Metastasis is the leading cause of mortality from breast cancer, but effective therapies to inhibit metastasis have not been identified. Identifying novel targets to limit breast cancer metastasis would therefore fill an important unmet clinical need. Metastasis involves the escape of tumor cells from the primary site into the circulation and invasion of these cells into secondary sites. Both of these processes depend upon tumor cell invasiveness, a process in which V-ATPases have recently been implicated. The long-term objective of this research is to determine the role of V-ATPases in breast tumor metastasis. V-ATPases are ATP-dependent proton pumps that function in both intracellular compartments and the plasma membrane in a variety of cellular processes. Targeting of V-ATPases to different cellular membranes is controlled by isoforms of subunit a (a1- a4), with a3 and a4 capable of targeting V-ATPases to the plasma membrane. We have identified V-ATPases in the plasma membrane of highly invasive breast tumor cells (MDA-MB231, MCF10CA1a, 4T1) but not in poorly invasive lines (MCF10a, MCF7). Moreover, the in vitro invasiveness of only the highly invasive lines is inhibited by specific V-ATPase inhibitors. In addition, highly invasive cells express higher levels of the a3 or a4 isoforms, and knock-down of these isoforms using isoform-specific siRNAs inhibits both invasion and plasma membrane localization of the V-ATPase. Importantly, overexpression of a3 in non-invasive MCF10a cells significantly increases plasma membrane V-ATPases and invasiveness. Recently, we have shown that selective inhibition of plasma membrane V-ATPases inhibits invasion of MB231 cells, that a3 is up-regulated in human breast tumor samples and is expressed at the highest levels in invasive breast carcinoma relative to solid tumors and normal tissue. These results suggest that up-regulation of a3 or a4 in breast tumor cells targets V-ATPases to the plasma membrane, where they function to increase invasiveness. Our first objective is to prepare antibodies directed against extracellular epitopes of the V-ATPase that are capable of inhibiting cell surface V-ATPases. Our previous studies employing inhibitory antibodies directed against extracellular epitope tags provide a strong proof of principal for this aim. We predict that these inhibitory antibodies will be effective at reducing breast tumor cell invasiveness. Our second objective is to test the hypothesis that plasma membrane and a3 or a4-containing V-ATPases function in breast tumor metastasis in vivo. This will be accomplished using a mouse xenograft model of breast cancer metastasis in collaboration with Dr. Charlotte Kuperwasser, co-Principal Investigator, who is an expert in the analysis of breast cancer metastasis using such models. We will determine the effect on metastasis of the inhibitory antibodies prepared in Aim 1 as well as disruption of a subunit isoforms in highly invasive breast cancer cell lines using CRISPR/Cas9. Accomplishment of these aims will provide a critical test of plasma membrane and a3 or a4-containing V- ATPases as novel therapeutic targets to limit breast cancer metastasis.
The proposed research is relevant to human health because metastasis is the leading cause of death from breast cancer, but effective therapies to limit metastasis have not been identified. Our data indicate that the V- ATPase, a proton pump involved in a number of basic cellular processes, functions at the plasma membrane of breast tumor cells to increase their invasiveness, and thus represents an important and novel anti-metastatic target to limit breast cancer metastasis. The proposed research will directly test the function of plasma membrane and a3 or a4-containing V-ATPases in in vivo metastasis of breast tumor cells and will develop anti- V-ATPase inhibitory antibodies as potential anti-metastatic therapeutic agents to limit breast cancer metastasis, making this work highly relevant to the mission of NCI.
