WO2009030789A1 - Method for the diagnosis and/or prognosis of breast cancer - Google Patents

Abstract

The invention relates to a method for the diagnosis and/or prognosis of breast cancer, by determining the expression level of transcription factor Snail 1 in a biological sample isolated from a subject and comparing the results obtained to reference values. The invention relates to the use of Snail1 interference oligonucleotides in the preparation of a drug intended for the treatment of breast cancer as well as pharmaceutical compositions based on a therapeutically effective quantity of said oligonucleotides.

Description

 METHOD OF DIAGNOSIS AND / OR FORECAST OF BREAST CANCER

DESCRIPTION

FIELD OF THE INVENTION

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.

BACKGROUND OF THE INVENTION

The local tumor invasion represents the first stage of the metastatic cascade of carcinomas. The invasion of carcinoma cells requires profound changes in the migratory, polarity and cell adhesion properties of tumor cells, collectively known as epithelial-mesenchymal transition (TEM).

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).

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).

In recent years, several repressors of the transcription of the cadherin-E gene have been characterized. Among these, two members of the Snail zinc finger family, SnaiM (Snail) and Snail2 (Slug), have emerged as principal regulators of TEM during tumor development and progression (Thiery JP. Et al. Complex networks orchestrate epithelial- mesenchymal transitions Nat. Rev. Mol. CeII Biol. 2006;

7 (2): 131-142; Hairstyle H. et al. Snail, Zeb and bHLH factors in tumour progression: an alliance against the epithelial phenotype? Nat Rev Cancer 2007; 7 (6): 415-428; Grandson MA. The snail superíamily of zinc-finger transcription factors. Nat Rev Mol CeII Biol 2002; 3 (3): 155-166; Hairstyle H. et al. Transcriptional regulation of cadherins during development and carcinogenesis. Int. J. Dev. Biol 2004; 48 (5-6): 365-375), as well as in other pathologies, such as renal fibrosis (Boutet A. et al. 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.

Genes Dev 2004; 18 (10): 1131-1143), the induction of movement and cell survival (Barrallo-Gimeno A. et al. The Snail genes as inducers of cell movement and survival: implications in development and cancer. Development 2005 ; 132 (14): 3151-3161).

The expression of 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. Oncogene 2003; 22 (27): 4205-4211) by means of repression of the target of p53, PUMA, a Bcl-2 antagonist (Wu WS. et al. Slug antagonizes p53-mediated apoptosis of hematopoietic progenitors by repressing puma. Cell 2005; 123 (4): 641-653). SnaiH expressing cells survive serum deprivation and are resistant to apoptosis induced by pro-apoptotic stimuli or by genotoxic agents (Vega S. et al. Snail blocks the cell cycle and confers resistance to cell death. Genes Dev 2004 ; 18 (10): 1131-1143; Kajita M. et al. Aberrant expression of the transcription factors snail and slug alters the response to genotoxic stress. Molecular and cellular biology 2004; 24 (17): 7559-7566). The full action of SnaiH can have a main effect on the growth, survival and / or invasive behavior of tumor cells, as well as on tumor progression (Thiery JP et al. Complex networks orchestrate epithelial-mesenchymal transitions. Nat. Rev Mol. Cell Biol. 2006; 7 (2): 131-142; Peinado H. et al. Snail, Zeb and bHLH factors in tumor progression: an alliance against the epithelial phenotype? Nat Rev Cancer 2007; 7 (6): 415-428; Barrallo-Gimeno A. et al. The Snail genes as inducers of cell movement and survival: implications in development and cancer. Development 2005; 132 (14): 3151-3161).

Based on these findings, documents ES2161612, ES2161655 and ES2234619 describe the use of the SnaiH transcription factor as a diagnostic and tumor progression marker, by determining the invasive and metastatic capacity of an epithelial tumor.

Recently, evidence has been provided in favor of a major role of SnaiH in tumorigenesis, since stable silencing of SnaiH in epidermal carcinoma cell lines of mouse skin leads to a more differentiated and less invasive phenotype, with a reduction Drastic potential for tumor growth in vivo (Olmeda D. et al. Snail silencing effectively suppresses tumor growth and invasiveness. Oncogene 2007,26 (13): 1862-187 '4).

In the specific case of breast cancer, the expression of SnaiH and / or its counterpart Snail2 (Slug) has been associated with the repression of cadherin-E in different series of tumors. However, the specific contribution of any factor to the advancement of breast cancer has not been determined. Thus, there are studies that suggest that 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) . Later studies, in primary breast carcinomas and metastasis, support the role of Snail2, together with Snaih or alone, in different types of metastatic spread of breast carcinomas (Martin TA. et al. Expression of the transcription factors snail, slug and twist and their clinical sigriif ¡canee in human breast cancer Ann Surg Oncol 2005; 12 (6): 488-496; Elloul S. et al. Snail, slug and Smad-interacting protein 1 as novel parameters of disease aggresiveness in metastatic ovarian and breast carcinoma. Cancer 2005; 103 (8): 1631-1643) as well as the association between the expression of Snail2 and recurrence of breast carcinomas (Come C. et al. Snail and Slug play a distinct roles during breast carcinoma progression. 2006. p 5395-5402). Likewise, other studies have shown the expression of other cadherin-E repressors in breast carcinomas and their association with tumor progression. The 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. CeII, 117, 927-939, 2004), as well as the ability to invade and angiogenesis (Mironchik et al. Twist overexpression induces in vivo angiogenesis and correlates with chromosomal instability in breast cancer, Cancer Res, 65, 10808-10809,2005) and / or worse prognosis of ductal carcinomas of the breast (Martin TA. et al. Expression of the transcription factors snail, slug and twist and their clinical signif canee in human breast cancer Ann Surg Oncol 2005; 12 (6) A88-496). Therefore, the previous references show the joint participation of SnaiH and other E-cadherin repressors in the progression of breast carcinomas.

