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Research Article

Hypoxia-Inducible Carbonic Anhydrase IX and XII Promote Tumor
Cell Growth by Counteracting Acidosis through the Regulation
of the Intracellular pH
Johanna Chiche, Karine Ilc, Julie Laferrière, Eric Trottier, Frédéric Dayan, Nathalie M. Mazure,
M. Christiane Brahimi-Horn, and Jacques Pouysségur
Institute of Developmental Biology and Cancer Research University of Nice, Centre National de la Recherche Scientifique UMR 6543,
Centre A. Lacassagne, Nice, France

apoptosis (5–7). Because numerous intracellular processes require
close regulation of pHi, most mammalian cells, particularly
hypoxic tumor cells, have developed key strategies to regulate
their pHi. Activation of the hypoxia-inducible factor-1 (HIF-1) in
hypoxia plays a major role in regulating pH homeostasis by
enhancing expression of membrane located transporters,
exchangers, pumps and ecto-enzymes (8). To survive in an acidic
environment, the pHi-regulating system of tumor cells actively
extrudes acids via the growth factor–activated Na+/H+ exchanger
1 (NHE-1; refs. 9–12) and the monocarboxylate transporters
(MCT1 and MCT4; ref. 13). We showed previously that NHE-1
plays a key role in tumor development particularly for cells
producing large amounts of lactic acid (14). In the opposite
direction to H+ extrusion, HCO3 influx through Na+-HCO3
cotransporters (NBC) and Cl /HCO3 exchangers (AE) contributes
to cytoplasmic alkalinization (15–17).
Carbonic anhydrases (CA), which catalyze the reversible
hydration of cell-generated carbon dioxide into protons and
bicarbonate ions, have also been proposed to contribute to
cellular alkalinization (18–20). The direction of the reaction is
dependent on the form, CO2 or bicarbonate and protons, that
predominates. Mammalian cells express 13 active isoforms of
CAs, with a conserved active site and variable levels of activity,
and 3 inactive isoforms. They differ in their tissue distribution
and cellular localization. The expression of the membraneassociated CAIX and CAXII is tightly controlled by oxygen levels
in multiple epithelial tumor types (21–23), and CAIX has a higher
extracellular activity than CAXII (23–25). CAIX is highly induced
in an HIF-1–dependent manner (26) and is constitutively
expressed in von Hippel-Lindau (VHL)–defective cells. CAXII is
up-regulated in VHL-defective renal tumors and induced in
hypoxia in tumor cells, but its dependence on HIF is not well
established (22). Whereas tumor expression of HIF-1a and CAIX
correlate with poor patient survival (19, 27), the significance of
CAXII, which lacks the extracellular proteoglycan domain of CAIX
implicated in cell adhesion (28–30), is less obvious (24). CAIX may
be functionally linked to the regulation of the tumor pH, because
it contributes to extracellular acidification (31) and forced CAIX
expression in three-dimensional cultured aggregates influences
pHi homeostasis (32). However, direct evidence of the conjugated
roles of CAIX and CAXII in pHi regulation in cell lines and in
tumor growth is still missing.
In this study, we show that the hypoxia-inducible CAIX and
CAXII proteins promote cell survival and growth through pHi
maintenance. We conclude that CAIX and CAXII constitute a
robust pHi-regulating system able to confer a tumor growth and
survival advantage on cells exposed to a hypoxic and acidic
microenvironment. Finally, as very often hypothesized, but not

Abstract
Acidosis of the tumor microenvironment is typical of a
malignant phenotype, particularly in hypoxic tumors. All cells
express multiple isoforms of carbonic anhydrase (CA),
enzymes catalyzing the reversible hydration of carbon dioxide
into bicarbonate and protons. Tumor cells express membranebound CAIX and CAXII that are controlled via the hypoxiainducible factor (HIF). Despite the recognition that tumor
expression of HIF-1A and CAIX correlates with poor patient
survival, the role of CAIX and CAXII in tumor growth is not
fully resolved. To understand the advantage that tumor cells
derive from expression of both CAIX and CAXII, we set up
experiments to either force or invalidate the expression of
these enzymes. In hypoxic LS174Tr tumor cells expressing
either one or both CA isoforms, we show that (a) in response
to a ‘‘CO2 load,’’ both CAs contribute to extracellular
acidification and (b) both contribute to maintain a more
alkaline resting intracellular pH (pHi), an action that
preserves ATP levels and cell survival in a range of acidic
outside pH (6.0–6.8) and low bicarbonate medium. In vivo
experiments show that ca9 silencing alone leads to a 40%
reduction in xenograft tumor volume with up-regulation of
ca12 mRNA levels, whereas invalidation of both CAIX and
CAXII gives an impressive 85% reduction. Thus, hypoxiainduced CAIX and CAXII are major tumor prosurvival pHiregulating enzymes, and their combined targeting shows that
they hold potential as anticancer targets. [Cancer Res
2009;69(1):358–68]

Introduction
Adaptation of tumor cells to hypoxia and acidosis is a critical
driving force in tumor progression and metastasis (1, 2). Cancer
cells produce a large amount of lactic acid (3), which is generated
through glucose metabolism and inefficient vascular clearing,
resulting in an acidic microenvironment within many solid
tumors (4). Extracellular acidosis represents a threat to cell
survival by modifying the intracellular pH (pHi), wherein a 0.1 pHi
variation can disrupt multiple biological functions, including ATP
production, protein synthesis, cell proliferation, migration, and

Note: Supplementary data for this article are available at Cancer Research Online
(http://cancerres.aacrjournals.org/).
Requests for reprints: Jacques Pouyssegur, Centre National de la Recherche
Scientifique UMR 6543, 33 Avenue Valombrose, Nice 06189, France. Phone: 33-492-031222; Fax: 33-492-03-1225; E-mail: pouysseg@unice.fr.
I2009 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-08-2470

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DOI:10.1158/0008-5472.CAN-08-2470
CAIX and XII Promote Tumor Growth by Regulating pHi

shown (23, 24), we validate here that CAIX and CAXII constitute
two new anticancer therapeutic targets.

