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Proc Natl Acad Sci U S A. Oct 4, 2011; 108(40): 16663–16668.
Published online Sep 19, 2011. doi:  10.1073/pnas.1106123108
PMCID: PMC3189052
Cell Biology

CD147 subunit of lactate/H+ symporters MCT1 and hypoxia-inducible MCT4 is critical for energetics and growth of glycolytic tumors

Abstract

Malignant tumors exhibit increased dependence on glycolysis, resulting in abundant export of lactic acid, a hypothesized key step in tumorigenesis. Lactic acid is mainly transported by two H+/lactate symporters, MCT1/MCT4, that require the ancillary protein CD147/Basigin for their functionality. First, we showed that blocking MCT1/2 in Ras-transformed fibroblasts with AR-C155858 suppressed lactate export, glycolysis, and tumor growth, whereas ectopic expression of MCT4 in these cells conferred resistance to MCT1/2 inhibition and reestablished tumorigenicty. A mutant-derivative, deficient in respiration (res) and exclusively relying on glycolysis for energy, displayed low tumorigenicity. These res cells could develop resistance to MCT1/2 inhibition and became highly tumorigenic by reactivating their endogenous mct4 gene, highlighting that MCT4, the hypoxia-inducible and tumor-associated lactate/H+ symporter, drives tumorigenicity. Second, in the human colon adenocarcinoma cell line (LS174T), we showed that combined silencing of MCT1/MCT4 via inducible shRNA, or silencing of CD147/Basigin alone, significantly reduced glycolytic flux and tumor growth. However, both silencing approaches, which reduced tumor growth, displayed a low level of CD147/Basigin, a multifunctional protumoral protein. To gain insight into CD147/Basigin function, we designed experiments, via zinc finger nuclease-mediated mct4 and basigin knockouts, to uncouple MCTs from Basigin expression. Inhibition of MCT1 in MCT4-null, Basiginhigh cells suppressed tumor growth. Conversely, in Basigin-null cells, in which MCT activity had been maintained, tumorigenicity was not affected. Collectively, these findings highlight that the major protumoral action of CD147/Basigin is to control the energetics of glycolytic tumors via MCT1/MCT4 activity and that blocking lactic acid export provides an efficient anticancer strategy.

Keywords: Warburg effect, lactate transport, EMMPRIN, Embigin, cancer metabolism

Interest in the “Warburg effect” has been intensely revived in recent years (16). The fact that rapidly proliferating cancer cells have adopted fermentative glycolysis rather than oxidative phosphorylation (OXPHOS) to supply energy is not unique to cancer but is shared by highly proliferating normal cells. Recent work has highlighted an intimate coupling of PFKFB3, a key regulator of glycolysis, with the G1-phase cell cycle machinery (7). It is now realized that fermentative glycolysis, although generating a low yield of ATP per glucose molecule consumed, is the pathway of choice for cell division because it generates carbon source intermediates for anabolic reactions (3). 18FDG-PET scan imaging reveals the simplest cancer signature of intense proliferation, reflecting a sort of “glucose addiction” for cell division. In consequence of rapid growth, these tumors have to face a hypoxic and acidic microenvironment linked to a restricted and often chaotic vascularization (8). Through HIF-1 activation, hypoxic tumor cells have evolved adaptative mechanisms to survive nutrient-deprived conditions such as induction of autophagy via BNIPs, induction and mobilization of glycogen stores (9) and increased migration via EMT induction (10). Acidic burden is the price paid by intense metabolism, primarily lactic acid, as the obligatory byproduct of fermentative glycolysis. Although normal proliferating cells use glycolysis, some human tumor cells are so “addicted” to this metabolic pathway that targeting glycolysis directly has become a promising therapeutic strategy in oncology (1115).

In our studies we have focused on exploring the role of intracellular pH (pHi) as a therapeutic strategy to target glycolytic tumors (8, 16, 17). Within an acidic microenvironment, “successful” tumors cells are those capable of actively extruding protons. Cancer cells have acquired several solutions like activation of a robust Na+/H+ exchanger (18), the HIF-1 induction of membrane-bound carbonic anhydrases CAIX and XII (19), and the efficient export of lactic acid through the HIF-1 induction of monocarboxylate transporter 4 (MCT4) (20). Here, we have further explored a pHi-anticancer strategy by analyzing blockade of lactic acid export (21). Four MCT family members (MCT1–MCT4) have been functionally characterized as H+/lactate symporters capable of mediating bidirectional transport of lactic acid across the plasma membrane (22). MCT1 is widely expressed, whereas the expression of the hypoxia-inducible MCT4 lactate transporter tends to be restricted to “glycolytic” tissues such as muscle and increased expression has been reported in several malignancies (20). A key glycoprotein, CD147/Basigin/EMMPRIN assists MCT1 and MCT4 in folding, stability, membrane expression, and functionality (23). CD147/Basigin/EMMPRIN is an evolutionary conserved member of the Ig superfamily that has gained considerable attention before being identified as an MCT subunit (24, 25). It was first described as a potent protumoral factor capable of promoting tumor invasion (26) via the induction of matrix metalloproteinases (MMPs) and has been reported to be involved in multiple physiological functions including embryo implantation, spermatogenesis, and vision (27). Here, we addressed the following questions: Is CD147/Basigin's protumoral action linked or independent of its role in lactic acid export? Can these two actions be uncoupled? Can we suppress tumor growth by simply inhibiting MCTs? With the use of a specific MCT inhibitor (28) and inducible MCT1/4 shRNAs, we established that growth of glycolytic tumors can be arrested through inhibition of lactate transport. Inducible knockdown of CD147/Basigin was sufficient to phenocopy these antitumor effects. Finally, by a genetic approach, we succeeded in uncoupling MCT1/MCT4 inhibition from CD147/Basigin expression. We propose that the protumoral action of CD147/Basigin is due to its major action on energy metabolism by controlling lactic acid export. Taken together, these studies suggest that the hypoxia-regulated lactate transporter MCT4 and its ancillary protein CD147/Basigin are important anticancer targets for hypoxic and rapidly growing tumors.

