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The Forgac Laboratory studies the vacuolar ATPases (V-ATPases), a family of ATP-dependent proton pumps that play a variety of critical roles in eukaryotic cells. The V-ATPases are involved in processes such as receptor-mediated endocytosis, protein trafficking and degradation, the loading of secretory vesicles and the entry of many animal viruses, including influenza and Ebola. V-ATPases are also present on the plasma membrane of specialized cells, such as osteoclasts and renal intercalated cells, where they transport protons to the extracellular space. The membrane-embedded subunit a contains the information necessary to localize the pump to specific membranes. Mammals have four subunit a isoforms, a1-a4. a1 and a2 predominantly localize the V-ATPase to intracellular membranes, while a3 and a4 localize the pump to the plasma membranes of osteoclasts and renal intercalated cells, respectively.
Cancer metastasis, or the spread of a primary tumor to secondary sites in the body, is the leading cause of death from cancer, and there are no effective treatments that target this process. Identifying key components that contribute to the metastatic cascade is crucial in order to develop anti-metastatic therapeutics. Kristina Cotter, a PhD candidate in Cellular & Molecular Physiology, is studying the role of the V-ATPase in breast cancer cell invasion and migration. The Forgac lab and other laboratories have shown that the V-ATPase is present on the plasma membrane of invasive breast cancer cells, but not noninvasive cells. Inhibition of V-ATPases with membrane-permeable inhibitors reduces the ability of highly invasive breast cancer cells to invade and migrate in vitro. Furthermore, the Forgac lab has shown that subunit a3 is overexpressed in and critical for invasion of these highly invasive breast cancer cells. Importantly, overexpression of subunit a3 in non-invasive breast epithelial cells induces invasiveness and plasma membrane localization of the V-ATPase. These results suggest that plasma membrane and subunit a3-containing V-ATPases are critical for cancer invasion.
Because of the limited expression of both plasma membrane and a3-containing V-ATPases in normal cells, they represent potentially important new targets to prevent metastasis. To more directly assess whether plasma membrane V-ATPases are critical for invasion and migration, Kristina utilized two methods to specifically ablate V-ATPases at the cell surface. First, a membrane-embedded subunit of the pump was epitope-tagged and expressed in invasive breast cancer cells such that the epitope was present extracellularly in plasma membrane V-ATPases. Treatment these cells with an antibody specific for the epitope ablated plasma membrane V-ATPase activity and significantly reduced invasion and migration. Next, a biotin-conjugated form of the inhibitor bafilomycin was bound to streptavidin to make the compound membrane impermeable. Treatment of breast cancer cells with this compound also significantly inhibited invasion and migration. These results demonstrate that plasma membrane V-ATPases are critical for the invasion and migration of breast tumor cells.
Kristina’s ongoing work in the lab focuses on assessing the localization and expression of subunit a3 in human breast cancer. It remains unknown whether plasma membrane V-ATPases contain a3, and thus the localization of a3 in breast epithelial and breast cancer cells is being examined using an a3-specific monoclonal antibody. In addition, the levels of mRNA for a3 in samples of human breast tumors of different invasiveness are being determined by quantitative RT-PCR, and expression of a3 in sections of human breast cancer tissue are being assessed by immunohistochemistry . These experiments will further test a3-containing V-ATPases as an exciting and novel target to inhibit breast cancer metastasis. Future experiments in our lab will directly test the effect of a3-knockdown on breast cancer metastasis in vivo in a mouse model.
Cotter K, Stransky L, McGuire C, Forgac M. 2015. Recent insights into the structure, regulation, and function of the V-ATPases. Trends Biochem Sci. 40: 611-622.
Cotter K, Capecci J, Sennoune S, Huss M, Maier M, Martinez-Zaguilan R, Forgac M. 2014. Activity of plasma membrane V-ATPases is critical for the invasion of MDA-MB231 breast cancer cells. J Biol Chem. 290: 3680-3692.