Immunofluorescence
Cells at sparse density were grown on glass coverslips and fixed with 3%
paraformaldehyde for 30 min followed by saturation for 30 min in PBS–2%
gelatin and PBS–1% bovine serum albumin. Cells were then incubated for
1 h with the CAIX antibody without permeabilization, followed by
incubation for 1 h with an anti-mouse Alexa 594–conjugated IgG antibody
(Invitrogen). Cells were mounted onto slides with citifluor and analyzed
with a Leica microscope (objective, 100).

Materials and Methods
Cell Culture and Hypoxic Exposure
Chinese hamster lung CCL39 fibroblasts (American Type Culture
Collection) and the CCL39-derived mutant PS120 cells, lacking the
amiloride-sensitive Na+/H+ exchanger (6), were maintained in DMEM
(Sigma) supplemented with 7.5% FCS in a humidified atmosphere of 5%
CO2, 95% air, or 100% air at 37jC. The colon adenocarcinoma cell line
LS174Tr expressing the tetracycline (Tet) repressor was provided by Dr. van
de Wetering (33) and maintained in DMEM supplemented with 10% FCS.
Other human tumor cell lines were likewise cultured. Incubation in hypoxia
at 1% O2 was carried out at 37jC in 95% humidity and 5% CO2/94% N2 in a
sealed anaerobic workstation (Ruskinn).

In vitro Determination of CA Activity
Cells incubated in normoxia (PS120-pca9 cells) or hypoxia (to induce CAIX
and CAXII expression in LS174Tr cells) were placed on ice in normoxia, scrapped
into ice-cold PBS, to obtain intact membrane-bound CAIX and CAXII. The cell
suspension was immediately centrifuged and resuspended in a bicarbonate-free
medium (Sigma) buffered at outside pH (pHo) 7.4 with 30 mmol/L HEPES. A 0.1
volume of this cell suspension was added to a 3 mmol/L HEPES-buffered
solution (HBS) adjusted to pHo 8.2 before rapid addition to a CO2-saturated
nonbuffered solution and pH determined over time (microelectrode, Schott
Instrument) to monitor the rapid hydration of CO2 to carbonic acid. For
inhibition of the total CA activity, 100 Amol/L acetazolamide (ACTZ; Sigma) was
added to the cell suspension in normoxia 15 min before the experiment.

Plasmids
Full-length human ca9 cDNA was obtained from hypoxic HeLa mRNA
extracts by PCR using the following specific primers: forward 5¶CGGGGTACCGCCGCCACCATGGCTCCCCTGTGCCCC-3¶ and reverse 5¶GCTCTAGACTAGGCTCCAGTCTCGGC-3¶. ca9 cDNA was ligated into the
pTREX-A (pcDNA4/TO/myc-His A; Invitrogen) vector (pca9) between the
KpnI and XbaI sites. The short hairpin RNA (shRNA)–ca9 (shca9) was
obtained with oligonucleotide sequences forward 5¶-AGTTAAGCCTAAATCAGAA-3¶ and reverse 5¶TTCTGATTTAGGCTTAACT-3¶ and inserted into
the pTER vector. The shRNA–hif-1a (shhif-1a) was obtained with
oligonucleotide sequences forward 5¶-CTGATGACCAGCAACTTGA-3¶ and
reverse 5¶-TCAAGTTGCTGGTCATCAG-3¶ and inserted into the pTER
vector. Lentivirus particles for two independent sequences (1 and 2) of
pLKO.1-Puro Vector shRNA targeting ca12 (ca12 ) and nontarget shRNA
(ctl ) were from Sigma (TRCN0000116249, TRCN0000116251, and
SHC002V).

Resting pHi Measurement

[14C]Benzoic acid. The pHi was measured using the technique of
distribution of the weak acid [7-14C]benzoic acid (Amersham Biosciences)
in intracellular and extracellular spaces for exponentially growing cells (35).
Diisothiocyanatostilbene-2¶,2-disulfonic acid (DIDS; 1 mmol/L, Sigma) was
used as an inhibitor of HCO3 transporters.
BCECF-AM probe. Exponentially growing cells seeded on glass coverslips
were incubated for 25 min in bicarbonate-free HBS (pHo 7.4), MES-buffered
solution (MBS; pHo 6.6), or bicarbonate-buffered solution (BBS; adjusted to
pHo 7.4). The pH-sensitive fluorescent dye BCECF-AM 1 Amol/L (Sigma) was
then added for 5 min at room temperature. Cells were then quickly washed
with the appropriate HBS, MBS, or BBS solution and transferred to a laminar
flow cell chamber perfused with the same solution. Ratiometric measurement of the fluorescence of 50 randomly selected individual cells per
coverslip was performed in a workstation (Acquacosmos). The pHi was
estimated by an in situ two-point calibration (pHo 6.6–7.6) with perfusion of a
high K+ buffer solution containing 130 mmol/L KCl, 2 mmol/L CaCl2, 1 mmol/
L MgCl2, 10 mmol/L glucose in 20 mmol/L HEPES-Tris (or MES-Tris), and
25 Amol/L nigericin to allow pHi to equilibrate with the external pHo.

Stable Transgenic Cells
CCL39, PS120, and LS174Tr cells were transfected with pca9, whereas
only LS174Tr cells were transfected with shca9 or shhif-1a, using
Polyfectamine (HiPerFect Transfection Reagent, Qiagen) according to the
manufacturer’s instructions. Isolated clones were maintained under zeocin
(500 Ag/mL, Invitrogen). Tet (10 Ag/mL) induces CAIX expression or ca9 or
hif-1a silencing. LS-shca9 cells were also transduced with lentiviral particles
containing shRNA-ca12 (ca12 ; Sigma) or nontarget shRNA (ctl) according
to the manufacturer’s instructions and named, respectively, LS-shca9/ca12
and LS-shca9/ctl.

DNA Synthesis
Cells (5,000) were plated on 96-well dishes before transfer to a CO2-free
incubator for 24 h in HCO3 free DMEM buffered with 30 mmol/L MES or
HEPES adjusted to different pHo (6.2–7.4), supplemented with 10% dialyzed
serum, hypoxanthine (0.1 mmol/L), and UTP (0.1 mmol/L). DNA synthesis
was measured using an ELISA colorimetric kit (Roche Diagnostics) based on
a 2-h incorporation of BrdUrd (20 Amol/L).