Results

Pharmacological Inhibition of MCT1/2 Suppressed Glycolysis and Tumor Growth of Ras-Transformed Fibroblasts Not Expressing MCT4.

We first analyzed the expression of MCT1–MCT4 isoforms in the Ras-transformed fibroblast CCL39 cell line for which we possess mutants impaired either in glycolysis (CCL39-gly) (29) or in respiration (CCL39-res) (30). By quantitative RT-PCR and immunoblotting, we found that the CCL39 fibroblast series expressed only MCT1 (Fig. 1A) and MCT2 (Fig. S1A). Uptake of 14C-lactic acid is totally suppressed in CCL39 cells treated with the MCT1/2 inhibitor AR-C155858(28) but not if they express MCT4 (CCL39-mct4) (Fig. S1 AC). This experiment, as well as the intracellular lactate accumulation (Fig. 1F), confirmed the specificity of this inhibitor for MCT1 and MCT2 (31). The growth and survival (colony size and number, Fig. 1B, Left) of CCL39-ev cells were not affected by the MCT1/2 inhibitor in normoxia (N), but it was severely reduced in hypoxia 1% O2 (Hx) and abolished when cells are strictly relying on glycolysis in response to oligomycin treatment (N + oligo). Fig. 1B, Right shows that ectopic expression of MCT4 (CCL39-mct4) eliminated all effects of the MCT1/2 inhibitor in all of the conditions analyzed (hypoxia and oligomycin treatment). By inhibiting lactic acid export, AR-C155858 strongly reduced the rate of glycolysis in the three conditions tested (Fig. 1C, Left). This inhibition of glycolysis did not impact on cell growth in normoxia. In contrast, in hypoxia or in oligomycin-treated cells, inhibiting lactic acid export disrupted pHi control (Fig. S1D). As expected, CCL39 cells expressing MCT4 (CCL39-mct4; Fig. 1C, Right) became fully resistant to the MCT1/2 inhibitor and, interestingly, their rate of glycolysis increased, particularly in cells incubated with oligomycin or in hypoxia 1% O2. These findings on the MCT1/2 inhibitor action were fully supported by the growth features of the CCL39-derived metabolic mutants. In vitro growth of glycolysis-defective cells (CCL39-gly) is not affected by MCT1/2 inhibition in normoxia and hypoxia conditions, whereas the respiratory-deficient cells (CCL39-res), which rely only on glycolysis, do not grow and die in the presence of the MCT1/2 inhibitor (Fig. S2B).

Fig. 1.
Pharmacological inhibition of MCT1/2 of Ras-transformed fibroblasts expressing or not MCT4. Impact on glycolysis and tumor growth. (A) Total extracts from Ras-transformed fibroblasts CCL39-ev and CCL39-mct4 exposed to normoxia were analyzed by immunoblotting ...

The impact of MCT1/2 inhibition on tumor growth in vivo was assessed by using the Ras-transformed CCL39 fibroblasts (CCL39-ev) implanted in nude mice (Fig. 1D). In contrast to tumor control mice injected with PBS-Tween (PBS-T), tumors from mice receiving 30 mg/kg AR-C155858 (iMCT1/2) twice daily (b.i.d.) did not develop during the 6 d of treatment. At the end of treatment, tumors reestablished a similar growth rate to the untreated controls. Ectopic expression of MCT4 in the same cells (CCL39-mct4) had two major consequences: (i) increased tumorigenicity (shorter tumor latency/increased growth rate), and (ii) total insensitivity to MCT1/2 inhibition (Fig. 1E). The same experiment conducted with the glycolytic-deficient mutant expressing only MCT1/2 showed full insensitivity to the MCT1/2 inhibitor (Fig. S2C), highlighting that the antitumor effects seen with the MCT1/2 inhibitor (Fig. 1D) were not due to side nonspecific toxic effects. We conclude that tumors, from Ras-transformed fibroblasts, experienced a hypoxic microenvironment in vivo, rendering these cells glycolytic and sensitive to the lactic acid export inhibitor.

