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[spa] Esta tesis consiste en el estudio de la regulación por microRNAs del proceso de metástasis de carcinomas ductales invasivos de mama. Hemos encontrado una expresión alterada de varios microRNAs en muestras tumorales, incluyendo las familia miR-200 y miR-181, miR-210, miR-101 y miR-10b, a lo largo de la progresión desde tumores primarios a metástasis regionales y a distancia. miR-7, miR-30, miR-148 y miembros de la familia let-7 se encontraron diferencialmente expresados entre tumores primarios no metastáticos vs primarios metastáticos a ganglios linfáticos. Además, los miembros del cluster 1 de la familia miR-200 se econtraban sobreexpresados en las metástasis ganglionares, las cuales mantenían o tenían una expresión más elevada de E-cadherina respecto de sus correspondientes tumores primarios. Adicionalmente y de acuerdo con la tendencia observada en los tejidos tumorales, observamos niveles elevados de miR-200b y miR-7 en la sangre de pacientes con metástasis regional o a distancia en comparación con pacientes con tumores primarios no metastáticos en el momento del diagnóstico. Centrándonos en la significación biológica de la mayor expresión de los miembros de la familia miR-200 en asociación con la progresión metastática, utilizamos el modelo celular MCF10CA1h para inducir la expresión de miR-200. Observamos que miR-200 promovía una transición mesénquima-epitelio que conllevaba la inducción de un marcado programa epitelial. Éste era acompañado de un fuerte incremento de la actividad aldehido deshidrogenasa (ALDH), mayor capacidad de crecimiento en mamoesferas y la transición de un immunofenotipo CD44+ CD24- a CD44+ CD24+, lo cual era indicativo de la adquisición de características de progenitor luminal por parte de células que expresaban miR-200. Además, las células MCF10CA1h que expresaban miR-200 desarrollaron la capacidad de diferenciarse y organizarse en estructuras tubuloalveolares ramificadas similares a las de la mama normal. Se observó además una mayor capacidad de crecimiento tumoral y de colonización metastática in vivo. Además de la expresión de marcadores epiteliales, la expresión de miR-200 resultó en la inducción de la expresión de marcadores basales y de diferenciación luminal in vitro y de forma más prominente in vivo. Todo ello refueza la idea de que miR-200 induce características de progenitor luminal así como de agresividad en células tumorales mamarias. A nivel mecanístico encontramos que la mayor capacidad de autorrenovación promovida por miR-200 era debida en parte a la inhibición de su ya conocidad diana ZEB2 así como de una activación de la ruta de señalización PI3K-AKT. Finalmente, la morfología de los tumores formados in vivo por células que expresaban miR-200 recordaba a la de los carcinomas metaplásicos mamarios, por lo que decidimos estudiarlos. De hecho, observamos que el componente epitelial de tumores metaplásicos expresaban niveles significativamente superiores de miR-200 que el componente mesenquimal y además, mostraban un perfil de marcadores compatible con el de células luminales progenitoras. En vista de los resultados, proponemos que los microRNAs de la familia miR-200 inducen características de células progenitoras luminales, las cuales asociadas a un fenotipo epitelial, promueven la capacidad metastática de las células tumorales., [eng] The maintenance of an epithelial gene program is essential for the metastatic growth of epithelial cancers, including breast cancer. However, little is known of the molecular and cellular mechanisms leading to the enhanced proliferative and survival properties of metastatic epithelial cells. We report here that forced expression of miR-200s in MCF10CA1h mammary cells induced not only a strong epithelial program but also aldehyde dehydrogenase (ALDH) activity, mammosphere growth and ability to form branched tubuloalveolar structures while promoting orthotopic tumor growth and lung colonization in vivo. This was accompanied with the expression of luminal differentiation markers in vitro and in vivo, the overall phenotype being compatible with a promotion of luminal progenitor traits. Interestingly, the morphology of tumors formed in vivo by MCF10CA1h cells expressing miR-200s was reminiscent of metaplastic breast cancer (MBC) and the epithelial components of MBC samples expressed significantly higher levels of miR-200s than their mesenchymal components and displayed a marker profile compatible with luminal progenitor cells. Additionally, the miR-200 family showed enhanced expression along progression of invasive ductal breast cancer (IDC) from primary tumors to distant metastases, further reflected in higher blood levels of miR-200b in patients with regional or distant metastases relative to patients with primary node-negative tumors. We propose that the expression of epithelial gene programs through miR-200 family microRNAs promote traits of highly proliferative mammary luminal progenitor cells, thereby exacerbating the growth and metastatic properties of transformed mammary epithelial cells.

