Molecular Aspects of Medicine 47–48 (2016) 3–14

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Molecular Aspects of Medicine

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / m a m

Review

Hypoxia optimises tumour growth by controlling nutrient

import and acidic metabolite export
Scott K. Parks a,*, Yann Cormerais a, Ibtissam Marchiq b, Jacques Pouyssegur a,b
a
Centre Scientifique de Monaco (CSM), Monaco
b Institute for Research on Cancer & Aging (IRCAN), CNRS, INSERM, University of Nice-Sophia Antipolis, Nice, France

A R T I C L E I N F O A B S T R A C T

Article history: In their quest for survival and successful growth, cancer cells optimise their cellular pro-
Available online 23 December 2015 cesses to enable them to outcompete normal cells in their microenvironment. In essence
cancer cells: (i) enhance uptake of nutrients/metabolites, (ii) utilise nutrients more effi-
Keywords: ciently via metabolic alterations and (iii) deal with the metabolic waste products in a way
hypoxia that furthers their progression while hampering the survival of normal tissue. Hypoxia In-
tumour metabolism
ducible Factors (HIFs) act as essential drivers of these adaptations via the promotion of
amino-acid transport (LAT1)
numerous membrane proteins including glucose transporters (GLUTs), monocarboxylate
tumour pH regulation
carbonic anhydrases (CAs) transporters (MCTs), amino-acid transporters (LAT1, xCT), and acid-base regulating car-
monocarboxylate transporters (MCTs) bonic anhydrases (CAs). In addition to a competitive growth advantage for tumour cells,
HCO3− transport these HIF-regulated proteins are implicated in metastasis, cancer ‘stemness’ and the immune
response. Current research indicates that combined targeting of these HIF-regulated mem-
brane proteins in tumour cells will provide promising therapeutic strategies in the future.
© 2015 Elsevier Ltd. All rights reserved.

Contents

1. General introduction .............................................................................................................................................................................................................. 3

2. Hypoxia enhanced tumour cell nutrient supply .......................................................................................................................................................... 4
2.1. Glucose transport ...................................................................................................................................................................................................... 4
2.2. Amino acid transport ............................................................................................................................................................................................... 5
3. Glycolysis, “Warburg Effect” and acid-base regulation .............................................................................................................................................. 6
3.1. Monocarboxylate transporters (MCTs) ............................................................................................................................................................... 6
3.2. Acid-base regulation ................................................................................................................................................................................................ 7
3.2.1. H+ extruding mechanisms (NHEs, MCTs) ......................................................................................................................................... 8
3.2.2. Carbonic anhydrases (CAs) .................................................................................................................................................................... 8
3.2.3. HCO3− transport ......................................................................................................................................................................................... 9
3.2.4. Acid-base regulators in migration, metastases and tumour growth ................................................................................... 10
4. Future perspectives .............................................................................................................................................................................................................. 10
Acknowledgements .............................................................................................................................................................................................................. 11
References ............................................................................................................................................................................................................................... 11

1. General introduction
Molecular Aspects of Medicine special issue: “Hypoxia in Health and
Disease”
* Corresponding author. Centre Scientifique de Monaco (CSM), Monaco.
Contemplating biological function in low oxygen
Tel.: +377 97 77 44 07; fax: +377 97 77 44 01. (hypoxia) conditions often evokes an image of mountain
E-mail address: sparks@centrescientifique.mc (S.K. Parks). climbers struggling to scale high elevation peaks such as

http://dx.doi.org/10.1016/j.mam.2015.12.001
0098-2997/© 2015 Elsevier Ltd. All rights reserved.
4 S.K. Parks et al. / Molecular Aspects of Medicine 47–48 (2016) 3–14

Mount Everest. These extreme altitudes present environ- 2. Hypoxia enhanced tumour cell nutrient supply
ments with close to 70% less oxygen (O2) content compared
to sea level and the resulting effect on human physiology Rapidly proliferating tumour cells require an abnormal-
can be drastic. Tissues however are already optimised to ly enhanced nutrient supply to support their increased
function with a relatively low O2 content compared to that bioenergetics and demand for carbon building blocks com-
of inspired air after delivery from the circulatory system. pared to normal cells (McCracken and Edinger, 2013). This
Pathophysiological conditions push the limits of hypoxia ex- nutrient requirement must be overcome in an environ-
posure to the extremes with tumours experiencing O2 levels ment that lacks regular perfusion of nutrients due to a
in the 1% to near anoxia range. Contrary to that observed chaotic vasculature. Hypoxia has been revealed to play a
for high altitude physiology in humans, hypoxia in patho- major role in order to compensate for the increased nutri-
physiology leads to numerous advantages for areas such as ent demand of tumour cells (Fig. 1). Here we will focus
tumour cell biology. These cellular advantages, particular- primarily on the role of glucose and amino acid transport
ly for survival and growth, will be the focus of this review. in the nutrient supply chain of the hypoxic tumour
Cancer cells are well established to have an altered me- environment.
tabolism compared to normal cells, often referred to as the
‘Warburg-effect’ (Gatenby and Gillies, 2004; Kroemer and 2.1. Glucose transport
Pouyssegur, 2008; Vander Heiden et al., 2009; Warburg,
1956). These metabolic differences are initiated by driver The majority of cancer cells are commonly described as
mutations (oncogenes) and then perpetuated by physio- ‘glucose addicts’. There is considerable debate in the liter-
logical alterations of the tumour environment. O2 levels are ature with respect to the ultimate role of this glucose
a major player in driving the pathophysiology of tumour pro- addiction. On the surface it appears that the obvious selec-
gression. This is due to the incomplete/chaotic vasculature tion pressure is to provide more cellular energy (ATP) for
that associates with tumour growth resulting in hypoxic proliferation purposes. However, there is mounting evi-
zones and a reduction in nutrient availability within the dence that enhanced glycolysis rates in tumour cells, which
tumour tissue (Fig. 1). Sensing low O2 levels and constitu- consume large amounts of glucose for low yields of ATP, play
tive activation of mTORC1 have proven to be essential for a more essential role in the production of intermediates and
the maintenance of altered tumour cell metabolism through macromolecules required for cellular proliferation (Vander
the hypoxia inducible factors (HIFs) signalling pathway, Heiden et al., 2009). Evidently, there is a combined impor-
which influences the expression of more than one-thousand tance of both ATP and production of carbon precursor
genes (Semenza, 2013). Of these HIF targets, a number of metabolites but the point remains that enhanced glucose
proteins residing at the cellular membrane play essential uptake is a hallmark of aggressive tumours and FDG PET-
roles in tumour cell metabolism and progression (Fig. 1). We scanning is routinely used to provide valuable clinical
wish to convey the role of a selection of these proteins in diagnoses (Szablewski, 2013).
the ‘big-picture’ view of the tumour cell lifespan. In this Glucose membrane transporters (GLUTs/SLC2A family,
respect we will discuss the importance of HIF controlled Fig. 2) consist of 14 family members with GLUT1 receiv-
membrane proteins that enhance (i) nutrient uptake, (ii) ing the most attention in tumour biology (Szablewski, 2013).
utilisation, and (iii) waste handling in the aggressive tumour Numerous studies have demonstrated that GLUT expres-
tissues that develop distant from the vasculature. sion corresponds with poor prognosis and metastases in

