ES2322834A1 - Method for the diagnosis and/or prognosis of breast cancer - Google Patents

Method for the diagnosis and/or prognosis of breast cancer Download PDF

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ES2322834A1
ES2322834A1 ES200702342A ES200702342A ES2322834A1 ES 2322834 A1 ES2322834 A1 ES 2322834A1 ES 200702342 A ES200702342 A ES 200702342A ES 200702342 A ES200702342 A ES 200702342A ES 2322834 A1 ES2322834 A1 ES 2322834A1
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snail1
oligonucleotide
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pharmaceutical composition
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ES2322834B1 (en
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David Olmeda Casadome
Gema Moreno Bueno
Francisco Portillo Perez
Amparo Cano Garcia
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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
    • A61K48/0066—Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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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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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

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

Método de diagnóstico y/o pronóstico de cáncer de mama.Method of diagnosis and / or prognosis of cancer of breast

Campo de la invenciónField of the Invention

La presente invención tiene su campo de aplicación dentro del sector sanitario, principalmente aquel relacionado con el cáncer. En concreto está dirigida a métodos de diagnóstico y/o pronóstico del cáncer de mama basados en la determinación del factor de transcripción Snail1.The present invention has its field of application within the health sector, mainly that Cancer related. Specifically it is aimed at methods of diagnosis and / or prognosis of breast cancer based on determination of the Snail1 transcription factor.

Antecedentes de la invenciónBackground of the invention

La invasión tumoral local representa la primera etapa de la cascada metastásica de los carcinomas. La invasión de las células de carcinoma requiere cambios profundos en las propiedades migratorias, de polaridad y de adhesión celular de las células tumorales, conocidos colectivamente como transición epitelio-mesénquima (TEM).The local tumor invasion represents the first stage of the metastatic cascade of carcinomas. The invasion of carcinoma cells require profound changes in the migratory, polarity and cell adhesion properties of tumor cells, collectively known as transition epithelium-mesenchyme (TEM).

La TEM es un proceso principal del desarrollo, que permite la remodelación tisular mediante la alteración de la adhesión celular y permitiendo la migración celular. Pero además, este proceso, esencial para la formación del mesodermo durante la embriogénesis, también lo pueden usar las células tumorales para escapar del entorno de alta presión encontrado en el tumor primario y que se extiende hacia los tejidos distantes formando metástasis (Thiery JP. Epithelial-mesenchymal transitions in tumour progression. Nat. Rev. Cancer 2002; 2(6): 442-454; Mehlen P. et al. Metastasis: a question of life or death. Nat. Rev. Cancer 2006; 6(6): 449-458).TEM is a main process of development, 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 tumor progression Nat. Rev. Cancer 2002; 2 (6): 442-454; Mehlen P. et al. Metastasis: a question of life or death. Nat. Rev. Cancer 2006; 6 (6): 449-458) .

La pérdida de cadherina-E funcional es un acontecimiento esencial para la TEM, considerándose uno de sus sellos distintivos (Thiery JP. Epithelial-mesenchymal transitions in tumour progresión. Nat. Rev. Cancer 2002; 2(6): 442-454; Thiery JP. Et al. Complex networks orchestrate epithelial-mesenchymal transitions. Nat. Rev. Mol. Cell Biol. 2006; 7(2):131-142). Esta pérdida se detecta en la mayoría de los carcinomas invasivos y metastásicos y su estudio ha conducido a la caracterización de los dos mecanismos moleculares principales implicados en su regulación a la baja: los cambios epigenéticos y la represión de la transcripción (Peinado H. et al. Transcriptional regulation of cadherins during development and carcinogenesis. Int. J. Dev. Biol 2004; 48(5-6):365-375).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. Cell Biol. 2006; 7 (2): 131-142) . This loss is detected in most 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) .

En los últimos años se han caracterizado varios represores de la transcripción del gen de cadherina-E. Entre éstos, dos miembros de la familia de dedos de zinc Snail, Snail1 (Snail) y Snail2 (Slug), han surgido como reguladores principales de la TEM durante el desarrollo y progresión tumoral (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 tumour progression: an alliance against the epithelial phenotype? Nat Rev Cancer 2007;7(6):415-428; Nieto MA. The snail superfamily of zinc-finger transcription factors. Nat Rev Mol Cell Biol 2002;3(3):155-166; Peinado H. et al. Transcriptional regulation of cadherins during development and carcinogenesis. Int. J. Dev. Biol 2004; 48(5-6):365-375), así como en otras patologías, tales como la fibrosis renal (Boutet A. et al. Snail activation disrupts tissue homeostasis and induces fibrosis in the adult kidney. Embo J 2006). Los factores Snail también desempeñan papeles importantes en otros procesos biológicos significativos, incluyendo la regulación del ciclo celular (Vega S. et al. Snail blocks the cell cycle and confers resistance to cell death. Genes Dev 2004;18(10):1131-1143), la inducción del movimiento y la supervivencia celular (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).In recent years, several transcription repressors of the cadherin-E gene have been characterized. Among these, two members of the Snail zinc finger family, Snail1 (Snail) and Snail2 (Slug), have emerged as major regulators of TEM during tumor development and 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 tumour progression: an alliance against the epithelial phenotype? Nat Rev Cancer 2007 ; 7 (6): 415-428; Nieto MA. The snail superfamily of zinc-finger transcription factors. Nat Rev Mol Cell Biol 2002; 3 (3): 155-166; Peinado 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 ) , 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) .

La expresión de Snail2 confiere resistencia a la muerte celular inducida por radiación a los progenitores hematopoyéticos (Inoue A. et al. Slug a highly conserved zinc finger transcriptional represor, protects hematopoietic progenitor cells from radiation-induced apoptosis in vivo. Cancer Cell 2002;2(4):279-288; Perez-Losada J. et al. The radioresistance biological function of the SCF/kit signaling pathway is mediated by the zinc-finger transcription factor Slug. Oncogene 2003; 22(27):4205-4211) mediante la represión de la diana de p53, PUMA, un antagonista de Bcl-2 (Wu WS. et al. Slug antagonizes p53-mediated apoptosis of hematopoietic progenitors by repressing puma. Cell 2005;123(4): 641-653).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; Perez-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 repression of the p53 target, 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) .

Las células que expresan Snail1 sobreviven a la privación de suero y son resistentes a la apoptosis inducida por los estímulos pro-apoptóticos o mediante agentes genotóxicos (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 expresión of the transcription factors snail and slug alters the response to genotoxic stress. Molecular and cellular biology 2004; 24(17):7559-7566). La acción completa de Snail1 puede tener un efecto principal sobre el crecimiento, la supervivencia y/o el comportamiento invasivo de las células tumorales, así como en la progresión tumoral (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 tumour progresión: 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).Snail1-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 complete action of Snail1 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) .

En base a estos hallazgos, en los documentos ES2161612, ES2161655 y ES2234619 se describe el empleo del factor de transcripción Snail1 como marcador de diagnóstico y de progresión tumoral, mediante la determinación de la capacidad invasiva y metastásica de un tumor epitelial.Based on these findings, in the documents ES2161612, ES2161655 and ES2234619 describe the use of the factor of Snail1 transcription as a diagnostic marker and of tumor progression, by determining capacity invasive and metastatic epithelial tumor.

Recientemente, se han proporcionado pruebas a favor de un papel principal de Snail1 en la tumorigénesis, puesto que el silenciamiento estable de Snail1 en líneas de células de carcinoma epidérmico de la piel del ratón conduce a un fenotipo más diferenciado y menos invasivo, con una reducción drástica en el potencial del crecimiento tumoral in vivo (Olmeda D. et al. Snail silencing effectively suppresses tumour growth and invasiveness Oncogene 2007;26(13):1862-1874). Recently, evidence has been provided in favor of a major role of Snail1 in tumorigenesis, since stable silencing of Snail1 in epidermal carcinoma cell lines of the 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): 1862-1874).

En el caso concreto del cáncer de mama, la expresión de Snail1 y/o de su homólogo Snail2, (Slug) se ha asociado con la represión de cadherina-E en diferentes series de tumores. Sin embargo, no se ha determinado la contribución específica de cualquier factor al avance del cáncer de mama. Así, existen estudios que plantean que Snail2 es el represor esencial de la cadherina-E (Hajra, K.M. et al. The slug zinc-finger protein represses E-cadherin in breast cancer. Cancer. Res. 62,1613-1618,2002). Estudios posteriores, en carcinomas de mama primarios y metástasis, apoyan el papel de Snail2, junto con Snail1 o por sí solo, en diferentes tipos de diseminación metastásica de carcinomas de mama (Martin TA. et al. Expression of the transcription factors snail, slug and twist and their clinical significance 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) así como la asociación entre la expresión de Snail2 y recurrencia de carcinomas de mama (Come C. et al. Snail and Slug play a distinct roles during breast carcinoma progresión.2006. p 5395-5402). In the specific case of breast cancer, the expression of Snail1 and / or its counterpart Snail2, (Slug) has been associated with repression of cadherin-E in different series of tumors. However, the specific contribution of any factor to the progression 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 metastases, support the role of Snail2, together with Snail1 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 significance 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 Snail2 expression and recurrence of breast carcinomas (Come C. et al. Snail and Slug play a distinct roles during breast carcinoma progression. 2006 p 5395-5402).

Asimismo, otros estudios han mostrado la expresión de otros represores de cadherina-E en carcinomas de mama y su asociación con progresión tumoral. El factor Twist, de la familia bHLH, en virtud de su actividad represora sobre cadherina-E, se ha asociado a la capacidad de intravasación y metástasis en un modelo de ratón de carcinoma de mama, y su expresión se ha asociado a carcinomas de mama lobulillares (Yang et al. Twist, a master regulador of morphogenesis, plays an essential role in tumor metastasis. Cell, 117, 927-939, 2004), así como a la capacidad de invasión y angiogénesis (Mironchik et al. Twist overexpression induces in vivo angiogenesis and correlates with chromosomal instability in breast cancer. Cancer Res, 65, 10808-10809,2005) y/o peor pronóstico de carcinomas ductales de mama (Martin TA. et al. Expression of the transcription factors snail, slug and twist and their clinical significance in human breast cancer. Ann Surg Oncol 2005;12(6):488-496). Por consiguiente, las referencias anteriores evidencian la participación conjunta de Snail1 y otros represores de cadherina-E en la progresión de carcinomas de mama.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. Cell, 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 significance in human breast cancer Ann Surg Oncol 2005; 12 (6): 488-496) . Therefore, the previous references show the joint participation of Snail1 and other E-cadherin repressors in the progression of breast carcinomas.

Por otra parte, la expresión de Snail1 se ha asociado con la represión de cadherina-E, el estado de los ganglios linfáticos y la metástasis, mediante estudios de correlación estadística (Zhou BP. et al. Dual regulation of Snail by GSK-3beta-mediated phosphorylation in control of epithelial-mesenchymal transition. Nature cell biology 2004;6(10):931-940; Blanco MJ et al. Correlation of Snail expresión with histological grade and lymph node status in breast carcinomas. Oncogene 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 progresión. 2006.p 5395-5402), así como con la recurrencia del tumor en un modelo de ratón de cáncer de mama y, de manera importante, algunos análisis de expresión génica de micromatrices del cáncer de mama (disponibles en bases de datos) correlacionaron la expresión elevada de Snail1 con la disminución de la supervivencia libre de recidiva (Moody SE. et al. The transcriptional repressor Snail promotes mammary tumour recurrent. Cancer cell 2005;8(3):197-209). Asimismo, en el documento WO2007025231 se define el factor de transcripción Snail1 como marcador del riesgo de recurrencia de cáncer de mama en un modelo de ratón.On the other hand, the expression of Snail1 has been associated with repression of cadherin-E, lymph node status and metastasis, through statistical correlation studies (Zhou BP. Et al. Dual regulation of Snail by GSK-3beta- 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. Oncogene 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 tumor recurrence in a mouse model of breast cancer and, importantly, some gene expression analyzes of Breast cancer microarrays (available in bases of data) correlated the high expression of Snail1 with the decrease in recurrence-free survival (Moody SE. et al. The transcriptional repressor Snail promotes mammary tumour recurrent. Cancer cell 2005; 8 (3): 197-209) . Also, in document WO2007025231 the Snail1 transcription factor is defined as a marker of the risk of recurrence of breast cancer in a mouse model.

Sin embargo, en ninguno de los documentos del estado de la técnica se ha demostrado experimental y/o funcionalmente que la expresión de Snail1 sea suficiente para incidir en el desarrollo y/o progresión de los carcinomas de mama, ya que la falta de anticuerpos apropiados frente a Snail1 ha impedido hasta ahora el establecimiento de una relación directa entre la expresión de Snail1 y los hallazgos histopatológicos. Además, hasta ahora no se han descrito estudios funcionales sobre el papel de Snail1 in vivo en las células de carcinoma de mama humano.However, none of the state-of-the-art documents have demonstrated experimentally and / or functionally that Snail1 expression is sufficient to influence the development and / or progression of breast carcinomas, since the lack of appropriate antibodies versus Snail1 has so far prevented the establishment of a direct relationship between the expression of Snail1 and histopathological findings. In addition, no functional studies on the role of Snail1 in vivo in human breast carcinoma cells have been described so far.

En base a las necesidades del estado de la técnica, los autores de la presente invención han realizados importantes estudios de experimentación de pérdida de la función en líneas celulares de carcinoma de mama en humanos, mediante el silenciamiento estable de Snail1.Based on the needs of the state of the technique, the authors of the present invention have performed important studies of experimentation of loss of function in human breast carcinoma cell lines, through the stable silencing of Snail1.

Así, por primera vez, los autores de la presente invención han determinado una relación directa entre la expresión de Snail1 y el desarrollo y progresión de los carcinomas de mama, demostrando, mediante análisis in vivo, el papel principal y esencial de SNAIL1 en el potencial del crecimiento tumoral de las células de carcinoma de mama.Thus, for the first time, the authors of the present invention have determined a direct relationship between the expression of Snail1 and the development and progression of breast carcinomas, demonstrating, through in vivo analysis, the main and essential role of SNAIL1 in the potential of tumor growth of breast carcinoma cells.

Estos resultados han permitido desarrollar un método de diagnóstico y/o pronóstico de cáncer de mama donde la determinación del factor de transcripción Snail1 permite evaluar la capacidad de crecimiento, de recurrencia y de desarrollo de metástasis, local y a distancia, del tumor, así como evaluar la capacidad de respuesta del tumor a agentes quimioterapéuticos.These results have allowed to develop a method of diagnosis and / or prognosis of breast cancer where the determination of the transcription factor Snail1 allows to evaluate the capacity for growth, recurrence and development of metastasis, local and distant, of the tumor, as well as assess the Tumor responsiveness to chemotherapeutic agents.