This Doctoral Thesis project is part of a broader research program whose global aim is to better understand key molecular and cellular events in tumor metastasis, in particular those associated with prostate cancer, in order to identify regulatory molecules, gene networks and biochemical pathways, to propose relevant biomarkers and, finally, to provide proof-of-concept validations for potential therapeutic targets. As a starting point, this particular project has relied on comparative analyses between isogenic tumor cells with high vs. low metastatic potentials. The results of this Thesis project are reflected in two different but closely related studies. Article 1, the main publication of this project, focuses on how phenotypic switches and interactions between distinct tumor cell subpopulations engage or suppress metastasis, and which are the genetic programs that may govern such transformations. Article 2 is more focused on specific molecular post-transcriptional regulatory mechanisms that influence the latter steps of metastatic colonization. 1) Malignant progression in cancer requires populations of tumor-initiating cells (TICs) endowed with unlimited self-renewal, survival under stress and establishment of distant metastases. Additionally, the acquisition of invasive properties driven by epithelial-mesenchymal transition (EMT) is critical for the evolution of neoplastic cells into fully metastatic populations. Here we characterize cellular models derived from prostate and bladder cancer cell lines in which tumor subpopulations expressing a strong epithelial gene program are enriched in highly metastatic TICs, while a second subpopulation with stable mesenchymal traits is impoverished in TICs. Constitutive overexpression of the transcription factor Snai1 in the epithelial/TIC-enriched populations engaged a mesenchymal gene program and suppressed their self-renewal and metastatic phenotypes. Conversely, knockdown of EMT factors in the mesenchymal-like prostate cancer cell subpopulation caused a gain in epithelial features and properties of TICs. Both tumor cell subpopulations cooperated among them, such that the non-metastatic mesenchymal-like prostate cancer subpopulation enhanced the in vitro invasiveness of the metastatic epithelial subpopulation and, in vivo, promoted the escape of the latter from primary implantation sites and accelerated their metastatic colonization. Our models provide new insights into how dynamic interactions between epithelial, self-renewal and mesenchymal gene programs determine the plasticity of epithelial tumor-initiating cells. 2) Although the role of miR-200s in regulating E-cadherin expression and epithelial-to-mesenchymal transition is well established, their influence on metastatic colonization remains controversial. Here we have used clinical and experimental models of breast cancer metastasis to discover a pro-metastatic role of miR-200s that goes beyond their regulation of E-cadherin and epithelial phenotype. Overexpression of miR-200s is associated with increased risk of metastasis in breast cancer and promotes metastatic colonization in mouse models, phenotypes that cannot be recapitulated by E-cadherin expression alone. Genomic and proteomic analyses revealed global shifts in gene expression upon miR-200 overexpression toward that of highly metastatic cells. miR-200s promote metastatic colonization partly through direct targeting of Sec23a, which mediates secretion of metastasis-suppressive proteins, including Igfbp4 and Tinagl1, as validated by functional and clinical correlation studies. Overall, these findings suggest a pleiotropic role of miR-200s in promoting metastatic colonization by influencing E-cadherin–dependent epithelial traits and Sec23a-mediated tumor cell secretome. Both studies, when considered together, share important and novel views of metastasis, mainly in relationship with the last steps of the metastatic cascade, strongly suggesting that prostate, bladder and breast tumor cells with an epithelial non-secretory phenotype are more prone to metastatize. We propose that mesenchymal-like tumor cells with a relatively stable EMT derive from epithelial-CSC cells at the primary tumor site, largely in response to microenvironmental influences. As we have shown in the first study, both tumor cell populations, epithelial-CSC or TIC and mesenchymal-like, interact with each other, one consequence being a transient EMT of the epithelial-CSC population that had retained their epithelial state. Thus, in a given tumor microenvironment, there is a strong pressure for epithelial-CSC to drift from epithelial to mesenchymal-like phenotypes. Together, these heterogeneous populations of tumor cells are effective at overcoming the initial barriers to