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 membrane-bound CAIX and CAXII that are controlled via the hypoxia-inducible factor (HIF). Despite the recognition that tumor expression of HIF-1α 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, hypoxia-induced CAIX and CAXII are major tumor prosurvival pHi-regulating enzymes, and their combined targeting shows that they hold potential as anticancer targets. [Cancer Res 2009;69(1):358–68]
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 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 membrane-associated 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-1α 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 shown (23, 24), we validate here that CAIX and CAXII constitute two new anticancer therapeutic targets.
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 37°C. 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 37°C in 95% humidity and 5% CO2/94% N2 in a sealed anaerobic workstation (Ruskinn).
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-1α (shhif-1α) 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).
Stable Transgenic Cells
CCL39, PS120, and LS174Tr cells were transfected with pca9, whereas only LS174Tr cells were transfected with shca9 or shhif-1α, using Polyfectamine (HiPerFect Transfection Reagent, Qiagen) according to the manufacturer's instructions. Isolated clones were maintained under zeocin (500 μg/mL, Invitrogen). Tet (10 μg/mL) induces CAIX expression or ca9 or hif-1α 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.
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 μg) was added to a 20 μL 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.
Cells were lysed in SDS sample buffer. Proteins (40 μg) 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-1α 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).
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×).
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 μmol/L acetazolamide (ACTZ; Sigma) was added to the cell suspension in normoxia 15 min before the experiment.
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 μmol/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 μmol/L nigericin to allow pHi to equilibrate with the external pHo.
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 μmol/L).
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.
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).
Cell Proliferation in Three Dimensions
To grow spheroids, 1,600 cells were seeded in drops in 20 μL 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 were dissociated in Accutase (Life Technologies) to determine the number of individualized living cells.
Nude Mice Tumorigenicity and Immunohistochemistry
Cells (1 × 106) suspended in 500 μL 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 μg/mL; Sigma) was given in the drinking water to induce silencing of hif-1α or ca9. Five mice were used for each experimental condition. The tumor volume was determined using the formula: (4π / 3) × L / 2 × W / 2 × H / 2 (L, length; W, width; H, height). When the tumor volume reached ∼1,500 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×).
The Student's t test was used wherein P values of <0.05 were considered significant.
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 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 higher for CAIX-expressing PS120 cells (Fig. 1A) and CAIX-expressing CCL39 cells (Supplementary Fig. S1) than for control cells (pev). The CA inhibitor ACTZ reduced the activity of CAIX-expressing 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 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–CAIX-expressing 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-1α with shRNA. In LS-shhif-1α cells, comparable levels of expression of hif-1α mRNA were detected in normoxia and hypoxia, confirming absence of transcriptional regulation of hif-1α by oxygen. In contrast, incubation of hypoxic LS-shhif-1α cells with Tet resulted in a substantial decrease in the mRNA level of hif-1α (Fig. 3A,, top). Expression of ca9 and ca12 was significantly decreased in hypoxic LS-shhif-1α cells when hif-1α was silenced (+Tet; Fig. 3A,, bottom). The LS174Tr cells endogenously express both membrane-bound 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 hypoxia-inducible 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-1α, 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-1α showed a substantial decrease in the size of tumors (Fig. 5A,, bottom). The constitutive silencing of ca12 in an 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 LS-shhif-1α, 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-1α 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-1α 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-1α silenced LS-shhif-1α cells (Supplementary Fig. S8B) revealed a significant decrease in HIF-1α, CAIX, and CAXII in hif-1α 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.
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 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-1α (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-1α, 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 (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 hypoxia-inducible 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.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).
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).