Además, los desarrollos realizados por los autores de la presente invención, dan lugar al empleo del silenciamiento de Snail1 como agente terapéutico, no sólo en tratamientos anti-metastásicos, sino también en tratamientos de sensibilidad tumoral, aumentando la quimiosensibilidad de las células de carcinoma de mama humano frente a agentes quimioterapéuticos clínicamente relevantes en el cáncer de mama.In addition, the developments made by the authors of the present invention, give rise to the use of silencing of Snail1 as a therapeutic agent, not only in anti-metastatic treatments, but also in Tumor sensitivity treatments, increasing the chemosensitivity of human breast carcinoma cells versus to clinically relevant chemotherapeutic agents in cancer of breast

Descripción de las figurasDescription of the figures

Figura 1. Análisis de la expresión de cadherina-E y de la regulación de marcadores mesenquimáticos tras el silenciamiento de Snail-1. A) Análisis RT-PCR de los niveles de RNAm de Snail1 humano (hSNAI1) y Snail1 de ratón (mSnai1) en células MDA-MB-231 originales, dos clones estables generados tras la transfección de shSNAI1 (shSNAI1-C2 y shSNAI1-C4) y dos clones estables generados tras la expresión de una versión mutante de Snail1 de ratón (mutS), no reconocida por el shSNAI1, en células shSNAI1-C2 y shSNAI1-C4 (shSNAI1-C2+mutS, shSNAI1-C4-mutS). Se incluyen como controles las células MDA-MB-231 transfectadas de manera estable con shEGFP (MDA-MB-231-shEGFP). Los niveles de RNAm de GAPDH se muestran como control de carga. B) Análisis qRT-PCR de los niveles de RNAm de Snail1 humano (hSNAI1), Snail2 humano (hSNAI2) y cadherina-E humana (hE-CD) de las líneas celulares descritas en A. C) Análisis de inmunotransferencia de tipo Western de los niveles de proteína de Snail1 humana/de ratón (h/mSNAI1), Snail2 humana (hSNAI2) y los marcadores mesenquimáticos fibronectina y vimentina en clones. independientes y células control indicadas en A y B. La inmunotransferencia de tipo Western de \alpha-tubulina se muestra como control de carga.Figure 1. Analysis of cadherin-E expression and regulation of mesenchymal markers after Snail-1 silencing . A) RT-PCR analysis of human Snail1 (hSNAI1) and mouse Snail1 (mSnai1) mRNA levels in original MDA-MB-231 cells, two stable clones generated after transfection of shSNAI1 (shSNAI1-C2 and shSNAI1-C4 ) and two stable clones generated after the expression of a mutant version of mouse Snail1 (mutS), not recognized by shSNAI1, in shSNAI1-C2 and shSNAI1-C4 (shSNAI1-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 mRNA levels of human Snail1 (hSNAI1), 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 Snail1 (h / mSNAI1), human Snail2 (hSNAI2) and mesenchymal markers fibronectin and vimentin in clones. independent and control cells indicated in A and B. Western blot of α-tubulin is shown as loading control.

Figura 2: Análisis de la expresión y organización de vimentina y fibronectina tras el silenciamiento estable de Snail1 en células MDA-MB-231. Análisis de inmunofluorescencia de los niveles y localización subcelular de fibronectina, vimentina, y Snail1-HA en células MDA-MB-231 y MDA-MB-231-shEGFP (shEGFP) control, dos clones estables generados tras la transfección con shSNAI1 (shSNAI1-C2 y shSNAI1-C4) y dos clones estables generados tras la expresión de una versión mutante de Snail1 de ratón (mutS) (clones shSNAI1-C2/C4+mutS). Barras, 20 \mum.Figure 2: Analysis of the expression and organization of vimentin and fibronectin after stable silencing of Snail1 in MDA-MB-231 cells . Immunofluorescence analysis of the levels and subcellular localization of fibronectin, vimentin, and Snail1-HA in MDA-MB-231 and MDA-MB-231-shEGFP (shEGFP) control cells, two stable clones generated after transfection with shSNAI1 (shSNAI1- C2 and shSNAI1-C4) and two stable clones generated after the expression of a mutant version of mouse Snail1 (mutS) (shSNAI1-C2 / C4 + mutS clones). Bars, 20 µm.

Figura 3. Análisis de la capacidad invasiva de las células MDA-MB-231 tras el silenciamiento estable de Snail1. A) Análisis qRT-PCR de los niveles de RNAm de MMP2, SPARC, ID1 e ID2 en células MDA-MB-231 originales y MDA-MB-231-shEGFP control (shEGFP), clones estables de shSNAI1 (shSNA1-C2 y -C4) y clones estables obtenidos tras la expresión del mutante silencioso de Snail de ratón (mutS) en células shSNA1-C2 y -C4. B) Análisis de inmunotransferencia de tipo Western de los niveles de proteína total de ID1 y SPARC humanas en los clones independientes y las células control, indicado como en A. La inmunotransferencia de tipo Western de \alpha-tubulina se muestra como control de carga. C) Ensayo de zimografía de la actividad de MMP-9 secretada realizada en el medio condicionado de las células descritas en A. La actividad de MMP-2 se muestra como control. D) Análisis del fenotipo invasivo de las líneas celulares indicadas en A, crecidas sobre filtros recubiertos con una matriz de colágeno de tipo IV. Los resultados muestran la media \pm DE de tres ensayos independientes. Análisis ANOVA; ** p<0,01.Figure 3. Analysis of the invasive capacity of MDA-MB-231 cells after stable silencing of Snail1. 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, shSNAI1 stable shones (shSNA1-C2 and - C4) and stable clones obtained after expression of the mouse Snail silent mutant (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 loading control. C) Zymography assay 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.

Figura 4. Análisis del potencial tumorigénico de las células MDA-MB-231 tras silenciamiento de Snail1. A) Análisis del potencial de crecimiento tumoral (latencia del tumor y tasa de crecimiento) de las líneas celulares MDA-MB-231, \rombonegrotachadolargo shEGFP \romboblancosintacharlargo, shSNAI1-C2 \cuadradonegrotachadolargo, shSNAI1C4 \cuadradoblancosintacharlargo, shSNAI1-C2+mutS \circulonegrotachadolargo y shSNAI1-C4+mutS \circuloblancosintacharlargo, tras su inyección ortotópica en el panículo adiposo mamario de los ratones inmunodeprimidos. Los resultados muestran la media \pm DE de dos ensayos independientes realizados con 5 ratones/línea celular cada uno. Análisis ANOVA: *** p<0,001. B) Análisis RT-PCR de la expresión de Snail1 y Snaii2 humanos (hSNAI1 y hSNAI2) y de Snail1 de ratón (mSnail) realizado en muestras de RNA aisladas de los tumores individuales generados por las líneas celulares indicadas en A. Se muestran dos tumores de cada tipo celular indicado. Los niveles de RNAm de GAPDH se incluyen como: control de carga. C) Análisis qRT-PCR de los niveles de RNAm de cadherina-E (hE-CD), SPARC, ID1 e ID2 humanos realizado en muestras de RNA aisladas de tumores individuales generados por los clones indicados anteriormente. Los resultados muestran la media \pm De de dos tumores independientes para cada clon.Figure 4. Analysis of the tumorigenic potential of MDA-MB-231 cells after Snail1 silencing. A) Analysis of the tumor growth potential (tumor latency and growth rate) of the MDA-MB-231 cell lines, shEGFP \ romboblancosintacharlargo, shSNAI1-C2 \ quadradonegrotachadolargo, shSNAI1C4 \ squareblancosintacharlargo, shSNAI1-C2culculus and shSNAI1-C4 + mutS \ circuloblancosintacharlargo, after orthotopic injection into the mammary adipose panicle of immunocompromised mice. The results show the mean ± SD of two independent assays performed with 5 mice / cell line each. ANOVA analysis: *** p <0.001. B) RT-PCR analysis of the expression of human Snail1 and Snaii2 (hSNAI1 and hSNAI2) and mouse Snail1 (mSnail) performed on RNA samples isolated from individual tumors generated by the cell lines indicated in A. Two tumors are shown of each cell type indicated. GAPDH mRNA levels are included as: load control. C) qRT-PCR analysis of levels of mRNA of cadherin-E (hE-CD), SPARC, ID1 and 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.

Figura 5. Análisis del efecto del silenciamiento estable de Snail1 sobre el fenotipo de los tumores inducidos por MDA-MB-231 A) Análisis histológico y de proliferación de tumores inducidos por células MDA-MB-231-shEGFP control, un clon en el que se interfiere SNAI1 representativo (shSNAI1-C2) y su correspondiente control tras la expresión estable del mutante silencioso de Snail1 (shSNAI1-C2+mutS). Se muestran imágenes de bajo aumento (a-c) y de alto aumento (d-f). Las zonas necróticas se indican mediante flechas. Se muestra la inmunotinción del antígeno de proliferación Ki67 en los paneles g-i. B) Análisis de inmunofluorescencia de las secciones de tumor de las líneas celulares indicadas anteriormente. Las secciones muestran tinción para MMP9 (a-c), CD31 (d-f) e ID2 (g-i). Detección de células apoptóticas mediante ensayo de TUNEL (j-l). Barras, 50 \mum.Figure 5. Analysis of the effect of stable silencing of Snail1 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 SNAI1 (shSNAI1-C2) and its corresponding control are interfered with after stable expression of the Snail1 silent mutant (shSNAI1-C2 + mutS). Low magnification (ac) and high magnification (df) images are shown. Necrotic areas are indicated by arrows. Immunostaining of Ki67 proliferation antigen in gi panels is shown. B) Immunofluorescence analysis of the tumor sections of the cell lines indicated above. Sections show staining for MMP9 (ac), CD31 (df) and ID2 (gi). Detection of apoptotic cells by TUNEL assay (jl). Bars, 50 µm.

Figura 6. Caracterización de líneas celulares derivadas de ganglios linfáticos en ratones nu/nu. A) Representación esquemática del diseño experimental; 1. Crecimiento del tumor y extracción; 2. Selección de células MDA-MB-231 resistentes a antibióticos; 3. Líneas celulares obtenidas a partir de ganglios linfáticos de las extremidades contralaterales. B) Análisis de PCR para determinar la presencia de alelo de amelogenina humano en las líneas celulares indicadas en A. C) Análisis de PCR de la presencia del casette de expresión H1 del vector pSuperior-shRNA en las líneas celulares indicadas. Se indican los tamaños esperados del pSuperior-shRNA control (281 pb) y pSuperior-shRNA que contiene shEGFP o shSNAI1 (345 pb).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 shSNAI1 (345 bp) are indicated.

Figura 7. Análisis de la expresión de Snail1 en líneas celulares derivadas de ganglio linfático. A) Análisis qRT-PCR de los niveles de RNAm de Snail1 humano (hSNAI1) y Snail2 humano (hSNAI2) de células MDA-MB-231 originales, dos líneas celulares derivadas de metástasis de ganglios linfáticos a partir de células MDA-MB-231-shEGFP control (shEGFP-01 y shEGFP-2D), una línea celular derivada de metástasis de ganglios linfáticos a partir de la línea celular MDA-MB-231-shSNAI1-C2 (shSNAI1-C2-2D) y dos líneas celulares derivadas de metástasis de ganglios linfáticos a partir de MDA-MB-231-shSNAI1-C4 (shSNAI1-C4-SM y shSNAI1-C4-01). B) Análisis de inmunotransferencia de tipo Western de los niveles de proteínas de Snail1 (SNAI1) y Snail2 (SNAI2) humanas en células MDA-MB-231 originales y clones derivados de ganglios linfáticos independientes según se indica en A. La inmunotransferencia de tipo Western de la \alpha-tubulina se muestra como control de carga. C) Análisis del potencial tumorigénico de las células 231-shSNA-C2-2D y 231-shSNA-C4-SM comparado con las MDA-15 (\circulonegrotachadolargo): MB-231 originales 100, mediante inyección ortotópica en el panículo adiposo mamario de ratones inmunodeprimidos (\cuadradonegrotachadolargo).Figure 7. Analysis of Snail1 expression in lymph node derived cell lines . A) qRT-PCR analysis of human Snail1 (hSNAI1) and Human Snail2 (mSNAI2) mRNA levels from original MDA-MB-231 cells, two cell lines derived from lymph node metastases from MDA-MB-231 cells -shEGFP control (shEGFP-01 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-C4-01). B) Western blot analysis of human Snail1 (SNAI1) and Snail2 (SNAI2) protein levels in original MDA-MB-231 cells and clones derived from independent lymph nodes as indicated in A. Western blot of the α-tubulin is shown as load control. C) Analysis of the tumorigenic potential of the 231-shSNA-C2-2D and 231-shSNA-C4-SM cells compared to the MDA-15 (?): Original MB-231 100 , by orthotopic injection into the mammary adipose panicle of immunocompromised mice (λ).

Figura 8: Análisis de las propiedades proliferativas de células con Snail1 silenciado mediante la incorporación de BrdU en presencia o ausencia de suero en las línea celulares MDA-MB-231, shEGFP, shSNAI1-C2, shSNAI1-C2+mutS, shSNAI1-C4 y shSNAI1-C4+mutS. Figure 8: Analysis of the proliferative properties of cells with Snail1 silenced by incorporation of BrdU in the presence or absence of serum in the MDA-MB-231, shEGFP, shSNAI1-C2, shSNAI1-C2 + mutS, shSNAI1-C4 and shSNAI1-C4 + mutS.

Figura 9. Análisis de la apoptosis inducida tras un tratamiento de 48 h con docetaxel 100 nM (A) o gemcitabina 6 pM (B) en células MDA-MB-231 originales y MDA-MB-231-shEGFP control, clones en los que se interfiere Snail1 de manera estable (shSNAI1-C2 y -C4) y clones estables obtenidos tras la expresión de Snail1 de ratón mutante (mutS). Los resultados muestran la media \pm DE de tres ensayos independientes. Análisis ANOVA: ** p < 0,01; *** p < 0,001.Figure 9. Analysis of apoptosis induced after a 48 h treatment with 100 nM docetaxel (A) or 6 pM gemcitabine (B) in original MDA-MB-231 and control MDA-MB-231-shEGFP cells, clones in which Snail1 is stably interfered (shSNAI1-C2 and -C4) and stable clones obtained after the expression of mutant mouse Snail1 (mutS). The results show the mean ± SD of three independent trials. ANOVA analysis: ** p <0.01; *** p <0.001.

Figura 10. Análisis de la apoptosis inducida tras el tratamiento de 48 h con docetaxel 100 nM (A) o gemcitabina 6 pM (B) en líneas celulares derivadas de ganglios linfáticos: MDA-MB-231 originales, dos líneas celulares derivadas de metástasis de ganglios linfáticos a partir de células MDA-MB-231-shEGFP control (shEGFP-01 y shEGFP-2D), una línea celular derivada de metástasis de ganglios linfáticos a partir de la línea celular MDA-MB-231-shSNAI1-C2 (shSNAI1-C2-2D) y dos líneas celulares derivadas de metástasis de ganglios linfáticos a partir de MDA-MB-231-shSNAI1-C4 (shSNAI1-C4-SM y shSNAI1-C4-01).Figure 10. Analysis of apoptosis induced after 48 h treatment with 100 nM docetaxel (A) or 6 pM gemcitabine (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-01 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-C4-01). 

