1. School of Medical Science and Laboratory Medicine, Jiangsu University
2. State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine
Address correspondence to:
State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital Shanghai Jiao Tong University School of Medicine, No. 25/Ln 2200 Xie-Tu Road, Shanghai 200032, China
Vacuolar H+-ATPase (V-ATPase) is a highly evolutionarily conserved enzyme, which is distributed within the plasma membranes and the membranes of some organelles such as endosome, lysosome and secretory vesicle. The mayor function of V-ATPase is to pump protons across the cell membrane to extracellular milieu or across the organelle membrane to intracellular compartments. V-ATPases located in cell surface act as important proton transporters that regulate the cytosolic pH to ~7.0 which is essential for most physiological processes, whereas V-ATPases within intracellular membrane are involved in cellular processes as receptor-mediated endocytosis, membrane trafficking, protein processing or degradation, and nutrients uptake (Nishi et al., 2002; Forgac et al., 2007; Toei et al., 2010; Cruciat et al., 2010). Malfunctioned V-ATPase is closely related to several diseases including tumor. More and more evidences indicate that V-ATPase is an enhancer for carcinogenesis and cancer progression, such as malignant transformation, growth and proliferation, invasion and metastasis, acquirement of multi-drug resistance, etc., which strongly supports that V-ATPase should be an effective target of anticancer strategy (Fais et al., 2007).
The structure of V-ATPases and its expression in tumor cells
The molecular structure of normal V-ATPase of yeast and mammalian cells has been well studied. V-ATPase is a delicate complex which is composed of a cytosolic catalytic domain V1 and an integral domain V0, the former responsible for ATP hydrolysis and the latter providing transmembraneous proton channel (Nishi et al., 2002; Yokoyama et al., 2005; Wang al., 2007). The core of the V1 section is composed of a hexameric arrangement of alternating A and B subunits, which participate in ATP binding and hydrolysis. Other subunits of V1 include three copies of E and G subunits which are the stator, one copy of the regulatory C and H subunits, one copy of subunits D and F which form a central rotor axle. The V0 section includes a ring of proteolipid subunits (c, c' and c") that are adjacent to subunits a and e. Subunits D and F of V1 and subunit a of V0 form the central stalk, whereas the multiple peripheral stalks are composed of subunits C, E, G, H and the N-terminal domain of subunit a. V1 and V0 is connected by both stalks. Several subunits like a, d, e, C, G, H, D and F contain slice variants as to spatial and temporal expression pattern in different cell types (Forgac et al., 2007; Miranda et al., 2010). As for tumor cells, especially those with high metastatic potential, the V-ATPases are usually excessively agitated. The altered structures of V-ATPase of tumor cells may include the increased level of subunit expressions and unique spliced variants of some subunits.
The level of the subunit c expression was found to be related to the metastasis potentials in tumors. One of the studies is the comparison of subunit c expression between normal and pancreatic carcinoma tissues and between invasive and non-invasive pancreatic cancers, which immunohistochemical data showed the notable difference - 92% invasive ductal cancers (42/46) were mild to marked subunit c positive in the cytoplasm, whereas neither non-invasive ductal cancers nor benign cystic neoplasms expressed detectable immunoreactive proteins (Ohta et al., 1996). Subunit c seems to be one of the V-ATPase subunit which significantly influence the proliferation and metastasis of tumor cells. The inhibition of the V-ATPase subunit c via siRNA resulted in the suppression of growth and metastasis of a hepatocellular carcinoma cell line in vitro and in mice model (Lu et al., 2005), which is according to another result of the suppression of subunit c in Hela cell via antisense oligonucleotides (Zhan et al., 2003). But in oral squamous cell carcinoma cells, subunit C1 was the most strongly over-expressed gene at the mRNA level compared to other genes of the V-ATPase complex (Otero-Rey et al., 2008).
Specific spliced variants of subunit have been observed in tumors. A study of expression of subunit a of V-ATPase in breast cancer cell lines displayed the metastasis-specific subunit a isoform expression profile. In highly metastatic breast cancer cell line compared with its lowly metastatic parallel, levels of a3 and a4 were much higher although all the four a isoforms - a1-4 can be detectable. They distribute differently, and especially, a4-containing v-ATPases were located mainly in the plasma membrane of higher metastatic breast cancer cell, seeming to be involved in the formation of the leading surface of the cells due to the combination with F-actin and closely correlated to the potency of invasion. a3-containing V-ATPases were located in intracellular compartment membrane, which regulated the pH of the cytosol and intracellular compartments and also involved in invasion (Hinton et al., 2009). In accordance with this data, the strongly expressed a3 isoform were observed in high-metastatic melanoma cells and in bone metastases (Nishisho et al., 2011). Other tumor-relevant spliced variants are yet to be found.
