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WO2009030789A1 - Procédé de diagnostic et/ou de pronostic du cancer du sein - Google Patents

Procédé de diagnostic et/ou de pronostic du cancer du sein Download PDF

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WO2009030789A1
WO2009030789A1 PCT/ES2008/000563 ES2008000563W WO2009030789A1 WO 2009030789 A1 WO2009030789 A1 WO 2009030789A1 ES 2008000563 W ES2008000563 W ES 2008000563W WO 2009030789 A1 WO2009030789 A1 WO 2009030789A1
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snaih
oligonucleotide
cells
expression
breast cancer
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David OLMEDA CASADOMÉ
Gema Moreno Bueno
Francisco PORTILLO PÉREZ
Amparo CANO GARCÍA
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Consejo Superior de Investigaciones Cientificas CSIC
Universidad Autonoma de Madrid
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Universidad Autonoma de Madrid
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    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
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    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention has its field of application within the health sector, mainly that related to cancer. Specifically, it is aimed at methods of diagnosis and / or prognosis of breast cancer based on the determination of the SnaiH transcription factor.
  • the local tumor invasion represents the first stage of the metastatic cascade of carcinomas.
  • carcinoma cells require profound changes in the migratory, polarity and cell adhesion properties of tumor cells, collectively known as epithelial-mesenchymal transition (TEM).
  • TEM epithelial-mesenchymal transition
  • TEM is a main development process, which allows tissue remodeling by altering cell adhesion and allowing cell migration. But in addition, this process, essential for the formation of the mesoderm during embryogenesis, can also be used by tumor cells to escape the high-pressure environment found in the primary tumor and which extends to distant tissues forming metastases (Thiery JP. Epithelial -mesenchymal transitions in tumour progression Nat. Rev. Cancer 2002; 2 (6): 442-454; Mehlen P. et al. Metastasis: a question oflife or death. Nat. Rev. Cancer 2006; 6 (6): 449 -458).
  • cadherin-E The loss of functional cadherin-E is an essential event for TEM, being considered one of its hallmarks (Thiery JP. Epithelial-mesenchymal transitions in tumour progression. Nat. Rev. Cancer 2002; 2 (6): 442-454; Thiery JP, Et al. Complex networks orchestrate epithelial-mesenchymal transitions. Nat. Rev. Mol. CeII Biol. 2006; 7 (2): 131-142).
  • This loss is detected in the majority of invasive and metastatic carcinomas and its study has led to the characterization of the two main molecular mechanisms involved in its downward regulation: epigenetic changes and repression of transcription (Peinado H. et al. Transcriptional regulation of cadherins during development and carcinogenesis. Int. J. Dev. Biol 2004; 48 ( 5-6): 365-375).
  • Snail activation disrupts tissue homeostasis and induces fibrosis in the adult kidney. Embo J 2006). Snail factors also play important roles in other significant biological processes, including cell cycle regulation (Vega S. et al. Snail blocks the cell cycle and confers resistance to cell death.
  • Snail2 confers resistance to radiation-induced cell death to hematopoietic progenitors (Inoue A. et al. Slug a highly conserved zinc finger transcriptional repressor, protects hematopoietic progenitor cells from radiation-induced apoptosis in vivo. Cancer Cell 2002; 2 (4): 279-288; Pérez-Losada J. et al. The radioresistance biological function of the SCF / signaling pathway kit is mediated by the zinc-finger transcription factor Slug.
  • SnaiH and / or its counterpart Snail2 has been associated with the repression of cadherin-E in different series of tumors.
  • Slug Snail2 is the essential repressor of cadherin-E (Hajra, KM et al.
  • the slug zinc-finger protein represses E-cadherin in breast cancer. Cancer. Res. 62, 1613-1618,2002) .
  • Twist factor of the bHLH family, by virtue of its repressive activity on cadherin-E, has been associated with the ability of intravasation and metastasis in a mouse model of breast carcinoma, and its expression has been associated with breast carcinomas lobular (Yang et al. Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis.
  • the Snail 1 transcription factor is defined as a marker of breast cancer recurrence risk in a mouse model.