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Objeto de la invenciónObject of the invention

En primer lugar, la invención se refiere a un método de diagnóstico y/o pronóstico de cáncer de mama basado en la determinación del nivel de expresión del factor de transcripción Snail1 en una muestra biológica aislada de un sujeto.First, the invention relates to a Method of diagnosis and / or prognosis of breast cancer based on determination of the level of expression of the transcription factor Snail1 in an isolated biological sample of a subject.

Es también objeto de la presente invención el empleo de un oligonucleótido interferente de Snail1 en la preparación de un medicamento destinado al tratamiento de cáncer de mama.The object of the present invention is also the use of an interfering oligonucleotide of Snail1 in the preparation of a medicine intended for the treatment of cancer of mom.

Finalmente, es objeto de la invención una composición farmacéutica que comprende una cantidad terapéuticamente efectiva de un oligonucleótido interferente de Snail1, para su uso en el tratamiento del cáncer de mama.Finally, an object of the invention is pharmaceutical composition comprising an amount therapeutically effective of an interfering oligonucleotide of Snail1, for use in the treatment of breast cancer. 

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Descripción de la invenciónDescription of the invention

Debido principalmente a la falta de anticuerpos apropiados frente al factor de transcripción Snail1, hasta ahora no se habían descrito estudios funcionales sobre el papel de Snail1 in vivo en células de carcinoma de mama humano, lo que no ha permitido establecer una relación directa entre la expresión de Snail1 y los hallazgos histopatológicos en cáncer de mama humano.Due mainly to the lack of appropriate antibodies against the Snail1 transcription factor, no functional studies on the role of Snail1 in vivo in human breast carcinoma cells have been described so far, which has not allowed establishing a direct relationship between expression of Snail1 and histopathological findings in human breast cancer.

Los autores de la presente invención, mediante el silenciamiento estable de la expresión del factor de transcripción Snail1 en líneas celulares de cáncer de mama humano, han demostrado que Snail1 desempeña un papel directo en el cáncer de mama humano. Así, han demostrado que el silenciamiento estable de Snail1 disminuye significativamente el fenotipo invasivo de líneas celulares de carcinoma de mama humano. Además, los experimentos realizados han demostrado que la expresión de Snail1 es necesaria, no sólo para la invasión local y la metástasis, como ya se había descrito para otro tipo de tumores, sino lo que es más importante, para el crecimiento tumoral primario y han confirmado su papel en la recurrencia del tumor en cáncer de mama humano.The authors of the present invention, by stable silencing of factor expression Snail1 transcription in human breast cancer cell lines, have shown that Snail1 plays a direct role in cancer of human breast. Thus, they have shown that stable silencing of Snail1 significantly decreases the invasive phenotype of human breast carcinoma cell lines. In addition, the Experiments performed have shown that Snail1 expression is necessary, not only for local invasion and metastasis, as already had been described for other types of tumors, but what is more important, for primary tumor growth and have confirmed their  Role in tumor recurrence in human breast cancer.

En base a estos avances, han desarrollado nuevas aplicaciones en el campo del diagnóstico, pronóstico y terapia del cáncer de mama, proporcionando un diagnóstico más sensible, y facilitando el pronóstico y la toma de decisiones respecto a actuaciones terapéuticas adecuadas.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 forecasting and decision making regarding adequate therapeutic actions.

Así, un aspecto principal de la invención se refiere a un método para el diagnóstico y/o pronóstico de cáncer de mama basado en la determinación del nivel de expresión del factor de transcripción Snail1 (a nivel de proteína y/o mRNA) en una muestra biológica aislada de un sujeto, comparando los resultados obtenidos con valores de referencia previamente establecidos.Thus, a main aspect of the invention is refers to a method for the diagnosis and / or prognosis of cancer of breast based on the determination of the level of factor expression of Snail1 transcription (at the level of protein and / or mRNA) in a isolated biological sample of a subject, comparing the results obtained with previously established reference values.

El término "sujeto", tal como se emplea en la presente invención, incluye seres humanos y animales, preferiblemente mamíferos.The term "subject", as used in The present invention includes humans and animals, preferably mammals.

La muestra biológica procede de un tejido tumoral de un sujeto con cáncer de mama.The biological sample comes from a tissue Tumor of a subject with breast cancer.

El factor Snail1 se refiere al anteriormente denominado Snail, mientras que Snail2 se refiere a Slug. De forma general, en la presente invención se emplea la denominación Snail1/Snail2. Al referirse a los factores humanos se utiliza la forma SNAIL1/SNAIL2 o de forma abreviada, SNAI1 y SNAI2, respectivamente. Asimismo, la forma abreviada Snail se utiliza para el factor Snail1 de ratón.The Snail1 factor refers to the above called Snail, while Snail2 refers to Slug. So In general, in the present invention the designation is used Snail1 / Snail2. When referring to human factors, the SNAIL1 / SNAIL2 form or in short, SNAI1 and SNAI2, respectively. Also, the abbreviated form Snail is used to the mouse Snail1 factor.

Como "método de diagnóstico" se entiende un ensayo realizado sobre un sujeto que presenta síntomas que podrían ser de cáncer de mama. Como "método de pronóstico" se entiende un método que ayuda a predecir, al menos en parte, el curso de la enfermedad. En este sentido, se puede analizar un sujeto al que se le ha diagnosticado previamente cáncer de mama para conocer el progreso de la enfermedad, así como la posibilidad de que responda favorablemente a un tratamiento terapéutico concreto.A "diagnostic method" means a trial conducted on a subject that has symptoms that could Be from breast cancer. As "forecasting method" is understood a method that helps predict, at least in part, the course of disease. In this sense, you can analyze a subject that you have previously been diagnosed with breast cancer to know the disease progress, as well as the possibility of responding favorably to a specific therapeutic treatment.

En una realización particular de la invención, la determinación del nivel de expresión del factor de transcripción Snail1 permite evaluar la capacidad de recurrencia del tumor. Además, la implicación directa de Snail1 en el crecimiento del tumor primario implica que la detección de Snail1 en un carcinoma de mama en el momento del diagnóstico puede predecir la velocidad de crecimiento del tumor primario, no sólo de su recurrencia, aumentando considerablemente la sensibilidad del diagnóstico y la toma de decisiones.In a particular embodiment of the invention, the determination of the level of expression of the transcription factor Snail1 allows to evaluate the recurrence capacity of the tumor. In addition, the direct involvement of Snail1 in tumor growth Primary implies that the detection of Snail1 in a breast carcinoma at the time of diagnosis you can predict the speed of primary tumor growth, not only of its recurrence, considerably increasing the sensitivity of the diagnosis and the decision making.

Así, en otra realización particular de la invención, la determinación del nivel de expresión del factor de transcripción Snail1, en el método definido, permite evaluar la capacidad de crecimiento del tumor.Thus, in another particular embodiment of the invention, the determination of the level of expression of the factor of Snail1 transcription, in the defined method, allows to evaluate the tumor growth capacity.

En otra realización particular, la determinación del nivel de expresión del factor de transcripción Snail1 permite evaluar la capacidad de desarrollo de metástasis local del tumor. Además, la implicación directa de Snail1 en la capacidad invasiva y metastásica permite predecir la capacidad de desarrollo de metástasis a distancia del tumor, facilitando el pronóstico y la toma de decisiones respecto a actuaciones terapéuticas adecuadas.In another particular embodiment, the determination of the level of expression of the transcription factor Snail1 allows evaluate the capacity of local tumor metastasis development. In addition, the direct involvement of Snail1 in invasive capacity and metastatic allows to predict the development capacity of distant metastasis of the tumor, facilitating prognosis and decision making regarding therapeutic actions adequate.

La detección de Snail1 en un carcinoma de mama en el momento del diagnóstico permite además predecir y evaluar la capacidad de respuesta del tumor a agentes quimioterapéuticos utilizados en el tratamiento del carcinoma de mama, incluyendo, pero no de forma exclusiva, a docetaxel y gemcitabine, utilizados en clínica para el tratamiento de diversos tipos tumorales en estadios localmente avanzados y metastásicos, entre ellos el cáncer de mama, habiéndose demostrado su utilidad en el tratamiento, primero en situación metastásica y, posteriormente, en la situación más favorable de adyuvancia.Detection of Snail1 in a breast carcinoma at the time of diagnosis it also allows to predict and evaluate the tumor responsiveness 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 stages locally advanced and metastatic, including breast cancer, having proven its usefulness in treatment, first in metastatic situation and subsequently in the most favorable adjuvant.

En otro aspecto principal de la invención se contempla el uso de un oligonucleótido interferente de Snail1 en la preparación de un medicamento destinado al tratamiento de cáncer de mama.In another main aspect of the invention, contemplates the use of an interfering oligonucleotide of Snail1 in the preparation of a medicine intended for the treatment of cancer of mom.

En la presente invención, el término oligonucleótido se refiere tanto a secuencias de RNA interferente de Snail1 (siRNA) como a secuencias de DNA que, incorporadas en un vector de expresión, son capaces de dirigir, una vez dentro de la célula, la síntesis de un shRNA.In the present invention, the term oligonucleotide refers to both interfering RNA sequences of Snail1 (siRNA) as to DNA sequences that, incorporated into a expression vector, are able to direct, once inside the cell, the synthesis of a shRNA.

siRNA es una molécula de RNA de doble cadena, generalmente de 19 pb cada una con 2 bases desapareadas en cada extremo ("overhangs"). Así, se encuentra de forma natural en las células. shRNA es RNA monocadena que forma un "lazo" (hairpin) plegándose sobre sí mismo para dar una estructura de doble cadena. Este "lazo" es cortado en el citoplasma por la enzima DICER dando lugar a un siRNA de doble cadena. Por tanto, los shRNA son precursores sintéticos de siRNA que se transcriben a partir de un plásmido/virus exógeno introducido en la célula para tal
fin.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) by folding over itself to give a double-stranded 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 transcribed from an exogenous plasmid / virus introduced into the cell for such
finish.

En una realización preferida, el oligonucleótido empleado es RNA interferente de Snail1, de doble cadena, preferiblemente con secuencias mostradas en SEQ ID NO 1 y SEQ ID NO 2.In a preferred embodiment, the oligonucleotide employee is Snail1 interfering RNA, double stranded, preferably with sequences shown in SEQ ID NO 1 and SEQ ID NO 2.

En otra realización preferida, el oligonucleótido empleado es DNA, preferiblemente con secuencia mostrada en SEQ ID NO 3.In another preferred embodiment, the oligonucleotide used is DNA, preferably with sequence shown in SEQ ID NO 3.

El silenciamiento específico de Snail1 mediante la interferencia estable del RNA induce a una disminución en los marcadores mesenquimáticos (fibronectina, vimentina) y pro-invasivos (MMP9, ID1, SPARC) en las células de cáncer de mama (p.ej. MDA-MB-231), de manera concomitante con una disminución en su comportamiento invasivo in vitro. Y lo que es más importante, la interferencia estable de Snail1 en dichas células conduce a una drástica reducción en la incidencia tumoral in vivo y a un aumento de dos veces en la latencia tumoral. Los tumores inducidos por las células silenciadas muestran regiones necróticas extensas y una disminución significativa en los marcadores invasivos y angiogénicos.Specific silencing of Snail1 through 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 more importantly, the stable interference of Snail1 in these 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.

Así, en realizaciones particulares de la invención, se contempla el empleo de un oligonucleótido interferente de Snail1, en la preparación de un medicamento destinado a la disminución de la formación de tumores primarios en cáncer de mama.Thus, in particular embodiments of the invention, the use of an oligonucleotide is contemplated interfering with Snail1, in the preparation of a medicine aimed at decreasing the formation of primary tumors in breast cancer

En otra realización particular, se emplea dicho oligonucleótido en la preparación de un medicamento destinado a la disminución de la recurrencia de tumores primarios en cáncer de mama.In another particular embodiment, said oligonucleotide in the preparation of a medicament intended for decreased recurrence of primary tumors in cancer of mom.

En otra realización particular, se contempla el uso del oligonucleótido en la preparación de un un medicamento destinado a la disminución de la capacidad invasiva del tumor en cáncer de mama.In another particular embodiment, the use of the oligonucleotide in the preparation of a medicine intended to decrease the invasive capacity of the tumor in breast cancer

En otra realización particular, el oligonucleótido se emplea en la preparación de un medicamento destinado a la disminución de la formación de metástasis local en cáncer de mama.In another particular embodiment, the oligonucleotide is used in the preparation of a medicine aimed at decreasing the formation of local metastases in breast cancer

En otra realización particular se contempla el uso del oligonucleótido en la preparación de un medicamento destinado a la disminución de la formación de metástasis a distancia en cáncer de mama.In another particular embodiment the use of the oligonucleotide in the preparation of a medicine destined to decrease the formation of distant metastases in breast cancer

Además, el silenciamiento de Snail1 en las células de cáncer de mama aumenta la sensibilidad a los agentes quimioterapéuticos relevantes en los tratamientos del cáncer de mama, por lo que en otra realización particular, el oligonucleótido se emplea en la preparación de un medicamento destinado al aumento de la sensibilidad a agentes quimioterapéuticos, preferiblemente gemcitabina y docetaxel.In addition, the silencing of Snail1 in the breast cancer cells increases sensitivity to agents relevant chemotherapeutic agents in cancer treatments of breast, so in another particular embodiment, the oligonucleotide it is used in the preparation of a medicine intended for augmentation of sensitivity to chemotherapeutic agents, preferably gemcitabine and docetaxel.

Finalmente, en otro aspecto principal de la invención se contempla una composición farmacéutica que comprende una cantidad terapéuticamente efectiva de un oligonucleótido interferente de Snail1, para su uso en el tratamiento del cáncer de mama.Finally, in another main aspect of the invention is contemplated a pharmaceutical composition comprising a therapeutically effective amount of an oligonucleotide interference of Snail1, for use in the treatment of cancer mom.

En la presente invención, la expresión "cantidad terapéuticamente efectiva" se refiere a la cantidad de secuencia de un oligonucleótido de la invención (siRNA) o a la cantidad de una construcción génica (capaz de generar shRNA) que permita su expresión intracelular calculada para producir el efecto deseado y, en general, vendrá determinada, entre otras causas, por las características propias de dichas secuencias y construcciones y el efecto terapéutico a conseguir.In the present invention, the term " 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.

En una realización particular, el oligonucleótido incluido en la composición farmacéutica es RNA interferente de Snail1 (siRNA), de doble cadena, preferentemente de secuencias SEQ ID NO 1 y 2.In a particular embodiment, the oligonucleotide included in the pharmaceutical composition is RNA interfering Snail1 (siRNA), double stranded, preferably of sequences SEQ ID NO 1 and 2.

Los oligonucleótidos de RNA pueden emplearse "desnudos" (naked siRNA) en su estado nativo o con modificaciones que mejoren su estabilidad en condiciones fisiológicas.RNA oligonucleotides can be used "naked" (naked siRNA) in its native state or with modifications that improve your stability in conditions physiological

De manera preferida, las modificaciones son químicas. Por modificaciones químicas se entiende toda modificación en la composición química del nucleótido de siRNA que conduzca a incrementar la estabilidad del siRNA en suero, incrementar la estabilidad termodinámica, tropismo celular, potencia de silenciamiento y propiedades farmacocinéticas.Preferably, the modifications are chemical Chemical modifications means any modification. in the chemical composition of the siRNA nucleotide that leads to increase the stability of serum siRNA, increase the thermodynamic stability, cell tropism, power of silencing and pharmacokinetic properties.

