Investigating the role of Annexin A2 in Epidermal Growth Factor (EGF) induced signalling in cancer

Dissertação de mestrado, Qualidade em Análises, Faculdade de Ciências e Tecnologia, Universidade do Algarve; Universitat de Barcelona; Gdansk University of Technology, Universidad de Cádiz, Universitas Bergensis; 2015

Reactive oxygen species (ROS) are produced as a consequence of cellular metabolism and can also be produced by the cell in response to growth factor/growth factor receptor stimulation to function as second messengers in major signalling pathways. Recently, increasing evidence has revealed that the ROS, H2O2 is an important second messenger in cellular signal transduction, because of its high diffusion and ability to selectively target reactive cysteine residues in proteins. Currently, H2O2-mediated signalling has been implicated in several fundamental physiological processes such as cell proliferation, differentiation, migration and apoptosis. EGF/EGFR is one of the most mutated GF/GFR associations in cancer thanks to its role in tumorigenesis. The binding of EGF to EGFR induces downstream events leading to intracellular production of H2O2 for signalling. Cancer cells characteristically exhibit increased ROS levels compared to normal counterparts that gives them a proliferative advantage and promotes cancer progression. To balance the advantage of low ROS levels (nanomolar concentration for proliferative signalling pathways) against its damaging effect (as a specific oxidant at high concentrations), cancer cells induce the cellular antioxidant response. Our laboratory identified a novel redox regulatory protein, annexin A2 (ANXA2) and showed that its antioxidant function plays a crucial role in supporting tumour growth and chemoresistance. As a logical follow up to this research we investigated the role played by ANXA2 in oncogenic signalling pathways induced by EGF. Here, the activation of signalling pathways in pre-established MDA MB 231 breast cancer cell lines with knockdown for ANXA2 and respective control cells was investigated by western blotting upon treatment of these cells with 50 ng/ml of EGF at different time points. Biotinylated Iodoacetamide (BIAM) assays were done to assess the oxidation of reactive Cys residues in redox sensitive proteins upon treatment with EGF. Intracellular ROS levels and cell proliferation were also analysed in ANXA2 depleted versus control cancer cells upon treatment with EGF. Our results show that ANXA2 depletion in MDA MB 231 cancer cells leads to enhanced activation of the pro-survival and pro-proliferative PI3K/Akt signalling pathway, enhanced ROS production and increased proliferation rate upon EGF treatment compared to control cells. EGF treatment also led to oxidation/inhibition of the main regulator of the PI3K/Akt pathway, PTEN. Interestingly, we observed upregulation of PRDX II (a redox regulatory protein) in ANXA2 depleted MDA MB 231 cells. Taken together, our results demonstrate that ANXA2 plays a redox regulatory role in EGF induced ROSmediated PI3K/Akt signalling and that ANXA2 knockdown cells might be upregulating PRDX II to compensate for the loss of the ANXA2 redox regulatory protein.