Biophysical membrane responses of hypoxic prostate cancer cells depend on kindlin-2.

Lethal prostate cancer migrates through hypoxic regions and contractile muscle using mechanosensitive integrin receptors (specifically, β1 integrin heterodimers) to escape the organ. Kindlin-2 (K2) binds and activates β1 integrin for focal adhesion (FA) assembly and is central to these functions. In live cells, we assessed K2's role in tumor cell-cell and cell-ECM (extracellular matrix) resistance and capacitance using electric cell-substrate impedance sensing (ECIS) in hypoxia (1% oxygen), before and after wounding. Reduction of K2 expression by 44% (DU145-shK2) increased the wound closure rates detected by ECIS under hypoxia-133% for cell-ECM adhesion and 127% for cell-cell adhesion. Despite increased response rates, restoration of cell-cell resistance after the wound to the pre-wound levels did not occur under hypoxia if K2 was limited. Since the wound closure rate was accelerated when K2 was limited under hypoxia, we determined the dynamics and size of integrin:K2 complexes and K2-containing FA structures under hypoxia. During the early response to hypoxia (4-8 h), α6β1:K2 complexes increased 2-fold and then returned to normal levels. The α5β1:K2 complexes remained constant until increasing at 12-16 h in hypoxia. High-resolution immunofluorescence microscopy confirmed α6β1:K2 colocalization at lamellipodial protrusions during the early response to hypoxia, with elevated α5β1:K2 complexes observed in FAs 12 h post exposure. FA abundance increased 2-fold as determined by paxillin staining, but FA size decreased up to 45% in hypoxia, persisting up to 16 h. Our collective findings suggest that under hypoxia, the biophysical cell-cell and cell-ECM interactions of cancer cells depend on K2, contain dynamic assemblies of α6β1 and α5β1 integrin:K2 complexes, and favor smaller and more numerous FAs.