RNA Extraction and Relative and Absolute Real-Time
Quantitative PCR
Total RNA was extracted from cells using the RNA extraction kit (Qiagen)
according to the manufacturer’s instructions. Total RNA (2 Ag) was added to
a 20 AL reverse transcription–PCR reaction using the Omniscript kit
(Qiagen). The relative expression level of ca9 and ca12 was quantified by
real-time quantitative PCR (qPCR), as reported previously (26). The absolute
quantification of ca9 and ca12 mRNA was obtained by absolute real-time
quantification. A standard curve was prepared using dilutions of the
pTREX-ca9 and pTREX-ca12 vectors from 30 to 3 108 copies in triplicate.
The cycle number of ca9 or ca12 amplification of each extract was
compared with the standard curve obtained respectively with pTREX-ca9 or
pTREX-ca12 vectors.

ATP Determination
Cells were seeded in DMEM and grown at different pHo, as described for
DNA synthesis. ATP levels were measured using a Cell Titer Glo kit (Promega)
according to the manufacture’s instructions. The relative luminescence unit
was normalized to the quantity of protein.

Clonogenicity Assay
Cells (1,000) were seeded onto 60-mm dishes. Once attached, the medium
was replaced by HCO3 free DMEM buffered at pHo 6.4 (30 mmol/L MES) or
at pHo 7.4 (30 mmol/L HEPES), supplemented with 10% dialyzed serum,
hypoxanthine (0.1 mmol/L), and UTP (0.1 mmol/L) for growth in the
absence of CO2/HCO3 and transferred to a CO2-free atmosphere for 24 h.
Dishes were then returned to 5% CO2 in a regular media for 7 d before
staining with Giemsa (Fluka).

Immunoblotting
Cells were lysed in SDS sample buffer. Proteins (40 Ag) were separated on
7.5% SDS polyacrylamide gels and transferred onto polyvinylidene difluoride
membranes (Millipore). Membranes were blotted with the M75 antibody to
CAIX (Bayer), Hsp90 (Abcam), a polyclonal antibody to recombinant CAXII
(Sigma), and a polyclonal antibody to HIF-1a prepared and validated in our
laboratory (34). Immunoreactive bands were detected with a horseradish
peroxidase (HRP) anti-mouse or anti-rabbit antibody (Promega) by
enhanced chemiluminescence (Amersham Biosciences).

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Cell Proliferation in Three Dimensions
To grow spheroids, 1,600 cells were seeded in drops in 20 AL of
HCO3 free DMEM buffered with 30 mmol/L HEPES adjusted to pHo 7.4
supplemented with 10% FCS. After 12 d, spheroids were collected and cells

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Figure 1. Forced expression of catalytically active CAIX induced extracellular acidification and cytoplasmic alkalinization. A, top, stable expression of human CAIX in
nonneoplastic PS120 fibroblasts lacking the Na+/H+ exchanger (NHE-1). Inset 1, immunoblot to CAIX of lysates of control, empty plasmid vector (PS120-pev ), or
human CAIX stable expressing cells (PS120-pca9 ). The Hsp90 protein level was used as a loading control. Inset 2, immunofluorescence to CAIX in PS120-pev
and PS120-pca9 cells. Bottom, rate of extracellular acidification in response to a CO2 load without cells (no cells ) or with samples of intact normoxic PS120-pev or
PS120-pca9 cells. The rate of acidification reflects an estimation of the CA activity at the cell surface evaluated with or without a specific inhibitor of CAs (100 Amol/L
ACTZ). B, CAIX expression increases the resting pHi. pHi of PS120-pev and PS120-pca9 cells was determined with pH-sensitive fluorescent dye BCECF-AM.
Histograms indicate the mean basal pHi for cells incubated for 30 min in either a 25 mmol/L HCO3 -buffered solution (+HCO3 ) or HCO3 –free MES or HBS (-HCO3 )
adjusted to a pHo of 6.6 or 7.4, respectively. Just before their transfer to a laminar flow cell chamber, BCECF-AM was added to the cells for 5 min. Cells were then
perfused with the respective solutions, and a three-point calibration curve was created with high KCl/nigericin solutions to measure pHi as a function of the fluorescence
ratio. Significant differences based on the Student’s t test (***, P < 0.001; n = 200 cells for each PS120-pev and PS120-pca9 cells at pHo 6.6, n = 3000 cells for pHo 7.4
with or without HCO3 ). In the absence of HCO3 , PS120-pca9 cells had a statistically higher resting pHi than PS120-pev cells perfused at either a pHo of 6.6 or 7.4.
C, CAIX expression increases pHi over a range of pHo. The pHi was determined in PS120-pev and PS120-pca9 cells as a function of pHo with [14C]benzoic acid.
Exponentially growing cells of two independent clones for PS120-pca9 and PS120-pev cells were equilibrated for 15 min in a nominally HCO3 free solution with pHo
varying from 6.2 to 7.4. Cells were then shifted for 15 min to the same solution containing [14C]benzoic acid at the specific activity of 1 ACi/mL. pHi was calculated as
described under Materials and Methods. Determinations were done in quadruplicate for each condition, and the experiment was repeated at least thrice. Significant
differences are based on the Student’s t test (**, P < 0.01; ***, P < 0.005). D, DIDS, an inhibitor of HCO3 transporters, abolishes the differential in CAIX-induced
alkalinization. The steady-state pHi of PS120-pev and PS120-pca9 cells was measured with [14C]benzoic acid. Exponentially growing cells were incubated for 15 min in
a nominally HCO3 free MBS at a pHo of 6.6 without (DMSO ) or with 1 mmol/L DIDS. Cells were then shifted for 15 min to the same medium containing [14C]benzoic
acid at a specific activity of 1 ACi/mL. pHi was calculated as described under Materials and Methods. The mean of quadruplicate determinations is given. Significant
differences are based on the Student’s t test (**, P < 0.01).

