The a3 isoform of subunit a of the vacuolar ATPase localizes to the plasma membrane of invasive breast tumor cells and is overexpressed in human breast cancer

Abstract

The vacuolar (H+)-ATPases (V-ATPases) are a family of ATP-driven proton pumps that acidify intracellular compartments and transport protons across the plasma membrane. Previous work has demonstrated that plasma membrane V-ATPases are important for breast cancer invasion in vitro and that the V-ATPase subunit a isoform a3 is upregulated in and critical for MDA-MB231 and MCF10CA1a breast cancer cell invasion. It has been proposed that subunit a3 is present on the plasma membrane of invasive breast cancer cells and is overexpressed in human breast cancer. To test this, we used an a3-specific antibody to assess localization in breast cancer cells. Subunit a3 localizes to the leading edge of migrating breast cancer cells, but not the plasma membrane of normal breast epithelial cells. Furthermore, invasive breast cancer cells express a3 throughout all intracellular compartments tested, including endosomes, the Golgi, and lysosomes. Moreover, subunit a3 knockdown in MB231 breast cancer cells reduces in vitro migration. This reduction is not enhanced upon addition of a V-ATPase inhibitor, suggesting that a3-containing V-ATPases are critical for breast cancer migration. Finally, we have tested a3 expression in human breast cancer tissue and mRNA prepared from normal and cancerous breast tissue. a3 mRNA was upregulated 2.5-47 fold in all breast tumor cDNA samples tested relative to normal tissue, with expression generally correlated to cancer stage. Furthermore, a3 protein expression was increased in invasive breast cancer tissue relative to noninvasive cancer and normal breast tissue. These studies suggest that subunit a3 plays an important role in invasive human breast cancer.

Isoform-specific gene disruptions reveal a role for the V-ATPase subunit a4 isoform in the invasiveness of 4T1-12B breast cancer cells

“…This was the first study showing that a3-containing V-ATPases localize to the leading edge of highly invasive, migrating breast cancer cells but not of noninvasive breast epithelial cells. Our laboratory also showed that a3 mRNA is overexpressed in 43 of 43 human breast tumor samples relative to normal breast tissue by 2.5-50-fold and that expression of the a3 protein is highest in invasive human breast carcinoma relative to both noninvasive solid tumors and normal breast tissue (25).…”

“…Recently, a3-containing V-ATPases were localized to the plasma membrane of a number of different invasive breast cancer cell lines using isoform-specific antibodies against a3 (25). This was the first study showing that a3-containing V-ATPases localize to the leading edge of highly invasive, migrating breast cancer cells but not of noninvasive breast epithelial cells.…”

“…ing V-ATPase activity using either pharmacological inhibitors or genetic approaches significantly reduces the invasiveness and migratory capacity of numerous cancer cell types (5,18,(21)(22)(23)(24)(25)(26)(27)(28). Specifically, plasma membrane V-ATPases appear to play a central role in cancer cell invasiveness (6).…”

“…Mammals express four different subunit a-isoforms, a1-a4, where a3 and a4 are known to target the V-ATPase to the plasma membrane of osteoclasts and renal intercalated cells, respectively (7,8,11,13). Subunit a3 is overexpressed at the mRNA level in a number of cancer types, including melanoma, breast, pancreatic, and ovarian cancers (18,21,(23)(24)(25)33). Similarly, a4 is overexpressed in glioma (35).…”

Vacuolar ATPase as a potential therapeutic target and mediator of treatment resistance in cancer

“…Other studies have described expression of V‐ATPase/specific subunits in pancreatic cancer,32, 33 nonsmall cell lung cancer,34 oral squamous cell carcinoma,35, 36 ovarian cancer,37 cervical cancer,38 breast cancer,39, 40 gliomas,41 and hepatocellular carcinoma 42. A summary of studies investigating clinical significance of V‐ATPase in cancer is provided in Table 1.…”