On the other hand, the expression of Snail 1 has been associated with the repression of cadherin-

E, lymph node status and metastasis, through statistical correlation studies (Zhou BP. Et al. Dual regulation of Snail by GSKSbetamed mediated phosphorylation in control of epithelial-mesenchymal transition. Nature cell biology 2004.6 (10) : 931-940; Blanco MJ et al. Correlation of Snail expression with histological / grade and lymph node status in breast carcinomas.

2002; 21 (20): 3241-3246; Chen CW. et al. Mechanisms of inactivation of E-cadherin in breast carcinoma: modification of the two hit hypothesis of tumor suppressor gene. Oncogene 2004; 20 (29): 3814-3823; Come C. et al. Snail and Slug play a distinct roles during breast carcinoma progression. 2006. p 5395-5402), as well as with The recurrence of the tumor in a mouse model of breast cancer and, importantly, some analysis of gene expression of breast cancer microarrays (available in databases) correlated the elevated expression of SnaiH with the decrease in free survival of recurrence (Moody SE. et al. The transcriptional repressor Snail promotes mammary tumour recurrent. Cancer cell

2005; 8 (3): 197-209). Likewise, in document WO2007025231 the Snail 1 transcription factor is defined as a marker of breast cancer recurrence risk in a mouse model.

However, none of the documents of the state of the art has demonstrated experimentally and / or functionally that the expression of SnaiH is sufficient to influence the development and / or progression of breast carcinomas, since the lack of appropriate antibodies against SnaiH has so far prevented the establishment of a direct relationship between the expression of Snail 1 and histopathological findings. In addition, no functional studies on the role of Snail 1 ih live in human breast carcinoma cells have been described so far.

Based on the needs of the state of the art, the authors of the present invention have conducted important studies of experimentation of loss of function in breast carcinoma cell lines in humans, by means of stable silencing of Snail 1.

Thus, for the first time, 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.

These results have allowed to develop a method of diagnosis and / or prognosis of breast cancer where the determination of the Snail 1 transcription factor allows to evaluate the capacity of growth, recurrence and development of metastasis, local and distant, of the tumor, as well as evaluate the ability of the tumor to respond to chemotherapeutic agents. In addition, the developments made by the authors of the present invention, give rise to the use of SnaiH silencing as a therapeutic agent, not only in anti-metastatic treatments, but also in treatments of tumor sensitivity, increasing the chemosensitivity of the carcinoma cells of Human breast versus clinically relevant chemotherapeutic agents in breast cancer.

DESCRIPTION OF THE FIGURES

Figure 1. Analysis of the expression of cadherin-E and the regulation of mesenchymal markers after the silencing of Snail-1. A) RT-PCR analysis of the levels of human SnaiH mRNA (hSNAM) and mouse SnaiH (mSnail) in original MDA-MB-231 cells, two stable clones generated after the transfection of shSNAM (shSNAI1-C2 and shSNAI1-C4 ) and two stable clones generated after the expression of a mutant version of mouse SnaiH (mutS), not recognized by ShSNAH, in shSNAI1-C2 and shSNAI1-C4 (shSNAM-C2 + mutS, shSNAI1-C4-mutS) cells. MDA-MB-231 cells stably transfected with shEGFP (MDA-MB-231-shEGFP) are included as controls. GAPDH mRNA levels are shown as load control. B) qRT-PCR analysis of the levels of human SnaiH mRNA (hSNAH), human Snail2 (hSNAI2) and human cadherin-E (hE-CD) of the cell lines described in A. C) Western blot analysis of Protein levels of human / mouse SnaiH (h / mSNAI1), human Snail2 (hSNAI2) and mesenchymal markers fibronectin and vimentin in independent clones and control cells indicated in A and B. Western blotting of α-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.

Figure 3. Analysis of the invasive capacity of MDA-MB-231 cells after the stable silencing of SnaiM. A) qRT-PCR analysis of MMP2, SPARC, ID1 and ID2 mRNA levels in original MDA-MB-231 and MDA-MB-231-shEGFP control (shEGFP) cells, shSNAM stable clones (shSNA1-C2 and - C4) and stable clones obtained after the expression of the silent mutant of Mouse Snail (mutS) in shSNA1-C2 and -C4 cells. B) Western blot analysis of the total protein levels of human ID1 and SPARC in independent clones and control cells, indicated as in A. Western blot of α-tubulin is shown as load control. C) Zymography test of the activity of secreted MMP-9 performed in the conditioned medium of the cells described in A. The activity of MMP-2 is shown as a control. D) Analysis of the invasive phenotype of the cell lines indicated in A, grown on filters coated with a type IV collagen matrix. The results show the mean ± SD of three independent trials. ANOVA analysis; ** p <0.01. Figure 4. Analysis of the tumorigenic potential of MDA-MB-231 cells after SnaiH silencing. A) Analysis of the tumor growth potential (tumor latency and growth rate) of the MDA-MB-231 »shEGFP -o—, ShSNAI 1-C2 -" -, shSNAI1C4 -o—, shSNAI1-C2 + mutS cell lines → - and shSNAI1-C4 + mutS - ° -, after orthotopic injection into the mammary adipose panicle of immunocompromised mice.The results show the mean ± SD of two independent tests performed with 5 mice / cell line each. ANOVA analysis: ^*** p <0.001. B) RT-PCR analysis of the expression of human SnaiH and Snail2 (hSNAM and hSNAI2) and mouse SnaiH (mSnail) performed on RNA samples isolated from individual tumors generated by the indicated cell lines in A. Two tumors of each cell type indicated are shown. GAPDH mRNA levels are included as a load control. C) qRT-PCR analysis of cadherin-E mRNA levels (hE-CD), SPARC, ID1 and human ID2 performed on RNA samples isolated from individual tumors generated by the clones indicated above. The results show the mean ± Of two independent tumors for each clone.