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CAIX and XII Promote Tumor Growth by Regulating pHi

Figure 2. CAIX expression promotes survival and growth of PS120 fibroblasts impaired in NHE-1 in vitro in an acidic microenvironment. A, proliferation of PS120-pev
and PS120-pca9 cells (two independent clones for each type) was measured after a 24-h incubation in normoxia in a CO2/HCO3 free microenvironment for media
buffered with either 30 mmol/L MES for a pHo of 6.2, 6.4, 6.6, and 6.8 or with 30 mmol/L HEPES for a pHo of 7.4 and supplemented with 100 Amol/L hypoxanthine/100
Amol/L uridine/10% dialyzed FCS. A cell proliferation ELISA colorimetric kit, which measures BrdUrd incorporation into newly synthesized DNA, was used. Data
are expressed as an average of three independent experiments performed in triplicate using two independent clones. Significant differences are based on Student’s
t test (***, P < 0.005). B, quantification of ATP levels in PS120-pev and PS120-pca9 cells (two independent clones for each type) grown in normoxia in a
CO2/HCO3 free environment with varying pHo from 6.2 to 7.4 for 24 h using a luciferin/luciferase-based assay, with results expressed as relative luminescence units.
Determinations were done in triplicate, and the entire experiment was done thrice using two independent clones. Significant differences are based on the Student’s t test
(***, P < 0.005). C, viability assay of PS120-pev and PS120-pca9 cells in normoxia as function of pHo. Top, cells (1,000) were seeded in 60-mm dishes. Once
attached, cells were incubated in HCO3 free media adjusted to pHo 6.4 or 7.4 for 24 h in a CO2-free atmosphere. Fresh medium containing HCO3 (40 mmol/L) at
pHo 7.4 was then added to the dishes that were maintained for 10 d in a 5% CO2 incubator before staining for visualization of the colonies. Bottom, the colony
number per plate was counted by eye for two independent PS120-pca9 and PS120-pev clones. The experiments were repeated thrice using two independent clones.
Student’s t test (*, P < 0.05).

were dissociated in Accutase (Life Technologies) to determine the number
of individualized living cells.

Statistical Analysis
The Student’s t test was used wherein P values of <0.05 were considered
significant.

Nude Mice Tumorigenicity and Immunohistochemistry

Cells (1 106) suspended in 500 AL of serum-free DMEM supplemented
with insulin-transferrin-selenium (Life Technologies) were s.c. injected into
the back of 4-wk-old male athymic mice (Harlan). Animal studies were
conducted according to Centre National de la Recherche Scientifique
institutional guidelines. Food and water were given ad libitum. Doxycycline
(Dox; 750 Ag/mL; Sigma) was given in the drinking water to induce
silencing of hif-1a or ca9. Five mice were used for each experimental
condition. The tumor volume was determined using the formula: (4p / 3)
L / 2 W / 2 H / 2 (L, length; W, width; H, height). When the tumor
volume reached f1,500 mm3, mice were injected i.p. with hypoxyprobe (60
mg/kg; Chemicon) 4 h before sacrifice. Tumors were collected for RNA,
protein, and immunohistochemical analysis, as described (34). Sections
were incubated with antibodies to hypoxyprobe or CAXII for 1.5 h followed
by incubation with anti-mouse or anti-rabbit IgG-HRP antibodies. Analysis
was performed with a Leica microscope (objective, 20).

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Results
Forced expression of catalytically active CAIX-induced
extracellular acidification and cytoplasmic alkalinization.
Nonneoplastic Chinese hamster CCL39 lung fibroblasts and
CCL39-derived PS120 mutant cells defective in the Na+/
H+exchanger, which do not express endogenous CAIX or CAXII
in normoxia or in hypoxia, were selected for expression of
human CAIX and examined for their contribution to pHi with
(CCL39 cells) or without (PS120 cells) interference from pHi
regulation by NHE-1. Stable expression of human CAIX in
normoxia was found to be plasma membrane located in PS120
(Fig. 1A) and CCL39 cells (Supplementary Fig. S1). The level of
expression of transfected human CAIX in these fibroblasts in

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Figure 3. The hypoxia-induced CAIX and CAXII activity contributes to cytoplasmic alkalinization in an acidic microenvironment. A, the genes ca9 and ca12 are
HIF-1–dependent in human colon adenocarcinoma LS174Tr cells. Expression of the mRNA of hif-1a (top ), ca9 , and ca12 (bottom ) was determined by real-time qPCR
in the stable LS-shhif-1 a clone, in which Tet induces shRNA of hif-1a . Cells were incubated without ( Tet ) or with (+Tet ) Tet for 4 d and maintained in either normoxia
21% O2 (N ) or hypoxia 1% O2 (H 1%) for 48 h. Each difference in gene expression was estimated within the limits of a 95% confidence interval. The results are
representative of at least three separate experiments. B, hypoxia increases the extracellular CA activity, as reflected in an increase in the rate of extracellular
acidification compared with normoxic conditions. Top, LS174Tr cells were incubated either in normoxia or hypoxia 1% O2 for 48 h. The rate of extracellular acidification
in response to a CO2 load was measured without cells (no cells ) or with intact LS174Tr cells. Bottom, The LS-shhif-1a clone was incubated without ( Tet ) or with
(+Tet ) Tet to allow for hif-1a silencing for 4 d before incubation in hypoxia 1% O2 for 48 h without ( Tet ) or with (+Tet ) Tet. The rate of acidification in response to a
CO2 load for intact hypoxic LS-shhif-1a cells silenced (+Tet ) or not ( Tet) for hif-1a was measured. ACTZ (+ACTZ ) at 100 Amol/L was added to a suspension of
hypoxic LS-shhif-1a Tet cells for 15 min before CO2 addition. C, determination of resting pHi with [14C]benzoic acid as a function of pHo in LS174Tr cells exposed to
either normoxia or hypoxia 1% O2 for 48 h. Exponentially growing LS174Tr cells incubated in normoxia or hypoxia were equilibrated for 15 min in a nominally
HCO3 free HBS or MBS with a pHo varying from 5.8 to 7.4. Cells were incubated for 15 min in the same appropriate equilibration solution containing [14C]benzoic acid
at a specific activity of 1 ACi/mL. The pHi was calculated as described under Materials and Methods. The experiment was repeated at least thrice, and each point
represents the average of quadruplicates for each experiment (**, P < 0.01; ***, P < 0.005). D, stable Tet-inducible expression of CAIX in LS174Tr cells in normoxia
maintains a higher pHi in an acidic and HCO3 free media. Top, inset 1, immunoblot of inducible expression of CAIX in normoxia in LS174Tr cells (LS-pca9 )
without ( Tet) or with (+Tet ) Tet for 4 d. Total extracts were analyzed by immunoblotting with antibodies against CAIX and Hsp90. The latter was used as a loading
control. Inset 2, immunofluorescence of inducible expression of CAIX expression (LS-pca9 ) in LS174Tr cells in the absence ( Tet) or presence (+Tet ) of Tet for
4 d. Bottom, determination of resting pHi with [14C]benzoic acid in as a function of pHo in Tet-inductible LS-pca9 clone expressing CAIX in normoxia under treatment
for 4 d with (+Tet ) or without ( Tet ) Tet. Determinations were done in quadruplicate for each condition, and each experiment was repeated at least thrice. Results
represent the average of quadruplicates for each experiment (**, P < 0.01; ***, P < 0.005).