Fig. 1. Generalised model of the impact of hypoxia on nutrient uptake, utilisation and waste removal in tumour cells. A 3-dimensional model is depicted
with a chaotic vasculature leading to the development of a hypoxia gradient within the solid tumour. The hypoxia-inducible factor (HIF) responds to hypoxia
to regulate gene expression that ultimately regulates membrane transporters in the enhancement of nutrient uptake, cellular metabolism and metabolic
waste removal.
 S.K. Parks et al. / Molecular Aspects of Medicine 47–48 (2016) 3–14 5

indicates that this glucose uptake mechanism becomes even

further important in the context of the tumour microen-
vironment. This reveals that targeting GLUT transporters
would be beneficial at each stage of tumour progression as
it appears there is a strong glucose addiction in both early
and late tumour progression (advanced clinical stage).
Current therapeutic development strategies targeting GLUT
include decreasing mRNA expression (Szablewski, 2013), ap-
plying antibodies (Rastogi et al., 2007) and direct
pharmacological blockers (Koch et al., 2015). Further-
more, GLUT1 is being implicated in the growth, survival and
maintenance of stemness in cancer stem cells, which broad-
ens its potential benefit as a future therapeutic target
(Shibuya et al., 2015).

2.2. Amino acid transport

Amino acids (AAs) represent an important class of nu-

Fig. 2. Hypoxia enhances nutrient uptake in tumour cells. Due to their ex-
acerbated metabolism, tumour cells require an enhanced nutrient supply
trients that are obligatory for survival of any cell. These
and this is maintained by hypoxia to a certain extent with induction of nutrients are most commonly known as the building blocks
glucose and amino acid transporters. HIF induces GLUT1 expression to of proteins but they also act as essential metabolites in the
supply the ‘glucose addiction’ of tumour cells while LAT1 (HIF-2) is also biosynthesis of fatty acids, membrane lipids and nucle-
increased to provide essential amino acids and maintain mTORC1 activ-
otides. Thus in combination with their need for increased
ity for protein synthesis. xCT is an amino acid transporter regulated by HIF-1
that contributes to tumour cell progression via providing cystine for the glucose uptake, rapidly growing tumour cells require an en-
synthesis of the antioxidant glutathione. hanced uptake of AAs to face the increased demand of
exacerbated metabolism and biosynthetic processes
(McCracken and Edinger, 2013). AAs, like all hydrophilic nu-
cancers such as lung, colon (Younes et al., 1996, 1997), breast trients, cannot cross the plasma membrane without carrier
and others (reviewed by Macheda et al., 2005; McCracken proteins. To date, approximately thirty AA transporters have
and Edinger, 2013; Szablewski, 2013). Tumours experi- been described for mammalian cells, which are expressed
ence a dual regulation on glucose transport to satisfy in a tissue-specific manner (Bhutia et al., 2015; Broer, 2002;
heightened energy demand. Independent of hypoxia, GLUT1 Makrides et al., 2014). Interestingly, only two AA transport-
expression is demonstrated to play a role in early tumour ers are known to be overexpressed in cancer and up-
progression due to Ras and Myc oncogenes or HPV infec- regulated by hypoxia: xCT (SLC7A11) through HIF-1 (Lu et al.,
tion (reviewed by Macheda et al., 2005). However, it is not 2015) and LAT1 (SLC7A5) by HIF-2 (Elorza et al., 2012).
clear if this response is initiated by oncogenic signalling re- LAT1 (L-type AA transporter 1) is responsible for Na+-
sulting in increased GLUT expression to increase energy independent transport of large neutral and essential AAs
supply or as a secondary response to increased glucose con- (Fig. 2). It is an obligatory exchanger with the uptake of one
sumption via glycolysis. Nonetheless, the induction of GLUT1 AA being coupled with the efflux of another AA (Kanai et al.,
by growth factors or oncogenic transformation is a direct 1998; Yanagida et al., 2001). LAT1 forms a heterodimer with
consequence of c-Myc induction of GLUT1 as is the case for the CD98 glycoprotein which acts as a chaperone promot-
other glycolytic enzymes (Osthus et al., 2000). ing stabilisation, trafficking and functional insertion of LAT1
Following tumour initiation, GLUTs, like other glyco- to the plasma membrane (Yanagida et al., 2001). LAT1 is
lytic enzymes induced by c-Myc, are further regulated by overexpressed in a wide range of tumour types including
hypoxia (Kroemer and Pouyssegur, 2008; Stine et al., 2015). prostate (Sakata et al., 2009), lung (Kaira et al., 2008), gliomas
Early studies on HIF gene regulation provided evidence for (Haining et al., 2012), and renal cell carcinomas (Betsunoh
HREs (Hypoxia response elements) for GLUT1 and binding et al., 2013; Elorza et al., 2012). LAT1 is therefore an attrac-
of the HIF complex to the promoter of both GLUT1 and tive and potential therapeutic target currently in a phase I
GLUT3 (Ebert et al., 1995; Zelzer et al., 1998). Although clinical trial for patients treated with a specific LAT1 inhib-
further extensive analysis of the hypoxia-regulating com- itor JPH203 (Rosilio et al., 2015; Yun et al., 2014).
ponent has not been performed, GLUT1 is routinely Recently, it was demonstrated that LAT1 is up-regulated
considered as the essential contributor to glucose uptake by HIF-2 to promote cell proliferation in clear cell renal car-