Figure 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. B) Immunofluorescence analysis of the tumor sections of the cell lines indicated above. Sections show staining for MMP9 (a-c), CD31 (d-f) and ID2 (g-i). Detection of apoptotic cells by TUNNEL test (j-l). Bars, 50 Dm. Figure 6. Characterization of cell lines derived from lymph nodes in nu / nu mice. A) Schematic representation of the experimental design; 1. Tumor growth and extraction; 2. Selection of antibiotic resistant MDA-MB-231 cells; 3. Cell lines obtained from lymph nodes of the contralateral limbs. B) PCR analysis to determine the presence of human amelogenin allele in the cell lines indicated in

A. C) PCR analysis of the presence of the H1 expression cassette of the pSuperior-shRNA vector in the indicated cell lines. The expected sizes of the pSuperior-shRNA control (281 bp) and pSuperior-shRNA containing shEGFP or ShSNAM (345 bp) are indicated.

Figure 7. Analysis of SnaiM expression in cell lines derived from lymph nodes. A) qRT-PCR analysis of the levels of human SnaiH mRNA (hSNAH) and human Snail2 (hSNAI2) of original MDA-MB-231 cells, two cell lines derived from lymph node metastases from MDA-MB-231 cells -shEGFP control (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 metastasis from MDA-MB-231-shSNAI1-C4 (shSNAI1-C4-SM and shSNAI1-C4-Ol). B) Western blot analysis of protein levels of SnaiH (SNAI1) and Snail2 (SNAI2) human in original MDA-MB-231 cells and clones derived from independent lymph nodes as indicated in A. The Western blot of D-tubulin is shown as load control. C) Analysis of the tumorigenic potential of 231-shSNA-C2-2D and 231-shSNA-C4-SM cells compared to the original MDA-MB-231 by orthotopic injection in the mammary adipose panicle of immunocompromised mice.

Figure 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

C2 + mutS, shSNAM -C4 and shSNAI1-C4 + mutS.

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

SnaiH stably (shSNAI1-C2 and -C4) and stable clones obtained after the expression of mutant mouse SnaiH (mutS). The results show the mean ± SD of three independent trials. ANOVA analysis: ^** p <0.01; * ^* * p <0.001.

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).

OBJECT PE THE INVENTION In the first place, 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.

The use of an interfering oligonucleotide of SnaiH in the preparation of a medicament for the treatment of breast cancer is also object of the present invention.

Finally, a pharmaceutical 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.

DESCRIPTION OF THE INVENTION

Due mainly to the lack of appropriate antibodies against the SnaiH transcription factor, until now no functional studies on the role of SnaiH in vivo in human breast carcinoma cells have been described, which has not allowed establishing a direct relationship between the expression of SnaiH and histopathological findings in human breast cancer.

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.

Based on these advances, they have developed new applications in the field of diagnosis, prognosis and therapy of breast cancer, providing a more sensitive diagnosis, and facilitating the prognosis and decision making regarding appropriate therapeutic actions. Thus, 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.

The term "subject", as used in the present invention, 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. In general, the designation Snail1 / Snail2 is used in the present invention. When referring to human factors, the form is used

SNAIL1 / SNAIL2 or in short, SNAM and SNAI2, respectively. Likewise, the abbreviated 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.

In a particular embodiment of the invention, the determination of the level of expression of the Snail 1 transcription factor allows the tumor recurrence capacity to be evaluated. In addition, 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. Thus, in another particular embodiment of Ia invention, 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.

In another particular embodiment, the determination of the level of expression of the SnaiH transcription factor allows to evaluate the capacity of local tumor metastasis development. In addition, 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.

The detection of 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.

In another main aspect of the invention, the use of an interfering oligonucleotide of SnaiH in the preparation of a medicament for the treatment of breast cancer is contemplated.

In the present invention, the term 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"

(hairpin) folding over itself to give a double chain structure. This "loop" is cut in the cytoplasm by the enzyme DICER resulting in a double stranded siRNA. Therefore, shRNAs are synthetic siRNA precursors that are transcribe from an exogenous plasmid / virus introduced into the cell for this purpose.

In a preferred embodiment, the oligonucleotide employed is interfering RNA of

SnaiH, double stranded, preferably with sequences shown in SEQ ID NO 1 and SEQ ID NO 2.

In another preferred embodiment, the oligonucleotide employed is DNA, preferably with sequence shown in SEQ ID NO 3.

Specific silencing of SnaiH by stable RNA interference induces a decrease in mesenchymal (fibronectin, vimentin) and pro-invasive (MMP9, ID1, SPARC) markers in breast cancer cells (eg MDA-MB -231), concomitantly with a decrease in invasive behavior in vitro. And what is more important, the stable interference of SnaiH in said cells leads to a drastic reduction in tumor incidence in vivo and a two-fold increase in tumor latency. Tumors induced by silenced cells show extensive necrotic regions and a significant decrease in invasive and angiogenic markers.

Thus, in particular embodiments of the invention, the use of an interfering oligonucleotide of SnaiH is contemplated, in the preparation of a medicament intended to reduce the formation of primary tumors in breast cancer.

In another particular embodiment, said oligonucleotide is used in the preparation of a medicament intended to reduce the recurrence of primary tumors in breast cancer.

In another particular embodiment, 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. In another particular embodiment, the oligonucleotide is used in the preparation of a medicament intended to reduce the formation of local metastases in breast cancer.

In another particular embodiment, the use of the oligonucleotide in the preparation of a medicament intended to reduce the formation of distant metastases in breast cancer is contemplated.

In addition, 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.

Finally, in another main aspect of the invention a pharmaceutical composition comprising a therapeutically effective amount of an interfering oligonucleotide of SnaiH is contemplated, for use in the treatment of breast cancer.

In the present invention, 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.

In a particular embodiment, the oligonucleotide included in the pharmaceutical composition is SnaiH interfering RNA (siRNA), double stranded, preferably of sequences SEQ ID NO 1 and 2.

RNA oligonucleotides can be used "naked" (naked siRNA) in their native state or with modifications that improve their stability under physiological conditions.

Preferably, the modifications are chemical. By 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.

