tumor volumes were graphed (means ⁇ s.d.). *: p ⁇ 0.05 by a 2-way ANOVA.
  • FIG. 6 shows CNTN1 increases lung metastases in mice.
  • FIG. 7 shows CNTN1 associates with prostate cancer progression and biochemical recurrence.
  • Figure 8 shows CNTN1 affects gene expressions mat function in tumorigenesis.
  • D analysis of PTEN, BCHE and TMPRSS2-ERG transcripts using NanoString technology for 7 paired non-tumor and prostate cancer tissues.
  • Figure 9 shows the number of differentially expressed genes for A) DU145
  • Figure 10 shows CNTN1 represses BCHE expression. UNA was isolated from
  • DU145 monolayer, sphere cells A
  • DU145 EV and CNTN1 cells B
  • LNCaP C4-2 EV and CNTN1 cells B
  • A Microarray analysis on gene expression between DU145 monolayer and DU145 PCSCs revealed a 63 fold down-regulation of BCHE in DU145 PCSCs. *:p ⁇ 0.05 by a two tailed student t-test.
  • B real-time PCR analysis for BCHE and ⁇ -actin on overexpression cell lines. * :p ⁇ 0.05 by a two tailed student t-test.
  • Figure 11 shows a robust elevation of blood CNTN1 in a patient with castration resistant prostate cancer.
  • Blood samples were collected from three volunteers and a patient with castration resistant prostate cancer.
  • Total RNA was purified from blood cells (Buffy coat) and serum.
  • Real time PCR reactions were performed to amplify CNTN1 and actin.
  • the respective CNTN1 mRNA was normalized to the corresponding actin mRNA.
  • Patient's CNTN1 was expressed as fold changes in reference to normal individuals.
  • Figure 12 shows the expression of CNTN1 in a panel of breast cancer cell lines.
  • Figure 13 shows that ectopic CNTN1 confers MCF7 cells resistant to tamoxifen- induced cytotoxicity by graphically illustrating quantification of surviving MCF7 EV and CNTN1 cells in response to tamoxifen treatment from 3 independent repeats. *p ⁇ 0.05 by 2-tailed student t-test.
  • Figure 14 shows elevation of CNTN1 in tamoxifen resistant MCF7 cells.
  • Tamoxifen resistant MCF7 cells (Tam-R) were produced by culturing in the presence of 0.1 uM tamoxifen for 12 months. The status of tamoxifen resistance was confirmed. Real time PCR were performed on both MCF7 and MCF7 Tam-R cells to analyze CNTN1 expression. The level of CNTN1 in MCF7 Tam-R cells was expressed as fold changes to it in MCF7 cells.
  • Figure 15 illustrates the amino acid sequence of A) human CNTN1 isoform 1, B) human CNTN1 isoform 3, C) mouse CNTN1, and the mRNA sequences for D) human CNTN1 isoform 1, and E) mouse CNTN1.
  • a method of determining prostate cancer in a male mammal comprises the steps of determining in a biological sample obtained from the mammal the expression level of CNTN1, comparing the CNTN1 expression level to a control level, and determining that the mammal has prostate cancer when the level of CNTN1 in the sample is greater than the control level.
  • Contactin-1 refers to a neural cell adhesion protein having the amino acid sequence shown in Fig. 20A, e.g. isoform 1, including functionally equivalent variants such as other isoforms, e.g. isoform 2 in which residues 11-21 of isoform 1 are missing; and orthologs such as CNTN1 from other mammals, e.g. mouse CNTN1 ortholog as shown in Fig. 20, and other functionally equivalent variants, including fragments.
  • functionally equivalent refers to CNTN-1 variant proteins that retain the same or similar function and/or activity as human CNTN-1 (isoform 1) and which, as a result, would similarly be expressed in connection with prostate cancer.
  • Functionally equivalent variants may include a native CNTN1 including one or more amino acid deletions, insertions or substitutions (such as, but not limited to, conservative amino acid substitutions in which one or more amino acids is replaced with a similarly charged amino acid, e.g. substitution of a basic amino acid such as histidine, lysine and arginine; a negatively charged amino acid such as glutamic and aspartic acid; a hydrophobic amino acid such as alanine, valine, leucine, isoleucine, tryptophan, methionine, and isoleucine; and substitution of an uncharged amino acid such as threonine, glutamine, asparagine and serine).
  • conservative amino acid substitutions in which one or more amino acids is replaced with a similarly charged amino acid, e.g. substitution of a basic amino acid such as histidine, lysine and arginine; a negatively charged amino acid such as glutamic and aspartic acid; a hydrophobic amino acid such
  • CNTN-1 variants include CNTN-1 in which the proline at position 794 is changed to histidine, or in which the valine at position 798 is changed to leucine, or a variant in which the glutamic acid at position 824 is changed to glycine.
  • Proteolytic fragments of CNTN-1 may result from its proteolytic breakdown, and thus, may be detectable in blood as representative of CNTN-1 expression.
  • prostate cancer refers to any cancer of the prostate gland, including but not limited to, adenocarcinoma and neuroendocrine prostate cancer, which may become castration resistant following androgen deprivation therapy.
  • a biological sample is obtained from a male mammal.
  • biological sample is meant to encompass any human sample that may contain an increased expression level of CNTN1, protein or nucleic acid (genomic or mRNA), including circulating biological fluids such as, but not limited to, blood, plasma/serum, urine and cerebrospinal fluid, lymphatic fluid, bone marrow, lymph nodes and tissue biopsies, e.g. prostate tumour or prostate tissue, may also be used.
  • the sample is obtained in a manner well-established in the art.
  • mamammal is used herein to refer to both male human and non-human mammals, such as domestic animals such as dogs, cats, horses and the like, as well as other animals.
