Functional characterization of the transcription factor ZEB1 in epithelial to mesenchymal transition and cancer progression

Epithelial to mesenchymal transition (EMT) is implicated in the progression of primary tumours towards metastasis and is likely caused by a pathological activation of transcription factors regulating EMT in embryonic development. To analyse EMT-causing pathways in tumourigenesis, transcriptional targets of the E-cadherin repressor ZEB1 in invasive human cancer cells were identified. We show that ZEB1 repressed multiple key determinants of epithelial differentiation and cell–cell adhesion, including the cell polarity genes Crumbs3, HUGL2, PKP3 and Pals1-associated tight junction protein. ZEB1 associated with their endogenous promoters in vivo, and strongly repressed promoter activities in reporter assays. ZEB1 downregulation in undifferentiated cancer cells by RNA interference was sufficient to upregulate expression of these cell polarity genes on the RNA and protein level, to re-establish epithelial features and to impair cell motility in vitro. In human colorectal cancer, ZEB1 expression was limited to the tumour–host interface and was accompanied by loss of intercellular adhesion and tumour cell invasion. EMT-inducing transcriptional repressor ZEB1 promotes colorectal cancer cell metastasis and loss of cell polarity. Thereby, ZEB1 suppresses the expression of cell polarity factors, in particular of Lgl2, which was found to be reduced in colorectal and breast cancers. In invasive ductal and lobular breast cancer, upregulation of ZEB1 was stringently coupled to cancer cell dedifferentiation. The invasion potential of MDA-MB-231, a highly invasive breast cancer cell line, is shown to be under the control of ZEB1. Over-expression of ZEB1down-regulates and relocalizes E-Cadherin in MCF7 breast cancer cells; moreover, ZEB1 over-expression results in reduced proliferation rate of these cells. Most importantly, we show that ZEB1 mediated downregulation of E-cadherin involves chromatin modifications. Markers of transcriptionally active chromatin Acetylated H3 and Acetylated H4 were increased upon ZEB1 knock down in MDA-MB-231 cells, while repressive marks like H3K9me2 and H3K27me2 were obseverved to disappear in the same cells. Collectively, the data presented in this thesis show that ZEB1 represents a key player in pathologic EMTs associated with tumour progression.Epithelial to mesenchymal transition (EMT) is implicated in the progression of primary tumours towards metastasis and is likely caused by a pathological activation of transcription factors regulating EMT in embryonic development. To analyse EMT-causing pathways in tumourigenesis, transcriptional targets of the E-cadherin repressor ZEB1 in invasive human cancer cells were identified. We show that ZEB1 repressed multiple key determinants of epithelial differentiation and cell–cell adhesion, including the cell polarity genes Crumbs3, HUGL2, PKP3 and Pals1-associated tight junction protein. ZEB1 associated with their endogenous promoters in vivo, and strongly repressed promoter activities in reporter assays. ZEB1 downregulation in undifferentiated cancer cells by RNA interference was sufficient to upregulate expression of these cell polarity genes on the RNA and protein level, to re-establish epithelial features and to impair cell motility in vitro. In human colorectal cancer, ZEB1 expression was limited to the tumour–host interface and was accompanied by loss of intercellular adhesion and tumour cell invasion. EMT-inducing transcriptional repressor ZEB1 promotes colorectal cancer cell metastasis and loss of cell polarity. Thereby, ZEB1 suppresses the expression of cell polarity factors, in particular of Lgl2, which was found to be reduced in colorectal and breast cancers. In invasive ductal and lobular breast cancer, upregulation of ZEB1 was stringently coupled to cancer cell dedifferentiation. The invasion potential of MDA-MB-231, a highly invasive breast cancer cell line, is shown to be under the control of ZEB1. Over-expression of ZEB1down-regulates and relocalizes E-Cadherin in MCF7 breast cancer cells; moreover, ZEB1 over-expression results in reduced proliferation rate of these cells. Most importantly, we show that ZEB1 mediated downregulation of E-cadherin involves chromatin modifications. Markers of transcriptionally active chromatin Acetylated H3 and Acetylated H4 were increased upon ZEB1 knock down in MDA-MB-231 cells, while repressive marks like H3K9me2 and H3K27me2 were obseverved to disappear in the same cells. Collectively, the data presented in this thesis show that ZEB1 represents a key player in pathologic EMTs associated with tumour progression