  • the authors of the present invention have determined a direct relationship between the expression of Snail 1 and the development and progression of breast carcinomas, demonstrating, by means of in vivo analysis, the main and essential role of SNAIL1 in the tumor growth potential of breast carcinoma cells.
  • FIG. 1 Analysis of the expression of cadherin-E and the regulation of mesenchymal markers after the silencing of Snail-1.
  • MDA-MB-231 cells stably transfected with shEGFP are included as controls.
  • GAPDH mRNA levels are shown as load control.
  • Western blotting of ⁇ -tubulin is Shows as charge control.
  • Figure 2 Analysis of the expression and organization of vimentin and fibronectin after stable silencing of SnaiH in MDA-MB-231 cells. Immunofluorescence analysis of the levels and subcellular localization of fibronectin, vimentin, and SnaiH -HA in MDA-MB-231 and MDA-MB-231-shEGFP (shEGFP) control cells, two stable clones generated after transfection with shSNAH (shSNAM- C2 and shSNAI1-C4) and two stable clones generated after the expression of a mutant version of mouse SnaiH (mutS) (shSNAI1-C2 / C4 + mutS clones). Bars, 20 Dm.
  • FIG. 3 Analysis of the invasive capacity of MDA-MB-231 cells after the stable silencing of SnaiM.
  • FIG. 5 Analysis of the effect of Snaih's stable silencing on the phenotype of tumors induced by MDA-MB-231 A) Histological and proliferation analysis of tumors induced by control MDA-MB-231-shEGFP cells, a clone in which Representative SNAH (shSNAI1-C2) and its corresponding control are interfered after the stable expression of the SnaiM silent mutant (shSNAI1-C2 + mutS). Low magnification (a-c) and high magnification (d-f) images are shown. Necrotic areas are indicated by arrows. The immunostaining of the Ki67 proliferation antigen is shown in the g-i panels.
  • Figure 7 Analysis of SnaiM expression in cell lines derived from lymph nodes.
  • hSNAH human SnaiH mRNA
  • hSNAI2 human Snail2
  • FIG. 8 Analysis of the proliferative properties of cells with SnaiM silenced by the incorporation of BrdU in the presence or absence of serum in the MDA-MB-231, shEGFP, shSNAI1-C2, shSNAM- cell lines
  • Figure 9 Analysis of the apoptosis induced after a 48 h treatment with 100 nM docetaxel (A) or 6 pM gemcitabine (B) in original MDA-MB-231 and MDA-MB-231 -shEGFP control cells, clones in which interferes
  • Figure 10 Analysis of the apoptosis induced after 48 h treatment with docetaxel 100 nM (A) or gemcitabine 6 pM (B) in cell lines derived from lymph nodes: original MDA-MB-231, two cell lines derived from metastasis of lymph nodes from MDA-MB-231 -shEGFP control cells (shEGFP-OI and shEGFP-2D), a cell line derived from lymph node metastases from the MDA-MB-231-shSNAI1-C2 cell line (shSNAI1 -C2-2D) and two cell lines derived from lymph node metastases from MDA-MB-231-shSNAI1-C4 (shSNAI1-C4-SM and shSNAI1-C4-OI).
  • A docetaxel 100 nM
  • B gemcitabine 6 pM
  • the invention relates to a method of diagnosis and / or prognosis of breast cancer based on the determination of the level of expression of the SnaiM transcription factor in a biological sample isolated from a subject.
  • composition comprising a therapeutically effective amount of an interfering oligonucleotide of SnaiH, for use in the treatment of breast cancer is the subject of the invention.
  • the authors of the present invention by stable silencing of the expression of the SnaiH transcription factor in human breast cancer cell lines, have shown that SnaiH plays a direct role in human breast cancer. Thus, they have shown that stable silencing of SnaiH significantly decreases the invasive phenotype of human breast carcinoma cell lines. In addition, the experiments carried out have shown that SnaiH expression is necessary, not only for local invasion and metastasis, as already described for other types of tumors, but more importantly, for primary tumor growth and have confirmed its role in the recurrence of the tumor in human breast cancer.