  • CNTN1 CNTN1 -encoding nucleic acid
  • the sample Prior to analysis, the sample may be subject to processing such as extraction, filtration, centrifugation or other sample preparation techniques to provide a sample that is suitable for further analysis.
  • biological fluids may be filtered or centrifuged (e.g. uUracentrifugation) to remove solids from the sample to facilitate analysis.
  • Tissue samples may be subject to extractions in order to provide an analyzable sample.
  • CNTN1 expression level may be determined using one of several techniques established in the art that would be suitable for detecting a protein or nucleic acid encoding the protein in a biological sample, including mass spectrometry, chromatographic techniques such as high performance liquid and gas chromatography, immunoassay or enzyme-based assays with colorimetric, fluorescence or radiometric detection, electrophoretic techniques, and nucleic acid hybridization techniques which may include FCR.
  • the expression level CNTN1 in a sample may be measured by immunoassay using an antibody specific to CNTN1.
  • the antibody binds to the biomarker and bound antibody is quantified by measuring a detectable marker which may be linked to the antibody or other component of the assay, or which may be generated during the assay.
  • Detectable markers may include radioactive, fluorescent, phosphorescent and luminescent (e.g. chemiluminescent or bioluminescent) compounds, dyes, particles such as colloidal gold and enzyme labels.
  • the term "antibody” is used herein to refer to monoclonal or polyclonal antibodies, or antigen-binding fragments thereof, e.g. an antibody fragment that retains specific binding affinity for the target biomarker.
  • Antibodies to the target biomarkers may be raised using techniques conventional in the art. For example, antibodies may be made by injecting a host animal, e.g. a mouse or rabbit, with the antigen (target biomarker), and then isolating antibody from a biological sample taken from the host animal. Alternatively, CNTN1 antibodies may be commercially obtained, e.g. from Sigma Aldrich, Sino Biological Inc., Atlas Antibodies, etc.
  • CNTN1 expression level in a sample may be determined using indirect immunoassay in which CNTN1 is non-specifically immobilized on a surface; sandwich immunoassay in which the CNTN1 is specifically immobilized on a surface by linkage to a capture antibody bound to the surface; and a competitive binding immunoassay in which a sample is first combined with a known quantity of CNTN1 antibody, and then the sample is exposed to immobilized CNTN1 which competes with the sample to bind any unbound antibody.
  • Enzyme Linked Immunosorbent Assay ELISA
  • ELISA Enzyme Linked Immunosorbent Assay
  • CNTN1 is generally immobilized on a solid support, complexed with an antibody which is itself linked to an enzyme indicator, such as horseradish peroxidase (HRP), alkaline phosphatase (AP), ⁇ -galactosidase, acetylcholinesterase and catalase. Detection may then be accomplished by incubating this enzyme-complex with a substrate for the enzyme that yields a detectable product.
  • HRP horseradish peroxidase
  • AP alkaline phosphatase
  • ⁇ -galactosidase acetylcholinesterase
  • catalase catalase
  • sphere-forming cells are first isolated from the biological sample and determination of CNTN1 expression level in the sphere-forming cells is conducted as described.
  • Sphere-forming cells may be prostate cancer stem cells which are able to grow in suspension (i.e. floating) in these 3-D sphere-shaped structures and are associated with prostate cancer.
  • Sphere-forming cells may be isolated using established methods. For example, biopsied cells may be dissociated, and then cultured in a serum-free media supplemented with EGF and optionally other nutrients (e.g. BSA, vitamins) for a sufficient period of time, e.g. 10-12 days.
  • This isolation step functions to concentrate potential target sphere-forming or cancer stem cells to facilitate the analysis.
  • control level is the level of CNTN1 protein or nucleic acid detected in a sample from a healthy unaffected subject who does not have prostate cancer.
  • control value is a mean control value obtained from a healthy population of matched subjects (e.g. age-, and/or ethnically-matched to a population).
  • CNTN1 expression levels may also be compared to a control level of one or more housekeeping or reference genes.
  • housekeeping genes as used herein is meant to refer to genes that encode protein products that are not connected to, involved in or required for processes specific to prostate cancer cells, and thus, exhibit a fixed expression level in cancerous and non-cancerous cells.
  • housekeeping genes include, but are not limited to, genes encoding ACTB (Beta-actih), GAPDH (Glyceraldehyde 3-phosphate dehydrogenase), RPLPO (60S acidic ribosomal protein PO), GUSB (beta-glucuronidase), beta-tubulin, ATP- binding cassette, sub-family F member 1, and TFRC (transferring receptor 1).
  • ACTB Beta-actih
  • GAPDH Glyceraldehyde 3-phosphate dehydrogenase
  • RPLPO 60S acidic ribosomal protein PO
  • GUSB beta-glucuronidase
  • beta-tubulin ATP- binding cassette
  • sub-family F member 1 sub-family F member 1
  • TFRC transferring receptor 1
  • a statistically significant difference i.e. an increase in CNTN1 expression level, is an increase in the CNTN1 expression level from the control level of at least about 10%, or greater, e.g. at least about 20%, 30%, 40%, 50% or greater, for example, an increase in CNTN1 expression of 80% or greater as compared to the control.
  • the present method may also be used to predict disease progression and/or monitor treatment response to therapy.
  • the present method may subsequently be used to determine CNTN1 expression level as described.
  • the determination of CNTN1 expression level is determined on at least two occasions.
  • the difference in a first CNTN1 expression level from a control level (which may be a baseline level previously determined in the patient) is determined (a first difference) and compared to a subsequent difference (second difference) which is the difference between a subsequent determined CNTN1 expression level and the control level.
  • difference 1 is less than difference 2
  • no change in the difference of CNTN1 expression levels over time indicates that the disease is not progressing (or treatment may be effective)
  • a decrease in the difference of CNTN1 expression levels over time indicates disease remission (or treatment is effective).
