Dissertação para obtenção do Grau de Mestre em
BiotecnologiaCancer is one of the leading causes of death worldwide. Biological hallmarks such as induced and sustained angiogenesis are implicated in tumour progression, as well as invasion and metastasis which are the major causes of cancer-related mortality. E-Cadherin impairment on the cell membrane is intimately related with invasion and metastasis. Also, increased levels of vascular endothelial growth factor (VEGF), an angiogenic marker, and its receptor on the plasma membrane can be implicated in tumour progression.
This work was focused on how the inactivation of E-Cadherin, a molecule associated to an invasive phenotype can be related with angiogenesis, probably through VEGF-A expression.
Two different cell lines without expression of E-Cadherin and stably transduced to express wild-type (WT) E-Cadherin were used to carry out this study: AGS Par/WT (from stomach) and MDA-435 Mock/WT (from breast). Immunohistochemical staining was performed to determine the cellular localization and western blot analysis was performed to assess the expression levels of E-Cadherin. VEGFA mRNA levels were assessed by quantitative Real-time PCR. Additionally, we determined the levels phosphorylated (phospho) ERK1/2, as well as the expression levels of total ERK1/2. To study the angiogenic role of E-cadherin the chick embryo Chorioallantoic Membrane (CAM) assay was used. We characterise in vivo the different cell lines concerning both angiogenic and tumorigenic responses dependent on E-Cadherin.
Only cell lines stably expressing WT human E-Cadherin showed levels of expression of this protein at the cell membrane regardless of their tissue of origin. In vitro, AGS and MDA-435 cells expressing WT E-Cadherin revealed an increased expression of VEGFA in comparison to the control although not statically significant. In addition, both phospho-ERK1/2 and total ERK1/2 presented similar levels of expression regardless of the tissue of origin and E-Cadherin expression. Both angiogenic and tumorigenic responses in AGS WT was significantly increased in comparison to the control. The MDA-435 WT cells revealed increased tumorigenic response in comparison to the control. Overall, these results suggest that E-Cadherin expression is important for micro-tumour formation as well as for neovascularisation but this effect is dependent on the in vivo context