  • a main aspect of the invention refers to a method for the diagnosis and / or prognosis of breast cancer based on the determination of the level of expression of the SnaiM transcription factor (at the level of protein and / or mRNA) in a sample Biological isolation of a subject, comparing the results obtained with previously established reference values.
  • subject includes humans and animals, preferably mammals.
  • the biological sample comes from a tumor tissue of a subject with breast cancer.
  • the SnaiH factor refers to the previously called Snail, while Snail2 refers to Slug.
  • Snail1 / Snail2 is used in the present invention.
  • human factors the form is used
  • SNAIL1 / SNAIL2 or in short, SNAM and SNAI2, respectively.
  • Snail form is used for the mouse Snail 1 factor.
  • Diagnostic method means a trial conducted on a subject that has symptoms that could be breast cancer.
  • Prognostic method means a method that helps predict, at least in part, the course of the disease. In this sense, a subject who has previously been diagnosed with breast cancer can be analyzed to know the progress of the disease, as well as the possibility of responding favorably to a specific therapeutic treatment.
  • the determination of the level of expression of the Snail 1 transcription factor allows the tumor recurrence capacity to be evaluated.
  • the direct involvement of SnaiH in the growth of the primary tumor implies that the detection of SnaiM in a breast carcinoma at the time of diagnosis can predict the growth rate of the primary tumor, not only of its recurrence, considerably increasing the sensitivity of the diagnosis and decision making.
  • the determination of the level of expression of the SnaiH transcription factor, in the defined method allows to evaluate the growth capacity of the tumor.
  • the determination of the level of expression of the SnaiH transcription factor allows to evaluate the capacity of local tumor metastasis development.
  • the direct involvement of SnaiH in the invasive and metastatic capacity makes it possible to predict the ability to develop distant metastases from the tumor, facilitating the prognosis and decision making regarding appropriate therapeutic actions.
  • SnaiH in a breast carcinoma at the time of diagnosis also makes it possible to predict and evaluate the tumor's ability to respond to chemotherapeutic agents used in the treatment of breast carcinoma, including, but not exclusively, docetaxel and gemcitabine, used in clinic for the treatment of various tumor types in locally advanced and metastatic stages, including breast cancer, having proven its usefulness in the treatment, first in metastatic situation and, subsequently, in the most favorable adjuvant situation.
  • chemotherapeutic agents used in the treatment of breast carcinoma including, but not exclusively, docetaxel and gemcitabine
  • an interfering oligonucleotide of SnaiH in the preparation of a medicament for the treatment of breast cancer is contemplated.
  • oligonucleotide refers to both SnaiH interfering RNA sequences (siRNA) and DNA sequences that, incorporated into an expression vector, are capable of directing, once inside the cell, the synthesis of a shRNA.
  • siRNA is a double stranded RNA molecule, usually 19 bp each with 2 mismatched bases at each end ("overhangs"). Thus, it is found naturally in cells.
  • shRNA is a single-stranded RNA that forms a "loop"
  • shRNAs are synthetic siRNA precursors that are transcribe from an exogenous plasmid / virus introduced into the cell for this purpose.
  • the oligonucleotide employed is interfering RNA of
  • SnaiH double stranded, preferably with sequences shown in SEQ ID NO 1 and SEQ ID NO 2.
  • the oligonucleotide employed is DNA, preferably with sequence shown in SEQ ID NO 3.
  • an interfering oligonucleotide of SnaiH is contemplated, in the preparation of a medicament intended to reduce the formation of primary tumors in breast cancer.
  • said oligonucleotide is used in the preparation of a medicament intended to reduce the recurrence of primary tumors in breast cancer.
  • the use of the oligonucleotide in the preparation of a medicament intended to decrease the invasive capacity of the tumor in breast cancer is contemplated.
  • the oligonucleotide is used in the preparation of a medicament intended to reduce the formation of local metastases in breast cancer.
  • the use of the oligonucleotide in the preparation of a medicament intended to reduce the formation of distant metastases in breast cancer is contemplated.