Ejemplos de modificaciones químicas son las contempladas en la Tecnología LNA (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), los fosfotiatos (Crooke ST. Potential roles of antisense technology in cancer chemotherapy. Oncogene 2000;19(56):6651-6659), los residuos añadidos a la cadena de ribosas: 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. Cell cycle (Georgetown, Tex 2006;5(18):2103-2109; Ikeda Y, Taira K. Ligand-targeted delivery of therapeutic siRNA. Pharmaceutical research 2006;23(8): 1631-1640.Examples of chemical modifications are those contemplated in the LNA (Locked Nucleid Acid) Technology (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. Cell cycle (Georgetown, Tex 2006; 5 ( 18): 2103-2109; Ikeda Y, Taira K. Ligand-targeted delivery of therapeutic siRNA Pharmaceutical research 2006; 23 (8): 1631-1640.

Pueden emplearse cualquiera de los sistemas de vehiculización, disponibles o en desarrollo actual, para favorecer la entrada de los oligonucleotidos en la célula. En una realización particular se emplean liposomas, nanoparticulas (p.ej, quitosanos, polietilenglicol y oligodendrómeros), complejos peptídicos así como modificaciones de todos estos que permitan el direccionamiento de los oligonucleótidos a un tipo celular/tejido concreto.Any of the systems of vehiculization, available or in current development, to favor the entry of oligonucleotides in the cell. In one embodiment In particular, liposomes, nanoparticles (eg, chitosans, polyethylene glycol and oligodendrometers), peptide complexes as well as modifications of all these that allow addressing oligonucleotides to a specific cell / tissue type.

Por complejos peptídicos se entiende un conjunto de péptidos de composición variable. La secuencia peptídica se puede establecer de manera que favorezca el tropismo a tipos celulares determinados, en función de la presencia de receptores celulares específicos para dichos secuencia peptídica (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 Schiffelers RM, Molema G, Kok RJ. RGD-based strategies for selective delivery of therapeutics and imaging agents to the tumour vasculature. Drug Resist Updat 2005;8(6):381-402). Así, en una realización particular, los complejos peptídicos pueden contener secuencias de péptidos reconocibles por receptores de células de mama.Peptide complexes means a set of peptides of variable composition. The peptide sequence can be established in a way that favors tropism at specific 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 Society 2007; 35 (Pt 1): 44-46), (Ikeda Y, Taira K. Ligand-targeted delivery of therapeutic siRNA Pharmaceutical research 2006; 23 (8): 1631-1640), (Temming 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.

En otra realización particular de la invención, el oligonucleótido empleado en la elaboración de la composición farmacéutica es DNA en forma de plásmido, lineal o formando parte de un vector viral, que sea capaz de dirigir, una vez dentro de la célula, la síntesis de un shRNA. Preferiblemente, el DNA presenta la secuencia SEQ ID NO 3.In another particular embodiment of the invention, the oligonucleotide used in the preparation of the composition pharmaceutical is plasmid DNA, linear or 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.

Las secuencias de DNA no sólo han de entrar en la célula sino que han de llegar hasta el núcleo para que se pueda transcribir. Por esto, aparte de usar los mismos sistemas de vehiculización que los oligonucleótidos de RNA (liposomas, nanoparticulas (p.ej. quitosanos, polietilenglicol y oligodendrómeros) y complejos peptídicos), se emplean como vectores sistemas virales que contienen en su genoma la secuencia que permite la síntesis del shRNA deseado. Entre los sistemas utilizados están los Retrovirus, Lentivirus, Adenovirus, Virus asociados a Adenovirus y
Baculovirus.The DNA sequences not only have to 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 the sequence in their genome allows the synthesis of the desired shRNA. Among the systems used are Retroviruses, Lentiviruses, Adenovirus, Adenovirus associated viruses and
Baculovirus

Las composiciones farmacéuticas proporcionadas por la presente invención pueden ser administradas por cualquier vía de administración apropiada que dé como resultado una respuesta terapéutica adecuada frente al cáncer de mama, para lo cual dicha composición se formulará en la forma farmacéutica adecuada a la vía de administración elegida.The pharmaceutical compositions provided by the present invention can be administered by any appropriate route of administration that results in a response adequate therapy against breast cancer, for which said Composition will be formulated in the appropriate pharmaceutical form to the route of chosen administration. 

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Estos resultados han permitido desarrollar nuevas aplicaciones en relación con el diagnóstico, pronóstico y tratamiento del cáncer de mama, que podrían ser extensibles a otros tipos de carcinomas y melanomas, en base a la expresión de Snail1 en diferentes tipos de tumores.These results have allowed to develop new applications in relation to diagnosis, prognosis and breast cancer treatment, which could be extensible to others types of carcinomas and melanomas, based on the expression of Snail1 in Different types of tumors. 

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Ejemplos Examples

Los materiales y métodos empleados en la realización de los ejemplos se describen a continuación:The materials and methods used in the Execution of the examples are described below:

Cultivo celularCell culture

Se hicieron crecer células MDA-MB-231 de cáncer de mama humano y sus líneas celulares derivadas en DMEM (Gibco BRL; San Diego, CA) complementado con FBS al 10%, L-glutamina 2 mM y antibióticos, a 37ºC en una atmósfera de CO_{2} al 5% humidificada.Cells were grown MDA-MB-231 of human breast cancer and its derived DMEM cell lines (Gibco BRL; San Diego, CA) supplemented with 10% FBS, 2 mM L-glutamine and antibiotics, at 37 ° C in a 5% CO2 atmosphere humidified

Generación de vectores de expresión y líneas celulares establesGeneration of expression vectors and cell lines stable

La generación del vector pcDNA3-Snail1-HA ya ha sido descrita (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 se construyó usando pcDNA3-Snail1-HA como molde para mutagénesis dirigida al sitio siguiendo protocolos convencionales. Los oligonucleótidos usados para la amplificación por plásmidos, con cinco mutaciones puntuales silenciosas, fueron: SEQ ID NO 4 y SEQ ID NO 5. Recientemente se ha descrito (Jorda M. et al. Upregulation of MMP-9 in MDCK epithelial cell line in response to expresión of the Snail trasncription factor. J. Cell Sci 2005;118(Pt 15):3371-3385) la generación de shRNA, que contiene secuencias de oligonucleótidos específicas frente a EGFP (proteína verde fluorescente potenciada) o Snail1 de ratón/humano (SEQ ID NO 3), clonada en el vector pSuperior-Puro (Oligoengine, Seattle, WA). En dicho vector, la transcripción del shRNA está dirigida por el promotor H1. La secuencia completa de DNA que aparece en el vector para generación de shRNA se muestra en SEQ ID NO 6, que incluye la secuencia específica SEQ ID NO 3 y la secuencia que formará el "lazo" al transcribirse el DNA a RNA como shRNA. Todas las transfecciones se llevaron a cabo usando lipofectamina (Gibco BRL). Los vectores pSuperior-shEGFP (shEGFP) y pSuperior-shSnail1 (shSNAI1) se transfectaron en células MDA-MB-231, y se realizó la selección con puromicina 11 \mug/ml durante 2-4 semanas. pcDNA3-Snail1-mutS-HA se transfectó en las líneas celulares MDA-MB-231-shSNAI1-C2/C4 y se seleccionó con G418 400 gg/ml durante 4-6 semanas. Se aislaron diez clones tras la transfección de shRNA en cada tipo celular y se caracterizaron individualmente, o se recogieron como clones reunidos en las transfecciones control.The generation of the pcDNA3-Snail1-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 recently been described (Jorda M. et al. Upregulation of MMP-9 in MDCK epithelial cell line in response to expression of the Snail trasncription factor J. Cell 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 Snail1 (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-shSnail1 (shSNAI1) vectors were transfected into MDA-MB-231 cells, and 11 µg / ml puromycin selection was performed for 2-4 weeks. pcDNA3-Snail1-mutS-HA was transfected into MDA-MB-231-shSNAI1-C2 / C4 cell lines and selected with G418 400 gg / ml for 4-6 weeks. Ten clones were isolated after shRNA transfection in each cell type and characterized individually, or collected as clones pooled in the control transfections.

RT-PCR y RT-PCR cuantitativa (qRT-PCR)RT-PCR and quantitative RT-PCR (qRT-PCR)

Se aisló el RNA total de las diferentes líneas celulares y se llevaron a cabo análisis de RT-PCR usando cebadores específicos para Snail1 de ratón o humano y GAPDH (Gliceraldehido 3 fosfato deshidrogenasa) (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;2(2):76-83; Perez-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). La RT-PCR de los tumores se realizó tal como se describe en Peinado H. et al. A molecular role for lysyl oxidase-like enzyme in snail regulation and tumor progresión. Embo J 2005; 24(19):3446-3458). Para la qRT-PCR, el cDNA de las células y los tumores se sintetizó usando el kit High Capacity cDNA Archive (Applied Biosystems, Foster City, CA. La qRT-PCR se llevó a cabo en un sistema 7900HT Fast Real Time PCR (Applied Biosystems) según las instrucciones del fabricante.Total RNA was isolated from the different cell lines and RT-PCR analysis was carried out using primers specific for mouse or human Snail1 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. 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; 2 (2): 76-83; Perez-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 enzyme in snail regulation and tumor progression. Embo J 2005; 24 (19): 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 system (Applied Biosystems) according to the manufacturer's instructions.

Para RT-PCR de Snail2 humano, se usaron los cebadores directo (SEQ ID NO 7) e inverso (amplifica un fragmento de 810 pb) (SEQ ID NO 8).For RT-PCR of human Snail2, it used the primers direct (SEQ ID NO 7) and inverse (amplifies a 810 bp fragment) (SEQ ID NO 8).

Para el análisis de qRT-PCR de células y tumores, se usaron los siguientes cebadores: SNAI1 humano, SEQ ID NO 9 y SEQ ID NO 10; SNAI2 humano, SEQ ID NO 11 y SEQ ID NO 12; cadherina-E humana (CDH1), SEQ ID NO 13 y SEQ ID NO 14; SPARC humana, SEQ ID NO 15 y SEQ ID NO 16; MMP2 humana, SEQ ID NO 17 y SEQ ID NO 18. Se realizaron qRT-PCR para genes de ID tal como se describió anteriormente (Xu, et al. 2005).For the qRT-PCR analysis of cells and tumors, the following primers were used: SNAI1 human, SEQ ID NO 9 and SEQ ID NO 10; Human SNAI2, SEQ ID NO 11 and SEQ ID NO 12; human cadherin-E (CDH1), SEQ ID NO 13 and SEQ ID NO 14; Human SPARC, SEQ ID NO 15 and SEQ ID NO 16; MMP2 human, SEQ ID NO 17 and SEQ ID NO 18. They were performed qRT-PCR for ID genes as described previously (Xu, et al. 2005).

Extractos celulares y análisis de inmunotransferencia de tipo WesternCell extracts and immunoblot analysis Western

Se obtuvieron extractos de células completas usando el tampón RIPA (Tris-HCl 50 mM, pH 7,5, NaCl 150 mM, NP-40 al 1%, desoxicolato al 0,5%, SDS al 0,1%), que contiene inhibidores de proteasas. Para el análisis de inmunotransferencia de tipo Western, se cargaron 50 \mug de proteínas totales de los diferentes extractos en geles de SDS-PAGE al 7,5, 10 ó 12%. La transferencia, el bloqueo y la incubación con los anticuerpos apropiados se llevó a cabo tal como se describe en 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; Perez-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). Los anticuerpos primarios usados incluyeron: anti-\alpha-tubulina monoclonal de ratón (1:1000) (SIGMA Chemical Co, St. Louis; MO), anti-vimentina (1:2000) (Babco, Richmond, CA), anti-Snail1 (1:40) (Franci et al. 2006), anti-Snail2 (1:100) (Wu et al. 2005) y anti-SPARC 15G12, (1:100) (Novocastra Laboratories, Newcastle upon Tyne, RU), anti-fibronectina policlonal de conejo (1:4000) (SIGMA Chemical Co) y anti-ID1 (1:500) (Santa Cruz Biotechnology, Santa Cruz, CA).Whole cell extracts were obtained using the RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 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; Perez-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) . Primary antibodies used included: mouse monoclonal anti-α-tubulin (1: 1000) (SIGMA Chemical Co, St. Louis; MO), anti-vimentin (1: 2000) (Babco, 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). 

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Zimografía en gelatinaJello Zymography

La zimografía en gelatina se llevó a cabo tal como se describe en Olmeda D. et al. Snail silencing effectively suppresses tumour growth and invasiveness. Oncogene 2007;26(13):1862-1874 y Jorda M. et al. Upregulation of MMP-9 in MDCK epithelial cell line in response to expresión of the Snail trasncription factor. J. Cell Sci 2005;118(Pt 15):3371-3385). En resumen, se hicieron crecer las células en medios de crecimiento normal y se colocaron los medios libres de suero en cultivos confluentes durante 24 horas para recoger los medios condicionados. Se separaron 12 \mug de medio condicionado y se analizaron en geles de SDS-PAGE al 7,5% que contenían gelatina al 0,1% y se trataron tal como se describió anteriormente (Olmeda D. et al. Snail silencing effectively suppresses tumour growth and invasiveness. Oncogene 2007;26(13):1862-1874 y Jorda M. et al. Upregulation of MMP-9 in MDCK epithelial cell line in response to expresión of the Snail trasncription factor. J. Cell Sci 2005;118(Pt 15):3371-3385). Se tiñeron los geles con Azul Brillante de Coomassie R250 y se detectaron las actividades gelatinolíticas como bandas transparentes frente al fondo azul.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 line in response to expression of the Snail trasncription factor. J. Cell 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 line in response to expression of the Snail trasncription 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. 

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Ensayos de invasiónInvasion tests

Se llevaron a cabo ensayos de invasión sobre cámaras de Boyden modificadas recubiertas con gel de colágeno tipo IV, tal como se describe en 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; Perez-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; Olmeda D. et al. Snail silencing effectively suppresses tumour-growth and invasiveness. Oncogene 2007;26(13):1862-1874, comenzando con 1x10^{6} células sembradas y contando las células en la parte inferior del filtro 24 h tras la siembra.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; Perez-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; 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 bottom of the filter 24 hours after sowing. 

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Tumorigénesis, ensayos de metástasis espontánea y obtención de líneas celulares derivadas de ganglio linfático.Tumorigenesis, spontaneous metastasis tests and obtaining lymph node derived cell lines.

