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      Extracellular and Luminal pH Regulation by Vacuolar H +-ATPase Isoform Expression and Targeting to the Plasma Membrane and Endosomes*

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          Abstract

          Plasma membrane vacuolar H +-ATPase (V-ATPase) activity of tumor cells is a major factor in control of cytoplasmic and extracellular pH and metastatic potential, but the isoforms involved and the factors governing plasma membrane recruitment remain uncertain. Here, we examined expression, distribution, and activity of V-ATPase isoforms in invasive prostate adenocarcinoma (PC-3) cells. Isoforms 1 and 3 were the most highly expressed forms of membrane subunit a, with a 1 and a 3 the dominant plasma membrane isoforms. Correlation between plasma membrane V-ATPase activity and invasiveness was limited, but RNAi knockdown of either a isoform did slow cell proliferation and inhibit invasion in vitro. Isoform a 1 was recruited to the cell surface from the early endosome-recycling complex pathway, its knockdown arresting transferrin receptor recycling. Isoform a 3 was associated with the late endosomal/lysosomal compartment. Both a isoforms associated with accessory protein Ac45, knockdown of which stalled transit of a 1 and transferrin-transferrin receptor, decreased proton efflux, and reduced cell growth and invasiveness; this latter effect was at least partly due to decreased delivery of the membrane-bound matrix metalloproteinase MMP-14 to the plasma membrane. These data indicate that in prostatic carcinoma cells, a 1 and a 3 isoform populations predominate in different compartments where they maintain different luminal pH. Ac45 plays a central role in navigating the V-ATPase to the plasma membrane, and hence it is an important factor in expression of the invasive phenotype.

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          Most cited references72

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          A matrix metalloproteinase expressed on the surface of invasive tumour cells.

          Gelatinase A (type-IV collagenase; M(r) 72,000) is produced by tumour stroma cells and is believed to be crucial for their invasion and metastasis, acting by degrading extracellular matrix macro-molecules such as type IV collagen. An inactive precursor of gelatinase A (pro-gelatinase A) is secreted and activated in invasive tumour tissue as a result of proteolysis which is mediated by a fraction of tumour cell membrane that is sensitive to metalloproteinase inhibitors. Here we report the cloning of the complementary DNA encoding a new matrix metalloproteinase with a potential transmembrane domain. Expression of the gene product on the cell surface induces specific activation of pro-gelatinase A in vitro and enhances cellular invasion of the reconstituted basement membrane. Tumour cells of invasive lung carcinomas, which contain activated forms of gelatinase A, were found to express the transcript and the gene product. The new metalloproteinase may thus trigger invasion by tumour cells by activating pro-gelatinase A on the tumour cell surface.
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            Acidic extracellular pH promotes experimental metastasis of human melanoma cells in athymic nude mice.

            Extracellular pH (pH(e)) is lower in many tumors than in the corresponding normal tissue. The significance of acidic pH(e) in the development of metastatic disease was investigated in the present work. Human melanoma cells (A-07, D-12, and T-22) were cultured in vitro at pH(e) 6.8 or 7.4 (control) before being inoculated into the tail vein of BALB/c nu/nu mice for formation of experimental pulmonary metastases. Cell invasiveness was studied in vitro by using Matrigel invasion chambers and angiogenesis was studied in vivo by using an intradermal assay. Protein secretion was measured by ELISA and immunocapture assays. Cells cultured at acidic pH(e) showed increased secretion of proteinases and proangiogenic factors, enhanced invasive and angiogenic potential, and enhanced potential to develop experimental metastases. Acidity-induced metastasis was inhibited by treatment with the general matrix metalloproteinase (MMP) inhibitor GM6001, the general cysteine proteinase inhibitor E-64, or blocking antibody against vascular endothelial growth factor-A (VEGF-A) or interleukin-8 (IL-8). Our study indicates that acidic pH(e) promotes experimental pulmonary metastasis in A-07, D-12, and T-22 human melanoma cells by a common mechanism involving acidity-induced up-regulation of the proteolytic enzymes MMP-2, MMP-9, cathepsin B, and cathepsin L and acidity-induced up-regulation of the proangiogenic factors VEGF-A and IL-8. One consequence of this observation is that treatment strategies involving deliberate tumor acidification to improve the efficacy of chemotherapy, photodynamic therapy, and hyperthermia should be avoided. Moreover, the possibility that the pH(e) of the primary tumor may be an important prognostic parameter for melanoma patients merits clinical investigation.
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              Effect of proton pump inhibitor pretreatment on resistance of solid tumors to cytotoxic drugs.