The roles of the v-ATPase in the growth, proliferation or apoptosis in tumor cells
One of cancer hallmarks is the shift in energy production from oxidative phosphorylation to aerobic glycolysis, ie "Warburg effect", which produces excess intracellular acidosis (Gillies et al., 2008). However, cancer cells usually have neutral to alkaline intracellular pH in the acidized extracellular microenvironment. The V-ATPase is among the four major types of pH regulators (the other three are: Na+/H+ exchangers, bicarbonate transporters, proton/lactate symporters). Much data implies proton pump is essential in tumors and cells seem to render V-ATPases more than any other three transporters to regulate pH in cytosol (Torigoe et al., 2002). The ability to extrude intracellular protons and maintain the cytosol pH is critical for cancer cell survival from a cascade of self-digestion triggered by acidosis.
The inhibition of v-ATPase may induce apoptotic cell death in several human cancer cell lines including pancreatic cancer (Ohta et al., 1998; Hayash et al., 2006), liver cancer (Morimura et al., 2008), gastric cancer (Nakashima et al., 2003), B-cell hybridoma cells (Nishihara et al., 1995; De Milito et al., 2007) and breast cancer (McHenry et al., 2010).The deficiency of V-ATPase will decrease cytosol pH and increased lysosome pH, both of which might influence lysosome function. The apoptosis induced by V-ATPase inhibitors were in either lysosome-mediated or non-lysosome-mediated manner. In the first case, when lysosomal V-ATPase was defected, lysosomal pH and permeability will be increased, resulted in the release of cathepsin D and activation of caspase, with no significant impact on mitochondrial transmembrane potential (Nakashima et al., 2003). In the other case, mitochondria and lysosome might be together involved in V-ATPase-inhibitor-induced apoptosis via capsase pathway or ROS-dependant manner (Ishisaki et al., 1999; De Milito et al., 2007). The inhibition of V-ATPase could also induce apoptosis by suppressing anti-apoptotic Bcl-2 or Bcl-xL and facilitate the caspase-independent apoptotic pathway (Sasazawa et al., 2009). In order to survive from the apoptosis induced by acidosis resulted from glycolysis, tumor cells needs to extrude excessive acid, in which processes V-ATPase plays a crucial role. It is reasonable to postulate that the inhibition of proton extrusion may be more susceptible or vulnerable to cell death of cancer cells than normal cells.
Moreover, the slightly alkalized cytosolic pH favors the growth and proliferation of the cells. Some glycolysis-related enzymes or oncogenes are sensitive the narrow range of pH alteration. Alkalization of cytosol, which mainly regulated by V-ATPase in tumor cells, could activate glycolysis whereas repress oxidative phosphorylation, meanwhile also promote the transcription of oncogenes like HIF-1, akt, myc, ras, etc (Gillies et al., 2008; López-Lázaro, 2008). The cytosol pH of tumor cells was found to be higher than in untransformed controls (Busa et al.,1984; Casey et al., 2010) and increasing cytosol pH was sufficient to confer tumourigenicity to cultured fibroblasts (Perona et al., 1988). On the contrast, p53, the important tumor suppressor could be inactivated in the condition of alkalization (Xiao et al., 2003). It is much likely that the glucose metabolism shift and mutant V-ATPase may be the co-selectors in selecting those "adaptive phenotype", which may take the advantages for survival and proliferation during the initial stage of carcinogenesis.
The functions of the v-ATPase in cellular signals processing
V-ATPase is the important factor that regulates the process of internization and activation of cellular signals. It is mainly due that the V-ATPase is the main contributor of low intracellular vesicles pH, which is essential for various membrane traffic processes. V-ATPase activity influence endocytosis and degradation of molecule-receptor complex, recycling of the released receptor, recruitment of signal molecules, and their proper spatial intracellular distributions (Hurtado-Lorenzo et al., 2006; Marshansky et al., 2008), therefore exerts a profound effect on cell behavior such as growth, proliferation or metastasis via the modulated signals and their pathways. It has been reported that tumor-associated m-TOR (mammalian target of rapamycin) (O'Callaghan et al., 2009), Notch (Fortini and Bilder, 2009; Vaccari et al., 2010) or Wnt (Cruciat et al., 2010; Buechling et al., 2010) could be regulated by V-ATPase.
