engleski

The αv-integrin adhesome affects melanoma cell migration and sensitivity to microtubule poisons

Integrins are heterodimeric glycoproteins that bind cells to extracellular matrix proteins. Upon integrin clustering, multimolecular integrin adhesion complexes (IACs) are formed, facilitating the linkage between integrins and the actin cytoskeleton and permitting bidirectional signalling. To better understand the previously observed change in cell migration and sensitivity to microtubule poisons upon transient transfection with integrin αV-specific siRNA, the aim of this work was to assess αV-dependent changes in IAC composition in two melanoma cell lines MDA-MB- 435S and RPMI-7951. Two MDA-MB-435S-derived integrin αV-specific shRNA expressing cell clones, with decreased expression of integrin αV, expressing 15% (2αV) or 5% (3αV) of the control cells showed decreased migration and increased sensitivity to paclitaxel and vincristine. These results are consistent with previous results obtained following transient transfection with integrin αV-specific siRNA. Similarly, knockdown of integrin αV in the melanoma cell line RPMI-7951 using integrin αV- specific siRNA (RPMI-7951/si(αV)) decreased migration and increased sensitivity to paclitaxel and vincristine compared to cells transfected with the control siRNA (RPMI- 7951/si(-)). In both cell models the decreased expression of integrin αV influenced cell morphology, i.e. the cells were smaller than the control cells and had a lower number of focal adhesions as observed by interference reflection microscopy and immunofluorescence detection of phosphopaxillin. The molecular composition of isolated IACs from MDA-MB-435S cells and cell clones 2αV and 3αV, as well as from RPMI-7951/si(-) and RPMI- 7951/si(αV) cells, was analysed using mass spectrometry (MS)–based proteomics. MS analysis from MDA-MB- 435S and RPMI-7951/si(-) cell lines showed that these cells preferentially use integrin αVβ5 for the formation of IAC. As expected, in clones 2αV and 3αV or RPMI7951/si(αV), integrins αV and β5 were detected at much lower levels compared to control cells. When clones 2αV and 3αV were compared to MDA-MB-435S cells or RPMI-7951/si(αV) compared to RPMI- 7951/si(-), lower levels of talin-2, alpha- actinin-1 and -4, filamin-A and -B, plectin and vinculin were detected. These data will enable follow-up analyses of signalling mediated by integrin αVβ5 and therefore represent a valuable resource to improve our understanding of the mechanisms involved adhesion control of melanoma cell sensitivity to microtubule poisons and cell migration.

integrin alphav ; adhesome ; integrin adhesion complex ; melanoma

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