  • the silencing of SnaiH in breast cancer cells increases the sensitivity to the relevant chemotherapeutic agents in breast cancer treatments, so that in another particular embodiment, the oligonucleotide is used in the preparation of a medicament intended for augmentation. of the sensitivity to chemotherapeutic agents, preferably gemcitabine and docetaxel.
  • composition comprising a therapeutically effective amount of an interfering oligonucleotide of SnaiH is contemplated, for use in the treatment of breast cancer.
  • the expression "therapeutically effective amount” refers to the amount of sequence of an oligonucleotide of the invention (siRNA) or to the amount of a gene construct (capable of generating shRNA) that allows its intracellular expression calculated to produce the desired effect and, in general, will be determined, among other causes, by the characteristics of said sequences and constructions and the therapeutic effect to be achieved.
  • the oligonucleotide included in the pharmaceutical composition is SnaiH interfering RNA (siRNA), double stranded, preferably of sequences SEQ ID NO 1 and 2.
  • siRNA SnaiH interfering RNA
  • RNA oligonucleotides can be used "naked” (naked siRNA) in their native state or with modifications that improve their stability under physiological conditions.
  • the modifications are chemical.
  • chemical modifications It is understood any modification in the chemical composition of the nucleotide of siRNA that leads to increase the stability of serum siRNA, increase thermodynamic stability, cell tropism, silencing power and pharmacokinetic properties.
  • RNA New York, NY 2003; 9 (9): 1034-1048; Li CX, Parker A, Menocal E, Xiang S, Borodyansky L, Fruehauf JH. Delivery of RNA interference. CeII cycle (Georgetown, Tex 2006; 5 ( 18): 2103-2109; lkeda Y, Taira K. Ligand-targeted delivery of therapeutic siRNA. Pharmaceutical research 2006; 23 (8): 1631-
  • any of the vehiculization systems can be used to favor the entry of the oligonucleotides into the cell.
  • liposomes, nanoparticles eg, chitosans, polyethylene glycol and oligodendromers
  • peptide complexes as well as modifications of all these that allow the targeting of the oligonucleotides to a specific cell / tissue type.
  • Peptide complexes means a set of peptides of variable composition.
  • the peptide sequence can be established so as to favor tropism at certain cell types, depending on the presence of specific cell receptors for said peptide sequence ⁇ Crombez L, Charnet A, Morris MC, Aldrian-Herrada G, Heitz F, Divita G A non-covalent peptide-based strategy for siRNA delivery. Biochemical Society transactions 2007; 35 (Pt 1): 44-46), (Ikeda Y, Taira K. Ligand-targeted delivery of therapeutic siRNA. Pharmaceutical research 2006; 23 (8): 1631-1640), (Temming K, Schiffelers RM, Molema G, Kok RJ. RGD-based strategies for selective delivery of therapeutics and imaging agents to the tumor vasculature.
  • the peptide complexes may contain peptide sequences recognizable by breast cell receptors.
  • the oligonucleotide used in the preparation of the pharmaceutical composition is DNA in the form of a plasmid, linear or forming part of a viral vector, which is capable of directing, once inside the cell, the synthesis of a shRNA.
  • the DNA has the sequence SEQ ID NO 3.
  • RNA oligonucleotides liposomes, nanoparticles (eg chitosans, polyethylene glycol and oligodendrometers) and peptide complexes
  • viral systems are used as vectors that contain in their genome the sequence that allows the synthesis of the desired shRNA.
  • retroviruses Lentiviruses, Adenoviruses, Viruses associated with
  • Adenovirus and Baculovirus are Adenovirus and Baculovirus.
  • compositions provided by the present invention can be administered by any appropriate route of administration that results in an adequate therapeutic response against breast cancer, for which said composition will be formulated in the pharmaceutical form appropriate to the route of administration chosen.
  • MDA-MB-231 human breast cancer cells and their DMEM-derived cell lines (Gibco BRL; San Diego, CA) supplemented with 10% FBS, 2 mM L-glutamine and antibiotics were grown at 37 0 C in an atmosphere of CO 2 at 5% humidified.