Export of Lactic Acid via MCT1 and MCT4 Is Crucial for Tumor Growth.

As we have seen above, tumor growth from Ras-transformed CCL39 fibroblasts that express only MCT1/2 was extremely sensitive to blockade of lactic acid export. The respiration-defective mutant (CCL39-res) that relies only on glycolysis for energy supply is poorly tumorigenic (low tumor incidence and elongated tumor latency; Fig. 2A). Ectopic expression of MCT4 in these cells (see immunohistochemistry in circle inserts; Fig. 2A) restored full tumorigenicity (100% tumor incidence and reduced latency from ≈62 to 20 d; Fig. 2A). Moreover the res cells cultivated in the presence of 10 nM MCT1/2 inhibitor (iMCT1) died in 24 h, a result expected from the lack of respiration in these cells. The very rare resistant clones (frequency <10−3) that survived were analyzed for possible gain of function by point mutation or amplification of the mct1 gene (Fig. S3). Two resistant clones (A and C) were isolated after incubation in serial increases (10–1,000-fold) in MCT1/2 inhibitor (Fig. S3A). These resistant clones did not show any mutation or amplification of the mct1 gene (SLC16A1). In contrast, these two resistant clones demonstrated de novo expression of the hamster mct4 gene (Fig. S3 B and C). When analyzed for tumor incidence, these highly glycolytic MCT1/2 inhibitor-resistant cells (res-iMCT1/2R) became highly tumorigenic (Fig. S3D), a result reflecting the reactivation of endogenous mct4 gene.

Fig. 2.
Key role of hypoxia-induced MCT4 in glycolysis and tumor growth. (A) Tumorigenicity in nude mice of Ras-transformed fibroblasts CCL39-mct4 and mutant impaired in respiration and expressing with (res-mct4) or without (res) MCT4. Cells ...

These two independent findings indicate that lactate export via MCT1/2 is limiting for growth of highly glycolytic cells and points to the protumoral advantage conferred by the expression of hypoxia-inducible MCT4, an isoform highly expressed in rapidly growing human tumors.

Next, we analyzed the human colon adenocarcinoma cell line LS174T that expresses both MCT1 and MCT4 in normoxia. This cell line exhibits a three- to fivefold increase in MCT4 mRNA and protein expression in hypoxia 1% O2 (Fig. 2D, compare lanes 1 and 5). Introduction of a tetracycline (Tet)-inducible shRNA targeting MCT1 (LS174T shmct1in) reduced MCT1 expression by 90% in to the presence of Tet (compare Fig. 2B, lanes 1 and 2). This single MCT1 knockdown did not affect the rate of glycolysis, nor the rate of tumor growth (Fig. 2C). The infection of these LS174T shmct1in cells with a lentivirus shRNA targeting MCT4 (shmct4) strongly reduced expression of MCT4 (compare shctl with shmct4 in Fig. 2B). It is only by achieving a double MCT1/4 knockdown (shmct1in/shmct4; Fig. 2B, lane 4) that we could reduce the rate of xenograft tumor growth (double KD mct1, mct4; Fig. 2C). Collectively these results highlight the key role played by the two H+/lactate symporters and particularly the hypoxia-inducible MCT4 in optimizing the rate of glycolysis associated with rapid tumor growth.

Another interesting feature of this double MCT1/4 knockdown is the concomitant reduction in the accessory protein CD147/Basigin (Fig. 2D, compare lanes 1 and 4 in normoxia and lanes 5 and 8 in hypoxia for total expression of Basigin). Cell surface expression of Basigin was reduced by 26% (MCT4 knockdown) to 47% (MCT1/4 double knockdown; Fig. 2E, shmct4 + Tet). These findings prompted us to evaluate whether direct targeting of CD147/basigin could act as a simple strategy to reduce expression of both MCT1 and MCT4.

Knockdown of CD147/Basigin Reduces MCT1 and MCT4 Expression, Glycolysis, and Tumor Growth.