Early endosomes are important sites for signal molecules internalization and activation in mammalian cells. Studies of the effects of V-ATPases inhibitors on isolated rat hepatocytes and rat sinusoidal endothelial cells suggested that the pH gradient between the endocytic compartments and the cytoplasm was necessary for the receptor-mediated endocytosis (Harada et al., 1996; Harada et al., 1997). Inhibition of V-ATPases can retard recycling of transferrin receptor (Presley et al., 1997), impair the formation of endosomal carrier vesicle (Clague et al., 1994), and inhibit late endosome-lysosome fusion (van Weert et al., 1995). Although the significance of active V-ATPase in signal molecules endocytosis and processing on the behavior of tumor cells is not yet full elucidated for most data was gained from yeast or normal mammalian cells, it could be hypothesized that V-ATPase might regulate some signal pathways via modulating the recycling rate of receptor, which would be responsible for the sensitivity of tumor cells to some signal molecules, ie, the faster rate at which the receptor cycling in a V-ATPase-regulated membrane trafficking, the more efficiently the cells render the receptors, the more signal molecules could be recruited, and the stronger or more lasting response to the stimulation by the signal molecules could be expected.
For example, the activation of Notch, a common hallmark of an increasing number of cancers (Miele et al., 2006; Roy et al., 2007), is involved in V-ATPase-associated endosomal system (Yan et al., 2009; Vaccari et al., 2010). V-ATPase activity is required for Notch signaling. In V-ATPase mutant cells, Notch and its receptors are trapped in an expanded lysosome-like compartment, where they accumulate rather than being degraded and a substantial reduction expression in downstream gene of notch. V-ATPase regulates Notch via: i) endocytosis of Notch, for acidification of earlier endosomal compartments is required in this process and a reduced rate of Notch endocytosis was found in V-ATPase mutant cells ii) endosomal cleavage patterns of the protease that degrade the Notch in the accordingly forms, each of which process exerting its own activating potency (Vaccari et al., 2010) iii) regulating endosome-lysosome fusion and Notch intracellular re-distribution or the targeting to cell surface.
The V-ATPase-associated signal molecules processing itself may also be regulated by endosomal protein, for example, HRG-1(heme-regulated genes), a downstream gene of IGF-I (insulin-like growth factor) and having an interaction with subunit c. HRG-1 could promote endosomal acidification and receptor trafficking, enhance the proliferative and invasive phenotype of cancer cells. It was implied that the increased active V-ATPase by HRG-1 not only regulate the endocytosis and degradation of receptors that promote signaling for survival, growth, and migration of cancer tumor, but also facilitate micronutrient uptake necessary for tumor cellular metabolism (O'Callaghan et al., 2009).
The contributions of the V-ATPase in cancer metastasis
Invasion and metastasis is the relatively late event of development of malignant cells, which is the continuous process of breaking through the basement membrane, degrading extracellular matrix, angiogenesis, invading vascular system and redistributing in the distinct host sites. The activation of the proteases which break down extracellular matrix is required during the procedure. The invasive phenotype is closely related to its highly active V-ATPase. It has been reported that the improper activated V-ATPases correlates with an invasive phenotype of several types of tumors, including breast cancer (Sennoune et al., 2004; Hinton et al., 2009), pancreatic cancer (Chung et al., 2011) and melanoma (Nishisho et al., 2011). The tumor metastasis can be suppressed in vitro or in animal model by the inhibition of V-ATPase inhibitors or siRNA (Lu et al., 2005; Hinton et al., 2009; Supino et al., 2008). Subunit a isoform and c seem to be important factors in regulating the metastasis of cancer.
