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

Christina M Mcguire,Michael P Collins,Gehong Sun-Wada,Yoh Wada,Michael Forgac

doi:10.1074/jbc.ra119.007713

Abstract

The vacuolar H+-ATPase (V-ATPase) is an ATP-driven proton pump present in various intracellular membranes and at the plasma membrane of specialized cell types. Previous work has reported that plasma membrane V-ATPases are key players in breast cancer cell invasiveness. The two subunit a-isoforms known to target the V-ATPase to the plasma membrane are a3 and a4, and expression of a3 has been shown to correlate with plasma membrane localization of the V-ATPase in various invasive human breast cancer cell lines. Here we analyzed the role of subunit a-isoforms in the invasive mouse breast cancer cell line, 4T1-12B. Quantitation of mRNA levels for each isoform by quantitative RT-PCR revealed that a4 is the dominant isoform expressed in these cells. Using a CRISPR/Cas9-based approach to disrupt the genes encoding each of the four V-ATPase subunit a-isoforms, we found that ablation of only the a4-encoding gene significantly inhibits invasion and migration of 4T1-12B cells. Additionally, cells with disrupted a4 exhibited reduced V-ATPase expression at the leading edge, suggesting that the a4 isoform is primarily responsible for targeting the V-ATPase to the plasma membrane in 4T1-12B cells. These findings suggest that different subunit a-isoforms may direct V-ATPases to the plasma membrane of different invasive breast cancer cell lines. They further suggest that expression of V-ATPases at the cell surface is the primary factor that promotes an invasive cancer cell phenotype.

Highlights

The vacuolar H؉-ATPase (V-ATPase) is an ATP-driven proton pump present in various intracellular membranes and at the plasma membrane of specialized cell types
Whereas the V-ATPase has previously been shown to function in in vitro migration and invasion of various human breast cancer cell lines (5, 21, 24, 25), using a breast cancer cell line derived from mouse will be important in future studies employing an animal model of breast cancer in an immunocompetent host. 4T1 cells originate from a spontaneously formed mammary tumor in mouse and are commonly used as a model of breast cancer metastasis (36, 37)
Invasion is measured using wells coated with the extracellular matrix– like material Matrigel, whereas migration is measured using uncoated wells. 4T1-12B cells were treated with DMSO or the V-ATPase–specific inhibitor concanamycin A (ConA) at 1 nM, and invasion or migration was induced by the presence of serum in the trans compartment

Summary

The abbreviations used are

V-ATPase, vacuolar protein-translocating adenosine triphosphatase; ConA, concanamycin A; gRNA, guide RNA; ECAR, extracellular acidification rate; FBS, fetal bovine serum; PMSF, phenylmethylsulfonyl fluoride; DMEM, Dulbecco’s modified Eagle’s medium; DAPI, 4Ј,6-diamidino-2-phenylindole. Recent work from our laboratory using an in vitro transwell assay showed that specific inhibition of plasma membrane V-ATPases inhibited the invasion and migration of invasive MDA-MB-231 breast cancer cells to a similar degree as pan-VATPase inhibitors (5). This suggests that plasma membrane V-ATPases may play a key role in promoting an invasive phenotype in breast cancer cells. 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. Isoform plays a role in in vitro invasion and migration as well as expression of V-ATPases at the plasma membrane of an invasive mouse breast cancer cell line

Results

Discussion

Experimental procedures