  • the present method advantageously provides a means to determine risk of, progression of and/or metastasis of, prostate cancer in a patient.
  • CNTN1 exists at the surface of tumor cells in prostate cancer. Its surface presence and its association with prostate cancer stem cells, the source of the disease progression and metastasis, make CNTN1 an ideal candidate to be used to diagnose patients who are at risk of, or may have prostate cancer, and thus, are at risk of developing metastatic prostate cancer.
  • a method of treating prostate cancer in a mammal comprising administering to the mammal a CNTN1 inhibitor.
  • CNTN1 activity may be inhibited at the protein level, for example, using inhibitors designed to block CNTN1 either directly or indirectly.
  • CNTN1 inhibitors may include biological compounds, and synthetic small molecules or peptide mimetics, for example, based on such biological compounds.
  • Biological CNTN1 inhibitors also include immunological inhibitors such as monoclonal antibodies prepared using the well- established hybridoma technology as described above.
  • CNTN1 antibodies can also be conjugated with a toxic anti-tumour agent such that the antibody delivers the anti-tumour agent to CNTN1 - positive prostate cancer cells.
  • CNTN1 may also be inhibited at the nucleic acid level using nucleic acid-based inhibitors such as anti-sense inhibitors, and RNA interference inhibitors, e.g. siRNA, shRNA and the like.
  • nucleic acid-based inhibitors such as anti-sense inhibitors, and RNA interference inhibitors, e.g. siRNA, shRNA and the like.
  • CNTN1 -encoding nucleic acid molecules may be used to prepare antisense oligonucleotides effective to bind to CNTN1 nucleic and inhibit the expression thereof.
  • antisense oligonucleotide as used herein means a nucleotide sequence that is complementary to at least a portion of a target CNTN1 nucleic acid sequence.
  • oligonucleotide refers to an oligomer or polymer of nucleotide or nucleoside monomers consisting of naturally occurring bases, sugars, and intersugar (backbone) linkages.
  • the term also includes modified or substituted oligomers comprising non-naturally occurring monomers or portions thereof, which function similarly. Such modified or substituted oligonucleotides may be preferred over naturally occurring forms because of properties such as enhanced cellular uptake, or increased stability in the presence of nucleases.
  • the term also includes chimeric oligonucleotides which contain two or more chemically distinct regions. For example, chimeric oligonucleotides may contain at least one region of modified nucleotides that confer beneficial properties (e.g. increased nuclease resistance, increased uptake into cells) as well as the antisense binding region.
  • two or more antisense oligonucleotides may be linked to form a chimeric oligonucleotide.
  • the antisense oligonucleotides of the present invention may be ribonucleic or deoxyribonucleic acids and may contain naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil.
  • the oligonucleotides may also contain modified bases such as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and other alkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine, 8- aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyl adenine and other 8-substituted adenines, 8-halo guanines, 8 -amino guanine, 8-thiol guanine, 8-thiolalkyl guanines, 8-hydrodyl guanine and other 8-substituted guanines, other aza and deaza uracils, thymidines, cytosines, adenines, or guanines, 5-tri-fiuoro
  • antisense oligonucleotides of the invention may contain modified phosphorous, oxygen heteroatoms in the phosphate backbone, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages.
  • the antisense oligonucleotides may contain phosphorothioates, phosphotriesters, methyl phosphonates and phosphorodithioates.
  • the antisense oligonucleotides may contain a combination of linkages, for example, phosphorothioate bonds may link only the four to six 3 '-terminal bases, may link all the nucleotides or may link only 1 pair of bases.
  • the antisense oligonucleotides of the invention may also comprise nucleotide analogs that may be better suited as therapeutic agent.
  • An example of an oligonucleotide analogue is a peptide nucleic acid (PNA) in which the deoxribose (or ribose) phosphate backbone in the DNA (or RNA), is replaced with a polyamide backbone which is similar to that found in peptides.
  • PNA analogues have been shown to be resistant to degradation by enzymes and to have extended lives in vivo and in vitro. PNAs also form stronger bonds with a complementary DNA sequence due to the lack of charge repulsion between the PNA strand and the DNA strand.
  • oligonucleotide analogues may contain nucleotides having polymer backbones, cyclic backbones, or acyclic backbones.
  • the nucleotides may have morpholino backbone structures (U.S. Pat. No. 5,034,506).
  • Oligonucleotide analogues may also contain groups such as reporter groups, protective groups and groups for improving the pharmacokinetic properties of the oligonucleotide.
  • Antisense oligonucleotides may also incorporate sugar mimetics as will be appreciated by one of skill in the art.
  • Antisense nucleic acid molecules may be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art based on a given CNTN1 nucleic acid sequence such as that provided herein.
  • the antisense nucleic acid molecules of the invention, or fragments thereof, may be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed with mRNA or the native gene, e.g. phosphorothioate derivatives and acridine substituted nucleotides.
  • the antisense sequences may also be produced biologically.
  • an antisense encoding nucleic acid is incorporated within an expression vector that is then introduced into cells in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense sequences are produced under the control of a high efficiency regulatory region, the activity of which may be determined by the cell type into which the vector is introduced.
  • RNA silencing technology can be applied to inhibit expression of CNTN1.
  • Application of nucleic acid fragments such as siRNA and shRNA fragments that correspond with regions in a CNTN1 transcript and which selectively target a CNTN1 transcript may be used to block CNTN1 expression. Such blocking occurs when the siRNA or shRNA fragments bind to the CNTN1 transcript thereby preventing translation thereof to yield functional CNTN1.