in cancer cells. Na+-coupled glucose transporters also con- cinoma (Elorza et al., 2012). This control of LAT1 expression
tribute to cancer cell metabolism, however, as they are not by HIF-2 offers an explanation for the high expression of
regulated by hypoxia, it appears that GLUT1 is a marker of LAT1 in supporting tumour cell metabolism within hypoxic
more aggressive tumours in oxygenated and vast hypoxic areas but also for oxygenated cells, like pVHL-null, for which
zones. In the global sense, Na+-coupled glucose transport- the mechanism of HIF instability has been disrupted (Shen
ers must be considered as a viable avenue for glucose supply et al., 2011). Interestingly, HIF-2 dependent induction of LAT1
to cancer cells if GLUTs are disrupted. was shown to be essential to fully activate mTORC1 and
Therefore, as hypoxia arrives following tumour cell met- promote tumour growth (Elorza et al., 2012). Regulation of
abolic transformation and growth, HIF-regulation of GLUT1 mTORC1 by AAs is a very hot topic and in spite of recent
6 S.K. Parks et al. / Molecular Aspects of Medicine 47–48 (2016) 3–14

advances demonstrating the translocation of mTORC1 to the system and ‘cancer stemness’ that will evidently be the di-
lysosome (Efeyan et al., 2015) the mechanism is not fully rection of future research.
understood (Rebsamen et al., 2015; Wang et al., 2015). To
summarise, LAT1 dependent activation of mTORC1 appears 3.1. Monocarboxylate transporters (MCTs)
to occur from a dual AA signalling acting in cytoplasmic and
lysosomal compartments (Efeyan et al., 2015; Hara et al., Enhanced glycolytic activity generates large amounts of
1998). LAT1 transported leucine is detected by the tRNA acidic by-products, particularly lactic acid (DeBerardinis et al.,
charging enzyme Leucyl-tRNA Synthetase (Bonfils et al., 2007; Gatenby and Gillies, 2008; Lunt and Vander Heiden,
2012; Han et al., 2012) whereas LAPTM4b targets LAT1 to 2011). This threatens the intracellular pH (pHi) homeosta-
the lysosomal membrane (Milkereit et al., 2015). This HIF- sis and consequently cell growth and survival, as alterations
2-LAT1-mTORC1 signalling axis demonstrates an incredible in pHi may disturb a wide range of biosynthetic and sig-
capacity for cancer cells to ensure essential AA uptake under nalling processes (Gottlieb et al., 1996; Lagadic-Gossmann
hypoxic and nutrient depleted environments. et al., 2004; Webb et al., 2011). Cancer cells have devel-
Interestingly, recently another AA transporter, xCT, was oped adaptive strategies to extrude acid and regulate pHi
reported to be inducible by hypoxia (Lu et al., 2015). This that will be further discussed below. However, tumour cells
carrier is an Na+-independent obligatory exchanger of cystine also require an enhanced export of lactic acid to ensure a
(Cys-S-S-Cys) and glutamate (Fig. 2), and like LAT1 re- continuous glycolytic rate and ATP production. In cancer cells
quires association with CD98 (Fotiadis et al., 2013; Sato et al., this is achieved by lactate/H+ symporters belonging to the
1999). In contrast to other AA transporters which carry a monocarboxylate transporter (MCT) family (Halestrap, 2012,
family of substrates, xCT is unique in that it only trans- 2013). The MCT family (SLC16) includes 14 members. Four
ports cystine (Sato et al., 1999) acting not as a nutrient carrier isoforms (MCT1-4) provide a co-transport of protons and
but more as a defensive protein to protect cells against oxi- short chain carbohydrates such as pyruvate and L-lactate,
dative stress and chemotherapy (Okuno et al., 2003). Indeed, with MCT1 (SLC16A1) and MCT4 (SLC16A3) preferentially
intracellular reduction of cystine to form cysteine, which regulating the transport of lactic acid across the plasma
is an essential precursor of glutathione, provides one of the membrane (Halestrap, 2012, 2013) (Fig. 3). MCT export of
major intracellular antioxidants that play a key role in lactic acid not only contributes to the acidification of tumour
tumour chemo-resistance. Interestingly, HIF-1 induced xCT microenvironment, but also plays a key role in promoting
expression and glutathione synthesis has been implicated tumour cell migration and angiogenesis, as well as sup-
in an enrichment of the cancer stem cell population in triple pressing the anti-cancer immune defence (reviewed in
negative breast cancer (Lu et al., 2015). More develop- Goetze et al., 2011; Hirschhaeuser et al., 2011; Marchiq and
ment on this topic appears in an accompanying chapter of Pouyssegur, 2015).
this issue covered by Semenza’s group. MCT1 and MCT4 differ by their substrate affinity: MCT1
The hypoxia-induced chemo-resistance of tumours transports L-lactate (Km ≈ 1–3.5 mmol/L) and pyruvate
remains one of the most difficult parameters to deal with (Km ≈ 0.1–0.74 mmol/L) (Halestrap, 2012; Poole and
in the fight against cancer. HIF induced AA transporters Halestrap, 1993) with greater affinity compared to MCT4
provide attractive, but yet unexplored, targets for the design
and development of a new class of anticancer drugs. Thera-
pies targeting these nutrient transporters are being
developed that hold promise as both direct targets and
factors to synergise with chemotherapy (Harris et al., 2015;
Rosilio et al., 2015; Timmerman et al., 2013; Yun et al., 2014).
We thus feel that this will be an important theme in future
cancer discovery.