  • pcDNA3-Snail1-mutS-HA was constructed using pcDNA3-Snail1-HA as a template for site-directed mutagenesis following conventional protocols.
  • SEQ ID NO 6 The complete DNA sequence that appears in the vector for shRNA generation is shown in SEQ ID NO 6, which includes the specific sequence SEQ ID NO 3 and the sequence that will form the "loop" when the DNA is transcribed to RNA as shRNA. All transfections were carried out using lipofectamine (Gibco BRL). The pSuperior-shEGFP (shEGFP) and pSuperior-shSnaiH (shSNAM) vectors were transfected into MDA-MB-231 cells, and the selection was performed with puromycin 1 ⁇ g / ml for 2-4 weeks.
  • pcDNA3-Snail1-mutS-HA was transfected into the MDA-MB-231-shSNAI1-C2 / C4 cell lines and selected with G418 400 ⁇ g / ml for 4-6 weeks. Ten clones were isolated after shRNA transfection in each cell type and were individually characterized, or collected as clones pooled in the control transfections.
  • RT-PCR RT-PCR and quantitative RT-PCR ( ⁇ RT-PCR).
  • the transcription factor Slug represses E-cadherin expression and induces epithelial to mesenchymal transitions: a comparison with Snail and E47 repressors. CeII Sci 2003; 116 (Pt3): 499-511; Cano A. et al.
  • the transcription factor snail controls epithelial- mesenchymal transitions by repressing E-cadherin expression. Nature cell biology
  • PCR the cDNA of cells and tumors was synthesized using the High Capacity cDNA Archive kit (Applied Biosystems, Foster City, CA.) The qRT-PCR was carried out in a 7900HT Fast Real Time PCR (Applied Biosystems) system according to manufacturer's instructions
  • anti-vimentin (1: 2000) (Babeo, Richmond, CA), anti-Snail1 (1: 40) (Franci et al. 2006), anti-Snail2 (1: 100) (Wu et al. 2005) and anti-SPARC 15G12, (1: 100) (Novocastra Laboratories, Newcastle upon Tyne, UK), rabbit polyclonal anti-fibronectin (1: 4000) (SIGMA Chemical Co) and anti-ID1 (1: 500) (Santa Cruz
  • gelatin zymography was carried out as described in Olmeda D. et al.
  • Invasion assays were carried out on modified Boyden chambers coated with type IV collagen gel, as described in Cano A. et al.
  • the transcription factor snail controls epithelial-mesenchymal transitions by repressing E-cadherin expression.
  • Tumorigenesis spontaneous metastasis assays and obtaining lymph node derived cell lines.
  • Tumor growth was measured every two days by determining the two orthogonal external diameters using a calibrator. When the tumors reached a size of 0.4 cm 3 , they were surgically removed and treated for histology, immunofluorescence and RT-PCR analysis. A minimum of 10 tumors were generated per cell line and at least 4 different tumors derived from each cell line were analyzed.
  • the mice were left alive for another 6 months and then sacrificed.
  • the contralateral lymph nodes were then surgically removed, carefully dissected and cultured for 3-4 weeks in the presence of puromycin (except in the case of animal derivatives to which the original cells were injected) to select MDA-MB cells -231 human antibiotic resistant. Mice were housed and maintained under specific pathogen-free conditions and were used according to institutional guidelines and approved by the Use Committee for Animal Care.
  • one part of the tumor was fixed in formalin and embedded in paraffin and another was frozen in liquid nitrogen embedded in Tissue Tek OCT Compound (compound for optimum temperature cutting) and stored at - 70 0 C.