  • SiRNA, small interfering RNA molecules, or shRNA, small hairpin RNA molecules, corresponding to CNTN1 mRNA are made using well-established methods of nucleic acid syntheses as outlined above with respect to antisense oligonucleotides.
  • siRNA and shRNA to block CNTN1 expression can be confirmed using a CNTN1 -expressing cell line.
  • selected siRNA/shRNA may be incubated with a CNTN1 -expressing cell line under appropriate growth conditions. Following a sufficient reaction time, i.e. for the siRNA or shRNA to bind with CNTN1 mRNA to result in decreased CNTN1 expression, the reaction mixture is tested to determine if such a decrease has occurred. Suitable siRNA/shRNA will prevent processing of the CNTN1 transcript to yield functional CNTN1 protein. This can be detected by assaying for CNTN1 activity in a cell-based assay, for example, to identify expression of a reporter gene that is regulated by CNTN1 binding, as described in more detail herein.
  • siRNA/shRNA fragments useful in the present method may be derived from specific regions of CNTN1 -encoding nucleic acid which may provide more effective inhibition of gene expression, for example, the 3' end of the transcript, including the 3' untranslated portion.
  • useful siRNA fragments may not correspond exactly with a CNTN1 target gene, but may incorporate sequence modifications, for example, addition, deletion or substitution of one or more of the nucleotide bases therein, provided that the modified siRNA retains its ability to bind to the target CNTN1 gene.
  • Selected siRNA fragments may additionally be modified in order to yield fragments that are more desirable for use. For example, siRNA fragments may be modified to attain increased stability in a manner similar to that described for antisense oligonucleotides.
  • oligonucleotides determined to be useful to inhibit CNTN1 gene expression may be used in a therapeutic method to treat a mammal having prostate cancer.
  • a selected oligonucleotide may be introduced into tissues or cells of the mammal using techniques in the art including vectors (retroviral vectors, adenoviral vectors and DNA virus vectors) or by using physical techniques such as microinjection.
  • a CNTN1 inhibitor may be administered by any route suitable to come in contact with and inhibit target CNTN1.
  • Suitable administrable routes include, but are not limited to, oral, subcutaneous, intravenous, intraperitoneal, intranasal, enteral, topical, sublingual, intramuscular, intra-arterial, intramedullary, intrathecal, inhalation, ocular, transdermal, vaginal or rectal means.
  • the inhibitor may be coated or encased in a protective material to prevent undesirable degradation thereof by enzymes, acids or by other conditions that may affect the therapeutic activity thereof.
  • the selected CNTN1 inhibitor may be used alone or combined with at least one pharmaceutically acceptable adjuvant, in the treatment of metabolic syndrome in accordance with an embodiment of the invention.
  • pharmaceutically acceptable means acceptable for use in the pharmaceutical and veterinary arts, i.e. not being unacceptably toxic or otherwise unsuitable.
  • pharmaceutically acceptable adjuvants are those used conventionally with peptide- or nucleic acid- based drugs, such as diluents, excipients and the like. Reference may be made to "Remington's: The Science and Practice of Pharmacy", 21st Ed., Lippincott Williams & Wilkins, 200S, for guidance on drug formulations generally. The selection of adjuvant depends on the intended mode of administration of the composition.
  • the compounds are formulated for administration by infusion, or by injection either subcutaneously or intravenously, and are accordingly utilized as aqueous solutions in sterile and pyrogen-free form and optionally buffered or made isotonic.
  • the compounds may be administered in distilled water or, more desirably, in saline, phosphate- buffered saline or 5% dextrose solution.
  • compositions for oral administration via tablet, capsule or suspension are prepared using adjuvants including sugars, such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and derivatives thereof, including sodium carboxymethylcellulose, ethylcellulose and cellulose acetates; powdered tragancanth; malt; gelatin; talc; stearic acids; magnesium stearate; calcium sulfate; vegetable oils, such as peanut oils, cotton seed oil, sesame oil, olive oil and corn oil; polyols such as propylene glycol, glycerine, sorbital, mannitol and polyethylene glycol; agar; alginic acids; water; isotonic saline and phosphate buffer solutions.
  • sugars such as lactose, glucose and sucrose
  • starches such as corn starch and potato starch
  • Creams, lotions and ointments may be prepared for topical application using an appropriate base such as a triglyceride base. Such creams, lotions and ointments may also contain a surface active agent. Aerosol formulations may also be prepared in which suitable propellent adjuvants are used. Other adjuvants may also be added to the composition regardless of how it is to be administered, for example, anti-microbial agents may be added to the composition to prevent microbial growth over prolonged storage periods.
  • the amount of inhibitor administered to a mammal to treat prostate cancer will vary with a variety of factors such as the individual and condition being treated, as well as the inhibitor utilized.
  • a dosage of inhibitor including oligonucleotide or other inhibitors (such as antibody inhibitors or other inhibitors as above) sufficient to result in silencing of CNTN1 expression to result in inhibition of target prostate cancer cells is used, for example, an amount of oligonucleotide to result in at least about 50% CNTN1 gene silencing, or a greater amount of gene silencing, e.g. about 60%, 70%, 80%, 90% or more.
  • a CNTN1 inhibitor may be administered to a mammal in conjunction with a second therapy or therapeutic agent to facilitate treatment of the prostate cancer.
  • the second therapy may be administered simultaneously with the inhibitor, either in combination or separately.
  • the second therapy may be administered prior or subsequent to the administration of the CNTN1 inhibitor.
  • the second therapy is a chemotherapeutic agent that is also useful to treat prostate cancer, or to treat side effects associate therewith.
  • the second therapy may also be androgen deprivation therapy.
  • a method of determining tamoxifen resistance in breast cancer comprising determining the level of CNTN1 in breast cancer cells, comparing the CNTN1 level to a control level, and determining that the mammal has tamoxifen resistant breast cancer when the level of CNTN1 in the sample is greater than the control level.