3. Glycolysis, “Warburg Effect” and

acid-base regulation

We and others have previously summarised the well

known ‘Warburg Effect’ in tumour cells whereby glycoly-
sis becomes the predominant metabolic pathway utilised
in the presence or absence of oxygen (Gatenby and Gillies,
2004; Kroemer and Pouyssegur, 2008; Parks et al., 2013a;
Fig. 3. Hypoxia enhances glycolysis, acidifies pHe and regulates pHi. Tumour
Pouyssegur et al., 2006). A number of membrane proteins
cells experience an exacerbated glycolytic metabolism that is potenti-
that enhance glycolysis and deal with the production of ad- ated by the hypoxia induction of enzymes in the glycolytic pathway. MCT4
ditional metabolic wastes are regulated by hypoxia including induction by hypoxia ensures continued energy production via glycoly-
monocarboxylate transporters (MCTs) and carbonic anhy- sis by exporting lactic acid. MCT4 activity also contributes to the
drases (CAs). These hypoxic adaptations ensure that tumour maintenance of an alkaline pHi while acidifying the extracellular microen-
vironment. Hypoxia also strongly influences the CO2/HCO3− balance of
cells thrive and outcompete normal tissues for space in the tumour cells via induction of extracellular facing CAIX and CAXII. These
body. Recently, intriguing insights have been gained for these proteins assist in the regulation of pHi in co-ordination with Na+/HCO3−
proteins with respect to the interactions with the immune co-transporters (NBC) while further contributing to the acidification of pHe.
 S.K. Parks et al. / Molecular Aspects of Medicine 47–48 (2016) 3–14 7