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DOI:10.1158/0008-5472.CAN-08-2470
CAIX and XII Promote Tumor Growth by Regulating pHi

Figure 4. Combined invalidation of CAIX and CAXII reduces pHi and spheroids growth. A, silencing of ca9, ca12 , and ca9/ca12 in LS174 cells. Top left, expression of
the mRNA levels of ca9 determined by real-time qPCR. Stable LS-shca9/ctl and LS-shca9/ca12 clones were incubated with ( Tet ) or without (+Tet ) Tet for 4 d before
incubation in either normoxia (N ) or hypoxia 1% O2 (H 1%) for 48 h. Top right, expression of the mRNA levels of ca12 determined by real-time qPCR under the
conditions described below. LS-shca9/ctl and LS-shca9/ca12 clones were incubated with ( Tet ) or without (+Tet ) Tet for 4 d before incubation in either normoxia (N )
or hypoxia 1% O2 (H 1%) for 48 h. The results are representative of at least three separate experiments (**, P < 0.01). Bottom, total extracts were analyzed by
immunoblotting with antibodies against HIF-1a, CAIX, CAXII, and Hsp90. The latter was used as a loading control. B, top, rate of acidification of a medium set at
pH 8.2 in response to the addition of CO2 incubated without cells (no cells ) or with intact LS-shca9/ctl cells previously incubated for 4 d with ( Tet ) or without (+Tet )
Tet and exposed in hypoxia 1% O2 for 48 h. Control LS-shca9/ctl Tet and ca9 silenced LS-shca9/ctl +Tet cells were also treated with 100 Amol/L ACTZ 15 min before
addition of CO2. Bottom, rate of acidification in response to a CO2 load of intact LS-shca9/ctl and LS-shca9/ca12 cells incubated without ( Tet) or with (+Tet) Tet
for 4 d and then exposed to either normoxia (N ) or hypoxia 1% O2 (H 1% ) for 48 h. C, combined ca9 and ca12 invalidation (LS-shca9/ca12 +Tet) diminishes resting
pHi in an acidic (pHo 6.0), MES-buffered, nominally HCO3 free solution. Stable expressing LS-shca9/ca12 cells were constitutively invalidated for ca12, whereas
exponentially growing Tet-inducible LS174Tr (LS-shca9/ctl and LS-shca9/ca12 ) clones were invalidated for ca9 after a 4-d treatment with Tet. Cells were then
incubated in hypoxic incubation 1% O2 for 48 h. Dishes were then returned to normoxia for resting pHi determination in a 20 mmol/L MES-buffered, nominally
HCO3 free saline/glucose solution adjusted to pHo 6.0 or in a 20 mmol/L HEPES-buffered, nominally HCO3 free saline/glucose solution adjusted to pHo 7.4 (15 min
incubation to reach the equilibrium). For pHi determination, cells were shifted for 15 min to the same medium containing [14C]benzoic acid at a specific activity 1 ACi/mL.
The pHi was calculated as described under Materials and Methods. The experiment was done twice. Each point represents the average of quadruplicates for each
experiment (**, P < 0.01). D, silencing of ca9 and ca12 diminished proliferation of three-dimensional spheroids. Top, an inducible LS174Tr clone invalidated for
Tet) or both ca9 and ca12 (LS-shca9/ca12 +Tet) with a 4-d Tet treatment (+Tet ) were cultured as spheroids in the absence ( Tet) or
ca12 (LS-shca9/ca12
presence (+Tet ) of Tet for 12 d. Hypoxyprobe (pimonidazole) was added to the extracellular media for staining of hypoxic zones 4 h before formaldehyde fixation.
Bottom, spheroids were subjected to Accutase dissociation, and individualized live cells were counted using Trypan blue. The spheroid proliferation index was
calculated as the ratio of the cell number counted at day 12 to the cell number at day 0. Data represent the average of three independent experiments.

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higher for CAIX-expressing PS120 cells (Fig. 1A) and CAIXexpressing CCL39 cells (Supplementary Fig. S1) than for control
cells (pev). The CA inhibitor ACTZ reduced the activity of CAIXexpressing cells to the spontaneous basal level obtained with the
control cells with or without ACTZ. In addition, PS120-pca9 cells
incubated in normoxia or hypoxia (48 h, 1% O2) showed the

normoxia was comparable with the expression of CAIX in tumor
cells after 48 h in hypoxia 1% O2 (data not shown). The CA
activity associated with the plasma membrane was determined
by the rapid acidification of a minimally buffered medium in
response to addition of a CO2-saturated solution. In the presence
of cell suspensions, the rate and magnitude of acidification was

Figure 5. Inducible invalidation of CAIX and CAXII reduces the rate of xenograft tumor growth. Characterization of the growth properties of CAIX-deficient,
CAXII-deficient, and CAIX/CAXII-deficient xenograft tumors. At 4 d before injection of LS-shev/ctl , LS-shhif-1a , LS-shca9/ctl , and LS-shca9/ca12 cells, cells were
incubated without ( Tet) or with (+Tet ) Tet to silence hif-1a, ca9 or both ca9 and ca12, respectively. For the respective conditions, mice received Dox or not, a
semisynthetic Tet, in the drinking water 4 d before cell injection. In vivo xenograft assays were performed by injecting s.c. into the back of athymic nude mice (1 106)
viable and individual tumor cells LS-shev/ctl Tet, LS-shev/ctl +Tet, LS-shhif-1a Tet, LS-shhif-1a +Tet, LS-shca9/ctl Tet, LS-shca9/ctl +Tet, LS-shca9/ca12
Tet, and LS-shca9/ca12 +Tet. Xenograft growth was determined by measuring the tumor volume. A, top, Dox in the drinking water does not affect tumor growth of
control LS-shev/ctl cells; bottom, invalidation of hif-1a (LS-shhif-1a +Tet) showed a slight reduction in tumor growth compared with tumor growth of control cells
(LS-shhif-1a +Tet). B, cells defective for ca12 (LS-shca9/ca12
Tet) formed xenograft tumors at the same rate and of the same size as control cells not silenced for
ca12 (LS-shca9/ctl Tet). C, cells silenced for ca9 (LS-shca9/ctl +Tet) or for both ca9 and ca12 (LS-shca9/ca12 +Tet) grew slower and gave a smaller tumor volume
than control (LS-shca9/ctl Tet) cells. Five mice were studied per condition. In vivo experiments were repeated twice. D, level of mRNA expression of ca9 and ca12 in
the xenograft tumors of different transgenic cells, as determined by real-time qPCR. Results represent an average of four tumors for each condition.