Examples of chemical modifications are those contemplated in the LNA Technology (Locked Nucleid Acid) (Braasch DA, Jensen S, Liu Y, et al. RNA interference in mammalian cells by chemically-modified RNA. Biochemistry 2003; 42 (26): 7967- 7975), phosphothiates (Crooke ST. Potential roles of antisense technology in cancer chemotherapy. Oncogene 2000; 19 (56): 6651-6659), waste added to the ribose chain: Chiu YL, Rana TM. siRNA function in RNAi: a Chemical modification analysis. 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-

1640

Any of the vehiculization systems, available or in current development, can be used to favor the entry of the oligonucleotides into the cell. In a particular embodiment, 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. Drug Resist Updat 2005; 8 (6): 381-402). Thus, in a particular embodiment, the peptide complexes may contain peptide sequences recognizable by breast cell receptors. In another particular embodiment of the invention, 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. Preferably, the DNA has the sequence SEQ ID NO 3.

The DNA sequences must not only enter the cell but must reach the nucleus so that it can be transcribed. Therefore, apart from using the same vehiculization systems as 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. Among the systems used are Retroviruses, Lentiviruses, Adenoviruses, Viruses associated with

Adenovirus and Baculovirus.

The pharmaceutical 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.

These results have allowed the development of new applications in relation to the diagnosis, prognosis and treatment of breast cancer, which could be extensible to other types of carcinomas and melanomas, based on the expression of SnaiH in different types of tumors.

EXAMPLES

The materials and methods used in the realization of the examples are described below:

Cell culture

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.

Generation of expression vectors and stable cell lines

The generation of the pcDNA3-Sna¡M-HA vector has already been described (Peinado H. et al.

Snail mediates E-cadherin repression by the recruitment of the Sin3A / histone deacetylase 1 (HDAC1) / HDAC2 complex. Molecular and cellular biology 2004,24 (1): 306-319). pcDNA3-Snail1-mutS-HA was constructed using pcDNA3-Snail1-HA as a template for site-directed mutagenesis following conventional protocols. The oligonucleotides used for plasmid amplification, with five silent point mutations, were: SEQ ID NO 4 and SEQ ID NO 5. It has been recently described (Jorda M. et al. Upregulation of MMP-9 in MDCK epithelial cell Une in response to expression of the Snail transncription factor J. CeII Sci 2005; 118 (Pt 15) .3371-3385) the generation of shRNA, which contains specific oligonucleotide sequences against EGFP (enhanced fluorescent green protein) or mouse / human Snaih (SEQ ID NO 3), cloned into the pSuperior-Puro vector (Oligoengine, Seattle, WA). In said vector, shRNA transcription is directed by the H1 promoter. 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 μg / 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 μg / 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 and quantitative RT-PCR (αRT-PCR).

Total RNA was isolated from the different cell lines and RT-PCR analysis was carried out using primers specific for mouse or human Snail 1 and GAPDH (Gliceraldehyde 3 phosphate dehydrogenase) {Bolos V. et al. 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

2000; 2 (2): 76-83; Pérez-Moreno et al. A new role for E12 / E47 in the repression of E-cadherin expression and epithelial-mesenchymal transitions. J Biol Chem 2001; 276 (29): 27424-27431). The RT-PCR of the tumors was performed as described in Peinado H. et al. A molecular role for lysyl oxidase-like 2 enzyme in snail regulation and tumor progression. Embo J 2005; 24 (1 '9): 3446-3458). For the qRT-

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

For RT-PCR of human Snail2, the direct (SEQ ID NO 7) and reverse primers (amplify an 810 bp fragment) (SEQ ID NO 8) were used.

For the analysis of qRT-PCR of cells and tumors, the following primers were used: human SNAH, SEQ ID NO 9 and SEQ ID NO 10; Human SNAI2, SEQ ID NO 11 and SEQ ID NO 12; Human E-cadherin (CDH1), SEQ ID NO 13 and SEQ ID

NO 14; Human SPARC, SEQ ID NO 15 and SEQ ID NO 16; Human MMP2, SEQ ID NO 17 and SEQ ID NO 18. qRT-PCR was performed for ID genes as described above (Xu, et al. 2005).

Cell extracts and Western blot analysis. Whole cell extracts were obtained using the RIPA buffer (50 mM Tris-HCI, pH 7.5, 150 mM NaCI, 1% NP-40, 0.5% deoxycholate, 0.1% SDS), containing protease inhibitors For Western blot analysis, 50 μg of total proteins from the different extracts were loaded in 7.5, 10 or 12% SDS-PAGE gels. Transfer, blocking and incubation with appropriate antibodies was carried out as described in Bolos V. et al. The transcription factor Slug represses E-cadherin expression and induces epithelial to mesenchymal transitions: a comparison with Snail and E47 repressors. Cell 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 2000; 3 (3): 155-166; Pérez-Moreno et al. A new role for E12 / E47 in the repression of E-cadherín expression and epithelial-mesenchymal transitions. J Biol Chem 2001; 276 (29): 27424-27431). Primary antibodies used included: mouse monoclonal anti-D-tubulin (1: 1000) (SIGMA Chemical Co, St. Louis, MO), 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

Biotechnology, Santa Cruz, CA).

Jelly Zimopraphy

The gelatin zymography was carried out as described in Olmeda D. et al.

Snail silencing effectively suppresses tumour growth and invasiveness. Oncogene 2007; 26 (13): 1862-1874 and Jorda M. et al. Upregulation of MMP-9 in MDCK epithelial cell Une in response to expression of the Snail trasneription factor. J. CeII Sci 2005; 118 (Pt 15): 3371-3385). In summary, the cells were grown in normal growth media and serum-free media were placed in confluent cultures for 24 hours to collect the conditioned media. 12 μg of conditioned medium was separated and analyzed in 7.5% SDS-PAGE gels containing 0.1% gelatin and treated as described above (Olmeda D. et al. Snail silencing effectively suppresses tumour growth and invasiveness Oncogene 2007; 26 (13): 1862-1874 and Jorda M. et al. Upregulation of

MMP-9 in MDCK epithelial cell Une in response to expression of the Snail trasneription factor. J. Cell Sci 2005; 118 (Pt 15): 3371-3385). The gels were stained with Coomassie Brilliant Blue R250 and gelatinolytic activities were detected as transparent bands against the blue background.