  • CNTN1 has been determined to facilitate the development of tamoxifen resistance in breast cancer.
  • CNTN1 was detected in breast cancer cells, including an aggressive HER2+ breast cancer cell line (HCC1954).
  • Ectopic expression of CNTN1 increases tamoxifen resistance in tamoxifen sensitive cells, while tamoxifen resistant cells express higher levels of CNTN1 than a control level expressed in tamoxifen sensitive cells (e.g. by at least about 10% or greater, e.g. by 20%, 30%, 40%, 50% or greater.
  • Cell lines and plasmids All cell lines used was purchased from American Type Culture Collection (ATCC) and cultured in MEM (DU145), F12 (PC3), RPMI-1640 (LNCaP, HCC1954, BT549, BT474, T47D, ZR-75-1) and DMEM (MCF7, MDAMB453) supplemented with 10% FBS (Sigma Aldrich) and 1% Penicillin-Streptomycin (Life Technologies).
  • the androgen- independent LNCaP derivative C4-2 was kindly provided by Dr. Martin Gleave at The University of British Columbia, Vancouver, BC, Canada (as described in Thomas et al. Mol Cancer Ther 10, 347-59 (2011)).
  • C4-2 was cultured in RPMI-1640 media supplemented with 5% FBS and 1% Penicillin-Streptomycin.
  • CNTN1 shRNA was purchased from Santa Cruz Biotechnology (Santa Cruz) and CNTN1 isoform 3 cDNA was purchased from Open Biosystems.
  • DU145 prostate cancer stem-like cells were isolated and propagated as previously published (Rybak et al. Biochim Blophys Acta 1813, 683-694 (2011)). Briefly, DU145 monolayer cells were individualized with TrypLE Express solution (Life Technologies) and subsequently resuspended at a density of 5,000 cells/mL in serum-free (SF) media (DMEM/F12 at a 3:1 mixture) (Life Technologies) supplemented with 0.4% bovine serum albumin (BSA) (Bioshop Canada Inc) and 0.2x B27 minus Vitamin A (Life Technologies) in T75 flasks. Typical spheres were formed in 10 to 12 days.
  • SF serum-free
  • BSA bovine serum albumin
  • Hairpin shRNAs (control/Ctrl and CNTN1) were expressed by a retroviral -based shRNA vector (Santa Cruz Biotechnology). Knockdown of CNTN1 was carried out according to published conditions (Yan et al. PLoS One 8, e65463 (2013)). Briefly, a gag-pol-expressing vector, a rev-expressing vector and an envelope-expressing vector (VSV-G) (Stratagene) were transiently co-transfected with a designed retroviral plasmid into 293T cells. The virus- containing medium was harvested 48 hours later, filtered through a 0.45 ⁇ M filter, and centrifuged at 20,000 g for 120 minutes to concentrate the retrovirus. Polybrene (10 ug/ml, Sigma Aldrich) was added before infection and cells were selected for stable integration with puromycin (lpg/mL, Sigma Aldrich).
  • Human CNTN1 isoform 3 cDNA was further modified to generate the full length isoform 1 of CNTN1 as previously described (Yan et al. PLoS One 8, e65463 (2013)). Briefly, PCR primers were synthesized flanking the C-terminus fragment present in isoform 1, but missing in isoform 3. The C-terminus fragment was then ligated with the commercially purchased isoform 3. The full length isoform 1 cDNA for CNTN1 was subsequently cloned into a retroviral vector, pBabe. Overexpression of CNTN1 was carried out using a gag-pol expressing vector and an envelope expressing vector (VSV-G) (Stratagene, Mississauga, ON). All steps were carried out in the same manner described above for the knockdown of CNTN1. The pBabe vector without CNTN1 was used as an empty vector control.
  • VSV-G envelope expressing vector
  • Cell lysates were prepared in a buffer containing 20 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 25 mM sodium pyrophosphate, 1 mM NaF, 1 mM ⁇ -glycerophosphate, 0.1 mM sodium orthovanadate, 1 mM PMSF, 2 ⁇ g/ml leupeptin and 10 Mg/ml aprotinin (Sigma Aldrich). A total of 50 ⁇ g of cell lysates were separated on SDS-PAGE gel and transferred onto Amersham hybond ECL nitrocellulose membranes (Amersham).
  • Membranes were blocked with 5% skimmed milk and then incubated with the indicated antibodies at 4°C overnight. Appropriate HRP-conjugated secondary antibodies were incubated for one hour at room temperature. Signals were detected using an ECL Western Blotting Kit (Amersham).
  • the primary and secondary antibodies and the concentrations used were: anti- CNTN1 (1:200, R&D systems, AF904), Anti-AKT Ser473 phosphorylation (1:1000, Cell Signaling, 9271), anti-AKT (1:1000, Santa Cruz Biotechnology, 1618), anti-E-cadherin (1 :2500, BD Biosciences, 610181), anti-actin (1 :1000, Santa Cruz Biotechnology, 1615), anti-goat (1 :3000, Santa Cruz Biotechnology, sc2473), anti-mouse (1 :3000, GE Healthcare, NA931 V) and anti-rabbit (1 :3000, GE Healthcare, NA934V).
  • Prostate tissues were collected from patients who underwent prostate biopsies or radical prostatectomy at St. Joseph's Hospital in Hamilton, Ontario, Canada under the approval from the local Research Ethics Board (REB# 11-3472) and with consent from patients.
  • TMA 2 and TMA 5 were obtained from the Cooperative Prostate Cancer Tissue Resource.
  • TMA2 was organized according to Gleason score and consisted of 1,128 prostate tissue cores that were derived from 250 PC tumors, 32 non-tumor cases, and 58 high grade prostatic intra-epithelial neoplasia (HGPIN) cases.
