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Vacuolar H(+)-ATPase in Cancer Cells: Structure
and Function
Xiaodong Lu, Wenxin Qin
School of Medical Science and Laboratory Medicine, Jiangsu University, China (XL), State Key Laboratory
of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University
School of Medicine, China (WQ)
Published in Atlas Database: September 2011
Online updated version : http://AtlasGeneticsOncology.org/Deep/V-ATPaseInCancerID20104.html
DOI: 10.4267/2042/47290
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence.
© 2012 Atlas of Genetics and Cytogenetics in Oncology and Haematology
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).
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
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
Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3)
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Vacuolar H(+)-ATPase in Cancer Cells: Structure and Function
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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 VATPase 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.
(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 nonlysosome-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 VATPase 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 glycolysisrelated 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 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 VATPases more than any other three transporters to
regulate pH in cytosol (Torigoe
The functions of the v-ATPase in
cellular signals processing
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
Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3)
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. VATPase activity influence endocytosis and degradation
of molecule-receptor complex, recycling of the released
receptor, recruitment of signal molecules, and their
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Vacuolar H(+)-ATPase in Cancer Cells: Structure and Function
Lu X, Qin W
proper spatial intracellular distributions (HurtadoLorenzo 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 VATPase 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 VATPase 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-ATPaseassociated 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 VATPase 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).
Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3)
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 VATPases 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 VATPases 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). VATPase 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 (MartínezZaguilá 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
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Vacuolar H(+)-ATPase in Cancer Cells: Structure and Function
Lu X, Qin W
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 proteasescontaining 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-12192treated 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 VATPase 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).
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 VATPase 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.
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
Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3)
255
Vacuolar H(+)-ATPase in Cancer Cells: Structure and Function
Lu X, Qin W
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.
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).
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.
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