LS174T cells were stably transfected with a Tet-inducible shRNA targeting Basigin (LS174T shbsgin) and the total expression of Basigin, MCT1, and MCT4 in response to tetracycline (+Tet and −Tet) in either normoxic or hypoxic conditions was analyzed by immunoblotting (Fig. S4A). The expression level of the upper glycosylated form of Basigin reproducibly increases in hypoxia (compare Fig. 2D, lanes 1 and 5 and compare Fig. S4A, lanes 1 and 3). This hypoxia-induced Basigin is not associated with an increase in mRNA, and we propose that this Basigin increase is an indirect effect of MCT4 up-regulation. In support of this conclusion, MCT4 is induced by hypoxia, whereas MCT1 remains unchanged (Fig. 2D and Fig. S4A). Knockdown of Basigin (+Tet; Fig. S4A), reduced the cell surface expression of Basigin by 64% (Fig. S4B). Furthermore Basigin knockdown resulted in a pronounced reduction in MCT1 and MCT4 expression in normoxia and hypoxia (compare Fig. S4A, lanes 1 and 2 or lanes 3 and 4). This observation was reproduced with three independent shRNA sequences against CD147/Basigin with a strong correlation between the level of Basigin ablation and concomitant reduction in MCT1 and MCT4 expression (Fig. S5A). Knockdown of Basigin reduced the glycolytic rate by 50% (Fig. S4C, compare −Tet and +Tet); this effect was further enhanced in the presence of the MCT1/2 inhibitor (to ≈75% control levels; Fig. S4C). Furthermore, knockdown of Basigin results in an increase in intracellular lactate (Fig. S4F), indicating that the reduced glycolytic capacity is a direct effect of loss of lactate transport. This reduced glycolytic capacity results in a significant antiproliferative effect, following Basigin knockdown (+Tet) (Fig. S4D, Fig. S4D Inset, and Fig. S5B) and a striking reduction in xenograft tumor growth in vivo as seen in Fig. S4E and Fig. S5C (compare −Dox and +Dox). Taken together, these findings reinforce the notion that blunting export of lactic acid has a profound anticancer action seen here in colon adenocarcinoma and reported in pancreatic cancer (32).

Major Protumoral Action of CD147/Basigin Is Mediated Through the Control of Lactic Acid Export.

The antitumor efficacy demonstrated in Fig. S4E and Fig. S5C are mediated by direct silencing of CD147/Basigin. Is the protumoral activity of CD147/Basigin intimately linked to its lactic acid export function and, therefore, associated to metabolic energy? Can we uncouple the metabolic function of CD147/Basigin from its widely recognized protumoral and invasive function? To answer these questions, we designed two experiments by using the zinc finger nuclease (ZFN) targeting approach (33) (Fig. 3 A and D). First, we disrupted the mct4 gene in the LS174T shmct1in cell line (Fig. 3A). In this cell line, referred to as mct4−/−, the ablation of MCT4 did not modify the total expression levels of either MCT1 or CD147/Basigin in normoxia (Fig. S6A), whereas in hypoxia, CD147/Basigin increased ≈30–40% to accommodate the HIF1-induced expression of MCT4 in control cells only (Fig. S6A, Hx). In vitro growth rates of control and mct4−/− cells in normoxia (Fig. S6C) or in hypoxia (Fig. S6D) were identical. However, pharmacological inhibition of MCT1/2, which did not impact on cell surface expressed Basigin (71%, Fig. S6B), did not affect the in vitro growth of control cells in hypoxic conditions but dramatically reduced the growth, the rate of glycolysis, and the tumorigenicity of mct4−/− cells (Fig. S6 DF). To further increase cell surface Basigin, we expressed in mct4−/− cells, MCT4mut-L40R, a catalytically inactive MCT4 form based on MCT1 conservation of Lysine38 (Fig. S7 and Fig. 3B), a key residue for lactate transport (34). The lactate transport experiment demonstrated that MCT4mut-L40R expressed on the plasma membrane, as judged by the induced expression of Basigin, is catalytically dead (compare Fig. S7, a and c). Silencing MCT1 reduces Basigin expression in control cells (Fig. 3B, lane 2), whereas Basigin was maintained high in MCT4mut-L40R cells (compare Fig. 3B, lanes 2 and 4). Interestingly the decreased tumorigenicity correlated with low MCT activity (+Dox; Fig. 3C) but remained independent of the Basigin expression level (Fig. 3 B and C). In the second experiment, we knockout basigin in a LS174T-derivative clone expressing rat Embigin (Fig. 3D). Embigin is a paralog of Basigin that, with the exception of the conserved transmembrane segment, shares <30% homology. Furthermore, unlike Basigin, Embigin has not been reported as a protumoral protein and does not mediate cell aggregation by homophilic interactions, suggesting different modes of function for the two (35). Interestingly, Embigin maintained expression of MCTs in basigin-knockout cells (compare Fig. 3E, lanes 2 and 4) and more importantly, ablation of Basigin did not affect the rate of tumorigenicity (Fig. 3F).

Fig. 3.
ZFN-mediated mct4 and basigin gene knockouts and uncoupling MCT1 and MCT4 activity from Basigin expression. (A) Knockout of mct4 in LS174T cell line via ZFN gene targeting. Schematic representation of the membrane organization of the MCT4 lactic acid ...

These are key experiments demonstrating that we can disable tumor growth of the colon adenocarcinoma cell line LS174T simply by directly blocking lactic acid export while maintaining a high level of CD147/Basigin. Conversely, as long as MCT activity is sustained, Basigin ablation does not suppress the tumoral potential. We therefore conclude that a major protumoral action of CD147/Basigin is mediated by its direct action in energy metabolism by controlling the export of lactic acid in glycolytic tumors.