  • TMA5 is a large patient cohort mat was organized according to the biochemical recurrence of PC.
  • Biochemical recurrence in Cooperative Prostate Cancer Tissue Resource was defined as an increase in serum PSA levels > 0.6 ng/ml (single value) or consecutive rise in serum PSA levels between 0.4 and 0.6 ng/ml after radical prostatectomy.
  • TMA5 contained 1,616 PC tissue cores that were derived from 404 patients based on their PC biochemical recurrence. IHC was carried out as described above with the following modifications. Primary antibody specific for CNTN1 (1:3000, Sigma Aldrich, HPA041060) was incubated with the sections overnight at 4°C. This was followed by the use of a TSATM Plus Biotin kit (Perkin Elmer) according to the manufacturer's protocol. TMA slides were scanned using a ScanScope and analyzed using ImageScope software (Aperio). All spots (stained cores) were also manually examined to exclude those spots that were either scratched or contained no prostate glands (only stromal tissue). All spots were also manually scored.
  • H-Score % positive X (intensity + 1)]. H-Scores were subtracted from the H-S cores obtained from stroma regions which were used for background subtraction. Scores were assigned to a scale of 0-3 (0- negative or background staining, 1-weak staining, 2-mod. staining, 3-strong staining). Positive staining was classified as moderate to strong staining with an H-score > 20.
  • DU145 spheres were prepared onto slides by cytospin and allowed to air dry for one hour prior to staining.
  • Cells were washed with lx PBS and fixed with 4% paraformaldehyde.
  • Primary antibody goat anti-CNTN1 (1:50, R&D systems, AF904) was incubated overnight at 4°C, washed with lxPBS and incubated with subsequent secondary antibodies for one hour.
  • Cells were mounted with DAPI mounting medium (Vector Labs). Images were taken with a fluorescence microscope (Carl Zeiss, Axiovert 200).
  • a total of 1000 cells of DU145 EV and DU145 CNTN1 cells was seeded into a 96 cell plate and incubated at 37°C for 5 days. Proliferation was measured using the WST-1 cell proliferation assay kit (Millipore) according to the manufacturer's instructions. Absorbance readings were measured with a plate reader at 420nm.
  • Modified boyden chambers were commercially purchased consisting of inserts with an 8 ⁇ m pore membrane coated with Matrigel (BD Biosciences) placed in a 24-well plate. Invasion assays were performed according to the manufacturer's procedure. Briefly, matrigel inserts were given 2 hour to rehydrate at 37°C prior to use in the presence of 0.5 ml of medium. Complete medium (0.5 ml) supplemented with 10% fetal bovine serum (FBS) was placed in the lower chamber. A total of 5 x 10 4 cells were seeded into the top chamber of the insert in 0.5 ml of serum-free medium for 22 hours. Cells that passed through the membranes were fixed and stained with crystal violet (0.5%, Sigma Aldrich). Percentage of invasive cells was calculated by dividing the number of cells passing through the 8 um pore size matrigel membrane by the number of cells migrating through the control membrane and multiplying by 100.
  • FBS fetal bovine serum
  • CNTN1 promotes prostate cancer cell invasion but not proliferation
  • CSCs cancer stem cells
  • Sphere cells were isolated from DU145 cells. These sphere cells are likely PCSCs because of their elevated (100 fold) ability for tumor initiation in NOD/SCID mice, as well as spheres can be re-isolated from xenograft tumors.
  • PCSCs DU145 sphere cells
  • gene expression between DU145 non-stem (monolayer) and PCSCs was profiled. Among a set of genes whose expression was altered was a top candidate, CNTN1. Its expression was increased approximately 10 fold in sphere cells compared to monolayer cells (Fig 1A).
  • PC3-SF serum free
  • Fig IE PC3-SF cells
  • PC3-SF cells do not proliferate slower than PC3 cells cultured in a FBS-containing medium (data not shown), adopted a different morphology, and formed significantly more colonies, suggesting that PC3-SF cells acquired PCSC characteristics.
  • LNCaP did not survive (data not shown).
  • CNTN1 The apparent cell surface localization of CNTN1 in DU145 spheres suggests CNTN1 as being functional.
  • ectopic CNTN1 was expressed in DU145 monolayer cells to a level comparable to the endogenous levels in DU145 sphere cells (Fig IF).
  • ectopic CNTN1 did not alter cell proliferation (Fig 2), but significantly enhanced DU145 cell invasion (Fig 1G).
  • a CNTN1 -expressing stable line was also constructed in LNCaP C4-2 cells (Fig 3A).
  • CNTN1 promotes prostate cancer cell invasion. CNTN1 enhances AKT activation and reduces E-cadherin expression in PC cells
  • CNTN1 - affected AKT activation during prostate tumorigenesis the status of AKT activation in xenograft tumors produced by DU145 cells was examined and it was found that CNTN1 expression was modulated. In comparison to DU145 EV cell-derived xenograft tumors, a clear elevation of AKT activation was detected in DU145 CNTN1 monolayer cell-produced xenograft tumors. Furthermore, reduction of AKT activation in xenograft tumors produced by CNTN1 -knocked down DU145 sphere cells was observed.
  • CNTN1 may reduce E-cadherin levels is in line with the above observation in which CNTN1 activated AKT and that AKT plays a role in E-cadherin downregulation.
  • overexpression of CNTN1 reduced E-cadherin levels in DU145 monolayer cells in comparison to DU145 EV cells (Fig 4C); conversely, knockdown of CNTN1 in DU145 sphere cells led to E-cadherin upregulation (Fig 4D).
  • DU145 CNTN1 cell-produced xenograft tumors exhibited lower levels of E-cadherin than DU145 EV cell-originated xenograft tumors; DU145 sphere cells in which CNTN1 was knocked down produced xenograft tumors with elevated levels of E- cadherin.
