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. 2013 Nov 8;288(45):32731-32741.
doi: 10.1074/jbc.M113.503771. Epub 2013 Sep 26.

The function of vacuolar ATPase (V-ATPase) a subunit isoforms in invasiveness of MCF10a and MCF10CA1a human breast cancer cells

Joseph Capecci  1 , Michael Forgac  2
Affiliations

The function of vacuolar ATPase (V-ATPase) a subunit isoforms in invasiveness of MCF10a and MCF10CA1a human breast cancer cells

Joseph Capecci et al. J Biol Chem. .

Abstract

The vacuolar H(+) ATPases (V-ATPases) are ATP-driven proton pumps that transport protons across both intracellular and plasma membranes. Previous studies have implicated V-ATPases in the invasiveness of various cancer cell lines. In this study, we evaluated the role of V-ATPases in the invasiveness of two closely matched human breast cancer lines. MCF10a cells are a non-invasive, immortalized breast epithelial cell line, and MCF10CA1a cells are a highly invasive, H-Ras-transformed derivative of MCF10a cells selected for their metastatic potential. Using an in vitro Matrigel assay, MCF10CA1a cells showed a much higher invasion than the parental MCF10a cells. Moreover, this increased invasion was completely sensitive to the specific V-ATPase inhibitor concanamycin. MCF10CA1a cells expressed much higher levels of both a1 and a3 subunit isoforms relative to the parental line. Isoforms of subunit a are responsible for subcellular localization of V-ATPases, with a3 and a4 targeting V-ATPases to the plasma membrane of specialized cells. Knockdown of either a3 alone or a3 and a4 together using isoform-specific siRNAs inhibited invasion by MCF10CA1a cells. Importantly, overexpression of a3 but not the other a subunit isoforms greatly increased the invasiveness of the parental MCF10a cells. Similarly, overexpression of a3 significantly increased expression of V-ATPases at the plasma membrane. These studies suggest that breast tumor cells employ particular a subunit isoforms to target V-ATPases to the plasma membrane, where they function in tumor cell invasion.

Keywords: Bioenergetics; Breast Cancer; Invasion; Proton Pumps; Proton Transport; Vacuolar ATPase; a Subunit Isoforms.

