Gene editing of α6 integrin inhibits muscle invasive networks and increases cell-cell biophysical properties in prostate cancer.

Human prostate cancer (PCa) confined to the gland is indolent (low-risk) but tumors outside the capsule are aggressive (high-risk). Extracapsular extension requires invasion within and through a smooth muscle-structured environment. Since integrins respond to biomechanical cues, we used a gene editing approach to determine if a specific region of laminin-binding α6β1 integrin was required for smooth muscle invasion both in vitro and in vivo. Human tissue specimens showed PCa invasion through smooth muscle and tumor co-expression of α6 integrin and E-cadherin in a cell-cell location and α6 integrin in a cell-ECM distribution. PCa cells expressing α6 integrin (DU145 α6WT) produced a 3D invasive network on laminin-containing Matrigel and invaded into smooth muscle both in vitro and in vivo. In contrast, cells without α6 integrin (DU145 α6KO) and cells expressing an integrin mutant (DU145 α6AA) did not produce invasive networks, could not invade muscle both in vitro and in vivo, and surprisingly formed 3D cohesive clusters. Using ECIS (electric cell-substrate impedance) testing, cohesive clusters had up to a thirty-fold increase in normalized resistance at 400Hz (cell-cell impedance) as compared to the DU145 α6WT cells. In contrast, measurements at 40,000 Hz (cell-ECM coverage) showed that DU145 α6AA cells were two-fold decreased in normalized resistance and were defective in restoring resistance after a 1uM S1P challenge as compared to the DU145 α6WT cells. The results suggest that gene editing of a specific α6 integrin extracellular region, not required for normal tissue function, can generate a new biophysical cancer phenotype unable to invade the muscle, presenting a new therapeutic strategy for metastasis prevention in PCa.