Characterising the role of Zeb1 in the adult haematopoietic system

Zeb1, a zinc finger E-box binding EMT transcription factor, acts as a critical regulator of cell plasticity and confers properties of ‘stemness’, such as self-renewal, in cancer. Yet little is known about the function of Zeb1 in somatic stem cells. We have used the haematopoietic system, as a well-established paradigm of stem cell biology, to explore Zeb1 mediated regulation of somatic stem cells. Here, we employed a conditional genetic approach using the Mx1-Cre system to specifically knockout (KO) Zeb1 in adult haematopoietic stem cells (HSCs) and their downstream progeny. Long-term loss of Zeb1 resulted in an expansion of HSCs and MPPs that impacted the differentiation to downstream progenitors, while acute deletion of Zeb1 resulted in a reduction of lymphoid progenitors in BM and no change was observed in HSCs. Transplantation of HSCs after acute and chronic loss of Zeb1 resulted in a profound self-renewal defect and multi-lineage differentiation block. Acute loss of Zeb1 in HSCs activates a transcriptional program of deregulated HSC maintenance and multi-lineage differentiation genes, and of cell polarity, consisting of cytoskeleton, lipid metabolism and cell adhesion related genes. Notably, Epithelial cell adhesion molecule (EpCAM) expression was prodigiously upregulated in Zeb1 KO HSCs. Furthermore, acute deletion of Zeb1 led to a rapid onset thymic atrophy and cell autonomous loss of thymocytes and T cells. This defect in thymocytes was associated with increased cell death and changes in cell cycle kinetics as well as perturbations of memory CD8+ T cell homeostasis. Thus, Zeb1 acts as a crucial transcriptional repressor in haematopoiesis, co-ordinating HSC self-renewal and multi-lineage differentiation fates

Caracterización fucnional del factor de transcripción ZEB1 en el programa de transición epitelio mesenquimal y la progresión tumoral

Tesis (Doctora en Ciencias Químicas) - - Universidad Nacional de Córdoba. Facultad de Ciencias Químicas, 2017.La Transición Epitelio-Mesenquimal (EMT) es un programa esencial de la embriogénesis y un evento crítico en la progresión tumoral. Durante la EMT las células epiteliales experimentan cambios moleculares que promueven un fenotipo mesenquimal, caracterizado por la pérdida de la polaridad celular y uniones intercelulares y la ganancia de propiedades migratorias e invasivas. Este programa es finamente regulado a diversos niveles por factores de transcripción, entre los que se incluyen los miembros de las familias ZEB (Zinc Finger E-box Binding Homeobox), SNAIL y TWIST. Estudios recientes demuestran que ZEB1, uno de los miembros de la familia ZEB, tiene una activa participación en la inducción de EMT en diversos tumores epiteliales, tales como colon, próstata y mama. Sin embargo, existen pocos reportes respecto a las señales extracelulares y los mecanismos que regulan a ZEB1 durante el programa EMT. Bajo la hipótesis de que la función de ZEB1 está regulada a distintos niveles (transcripcional, postranscripcional, traduccional y postraduccional) por elementos presentes en el entorno intracelular o extracelular, el objetivo general de esta Tesis Doctoral fue caracterizar los mecanismos moleculares y las vías de transducción de señales que tienen impacto sobre la regulación funcional de ZEB1 y su relevancia en el programa EMT y la progresión tumoral. Durante el desarrollo de esta Tesis Doctoral, en primer lugar, se estudió el rol de la región amino terminal de ZEB1 en el programa EMT, se identificó la porción mínima de ZEB1 (NZEB1) que recapitula la función de ZEB1 wild type (wt) en la EMT en una línea celular de epitelio mamario murino no transformado (NMuMG). En segundo lugar, a través del estudio de múltiples cascadas de transducción de señales se estableció que las vías del receptor del factor de crecimiento similar a la insulina 1 (IGF-1R) y de las proteínas quinasa C (PKCs) regulan los niveles proteicos de ZEB1 y consecuentemente el programa EMT. En este sentido, se demostró que la vía de IGF-1R regula la función de NZEB1 mediante la modulación de su estabilidad proteica por un mecanismo dependiente del proteasoma. En tercer lugar, se evaluó la participación de la vía de PKCs en la regulación de la función biológica de ZEB1 utilizando 10 líneas celulares de mama con diferente grado de agresividad. Esto permitió establecer una correlación lineal entre los niveles de expresión de PKCα, ZEB1 y el estado de activación de la EMT. Mediante ensayos con ARNi y el uso de inhibidores farmacológicos se estableció que PKCα regula los niveles de ZEB1, revirtiendo el fenotipo migratorio e invasivo de células MDA-MB-231. De esta manera se demostró por primera vez que la vía de transducción de señales de PKCs regula la función biológica de ZEB1, definiendo un nuevo eje regulatorio del programa EMT en células MDA-MB-231. Palabras clave: ZEB1, EMT, cáncer de mama, vías de transducción de señales, PKCα, IGF-1R.Llorens de los Ríos, Candelaria. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas; Argentina.Cabanillas, Ana María de Los Angeles. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina.Bocco, José Luis. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina.Daniotti, José Luis. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica; Argentina.Lopez, Pablo Héctor Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentina.Simian, Marina. Universidad Nacional de San Martín. Instituto de Nanosistemas; Argentina