O câncer de mama é a principal causa de morte por câncer entre as mulheres no mundo. Fatores genéticos, comportamentais e ambientais afetam o risco de aparecimento dessa doença e seu desenvolvimento e progressão ocorrem pelo acúmulo de alterações genéticas/epigenéticas que levam a manutenção de sinais proliferativos nas células, fuga dos agentes supressores de crescimento e resistência à morte celular. O gene PHLDA1 (de Pleckstrin Homology-Like Domain, Family A, Member 1) codifica uma proteína de 401 aminoácidos que já foi descrita envolvida em distintos processos biológicos incluindo morte celular e, dessa forma, é frequentemente associada ao câncer. Perda progressiva de PHLDA1 já foi descrita em melanoma primário e metastático enquanto sua superexpressão foi descrita para tumores intestinais e pancreáticos. Em dados prévios de nosso grupo de pesquisa o gene PHLDA1 foi encontrado diferencialmente expresso em tumores de mama onde sua ausência estava relacionada com sobrevida livre de doença e sobrevida global reduzidas nas pacientes. Estudos do gene PHLDA1 em linhagens de mama são escassos e a compreensão de seu papel funcional e de como sua ausência pode estar relacionada com a redução da sobrevida em pacientes com câncer de mama permanecem obscuros. Com o objetivo de compreender a função de PHLDA1 em células epiteliais de mama, nós investigamos os efeitos da supressão do gene PHLDA1 em células MCF10A. A redução da expressão foi alcançada a partir de transfecção das células com vetores plasmidiais contendo shRNAs específicos para o transcrito de PHLDA1 e subsequentemente foram realizados ensaios funcionais. A expressão diminuida de PHLDA1 foi capaz de induzir acentuadas alterações morfológicas e comportamentais nas células MCF10A, incluindo mudança no padrão de ancoragem célula-célula e reorganização nos filamentos de actina, além de maior taxa de proliferação, migração e invasão das células. Além disso, em condições de baixa ancoragem, as células com expressão reduzida de PHLDA1 apresentaram mamosferas de formato irregular em comparação às células controle. Em conjunto, nossos resultados mostram que a diminuição da expressão de PHLDA1 em células MCF10A está relacionada a um comportamento agressivo e acentuadas alterações morfológicas. Estes dados são consistentes com atividade supressora tumoral de PHLDA1 em células epiteliais de mama / Breast cancer is the leading cause of cancer death among women worldwide. Genetic, behavioral and environmental factors affect the risk of onset of the disease. Breast cancer development and progression involves the accumulation of genetic/epigenetic changes that lead to maintenance of proliferative signals, evasion of growth suppressors and resistance to cell death. The PHLDA1 gene (Pleckstrin Homology-like domain, Family A, member 1) encodes a 401 amino acids protein that has been described involved in different biological processes including cell death and thus, is often associated with cancer. Progressive loss of PHLDA1 has been described in primary and metastatic melanoma while their overexpression has been reported for intestinal and pancreatic tumors. In previous data from our research group the PHLDA1 gene was found differentially expressed in breast tumors where its downregulation was related to shorter disease-free survival and overall survival of the patients. Literature regarding PHLDA1 in mammary epithelial cell lines is scarce and the understanding of their functional role and how its downregulation can be related to poor prognosis in breast cancer patients remain unclear. In order to understand the PHLDA1 function in breast epithelial cells, we investigated the effects of downregulation of PHLDA1 in MCF10A cells. The reduced expression was achieved from transfection of cells with plasmid vectors containing shRNAs for the specific transcript of PHLDA1 followed by functional assays. The decreased expression of PHLDA1 was sufficient to induce marked morphological and behavioral changes in MCF10A cells, including changes in cell-to-cell attachment pattern and actin reorganization, increased proliferation, migration and invasion rate of cells. Furthermore, in independent of attachment condition, cells with reduced expression of PHLDA1 formed mammospheras whit irregular shape compared to control cells. Taken together, our results showed that the decreased expression of PHLDA1 in MCF10A cells is related to aggressive behavior and marked morphological changes. These data are consistent with tumor suppressor activity for PHLDA1 in breast epithelial cells

O câncer de mama é a principal causa de morte por câncer entre as mulheres no mundo. Fatores genéticos, comportamentais e ambientais afetam o risco de aparecimento dessa doença e seu desenvolvimento e progressão ocorrem pelo acúmulo de alterações genéticas/epigenéticas que levam a manutenção de sinais proliferativos nas células, fuga dos agentes supressores de crescimento e resistência à morte celular. O gene PHLDA1 (de Pleckstrin Homology-Like Domain, Family A, Member 1) codifica uma proteína de 401 aminoácidos que já foi descrita envolvida em distintos processos biológicos incluindo morte celular e, dessa forma, é frequentemente associada ao câncer. Perda progressiva de PHLDA1 já foi descrita em melanoma primário e metastático enquanto sua superexpressão foi descrita para tumores intestinais e pancreáticos. Em dados prévios de nosso grupo de pesquisa o gene PHLDA1 foi encontrado diferencialmente expresso em tumores de mama onde sua ausência estava relacionada com sobrevida livre de doença e sobrevida global reduzidas nas pacientes. Estudos do gene PHLDA1 em linhagens de mama são escassos e a compreensão de seu papel funcional e de como sua ausência pode estar relacionada com a redução da sobrevida em pacientes com câncer de mama permanecem