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Integrin switching in human tongue squamous carcinoma cells CAL27: de novo expression of ΑvΒ3 results in increased expression of integrins αVβ5 and α6β4

Integrins are heterodimeric transmembrane proteins that lack enzymatic activity, instead, signalling is induced by binding to extracellular matrix (ECM) and forming integrin adhesion complexes, composed various structural and signalling proteins, referred to as the integrin adhesome. Integrins play key role in the regulation of tumor cell adhesion, survival, motility and drug sensitivity. We have previously shown in Cal27 tongue squamous carcinoma cells that transfection of integrin β3, giving rise to cell clone 2B1, led to de novo integrin αVβ3 expression and increased expression of integrin a αVβ5. 2B1 cells displayed increased migration and invasion, and were resistant to multiple antitumor drugs as a consequence of integrin αVβ3-mediated loss of pSrc(Y418). The goal of this study was to analyse the adhesome composition of Cal27 and 2B1 cells by mass spectrometry (MS)-based proteomics. MS analysis identified 639 proteins including 19 out of the 60 consensus adhesome. Based on DAVID analysis, focal adhesion proteins accounted for 17% of identified proteins, whereas 13.8% were ECM components. Unexpectedly, in 2B1 the integrin B3 subunit was not detected, whilst integrin β5 and αV subunits were only identified in one out of five sets of replicates. Interestingly, the MS analysis suggested that Cal27 and 2B1 formed focal adhesions through integrin α6β4. Moreover, an integrin switching effect, i.e. upregulation of integrin α6β4 in2B1 as compared to parental Cal27 cells was observed. Additionally, in focal adhesions isolated from 2B1 cells we found increased abundance of seven focal adhesion proteins (filamin A, filamin B, talin-1, α- actinin-1, Ras GTPase-activating-like protein, tensin-3, vinculin) and eight ECM proteins (trombospondin-1, EGF-like repeat and discoidin I- like domain-containing protein 3, laminin subunit a5, tubulin β chain, perlecan, transforming growth factor beta-induced protein ig-h3 and tenascin C) as compared to Cal27. Of particular interest was the upregulation of both integrin α6 and β4 subunits and the α6β4 ligand laminin 5 in the more drug resistant and motile 2B1 cells since the upregulation of these genes was already found to be significant in the squamous cell carcinoma of the head and neck from patient samples. In conclusion, MS-based proteomic analysis of the integrin adhesome may be a valuable tool for identifying key changes in signalling pathways which can be potentially exploited for diagnostic or therapy purposes.

tongue squamous carcinoma ; integrins ; integrin switching ; adhesome

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