Invasion tests

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. Nature cell biology 2000; 3 (3): 155-166; Pérez-Moreno et al. A new role for E12 / E47 in the repression of E-cadherin expression and epithelial- mesenchymal transitions. J Biol Chem 2001; 276 (29): 27 '424-27'431; Olmeda D. et al. Snail silencing effectively suppresses tumour growth and invasiveness.

Oncogene 2007; 26 (13): 1862-1874, starting with 1x10 ⁶ seeded cells and counting the cells in the lower part of the filter 24 hours after planting.

Tumorigenesis, spontaneous metastasis assays and obtaining lymph node derived cell lines.

Original human MDA-MB-231 cells and clones derived from subconfluent cultures (1x10 ⁶ in 0.05 ml of serum-free growth medium) were injected orthotopically into the breast adipose panicle of female Balb / c nude immunocompromised mice, 8 weeks old of age (Charles River, Wilmington, MA).

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. For the spontaneous metastasis test, after the surgical removal of the primary tumor, 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.

Immunohistochemical analysis, RT-PCR and TUNNEL of primary tumors

For immunohistochemical staining, 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)

(Chemicon, Billerica, MA), rabbit polyclonal anti-ID2 (1: 100) (Santa Cruz Biotechnology) and appropriate secondary antibodies, as described in Peinado H. et al. A molecular role for lysyl oxidase-like 2 enzyme In snail regulation and tumor progression. Embo J 2005; 24 (19) .3446-3458. For DNA fragmentation analysis, cryostat sections fixed with paraformaldehyde were analyzed by the TUNNEL method using the CeII Death Detection in situ kit (Roche, Basel, Switzerland) according to the manufacturer's protocol. RT-PCR analysis was performed on tumor sections frozen in liquid nitrogen after careful removal of all surrounding skin, as described in Olmeda D. et al. Snail silencing effectively suppresses tumour growth and invasiveness.

Oncogene 2007; 26 (13): 1862-1874 and in Peinado H. et al. A molecular role for lysyl oxidase-like 2 enzyme in snail regulation and tumor progression. Embo J 2005; 24 (19): 3446-3458.

Drug sensitivity tests

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)

(Hernández-Vargas, et al. Molecular profiling of docetaxel cytotoxicity in breast cancer cells: uncoupling of aberrant mitosis and apoptosis. Oncogene 2007; 26 (20): 2902-2913) or gemcitabine 6 pM (Gemzar®, Lilly SA, Indianapolis, IN) (Hernández-Vargas H. et al. Gene expression profiling of breast cancer cells in response to gemcitabine: NF-kappaB pathway activation as a potential mechanism of résistance. Breast cancer research and treatment 2007; 102 (2): 157-172 ) for 48 hours. 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.

To directly analyze the role of SnaiH in the metastasis and tumor growth of breast carcinomas, the expression of

SNAH, without affecting the expression of Snail2, in 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. In order to obtain clones with stably silenced SnaiH, MDA-MB- cells were transfected

231 with a pSuperior-shSNAH vector designed to recognize both human and mouse SnaiH mRNAs and recently described as capable of effectively silencing SnaiH in mouse carcinoma cells (Olmeda D. et al. Snail silencing effectively suppresses tumour growth and invasiveness Oncogene 2007.26 (13): 1862-1874). After the selection with the puromycin selection antibiotic, 1 Dg / ml, at least ten clones were isolated and characterized to determine the expression of SnaiH; among them, clones MDA-MB-231-shSNAI1-C2 and -C4 (hereinafter referred to as shSNAI1-C2 and shSNAI1-C4) were selected as the most representative (Figure 1A, B). As a control, a mutant form of mouse SnaiH was stably transfected in the shSNAI1-C2 and shSNAI2-C4 clones. 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). As shown in Figure 1A, the stable expression of shSNAM led to the almost complete inhibition of SnaiH expression, both at the level of mRNA, and of the protein in the cells MDA-MB-231, without significantly affecting the expression of Snail2 (Figure 1 B and C, comparison of the original control cells and MDA-MB-231 with shSNA1-C2 and C4). Overexpression of the mutant form of mouse SnaiH (mutS) in the shSNA1-C2 + mutS and shSNA1-C4 + mutS cells was confirmed at the mRNA and protein levels, obtaining similar or even higher levels of SnaiH expression to those observed in the original MDA-MB-231 cells (Figure 1 AC). Due to the high conservation of the mouse and human SnaiM sequences (Nieto MA. The snail superfamily of zinc-finger transcription factors. Nat Rev Mol CeII Biol 2002; 3 (3): 155-166; Manzanares M et al. The increasing complexity of the Snail gene superfamily in metazoan evolution. Trenos Genet 2001; 17 (4): 178-181), mouse 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). Since 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). Importantly, the increase in cadherin-E transcripts detected in the shSNAI1-C2 and -C4 clones was completely reversed by the overexpression of the mutS allele (Figure 1B, compare the levels of shENAI1-C2 hE-CD and - C4 with shSNAI1-C2 + mutS and -C4 + mutS, respectively). Despite the increase in mRNA levels, the expression of the cadherin-E protein could not be detected in the clones in which SnaiH interferes. On the contrary, a strong decrease in the expression and / or organization of the fibronectin and vimentin mesenchymal markers was observed in the shSNAI1-C2 and -C4 clones (Figure 1C and Figure 2), an effect also suppressed by the overexpression of the silent mouse mutant, mutS-Snail (Figure 1C and Figure 2). The detection of SnaiH muts, which carries the HA epitope, was only detected in the nucleus of shSNAI1-C2-mutS and shSNAI1-C4-mutS cells, as expected (Figure 2, panels on the right). Taken together, these results confirmed that SnaiH silencing induces a transition from mesenchyme to partial epithelium in MDA-MB-231 cells.