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Figures

FIGURE 1.
FIGURE 1.
In vitro invasion of MCF10a and MCF10CA1a cells after concanamycin treatment. In vitro invasion was assayed using MatrigelTM-coated FluoroBlokTM inserts as described under “Experimental Procedures.” Cells were treated with or without 100 nm concanamycin, allowed to invade toward a chemoattractant, and stained with calcein-AM. An equal amount of solvent (dimethyl sulfoxide) was added to untreated cells. Cells that had migrated to the trans-side were counted, with three wells analyzed per sample and 15 images analyzed per well. Values are the mean ± S.D. of three independent experiments. *, p < 0.01 compared with the untreated control.
FIGURE 2.
FIGURE 2.
mRNA levels of a subunit isoforms in MCF10a and MCF10CA1a cells. mRNA levels were determined using quantitative RT-PCR for each a subunit isoform on mRNA isolated from each cell line as described under “Experimental Procedures.” Plasmids expressing the cDNA of each a subunit isoform were used to establish a standard curve. Values were normalized to the total mRNA loaded and are the mean of four separate experiments. Error bars show standard deviation. A, a subunit isoform levels in MCF10a cells reported as the ratio of a subunit isoform mRNA to the total mRNA. B, ratio of a subunit isoform mRNA levels in MCF10CA1a cells versus MCF10a cells. *, p < 0.01 compared with the MCF10a mRNA level.
FIGURE 3.
FIGURE 3.
In vitro invasion of MCF10CA1a cells after siRNA treatment. A, mRNA levels of a subunit isoforms in MCF10CA1a cells after siRNA treatment. Cells were exposed to a subunit isoform-specific siRNA pools for 96 h prior to measuring mRNA levels, as described under “Experimental Procedures.” Quantitative RT-PCR was conducted as described in the legend for Fig. 2. Knockdown is reported as the ratio of a subunit isoform mRNA in cells treated with siRNA versus a subunit isoform mRNA in untreated cells. Values are the mean of three separate experiments. Error bars indicate S.D. B, Cells were exposed to a subunit isoform-specific siRNAs for 81 h prior to measuring invasion through MatrigelTM-coated FluoroBlokTM wells. Invasion is reported as the percentage of invasion observed for siRNA-treated cells relative to untreated cells (Mock). Three wells were counted per sample, with 15 images analyzed per well. Values are the mean ± S.D. of two or three independent experiments. *, p < 0.005.
FIGURE 4.
FIGURE 4.
In vitro invasion assay of MCF10a cells selectively overexpressing each a subunit isoform. Each a subunit isoform was separately cloned into an overexpression vector, and the vectors were individually stably transfected into MCF10a cells. A, mRNA levels of a subunit isoforms in MCF10a cells expressing a subunit isoform overexpression vectors. mRNA levels were determined using quantitative RT-PCR for each a subunit isoform with mRNA isolated from each stable cell line. Plasmids expressing the cDNA of each a subunit isoform were used to establish a standard curve. The values reported are the ratio of isoform-specific mRNA to total mRNA. Values represent the mean ± S.D. of two or three experiments. B, in vitro invasion of MCF10a cells selectively overexpressing each a subunit isoform through MatrigelTM-coated FluoroBlokTM wells. Invasion is reported as the percentage of invasion observed for cells overexpressing particular a subunit isoforms relative to cells expressing empty vector. Three wells were counted per sample, with 15 images analyzed per well. Values are the mean ± S.D. of three independent experiments. pTracer, MCF10a cells transfected with an empty pTracer vector; a1, MCF10a cells overexpressing a1; a2, MCF10a cells overexpressing a2; a3, MCF10a cells overexpressing a3; a4, MCF10a cells overexpressing a4. *, p < 0.01 compared with pTracer.
FIGURE 5.
FIGURE 5.
Protein levels of subunit A in MCF10a cells overexpressing various a subunit isoforms. The Western blot analysis shows protein levels of subunit A in MCF10a cells overexpressing various subunit a isoforms. pT, MCF10a cells transfected with the empty pTracer vector; a1, MCF10a cells overexpressing a1; a2, MCF10a cells overexpressing a2; a3, MCF10a cells overexpressing a3; a4, MCF10a cells overexpressing a4. The Western blot displayed is representative of data obtained from two separate experiments.
FIGURE 6.
FIGURE 6.
Immunostaining of MCF10a and MCF10CA1a cells using an antibody against V-ATPase. MCF10a and MCF10CA1a cells were grown as a monolayer on coverslips in 6-well plates. Cells were immunostained using an antibody against subunit A of V-ATPase (part of the V1 domain, which stains all V-ATPases in the cell) as well as phalloidin to stain actin. Images were taken with identical exposure times and antibody concentrations. A, MCF10a (top row) and MCF10CA1a (bottom row) cells showing fluorescence staining of actin (left column), V-ATPase (center column), and the merge (left column). B, quantification of plasma membrane staining in cells from immunostained images. 60 cells from each of three separate batches of immunostained images were counted, and the number of cells showing plasma membrane V-ATPase localization (arrows in A) was determined. The values represent the mean percentage ± S.D. of cells displaying plasma membrane staining. *, p < 0.01.
FIGURE 7.
FIGURE 7.
Immunostaining of MCF10a cells overexpressing subunit a isoforms using an antibody against V-ATPase. MCF10a cells were grown as a monolayer on coverslips in 6-well plates. Cells were immunostained using an antibody against subunit A of V-ATPase. Images were taken with identical exposure times and antibody concentrations. A, MCF10a cells expressing the empty pTracer vector or overexpressing the subunit a isoform indicated. B, quantification of plasma membrane staining in cells from immunostained images. 60 cells from each of two or three separate batches of immunostained images were counted, and the number of cells showing plasma membrane V-ATPase localization was determined. The values represent the mean percentage ± S.D. of cells displaying plasma membrane staining. a1, MCF10a cells overexpressing a1; a2, MCF10a cells overexpressing a2; a3, MCF10a cells overexpressing a3; a4, MCF10a cells overexpressing a4.
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