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Integrins are heterodimeric cell surface glycoproteins that bind cells to extracellular matrix (ECM) and regulate tumour cell proliferation, migration and survival. We have previously observed that de novo expression of integrin αVβ3 in Cal27-derived cell clone 2B1 conferred resistance to several anticancer drugs (cisplatin, mitomycin C and doxorubicin) through the mechanism which involves downregulation of pSrc(Y418) and increased cell migration and invasion. In addition, in 2B1 cells we observed increased expression of integrin αVβ5 whose expression was unrelated to drug resistance (Stojanović et al., BBA-MCR, 2016). Upon integrin clustering, multimolecular integrin adhesion complexes (IACs) are formed, creating links to the cell cytoskeleton. Functional and morphological analyses have defined major forms of IACs, including focal adhesions, fibrillar adhesions, hemidesmosomes and reticular adhesions. Several protocols have been developed to purify ventral IACs from 2D cell cultures, but most preparations have been isolated from cells seeded on fibronectin which led to the definition of a FN- induced adhesion components. In order to further analyse the adhesion components whose differential expression contributes to the observed changes in Cal27 and 2B1 cell adhesion and anticancer drug resistance, we isolated IACs from both cell lines cultured on dishes without prior coating with ECM proteins enabling us to detect adhesion components and various ECM proteins secreted by the cells themselves. Mass spectrometry (MS)-based proteomics analysis showed that both cell lines preferentially use integrin α6β4 for adhesion. Anticancer drug resistant cell clone 2B1 show increased level of α6β4 accompanied with increased deposition of laminin 332 but decreased level of keratins 5 and 14 (KRT-5/14). Validation of MS data using western blot, immunofluorescence and electron microscopy experiments demonstrated formation of type II hemidesmosomes which are not linked to KRT-5/14. Keratins 5/14 are very likely part of desmosomes whose levels are reduced in 2B1 cells. The downregulation of integrin α6β4 heterodimer achieved by β4 subunit knockdown conferred resistance to anticancer drugs of both, Cal27 and 2B1 cells, revealing another mechanism of drug resistance. Taken together, our results show multiple integrin crosstalk events triggered by αVβ3 expression i.e. upregulation of αVβ5 and α6β4, and the role of α6β4 in regulating anticancer drug sensitivity.