Se inyectaron ortotópicamente células MDA-MB-231 humanas originales y clones derivados de cultivos subconfluentes (1x10^{6} en 0,05 ml de medio de crecimiento libre de suero) en el panículo adiposo mamario de ratones inmunocomprometidos desnudos hembra Balb/c, de 8 semanas de edad (Charles River, Wilmington, MA). El crecimiento de los tumores se midió cada dos días mediante la determinación de los dos diámetros externos ortogonales usando un calibrador. Cuando los tumores alcanzaron un tamaño de 0,4 cm^{3}, se extirparon quirúrgicamente y se trataron para análisis de histología, inmunofluorescencia y RT-PCR. Se generó un mínimo de 10 tumores por cada línea celular y, al menos, se analizaron 4 tumores diferentes derivados de cada línea celular. Para el ensayo de metástasis espontánea, tras la extirpación quirúrgica del tumor primario, los ratones se dejaron vivos durante otros 6 meses y después se sacrificaron. Los ganglios linfáticos contralaterales se extirparon entonces quirúrgicamente, se disecaron cuidadosamente y se cultivaron durante 3-4 semanas en presencia de puromicina (excepto en el caso de los derivados de animales a los que se inyectaron las células originales) para seleccionar las células MDA-MB-231 humanas resistentes a antibióticos. Los ratones se alojaron y se mantuvieron en condiciones específicas libres de patógenos y se usaron según las directrices institucionales y aprobadas por el Use Committee for Animal Care. Original human MDA-MB-231 cells and clones derived from subconfluent cultures (1x10 6 in 0.05 ml of serum-free growth medium) were orthotopically injected into the breast adipose panicle of female Balb / c nude immunocompromised mice, 8-week-old (Charles River, Wilmington, MA). Tumor growth was measured every two days by determining the two orthogonal outer diameters using a caliper. When the tumors reached a size of 0.4 cm3, 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 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. 

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Análisis de inmunohistoquímica, RT-PCR y TUNEL de los tumores primariosImmunohistochemical analysis, RT-PCR and TUNEL of primary tumors

Para la tinción inmunohistoquímica, una parte del tumor se fijó en formalina y se embebió en parafina y otra se congeló en nitrógeno líquido embebido en Tissue Tek OCT Compound (compuesto para corte a temperatura óptima) y se almacenó a -70ºC. Se tiñeron las secciones en parafina con hematoxilina y eosina o se inmunotiñeron con anti-Ki67 monoclonal de conejo (1:200) (Clone SP6, Lab Vision Corporation, CA, EE.UU.); las secciones congeladas se inmunotiñeron simultáneamente con anti-MMP-9 de conejo (1:200), anti-CD31 de rata (1:300) (Chemicon, Billerica, MA), anti-ID2 policlonal de conejo (1:100) (Santa Cruz Biotechnology) y anticuerpos secundarios apropiados, tal como se describe en Peinado H. et al. A molecular role for lysyl oxidase-like 2 enzyme in snail regulation and tumor progresión. Embo J 2005; 24(19):3446-3458. Para el análisis de fragmentación del ADN, se analizaron secciones de criostato fijadas con paraformaldehído mediante el método TUNEL usando el kit Cell Death Detection in situ (Roche, Basilea, Suiza) según el protocolo del fabricante. Se realizaron análisis de RT-PCR en las secciones tumorales congeladas en nitrógeno líquido tras la extirpación cuidadosa de toda la piel circundante, tal como se describe en Olmeda D. et al. Snail silencing effectively suppresses tumour growth and invasiveness. Oncogene 2007;26(13):1862-1874 y en Peinado H. et al. A molecular role for lysyl oxidase-like 2 enzyme in snail regulation and tumor progresión. Embo J 2005; 24(19):3446-3458. 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 optimal temperature cutting) and stored at -70 ° C. The paraffin sections were stained with hematoxylin and eosin or immunostained with rabbit monoclonal anti-Ki67 (1: 200) (Clone SP6, Lab Vision Corporation, CA, USA); the frozen sections were immunostained simultaneously 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 using the TUNEL method using the Cell 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. 

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Ensayos de sensibilidad a fármacosDrug sensitivity tests

La sensibilidad a fármacos se analizó con la concentración mínima de fármacos que induce una reducción del 80% en el crecimiento celular en comparación con las células no tratadas (CI80). Las células que crecían exponencialmente se trataron con docetaxel 100 mM (Taxotere®, Aventis Pharma SA, París, Francia) (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) o gemcitabina 6 pM (Gemzar®, Lilly S.A, Indianápolis, IN) (Hernández-Vargas H. et al. Gene expression profiling of breast cancer cells in response to gemcitabine: NF-kappaB pathway activation as a potencial mechanism of resistance. Breast cancer research and treatment 2007; 102(2):157-172) durante 48 horas. Las células tratadas y control se trataron entonces con tripsina y, tras centrifugación y lavado dos veces con PBS, se tiñeron con anexina-V-FITC/yoduro de propidio usando el kit Annexin V/FITC (MBL internacional, Woburn, MA) según las instrucciones del fabricante. Las células teñidas se analizaron mediante citometría de flujo. Los resultados muestran la media \pm DE de tres ensayos independientes.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 6 pM gemcitabine (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 resistance 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 to manufacturer's instructions Stained cells were analyzed by flow cytometry. The results show the mean ± SD of three independent trials. 

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Ejemplo 1Example one

El silenciamiento estable de Snail1 en las células MDA-MB-231 aumenta los transcritos de cadherina-E y disminuye la expresión de los marcadores mesenquimáticos.Stable silencing of Snail1 in cells MDA-MB-231 increases transcripts of cadherin-E and decreases the expression of mesenchymal markers.

Para analizar directamente el papel de Snail1 en la metástasis y el crecimiento tumoral de los carcinomas de mama, se silenció de manera estable la expresión de SNAI1, sin afectar a la expresión de Snail2, en la línea celular de cáncer de mama humano desdiferenciada MDA-MB-231. Las células MDA-MB-231 originales son células altamente tumorigénicas y débilmente metastásicas, que presentan altos niveles de Snail1 y Snail2 y sin expresión de cadherina-E. Con el fin de obtener clones con Snail1 silenciado de manera estable, se transfectaron células MDA-MB-231 con un vector pSuperior-shSNAI1 diseñado para reconocer a los mRNA de Snail1 tanto humano como de ratón y recientemente descrito como capaz de silenciar eficazmente Snail1 en células de carcinoma de ratón (Olmeda D. et al. Snail silencing effectively suppresses tumour growth and invasiveness. Oncogenee 2007;26(13):1862-1874). Tras la selección con el antibiótico de selección puromicina, 1 \mug/ml, se aislaron al menos diez clones y se caracterizaron para determinar la expresión de Snail1; entre ellos, se seleccionaron los clones MDA-MB-231-shSNAI1-C2 y -C4 (denominados de aquí en adelante shSNAI1-C2 y shSNAI1-C4) como los más representativos (figura 1A, B). Como control, se transfectó establemente una forma mutante de Snail1 de ratón en los clones shSNAI1-C2 y shSNAI2-C4. Esta forma mutante de Snail1, fue obtenida por ingeniería genética para que no se reconociera por el siRNA de Snail1 al llevar 5 mutaciones puntuales silenciosas (mutS), tal como se describe en el apartado de "Generación de vectores". Tras la selección con antibiótico (G480, 400 \mug/ml), se reunieron las células (shSNAI1-C2+mutS y shSNAI1-C4+mutS). Como control adicional, se transfectó establemente una secuencia de shRNA irrelevante frente a EGFP en células MDA-MB-231 (células shEGFP). Tal como se muestra en la figura 1A, la expresión estable de shSNAI1 condujo a la inhibición casi completa de la expresión de Snail1, tanto al nivel del mRNA, como de la proteína en las células MDA-MB-231, sin afectar significativamente a la expresión de Snail2 (figura 1B y C, comparación de las células control y MDA-MB-231 originales con shSNA1-C2 y C4). La sobreexpresión de la forma mutante de Snail1 de ratón (mutS) en las células shSNA1-C2+mutS y shSNA1-C4+mutS se confirmó a los niveles del mRNA y de la proteína, obteniendo niveles similares o incluso superiores de la expresión de Snail1 a los observados en las células MDA-MB-231 originales (figura 1 A-C). Debido a la alta conservación de las secuencias de Snail1 de ratón y ser humano (Nieto MA. The snail superfamily of zinc-finger transcription factors. Nat Rev Mol Cell Biol 2002;3(3):155-166; Manzanares M et al. The increasing complexity of the Snail gene superfamily in metazoan evolution. Trends Genet 2001; 17(4):178-181), se detecta mRNA de Snail1 mutS de ratón en las reacciones de PCR y qRT-PCR que utilizan oligonucleótidos frente a Snail1 humano (figura 1A, panel superior y figura 1B, panel izquierdo), así como en la reacción cruzada de los anticuerpos frente a Snail1 (figura 1C, panel superior), lo que explica los niveles aumentados de Snail1 en las células que llevan el alelo Snail1 mutante, aunque no puede descartarse completamente la modesta expresión de nuevo de Snail1 humano endógeno en estas células. También se confirmó la:: expresión y la localización nuclear de la proteína Snail1 mutS en los clones de:. sobreexpresión mediante inmunofluorescencia con anticuerpos anti-HA (figura 2). Puesto. que Snail1 es un inductor auténtico de la 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;) se analizó el efecto del silenciamiento de Snail1 en las células MDA-MB-231 en algunos marcadores epiteliales (cadherina-E) (hE-CD) y mesenquimáticos (fibronectina y vimentina). El silenciamiento estable de Snail1 en las células MDA-MB-231 indujo un modesto aumento en los niveles de mRNA de cadherina-E de hasta 2,5 veces, medido mediante qRT-PCR (figura 1, derecha). De manera importante, el aumento en los transcritos de cadherina-E detectado en los clones shSNAI1-C2 y -C4 se invirtió completamente mediante la sobreexpresión del alelo mutS (figura 1B, comparar los niveles de hE-CD de shSNAI1-C2 y -C4 con shSNAI1-C2+mutS y -C4+mutS, respectivamente). Pese al aumento en los niveles de mRNA, no pudo detectarse la expresión de la proteína cadherina-E en los clones en los que se interfiere Snail1. Por el contrario, se observó una fuerte disminución en la expresión y/u organización de los marcadores mesenquimáticos fibronectina y vimentina en los clones shSNAI1-C2 y -C4 (figura 1C y figura 2) un efecto también suprimido por la sobreexpresión del mutante silencioso de ratón, mutS-Snail (figura 1 C y figura 2). La detección de Snail1 muts, que porta el epitopo HA, solo se detectó en el núcleo de las células shSNAI1-C2-mutS y shSNAI1-C4-mutS, como cabía esperar (figura 2, paneles de la derecha). Tomados juntos, estos resultados confirmaron que el silenciamiento de Snail1 induce una transición de mesénquima a epitelio parcial en las células MDA-MB-231.To directly analyze the role of Snail1 in the metastasis and tumor growth of breast carcinomas, the expression of SNAI1 was stably silenced, without affecting the expression of Snail2, in the MDA-dedifferentiated human breast cancer cell line. MB-231 The original MDA-MB-231 cells are highly tumorigenic and weakly metastatic cells, which have high levels of Snail1 and Snail2 and no cadherin-E expression. In order to obtain clones with stably silenced Snail1, MDA-MB-231 cells were transfected with a pSuperior-shSNAI1 vector designed to recognize both human and mouse Snail1 mRNAs and recently described as capable of effectively silencing Snail1 in Mouse carcinoma cells (Olmeda D. et al. Snail silencing effectively suppresses tumor growth and invasiveness. Oncogenee 2007; 26 (13): 1862-1874) . After selection with the puromycin selection antibiotic, 1 µg / ml, at least ten clones were isolated and characterized to determine Snail1 expression; 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 Snail1 was stably transfected in the shSNAI1-C2 and shSNAI2-C4 clones. This mutant form of Snail1 was obtained by genetic engineering so that it was not recognized by the Snail1 siRNA by carrying 5 silent point mutations (mutS), as described in the " Generation of vectors " section. After antibiotic selection (G480, 400 µg / ml), the cells (shSNAI1-C2 + mutS and shSNAI1-C4 + mutS) were pooled. 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 shSNAI1 led to the almost complete inhibition of Snail1 expression, both at the level of mRNA, and of the protein in MDA-MB-231 cells, without significantly affecting the Snail2 expression (Figure 1B and C, comparison of the original control cells and MDA-MB-231 with shSNA1-C2 and C4). Overexpression of the mutant form of mouse Snail1 (mutS) in shSNA1-C2 + mutS and shSNA1-C4 + mutS cells was confirmed at mRNA and protein levels, obtaining similar or even higher levels of Snail1 expression to those observed in the original MDA-MB-231 cells (Figure 1 AC). Due to the high conservation of the mouse and human Snail1 sequences (Nieto MA. The snail superfamily of zinc-finger transcription factors. Nat Rev Mol Cell Biol 2002; 3 (3): 155-166; Manzanares M et al. The increasing complexity of the Snail gene superfamily in metazoan evolution Trends Genet 2001; 17 (4): 178-181) , mouse Snail1 mutS mRNA is detected in PCR and qRT-PCR reactions using oligonucleotides against human Snail1 (Figure 1A, upper panel and Figure 1B, left panel), as well as in the cross-reaction of antibodies against Snail1 (Figure 1C, upper panel), which explains the increased levels of Snail1 in the cells carrying the Snail1 allele mutant, although the modest expression of endogenous human Snail1 in these cells cannot be completely ruled out. The :: expression and nuclear localization of the Snail1 mutS protein in the clones of :. overexpression by immunofluorescence with anti-HA antibodies (figure 2). Market Stall. that Snail1 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;) the effect of Snail1 silencing in MDA-MB-231 cells in some epithelial (cadherin-E) (hE-CD) and mesenchymal (fibronectin and vimentin) markers. Stable silencing of Snail1 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 hE-CD levels of shSNAI1-C2 and -C4 with shSNAI1-C2 + mutS and -C4 + mutS, respectively). Despite the increase in mRNA levels, the expression of cadherin-E protein could not be detected in the clones in which Snail1 interferes. On the contrary, a strong decrease in the expression and / or organization of 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 mutant of mouse, mutS-Snail (figure 1 C and figure 2). The detection of Snail1 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, right panels). Taken together, these results confirmed that Snail1 silencing induces a transition from mesenchyme to partial epithelium in MDA-MB-231 cells. 