  • CNTN1 reduces E-cadherin expression concurrently with the upregulation of AKT activation.
  • CSCs are defined by their elevated ability for cancer initiation in immunocompromised mice.
  • the specific expression of CNTN1 in DU145-derived PCSCs and its role in promoting PC cell invasion and AKT activation as observed above collectively indicatae a role of CNTN1 in DU145 PCSCs-associated tumor initiation.
  • CNTN1 was knocked down in DU145 sphere cells (Fig 4B). CNTN1 downregulation did not have an apparent effect on the cell's ability to form spheres (data not shown). However, in comparison to DU145 Ctrl shRNA sphere cells, knockdown of CNTN1 significantly reduced the cell's ability on tumor initiation (Fig SA).
  • CNTN1 expression was significantly lower in xenograft tumors produced by CNTN1 knockdown sphere cells. Additionally, overexpression of CNTN1 in DU145 monolayer cells enhanced xenograft tumor formation (Fig SB). CNTN1 levels remained higher in DU145 CNTN1 cell-produced xenograft tumors than DU145 EV cell-derived tumors. CNTN1 was clearly detected at the cell surface in DU145 CNTN1 monolayer and DU145 Ctrl sphere cell produced xenograft tumors. Interestingly, despite DU145 EV monolayer cells expressing no detectable levels of CNTN1 in vitro, clusters of CNTN1 -positive cells with membrane localization were clearly detected.
  • CNTN1 -negative clusters of cells were also present in DU145 CNTN1 monolayer and DU145 Ctrl shRNA sphere cell-produced xenograft tumors.
  • CNTN1 enhances prostate cancer metastasis
  • CNTN1 promotes DU145 cell invasion (Fig 1G) suggests a role of CNTN1 in PC metastasis.
  • DU145 EV and CNTN1 cells were administrated into NOD/SCID mice via the tail vein. While DU145 EV cells formed lung metastases, it was clear that DU145 CNTN1 produced larger lung nodules, especially in the 16mm 3 or larger (16+mm 3 ) tumor group (Fig 6A).
  • CNTN1 -positive cell clusters were detected in both DU145 EV and CNTN1 -produced lung metastases, but the latter contained more CNTN1 -positive cells with higher levels.
  • a greater number of lung nodules were generated in both the ⁇ 15mm 3 and 16+mm 3 categories for DU145 CNTN1 cells (Fig 6B).
  • CNTN1 associates with prostate cancer progression
  • CNTN1 To further support a role of CNTN1 in promoting prostate cancer tumorigenesis, its expression in primary prostate cancer tissues was examined.
  • An anti- CNTN1 (Sigma) antibody specifically recognized tumor-associated CNTN1 in IHC staining, evidenced by no detectable staining with control IgG (data not shown) and the positive signals could be competed out by a CNTN1 peptide.
  • GS Gleason Score
  • GS 8-10 three pairs of local and lymph node metastasis, and 9 bone metastases were examined; CNTN1 was stained either negative or at very low levels in normal prostate glands and positively in advanced (GS8-10) prostate carcinomas.
  • CNTN1 expression was examined using two large tissue microarrays (TMAs), TMA2 and TMA5, obtained from the Cooperative Prostate Cancer Tissue Resource. TMA2 was organized according to PC progression; TMAS was arranged based on outcome. TMA2 had 1,128 prostate tissue cores covering 250 patients; TMAS contained 1,616 prostate tissue cores derived from 404 patients (http://www.cpctr.net/tma.html). All tissue cores were visually inspected.
  • Typical staining for non-tumor tissue, high grade PIN (HGPIN), GS6 and GS9 carcinomas shows the trends of elevating CNTN1 expression following PC progression. Quantification of CNTN1 staining was derived from 637 patients from those whose GS score was obtained, and revealed that levels of CNTN1 expression increased following prostate cancer progression from GS5/6 carcinomas to GS7, and to GS8/9 carcinomas (Fig 7A). The number of CNTN1 -positive cases was also increased following the progression from non-tumor prostate tissues to HGPINs and to carcinomas (Table 1).
  • CNTN1 affects pathways that are important for tumorigenesis
  • Prostate cancer progression and metastasis is a complex process involving numerous factors.
  • CNTN1 has been found to enhance tumor initiation and metastasis, and to be associated with PC progression and biochemical recurrence indicating that CNTN1 may alter the expression of tumorigenesis-relevant genes.
  • analysis of gene expression using the Affymetrix platform revealed the alterations of the expression of numerous genes by >1.5 fold following ectopic expression of CNTN1 in DU145 monolayer cells and knockdown of CNTN1 in DU145 spheres (Fig 8A, B; Table 1). The expected changes of CNTN1 in the overexpression and knockdown cells were confirmed (Fig 8 A, B; Table 1).
  • CNTN1 Knockdown of CNTN1 in spheres predominantly affected the disease of cancer, while overexpression of CNTN1 in DU145 monolayer cells alerted genes involved in cancer, immunological disorders, and inflammation (Fig 8C, top panels); the latter two processes are well known to impact tumorigenesis.
  • the main physiological processes affected by these candidate genes are organism and organ development (Fig 8C, middle panels). Consistent with CNTN1 as a neural cell adhesion protein, these genes contribute to neurological disorders (Fig 8C, top right panel) and nervous system development (Fig 8C, middle left panel).
  • CNTN1 in DU145 sphere cells affected the expression of genes functioning in cell-cell communications and maintenance, is consistent with CNTN1 being a cell surface protein and with PCSCs' role in maintaining a tumor mass (Fig 8C, bottom left panel).