(with Km ≈ 28 mmol/L and 153 mmol/L, respectively) absence of MCT4 (Le Floch et al., 2011; Marchiq et al., 2015).
(Dimmer et al., 2000; Manning Fox et al., 2000). These dif- MCT4 has also been implicated as a pH-regulating system
ferences are indicative of their respective metabolic roles in vivo that maintains the reversed pH gradient (pHi > pHe)
and tissue expression patterns (Fishbein et al., 2002). MCT4’s to increase tumour progression (Chiche et al., 2012). More-
low affinity for pyruvate prevents its release from glyco- over, highly aggressive tumours (i.e. triple-negative breast
lytic cells and thus facilitates the conversion of pyruvate into cancers (Doyen et al., 2014)) are reported to predomi-
lactate required to regenerate cytosolic NAD+ (Dimmer et al., nantly express the hypoxia-induced MCT4. Furthermore,
2000). Consequently, MCT4 is restricted to highly glyco- MCT4 is essential for lactate secretion, pH homeostasis and
lytic tissues such as white skeletal muscle fibres and survival of clear cell renal cell carcinoma (ccRCC) cell lines
astrocytes. In contrast, the high affinity of the ubiquitous (Gerlinger et al., 2012). High levels of MCT4 in primary ccRCC
MCT1 for lactate may be important for lactate influx and tumours correlated with poor relapse-free survival and meta-
thereby it is highly expressed in red skeletal muscle, the static lesions exhibited higher MCT4 expression than primary
heart, red blood cells, and the liver, where it plays a role in tumours. Similarly, recent studies have shown that MCT4
lactate shuttle (reviewed in Halestrap, 2013). is over-expressed in a highly glycolytic subtype of pancre-
MCT4 was identified as an HIF-1 up-regulated gene via atic adenocarcinoma (PDA) (Baek et al., 2014). MCT4
binding of two HREs (Ullah et al., 2006). In breast cancer, attenuation in PDA resulted in a substantial reduction of gly-
this hypoxia-induced expression of MCT4 was recently re- colytic flux, which evoked compensatory mechanisms
ported to be dependent on growth factors, including insulin including increased oxidative phosphorylation, macro-
and EGF (Baenke et al., 2015). However, while there is no pinocytosis and autophagy. Consistent with this, an unbi-
evidence of an HRE on the MCT1 gene, other transcrip- ased functional RNA interference screen has recently shown
tional factors such as MYC and PGC-1α have been described that MCT4 is an important regulator of breast cancer cell
to regulate MCT1 expression (Doherty et al., 2014; survival (Baenke et al., 2015). Similar to PDA cell lines, MCT4-
Hashimoto et al., 2007). Based on the activity of these tran- depleted breast cancer cells revealed reduced glycolytic flux
scriptional factors, it is not surprising that MCT1 and MCT4 resulting in increased uptake of AAs, particularly gluta-
have been shown to be up-regulated in several cancers, in- mine, and enhanced entry of glucose-derived pyruvate into
cluding cervical, glioblastoma, melanoma, breast, colorectal, the TCA cycle. In spite of these adaptations, MCT4 deple-
kidney and lung (Doyen et al., 2014; Gerlinger et al., 2012; tion reduced tumour growth and increased cell death due
Pinheiro et al., 2012). Conversely, the distribution pattern to elevation of reactive oxygen species in some tumour cells
of these two MCTs is different, not only between distinct and decreased pHi. Loss of cell viability was enhanced by
cancer types but also between different intra-tumoural com- treatment with inhibitors of macropinocytosis (EIPA), au-
partments and cell types. MCT4 was reported to be up- tophagy (Chloroquine), glutaminolysis (BPTES), and
regulated in hypoxic tumour cells (Meijer et al., 2012; mitochondrial complex I (metformin/phenformin) (Baek
Rademakers et al., 2011) and tumour associated fibro- et al., 2014; Baenke et al., 2015; Marchiq et al., 2015).
blasts (Pavlides et al., 2009; Whitaker-Menezes et al., 2011; High MCT4 expression in the aggressive mesenchymal
Witkiewicz et al., 2012) where it supports lactate release, subset of glioblastoma (GBM) was shown to be inversely cor-
whereas MCT1 was highly expressed in oxidative tumour related with overall survival of GBM patients (Lim et al.,
cells and angiogenic endothelial cells (Sonveaux et al., 2012; 2014). MCT4 knockdown dramatically reduced the stem-
Vegran et al., 2011) that recapture lactate and convert it into like CD133-positive population, leading to reduced
pyruvate to feed the TCA cycle, thereby increasing glucose proliferation in vitro and intracranial tumour xenograft for-
availability for hypoxic cells (Bonuccelli et al., 2010). mation in vivo (Lim et al., 2014). However, these effects seem
Information summarised above combined with studies to be independent of MCT4 transport activity, suggesting
showing that elevated lactate concentrations are associ- an unidentified function of the transporter. Moreover, de-
ated with poor outcome (Marchiq and Pouyssegur, 2015) pletion of MCT4 in GBM dramatically reduced HIF1α
has resulted in increased interest in targeting MCTs for cancer transcriptional activity implying that more studies are
therapy. Inhibiting MCT1 has shown striking impairment of needed to clearly determine the mechanisms regulating
tumour cell growth in HRas-transformed fibroblasts (Le Floch MCT4 expression and function (Lim et al., 2014).
et al., 2011), Burkitt lymphoma, MCF7 breast cancer cells Finally, MCT4 activity is reported to be significantly in-
(Doherty et al., 2014), small cell lung cancer (SCLC) and creased in hypoxia through up-regulation of CAIX which,
gastric cancer cell lines (Polanski et al., 2014). Targeting like CAII for MCT1, functions as an ‘H+-distributing antenna’
CD147/BASIGIN, which is a required ancillary protein for that dissipates local proton micro-domains and facilitates
proper folding and trafficking to the cell surface of both H+/lactate co-transport (Becker et al., 2010; Jamali et al.,
MCT1 and MCT4, has also shown promising anti-tumour 2015). Combined, these new developments in MCT re-
effects (Baba et al., 2008; Granja et al., 2015; Le Floch et al., search may be useful for developing novel anti-cancer
2011; Marchiq et al., 2015; Schneiderhan et al., 2009). strategies that will interfere with MCT4 expression and H+/
However, in the context of the hypoxic tumour environ- lactate efflux in hypoxic areas of tumour tissues.
ment, emerging evidence points to a crucial role of MCT4
in tumour progression highlighting the necessity of devel- 3.2. Acid-base regulation
oping MCT4-specific inhibitors. Indeed, due to functional
redundancy between MCT1 and MCT4, it has been shown A common distinguishing feature of solid tumours is the
that silencing or pharmacological inhibition of MCT1 in presence of an acidic extracellular environment. As men-
human colon adenocarcinoma cells was only effective in the tioned above, this arrives via the excretion of lactic acid
8 S.K. Parks et al. / Molecular Aspects of Medicine 47–48 (2016) 3–14

