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In vitro analysis of Src family kinases in the metastatic process of MDA-MB-231 cell line

AutorSánchez-Bailón, María Pilar
DirectorMartín-Pérez, Jorge ; Calcabrini, Annarica
Fecha de publicación2014
EditorUniversidad Autónoma de Madrid
Resumen[EN]: Breast cancer is the leading cause of cancer death of women in developed countries. In particular, triple-negative breast cancer is highly aggressive with reduced therapeutic options since lacks well-defined molecular targets and is very heterogeneous. It is defined by the absence of Estrogen receptor (ER), Progesterone receptor (PR), as well as, Human epidermal growth factor receptor 2 (HER2) and constitutes 10 – 20% of all breast cancer. Besides, it has been described that Src family kinases (SFKs) are involved in the development of human cancers, including breast cancer. Increases in SFKs activity is correlated with the progression of malignancy. Overexpression of c-Src or aberrant activation has been identified in various human cancers. Src is an essential component of several signalling pathways that regulate proliferation, survival, angiogenesis and metastasis. This Doctoral Thesis is focused on unrevealing the mechanisms that depend on SFKs catalytic activity or on c-Src that lead to metastasis of the triple-negative/basallike breast cancer cell line, MDA-MB-231. To accomplish this objective we have used three complementary approaches: inhibition of SFKs catalytic activity by using three selective inhibitors, Dasatinib, PP2 and SU6656; conditional expression of shRNA-cSrc or conditional expression of dominant-negative Src (SrcDN; K295M/Y527F). We found that SFKs catalytic activity controlled cell proliferation at G1-S phase transition by regulating p27Kip1 and c-Myc expressions, by using Dasatinib and PP2. In contrast, SU6656 provoked polyploid cells, a SFKs-independent effect caused by concomitant inhibition of Aurora B kinase. Furthermore, SFKs inhibition and c-Src depletion reduced cell migration and invasion capabilities of MDA-MB-231 cells by reducing phosphorylation of Src substrates, Y925-Fak, p130CAS, Y118-paxillin and Y14-caveolin 1, required for focal adhesion turnover and cell motility. The expression of SrcDN showed an additional pathway, through Akt activation, involved in cell migration and invasion. Besides, c-Src modulated the secretion of proteins involved in breast cancer progression and metastasis, including Cyr61, IGFBP4 and CTGF. We found a new exosomal protein, Cyr61, which regulated invasion and transendothelial migration, important for extravasation of tumour cells. Furthermore, c-Src modulated the survival of MDA-MB-231 cells in anchorage-independent conditions, as well as, was required to maintain the breast cancer stem/progenitor cell subpopulation by regulating the expression of pluripotency-associated transcription factors Oct3/4, Nanog and Sox2.
[EN]: Breast cancer is the leading cause of cancer death of women in developed countries. In particular, triple-negative breast cancer is highly aggressive with reduced therapeutic options since lacks well-defined molecular targets and is very heterogeneous. It is defined by the absence of Estrogen receptor (ER), Progesterone receptor (PR), as well as, Human epidermal growth factor receptor 2 (HER2) and constitutes 10 – 20% of all breast cancer. Besides, it has been described that Src family kinases (SFKs) are involved in the development of human cancers, including breast cancer. Increases in SFKs activity is correlated with the progression of malignancy. Overexpression of c-Src or aberrant activation has been identified in various human cancers. Src is an essential component of several signalling pathways that regulate proliferation, survival, angiogenesis and metastasis. This Doctoral Thesis is focused on unrevealing the mechanisms that depend on SFKs catalytic activity or on c-Src that lead to metastasis of the triple-negative/basallike breast cancer cell line, MDA-MB-231. To accomplish this objective we have used three complementary approaches: inhibition of SFKs catalytic activity by using three selective inhibitors, Dasatinib, PP2 and SU6656; conditional expression of shRNA-cSrc or conditional expression of dominant-negative Src (SrcDN; K295M/Y527F). We found that SFKs catalytic activity controlled cell proliferation at G1-S phase transition by regulating p27Kip1 and c-Myc expressions, by using Dasatinib and PP2. In contrast, SU6656 provoked polyploid cells, a SFKs-independent effect caused by concomitant inhibition of Aurora B kinase. Furthermore, SFKs inhibition and c-Src depletion reduced cell migration and invasion capabilities of MDA-MB-231 cells by reducing phosphorylation of Src substrates, Y925-Fak, p130CAS, Y118-paxillin and Y14-caveolin 1, required for focal adhesion turnover and cell motility. The expression of SrcDN showed an additional pathway, through Akt activation, involved in cell migration and invasion. Besides, c-Src modulated the secretion of proteins involved in breast cancer progression and metastasis, including Cyr61, IGFBP4 and CTGF. We found a new exosomal protein, Cyr61, which regulated invasion and transendothelial migration, important for extravasation of tumour cells. Furthermore, c-Src modulated the survival of MDA-MB-231 cells in anchorage-independent conditions, as well as, was required to maintain the breast cancer stem/progenitor cell subpopulation by regulating the expression of pluripotency-associated transcription factors Oct3/4, Nanog and Sox2.
DescripciónMemoria presentada en la Universidad Autónoma de Madrid para optar por al grado de Doctor con Mención Internacional por María Pilar Sánchez Bailón, licenciada en Bioquímica y Biología, y realizada en el Instituto de Investigaciones Biomédicas Alberto Sols (CSIC-IIBM).-- Esta obra está bajo una licencia Creative Commons Reconocimiento-No Comercial-Sin Obra Derivada 4.0 Internacional.
URIhttp://hdl.handle.net/10261/126100
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