The vacuolar H+ ATPases (V-ATPases) are ATP-driven proton pumps that transport protons across both intracellular and plasma membranes. Previous studies have implicated V-ATPases in the invasiveness of various cancer cell lines. In the present study, we evaluated the role of V-ATPases in the invasiveness of two closely matched human breast cancer lines. MCF10a cells are a non-invasive, immortalized breast epithelial cell line and MCF10CA1a cells are a highly invasive, H-Ras transformed derivative of MCF10a cells selected for their metastatic potential. Using an in vitro Matrigel assay, MCF10CA1a cells showed much higher invasion than the parental MCF10a cells. Moreover, this increased invasion was completely sen... More
The vacuolar H+ ATPases (V-ATPases) are ATP-driven proton pumps that transport protons across both intracellular and plasma membranes. Previous studies have implicated V-ATPases in the invasiveness of various cancer cell lines. In the present study, we evaluated the role of V-ATPases in the invasiveness of two closely matched human breast cancer lines. MCF10a cells are a non-invasive, immortalized breast epithelial cell line and MCF10CA1a cells are a highly invasive, H-Ras transformed derivative of MCF10a cells selected for their metastatic potential. Using an in vitro Matrigel assay, MCF10CA1a cells showed much higher invasion than the parental MCF10a cells. Moreover, this increased invasion was completely sensitive to the specific V-ATPase inhibitor concanamycin. MCF10CA1a cells expressed much higher levels of both a1 and a3 subunit isoforms relative to the parental line. Isoforms of subunit a are responsible for subcellular localization of V-ATPases, with a3 and a4 targeting V-ATPases to the plasma membrane of specialized cells. Knockdown of either a3 alone or a3 and a4 together using isoform-specific siRNAs inhibited invasion by MCF10CA1a cells. Importantly, overexpression of a3 but not the other a subunit isoforms greatly increased the invasiveness of the parental MCF10a cells. Similarly, overexpression of a3 significantly increased expression of V-ATPases at the plasma membrane. These studies suggest that breast tumor cells employ particular a subunit isoforms to target V-ATPases to the plasma membrane where they function in tumor cell invasion.