(MCTs) but is also enhanced by CO2 hydration via the ex- et al., 2000). It is now well established that this enzyme is
tracellular facing CAs. Thus tumour cells are required to deal key in the acidification of the extracellular space (pHe)
efficiently with increased metabolic waste production to (Svastova et al., 2004; Swietach et al., 2009) and mainte-
survive in their potentially hostile environment. This is nance of an alkaline pHi (Chiche et al., 2009; Swietach et al.,
enabled by an extremely efficient pHi regulating mecha- 2008, 2009). The underlying hypothesis is that CA IX con-
nism that maintains tumour cell pHi in the alkaline range verts CO2 to H+ + HCO3− with the HCO3− being re-absorbed
despite the acidic surroundings (for review see Parks et al., by the cell to buffer pHi while H+ diffuses to the blood-
2013a). We have recently re-enforced that hypoxia pro- stream for clearance (Fig. 3).
vides a definite survival advantage for tumour cells CAIX has become an important clinical marker or poor
experiencing acidosis (Parks et al., 2013b). This improved prognosis in a number of tumour types (for an extensive
survival in hypoxia is a combination of enhanced pHi regu- summary refer to Chiche et al., 2010; Neri and Supuran,
lating capacities that can maintain sufficient ATP levels for 2011; Sedlakova et al., 2014). Numerous pre-clinical studies
cellular function. Tumour cell pHi regulation follows the clas- have validated that disruption of CAIX alters both pHe and
sical models of either H+ extrusion or HCO3− buffering of the pHi and gene knockdown has been effective in slowing or
cytoplasm (Swietach et al., 2014). A redundancy of pHi regu- even stopping the growth of tumour cells in both in vitro
lating proteins is evident in tumour cells with alterations and in vivo models (Chiche et al., 2009; Lock et al., 2013;
in a few key players under hypoxia appearing to give tumour Lou et al., 2011; McIntyre et al., 2012; Svastova et al., 2004;
cells their pHi regulating advantage over normal cells (for Swietach et al., 2008, 2009). Consequently, there has been
extensive review see Parks et al., 2011, 2013a). an intense focus on pharmacological inhibitor develop-
ment, which has led to current on-going clinical trials for
3.2.1. H+ extruding mechanisms (NHEs, MCTs) this promising anti-cancer target (Neri and Supuran, 2011).
The ubiquitous Na+/H+ exchanger (NHE1) is implicated Encouraging results for other clinical applications have dem-
in regulating tumour cell pHi (Beltran et al., 2008; Lagarde onstrated increased sensitivity to ionising radiation with
et al., 1988; Luo and Tannock, 1994; Reshkin et al., 2000). CAIX disruption (Doyen et al., 2012; Dubois et al., 2011;
However, despite original indications that NHE1 was regu- Duivenvoorden et al., 2015). CAIX targeting has been shown
lated by hypoxia (Rios et al., 2005; Shimoda et al., 2006) this to improve chemotherapeutic and antibody treatments such
has not proven to be a feature of tumour cells. Analysis of as bevacizumab (McIntyre et al., 2012) and is continuing to
NHE function in various cell lines revealed differential effects be an important target in therapeutic development (Pore
depending on oxygenation levels and cell lines which further et al., 2015; Ward et al., 2015). In addition, CAIX disrup-
questions a direct role of hypoxia in NHE1 function (Hulikova tion can be important in the stroma where induction in
et al., 2013). Therefore we must be cognisant of the con- prostate stromal fibroblasts has been demonstrated
tribution that NHE1 plays in tumour cell pHi regulation, but to play an important role in tumour progression and
in the discussion of the hypoxia advantage for survival and metalloprotease activity (Fiaschi et al., 2013). More re-
proliferation in acidosis we assume that influence from cently, focus has been placed on monitoring the in vivo
changes in NHE1 expression does not occur. To date, other function of CAIX. These results reinforce that the primary
NHE isoforms have not been strongly implicated in either role of CAIX in vivo is to acidify the extracellular tumour
tumour pHi regulation or the hypoxia response. space, while in turn this acidification limits its catalytic ac-
As mentioned above, MCTs extrude H+ from the cell in tivity thus necessitating the increased expression that is
combination with lactate. Although this transporter does not observed with hypoxia (Gallagher et al., 2015).
directly respond to pHi changes (it is driven by lactate pro- The last 2–3 years have observed a divergence from the
duction), the high glycolytic rate of tumour cells and original focus on the pH physiology of CAIX to observe its
enhanced MCT extrusion activity contributes to the regu- interactions with other components of tumour progres-
lation of pHi (Fig. 3). Indeed it has been demonstrated that sion. These include interactions with immune factors and
tumour cells lacking the hypoxia inducible MCT4 experi- the influence on the prevalence of cancer stem cells (CSCs)
ence an acidic pHi compared to wild-type cells (Chiche et al., in the tumour environment. Dedhar’s group was the first
2012; Marchiq et al., 2015). Therefore evidence is mount- to provide evidence for the role of CAIX and hypoxia in the
ing for MCTs to be considered as “non-classical pH i enrichment of breast CSCs (Lock et al., 2013). Utilisation of
regulating” proteins which make an important contribu- a small molecule inhibitor for CAIX decreased the CSC
tion to the pHi balance of tumour cells in the face of altered population resulting in diminished tumour growth and me-
metabolic states. tastases. This work was complemented by another group
Finally, H+ extrusion could occur via direct pumping of showing a role for CAIX overexpression in MCF7 breast
H+-ATPases (for review see Neri and Supuran, 2011). This cancer CSCs (Papi et al., 2013). CAIX has now been linked
mechanism is not an obvious candidate as convincing ev- to priming of the stem cell niche via its interactions with
idence for localisation at the plasma membrane is lacking. cytokines such as G-CSF and CCL5 (Chafe et al., 2015). Re-
Furthermore, hypoxia does not regulate H+-ATPase expres- cently Harris’ group sorted CAIX −/+ expressing cells from
sion so it will not be a focus here. hypoxia exposure (0.1% O2) based on chromatin organisation
and found that gene markers of stem cells (such as ALDH1,
3.2.2. Carbonic anhydrases (CAs) TWIST1, SOX2) were enriched in the CAIX (+) expressing cells
Extracellular CAIX is one of the most sensitive hypoxia (Ledaki et al., 2015). Importantly, only the CAIX (+) cells were
markers and has received intense scrutiny over the past 15 able to exhibit functional properties of CSCs such as ALDH
years since its original hypoxia-induced description (Wykoff enzymatic activity and mammosphere production. To
 S.K. Parks et al. / Molecular Aspects of Medicine 47–48 (2016) 3–14 9