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Ejemplo 2Example 2

El silenciamiento estable de Snail1 inhibe el comportamiento invasivo de las células MDA-MB-231Stable silencing of Snail1 inhibits behavior invasive cells MDA-MB-231

A continuación se estudió el efecto del silenciamiento de Snail1 en el comportamiento invasivo de las células MDA-MB-231. Para este fin, se analizó en primer lugar, mediante. qRT-PCR, los niveles de expresión de genes diana Snail1, descritos recientemente, implicados en la invasión y la metástasis, tales como MMP2, SPARC, ID1 y la proteína íntimamente relacionada ID2 (Jorda M. Et al. Id-1 is induced in MDCK epithelial cells by activated ErWMAPK pathway in response to expression of the Snail and E47 trasncription factors. Exp Cell Res 2007; Minn AJ. Et al. Genes that mediate breast cancer metastasis to lung. Nature 2005; 436(7050):518-524; Moreno-Bueno. Et al. Genetic profiling of epithelial cells expressing e-cadherin repressors reveals 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 expresión correlatos 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) en las células MDA-MB-231 con Snail1 silenciado. El silenciamiento de Snail1 indujo una fuerte disminución en la expresión de SPARC e ID1 en el mRNA (figura 3A) y en los niveles de proteína total (figura 3B). Por el contrario, los niveles de mRNA de ID2 aumentaron fuertemente tras el silenciamiento de Snail1, De manera importante, los efectos del silenciamiento de Snail1 en la expresión de los genes SPARC e ID se invirtió completamente tras la sobreexpresión del alelo mutS-Snail1 (figura 3 A, B; comparar shSNAI1-C2 y -C4 con shSNAI1-C2+mutS y -C4+mutS y las células MDA-MB-231 originales), lo que apoya un efecto específico del silenciamiento de Snail1 sobre estos genes.Next, the effect of Snail1 silencing on the invasive behavior of MDA-MB-231 cells was studied. For this purpose, it was analyzed first, by. qRT-PCR, the expression levels of Snail1 target genes, recently described, involved in invasion and metastasis, such as MMP2, SPARC, ID1 and the closely related protein ID2 (Jorda M. Et al. Id-1 is induced in MDCK epithelial cells by activated ErWMAPK pathway in response to expression of the Snail and E47 transncription factors. Exp Cell Res 2007; Minn AJ. Et al. Genes that mediate breast cancer metastasis to lung. Nature 2005; 436 (7050): 518-524 ; Moreno-Bueno. Et al. Genetic profiling of epithelial cells expressing e-cadherin repressors reveals 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 cells 231 with Snail1 silenced. Snail1 silencing induced a sharp decrease in the expression of SPARC and ID1 in the mRNA (Figure 3A) and in total protein levels (Figure 3B). In contrast, ID2 mRNA levels increased strongly after Snail1 silencing. Importantly, the effects of Snail1 silencing on the expression of the SPARC and ID genes were completely reversed following the overexpression of the mutS-Snail1 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 Snail1 silencing on these genes.

Por otra parte, los niveles de transcrito de otra diana de Snail1 notificada, MMP2 (Miyoshi A. et al. Snail and SIP1 increase cancer invasión by upregulating MMP family in hepatocellular carcinoma cells. British journal of cancer 2004; 90(6):1265-1273; Yokoyama K. et al. Increased invasión and matriz metalloproteinase-2 expression by Snail induced mesenchymal transition in squoamous cell carcinomas. Internacional journal of oncology 2003;22(4):891-898) no variaron tras el silenciamiento de Snail1 o la sobreexpresión de mutS-Snail1 en las células MDA-MB-231 (figura 3A, panel superior), porque los niveles de MMP2 secretados en las células MDA-MB-231 originales fueron muy bajos (figura 3C). Por el contrario, las células MDA-MB-231 originales mostraron altos niveles de actividad de metaloproteinasa MMP9, otra diana de Snail1 (Jorda M. et al. Upregulation of MMP-9 in MDCK epithelial cell line in response to expression of the Snail transcription factor. J. Cell Sci 2005;118(Pt 15):3371-3385), y el silenciamiento de Snail1 redujo de hecho la actividad de MMP9 (figura 3C), un efecto revertido completamente por la sobreexpresión del alelo mutS-Snail1 (figura 3C, comparar shSNAI1-C2 y C4 con C2-mutS y C4+mutS, respectivamente, y las células originales), lo que confirmó el efecto específico del silenciamiento Snail1 sobre la regulación de MMP9.On the other hand, the transcript levels of another reported Snail1 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 change after the silencing of Snail1 or Overexpression of mutS-Snail1 in MDA-MB-231 cells (Figure 3A, upper panel), because the levels of MMP2 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 Snail1 (Jorda M. et al. Upregulation of MMP-9 in MDCK epithelial cell line in response to expression of the Snail transcription factor J. Cell Sci 2005; 118 (Pt 15): 3371-3385) , and the silencing of Snail1 actually reduced the activity of MMP9 (Figure 3C), an effect completely reversed by the overexpression of the mutS-Snail1 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 Snail1 silencing on MMP9 regulation.

Para comprobar que los cambios moleculares observados tras el silenciamiento de Snail1 en las células MDA-MB-231 podrían influir en su fenotipo invasivo, se realizaron ensayos de invasión in vitro en cultivos crecidos sobre filtros (0,8 \mum de poro) (transwells):, recubiertos con una matriz de colágeno tipo IV. Las células se sembraron en placa por encima de la matriz y se permitió que migraran durante 24 horas. Tras este tiempo, las células que habían migrado hacia la cámara inferior se sometieron a tratamiento con tripsina y se contaron. Los resultados muestran la media \pm DE de tres ensayos independientes. Análisis ANOVA; ** p<0,01. Tal como se muestra en la figura 3D, el silenciamiento de Snail1 redujo drásticamente la capacidad de las células MDA-MB-231 para migrar en matrices de colágeno IV y se logró una reversión de más del 50% en la capacidad de invasión tras la expresión de mutS-Snail1en los clones con Snail1 silenciado (figura 3D, comparar shSNAI1-C2 y -C4 con shSNAI1-C2-mutS y -C4-mutS, respectivamente, y con las células originales), lo que confirma que el silenciamiento específico de Snail1 confiere un fenotipo menos invasivo en las células MDA-MB-231.To verify that the molecular changes observed after Snail1 silencing in MDA-MB-231 cells could influence its invasive phenotype, in vitro invasion assays 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 Snail1 drastically 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-Snail1 in the clones with silenced Snail1 (3D figure, compare shSNAI1-C2 and -C4 with shSNAI1-C2-mutS and -C4-mutS, respectively, and with the original cells), confirming that the specific silencing of Snail1 confers a less invasive phenotype in MDA-MB-231 cells. 

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Ejemplo 3Example 3

La interferencia de Snail1 disminuye drásticamente las propiedades tumorigénicas de las células MDA-MB-231Snail1 interference dramatically decreases the tumorigenic properties of cells MDA-MB-231

Los dos clones con Snail1 silenciado independientes, shSNAI1-C2 y C4 y las células derivadas tras la expresión de mutS-Snail1 (C2+mutS y C4+mutS), junto con las células MDA-MB-231 originales y MDA-MB-231-shEGFP control se inyectaron ortotópicamente en el panículo adiposo mamario de ratones desnudos. En la tabla 1 se muestran datos comparativos de la incidencia del tumor de las células MDA-MB-231 originales, MDA-MB-231-shEGFP control, clones en los que se interfiere Snail1 de manera estable (C2 y C4) y clones estables obtenidos tras la expresión del mutante silencioso de Snail1 de ratón (mutS). Las líneas celulares indicadas se inyectaron en el panículo adiposo mamario de ratones desnudos hembra de 8 semanas de edad. La incidencia se representa como el porcentaje de los tumores inducidos por ratón a los 2 meses tras la inyección. Tal como se muestra en la tabla 1, la incidencia de tumores primarios inducida por las células MDA-MB-231 originales y las células MDA-MB-232-shEGFP control era muy alta (80-90% de los sitios inyectados), pero las células MDA-MB-231-shSNAI1 indujeron tumores con frecuencias mucho menores (sólo el 30-40% de los sitios inyectados). De manera significativa, la incidencia de los tumores primarios se recuperó completamente tras la expresión de la forma mutS-Snail1 (tabla 1, comparar shSNAI1-C2 o C4 con shSNAI1-C2+mutS y -C4+mutS y las células control).The two clones with Snail1 silenced independent, shSNAI1-C2 and C4 and cells derived after mutS-Snail1 expression (C2 + mutS and C4 + mutS), together with the cells MDA-MB-231 originals and MDA-MB-231-shEGFP control were injected orthotopically into the adipose panicle breast of nude mice. Table 1 shows data. Comparisons of the incidence of tumor cells MDA-MB-231 originals, MDA-MB-231-shEGFP control, clones in which Snail1 interferes stably (C2 and C4) and stable clones obtained after mutant expression Silent Mouse Snail1 (mutS). Cell lines indicated were injected into the mammary fat panicle of mice Nude female 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 cell-induced tumors MDA-MB-231 original and cells MDA-MB-232-shEGFP control was very high (80-90% of sites injected), but the cells MDA-MB-231-shSNAI1 induced tumors with much lower frequencies (only the 30-40% of injected sites). By way of significant, the incidence of primary tumors recovered completely after the expression of the form mutS-Snail1 (table 1, compare shSNAI1-C2 or C4 with shSNAI1-C2 + mutS and -C4 + mutS and control cells).

TABLA 1TABLE 1 Datos comparativos de incidencia tumoralComparative Data of Incidence tumor 

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Además, se detectó una reducción drástica en la tasa de crecimiento de los tumores inducidos por las células con Snail1 silenciado (figura 4A). Se observó una reducción demás del 95% en el volumen de los tumores inducidos por los clones shSNA1-C2 y C4 a los 40 días tras la inyección, junto con un aumento de 2 veces en la latencia del tumor (días hasta alcanzar un tamaño de tumor de 0,1 cm^{3}) en comparación con los tumores inducidos por las células originales y control. De manera importante, la tasa de crecimiento tumoral se recuperó casi completamente tras la expresión de nuevo del alelo mutS-Snail1 (figura 4A, comparar shSNA1-C2 o shSNA1-C4 con C2+mutS y C4+mutS, respectivamente, y con las células control).In addition, a drastic reduction in the growth rate of cell-induced tumors with Snail1 silenced (figure 4A). A further reduction of the 95% in the volume of clone-induced tumors shSNA1-C2 and C4 at 40 days after injection, along with a 2-fold increase in tumor latency (days until reaching a tumor size of 0.1 cm3) in comparison with tumors induced by the original and control cells. From importantly, the tumor growth rate recovered almost completely after the expression of the allele again mutS-Snail1 (figure 4A, compare shSNA1-C2 or shSNA1-C4 with C2 + mutS and C4 + mutS, respectively, and with the control cells).

El análisis mediante RT-PCR de los tumores derivados de las diferentes líneas celulares indicó que los transcritos de Snail1 permanecían silenciados en todos los tumores derivados de los clones shSNAI1, mientras que no se observaron cambios significativos en la expresión del gen Snail2 homólogo (figura 4B). De hecho, el silenciamiento de Snail2 en las células MDA-MB-231 no produjo ninguna diferencia en la tasa de crecimiento o incidencia del tumor, lo que apoya adicionalmente un efecto específico del silenciamiento de Snail1 en el comportamiento tumorigénico de las células MDA-MB-231 de carcinoma de mama.RT-PCR analysis of tumors derived from different cell lines indicated that Snail1 transcripts were silenced in all tumors derived from shSNAI1 clones, while not observed significant changes in the expression of the Snail2 gene counterpart (figure 4B). In fact, the silencing of Snail2 in MDA-MB-231 cells did not produce no difference in the growth rate or incidence of tumor, which additionally supports a specific effect of Snail1 silencing in the tumorigenic behavior of MDA-MB-231 carcinoma cells mom.

De manera interesante, se detectó un aumento de hasta 6 veces en los niveles de mRNA de cadherina-E y una fuerte disminución en los niveles de los tránscritos de SPARC en los tumores inducidos por las células shSNAI1-C2 y -C4 (figuras 4C, paneles superiores). De manera similar al comportamiento observado in vitro, estos cambios de expresión se anularon en los tumores inducidos por las células shSNA1 que expresan el alelo mutS-Snail1 (figura 4C, superior). La regulación de la expresión de ID2 también se mantuvo entre la situación ex vivo e in vitro con un aumento de hasta 1,8 veces en los niveles de mRNA detectados en los tumores inducidos mediante las células con Snail1 silenciado (figura 4C, paneles de ID2). De manera sorprendente, los niveles de mRNA de ID1 detectados en los tumores inducidos por los clones shSNA1 y las células derivadas mutS-Snail1 mostraron el patrón inverso al observado en cultivo (figura 4C panel de ID1), lo que indica un mecanismo diferencial de la regulación para la expresión de ID1 entre la situación ex vivo e in vitro.Interestingly, an up to 6-fold increase in cadherin-E mRNA levels and a sharp decrease in SPARC transcript levels were detected in tumors induced by shSNAI1-C2 and -C4 cells (Figures 4C, upper panels). Similar to the behavior observed in vitro , these expression changes were canceled in tumors induced by shSNA1 cells expressing the mutS-Snail1 allele (Figure 4C, above). The regulation of ID2 expression was also maintained between the ex vivo and in vitro situation with an increase of up to 1.8 times in mRNA levels detected in tumors induced by cells with silenced Snail1 (Figure 4C, ID2 panels ). Surprisingly, the levels of ID1 mRNA detected in tumors induced by shSNA1 clones and mutS-Snail1 derived cells showed the inverse pattern to that observed in culture (Figure 4C panel of ID1), indicating a differential mechanism of regulation for the expression of ID1 between the ex vivo and in vitro situation .

Para comprender adicionalmente el efecto del silenciamiento por Snail1 sobre el fenotipo de los tumores inducidos por MDA-MB-231, se analizaron cortes en parafina de tumores de tamaño similar generados por los diferentes clones celulares mediante histología e inmunohistoquímica. No se detectaron diferencias significativas en la histología de los tumores inducidos por las células shSNAI1-C2 con respecto a los inducidos por las células C2 control o que sobreexpresan mutS-Snail1 (figura 5A, a-f). Sin embargo, se encontraron diferencias principales en el número y la extensión de zonas necróticas detectadas dentro de los tumores, que fueron más destacadas en los derivados de las células con Snail1 silenciado (figura 5A, a-c). De manera concomitante con el aumento en las zonas necróticas, se detectó un aumento en el número de células apoptóticas (hasta 27 veces) dentro de los tumores derivados de los clones con Snail1 silenciado, en comparación con los tumores de las células control (figura 5B, j-k). Además, la inmunotinción frente al marcador de la proliferación Ki67 mostró que el silenciamiento de Snail1 indujo una reducción drástica en el potencial proliferativo del tumor (figura 5A, g-h). Los análisis de inmunofluorescencia de los marcadores invasivos y angiogénicos en los tumores confirmaron los datos obtenidos mediante qRT-PCR. Se observó un fuerte aumento en la expresión de ID2 con marcada localización citoplasmática (figura 5B, g-i), mientras que se observó una reducción en el número de células positivas para MMP-9 y CD31 en los tumores inducidos por células shSNAI1-C2 (figura 5B, a-c y d-f, respectivamente). Los cambios en los marcadores anteriores y en el potencial tumoral proliferativo y anti- apoptótico se recuperaron completamente en los tumores inducidos tras la expresión del alelo mutS-Snail1 en las células shSNA1-C2 (figura 5A, B, comparar los paneles derechos con el resto), lo que confirmó el efecto específico del silenciamiento de Snail1 en el, comportamiento tumorigénico de las células MDA-MB-231.To further understand the effect of Snail1 silencing on the phenotype of induced tumors by MDA-MB-231, cuts were analyzed in paraffin from tumors of similar size generated by the different cell clones by histology and immunohistochemistry No significant differences were detected in histology of cell-induced tumors shSNAI1-C2 with respect to those induced by C2 control or overexpress mutS-Snail1 cells (Figure 5A, a-f). However, they found main differences in the number and extent of zones necrotic detected within the tumors, which were more highlighted in cell derivatives with silenced Snail1 (Figure 5A, a-c). Concomitantly with the increase in necrotic areas, an increase in the number was detected of apoptotic cells (up to 27 times) within tumors Clone derivatives with silenced Snail1, compared to control cell tumors (Figure 5B, j-k). In addition, immunostaining against the proliferation marker Ki67 showed that Snail1 silencing induced a reduction drastic in the proliferative potential of the tumor (Figure 5A, g-h). The 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 localization cytoplasmic (Figure 5B, g-i), while observed a reduction in the number of positive cells for MMP-9 and CD31 in cell-induced tumors shSNAI1-C2 (Figure 5B, a-c and d-f, respectively). The changes in previous markers and proliferative tumor potential and anti-apoptotic recovered completely in tumors induced after mutS-Snail1 allele expression in shSNA1-C2 cells (Figure 5A, B, compare the right panels with the rest), which confirmed the effect Specific Snail1 silencing in the behavior tumorigenic cells MDA-MB-231. 

