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

Abstract

Edited by Alex TokerThe 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. 3 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.

The Vacuolar H+ ATPase α3 Subunit Negatively Regulates Migration and Invasion of Human Pancreatic Ductal Adenocarcinoma Cells

“…In agreement with our findings under control conditions in PDAC cells, little if any V-ATPase expression was detectable in the plasma membrane of melanocytes [12]. In contrast, the introduction of a wound scratch elicited significant redistribution of a3 to the plasma membrane in BxPC-3 cells, consistent with previous findings [23]. This agrees well with previous work showing a role of a3-Rab7 interactions in secretory lysosome trafficking in osteoclasts [39].…”

“…Similar findings were reported in melanoma cells [20], whereas in prostate cancer cells, invasiveness was inhibited by bafilomycin in the absence of detectable V-ATPase expression in the plasma membrane [9,10]. In breast cancer cells, a3 knockdown (KD) reduced invasiveness, yet only by at most~25% [21,22], and a role for a4 rather than a3 in invasiveness was proposed in 4T1-12B breast cancer cells [23].…”

“…It is notable that also in MDA-MB-231 cells, KD of a4, but not of a3, reduced plasma membrane V-ATPase expression and a4 KD inhibited invasion more than did a3 KD [21]. Furthermore, in 4T1-12B breast cancer cells, CRISPR/Cas9 knockout of a3 had no effect on migration, invasion, and plasma membrane V-ATPase localization, whereas all of these parameters were inhibited by a4 knockout [23]. We therefore conclude that the evidence for a role of a3 in plasma membrane targeting and invasion of cancer cells is limited to a few cell types, and at least in some cases seems linked to the expression of a4.…”

“…The involvement of the V-ATPases per se, and the a subunits, in particular, in cancer, are still incompletely understood. This is exemplified by the striking yet unexplained differences between the roles of different a subunits in invasion/migration in different cancers [18,[20][21][22][23], but the roles of a3-containing V-ATPases in other cancer hallmarks, such as chemotherapy resistance [7] and macropinocytosis [15], should be explored in future studies.…”

Loss of a subunit of vacuolar ATPase identifies unexpected biological signatures of reduced organelle acidificationin vivo

“…Plasma membrane v-ATPases promote cancer cell survival and malignancy since (i) they maintain a more alkaline intracellular environment that stimulates the Warburg effect and promotes actin polymerization near the v-ATPases enhancing cell mobility and (ii) they decrease the extracellular pH that favors activation of matrix metalloproteases and cathepsins, promoting the invasiveness and metastatic potential of cancer cells [17,[61][62][63]. Accordingly, in vitro experiments suggested that v-ATPase inhibition can be used as a viable therapeutic strategy to reduce cancer invasion and cell migration, and to avoid multi-drug resistance in cancer cells [21].…”