complete the story they investigated HDAC (Histone Recently Swietach’s group has shown that CAII activity
deacetylase) inhibitors as they have been shown to reduce exacerbates pHi fluctuations during changes in cellular CO2
expression of stem cell markers (Karantzali et al., 2008; Park levels that results in large Ca2+ fluctuations and changes in
et al., 2011). HDAC inhibitors acted both to decrease CAIX mTORC1 cellular signalling (Hulikova et al., 2014). Thus
expression and to reduce cell growth in the CAIX (+) cells CA interactions between hypoxia and non-hypoxia regu-
only (Ledaki et al., 2015). Evidently, the role of CAIX in lated isoforms in the overall CO2/HCO3− balance of tumour
hypoxic CSCs is an exciting area of current study that may cells and the cellular signalling pathways that are initi-
provide promising additional therapeutic areas in the future. ated as a result are a current focus of our on-going research
CAXII is also an extracellular facing CA regulated by that we believe will prove valuable for therapeutic
hypoxia but it has received less attention compared to CAIX. development.
CAXII is often expressed with CAIX (Haapasalo et al., 2008;
Tafreshi et al., 2012; Wykoff et al., 2000), has been linked 3.2.3. HCO3− transport
with poor prognosis in various cancers (Chien et al., 2012; An elusive molecular component of the hypoxia driven
Haapasalo et al., 2008) and can contribute to the regula- pH-regulating system in tumour cells has been the HCO3−
tion of tumour pHe and pHi (Chiche et al., 2009; Kopecka transporter allegedly responsible for re-absorbing HCO3− to
et al., 2015). CAXII has been suggested to contribute to the maintain an alkaline pHi (Fig. 3). This is likely due in part
redundancy of CA activity in tumours by compensating for to the vast number of potential HCO3− transporters origi-
a loss of CAIX activity under experimental disruption (Chiche nating from both the SLC4 (9 HCO3− transporting members
et al., 2009). Some controversy exists in the literature as to (Romero et al., 2013)) and SLC26 (7 HCO3− transporting
the similarities between CAIX and CAXII for prognosis as members (Alper and Sharma, 2013)) gene families. In ad-
CAXII has also been described as a good prognostic factor dition, HCO 3 − transport inhibitors such as DIDS are
(Ilie et al., 2011; Watson et al., 2003). Recently, CAXII has notoriously non-specific and it has just been reported that
been described as a gene marker for thyroid malignancies, the NBC specific inhibitor S0859 inhibits MCT1/2/4 even
providing a valuable diagnostic tool in the clinic for this more effectively than NBC (Heidtmann et al., 2015). Thus
tissue that is difficult to diagnose (Zheng et al., 2015). achieving clear results via inhibitor studies has been diffi-
Increased scrutiny of CAXII will be encouraged in the cult. Historically, none of the HCO3− transporting members
context of two recent studies. A CAXII specific antibody presented a clear regulation by hypoxia in the literature. We
(6A10) was shown to block the catalytic activity of CAXII recently described that the electrogenic NBC (SLC4A4,
and prevent cell growth in vitro (Gondi et al., 2013). This NBCe1) is induced by hypoxia at the gene level in an HIF1α
was confirmed by in vivo xenografts (A549 cells) where 6A10 dependant manner (Parks and Pouyssegur, 2015). However,
application effectively slowed tumour growth. A point to this hypoxia induction was limited to one cell line (6 cell
consider in this study is that CAXII appeared to carry the lines tested in total) and a generalisation of the hypoxia reg-
dominate catalytic activity in these cells indicating that CAIX ulation of SLC4A4 in other cell lines remains to be
and/or CAXII expression patterns may need to be defini- determined. Nonetheless, knockdown of SLC4A4 proved to
tively understood depending on tumour type for treatment impact on important areas of tumour cell (colon and breast)
strategies. Further to this study, it has just been reported physiology including growth, migration, and pH regula-
that CAXII is up-regulated as a marker of chemo-resistance tion (Parks and Pouyssegur, 2015). This complements a series
in colon (HT29), lung (A549) and osteosarcoma (U2-OS) cells of studies that have described the importance of the
(Kopecka et al., 2015). This study demonstrated that the electroneutral NBC (NBCn1, SLC4A7) in a subset of breast
CAXII up-regulation in doxorubicin resistance was HIF1- cancers (Boedtkjer et al., 2013; Gorbatenko et al., 2014;
dependent and that CAXII contributed directly to pH Lauritzen et al., 2010, 2012; Lee et al., 2015b). The most
regulation. Increased CAXII expression was shown to be nec- recent advance in NBCn1 studies has demonstrated a delay
essary for the activity of Pgp (P-glycoprotein; a classical in the chemical induction of breast tumour development
marker of multidrug resistance) suggesting that CAXII in- in NBCn1 knockout mice (Lee et al., 2015a). Although well
hibition could be a promising avenue to overcome studied, NBCn1 has never proven to be regulated by hypoxia.
chemoresistance associated with Pgp (Kopecka et al., 2015). Therefore, the literature implicating HCO3− transporters in
In light of these important new findings for the role of CAXII the importance of regulating tumour cell pH is growing but
it will be of interest to observe if a generalised CA inhibi- a direct hypoxia regulation of HCO3− transport is still lacking.
tory approach will prove more successful compared to Cooperation between the strongly hypoxia regulated car-
inhibition of a single CA isoform in future studies. bonic anhydrases and HCO3− transporters as previously
No other CA members of the 15 total that exist have suggested (Svastova et al., 2012) will be an active area of
been implicated in the hypoxia response. The intracellular study as we progress to a more global appreciation of HCO3−
CAII has been proposed to play a role in certain instances regulation in the tumour cell pH regulating machinery.
(for review see Parks et al., 2011) but its overall contribu- A bicarbonate therapy approach has been developed for
tion to tumour cell physiology has not been determined. clinical use based on the context of the hypoxia-induced al-
In light of the expansive work by Boron’s group in the teration in pH and CO2/HCO3− levels due to the presence of
description of a CA “push and/or pull” mechanism for moving proteins such as CAIX. Predicting that HCO3− or other sys-
CO2/HCO3− across the membrane (Musa-Aziz et al., 2014a, temic buffers would neutralise the acidic pH of the tumour
2014b; Occhipinti et al., 2014) we predict that the role of environment, members of Gatenby and Gillies teams dem-
intracellular CAII and its interactions with extracellular onstrated impressive results on the inhibition of metastasis
CAs may prove to be more important than originally thought. with this simple therapeutic approach (Robey et al., 2009;
10 S.K. Parks et al. / Molecular Aspects of Medicine 47–48 (2016) 3–14