  • the overexpression of CNTN1 in DU145 monolayer cells altered the expression of genes functioning in cell movement (Fig 8C, bottom right panel) and supports its role in promoting DU145 cell's invasion (Fig 1G).
  • knockdown of CNTN1 in DU145 spheres affected genes largely functioning in tumorigenesis, while overexpression of CNTN1 in DU145 monolayer cells altered genes involved in multiple processes, including tumor cell invasion (Fig 9).
  • BChE butyrylcholinesterase
  • CTCs and DTCs are heterogeneous cell populations; the current detection of CTCs relies on the presence of epithelial specific cell surface proteins, especially EpCAM.
  • EMT epithelial-mesenchymal transition
  • CNTN1 is thus attractive for these applications.
  • CNTN1 is thus attractive for these applications.
  • a robust presence of CNTN1 in the blood of a patient with castration- resistant prostate cancer was clearly detected in comparison to 3 normal controls (Fig 11). CNTN1 contributes to the development of tamoxifen resistance in breast cancer
  • breast cancer is the major malignancy affecting women worldwide. The disease shares similarities with the male-specific prostate cancer. While prostate cancer depends on androgen, a predominant proportion of breast cancer requires estrogen signaling. The biosynthesis of both hormones are intimately related. Approximately 70% of breast cancers are estrogen receptor (ER.) positive; these cancers are commonly treated by the disruption of ER signaling using tamoxifen. However, resistance commonly occurs, and is a major clinical problem in breast cancer therapy. Unfortunately, the mechanisms leading to tamoxifen resistance remains unclear.
  • CNTN1 The demonstrated role of CNTN1 in promoting prostate cancer prompted an examination of its potential role in enhancing resistance to tamoxifen.
  • CNTN1 was clearly detected in some lines, including a HER2+ line, HCC1954 (Fig 12).
  • Ectopic expression of CNTN1 in CNTN1 -negative MCF7 cells substantially elevated tumour cell survival to the cytotoxicity imposed by tamoxifen treatment (Fig 13). Tamoxifen-resistant MCF7 cells were produced by prolonged culturing (12 months) of the cells in the presence of low levels of tamoxifen.
  • CNTN1 In comparison to tamoxifen-sensitive MCF7 cells, the resistant cells displayed 1.5 fold higher CNTN1 expression (Fig 14). Collectively, this indicates the role of CNTN1 in enhancing the resistance of breast cancer to tamoxifen.
  • CNTN1 DU145 cell-derived prostate cancer stem-like cells (PCSCs) and in PC3 cells cultured under PCSC conditions was found.
  • PCSCs DU145 cell-derived prostate cancer stem-like cells
  • CNTN1 enhanced cell invasion and AKT activation, and reduced E-cadherin expression.
  • CNTN1 promoted xenograft tumor formation and lung metastasis concurrently with the upregulation of AKT activation and the downregulation of E-cadherin.
  • PC primary prostate cancer
  • CNTN1 was detected in advanced PC, lymph node and bone metastases, and is associated with PC progression and the reduction of biochemical recurrence free survival.

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  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Mycology (AREA)

Abstract

La présente invention concerne un procédé de détermination d'un cancer de la prostate, ou de la progression, la récurrence ou la métastase d'un cancer de la prostate chez un mammifère mâle. Le procédé comprend les étapes consistant à déterminer, dans un échantillon biologique obtenu à partir du mammifère, le niveau d'expression de CNTN1 dans l'échantillon, à comparer le niveau d'expression de CNTN1 à un niveau témoin, et à déterminer que le mammifère souffre d'un cancer de la prostate, lorsque le niveau d'expression de CNTN1 dans l'échantillon est supérieur au niveau témoin.
PCT/CA2015/050663 2014-07-15 2015-07-15 Contactine-1 (cntn1) pour utilisation dans des procédés de diagnostic et de traitement du cancer de la prostate Ceased WO2016008051A1 (fr)

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US201462024716P 2014-07-15 2014-07-15
US62/024,716 2014-07-15

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112877420A (zh) * 2021-02-20 2021-06-01 台州市立医院 与视网膜病变相关的生物标志物及其应用
WO2022109441A1 (fr) * 2020-11-23 2022-05-27 Genentech, Inc. Méthodes de modulation des interactions de surfaces de cellules hôtes avec le sars-cov-2
CN115808522A (zh) * 2022-11-22 2023-03-17 郑州大学 一种基于细胞免疫荧光法的人CNTN1-Ab检测方法及试剂盒

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Title
SU JL ET AL.: "Knockdown of conatctin-1 expression suppresses invasion and metastasis of lung adenocarcinoma.", CANCER RES., vol. 66, no. 5, 1 March 2006 (2006-03-01), pages 2553 - 61 *
VINARSKAJA A ET AL.: "DNA Methylation and the HOXC6 Paradox in Prostate Cancer.", CANSERS (BASEL)., vol. 3, no. 4, 27 September 2011 (2011-09-27), pages 3714 - 25 *
YAN J ET AL.: "Contactin-1 reduces E-cadherin expression via activating AKT in lung cancer.", PLOS ONE., vol. 8, no. 5, 28 May 2013 (2013-05-28), pages e65463 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022109441A1 (fr) * 2020-11-23 2022-05-27 Genentech, Inc. Méthodes de modulation des interactions de surfaces de cellules hôtes avec le sars-cov-2
CN112877420A (zh) * 2021-02-20 2021-06-01 台州市立医院 与视网膜病变相关的生物标志物及其应用
CN112877420B (zh) * 2021-02-20 2022-05-27 台州市立医院 与视网膜病变相关的生物标志物及其应用
CN115808522A (zh) * 2022-11-22 2023-03-17 郑州大学 一种基于细胞免疫荧光法的人CNTN1-Ab检测方法及试剂盒

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