Isoform-specific gene disruptions reveal a role for the V-ATPase subunit a4 isoform in the invasiveness of 4T1-12B breast cancer cells
Isoform-specific gene disruptions reveal a role for the V-ATPase subunit a4 isoform in the invasiveness of 4T1-12B breast cancer cells
复制标题
DOI：
10.1074/jbc.ra119.007713
复制
发表时间：
2019-07-19
影响因子：
4.8
通讯作者：
Forgac, Michael
中科院分区：
文献类型：
--
作者：
McGuire, Christina M.;Collins, Michael P.;Forgac, Michael
液泡H+-ATP酶（V-ATP酶）是一种ATP驱动的质子泵，存在于各种细胞内膜和特殊细胞类型的质膜上。先前的工作已经报道质膜V-ATP酶是乳腺癌细胞侵袭的关键参与者。已知将V-ATP酶靶向质膜的两种亚基α-同种型是α 3和α 4，并且已显示α 3的表达与各种侵袭性人乳腺癌细胞系中V-ATP酶的质膜定位相关。在这里，我们分析了亚基a-亚型在侵袭性小鼠乳腺癌细胞系4 T1 - 12 B中的作用。通过定量RT-PCR对每种亚型的mRNA水平进行定量，结果表明α 4是这些细胞中表达的主要亚型。使用基于CRISPR/Cas9的方法来破坏编码四种V-ATP酶亚基α-同种型中的每一种的基因，我们发现仅切除α 4编码基因显著抑制4 T1 - 12 B细胞的侵袭和迁移。此外，具有破坏的α 4的细胞在前缘处表现出降低的V-ATP酶表达，表明α 4同种型主要负责将V-ATP酶靶向4 T1 - 12 B细胞中的质膜。这些发现表明，不同的亚基α-亚型可能直接V-ATP酶的质膜不同的侵袭性乳腺癌细胞系。他们进一步表明，V-ATP酶在细胞表面的表达是促进侵袭性癌细胞表型的主要因素。
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.