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CAIX and XII Promote Tumor Growth by Regulating pHi

and catalytically active CAs in a HIF-1–dependent manner
(Fig. 3B). Hypoxic induction was associated with significantly
enhanced alkalinization of the resting pHi of cells when exposed to
a low pHo in the absence of extracellular bicarbonate (Fig. 3C). To
study the contribution of CAIX to pHi regulation, hypoxia-induced
CAIX expression was mimicked in LS174Tr cells expressing a basal
level of endogenous CAXII in normoxia (Fig. 3D; Supplementary
Table S1; Supplementary Fig. S7). Forced Tet-inducible expression
of CAIX leads to enhanced alkalinization of the resting pHi in an
acidic and bicarbonate-free environment (Fig. 3D and Supplementary Fig. S7). Thus, CAIX expression plays a key role in maintaining
the resting pHi in an acidic and bicarbonate-limiting environment
in LS174Tr cells.
Combined invalidation of CAIX and CAXII reduces pHi and
spheroid growth. LS174Tr cells were selected for Tet-inducible
silencing of ca9 combined (LS-shca9/ca12 ) or not (LS-shca9/ctl)
with constitutive silencing of ca12. Tet addition to shca9 cells
(LS-shca9/ctl and LS-shca9/ca12 ) resulted in 95% invalidation of
ca9 mRNA (Fig. 4A, top) and protein (Fig. 4A, bottom), whereas 90% of
the ca12 mRNA (Fig. 4A, top) and protein (Fig. 4A, bottom) were
silenced in LS-shca9/ca12 cells. Note that an increase in hypoxiainducible expression of the mRNA and protein levels of CAXII was
observed when ca9 was silenced. The copy number of ca9 mRNA is
18-fold lower than that of ca12 in LS-shca9/ctl in normoxia
(Supplementary Table S2), whereas in hypoxia, the number of copies
of ca9 mRNA was only twice higher than that of ca12. Moreover,
when ca9 is suppressed in hypoxia, the number of ca12 mRNA copies
was almost similar to ca9 induced in hypoxia. In addition, when ca9
was silenced in hypoxia, the overall hypoxia-induced CA activity was
unchanged whereas ACTZ showed a marked reduction in acidification (Fig. 4B, top). When ca12 was silenced in hypoxia, no reduction
in extracellular acidification occurred (data not shown); however,
combined silencing of ca9 and ca12 in hypoxia (LS-shca9/ca12
+Tet) reduced the CA activity to the basal level (Fig. 4B, bottom).
We then determined the relative contributions of CAIX and CAXII
in hypoxia regarding pHi regulation. At a neutral pHo of 7.4, silencing
of ca9, ca12, or both together did not affect the resting pHi (Fig. 4C).
When cells were exposed to an acidic pHo of 6.0, only combined
invalidation of ca9 and ca12 resulted in a significantly lower resting
pHi (0.2 units), whereas silencing of either isoform alone had no
effect (Fig. 4C); results were obtained with two independent
sequences targeting ca12 (data not shown for the second sequence).
We then assessed the importance of this pHi regulating system in
hypoxia on cells grown as three-dimensional spheroids. In a
bicarbonate-limiting environment, a hypoxic gradient is established
(hypoxyprobe labeling; Fig. 4D, top) and, thus, lactic acid is
produced. When ca9 was silenced (LS-shca9/ctl +Tet) the proliferation index of hypoxic spheroids diminished compared with control
cells (LS-shca9/ctl Tet) and diminished further when both
isoforms were silenced (LS-shca9/ca12 +Tet; Fig. 4D, bottom).
These results indicate that both CAIX and CAXII play an important
role in the regulation of pHi recapitulating the protective effect of
hypoxia in promoting cell survival in an acidic environment.
Inducible invalidation of CAIX and CAXII reduces the rate
of xenograft tumor growth. To investigate the in vivo functional
consequence of CAIX and CAXII expression on tumor growth,
athymic mice were s.c. injected with Tet-inducible LS-shhif-1a,
LS-shca9/ctl, or LS-shca9/ca12 cells. Dox had no significant effect
on tumor growth of control cells (LS-shev/ctl; Fig. 5A, top), whereas
Dox silencing of hif-1a showed a substantial decrease in the size of
tumors (Fig. 5A, bottom). The constitutive silencing of ca12 in an