Silva et al., 2009). This work has been extended to other micro and macro migratory ability should be assessed in
models of acid-mediated invasion (Estrella et al., 2013) and future studies to improve the evaluation of their use as ther-
is being considered in the context of tumour hetero- apeutic targets.
geneity (Robertson-Tessi et al., 2015) in the continued de-
velopment for this promising buffer therapy in a clinical 4. Future perspectives
setting. Thus in the context of tumour hypoxia, CO2/HCO3−
balance, disruptions due to CA activity will continue to be Pathophysiological responses initiated by hypoxia have
an essential consideration in the overall models of tumour clearly given tumour cells a means to rapidly expand and
development moving forward. outcompete their surrounding normal tissues. Consider-
ing that hypoxia touches three key areas of cellular function
3.2.4. Acid-base regulators in migration, metastases and as we have summarised in this review including nutrient
tumour growth supply, utilisation, and waste removal it appears that
Membrane proteins controlling pH gradients have been hypoxia-regulated proteins present valuable targets for drug
shown to contribute to cell migration and invasion. Estab- development (Fig. 4). Additionally, as hypoxia is limited in
lishment of pH gradients across the cytoplasm is thought normal tissues, these targets have the appealing character-
to play a role with the re-distribution of pH regulating pro- istic of being tumour specific, the best case being represented
teins to the leading or trailing edge of the cell (Schwab et al., by CAIX. Others and we have previously proposed mecha-
2012). These proteins contribute to the phenomenon of acid- nisms of combined protein disruption that will lead to
mediated metastasis leading to poor patient prognosis metabolic disorders and ultimately tumour cell death. This
(Gatenby and Gillies, 2008; Gatenby et al., 2006; Gillies et al., synthetic lethality approach continues to be expanded in
2012; Rofstad et al., 2006). Extracellular acidification has the context of tumour cell metabolism and hypoxia. In light
been recently demonstrated to clearly direct tumour inva- of new data supporting a role in important clinical param-
sion (Estrella et al., 2013) while in vitro models have eters such as tumour cell stemness, we believe it is vital to
confirmed a role for hypoxia regulated CAIX and NBC in cel- continue these experimental approaches in the future. Since
lular migration/invasion (Parks and Pouyssegur, 2015; (i) pHi disruptions alter the function of most enzymes, (ii)
Svastova et al., 2012). The role that acidosis and these AA levels direct the activity of the mTORC1 growth signal-
hypoxia-regulated proteins play in metastases strength- ling pathway, and (iii) lactate expulsion is essential for
ens their potential for tumour specific therapeutic benefit maintaining the glycolytic propensity of tumour cells, a com-
as drug targets. bination therapy targeting components of these systems
Potentially, the role of pH regulating proteins in cell mi- should prove useful for combating tumour growth (Fig. 4).
gration has been under-appreciated for primary tumour For certain hypoxia-regulated proteins such as CAIX, the most
growth. Recently, an evolutionary study of tumour devel- dramatic anti-tumoural effects have been observed in syn-
opment has shown that cell migration (even on a small scale) geneic mouse models (Lou et al., 2011). Thus it appears that
is required to achieve an exponential and nodule tumour future studies must focus more keenly on these types of
expansion (Waclaw et al., 2015). The authors conclude that models coupled with their immune system interactions. Evi-
tumour cell migration is not only important for metasta- dently, the pursuit of cell membrane hypoxia-regulated drug
sis but also essential for exponential tumour growth. Thus targets will remain an important component of cancer dis-
the role of hypoxia controlled pH-regulating proteins in both covery in the coming years.

Fig. 4. Targeting hypoxia regulated membrane proteins for developing cancer therapies. Hypoxia provides three key areas of tumour cell progression that
hold promise for stopping tumour growth. Various studies are in progress to assess how disruptions of glycolytic energy production via MCT inhibition,
interfering with amino-acid transport, and blocking of CA activity/pHi regulation can stress the tumour cell sufficiently to arrest growth and potentially
lead to cell death in their microenvironment. These therapeutic developments also have the potential to disrupt cancer stemness and metastasis.
 S.K. Parks et al. / Molecular Aspects of Medicine 47–48 (2016) 3–14 11

Acknowledgements Doherty, J.R., Yang, C., Scott, K.E., Cameron, M.D., Fallahi, M., Li, W., et al.,
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The authors of this manuscript have received funding Doyen, J., Parks, S.K., Marcie, S., Pouyssegur, J., Chiche, J., 2012. Knock-down
from a variety of sources including the Centre Scientifique of hypoxia-induced carbonic anhydrases IX and XII radiosensitizes
de Monaco, the Ligue Nationale Contre le Cancer (LNCC; tumor cells by increasing intracellular acidosis. Front. Oncol. 2, 199.
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