Function of Plasma Membrane V-ATPases in Breast Tumor Cell Invasion.
vacuolar H+ ATPases (V-ATPases) are a family of ATP-driven proton pumps that couple ATP
hydrolysis with translocation of protons across membranes. V-ATPases are expressed in
intracellular compartments, such as endosomes and lysosomes, where they participate in
processes such as membrane trafficking and protein degradation. They are also present in
the plasma membrane of speciali... read morezed cells, such as osteoclasts and renal cells, where
they function in bone resorption and urinary acidification, respectively. Previous
studies have implicated V-ATPases in human cancer cell invasion. The a subunit, which
controls cellular targeting of V-ATPases, is expressed as four isoforms in mammalian
cells (a1-a4). The a3 and a4 isoforms target V-ATPases to the plasma membrane of
osteoclasts and renal intercalated cells, respectively. Prior work from our laboratory
comparing a subunit expression in human breast cancer cell lines has shown that both a3
and a4 are highly expressed in highly invasive MDA-MB231 cells compared to poorly
invasive MCF7 cells and that knockdown of either isoform using isoform-specific siRNAs
significantly inhibits invasion of MDA-MB231 cells. To further examine whether
expression of particular a subunit isoforms is critical to invasiveness of breast tumor
cells, two closely related cell lines have been examined. MCF10a is a non-invasive,
immortalized, human breast epithelial cell line and MCF10CA1a is a highly invasive,
H-Ras-transformed derivative of the MCF10a cell line that has been selected for its
ability to form metastases in mice. We find that inhibition of V-ATPase activity by
concanamycin reduced in vitro invasion of MCF10CA1a cells, but not the parental MCF10a
cells. MCF10CA1a cells expressed higher levels of the a3 isoform and higher levels of
plasma membrane V-ATPases relative to MCF10a cells, and knockdown of a3 (but not other
isoforms) using isoform-specific siRNAs inhibits invasion of MCF10CA1a cells.
Importantly, overexpression of the a3 isoform in the parental MCF10a cells significantly
increased both the level of plasma membrane V-ATPases and in vitro invasion. To
determine whether expression of V-ATPases at the plasma membrane is important in breast
cancer cell invasion, we have employed an inhibitory antibody that selectively blocks
plasma membrane V-ATPase activity. Inhibition of plasma membrane V-ATPases is shown to
significantly reduce invasion of MDA-MB231 cells. These studies suggest that human
breast tumor cells employ particular a subunit isoforms to target V-ATPases to the
plasma membrane, where they aid in tumor cell
Thesis (Ph.D.)--Tufts University, 2014.
Submitted to the Dept. of Cellular & Molecular Physiology.
Advisor: Michael Forgac.
Committee: Peter Juo, Brent Cochran, and Daniel Jay.
Keywords: Cellular biology, and Biochemistry.read less