same level of activity (data not shown). The resting pHi of
PS120-pca9 cells, with the BCECF-AM dye, showed it to be more
alkaline compared with control PS120-pev cells when incubated
in a nominally HCO3 /CO2-free solution set at a pHo of 6.6 to
7.4 (Fig. 1B). The difference was more pronounced for a pHo of
6.6 (0.4 units) compared with 7.4 (0.15 units). In the presence of
25 mmol/L bicarbonate, no difference in pHi was observed
between CAIX-expressing cells and control cells. The intracellular
alkalinization associated with CAIX expression was confirmed in
PS120 (Fig. 1C) and CCL39 (Supplementary Fig. S2) cells with
another technique that uses [14C]benzoic acid, indicating that
CAIX expression protects cells against cytoplasmic acidification.
In the presence of endogenous NHE-1 (CCL39 cells), CAIX was
able to restore a more alkaline pHi in acidic environments. This
was more marked in fibroblasts impaired in NHE-1 because
CAIX was able to compensate for the lack of NHE-1 in
maintaining pHi in acidic environments. The implication of
bicarbonate transport in changes in pHi is shown by its
suppression in the presence of the bicarbonate transport
inhibitor DIDS (Fig. 1D). This shows that the function of CAIX
as a pHi regulator is revealed only in the absence of added
extracellular bicarbonate.
CAIX-mediated cytoplasmic alkalinization and cell survival
in an acidic environment. To examine if hypoxia-induced CAs
protect cells from extracellular acidosis, we assessed the effect of
forced expression of CAIX in PS120 and CCL39 cells on
proliferation, ATP level, and cell survival. Cell proliferation
determined by BrdUrd incorporation into DNA was significantly
increased in normoxic CAIX-expressing cells at low pHo (Fig. 2A
and Supplementary Fig. S3), as was the production of ATP (Fig. 2B
and Supplementary Fig. S4) after 24 h in the absence of CO2/HCO3 .
Cell colony formation was not significantly different when
comparing CAIX-expressing and control cells incubated at a pHo
of 7.4; however, it was substantially diminished in non–CAIXexpressing cells at a low pHo of 6.4 (Fig. 2C and Supplementary Fig.
S5). Stable PS120 clones expressing human CAXII were also
obtained and showed similar characteristics to those expressing
CAIX: extracellular acidification, conserved pHi regulation, and
increase in cell survival in acidic conditions (data not shown).
However, the CAXII activity was slightly lower compared with that
of CAIX. These results suggest that CAIX and CAXII, by maintaining
a more alkaline resting pHi, sustain ATP levels, promoting cell
survival in a bicarbonate-free acidic microenvironment.
Hypoxia-induced CAIX and CAXII activity contributes to
cytoplasmic alkalinization in an acidic microenvironment. A
number of human tumor cell lines showed an increase in the
number of copies of ca9 and ca12 mRNA and CAIX and CAXII
protein expression in response to hypoxia 1% O2, including RCC4,
HeLa, A375Tr, A549, and LS174Tr cells (Supplementary Table S1;
Supplementary Fig. S6). To further investigate the contribution of
both CAIX and CAXII, we chose to use the Tet-inducible LS174Tr
human colorectal adenocarcinoma cells to silence hif-1a with
shRNA. In LS-shhif-1a cells, comparable levels of expression of
hif-1a mRNA were detected in normoxia and hypoxia, confirming
absence of transcriptional regulation of hif-1a by oxygen. In
contrast, incubation of hypoxic LS-shhif-1a cells with Tet resulted
in a substantial decrease in the mRNA level of hif-1a (Fig. 3A, top).
Expression of ca9 and ca12 was significantly decreased in hypoxic
LS-shhif-1a cells when hif-1a was silenced (+Tet; Fig. 3A, bottom).
The LS174Tr cells endogenously express both membrane-bound

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Figure 6. Immunohistologic confirmation of the expression of HIF-1a, CAIX, or CAXII in hypoxic regions of the corresponding tumor xenografts. Mice were injected with
ca9-deficient LS-shca9/ctl cells (A ) or ca9 - and ca12 -deficient LS-shca9/ca12 cells (B) and drinking water supplemented without ( Dox ) or with (+Dox ) Dox
was provided to mice. Serial sections of tumors of the same size were stained for hypoxic regions with antibodies to hypoxyprobe (pimonidazole), for nuclear staining
of HIF-1a, or for membrane staining of CAIX or CAXII. Costaining of tumor sections was done first using the immunofluorescence protocol for CAIX followed by
immunohistochemistry for CAXII in the same tumor section of LS-shca9/ctl cells (C ) and LS-shca9/ca12 (D ). Magnification, 20. V, major blood vessel of the tumor
lobe analyzed in serial sections.

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CAIX and XII Promote Tumor Growth by Regulating pHi

CAXII in pHi regulation, it is necessary to operate in nominally
bicarbonate-free solutions exposed only to ambient CO2.
We also examined CAIX regulation of pHi in fibroblasts impaired
in NHE-1 expression (PS120 cells) to ensure the absence of
interference by this major player in pHi homeostasis (12, 36–38).
Nonetheless, the effect of CAIX on pHi was also detected in CCL39
fibroblasts and in a human colon adenocarcinoma cell line
LS174Tr expressing endogenous NHE-1. Previous studies showed
that NHE–1–deficient cells fail to grow in a range of acidic pHo
(6.2–6.8) due to their inability to reach the permissive pHi values
required for DNA synthesis and ATP production (6, 7, 39). Forced
CAIX expression in this pHi regulation–deficient cell system was
able to restore viability of PS120 cells when exposed to a range of
acidic pHo (6.2–6.8). This shows the role of a pHi-threshold value
for growth confirming the role of CAIX in pHi control.
It is postulated that the mechanism by which membrane-bound
CAs regulate pHi occurs through the efficient uptake of HCO3
locally formed in the ‘‘mouth’’ of CAs through Cl /HCO3
exchangers and/or Na+/HCO3 cotransporters, forming tight
functional complexes (40). Although the ‘‘metabolon’’ is an
interesting concept, this notion has been challenged (41–43).
Interaction of the catalytic domain of CAIX with bicarbonate
transporters has also been reported and was shown to increase the
AE exchanger activity (21, 44). Future investigation is under way to
evaluate the key bicarbonate transporters coupled to CAIX and
CAXII in pHi regulation in hypoxic tumor cells.
In our study, invalidation of CAIX leads to partial compensation by up-regulation of CAXII. This may explain the maintenance
of the catalytic activity of ca9 invalidated cells in hypoxia and
suggest that a threshold level of activity is required for cell pH
homeostasis. In the recent study of Swietach and colleagues (32),
overexpression of CAIX down-regulated cytosolic CAII. This result
suggests that different tissues with different expression patterns
of CAs may bring into play different CA isoforms that would
‘‘communicate’’ in a yet unresolved network when confronted
with an acidic stress.
Hypoxic induction may not be the only mechanism by which
CAs regulate pH homeostasis in tumors. Signaling through the
epidermal growth factor pathway by phosphorylation of a
cytoplasmic tyrosine residue of CAIX may either activate CAIX or
enhance its expression by increasing translation of HIF-1a (30). In
addition, phosphorylation activates phosphatidylinositol 3-kinase,
resulting in phosphorylation of Akt and cell survival. The possible
implication of these CAs as signaling molecules, independent of
their function as pH-regulating enzymes, is another important
point that is under investigation and may explain reduced xenograft growth of ca9 silenced cells despite maintenance of CA
catalytic activity.
It has been proposed that an acidic microenvironment promotes
metastasis associated with poor patient survival (4, 45). However,
acidosis may not always favor metastasis (4, 46). When renal
carcinoma cells were treated with ACTZ, their capacity to invade
was diminished (47) but the invasion of carcinoma cells was not
influenced by CAIX in another study (48). Here, we not only show
that CAIX and CAXII promote survival in an acidic environment in
two cell culture systems but extend it in in vivo studies. The
combined silencing of CAIX and CAXII gave a dramatic decrease in
the rate of growth of xenograft tumors that was greater than the
invalidation of HIF-1a, which may reflect the pleotropic action of
HIF-1 on prosurvival and prodeath genes. This may suggest that
the interest being paid to HIF inhibitors as anticancer treatments