  • Paraffin sections were stained with hematoxylin and eosin or immunostained with rabbit monoclonal anti-Ki67 (1: 200) (Clone SP6, Lab Vision Corporation, CA 1 USA); the frozen sections were simultaneously immunostained with rabbit anti-MMP-9 (1: 200), rat anti-CD31 (1: 300)
  • Drug sensitivity was analyzed with the minimum concentration of drugs that induces an 80% reduction in cell growth compared to untreated cells (IC80). Exponentially growing cells were treated with 100 mM docetaxel (Taxotere®, Aventis Pharma SA, Paris, France)
  • the treated and control cells were then treated with trypsin and, after centrifugation and washed twice with PBS, stained with annexin-V-FITC / propidium iodide using the Annexin V / FITC kit (International MBL, Woburn, MA) according Manufacturer's instructions. Stained cells were analyzed by flow cytometry. The results show the mean ⁇ SD of three independent trials.
  • Example 1 Stable silencing of SnaiH in MDA-MB-231 cells increases cadherin-E transcripts and decreases the expression of mesenchymal markers.
  • MDA-MB-231 the dedifferentiated human breast cancer cell line MDA-MB-231.
  • the original MDA-MB-231 cells are highly tumorigenic and weakly metastatic cells, which have high levels of SnaiH and Snail2 and no cadherin-E expression.
  • MDA-MB- cells were transfected
  • This mutant form of SnaiH was obtained by genetic engineering so that it was not recognized by the Snail 1 siRNA by carrying 5 silent point mutations (mutS), as described in the "Generation of vectors" section. Following the selection antibiotic (G480, 400 Dg / ml), 'assembled cells (shSNAI1-C2 + mutS and shSNAI1-C4 + mutS). As an additional control, an irrelevant shRNA sequence against EGFP was stably transfected into MDA-MB-231 cells (shEGFP cells).
  • mice SnaiH mutS mRNA is detected in PCR and qRT-PCR reactions using oligonucleotides against human SnaiH ( Figure 1A, upper panel and Figure 1 B, left panel), as well as in the cross-reaction of the antibodies against SnaiH ( Figure 1C, upper panel), which explains the increased levels of SnaiM in the cells carrying the allele Mutant SnaiH, although the modest expression of endogenous human Snail 1 in these cells cannot be completely ruled out.
  • the expression and nuclear localization of the Snail 1 mutS protein in the overexpression clones was also confirmed by immunofluorescence with anti-HA antibodies ( Figure 2).
  • SnaiH is an authentic inducer of TEM (Cano A. et al. The transcription factor snail controls epithelial-mesenchymal transitions by repressing E-cadherin expression. Nature cell biology 2000; 2 (2): 76-83;) was analyzed the effect of SnaiH silencing on MDA-MB-231 cells in some epithelial (cadherin-E) (hE-CD) and mesenchymal (fibronectin and vimentin) markers. Stable SnaiH silencing in MDA-MB-231 cells induced a modest increase in E-cadherin mRNA levels up to 2.5 times, measured by qRT-PCR (Figure 1, right).
  • Example 2 Stable silencing of SnaiH inhibits the invasive behavior of MDA-MB-231 cells.
  • transcript levels of another reported SnaiH target MMP2 (Miyoshi A. et al. Snail and SIP1 increase cancer invasion by upregulating MMP family in hepatocellular carcinoma cells. British journal of cancer 2004; 90 (6): 1265 - 1273; Yokoyama K. et al. Increased invasion and matrix metalloproteinase-2 expression by Snail induced mesenchymal transition in squoamous cell carcinomas.
  • the indicated cell lines were injected into the mammary adipose panicle of female nude mice 8 weeks old.
  • the incidence is represented as the percentage of mouse-induced tumors at 2 months after injection.
  • Table 1 the incidence of primary tumors induced by the original MDA-MB-231 cells and the control MDA-MB-232-shEGFP cells was very high (80-90% of the injected sites), but MDA-MB-231-shSNAI1 cells induced tumors with much lower frequencies (only 30-40% of the injected sites).
  • Immunofluorescence analyzes of invasive and angiogenic markers in tumors confirmed the data obtained by qRT-PCR.
  • a strong increase in the expression of ID2 with marked cytoplasmic localization was observed ( Figure 5B, gi), while a reduction in the number of MMP-9 and CD31 positive cells was observed in tumors induced by shSNAI1-C2 cells ( Figure 5B, ac and df, respectively).