Example 2. Stable silencing of SnaiH inhibits the invasive behavior of MDA-MB-231 cells.

Next, the effect of SnaiH silencing on the invasive behavior of MDA-MB-231 cells was studied. To this end, the recently described levels of SnaiH gene expression, involved in the invasion and metastasis, such as MMP2, SPARC, ID1 and the intimately related protein ID2 (Jorda) were analyzed first by qRT-PCR. M. Et al. Ld-1 is induced in MDCK epithelial cells by activated Erk / MAPK pathway in response to expression of the Snail and E47 trasncription factors. Exp CeII Res 2007; Minn AJ. Et al. Genes that mediated breast cancer metastasis to lung. Nature 2005; 436 (7050): 518-524; Moreno-Bueno. Et al. Genetic profϊling of epithelial cells expressing e-cadherin repressors reveal a distinct role for snail, slug and e47 factors in epithelial-mesenchymal trasnition. Cancer Research 2006.66 (19): 9543-9556; Stighall M. et al. High ID2 protein expression correlates with a favourale prognosis in patients with primary breast cancer and reduces cellular invasiveness of breast cancer cells. International journal of cancer 2005: 115 (3 ): 403-411 ) in MDA-MB-231 cells with SnaiH silenced. The silencing of SnaiH induced a sharp decrease in the expression of SPARC and ID1 in the mRNA (Figure 3A) and in the levels of total protein (Figure 3B). In contrast, ID2 mRNA levels increased strongly after SnaiH silencing. Importantly, the effects of SnaiH silencing on the expression of the SPARC and ID genes were completely reversed after the overexpression of the mutS-SnaiH allele (Figure 3 A, B; compare shSNAI1-C2 and -C4 with shSNAI1-C2 + mutS and -C4 + mutS and the original MDA-MB-231 cells), which supports a specific effect of SnaiH silencing on these genes. On the other hand, 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.

International journal of oncology 2003; 22 (4): 891-898) did not vary after SnaiH silencing or the overexpression of mutS-SnaiH in MDA-MB-231 cells (Figure 3A, upper panel), because MMP2 levels secreted in the original MDA-MB-231 cells were very low (Figure 3C). In contrast, the original MDA-MB-231 cells showed high levels of MMP9 metalloproteinase activity, another target of SnaiH (Jorda M. et al. Upregulation of MMP- 9 in MDCK epithelial cell Une in response to expression of the Snail transcription factor J. Cell Sci 2005; 118 (Pt 15): 337 '1-3385), and SnaiH silencing actually reduced the activity of MMP9 (Figure 3C), an effect completely reversed by the overexpression of the mutS-SnaiH allele (Figure 3C, compare shSNAI1-C2 and C4 with C2-mutS and C4 + mutS, respectively, and the original cells), which confirmed the specific effect of Snail 1 silencing on the regulation of MMP9.

To verify that the molecular changes observed after SnaiM silencing in MDA-MB-231 cells could influence its invasive phenotype, in vitro invasion tests were performed on cultures grown on filters (0.8 μm pore) (transwells) coated with a matrix of type IV collagen. The cells were plated above the matrix and allowed to migrate for 24 hours. After this time, the cells that had migrated to the lower chamber were treated with trypsin and counted. The results show the mean ± SD of three independent trials. ANOVA analysis; ^** p <0.01. As shown in Figure 3D, the silencing of SnaiH dramatically reduced the ability of MDA-MB-231 cells to migrate in collagen IV matrices and a reversal of more than 50% in invasion capacity after expression was achieved. of mutS-SnaiMen the clones with SnaiH silenced (3D figure, compare shSNAH-

C2 and -C4 with shSNAI1-C2-mutS and -C4-mutS, respectively, and with the original cells), which confirms that SnaiH-specific silencing confers a less invasive phenotype on MDA-MB-231 cells. Example 3. SnaiH interference dramatically decreases the tumorigenic properties of MDA-MB-231 cells.

The two clones with independent silenced SnaiH, shSNAI1-C2 and C4 and the derived cells after the expression of mutS-SnaiH (C2 + mutS and C4 + mutS), together with the original MDA-MB-231 and MDA-MB-231 cells -shEGFP control were injected orthotopically into the mammary adipose panicle of nude mice. Table 1 shows comparative data on the incidence of the tumor of the original MDA-MB-231, MDA-MB-231-shEGFP control cells, clones in which SnaiH interferes stably (C2 and C4) and stable clones obtained after the expression of the silent mutant mouse SnaiH (mutS). 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. As shown in 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). Significantly, the incidence of the primary tumors recovered completely after the expression of the mutS-SnaiH form (table 1, compare shSNAI1-C2 or C4 with shSNAI1-C2 + mutS and -C4 + mutS and the control cells).

Table 1. Comparative tumor incidence data.

In addition, a drastic reduction in the growth rate of the Cell-induced tumors with silenced SnaiH (Figure 4A). A reduction of more than 95% in the volume of tumors induced by shSNA1-C2 and C4 clones was observed at 40 days after injection, together with a 2-fold increase in tumor latency (days to reach a size of tumor of 0.1 cm ³ ) compared to tumors induced by the original and control cells. Importantly, the tumor growth rate recovered almost completely after the expression of the mutS-SnaiH allele again (Figure 4A, compare shSNA1-C2 or shSNA1-C4 with C2 + mutS and C4 + mutS, respectively, and with the cells control).

The analysis by RT-PCR of the tumors derived from the different cell lines indicated that the SnaiH transcripts remained silenced in all tumors derived from the shSNAM clones, while no significant changes were observed in the expression of the homologous Snail2 gene (Figure 4B ). In fact, the silencing of Snail2 in the MDA-MB-231 cells did not produce any difference in the growth rate or incidence of the tumor, which additionally supports a specific effect of SnaiH silencing in the tumorigenic behavior of the MDA-MB cells. -231 breast carcinoma.