Discussion

Our previous findings on the disruption of the Na+/H+ exchanger (16, 36), a universal pHi-regulating system from bacteria to man, lead us to propose that disabling pHi control mechanisms could represent an alternative anticancer approach, particularly to highly proliferating, hypoxic, and glycolytic tumors. We proposed that HIF-1–mediated pHi control is a key determinant for cellular adaptation, tumor survival, and progression in a hypoxic microenvironment. We hypothesized that carbonic anhydrase IX (CAIX) and the H+/lactate symporter MCT4, induced by HIF-1, should be considered as two prominent candidates for pHi regulation and promising targets for anticancer strategies (8). The first target, CAIX, characterized by a very restricted pattern of tissue expression, is hijacked in many malignancies (37), and recent work from several groups has validated the hypothesis whereby CAIX controls pHi by accelerating H+ extrusion and promoting tumor growth (19, 38) and metastasis (39). For the second target, we recently demonstrated by monitoring tumor pHi in vivo by 31P-NMR spectrometry that tumors expressing MCT4 had a more alkaline pHi and lower external pH (pHe) than tumors expressing MCT1/2 only (40). We concluded that MCT4, which is an efficient lactate exporter, should contribute to increased glycolysis and tumor growth rate in a hostile acidic microenvironment.

Here, we reported that pharmacological inhibition of MCT1/2 with AR-C155858 of Ras-transformed fibroblasts that express MCT1/2 only, induced intracellular lactate pool, reduced glycolysis and growth in hypoxia, and prevented tumor growth in nude mice during the time of treatment. In contrast, this compound was unable to affect tumor growth of cells defective in glycolysis that use only OXPHOS (gly). Furthermore, in this and other unpublished studies, we have observed that cell lines that coexpress MCT4 (mouse embryonic stem cells, MEFs, res-MCT4, LS174T, MDA-MB-231) are insensitive to the MCT1/2 inhibitor in either lactate efflux or cell growth endpoints. These results confirm the specificity of AR-C155858 for MCT1 and MCT2, demonstrate that the compound is well-tolerated in mice at 30 mg/kg b.i.d. (no weight loss), and demonstrate that blocking lactic acid export blunts the growth of hypoxic glycolytic tumors. It is important to note that highly glycolytic cells mutated in OXPHOS (res), expressing only MCT1/2 and that display a very low tumorigenic potential, rapidly developed resistance in vitro to increasing concentrations (from 0.01 mM to 10 mM) of AR-C155858. The 1,000-fold acquired resistance was caused by the de novo expression of endogenous MCT4. These resistant cells, defective in respiration, now became highly tumorigenic (100% tumor incidence, reduced latency) like in res cells in which we ectopically expressed the human MCT4. This simple experiment highlights the unique protumoral role that MCT4 could exert in highly glycolytic tumors. Clearly, MCT4 with its relative low affinity for l-lactate (Km of 20–30 mM) but extremely low affinity for pyruvate (Km of 150 mM) appears to be exclusively designed for lactate efflux (41, 42). It is therefore not surprising that MCT4 expression emerges in most of the human aggressive malignancies representing a poor prognostic marker. In contrast, MCT1 and MCT2, which respectively have a 5- and 20-fold higher affinity for lactate and a very high affinity for pyruvate, are mostly appropriate for both lactate efflux/influx (MCT1) or lactate/pyruvate recapture only (MCT2) (41).

As yet no pharmacological inhibitor exists for MCT4 despite its close sequence homology to MCT1 and MCT2. We therefore investigated their respective roles by specific knockouts or shRNA silencing of MCT4, MCT1, and CD147/Basigin in the colon adenocarcinoma LS174T. Silencing of CD147/Basigin, which induced a concomitant down-regulation of MCT1 and MCT4 expression, had a profound effect in reducing tumorigenicity of LS174T cells. This study confirmed and extended the profound effect of CD147 silencing on the lactate transporters MCT1 and MCT4 and tumor growth recently reported for pancreatic cancer (32). The reciprocal loss of CD147/Basigin by cosilencing MCT1 and MCT4 would suggest that the major role of CD147/Basigin in cells is the cochaperoning of MCT1 and MCT4, an action going in both directions as suggested (43).