The main mechanisms by which overly active V-ATPases enhance the tumor invasion and metastasis may be that the extracellular milieu is acidized and it is suitable for optimal pH of proteases that degenerate extracellular matrix (ECM). The plasma membrane V-ATPases is responsible for pumping cytosol protons to the extracellular space resulting in a low extracelluar pH, which is required for the activation of several types of proteases including cathepsins, metalloproteases, and gelatinases. V-ATPase may influence the expression of proteases directly independent of the whole enzyme V-ATPase function. For example, transfectants which over express V-ATPase subunit c at the mRNA level showed an enhance invasiveness in vitro with a concomitant increases in secretion of matrix metalloproteinase-2 (Kubota et al., 2000). V-ATPase may also regulate metastasis by enhancing proteases activation. Cathepsin is an example, which is secreted by several types of tumor cells and related to invasion. Once the extracellular cathepsin is activated, it can both degrade extracellular matrix proteins and activate other secreted proteases involved in invasion, such as matrix metalloprotease (Joyce et al., 2004; Gocheva et al., 2007) and gelatinases (Martínez-Zaguilá et al., 1996). The plasma membrane V-ATPase appeared to be recruited at the proceeding edge of the cancer cell by the interaction with F-actin so as to give rise an acidic microenvironment by the edge (Hinton et al., 2009). Moreover, intracellular V-ATPases, the major contributor of acidity of intracellular compartment and membrane trafficking regulator, also facilitate in the invasion and metastasis, which is due to possible modulating proteolytic activation of cathepsins or matrix metalloproteases within lysosomes or secretory vesicles and targeting the proteases-containing secretory vesicles to the cell surface to be extracytosed (Hinton et al., 2009). The accumulation of acidity, concentration of plasma membrane V-ATPase and activated protease crown the proceeding surface of a metastatic cell, conferring the tumor cell a "cutting edge".
Mobility is crucial for spread of tumor cells to the distant sites. NiK-12192, one of V-ATPase inhibitor was shown able to reduce the migration/invasion of human lung cancer cells in vitro and significantly reduce the number of spontaneous metastases in the lung of nude mice implanted with a human lung carcinoma. After the treatment of NiK-12192, the lung cancer cells in vitro showed that actin fibers were broken, spots of aggregation were evident and no pseudopodia and regular structure for actin filaments could be seen, comparing to the control cells with long and regular fibers of tubulin in the cell cytoplasm and filaments of actin forming pseudopodia. NiK-12192-treated cells also demonstrate a reduction in the experiment of wound healing assay due to the retard of migration (Supino et al., 2008). V-ATPase subunit B and C appear to contain the binding sites to the actin cytoskeleton (Vitavska et al., 2003; Vitavska et al., 2005; Zuo et al., 2006). The interactions between V-ATPase and cytoskeleton implicate their involvement and regulation of cell mobility and membrane trafficking (Sun-Wada et al., 2009).
Angiogenesis, a consequence of the mutual interaction between cancer cells and the stoma cells of extracellular microenvironments, is another important step during metastasis, during which process, endothelial cells is mainly involved. It was documented that V- ATPases play a crucial role in growth and phenotypic modulation of myofibroblasts that contribute to neointimal formation in cultured human saphenous vein (Otani et al., 2000) The microvascular endothelial cells in tumor tissue also incline to render plasma membrane V-ATPase to cope with the acidic extracellular environment. The ability of migration of endothelial cell toward the adjacent tissue is required during angiogenesis, in which process V-ATPase plays a role, shown in the result that the penetration of basement membrane of endothelial cell was suppressed by bafilomycin treatment (Rojas et al., 2006).
The relations of V-ATPase and drug resistance in cancer
Acquired multidrug resistance (MDR) can limit therapeutic potential and one of the reasons of relapse. It is well known that MDR is correlate to the evolutionarily conserved family of the ATP binding cassette (ABC) proteins pg, yet it is documented that V-ATPase plays a role in MDR in a pg-independent manner, and the inhibition of V-ATPase could not only suppress tumor cells directly, but also sensitize the tumor cells to the chemical therapy (De Milito et al., 2005). It was documented that proton pump inhibitor (PPI) pretreatment sensitized tumor cell lines to the effects of cisplatin, 5-fluorouracil, and vinblastine significantly. PPI treatment will increases both extracellular pH and the pH of lysosomal organelles, which induced a marked increase in the cytoplasmic retention of the cytotoxic drugs, with clear targeting to the nucleus in the case of doxorubicin. In vivo experiments, oral pretreatment with omeprazole was able to induce sensitivity of human solid tumors to cisplatin (Lucian et al., 2004).