metastasis where invasion is required. After completing these initial steps, mesenchymal-like tumor cell populations are less critically required, while epithelial-CSC are more important and critical to finally grow metastastatic secondary tumors. There are many reasons to support that epithelial-CSC-unsecretory cells are more capable of metastatic colonization, including the facts that they can form clusters in the bloodstream for better survival, are resistant to anoikis, they show anchorage-independent growth and strong self-renewal capacity that permit them to adapt to different environments and initiate and grow secondary tumors. Very interestingly, such cells have a reduced secretion of metastatic suppressor factors which may interact in autocrine and paracrine fashion with tumoral and stromal cells at metastatic sites., La progressió maligna en càncer requereix de poblacions iniciadores de tumors (TICs) que tenen capacitat auto-renovadora il•limitada, supervivència baix estrès i d'establir metàstasis a distància. Addicionalment, l'adquisició de propietats invasives per transició epiteli-mesènquima (EMT) és crítica per l'evolució neoplàsica en la conversió a poblacions metastàtiques. Aquí caracteritzem models cel•lulars derivats de línies de càncer de pròstata i bufeta, en les quals unes subpoblacions expressen un programa epitelial enriquit en cèl•lules metastàtiques iniciadores de tumors (TICs), mentre que una segona població presenta característiques mesenquimals i és pobre en cèl•lules iniciadores de tumors (TICs). La sobreexpressió constitutiva del factor de transcripció Snai1 en les poblacions enriquides en cèl.lules epitelials-iniciadores de tumors, indueix el fenotip mesenquimal i suprimeix l'auto-renovador i metastàtic. D'altra banda, el knock-down de factors d'EMT en la subpoblació mesenquimal va provocar el guany de propietats epitelials i de cèl•lules iniciadores de tumors. Aquestes dues subpoblacions de càncer de pròstata poden cooperar entre elles. La subpoblació mesenquimal augmenta la seva invasió in vitro de les epitelials-TICs i in vivo promou la seva sortida del lloc de tumor primari accelerant així la colonització metastàtica. Els nostres models proveeixen nous coneixements en com la dinàmica entre programes epitelials auto-renovadors i mesenquimals, determinen la plasticitat de les cèl•lules epitelials iniciadores de tumors

[eng] This Doctoral Thesis project is part of a broader research program whose global aim is to better understand key molecular and cellular events in tumor metastasis, in particular those associated with prostate cancer, in order to identify regulatory molecules, gene networks and biochemical pathways, to propose relevant biomarkers and, finally, to provide proof-of-concept validations for potential therapeutic targets. As a starting point, this particular project has relied on comparative analyses between isogenic tumor cells with high vs. low metastatic potentials. The results of this Thesis project are reflected in two different but closely related studies. Article 1, the main publication of this project, focuses on how phenotypic switches and interactions between distinct tumor cell subpopulations engage or suppress metastasis, and which are the genetic programs that may govern such transformations. Article 2 is more focused on specific molecular post-transcriptional regulatory mechanisms that influence the latter steps of metastatic colonization. 1) Malignant progression in cancer requires populations of tumor-initiating cells (TICs) endowed with unlimited self-renewal, survival under stress and establishment of distant metastases. Additionally, the acquisition of invasive properties driven by epithelial-mesenchymal transition (EMT) is critical for the evolution of neoplastic cells into fully metastatic populations. Here we characterize cellular models derived from prostate and bladder cancer cell lines in which tumor subpopulations expressing a strong epithelial gene program are enriched in highly metastatic TICs, while a second subpopulation with stable mesenchymal traits is impoverished in TICs. Constitutive overexpression of the transcription factor Snai1 in the epithelial/TIC-enriched populations engaged a mesenchymal gene program and suppressed their self-renewal and metastatic phenotypes. Conversely, knockdown of EMT factors in the mesenchymal-like prostate cancer cell subpopulation caused a gain in epithelial features and properties of TICs. Both tumor cell subpopulations cooperated among them, such that the non-metastatic mesenchymal-like prostate cancer subpopulation enhanced the in vitro invasiveness of the metastatic epithelial subpopulation and, in vivo, promoted the escape of the latter from primary implantation sites and accelerated their metastatic colonization. Our models provide new insights into how dynamic interactions between epithelial, self-renewal and mesenchymal gene programs determine the plasticity of epithelial tumor-initiating cells. 