Interestingly, an up to 6-fold increase in cadherin-E mRNA levels and a sharp decrease in transcript levels were detected.

SPARC in tumors induced by shSNAI1-C2 and -C4 cells (Figures 4C, upper panels). Similar to the behavior observed in vitro, these expression changes were annulled in tumors induced by shSNAl cells expressing the mutS-SnaiH allele (Figure 4C, above). The regulation of the expression of ID2 was also maintained between the ex vivo and in vitro situation with an increase of up to 1.8 times in the levels of mRNA detected in tumors induced by the cells with silenced SnaiH (Figure 4C, panels of ID2 ). Surprisingly, the levels of ID1 mRNA detected in tumors induced by shSNAl clones and mutS-SnaiH derived cells showed the inverse pattern to that observed in culture (Figure 4C panel of ID1), which indicates a differential mechanism of Ia regulation for the expression of ID1 between the situation

'ex vivo and in vitro.

To further understand the effect of SnaiH silencing on the Phenotype of tumors induced by MDA-MB-231, paraffin sections of tumors of similar size generated by different cell clones were analyzed by histology and immunohistochemistry. No significant differences were detected in the histology of the tumors induced by shSNAI1-C2 cells with respect to those induced by control C2 cells or that overexpress mutS-

SnaiH (Figure 5A, a-f). However, main differences were found in the number and extent of necrotic areas detected within the tumors, which were more prominent in cell derivatives with silenced SnaiH (Figure 5A, a-c). Concomitantly with the increase in necrotic areas, an increase in the number of apoptotic cells (up to 27 times) was detected within tumors derived from clones with silenced SnaiH, compared to control cell tumors (Figure 5B , jk). In addition, immunostaining against the Ki67 proliferation marker showed that the silencing of SnaiH induced a drastic reduction in the proliferative potential of the tumor (Figure 5A, g-h). 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). The changes in the previous markers and in the proliferative and anti-apoptotic tumor potential were fully recovered in tumors induced after the expression of the mutS-SnaiH allele in shSNA1-C2 cells (Figure 5A, B, compare the right panels with the rest ), Which confirmed the specific effect of SnaiH silencing on the tumorigenic behavior of MDA-MB-231 cells.

Example 4. The expression of SnaiH favors the metastasis of distant lymph nodes.

Once the effect of SnaiH silencing on the tumorigenic behavior of MDA-MB-231 cells was established, the consequences of the decrease in SnaiH on the metastatic capacities of MDA-MB-231 cells were analyzed. For this purpose, it was carried carried out a spontaneous metastasis test after Ia surgical removal of primary tumors induced by the injection of control cells and MDA-MB-231 with SnaiH silenced in the mammary adipose panicle. Six months later, mice were sacrificed and organs (lung, liver and spleen) were analyzed to determine the appearance of metastatic lesions. No macrometástasis was detected in any organ according to the low spontaneous metastatic potential of the original MDA-MB-231 cells (Minn AJ. Et al. Genes that measured breast cancer metastasis to the moon. NAture 2005; 436 (7050): 518- 524; Kang Y. et al. A multigenic program mediatin breast cancer metastasis to bone. CeII Cancer 2003; 3 (6): 537-549). However, inflammation of the lymph nodes of the extremities was observed in the contralateral region in most of the animals. Therefore, the existence of metastases in the distant lymph nodes was analyzed by means of the surgical removal of the contralateral lymph node. In addition, lymph node cell lines were derived, after careful dissection and growth in culture for 3-4 weeks, in the presence of puromycin, to prevent the growth of mouse cells, and MDA-MB-231 cells were selected Antibiotic resistant human. Only 6 cell lines were obtained, out of a total of 24 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). To rule out the selection of immortalized mouse cells, the presence of the human amelogenin gene, frequently used in forensic tests for human DNA and in sex determination, was analyzed by PCR (Mitchell RJ. Et al. An investigation of sequence deletions of amelogenin (AMELY), a Y-chromosome locus commonly used for gender determination, Annals of human biology 2006; 33 (2): 227-240). All the selected cell lines, except 231-20, were positive for the female form of the amelogenin gene (Figure 6B), which confirmed their human origin and their derivation of the lymph node metastasis distant from the primary tumors induced by the cells derived from MDA-MB-231 indicated. The expected size of the PCR amelogenin amplicon is 977 bp for females and 788 bp for males. Next, 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 lack of SnaiH interference observed in shSNA1-C2-2D, shSNA1-C4-SM and shSNA1-C4-OI cells derived from lymph nodes could not be attributed to the loss of the shSNAM expression cassette through DNA reorganization, since that the presence of the pSuperior-shRNA vector was still detected, which contained the insert corresponding to the control of specific 64 nucleotides or to the SNAM hairpin by PCR in all cell lines derived from lymph nodes (Figure 6C). In addition, an increase in the levels of endogenous Snail2 expression was detected, both at the mRNA and protein level, in most of the analyzed lines derived from lymph nodes, compared to those detected in MDA-MB cells. 231 originals (figure 7A, B). For additional information, the tumorigenic potential of shSNAI1-C2-2D and shSNAI1-C4-SM cells derived from lymph nodes was analyzed, together with MDA-MB- cells.

231 originals, by orthotopic injection. Tumors induced by lymph node shSNAI1-C2-2D and shSNAI1-C4-SM cells grew at the same rate as those induced by the original MDA-MB-231 cells (Figure 7C), as opposed to tumor growth potential delayed MDA-MB-231-shSNAI1-C2 and -shSNAI1-C4 cells (Figure 5B). In fact, the analysis of the expression of cadherin-E, SPARC, ID1 and ID2 in tumors induced by lymph node derived cell lines revealed levels similar to those found in tumors induced by the original MDA-MB-231 cells.

Collectively, 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

To analyze whether the resistance to apoptosis induced by genotoxic lesion can be affected by the silencing of SnaiH in the MDA-MB-231 cells, the proliferative properties of the cells with silenced SnaiH were analyzed, not detecting significant differences compared to the cells. originals when grown in the presence or absence of serum in in vitro cultures, based on the incorporation of BrdU (Figure 8).