  • Example 4 The expression of SnaiH favors the metastasis of distant lymph nodes.
  • mice with dissected lymph nodes 1/6 of mice injected with MDA-MB-231 (named 231-20), 2/6 of mice in which injected MDA-MB-231-shEGFP (called shEGFP-OI and shEGFP-2D), 1/6 of mice injected with shSNAI1-C2 (called shSNAI1-C2-2D) and 2/6 of the mice in which shSNAI1-C4 (called shSNAI1-C4-SM and shSNAI1-C4-OI) was injected ( Figure 6A).
  • the expected size of the PCR amelogenin amplicon is 977 bp for females and 788 bp for males.
  • SnaiH expression was analyzed in the cell lines derived from the lymph node. Surprisingly, SnaiH expression was detected by Western-type RT-PCR and Western blotting, in the five lymph node derived cell lines, regardless of whether the origin of the primary tumor was from MDA-MB-231-shEGFP control cells or from the silenced clones MDA-MB-231-shSNAI1. The levels of SnaiM detected were similar or even higher than those observed in the original MDA-MB-231 cells ( Figure 7 A, B).
  • the in vivo data confirm the strong selective pressure in favor of the cells expressing SnaiM and Snail2 within the subpopulation of more aggressive / metastatic MDA-MB-231 breast carcinoma cells.
  • Example 5 The stable silencing of SnaiM confers sensitivity to Ia chemotherapy
  • lymph node derived cell lines shSNAI1-C2-2D, shSNAI1-C4-SM and shSNAI1-C4-OI
  • any of the drugs induced a low apoptotic response, similar to that shown by the original and control cells (Figure 10), which further confirms the association between SnaiH, tumorigenic / metastatic behavior and resistance to apoptosis in MDA-MB-231 cells.

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Abstract

La présente invention concerne un procédé de diagnostic et/ou de pronostic du cancer du sein fondé sur la détermination du niveau d'expression du facteur de transcription Snail 1 dans un prélèvement biologique isolé d'un sujet et sur la comparaison des résultats obtenus avec des valeurs de référence. L'invention concerne aussi l'utilisation d'oligonucléotides interférents de Snail 1 dans la préparation d'un médicament destiné au traitement du cancer du sein, ainsi que des compositions pharmaceutiques basées sur une quantité thérapeutiquement efficace de ces oligonucléotides.
PCT/ES2008/000563 2007-08-28 2008-08-19 Procédé de diagnostic et/ou de pronostic du cancer du sein Ceased WO2009030789A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060003956A1 (en) * 2004-03-03 2006-01-05 Casadome David O Materials and methods for the derepression of the E-cadherin promoter
WO2007025231A2 (fr) * 2005-08-26 2007-03-01 The Trustees Of The University Of Pennsylvania Methodes mettant en oeuvre un represseur transcriptionnel snail

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060003956A1 (en) * 2004-03-03 2006-01-05 Casadome David O Materials and methods for the derepression of the E-cadherin promoter
WO2007025231A2 (fr) * 2005-08-26 2007-03-01 The Trustees Of The University Of Pennsylvania Methodes mettant en oeuvre un represseur transcriptionnel snail

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
HERNANDEZ-VARGAS H. ET AL.: "Gene expression profiling of breast cancer cells in response to gemcitabine: NF-kB pathway activation as a potential mechanism of resistance", BREAST CANCER RESEARCH AND TREATMENT, vol. 102, April 2007 (2007-04-01), pages 157 - 172, XP019478136, ISSN: 0167-6806 *
OLMEDA D. ET AL.: "SNAI1 is required for tumour growth and lymph node metastasis of human breast carcinoma MDA-MB-231 cells", CANCER RESEARCH, vol. 67, no. 24, December 2007 (2007-12-01), pages 11721 - 11731, XP055352340, ISSN: 0008-5472 *
OLMEDA D. ET AL.: "Snail silencing effectively supresses tumour growth and invasiveness", ONCOGENE, vol. 26, no. 13, March 2007 (2007-03-01), pages 1862 - 1874, XP008091741, ISSN: 0950-9232 *

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