V-ATPase renders several mechanisms of multidrug resistance including: neutralized drug extracellularly or intracellularly, decreased drug internalization, altered DNA repair and inhibition of apoptosis. The pH of the tumor microenvironment may influence the uptake of anticancer drugs. Molecules diffuse passively across the cell membrane most efficiently in the uncharged form. Because the extracellular pH in tumors is low and the intracellular pH of tumor cells is neutral to alkaline, weakly basic drugs that have an acid dissociation constant of 7.5-9.5, such as doxorubicin, mitoxantrone, vincristine, and vinblastine, are protonated and display decreased cellular uptake (Raghunand et al., 1999; Gerweck et al., 2006; McCarty and Whitaker, 2010). The data in vitro or in animal models indicates that extracellular alkalinization leads to substantial improvement in the therapeutic effectiveness of antitumor drugs via enhanced the cellular drug uptake and cytotoxicity (Gerweck et al., 2006; Trédan et al., 2007).The reduced intracellular accumulation of anticancer drugs may also be due that V-ATPase has a role as cooperating factor of ATP-dependent membrane proteins that function as drug efflux pumps (Raghunand et al., 1999). Interestingly, the levels of V-ATPase subunit expressions can be up-regulated by anticancer drug. The treatment of cisplatin on human epidermoid cancer KB cells increased the protein levels of the majority of the subunits such as c, c", D, a, A, C and E, which indicates it may stimulate the expression of the V-ATPase complex as a whole. It is suggested that the V-ATPase expression may be a defensive response to the anticancer drug (Murakami et al., 2001; Torigoe et al., 2002). Still, there are also some controversial results on the relationship between the cationic drugs uptake and V-ATPase - the inhibition of V-ATPase decreased the uptake of the cationic drugs (Morissette et al., 2009; Marceau et al., 2009), which might be explained that the influence of V-ATPase on the drug uptake may also be depend upon the characteristics of the drugs and its relation to membrane trafficking.
That the defects of V-ATPase increase the sensitivity to drugs may be partly due to the decreased cytosolic pH, which were observed in the influence of cisplatin on the V-ATPase mutant yeast Saccharomyces cerevisiae (Liao et al., 2006) or increased toxicity of combined treatment of V-ATPase inhibition and anticancer drug on lung cancer cell, breast cancer or liver cancer cell lines (Wong et al., 2005; Farina et al., 2006; You et al., 2009). At low cytosolic pH, sensitivity to DNA damaging drugs or UV irradiation in V-ATPase mutants may be associated with altered DNA conformation or defective DNA damage repair mechanisms, rendering DNA more prone to damage (Robinson et al., 1992; Petrangolini et al., 2006; Liao et al., 2006).
The roles of V-ATPase in cancer cells. 1) Protons produced by glycolysis are pumped by plasma membrane V-ATPase (green circle: V0; blue circle: V1) which prevents the cell from acidosis-induced apoptosis and the slightly basic of cytosolic pH enhanced cell growth and proliferation; 2) Acidification of secretary vesicle, which is maintained by intracellular V-ATPase, is essential for protease secretion and activation (orange bars: active form; orange-red bars: inactive forms of protease). The interaction between V-ATPase and actin (green wave line) may contribute the recruitment of V-ATPase on plasma membrane. The accumulation of V-ATPase on the plasma membrane, the extracellular acidic-microenvironment and activated-protease appear to crown the tumor cell, conferring it a "cutting edge" at the proceeding surface which facilitates invasion and metastasis. Moreover, in acidic microenvironment, angiogenesis is enhanced; 3) V-ATPases might regulate signal pathway via controlling international of signal molecules (red circle), releasing and recycling the receptors, and processing signal molecules. Therefore, V-ATPases may exert effects on cell behavior via signal pathway; 4) V-ATPases contributes to acquirement of resistance of anticancer drug (green square) supported by the data that inhibition of V-ATPase sensitize the tumor cells to chemical therapy, which is partly due to the increased influx of anticancer drug when in a basic extracellular condition.
According to the roles V-ATPase in tumor cells, we conclude that alteration of V-ATPase is much likely the necessary initial step of transformation of the malignant cells and the malfunctional V-ATPase acts as a continual enhancer of carcinogenesis and tumor progression. Tumor cells take the advantages of disfunctioned plasma and intracellular V-ATPase in these aspects: enhanced proliferation and growth, evading apoptosis, facilitating metastasis and angiogenesis, and acquirement of the drug resistance. V-ATPase will be a prospective candidate for cancer diagnosis and treatment.