              Resistance to antitumor agents is a major cause of treatment failure in patients with cancer. Some mechanisms of tumor resistance to cytotoxic drugs may involve increased acidification of extracellular compartments. We investigated whether proton pump inhibitors (PPIs), currently used in the anti-acid treatment of peptic disease, could inhibit the acidification of the tumor microenvironment and increase the sensitivity of tumor cells to cytotoxic agents. We pretreated cell lines derived from human melanomas, adenocarcinomas, and lymphomas with the PPIs omeprazole, esomeprazole, or pantoprazole and tested their response to cytotoxic drugs in cell death assays. We also evaluated extracellular and intracellular pH and vacuolar-H+-ATPase (V-H+-ATPase) expression, distribution, and activity in PPI-pretreated cells by using western blot analyses, immunocytochemistry, laser scanning confocal analysis, and bioluminescence assays. Finally, we evaluated human melanoma growth and cisplatin sensitivity with or without omeprazole pretreatment in xenografted SCID/SCID mice. PPI pretreatment sensitized tumor cell lines to the effects of cisplatin, 5-fluorouracil, and vinblastine, with an IC50 value reduction up to 2 logs. PPI pretreatment was associated with the inhibition of V-H+-ATPase activity and increases in both extracellular pH and the pH of lysosomal organelles. PPI pretreatment induced a marked increase in the cytoplasmic retention of the cytotoxic drugs, with clear targeting to the nucleus in the case of doxorubicin. In in vivo experiments, oral pretreatment with omeprazole was able to induce sensitivity of human solid tumors to cisplatin. Our results open new possibilities for the treatment of drug-resistant tumors through combination strategies based on the use of well-tolerated pH modulators such as PPIs.
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                Author and article information

                Journal
                J Biol Chem
                J. Biol. Chem
                jbc
                jbc
                JBC
                The Journal of Biological Chemistry
                American Society for Biochemistry and Molecular Biology (11200 Rockville Pike, Suite 302, Rockville, MD 20852-3110, U.S.A. )
                0021-9258
                1083-351X
                15 April 2016
                24 February 2016
                24 February 2016
                : 291
                : 16
                : 8500-8515
                Affiliations
                From the []Endothelial Cell Biology Unit, School of Molecular and Cellular Biology and
                [§ ]School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom
                Author notes
                [5 ] To whom correspondence should be addressed: School of Biomedical Sciences, University of Leeds, Woodhouse Lane, Leeds LS2 9JT, United Kingdom. Tel.: 44-113-3437766; E-mail: m.a.harrison@ 123456leeds.ac.uk .
                [1]

                Recipient of British Heart Foundation Ph.D. Studentship FS/12/20/29462.

                [2]

                Present address: Manchester Collaborative Centre for Inflammation Research Flow Cytometry Facility, University of Manchester, Oxford Rd., Manchester M13 9PT, United Kingdom.

                [3]

                Medical Research Council Career Development Fellow supported by Grant G1000567.

                [4]

                Supported by grants from Heart Research UK, British Heart Foundation, and Leverhulme Trust.

                Article
                M116.723395
                10.1074/jbc.M116.723395
                4861423
                26912656
                b8cc0926-8a39-4591-98fe-90979ded8b01
                © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

                Author's Choice—Final version free via Creative Commons CC-BY license.

                History
                : 23 February 2016
                Funding
                Funded by: Medical Research Council http://dx.doi.org/10.13039/501100000265
                Award ID: G1000567
                Funded by: British Heart Foundation http://dx.doi.org/10.13039/501100000274
                Award ID: FS/12/2029462
                Categories
                Membrane Biology

                Biochemistry
                endocytosis,membrane trafficking,receptor internalization,receptor recycling,vacuolar atpase

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