The a3 Isoform Vacuolar Type H+-ATPase Promotes Distant Metastasis in the Mouse B16 Melanoma Cells

Abstract

Accumulating evidence indicates that the acidic microenvironments critically influence malignant behaviors of cancer including invasiveness, metastasis, and chemoresistance. Because the vacuolar-type H þ -ATPase (V-ATPase) has been shown to cause extracellular acidification by pumping protons, we studied the role of V-ATPase in distant metastasis. Real-time PCR analysis revealed that the high-metastatic B16-F10 melanoma cells strongly expressed the a3 isoform V-ATPase compared to the low-metastatic B16 parental cells. Consistent with this, B16-F10 cells created acidic environments in lung metastases by acridine orange staining and strong a3 V-ATPase expression in bone metastases by immunohistochemistry. Immunocytochemical analysis showed B16-F10 cells expressed a3 V-ATPase not only in cytoplasm but also plasma membrane, whereas B16 parental cells exhibited its expression only in cytoplasm. Of note, knockdown of a3 V-ATPase suppressed invasiveness and migration with reduced MMP-2 and MMP-9 expression in B16-F10 cells and significantly decreased lung and bone metastases, despite that tumor growth was not altered. Importantly, administration of a specific V-ATPase a3 inhibitor FR167356 reduced bone metastasis of B16-F10 cells. These results suggest that a3 V-ATPase promotes distant metastasis of B16-F10 cells by creating acidic environments via proton secretion. Our results also suggest that inhibition of the development of cancer-associated acidic environments by suppressing a3 V-ATPase could be a novel therapeutic approach for the treatment of cancer metastasis. Mol Cancer Res; 9(7); 845-55. Ó2011 AACR.

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

“…Increased cancer cell invasive activity is frequently associated with aberrant V‐ATPase plasma membrane localization suggesting that increased acidification of the extracellular space plays an important role 29, 33, 34, 37, 47, 48, 49, 50, 51, 52…”

“…For example, using 2 closely related human breast cancer cell lines, MCF10a and MCF10CA1a, Capecci et al found that the levels of mRNA encoding V o a1 and V o a3 were highest in the invasive MCF10CA1a cells 48. Furthermore, the V o a3 isoform was also expressed in lung and bone metastasis of B16‐F10 cells, suggesting this isoform might be relevant to the increased metastatic potential of melanoma cells 51. As previously discussed, V o a isoforms can be expressed in a tissue‐enriched pattern and are associated with plasma membrane localization.…”

Estimating the rotation rate in the vacuolar proton-ATPase in native yeast vacuolar membranes

“…Specific inhibitors of V-ATPase are important because the enzyme is a potential therapeutic target in certain diseases, e.g., osteoporosis, deafness and cancer (Linnett and Beechey 1979;Farina and Gagliardi 1999;Bowman and Bowman 2005;Lu et al 2005;Morimura et al 2008;Otero-Rey et al 2008;Supino et al 2008;Hinton et al 2009;Perez-Sayans et al 2009;McHenry et al 2010;Nishisho et al 2011). The macrolide antibiotics concanamycin A and bafilomycin are the most potent and most selective inhibitors of V-ATPase, with IC 50 values down to the nM region (Bowman et al 1988;Farina and Gagliardi 1999;Gagliardi et al 1999;Huss et al 2002;Dixon et al 2008).…”

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

“…Furthermore, blocking V-ATPase activity inhibits pancreatic cancer cell invasion and reduces matrix metalloproteinase 9 activity (27). The highly metastatic mouse melanoma cell line B16-F10 expresses more a3 than the less metastatic B16 cell line (28). B16-F10 cells also localize V-ATPases to the plasma membrane, and knockdown of a3 suppresses invasion.…”