AbstractAlmost all non-small cell lung cancer (NSCLC) patients initially responding to EGFR tyrosine kinase inhibitors (TKIs) develop acquired resistance. Since K_Ca3.1 channels, expressed in mitochondria and plasma membrane, regulate similar behavioral traits of NSCLC cells as EGFR, we hypothesized that their blockade contributes to overcoming EGFR-TKI resistance. Meta-analysis of microarray data revealed that K_Ca3.1 channel expression in erlotinib-resistant NSCLC cells correlates with that of genes of integrin and apoptosis pathways. Using erlotinib-sensitive and –resistant NSCLC cells we monitored the role of mitochondrial K_Ca3.1 channels in integrin signaling by studying cell-matrix adhesion with single-cell force spectroscopy. Apoptosis was quantified with fluorescence-based assays. The function of mitochondrial K_Ca3.1 channels in these processes was assessed by measuring the mitochondrial membrane potential and by quantifying ROS production. Functional assays were supplemented by biochemical analyses. We show that K_Ca3.1 channel inhibition with senicapoc in erlotinib-resistant NSCLC cells increases cell adhesion by increasing β1-integrin expression, that in turn depends on mitochondrial ROS release. Increased adhesion impairs migration of NSCLC cells in a 3D matrix. At the same time, the senicapoc-dependent ROS production induces cytochrome C release and triggers apoptosis of erlotinib-resistant NSCLC cells. Thus, K_Ca3.1 channel blockade overcomes EGFR-TKI resistance by inhibiting NSCLC motility and inducing apoptosis.