2) Although the role of miR-200s in regulating E-cadherin expression and epithelial-to-mesenchymal transition is well established, their influence on metastatic colonization remains controversial. Here we have used clinical and experimental models of breast cancer metastasis to discover a pro-metastatic role of miR-200s that goes beyond their regulation of E-cadherin and epithelial phenotype. Overexpression of miR-200s is associated with increased risk of metastasis in breast cancer and promotes metastatic colonization in mouse models, phenotypes that cannot be recapitulated by E-cadherin expression alone. Genomic and proteomic analyses revealed global shifts in gene expression upon miR-200 overexpression toward that of highly metastatic cells. miR-200s promote metastatic colonization partly through direct targeting of Sec23a, which mediates secretion of metastasis-suppressive proteins, including Igfbp4 and Tinagl1, as validated by functional and clinical correlation studies. Overall, these findings suggest a pleiotropic role of miR-200s in promoting metastatic colonization by influencing E-cadherin–dependent epithelial traits and Sec23a-mediated tumor cell secretome. Both studies, when considered together, share important and novel views of metastasis, mainly in relationship with the last steps of the metastatic cascade, strongly suggesting that prostate, bladder and breast tumor cells with an epithelial non-secretory phenotype are more prone to metastatize. We propose that mesenchymal-like tumor cells with a relatively stable EMT derive from epithelial-CSC cells at the primary tumor site, largely in response to microenvironmental influences. As we have shown in the first study, both tumor cell populations, epithelial-CSC or TIC and mesenchymal-like, interact with each other, one consequence being a transient EMT of the epithelial-CSC population that had retained their epithelial state. Thus, in a given tumor microenvironment, there is a strong pressure for epithelial-CSC to drift from epithelial to mesenchymal-like phenotypes. Together, these heterogeneous populations of tumor cells are effective at overcoming the initial barriers to metastasis where invasion is required. After completing these initial steps, mesenchymal-like tumor cell populations are less critically required, while epithelial-CSC are more important and critical to finally grow metastastatic secondary tumors. There are many reasons to support that epithelial-CSC-unsecretory cells are more capable of metastatic colonization, including the facts that they can form clusters in the bloodstream for better survival, are resistant to anoikis, they show anchorage-independent growth and strong self-renewal capacity that permit them to adapt to different environments and initiate and grow secondary tumors. Very interestingly, such cells have a reduced secretion of metastatic suppressor factors which may interact in autocrine and paracrine fashion with tumoral and stromal cells at metastatic sites., [cat] La progressió maligna en càncer requereix de poblacions iniciadores de tumors (TICs) que tenen capacitat auto-renovadora il•limitada, supervivència baix estrès i d'establir metàstasis a distància. Addicionalment, l'adquisició de propietats invasives per transició epiteli-mesènquima (EMT) és crítica per l'evolució neoplàsica en la conversió a poblacions metastàtiques. Aquí caracteritzem models cel•lulars derivats de línies de càncer de pròstata i bufeta, en les quals unes subpoblacions expressen un programa epitelial enriquit en cèl•lules metastàtiques iniciadores de tumors (TICs), mentre que una segona població presenta característiques mesenquimals i és pobre en cèl•lules iniciadores de tumors (TICs). La sobreexpressió constitutiva del factor de transcripció Snai1 en les poblacions enriquides en cèl.lules epitelials-iniciadores de tumors, indueix el fenotip mesenquimal i suprimeix l'auto-renovador i metastàtic. D'altra banda, el knock-down de factors d'EMT en la subpoblació mesenquimal va provocar el guany de propietats epitelials i de cèl•lules iniciadores de tumors. Aquestes dues subpoblacions de càncer de pròstata poden cooperar entre elles. La subpoblació mesenquimal augmenta la seva invasió in vitro de les epitelials-TICs i in vivo promou la seva sortida del lloc de tumor primari accelerant així la colonització metastàtica. Els nostres models proveeixen nous coneixements en com la dinàmica entre programes epitelials auto-renovadors i mesenquimals, determinen la plasticitat de les cèl•lules epitelials iniciadores de tumors