Gene Editing of α6 Integrin Inhibits Muscle Invasive Networks and Increases Cell–Cell Biophysical Properties in Prostate Cancer

Abstract

Human prostate cancer 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. Because integrins respond to biomechanical cues, we used a gene editing approach to determine if a specific region of laminin-binding a6b1 integrin was required for smooth muscle invasion both in vitro and in vivo. Human tissue specimens showed prostate cancer invasion through smooth muscle and tumor coexpression of a6 integrin and E-cadherin in a cell-cell location and a6 integrin in a cell-extracellular matrix (ECM) distribution. Prostate cancer cells expressing a6 integrin (DU145 a6WT) produced a 3D invasive network on laminin-containing Matrigel and invaded into smooth muscle both in vitro and in vivo. In contrast, cells without a6 integrin (DU145 a6KO) and cells expressing an integrin mutant (DU145 a6AA) did not produce invasive networks, could not invade muscle both in vitro and in vivo, and surprisingly formed 3D cohesive clusters. Using electric cell-substrate impedance testing, cohesive clusters had up to a 30-fold increase in normalized resistance at 400 Hz (cell-cell impedance) as compared with the DU145 a6WT cells. In contrast, measurements at 40,000 Hz (cell-ECM coverage) showed that DU145 a6AA cells were twofold decreased in normalized resistance and were defective in restoring resistance after a 1 mmol/L S1P challenge as compared with the DU145 a6WT cells. The results suggest that gene editing of a specific a6 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 prostate cancer. Significance: This study shows an innovative strategy to block prostate cancer metastasis and invasion in the muscle through gene editing of a specific a6 integrin extracellular region.

Role of secreted extracellular nicotinamide phosphoribosyltransferase (eNAMPT) in prostate cancer progression: Novel biomarker and therapeutic target

“…The Cultrex invasion assay was performed as previously described [32,33]. Prostate smooth muscle cells (PrSMC; Lonza, catalog no.…”

“…Prostate cancer dissemination requires invasion of smooth muscle tissues during vascular intravasation, extravasation and prostatic extracapsular extension [40]. A 3-D in vitro cell culture model of PCa cell invasion was used [33] involving placement of PCa cells on a layer of prostate smooth muscle cells (PrSMC) (Fig. 4A).…”

“…We next utilized a in vivo preclinical model of PCa invasion [33] with the inferior diaphragmatic muscle of the SCID mouse as the PCa invasion platform. PC3 cells were intraperitoneally injected into 2 groups of SCID mice with both groups sacrificed at 4 weeks.…”

Microfluidic-based human prostate-cancer-on-chip

“…Integrin α6, ITGα6, has been associated with migratory and invasive phenotype in human prostate cancer. A considerable amount of work has implied that ITGα6 plays an important role in tumor cell migration and modification of the tumor environment ( Rabinovotz et al, 1995 ; King et al, 2008 ; Goel et al, 2008 ; Rubenstein et al, 2019 ). In our experiments, the cancer marker TMPRSS2 is indeed overexpressed in the entire population of tumor cells, either EMP or C4-2, confirming their prostate cancer identity ( Figures 4A1–4A3 ).…”

Integrins and Epithelial-Mesenchymal Cooperation in the Tumor Microenvironment of Muscle-Invasive Lethal Cancers

“…In smooth muscle, the α7β1 integrin, which is specific for laminin binding to the ECM, is highly expressed; inactivating mutations of this integrin will cause loss of muscle integrity ( Burkin and Kaufman, 1999 ; Boppart et al, 2006 ; Liu et al, 2009 ; Okada et al, 2013 ; Harryman et al, 2021 ). The function of α7β1 integrin is to facilitate muscle cell interactions with laminin in the ECM, while the role of α6β1 integrin (also specific for laminin) is to mediate tumor cell–ECM interactions and smooth muscle invasion ( Rubenstein et al, 2019 ; Harryman et al, 2021 ), suggesting that there may be integrin cooperation between the invading tumor and the normal muscle during laminin-based invasion ( Harryman et al, 2021 ). There is still little known about the various elements that promote tumor migration from the primary organ into and through the smooth muscle barrier, and there likely will be a combination of biomechanical forces, chemoattractants, biochemical signals, CAFs, and hormonal factors that reduce resistance in the muscle to the invasive tumor ( Harryman et al, 2021 ).…”