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Ejemplo 4Example 4

La expresión de Snail1 favorece la metástasis de los ganglios linfáticos distantesSnail1 expression favors lymph node metastasis distant lymphatics

Una vez establecido el efecto del silenciamiento de Snail1 en el comportamiento tumorigénico de las células MDA-MB-231, se analizó las consecuencias de la disminución de Snail1 en las capacidades metastásicas de las células MDA-MB-231. Para este fin, se llevó a cabo un ensayo de metástasis espontánea tras la extirpación quirúrgica de tumores primarios inducidos por la inyección de células control y MDA-MB-231 con Snail1 silenciado en el panículo adiposo mamario. Seis meses después, se sacrificaron los ratones y se analizaron los órganos (pulmón, hígado y bazo) para determinar la aparición de lesiones metastásicas. No se detectó macrometástasis en ningún órgano de acuerdo con el bajo potencial metastásico espontáneo de las células MDA-MB-231 originales (Minn AJ. Et al. Genes that mediate breast cancer metastasis to luna. NAture 2005; 436(7050):518-524; Kang Y. et al. A multigenic program mediatin breast cancer metastasis to bone. Cancer Cell 2003; 3(6):537-549). Sin embargo, se observó inflamación de los ganglios linfáticos de las extremidades en la región contralateral en la mayoría de los animales. Por tanto, se analizó la existencia de metástasis en los ganglios linfáticos distantes mediante la extirpación quirúrgica del ganglio linfático contralateral. Además, se derivaron líneas celulares de los ganglios linfáticos, tras su disección cuidadosa y crecimiento en cultivo durante 3-4 semanas, en presencia de puromicina, para evitar el crecimiento de las células de ratón, y se seleccionaron las células MDA-MB-231 humanas resistentes a antibióticos. Sólo se obtuvieron 6 líneas celulares, de un total de 24 ratones con los ganglios linfáticos disecados: 1/6 de ratones a los que se inyectó MDA-MB-231 (denominadas 231-2O), 2/6 de ratones a los que se inyectó MDA-MB-231-shEGFP (denominadas shEGFP-OI y shEGFP-2D), 1/6 de ratones a los que se inyectó shSNAI1-C2 (denominadas shSNAI1-C2-2D) y 2/6 de los ratones a los que se inyectó shSNAI1-C4 (denominadas shSNAI1-C4-SM y shSNAI1-C4-OI) (figura 6A). Para descartar la selección de células de ratón inmortalizadas, se analizó mediante PCR la presencia del gen amelogenina humano, usado frecuentemente en las pruebas forenses para el DNA humano y en la determinación del sexo (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). Todas las líneas celulares seleccionadas, excepto 231-2O, fueron positivas para la forma femenina del gen amelogenina (figura 6B), lo que confirmó su origen humano y su derivación de la metástasis de los ganglios linfáticos distantes de los tumores primarios inducidos por las células derivadas de MDA-MB-231 indicadas. El tamaño esperado del amplicón de amelogenina de PCR es de 977 pb para hembras y 788 pb para machos.Once the effect of Snail1 silencing on the tumorigenic behavior of MDA-MB-231 cells was established, the consequences of the decrease in Snail1 on the metastatic abilities of MDA-MB-231 cells were analyzed. For this purpose, a spontaneous metastasis test was performed after the surgical removal of primary tumors induced by the injection of control cells and MDA-MB-231 with Snail1 silenced in the mammary adipose panicle. Six months later, the mice were sacrificed and the 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 mediate breast cancer metastasis to luna. NAture 2005; 436 (7050): 518- 524; Kang Y. et al. A multigenic program mediatin breast cancer metastasis to bone. Cancer Cell 2003; 3 (6): 537-549) . However, inflammation of the lymph nodes of the extremities in the contralateral region was observed in most animals. Therefore, the existence of metastases in distant lymph nodes was analyzed by 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 (called 231-2O), 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 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 selected cell lines, except 231-2O, were positive for the female form of the amelogenin gene (Figure 6B), which confirmed their human origin and their derivation of distant lymph node metastasis from primary cell-induced tumors. derived from MDA-MB-231 indicated. The expected size of the PCR amelogenin amplicon is 977 bp for females and 788 bp for males.

A continuación se analizó la expresión de Snail1 en las líneas celulares derivadas del ganglio linfático. Sorprendentemente, se detectó la expresión de Snail1 mediante RT-PCR e inmunotransferencia de tipo Western, en las cinco líneas celulares derivadas de ganglio linfático, independientemente de si el origen del tumor primario era a partir de células control MDA-MB-231-shEGFP o a partir de los clones silenciados MDA-MB-231-shSNAI1. Los niveles de Snail1 detectados fueron similares o incluso superiores a los observados en las células MDA-MB-231 originales (figura 7 A, B). La falta de interferencia de Snail1 observada en las células shSNA1-C2-2D, shSNA1-C4-SM y shSNA1-C4-OI derivadas de ganglios linfáticos no pudo atribuirse a la pérdida del casette de expresión de shSNAI1 mediante la reorganización del DNA, ya que todavía se detectó la presencia del vector pSuperior-shRNA, que contenía el inserto correspondiente al control de 64 nucleótidos específico o a la horquilla de SNAI1 mediante PCR en todas las líneas celulares derivadas de ganglios linfáticos (figura 6C).Next, Snail1's expression was analyzed in cell lines derived from the lymph node. Surprisingly, Snail1 expression was detected by Western-type RT-PCR and immunoblot, in the five cell lines derived from lymph nodes, regardless of whether the origin of the primary tumor was from of control cells MDA-MB-231-shEGFP or from silenced clones MDA-MB-231-shSNAI1. The levels of Snail1 detected were similar or even higher than those observed in cells MDA-MB-231 originals (figure 7 A, B). The lack of Snail1 interference observed in the cells shSNA1-C2-2D, shSNA1-C4-SM and shSNA1-C4-OI derived from ganglia lymphatics could not be attributed to the loss of the expression cassette of shSNAI1 by reorganizing the DNA, since it is still detected the presence of the pSuperior-shRNA vector, containing the insert corresponding to the control of 64 nucleotides  specific or to the fork of SNAI1 by PCR in all cell lines derived from lymph nodes (Figure 6C).

Además, también se detectó un aumento en los niveles de expresión de Snail2 endógeno, tanto a nivel del mRNA como de la proteína, en la mayoría de las líneas analizadas derivadas de ganglios linfáticos, en comparación con los detectados en las células MDA-MB-231 originales (figura 7A, B). Para obtener información adicional, se analizó el potencial tumorigénico de las células shSNAI1-C2-2D y shSNAI1-C4-SM derivadas de ganglios linfáticos, junto con las células MDA-MB-231 originales, mediante inyección ortotópica. Los tumores inducidos por las células shSNAI1-C2-2D y shSNAI1-C4-SM derivadas de ganglio linfático crecieron a la misma tasa que los inducidos por las células MDA-MB-231 originales (figura 7C), a diferencia del potencial de crecimiento tumoral retrasado de las células MDA-MB-231-shSNAI1-C2 y -shSNAI1-C4 (figura 5B). De hecho, el análisis de la expresión de cadherina-E, SPARC, ID1 e ID2 en los tumores inducidos por las líneas celulares derivadas de ganglios linfáticos revelaron niveles similares a los encontrados en los tumores inducidos por las células MDA-MB-231 originales.In addition, there was also an increase in Endogenous Snail2 expression levels, both at the mRNA level as of the protein, in most of the analyzed lines derived from lymph nodes, compared to those detected in MDA-MB-231 cells originals (figure 7A, B). For additional information, you analyzed the tumorigenic potential of the cells shSNAI1-C2-2D and shSNAI1-C4-SM derived from ganglia lymphatics, together with the cells Original MDA-MB-231, through orthotopic injection Cell-induced tumors shSNAI1-C2-2D and shSNAI1-C4-SM derived from ganglion lymphatic grew at the same rate as those induced by original MDA-MB-231 cells (Figure 7C), unlike tumor growth potential delayed cells MDA-MB-231-shSNAI1-C2 and -shSNAI1-C4 (Figure 5B). In fact, the analysis of the expression of cadherin-E, SPARC, ID1 and ID2 in the tumors induced by ganglion-derived cell lines lymphatics revealed levels similar to those found in the cell-induced tumors MDA-MB-231 originals.

Colectivamente, los datos in vivo confirman la fuerte presión selectiva en favor de las células que expresan Snail1 y Snail2 dentro de la subpoblación de células de carcinoma de mama MDA-MB-231 más agresivas/metastásicas.Collectively, the in vivo data confirm the strong selective pressure in favor of the cells expressing Snail1 and Snail2 within the subpopulation of more aggressive / metastatic MDA-MB-231 breast carcinoma cells. 

       \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip

Ejemplo 5Example 5

El silenciamiento estable de Snail1 confiere sensibilidad a la quimioterapiaThe stable silencing of Snail1 confers chemotherapy sensitivity

Para analizar si la resistencia a la apoptosis inducida por lesión genotóxica puede resultar afectado por el silenciamiento de Snail1 en las células MDA-MB-231, se analizaron las propiedades proliferativas de las células con Snail1 silenciado, no detectando diferencias significativas en comparación con las células originales cuando se las hacía crecer en presencia o ausencia de suero en cultivos in vitro, basándose en la incorporación de BrdU, (figura 8).To analyze whether apoptosis resistance induced by genotoxic lesion may be affected by the silencing of Snail1 in the MDA-MB-231 cells, the proliferative properties of the cells with silenced Snail1 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).

Para analizar si el silenciamiento de Snail1 podía conferir sensibilidad a la lesión genotóxica inducida, se trataron las células con dos fármacos quimioterápicos diferentes, docetaxel y gencitabina, usados comúnmente para tratar el cáncer de mama (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 expresión profiling of breast cancer cells in response to gemcitabine: NF-kappaB pathway activation as a potencial mechanism of resistance. Breast cancer research and treatment 2007; 102(2):157-172). Aunque ambos fármacos provocan un nivel modesto de apoptosis en las células MDA-MB-231, el silenciamiento de Snail1 indujo una respuesta 2 veces superior a la apoptosis bajo ambos tratamientos quimioterápicos, un efecto anulado tras la expresión del mutante silencioso mutS-Snail1 en los clones shSNA1-C2 y -C4 (figura 9). De manera importante, el tratamiento de las líneas celulares derivadas de ganglios linfáticos (shSNAI1-C2-2D, shSNAI1-C4-SM y shSNAI1-C4-OI) con cualquiera de los fármacos indujo una baja respuesta apoptótica, similar a la mostrada por las células JV originales y control (figura 10), lo que confirma adicionalmente la asociación entre Snail1, el comportamiento tumorigénico/metastásico y la resistencia a la apoptosis en las células MDA-MB-231.To analyze whether Snail1 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, Snail1 silencing induced a response 2 times higher than apoptosis under both chemotherapeutic treatments, an effect canceled after the expression of the silent mutant mutS-Snail1 in the shSNA1-C2 and -C4 clones (Figure 9). Importantly, 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 Original and control JV cells (Figure 10), which further confirms the association between Snail1, tumorigenic / metastatic behavior and apoptosis resistance in MDA-MB-231 cells.

Tomados conjuntamente, los datos in vivo e in vitro demostraron que la expresión de Snail1 confiere propiedades a las células del carcinoma de mama que van más allá de la represión de cadherina-E y la inducción de la TEM, confiriendo la ventaja de crecimiento tumoral, la resistencia a los fármacos quimioterápicos y la invasión y las capacidades metastásicas.Taken together, the in vivo and in vitro data demonstrated that Snail1 expression confers properties to breast carcinoma cells that go beyond cadherin-E repression and TEM induction, conferring the advantage of tumor growth, resistance to chemotherapeutic drugs and invasion and metastatic abilities.

<110> Universidad Autónoma de Madrid<110> Autonomous University of Madrid 

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<120> Método de diagnóstico y/o pronóstico de cáncer de mama<120> Diagnostic method and / or prognosis of breast cancer 

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<130> 165/07<130> 165/07 

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<170> PatentIn version 3.4<170> PatentIn version 3.4 

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<213> ratón/humano<213> mouse / human 

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\hskip-.1em\dddseqskip
gaugcacauc cgaagccac 
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19 
 \ hskip-.1em \ dddseqskip
gaugcacauc cgaagccac 
 \ hfill
19 

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<213> ratón/humano<213> mouse / human 

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<400> 2<400> 2 

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\hskip-.1em\dddseqskip
guggcuucgg augugcauc 
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19 
 \ hskip-.1em \ dddseqskip
guggcuucgg augugcauc 
 \ hfill
19 

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         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 3<210> 3 

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<212> DNA<212> DNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> ratón/humano<213> mouse / human 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 3<400> 3 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
gatgcacatc cgaagccac 
\hfill
19 
 \ hskip-.1em \ dddseqskip
gatgcacatc cgaagccac 
 \ hfill
19 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 4<210> 4 

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<212> DNA<212> DNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> cebador<223> primer 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 4<400> 4 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
tgcacatcag gtcgcacacg ctgccttgtg tctgcacgac ctg 
\hfill
43 
 \ hskip-.1em \ dddseqskip
tgcacatcag gtcgcacacg ctgccttgtg tctgcacgac ctg 
 \ hfill
43 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 5<210> 5 