endogenous ca9 background (LS-shca9/ca12
Dox) showed
similar tumor growth to that of the control cells (LS-shca9/ctl
Dox; Fig. 5B). However, the silencing of ca9, in the presence of
endogenous ca12 (LS-shca9/ctl +Dox), gave a slight but significant
reduction in tumor growth (40%) compared with control cells (Fig.
5C). Invalidation of both isoforms (LS-shca9/ca12 +Dox) resulted
in a spectacular decrease in tumor size (85%) as a result of slower
cell proliferation (Fig. 5C). Examination of the mRNA levels of ca9
and ca12 in the tumors confirmed almost complete invalidation of
ca9 in the invalidated cell lines when Dox was added (95% for LSshhif-1a, 94% for LS-shca9/ctl, and 92% for LS-shca9/ca12 ).
Whereas the expression of the ca12 mRNA was diminished by a
half in hif-1a invalidated cells (Figs. 4A and 5D), its expression
increased 1.5-fold when ca9 (LS-shca9/ctl +Dox) was silenced
(Fig. 5D). In the LS-shca9/ca12 cell line, the level of ca12 mRNA
was diminished (80%) whereas that of ca9 in the absence of Dox was
not significantly different. These results indicate that the respective
tumor types retain their invalidated phenotype and that the
expression of ca12 responds to the level of expression of ca9,
although the reverse is not the case.
Immunohistochemical and immunofluorescence analysis of
tumor sections from mice injected with LS-ca9/ctl (Fig. 6A and C)
and LS-ca9/ca12 cells (Fig. 6B and D) showed diminished levels of
CAIX in cells silenced for ca9 and for ca9 plus ca12 in Dox-treated
mice, despite the expression of HIF-1a in hypoxic zones (hypoxyprobe staining). CAXII expression is seen in all cells of the tumor
(slightly increased in the perinecrotic area) in contrast to the
expression of CAIX, which correlates with the hypoxic and
perinecrotic regions. It is important to note the increase in CAXII
expression when CAIX is silenced in vivo. Comparison of the
immunohistochemistry of tumor sections of control LS-shev/ctl cells
(Supplementary Fig. S8A) and hif-1a silenced LS-shhif-1a cells
(Supplementary Fig. S8B) revealed a significant decrease in HIF-1a,
CAIX, and CAXII in hif-1a silenced cells. For tumor histology, to
insure that the reduced labeling observed for CAIX in ca9/ca12
silenced tumors was indeed due to invalidation and not small-sized
tumors with minimal hypoxic zones, mice were maintained for a
longer time and sacrificed 27 days later than control mice when the
tumors were the same size as for control nonsilenced cells. Thus,
these results show that invalidation of both CAIX and CAXII brings
about a dramatic decrease in tumor xenograft cell growth.

Discussion
Investigation into the implication of hypoxia-inducible CAs in the
regulation of pH has concerned only CAIX and has been restricted
to monolayer or three-dimensional cell cultures (31, 32). During the
preparation of this manuscript, a study showed that ectopically
expressed CAIX in human bladder carcinoma RT112 cells was able
to spatially coordinate pHi, but only when cells are cultured as
three-dimensional spheroids (32). In agreement with this report,
CAIX expression in our study had no effect on pHi regulation in
isolated cells in a neutral and bicarbonate-buffered medium (25
mmol/L); however, when cells were exposed to a nominally
bicarbonate-free and acidic milieu, CAIX effected on the resting
pHi. Here, we conducted in vitro studies in the absence of
extracellular bicarbonate in order not to saturate bicarbonate
transporters at the cell surface. As we showed previously (6, 35), the
presence of a high bicarbonate level (25 mmol/L) totally blunts the
effect of NHE-1 on pHi regulation. Thus, we reasoned that, to
investigate the putative contribution of membrane-bound CAIX and

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Disclosure of Potential Conflicts of Interest

(49, 50) might be better directed to the inhibition of downstream HIF
target gene products, as we recently proposed (1) and, in particular
to CAIX together with CAXII, as novel and potentially efficient drug
approaches.
The present study highlights the role of CAIX and CAXII
expression in pHi regulation, a key event controlling cell viability,
and in in vivo tumor growth in a hostile acidic and hypoxic
microenvironment. The results herein call for the development of
specific ‘‘CA-antagonist’’ antibodies or cell impermeable drugs
specifically targeting the membrane-associated and hypoxiainducible CAs, taking into consideration the CA isoform profile
of a given tumor type. Such inhibitors are being actively
investigated at the molecular and cellular levels (23, 24) and may
hold promise as effective anticancer treatments.

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No potential conflicts of interest were disclosed.

Acknowledgments
Received 6/27/2008; revised 10/15/2008; accepted 11/8/2008.
Grant support: LNCC (Equipe labellisée), ANR, INCA, EUFP7 ‘METOXIA’, and
Canceropôle PACA. The laboratory is funded by Centre A. Lacassagne, Centre National
de la Recherche Scientifique, and Institut National de la Sante et de la Recherche
Medicale. J. Laferrière was a Research Fellow of the Terry Fox Foundation through an
award from the National Cancer Institute of Canada.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Dr. Wakabayashi of the National Cardiovascular Center Research
Institute, Japan, for assistance with pHi determination and Drs. Zavada, Pastorekova,
and Pastorek for providing the source of the M75 antibody to CAIX (Bayer).

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