Before being recognized as a MCT subunit, CD147/Basigin has been reported as a key protumoral cell surface glycoprotein. Forced expression of CD147 in breast cancer cells resulted in increased tumor growth and metastasis, an action correlated with its capacity to promote extracellular matrix metalloproteinase induction (alternative name EMMPRIN) and to modify the tumor microenvironment (26). This tumor invasive capacity was also associated more recently to increased angiogenesis via Basigin-mediated VEGF induction. The profound antitumoral effects induced by CD147/Basigin silencing raises the question of whether the effects seen are reflecting the impairment of energy metabolism by inhibition of lactate export or of the protumoral action initially reported for Basigin or both. Because of the interdependency of CD147/MCTs for plasma membrane expression, it is not easy to uncouple the expression/function of both proteins. However, we could provide an answer to this question with the two tumor models we reported in this study. The Ras-transformed CCL39 cells that express only MCT1 and MCT2 are extremely sensitive to the lactic acid transporter inhibitor (iMCT1/2) that fully blocks lactic transport (Fig. S1B), glycolysis, and xenograft tumor (Fig. 1 C and D), yet the level of the CD147/Basigin remained unchanged (Fig. 1D Inset). In the colon adenocarcinoma cell line, the knockout of the mct4 gene reduced cell surface expression of CD147/Basigin by only 29%. This mutant cell line, with a fully functional MCT1, was not affected for in vitro growth in normoxia or hypoxia 1% O2 (Fig. S6 C and D). However, the pharmacological inhibition of MCT1/2 in these mct4-null cells abolished lactic acid export and proliferation under conditions where the expression level of the glycosylated CD147/Basigin remained unchanged (Fig. S6D, Right). Finally, forced expression of a catalytically dead MCT4 (K40R) that ensured full maintenance of Basigin surface expression did not affect the reduced tumorigenicity when MCT activity was silenced (Fig. 3 B and C). Conversely, we succeeded in embigin-LS174T cells to maintain MCT activity and full tumorigenicity despite the genetic deletion of Basigin (Fig. 3 E and F). We therefore conclude that the major protumoral action of CD147/Basigin is mediated by its cardinal function of chaperoning MCTs, an action central in energy metabolism particularly in highly glycolytic tissues and aggressive malignancies. The question whether CD147/Basigin has additional protumoral functions, particularly through its interaction with signaling integrins, CD98/LAT1 complex or through promotion of MMPs in the acidic tumor microenvironment, remains an open question.

Increased glycolysis and cell migration go together in embryonic development, wound healing, and metastasis. In this context, it is interesting that the Basigin/MCT4 complex has been found to associate with integrin β1 and localize at the leading edge of migrating cells, an area very active for invasive growth and also very demanding in metabolic energy (44). More importantly Philp et al. (45) reported that silencing MCT4 but not MCT1 in retinal pigment epithelial cells slowed down their migration, a process highly controlled by pH. The key role of the H+/Lactate symporter MCT4 in promoting pHi regulation (40) and glycolysis would suggest a strong functional link between glycolysis and migration via the Basigin/MCT/integrin complex. On the same line, we would like to propose that many of the multiples functions previously attributed to Basigin/EMMPRIN and emphasized with bsg−/− mice (27) are tightly linked to its major role in metabolic energy.

In conclusion, CD147/Basigin, by controlling the stability and functional plasma membrane insertion of the H+/lactate symporters MCTs in many tissues, plays a determinant role in metabolic energy. Pharmacological agents able to disrupt theses complexes Basigin/MCTs or to inhibit MCT1 and/or MCT4, which are prominently expressed in glycolytic, aggressive and rapidly proliferating malignancies, would provide a unique avenue for anticancer therapy.

Materials and Methods

Cell Culture and Hypoxic Exposure.

Chinese hamster lung CCL39 fibroblasts (American Type Culture Collection) and the CCL39-derived mutants impaired either in respiration (res) or in glycolysis (gly), obtained as described (29, 30), were maintained in DMEM (Sigma) supplemented with 7.5% FCS, penicillin (10 units/mL) and streptomycin (10 μg/mL) in a humidified atmosphere of 5% CO2/95% air or 100% air at 37 °C. The colon carcinoma LS174T cells provided by Marc van de Wetering (Utrecht) expressing the tetracyclin (Tet) repressor were maintained in DMEM supplemented with 10% inactivated FCS and blasticidin (10 μg/mL; InvivoGen). Incubation in hypoxia at 1% O2 (Hx) was carried out at 37 °C in 95% humidity and 5% CO2/94% N2 in a sealed anaerobic workstation (Ruskinn).

See SI Materials and Methods for additional details.

Supplementary Material

Supporting Information:

Acknowledgments

We thank Prof. Andrew Halestrap for providing the vectors expressing human MCT4 and rat Embigin and Dr. Natalya Merezhinskaya for providing high affinity MCT4 antibodies. R.L.F., J.C., and T.N. were supported in part by the “METOXIA” and Agence Nationale de la Recherche grants. The laboratory received funds from the Ligue Nationale Contre le Cancer (Equipe labellisée), a European Union-Seventh Framework Programme METOXIA grant, the Association pour la Recherche contre le Cancer, the Institut National du Cancer, the Agence Nationale pour la Recherche, the Centre National de la Recherche Scientifique, the Centre A. Lacassagne, and the University of Nice.

Footnotes

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1106123108/-/DCSupplemental.

