Avaliação funcional do gene PIK3CA (fosfatidilinositol-3-quinase, subunidade catalítica alfa) em células endoteliais resistentes ao Anoikis

• Ano de defesa: 2019
• Autor(a) principal: Mesquita, Ana Paula De Sousa [UNIFESP]
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de São Paulo (UNIFESP)
• Palavras-chave em Português: Anoikis; PIK3CA; PI3K p110α
• Link de acesso: https://sucupira.capes.gov.br/sucupira/public/consultas/coleta/trabalhoConclusao/viewTrabalhoConclusao.jsf?popup=true&id_trabalho=8277057; https://repositorio.unifesp.br/handle/11600/59729

The development, differentiation and homeostasis of adherent cells are controlled by cell-cell, cell-extracellular matrix (ECM) and cell-soluble factors interactions. The loss of these contacts induces to anoikis, an apoptosis specific type. Tumor cells acquire resistance to anoikis, survival after loss of anchorage and invasion of the vascular system, colonizing distant organs. This process is called metastasis. Activation of the PI3K / Akt signaling pathway is the most common mechanism involved in the acquisition of anoikis resistance by cancer cells. Thus, this pathway represents an opportunity and a challenge for cancer therapy. Previous studies from our group reported that the acquisition of anoikis resistance by endothelial cells from rabbit aortic trigger morphological changes, high proliferation rate, poor adhesiveness to fibronectin, laminin and collagen IV, high invasiveness, low apoptosis rate, and dysregulation of cell cycle. In addition, anoikis resistant cell lines exhibit up-regulation of the Ras/ERK and PI3K/Akt pathways. This is accompanied by an extensive remodeling of the MEC. The role of PI3K in cancer was highlighted by the observation that the gene encoding the p110α catalytic subunit is often mutated in the most common human cancers. In this study, we aim to elucidate the role of the p110α catalytic subunit PI3K protein in the acquisition of the anoikis-resistant phenotype and in the regulation of cellular characteristics directly related to tumorigenesis such as apoptosis rate, cell proliferation, adhesiveness, invasiveness and PI3K p110α downstream cellular signaling. For this purpose, we performed the gene silencing of PIK3CA, that gene encodes the PI3K catalytic subunit p110α. Anoikis-resistant endothelial cells (Adh1-EC) subjected to PIK3CA gene silencing originated the cell lines miR-PIK3CA-Adh1-EC 1 e miR-PIK3CA-Adh1-EC 2. Those cell lines shown a reversal in characteristics associated with malignancy: decrease in anoikis resistance and cell proliferation rate, increase in apoptosis rate and caspases 3, 7, 8 activity, increased fibronectin adhesiveness, however, collagen IV and laminin adhesion rates were not altered. This was accompanied by a decrease in invasive capacity, reduction in gene and protein expression of Akt (phosphorylated on threonine 308 and serine 473), mTOR (phosphorylated on serine 2448), PTEN and H-Ras. A reduction in ERK gene expression was also observed, but this reduction xxi was not reflected in the ERK1/2 protein levels phosphorylated on the residues threonine 202 and tyrosine 204, threonine 185 and tyrosine 187. In summary, silencing of only one subunit of the PI3K family, p110α, showed good results in reversing the anoikis-resistant phenotype of endothelial cell. These results provide a molecular basis for the design of new therapies for the treatment of metastatic cancers. This strategy may help overcome apoptosis resistance in aberrantly PI3K / Akt pathway cancers.