engleski

Proteomic analysis of integrin αV-dependent adhesion complex in triple negative breast cancer cell line MDA-MB-231

Integrins are heterodimeric receptors that mediate signalling across the plasma membrane from the extracellular environment to the actin cytoskeleton. As they bind to the proteins of the extracellular matrix, a formation of integrin adhesion complexes (IACs), large protein aggregates on the cytoplasmic tails of integrins, is induced. The composition of these complexes has been named the adhesome, and some adhesion proteins have been identified as promising druggable candidates in cancer therapy. Our recently published data in triple negative breast cancer cell line MDA-MB-231 has shown that transient transfection with integrin αv-specific siRNA increased sensitivity to paclitaxel, vincristine and cisplatin, and decreased migration and invasion. Transient knockdown of integrin subunit β5, but not β3, has shown the same effect. The aim of this work was to assess integrin αV- dependent changes in IAC composition. Plasmid pSUPER.puro or pSUPER.puro containing integrin αV- specific shRNA were transfected into MDA-MB-231 cells using Lipofectamine, and two stably transfected cell clones 231sh (control plasmid) and 231shαV (plasmid containing integrin αV- specific shRNA) were selected using puromycin. Decreased expression of integrin subunit αV and integrin heterodimers αVβ3 and αVβ5 was confirmed using flow cytometry and the sensitivity of cells to antitumor drugs was determined by using MTT assay. Surprisingly, cell clone 231shαV was shown to be less sensitive to paclitaxel, vincristine and cisplatin as compared to 231sh which is not in line with the results obtained by transient transfection experiments. IACs were isolated following crosslinking and their molecular composition analysed using mass spectrometry (MS)– based proteomics. MS analysis of isolated IACs from control 231sh and 231shαV cells identified 426 proteins, including 27 out of 60 consensus adhesome proteins. As expected, in 231shαV clone, integrin αV was detected at much lower levels compared to 231sh. In addition, lower levels of various adhesion proteins such as actinin alpha (1 and 4), AHNAK, filamin (A, B, C), HSP70, myosin 9, plectin, IQGAP1, talin-1, tensin-3, vimentin and vinculin were detected. These data will potentiate further analyses of integrin-related signalling. Also, the biological basis for the contradictory results of sensitivity to antitumor drugs for transient versus stable knockdown warrants further exploration.

breast cancer ; integrins ; resistance ; adhesome

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