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TLN2 and KANK2 are potential targets for enhanced sensitivity to paclitaxel treatment in MDA-MB-435S cells

Despite development of targeted agents and immune- based therapies, a fraction of patients diagnosed with melanoma are still treated with conventional antitumor drugs such as microtubule (MT) poison paclitaxel (PTX). Since chemotherapy is usually not as effective for melanoma as it is for some other tumours, there is an urge to characterise novel therapeutic targets that can improve effectiveness of classic chemotherapies. One of the potential targets within the cell are proteins of integrin adhesion complexes (IACs) formed through binding and clustering of integrins to the extracellular matrix. IAC composition analysis in MDA-MB-435S melanoma cell line using mass spectrometry (MS)-based proteomics revealed integrin αVβ5 as the predominant integrin used for adhesion in long term cell culture. Concomitantly, (MS)-based proteomics upon integrin αV knockdown revealed components of integrin αVβ5 focal adhesions (FAs), i.e., talin (TLN) 1 and 2, and KANK 1 and 2. Since the integrin αV knockdown increased sensitivity to PTX we aimed to analyse the underlying mechanism. The immunofluorescence analysis of TLN1, TLN2, KANK1 and KANK2 demonstrated that KANK1 does not localise near integrin αVβ5 FAs and its expression does not change upon integrin αV knockdown. Conversely, we found KANK2 localised near αVβ5 FAs and integrin αV knockdown decreased its expression. Moreover, KANK2 knockdown mimicked the effect of αV depletion i. e. increased sensitivity to PTX and inhibited cell migration. TLN1 knockdown resulted in complete breakdown of integrin αVβ5 FAs, decreased proliferation and expression of KANK2 as well as changed cell morphology, actin and MT appearance. On the other hand, TLN2 knockdown did not affect the number of αVβ5 FAs, cell proliferation, KANK2 expression or localisation, but it increased sensitivity to PTX, decreased migration and altered the MT appearance. The velocity of MT growth, using transfection of fluorescently labelled end-binding protein 3, demonstrated increased velocity upon TLN1, TLN2 or KANK2 knockdown. Finally, the western blot analysis of isolated IACs indicated that TLN2 knockdown reduced KANK2 levels within IACs. Together, these results indicate that TLN2-KANK2 interaction controls MT dynamics and sensitivity of MDA-MB-435S cells to PTX. Therefore, our study identifies TLN2 and KANK2 as additional targets within αVβ5 FAs whose knockdown might be used to increase efficacy of PTX treatment and at the same time inhibit migration.

TLN ; KANK ; actin-microtubule crosstalk ; paclitaxel

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