References

1. Gatenby RA, Gillies RJ. A microenvironmental model of carcinogenesis. Nat Rev Cancer. 2008;8:56–61. [PubMed]
2. Kroemer G, Pouyssegur J. Tumor cell metabolism: Cancer's Achilles’ heel. Cancer Cell. 2008;13:472–482. [PubMed]
3. Vander Heiden MG, Cantley LC, Thompson CB. Understanding the Warburg effect: The metabolic requirements of cell proliferation. Science. 2009;324:1029–1033. [PMC free article] [PubMed]
4. Kaelin WG, Jr, Thompson CB. Q&A: Cancer: Clues from cell metabolism. Nature. 2010;465:562–564. [PubMed]
5. Semenza GL. HIF-1: Upstream and downstream of cancer metabolism. Curr Opin Genet Dev. 2010;20:51–56. [PMC free article] [PubMed]
6. Tennant DA, Durán RV, Gottlieb E. Targeting metabolic transformation for cancer therapy. Nat Rev Cancer. 2010;10:267–277. [PubMed]
7. Tudzarova S, et al. Two ubiquitin ligases, APC/C-Cdh1 and SKP1-CUL1-F (SCF)-beta-TrCP, sequentially regulate glycolysis during the cell cycle. Proc Natl Acad Sci USA. 2011;108:5278–5283. [PMC free article] [PubMed]
8. Pouysségur J, Dayan F, Mazure NM. Hypoxia signalling in cancer and approaches to enforce tumour regression. Nature. 2006;441:437–443. [PubMed]
9. Brahimi-Horn MC, Bellot G, Pouysségur J. Hypoxia and energetic tumour metabolism. Curr Opin Genet Dev. 2011;21:67–72. [PubMed]
10. Thiery JP, Acloque H, Huang RY, Nieto MA. Epithelial-mesenchymal transitions in development and disease. Cell. 2009;139:871–890. [PubMed]
11. Fantin VR, St-Pierre J, Leder P. Attenuation of LDH-A expression uncovers a link between glycolysis, mitochondrial physiology, and tumor maintenance. Cancer Cell. 2006;9:425–434. [PubMed]
12. Christofk HR, et al. The M2 splice isoform of pyruvate kinase is important for cancer metabolism and tumour growth. Nature. 2008;452:230–233. [PubMed]
13. Spoden GA, et al. Isotype-specific inhibitors of the glycolytic key regulator pyruvate kinase subtype M2 moderately decelerate tumor cell proliferation. Int J Cancer. 2008;123:312–321. [PubMed]
14. Sonveaux P, et al. Targeting lactate-fueled respiration selectively kills hypoxic tumor cells in mice. J Clin Invest. 2008;118:3930–3942. [PMC free article] [PubMed]
15. Le A, et al. Inhibition of lactate dehydrogenase A induces oxidative stress and inhibits tumor progression. Proc Natl Acad Sci USA. 2010;107:2037–2042. [PMC free article] [PubMed]
16. Pouyssegur J, Franchi A, Pages G. pHi, aerobic glycolysis and vascular endothelial growth factor in tumour growth. Novartis Found Symp. 2001;240:186–196. and discussion 196–198. [PubMed]
17. L'Allemain G, Paris S, Pouysségur J. Growth factor action and intracellular pH regulation in fibroblasts. Evidence for a major role of the Na+/H+ antiport. J Biol Chem. 1984;259:5809–5815. [PubMed]
18. Counillon L, Pouysségur J. The expanding family of eucaryotic Na(+)/H(+) exchangers. J Biol Chem. 2000;275:1–4. [PubMed]
19. Chiche J, et al. Hypoxia-inducible carbonic anhydrase IX and XII promote tumor cell growth by counteracting acidosis through the regulation of the intracellular pH. Cancer Res. 2009;69:358–368. [PubMed]
20. Ullah MS, Davies AJ, Halestrap AP. The plasma membrane lactate transporter MCT4, but not MCT1, is up-regulated by hypoxia through a HIF-1alpha-dependent mechanism. J Biol Chem. 2006;281:9030–9037. [PubMed]
21. Chiche J, Le Floch R, Roux D, Pouysségur J. The monocarboxylate transporter 1 (MCT1) and Hypoxia-induced MCT4 are key targets promoting tumor cell surviva. Eur J Cancer, Suppl. 2008;6:24.
22. Halestrap AP, Meredith D. The SLC16 gene family-from monocarboxylate transporters (MCTs) to aromatic amino acid transporters and beyond. Pflugers Arch. 2004;447:619–628. [PubMed]
23. Kirk P, et al. CD147 is tightly associated with lactate transporters MCT1 and MCT4 and facilitates their cell surface expression. EMBO J. 2000;19:3896–3904. [PMC free article] [PubMed]
24. Weidle UH, Scheuer W, Eggle D, Klostermann S, Stockinger H. Cancer-related issues of CD147. Cancer Genomics Proteomics. 2010;7:157–169. [PubMed]
25. Kennedy KM, Dewhirst MW. Tumor metabolism of lactate: The influence and therapeutic potential for MCT and CD147 regulation. Future Oncol. 2010;6:127–148. [PMC free article] [PubMed]