To analyze whether SnaiM silencing could confer sensitivity to induced genotoxic lesion, the cells were treated with two different chemotherapeutic drugs, docetaxel and gencitabine, commonly used to treat breast cancer (Hernández-Vargas, et al. Molecular profiling of docetaxel cytotoxicity in breast cancer cells: uncoupling of aberrant mitosis and apoptosis Oncogene 2007; 26 (20): 2902-2913; Hernández-Vargas H. et al. Gene profiling expression of breast cancer cells in response to gemcitabine: NF-kappaB pathway activation as a potential mechanism of resistance Breast cancer research and treatment 2007; 102 (2): 157-172). Although both drugs cause a modest level of apoptosis in MDA-MB-231 cells, SnaiH silencing induced a response 2 times higher than apoptosis under both chemotherapeutic treatments, an effect canceled after the expression of the mutS-SnaiH silent mutant in the shSNA1-C2 and -C4 clones (Figure 9). Importantly, the treatment of lymph node derived cell lines (shSNAI1-C2-2D, shSNAI1-C4-SM and shSNAI1-C4-OI) with 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.

Taken together, the in vivo and in vitro data showed that SnaiH expression confers properties to breast carcinoma cells that go beyond the repression of cadherin-E and the induction of TEM, conferring the advantage of tumor growth, The resistance to chemotherapeutic drugs and the invasion and metastatic abilities.

Claims

1. Method for the diagnosis and / or prognosis of breast cancer comprising: to. determine the level of expression of the SnaiH transcription factor in a biological sample isolated from a subject, and b. compare the results obtained in a) with reference values. 2. Method according to claim 1, wherein the biological sample comes from a tumor tissue of a patient with breast cancer. 3. Method according to claim 2, wherein the determination of the level of expression of the SnaiH transcription factor allows to evaluate the tumor growth capacity. 4. Method, according to claim 2, wherein the determination of the level of expression of the SnaiH transcription factor allows to assess the recurrence capacity of the tumor. 5. Method, according to claim 2, wherein the determination of the level of expression of the SnaiM transcription factor allows to evaluate the capacity of local tumor metastasis development. 6. Method according to claim 2, wherein the determination of the level of expression of the SnaiH transcription factor allows to evaluate the ability to develop distant metastasis of the tumor. 7. Method according to claim 2, wherein the determination of the level of expression of the SnaiH transcription factor allows to evaluate the capacity of the tumor to respond to chemotherapeutic agents. 8. Use of an interfering oligonucleotide of SnaiH, in the preparation of a medicament for the treatment of breast cancer. 9. Use of an oligonucleotide, according to claim 8, wherein said oligonucleotide is SnaiH interfering RNA. 10. Use of an oligonucleotide according to claim 9, wherein the RNA is double stranded with sequences SEQ ID NO 1 and SEQ ID NO 2. 11. Use of an oligonucleotide according to claim 8, wherein said oligonucleotide is DNA. 12. Use of an oligonucleotide according to claim 11, wherein the DNA has the sequence SEQ ID NO 3. 13. Use of an oligonucleotide, according to any of claims 8-12, in the preparation of a medicament intended to reduce the formation of primary tumors in breast cancer. 14. Use of an oligonucleotide, according to any of claims 8-12, in the preparation of a medicament intended to reduce the recurrence of primary tumors in breast cancer. 15. Use of an oligonucleotide, according to any of claims 8-12, in the preparation of a medicament intended to reduce the invasive capacity of the tumor in breast cancer. 16. Use of an oligonucleotide according to any of claims 8-12, in The preparation of a medicament intended to reduce the formation of local metastases in breast cancer. 17. Use of an oligonucleotide, according to any of claims 8-12, in the preparation of a medicament intended to reduce the formation of distant metastases in breast cancer. 18. Use of an oligonucleotide, according to any of claims 8-12, in the preparation of a medicament intended to increase the sensitivity to chemotherapeutic agents. 19. Use of an oligonucleotide according to claim 18, wherein the chemotherapeutic agents are gemcitabine and docetaxel. 20. Pharmaceutical composition comprising a therapeutically effective amount of a SnaiH interfering oligonucleotide for use in the treatment of breast cancer. 21. Pharmaceutical composition according to claim 20, wherein the oligonucleotide is SnaiH interfering RNA. 22. Pharmaceutical composition according to claim 21, wherein the RNA is double stranded with sequences SEQ ID NO 1 and SEQ ID NO 2. 23. Pharmaceutical composition according to claims 21 or 22, wherein the oligonucleotide has modifications that favor its stability. 24. Pharmaceutical composition according to any of claims 21-23, further comprising a pharmaceutically acceptable carrier. 25. Pharmaceutical composition according to claim 24, wherein the vehicle is selected from liposomes, nanoparticles or peptide complexes. 26. Pharmaceutical composition according to claim 25, wherein the nanoparticles are selected from chitosans, polyethylene glycol and oligodendrometers. 27. Pharmaceutical composition, according to claim 20, comprising a plasmid or linear DNA sequence comprising the sequence coding for the sequence of a Snaih interference RNA. 28. Pharmaceutical composition according to claim 27, wherein the DNA sequence comprises the sequence SEQ ID NO 3. 29. Pharmaceutical composition according to any of claims 27-28, further comprising a pharmaceutically acceptable carrier. 30. Pharmaceutical composition according to claim 29, wherein the vehicle is selected from liposomes, nanoparticles, peptide complexes or viruses. 31. Pharmaceutical composition according to claim 30, wherein the nanoparticles are selected from chitosans, polyethylene glycol and oligodendrometers. 32. Pharmaceutical composition according to claim 30, wherein the viruses are selected from retroviruses, lentiviruses, adenoviruses, adenovirus-associated viruses or baculoviruses. 33. Pharmaceutical composition according to claim 30 wherein the peptide complexes contain peptide sequences recognizable by breast cell receptors.