The V-ATPase-Inhibitor Archazolid Abrogates Tumor Metastasis via Inhibition of Endocytic Activation of the Rho-GTPase Rac1

Abstract

The abundance of the multimeric vacuolar ATP-dependent proton pump, V-ATPase, on the plasma membrane of tumor cells correlates with the invasiveness of the tumor cell, suggesting the involvement of V-ATPase in tumor metastasis. V-ATPase is hypothesized to create a proton efflux leading to an acidic pericellular microenvironment that promotes the activity of proinvasive proteases. An alternative, not yet explored possibility is that V-ATPase regulates the signaling machinery responsible for tumor cell migration. Here, we show that pharmacologic or genetic reduction of V-ATPase activity significantly reduces migration of invasive tumor cells in vitro. Importantly, the V-ATPase inhibitor archazolid abrogates tumor dissemination in a syngeneic mouse 4T1 breast tumor metastasis model. Pretreatment of cancer cells with archazolid impairs directional motility by preventing spatially restricted, leading edge localization of epidermal growth factor receptor (EGFR) as well as of phosphorylated Akt. Archazolid treatment or silencing of V-ATPase inhibited Rac1 activation, as well as Rac1-dependent dorsal and peripheral ruffles by inhibiting Rab5-mediated endocytotic/exocytotic trafficking of Rac1. The results indicate that archazolid effectively decreases metastatic dissemination of breast tumors by impairing the trafficking and spatially restricted activation of EGFR and Rho-GTPase Rac1, which are pivotal for directed movement of cells. Thus, our data reveals a novel mechanism underlying the role of V-ATPase in tumor dissemination. Cancer Res; 72(22); 5976-87. Ó2012 AACR.

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

“…For example, in a 4T1‐Luc mouse breast cancer xenograft model, archazolid treatment inhibited lung metastasis at a concentration (1 mg/kg i.v.) which did not cause signs of obvious toxicity 64. Similar results were obtained using baf‐A1, in which treatment led to the reduction of average tumor volume by 50% in MCF‐7 and MDA‐MB‐231 xenograft mouse models.…”

Activity of Plasma Membrane V-ATPases Is Critical for the Invasion of MDA-MB231 Breast Cancer Cells

“…Although a number of studies have implicated the V-ATPase in tumor cell invasion, the mechanisms by which the pump is involved in this process have not been elucidated (17)(18)(19)(20)(21)(22)(23)(24). Several reports have identified a correlation between cancer cell invasiveness and V-ATPase localization to the plasma mem- A, untransfected control and c-V5-transfected cells were incubated in the absence or presence of a monoclonal antibody against V5 and then loaded with SNARF-1.…”

“…A recent study has found that subunit C interacts with F-actin at the plasma membrane of invasive breast cancer cells and that loss of this subunit disrupts actin arrangement in these cells (54). Moreover, inhibiting V-ATPases with archazolids has been found to impair plasma membrane localization of EGFR and Rac1 in SKBR3 breast cancer cells, a critical process for cell migration (23). It has also been suggested that plasma membrane V-ATPases present in the leading edge of microvascular endothelial cells promote migration through a local increase in cytosolic pH, which facilitates actin polymerization (4,55).…”

“…Inhibitors such as bafilomycin and concanamycin A are membrane-permeable and thus inhibit all of the VATPases in the cell. Furthermore, knockdown of particular subunit a isoforms could alter plasma membrane localization of the V-ATPase or reduce secretion of proinvasive factors by disrupting membrane trafficking (23,24). Previous studies demonstrating that V-ATPase inhibitors and subunit a isoform knockdown reduce cancer cell invasion have thus been unable to determine whether plasma membrane, intracellular, or all cellular V-ATPases contribute to an invasive phenotype.…”

The Function of Vacuolar ATPase (V-ATPase) a Subunit Isoforms in Invasiveness of MCF10a and MCF10CA1a Human Breast Cancer Cells

“…Treatment of metastatic cells with the V-ATPase inhibitor archazolid limited leading edge localization of the epidermal growth factor receptor and blocked endocytic activation of Rac1, a member of the Rho family of small GTPases with known functions in cell migration (40). Inhibition of V-ATPases with bafilomycin or through siRNA silencing prevented peripheral localization of Rab27 secretory vesicles and blocked heat shock protein 90␣ secretion (41).…”