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<211> 42<211> 42 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> DNA<212> DNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> artificial<213> artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> cebador<223> primer 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 5<400> 5 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
agcgtgtgcg acctgatgtg catcttcaga gcgcccaggc tg 
\hfill
42 
 \ hskip-.1em \ dddseqskip
agcgtgtgcg acctgatgtg catcttcaga gcgcccaggc tg 
 \ hfill
42 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 6<210> 6 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 45<211> 45 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> DNA<212> DNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> ratón/humano<213> mouse / human 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 6<400> 6 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
gatgcacatc cgaagccact tcaagagtgg cttcggatgtgcatc 
\hfill
45 
 \ hskip-.1em \ dddseqskip
gatgcacatc cgaagccact tcaagagtgg cttcggatgtgcatc 
 \ hfill
Four. Five 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 7<210> 7 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 19<211> 19 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> DNA<212> DNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> cebador directo Snail2 humano para RT-PCR<223> human Snail2 direct primer for RT-PCR 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 7<400> 7 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
cgctccttcc tggtcaaga 
\hfill
19 
 \ hskip-.1em \ dddseqskip
cgctccttcc tggtcaaga 
 \ hfill
19 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 8<210> 8 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 18<211> 18 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> DNA<212> DNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> cebador inverso Snail 2 humano para RT-PCR<223> human Snail 2 reverse primer for RT-PCR 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 8<400> 8 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
ttgcgtcact cagtgtgc 
\hfill
18 
 \ hskip-.1em \ dddseqskip
ttgcgtcact cagtgtgc 
 \ hfill
18 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 9<210> 9 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 18<211> 18 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> DNA<212> DNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> cebador directo SNAI1 humano para qRT-PCR<223> human SNAI1 direct primer for qRT-PCR 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 9<400> 9 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
cactatgccg cgctcttc 
\hfill
18 
 \ hskip-.1em \ dddseqskip
cactatgccg cgctcttc 
 \ hfill
18 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 10<210> 10 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 19<211> 19 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> DNA<212> DNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> cebador inverso SNAI1 humano para qRT-PCR<223> human SNAI1 reverse primer for qRT-PCR 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 10<400> 10 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
ggtcgtaggg ctgctggaa 
\hfill
19 
 \ hskip-.1em \ dddseqskip
ggtcgtaggg ctgctggaa 
 \ hfill
19 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 11<210> 11 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 20<211> 20 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> DNA<212> DNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> cebador directo SNAI2 humano para qRT-PCR<223> human SNAI2 direct primer for qRT-PCR 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 11<400> 11 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
tggttgcttc aaggacacat 
\hfill
20 
 \ hskip-.1em \ dddseqskip
tggttgcttc aaggacacat 
 \ hfill
twenty 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 12<210> 12 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 19<211> 19 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> DNA<212> DNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> cebador inverso SNAI2 humano para qRT-PCR<223> human SNAI2 reverse primer for qRT-PCR 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 12<400> 12 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
gttgcagtga gggcaagaa 
\hfill
19 
 \ hskip-.1em \ dddseqskip
gttgcagtga gggcaagaa 
 \ hfill
19 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 13<210> 13 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 23<211> 23 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> DNA<212> DNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> cebador directo cadherina-E humana para qRT-PCR<223> direct primer human cadherin-E for qRT-PCR 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 13<400> 13 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
agaacgcatt gccacataca ctc 
\hfill
23 
 \ hskip-.1em \ dddseqskip
agaacgcatt gccacataca ctc 
 \ hfill
2. 3 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 14<210> 14 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 23<211> 23 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> DNA<212> DNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> cebador inverso cadherina-E humana para qRT-PCR<223> reverse primer human cadherin-E for qRT-PCR 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 14<400> 14 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
cattctgatc ggttaccgtg atc 
\hfill
23 
 \ hskip-.1em \ dddseqskip
cattctgatc ggttaccgtg atc 
 \ hfill
2. 3 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 15<210> 15 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 20<211> 20 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> DNA<212> DNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> cebador directo SPARC humana para qRT-PCR<223> human SPARC direct primer for qRT-PCR 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 15<400> 15 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
gtgcagagga aaccgaagag 
\hfill
20 
 \ hskip-.1em \ dddseqskip
gtgcagagga aaccgaagag 
 \ hfill
twenty 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 16<210> 16 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 20<211> 20 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> DNA<212> DNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> cebador inverso SPARC humana para qRT-PCR<223> human SPARC reverse primer for qRT-PCR 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 16<400> 16 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
tgtttgcagt ggtggttctg 
\hfill
20 
 \ hskip-.1em \ dddseqskip
tgtttgcagt ggtggttctg 
 \ hfill
twenty 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 17<210> 17 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 19<211> 19 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> DNA<212> DNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> cebador directo MMP2 humana para qRT-PCR<223> human MMP2 direct primer for qRT-PCR 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 17<400> 17 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
ataacctgga tgccgtcgt 
\hfill
19 
 \ hskip-.1em \ dddseqskip
ataacctgga tgccgtcgt 
 \ hfill
19 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<210> 18<210> 18 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<211> 21<211> 21 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<212> DNA<212> DNA 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<213> Artificial<213> Artificial 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<220><220> 

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<223> cebador inverso MMP2 humana para qRT-PCR<223> human MMP2 reverse primer for qRT-PCR 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip

<400> 18<400> 18 

         \vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
      

         \vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
      

\hskip-.1em\dddseqskip
aggcaccctt gaagaagtag c 
\hfill
21 
 \ hskip-.1em \ dddseqskip
aggcaccctt gaagaagtag c 
 \ hfill
twenty-one

Claims (33)

1. Método para el diagnóstico y/o pronóstico de cáncer de mama que comprende:1. Method for diagnosis and / or prognosis of breast cancer comprising:
a.to.
determinar el nivel de expresión del factor de transcripción Snail1 en una muestra biológica aislada de un sujeto, ydetermine the level of expression of Snail1 transcription factor in a biological sample isolated from a subject, and
b.b.
comparar los resultados obtenidos en a) con valores de referencia.compare the results obtained in a) With reference values.
2. Método, según la reivindicación 1, donde la muestra biológica procede de un tejido tumoral de un paciente con cáncer de mama.2. Method according to claim 1, wherein the biological sample comes from a tumor tissue of a patient with breast cancer 3. Método, según la reivindicación 2, para la evaluación de la capacidad de crecimiento del tumor.3. Method according to claim 2 for the Evaluation of tumor growth capacity. 4. Método, según la reivindicación 2, para la evaluación de la capacidad de recurrencia del tumor.4. Method according to claim 2 for the Evaluation of the recurrence capacity of the tumor. 5. Método, según la reivindicación 2, para la evaluación de la capacidad de desarrollo de metástasis local del tumor.5. Method according to claim 2 for the evaluation of the development capacity of local metastasis of the tumor. 6. Método, según la reivindicación 2, para la evaluación de la capacidad de desarrollo de metástasis a distancia del tumor.6. Method according to claim 2 for the evaluation of the ability to develop distant metastases of the tumor 7. Método, según la reivindicación 2, para la evaluación de la capacidad de respuesta del tumor a agentes quimioterapéuticos.7. Method according to claim 2 for the assessment of tumor responsiveness to agents Chemotherapeutic 8. Uso de un oligonucleótido interferente de Snail1, en la preparación de un medicamento destinado al tratamiento de cáncer de mama.8. Use of an interfering oligonucleotide of Snail1, in the preparation of a medicine intended for breast cancer treatment 9. Uso de un oligonucleótido, según la reivindicación 8, donde dicho oligonucleótido es RNA interferente de Snail1.9. Use of an oligonucleotide, according to the claim 8, wherein said oligonucleotide is interfering RNA of Snail1. 10. Uso de un oligonucleótido, según la reivindicación 9, donde el RNA es de doble cadena con secuencias SEQ ID NO 1 y SEQ ID NO 2.10. Use of an oligonucleotide, according to the claim 9, wherein the RNA is double stranded with sequences SEQ ID NO 1 and SEQ ID NO 2. 11. Uso de un oligonucleótido, según la reivindicación 8, donde dicho oligonucleótido es DNA.11. Use of an oligonucleotide, according to the claim 8, wherein said oligonucleotide is DNA. 12. Uso de un oligonucleótido, según la reivindicación 11, donde el DNA presenta la secuencia SEQ ID NO 3.12. Use of an oligonucleotide, according to the claim 11, wherein the DNA has the sequence SEQ ID NO 3. 13. Uso de un oligonucleótido, según cualquiera de las reivindicaciones 8-12, en la preparación de un medicamento destinado a la disminución de la formación de tumores primarios en cáncer de mama.13. Use of an oligonucleotide, according to any of claims 8-12, in the preparation of a medicine intended to decrease the formation of Primary tumors in breast cancer. 14. Uso de un oligonucleótido, según cualquiera de las reivindicaciones 8-12, en la preparación de un medicamento destinado a la disminución de la recurrencia de tumores primarios en cáncer de mama.14. Use of an oligonucleotide, according to any of claims 8-12, in the preparation of a medicine intended to reduce the recurrence of Primary tumors in breast cancer. 15. Uso de un oligonucleótido, según cualquiera de las reivindicaciones 8-12, en la preparación de un medicamento destinado a la disminución de la capacidad invasiva del tumor en cáncer de mama.15. Use of an oligonucleotide, according to any of claims 8-12, in the preparation of a medicine intended to decrease invasive capacity of the tumor in breast cancer. 16. Uso de un oligonucleótido, según cualquiera de las reivindicaciones 8-12, en la preparación de un medicamento destinado a la disminución de la formación de metástasis local en cáncer de mama.16. Use of an oligonucleotide, according to any of claims 8-12, in the preparation of a medicine intended to decrease the formation of Local metastasis in breast cancer. 17. Uso de un oligonucleótido, según cualquiera de las reivindicaciones 8-12, en la preparación de un medicamento destinado a la disminución de la formación de metástasis a distancia en cáncer de mama.17. Use of an oligonucleotide, according to any of claims 8-12, in the preparation of a medicine intended to decrease the formation of distant metastasis in breast cancer. 18. Uso de un oligonucleótido, según cualquiera de las reivindicaciones 8-12, en la preparación de un medicamento destinado al aumento de la sensibilidad a agentes quimioterapéuticos.18. Use of an oligonucleotide, according to any of claims 8-12, in the preparation of a medicine intended to increase the sensitivity to agents Chemotherapeutic 19. Uso de un oligonucleótido, según la reivindicación 18, donde los agentes quimioterapéuticos son gemcitabina y docetaxel.19. Use of an oligonucleotide, according to the claim 18, wherein the chemotherapeutic agents are gemcitabine and docetaxel. 20. Composición farmacéutica que comprende una cantidad terapéuticamente efectiva de un oligonucleótido interferente de Snail1 para su uso en el tratamiento del cáncer de mama.20. Pharmaceutical composition comprising a therapeutically effective amount of an oligonucleotide interference of Snail1 for use in the treatment of cancer mom. 21. Composición farmacéutica, según la reivindicación 20, donde el oligonucleótido es RNA interferente de Snail1.21. Pharmaceutical composition, according to claim 20, wherein the oligonucleotide is interfering RNA of Snail1. 22. Composición farmacéutica, según la reivindicación 21, donde el RNA es de doble cadena con secuencias SEQ ID NO 1 y SEQ ID NO 2.22. Pharmaceutical composition, according to claim 21, wherein the RNA is double stranded with sequences SEQ ID NO 1 and SEQ ID NO 2. 
         \newpage\ newpage
23. Composición farmacéutica, según las reivindicaciones 21 ó 22, donde el oligonucleótido presenta modificaciones que favorecen su estabilidad.23. Pharmaceutical composition, according to claims 21 or 22, wherein the oligonucleotide has modifications that favor its stability. 24. Composición farmacéutica, según cualquiera de las reivindicaciones 21-23, que comprende adicionalmente un vehículo farmacéuticamente aceptable.24. Pharmaceutical composition, according to any of claims 21-23, comprising additionally a pharmaceutically acceptable vehicle. 25. Composición farmacéutica, según la reivindicación 24, donde el vehículo se selecciona entre liposomas, nanopartículas o complejos peptídicos.25. Pharmaceutical composition, according to claim 24, wherein the vehicle is selected from liposomes, nanoparticles or peptide complexes. 26. Composición farmacéutica, según la reivindicación 25, donde las nanopartículas se seleccionan entre quitosanos, polietilenglicol y oligodendrómeros.26. Pharmaceutical composition, according to claim 25, wherein the nanoparticles are selected from chitosans, polyethylene glycol and oligodendrometers. 27. Composición farmacéutica, según la reivindicación 20, que comprende una secuencia de DNA plasmídico o lineal que comprende la secuencia codificante de la secuencia de un RNA de interferencia de Snail1.27. Pharmaceutical composition, according to claim 20, comprising a plasmid DNA sequence or linear comprising the sequence coding for the sequence of a RNA interference from Snail1. 28. Composición farmacéutica, según la reivindicación 27, donde la secuencia de DNA comprende la secuencia SEQ ID NO 3.28. Pharmaceutical composition, according to claim 27, wherein the DNA sequence comprises the sequence SEQ ID NO 3. 29. Composición farmacéutica, según cualquiera de las reivindicaciones 27-28, que comprende adicionalmente un vehículo farmacéuticamente aceptable.29. Pharmaceutical composition, according to any of claims 27-28, comprising additionally a pharmaceutically acceptable vehicle. 30. Composición farmacéutica, según la reivindicación 29, donde el vehículo se selecciona entre liposomas, nanopartículas, complejos peptídicos o virus.30. Pharmaceutical composition, according to claim 29, wherein the vehicle is selected from liposomes, nanoparticles, peptide complexes or viruses. 31. Composición farmacéutica, según la reivindicación 30, donde las nanopartículas se seleccionan entre quitosanos, polietilenglicol y oligodendrómeros.31. Pharmaceutical composition, according to claim 30, wherein the nanoparticles are selected from chitosans, polyethylene glycol and oligodendrometers. 32. Composición farmacéutica, según la reivindicación 30, donde los virus se seleccionan entre retrovirus, lentivirus, adenovirus, virus asociados a adenovirus o baculovirus.32. Pharmaceutical composition, according to claim 30, wherein the viruses are selected from retroviruses, lentivirus, adenovirus, adenovirus associated virus or baculovirus 33. Composición farmacéutica, según la reivindicación 30 donde los complejos peptídicos contienen secuencias de péptidos reconocibles por receptores de células de mama.33. Pharmaceutical composition, according to claim 30 wherein the peptide complexes contain peptide sequences recognizable by cell receptors mom.
ES200702342A 2007-08-28 2007-08-28 METHOD OF DIAGNOSIS AND / OR FORECAST OF CANCER OF MAMA. Active ES2322834B1 (en)

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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 (en) * 2005-08-26 2007-03-01 The Trustees Of The University Of Pennsylvania Methods using snail transcriptional repressor

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 (en) * 2005-08-26 2007-03-01 The Trustees Of The University Of Pennsylvania Methods using snail transcriptional repressor

Non-Patent Citations (4)

* 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"{} Abril 2007, BREAST CANCER RESEARCH AND TREATMENT; Vol. 102, páginas 157-172; ISSN 0167-6806; todo el documento. *
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" Abril 2007, BREAST CANCER RESEARCH AND TREATMENT; Vol. 102, páginas 157-172; ISSN 0167-6806; todo el documento. *
OLMEDA D., JORDA M., PEINADO H., FABRA A., CANO A.,"{}Snail silencing effectively supresses tumour growth and invasiveness"{} Marzo 2007, ONCOGENE Vol. 26, páginas 1862-1874; ISSN 0950-9232; todo el documento. *
OLMEDA D., JORDA M., PEINADO H., FABRA A., CANO A.,"Snail silencing effectively supresses tumour growth and invasiveness" Marzo 2007, ONCOGENE Vol. 26, páginas 1862-1874; ISSN 0950-9232; todo el documento. *

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