obscuros. Com o objetivo de compreender a função de PHLDA1 em células epiteliais de mama, nós investigamos os efeitos da supressão do gene PHLDA1 em células MCF10A. A redução da expressão foi alcançada a partir de transfecção das células com vetores plasmidiais contendo shRNAs específicos para o transcrito de PHLDA1 e subsequentemente foram realizados ensaios funcionais. A expressão diminuida de PHLDA1 foi capaz de induzir acentuadas alterações morfológicas e comportamentais nas células MCF10A, incluindo mudança no padrão de ancoragem célula-célula e reorganização nos filamentos de actina, além de maior taxa de proliferação, migração e invasão das células. Além disso, em condições de baixa ancoragem, as células com expressão reduzida de PHLDA1 apresentaram mamosferas de formato irregular em comparação às células controle. Em conjunto, nossos resultados mostram que a diminuição da expressão de PHLDA1 em células MCF10A está relacionada a um comportamento agressivo e acentuadas alterações morfológicas. Estes dados são consistentes com atividade supressora tumoral de PHLDA1 em células epiteliais de mama / Breast cancer is the leading cause of cancer death among women worldwide. Genetic, behavioral and environmental factors affect the risk of onset of the disease. Breast cancer development and progression involves the accumulation of genetic/epigenetic changes that lead to maintenance of proliferative signals, evasion of growth suppressors and resistance to cell death. The PHLDA1 gene (Pleckstrin Homology-like domain, Family A, member 1) encodes a 401 amino acids protein that has been described involved in different biological processes including cell death and thus, is often associated with cancer. Progressive loss of PHLDA1 has been described in primary and metastatic melanoma while their overexpression has been reported for intestinal and pancreatic tumors. In previous data from our research group the PHLDA1 gene was found differentially expressed in breast tumors where its downregulation was related to shorter disease-free survival and overall survival of the patients. Literature regarding PHLDA1 in mammary epithelial cell lines is scarce and the understanding of their functional role and how its downregulation can be related to poor prognosis in breast cancer patients remain unclear. In order to understand the PHLDA1 function in breast epithelial cells, we investigated the effects of downregulation of PHLDA1 in MCF10A cells. The reduced expression was achieved from transfection of cells with plasmid vectors containing shRNAs for the specific transcript of PHLDA1 followed by functional assays. The decreased expression of PHLDA1 was sufficient to induce marked morphological and behavioral changes in MCF10A cells, including changes in cell-to-cell attachment pattern and actin reorganization, increased proliferation, migration and invasion rate of cells. Furthermore, in independent of attachment condition, cells with reduced expression of PHLDA1 formed mammospheras whit irregular shape compared to control cells. Taken together, our results showed that the decreased expression of PHLDA1 in MCF10A cells is related to aggressive behavior and marked morphological changes. These data are consistent with tumor suppressor activity for PHLDA1 in breast epithelial cells

<p>Pleckstrin homology like domain family A, member 1 (PHLDA1) is a member of the PHLDA family of homologous proteins recognized for their role in apoptotic cell death. PHLDA1 was first reported as a proapoptotic factor involved in Fas-mediated T-cell apoptosis. The role of this protein with regards to apoptosis remains poorly understood, with literature demonstrating both proapoptotic and antiapoptotic functions in a cell and/or pathway specific manner. Intracellular localization may account for the apoptotic potential of this protein, with nuclear accumulation of PHLDA1 increasing its apoptotic potential. We hypothesize that the functional regions of PHLDA1 including its localization signals (pNLS/pNES), pleckstrin homology like domain (PHLD), and PQ region direct cellular localization of PHLDA1, thereby regulating its apoptotic potential.</p> <p>In this thesis, well-established molecular and cellular approaches were utilized to better define the functional regions within PHLDA1 and to gain further understanding of the role of its localization on apoptosis. Using an EGFP fusion construct and leptomycin B, we confirmed that PHLDA1 contains a weak, CRM1-responsive NES. Using an EGFP-β-galactosidase fusion protein we examined the putative NLS of PHLDA1 and determined that it was not sufficient to direct nuclear localization. However, the PHLD was found to direct cellular localization, mirroring the distribution and punctate patterning of full length PHLDA1. Evidence of association of the PHLD with the membrane was confirmed using fluorescence and electron microscopy, and changes in cell morphology indicative of EMT were apparent following overexpression of the PHLD.</p> <p>Although previous reports have suggested that the PQ region of PHLDA1 is responsible for its proapoptotic function, its cellular localization was not clearly defined. Nuclear accumulation of the PQ region was found to be highly cytotoxic, indicating that it is sufficient to induce apoptosis and that its proapoptotic activity occurs within the nucleus. The findings of this thesis provide fresh insight into the functional regions of PHLDA1 and their respective contributions to the protein’s intracellular localization and apoptotic function, demonstrating that localization dictates the apoptotic potential of PHLDA1. This data provides a solid foundation for identifying the cellular mechanisms by which PHLDA1 influences the progression of chronic human diseases including diabetes, cancer and obesity.</p> / Master of Science (MSc)