26. Biswas C, et al. The human tumor cell-derived collagenase stimulatory factor (renamed EMMPRIN) is a member of the immunoglobulin superfamily. Cancer Res. 1995;55:434–439. [PubMed]
27. Igakura T, et al. A null mutation in basigin, an immunoglobulin superfamily member, indicates its important roles in peri-implantation development and spermatogenesis. Dev Biol. 1998;194:152–165. [PubMed]
28. Murray CM, et al. Monocarboxylate transporter MCT1 is a target for immunosuppression. Nat Chem Biol. 2005;1:371–376. [PubMed]
29. Pouysségur J, Franchi A, Salomon JC, Silvestre P. Isolation of a Chinese hamster fibroblast mutant defective in hexose transport and aerobic glycolysis: Its use to dissect the malignant phenotype. Proc Natl Acad Sci USA. 1980;77:2698–2701. [PMC free article] [PubMed]
30. Franchi A, Silvestre P, Pouysségur J. A genetic approach to the role of energy metabolism in the growth of tumor cells: Tumorigenicity of fibroblast mutants deficient either in glycolysis or in respiration. Int J Cancer. 1981;27:819–827. [PubMed]
31. Ovens MJ, Davies AJ, Wilson MC, Murray CM, Halestrap AP. AR-C155858 is a potent inhibitor of monocarboxylate transporters MCT1 and MCT2 that binds to an intracellular site involving transmembrane helices 7-10. Biochem J. 2010;425:523–530. [PMC free article] [PubMed]
32. Schneiderhan W, et al. CD147 silencing inhibits lactate transport and reduces malignant potential of pancreatic cancer cells in in vivo and in vitro models. Gut. 2009;58:1391–1398. [PubMed]
33. Santiago Y, et al. Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases. Proc Natl Acad Sci USA. 2008;105:5809–5814. [PMC free article] [PubMed]
34. Wilson MC, Meredith D, Bunnun C, Sessions RB, Halestrap AP. Studies on the DIDS-binding site of monocarboxylate transporter 1 suggest a homology model of the open conformation and a plausible translocation cycle. J Biol Chem. 2009;284:20011–20021. [PMC free article] [PubMed]
35. Lain E, et al. A novel role for embigin to promote sprouting of motor nerve terminals at the neuromuscular junction. J Biol Chem. 2009;284:8930–8939. [PMC free article] [PubMed]
36. Pouysségur J, Sardet C, Franchi A, L'Allemain G, Paris S. A specific mutation abolishing Na+/H+ antiport activity in hamster fibroblasts precludes growth at neutral and acidic pH. Proc Natl Acad Sci USA. 1984;81:4833–4837. [PMC free article] [PubMed]
37. Pastorekova S, Kopacek J, Pastorek J. Carbonic anhydrase inhibitors and the management of cancer. Curr Top Med Chem. 2007;7:865–878. [PubMed]
38. Swietach P, et al. Tumor-associated carbonic anhydrase 9 spatially coordinates intracellular pH in three-dimensional multicellular growths. J Biol Chem. 2008;283:20473–20483. [PubMed]
39. Lou Y, et al. Targeting tumor hypoxia: Suppression of breast tumor growth and metastasis by novel carbonic anhydrase IX inhibitors. Cancer Res. 2011;71:3364–3376. correction (2011) 71:4733. [PubMed]
40. Chiche J, et al. In vivo pH in metabolic-defective Ras-transformed fibroblast tumors: Key role of the monocarboxylate transporter, MCT4, for inducing an alkaline intracellular pH. Int J Cancer. 2011 in press 10.1002/ijc.26125. [PubMed]
41. Manning Fox JE, Meredith D, Halestrap AP. Characterisation of human monocarboxylate transporter 4 substantiates its role in lactic acid efflux from skeletal muscle. J Physiol. 2000;529:285–293. [PMC free article] [PubMed]
42. Dimmer KS, Friedrich B, Lang F, Deitmer JW, Bröer S. The low-affinity monocarboxylate transporter MCT4 is adapted to the export of lactate in highly glycolytic cells. Biochem J. 2000;350:219–227. [PMC free article] [PubMed]
43. Gallagher SM, Castorino JJ, Wang D, Philp NJ. Monocarboxylate transporter 4 regulates maturation and trafficking of CD147 to the plasma membrane in the metastatic breast cancer cell line MDA-MB-231. Cancer Res. 2007;67:4182–4189. [PubMed]
44. Gallagher SM, Castorino JJ, Philp NJ. Interaction of monocarboxylate transporter 4 with beta1-integrin and its role in cell migration. Am J Physiol Cell Physiol. 2009;296:C414–C421. [PMC free article] [PubMed]
45. Stock C, Schwab A. Role of the Na/H exchanger NHE1 in cell migration. Acta Physiol (Oxf) 2006;187:149–157. [PubMed]

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