PO-205 Characterisation of the integrin alpha v-dependent adhesome in mda-mb-435s melanoma cells

Abstract

IntroductionIntegrins are heterodimeric glycoproteins that bind cells to extracellular matrix proteins. Upon integrin clustering, multimolecular integrin adhesion complexes (IACs) form that facilitate the linkage between integrins and the actin cytoskeleton and permit bidirectional signalling. The αV integrin is expressed in most tumour cells where it regulates an array of cellular functions and plays a role in anti-tumour drug resistance. The aim of our work was to assess αV-dependent changes in IAC composition in MDA-MB-435S melanoma cells in order to better understand the increased sensitivity to paclitaxel and vincristine upon integrin αV knockdown.Material and methodsIntegrin αV-specific shRNA was cloned into pSUPER.puro, transfected into MDA-MB-435S cells using Lipofectamine, and cell clones were selected using puromycin. The sensitivity of cells to antitumor drugs was determined using an MTT assay. Cell migration was monitored using a Transwell assay. IACs were isolated following crosslinking and their molecular composition analysed using mass spectrometry (MS)–based proteomics.Results and discussionsIn two MDA-MB-435S-derived cell clones with decreased expression of integrin αV, expressing 15% (2αV) or 5% (3αV) of the control cells amount, increased sensitivity to paclitaxel and vincristine, decreased sensitivity to cisplatin, and decreased migration were observed in line with previous results obtained following transient transfection with integrin αV siRNA. In cell clones 2αV and 3αV, which were smaller than control cells and lacked stress fibres, the number of focal adhesions was shown to be significantly lower as observed by interference reflection microscopy and immunofluorescence detection of phospho-paxillin, phospho-FAK and phospho-Src. MS analysis of isolated IACs from control MDA-MB-435S, 2αV and 3αV cells identified 282 proteins, including 36 out of 60 consensus adhesome proteins. As expected, in clones 2αV and 3αV, integrins αV, β3 and β5 were detected at much lower levels compared with control cells. In addition, lower levels of talin-1 and −2, vinculin, alpha-actinin-4, tensin-3, filamin-A and -B, liprin β1 and plectin were detected.ConclusionThese data will enable follow-up analyses of the mechanisms of signalling by integrins αVβ3/β5 and therefore represent a valuable resource to improve our understanding of the mechanisms involved in adhesion control of cell sensitivity to antitumor drugs and metastatic potential.