Cancer cells show constitutive upregulation of glycolysis, and the concentration of lactate thus produced correlates with prognosis. Here, we examined whether lactate concentration and lactate transporter expression are related to migration and invasion activity. We found that the expression of the monocarboxylate transporters MCT1 and MCT4, but not MCT5, in human lung cancer cell lines was significantly correlated with invasiveness. To clarify the effects of MCT1 and MCT4 expression on invasion, we performed migration and invasion assays after transfection with siRNA specific for MCT1 or MCT4. Knockdown of MCT1 or MCT4 did not influence cell migration but reduced invasion; this was also observed for knockdown of the lactate transporter-associated protein basigin. We also demonstrated that both expression and activity of MMP9 and MMP2 were not correlated with invasion activity and not regulated by MCT1, MCT4 and basigin. Furthermore, the addition of lactate did not increase migration and invasion activity, but low concentration of 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS), a general anion channel blocker, as well as other MCT inhibitors quercetin and simvastatin, inhibited cell invasion without influencing migration activity and the cellular expression of MCT1 and MCT4. This is the first report suggesting that lactate transporters are involved in human cancer cell invasiveness. As such, these proteins may be promising targets for the prevention of cancer invasion and metastasis. (Cancer Sci 2011; 102: 1007–1013)
Monocarboxylates, such as lactate and pyruvate, play a central role in cellular metabolism and metabolic communication between tissues.(1) In cancer cells, a steady source of metabolic energy is required to continue the uncontrolled growth and proliferation of these cells.(2) Most cancer cells rely on a high rate of aerobic glycolysis, a phenomenon termed “the Warburg effect”, to obtain sufficient ATP in a hypoxic microenvironment.(3) As a result of the Warburg effect, lactate is abundantly synthesized from pyruvate,(4) but lactic acid induces cellular acidosis, which triggers apoptosis. To avoid apoptosis, cancer cells must transport the lactate out of the cell. On the other hand, lactate is not just a waste product: it was recently identified as a major energy fuel in tumors.(4) Lactate is transported by monocarboxylate anion transporters (MCT; also called the solute carrier family 16 [SLC16]).(1) It is known that MCT4 (SLC16A3) transports lactate out of the cell(5) and MCT1 (SLC16A1) regulates the entry of lactate into tumor cells.(4)
Migration and invasion are two of the most important aspects of the malignant cancer phenotype; if they could be inhibited, the cancer prognosis would improve. Hypoxia and acidosis create a nurturing environment for tumor progression and the evolution of metastases, and invasiveness is abetted by acidosis, the result of shifting to an anaerobic glycolytic metabolism.(6) Basigin (BSG; also called EMMPRIN and CD147) is a multifunctional glycoprotein that can modify the tumor microenvironment by activating proteinases, inducing angiogenic factors in tumor and stromal cells. It also regulates the growth and survival of anchorage-independent tumor cells (micrometastases) and regulates multidrug resistance.(7,8) Basigin also modulates MMP and cancer progression,(9,10) and is a potential therapeutic target for metastatic prostate cancer.(11) It has been reported that BSG is tightly associated with the lactate transporters MCT1 and MCT4.(12) Accumulation of lactate within tumors has been correlated with poor clinical outcome.(13) However, the implications and consequences of lactate utilization by tumors are currently unknown. In this study, we found that cellular expression levels of MCT1 and MCT4 correlated with invasion activity. Although many factors are involved in tumor invasion, to our knowledge this is the first study to show that MCT expression is associated with tumor invasion, and that MCT may provide novel therapeutic targets.
Materials and Methods
Cell culture. The 11 lung cancer cell lines (B203L, PC9, A110L, A549, QG56, SQ1, B1203L, PC10, 904L, PC1, A529L) have been described previously.(14,15) Cell lines were cultured in RPMI-1640 medium (Nissui Seiyaku, Tokyo, Japan) and maintained in a 5% CO2 atmosphere at 37°C.
Antibodies and chemicals. Antibodies against MCT1 (sc-14916), MCT4 (sc-50329), MCT5 (sc-14932), BSG (sc-9753) and MMP9 (sc-6840) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-β-actin (A5441) antibody was purchased from Sigma (St Louis, MO, USA). 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS) (D3514), quercetin (Q4951), simvastatin (S6196) and Sodium l-lactate (L7022) were purchased from Sigma. Sodium lactate solution (195-02305) and Sodium DL-lactate solution (3160572) were purchased from Wako (Osaka, Japan) and Nacalai (Kyoto, Japan), respectively.
Lactate measurement. To measure lactate in the medium and cell lysates, Lactate Colorimetric Assay kits (ab65331) (AbCam, Cambridge, UK) were used according to the manufacturer’s instructions. Briefly, the concentration of lactate in 2 μL culture medium or cell lysates was detected by spectrophotometry at 450 nm using a standard curve generated with a known concentration of lactate solution. For the scattergram, the concentration of lactate in the medium without cells was subtracted. For the cellular lactate, A110L cells were sonicated with PBS and pmol/μg was calculated with the concentration of cell lysate.
Knockdown with siRNA. siRNA transfection was performed as previously described.(15) Briefly, the following double-stranded, 25-bp RNA oligonucleotides were commercially generated (Invitrogen, Carlsbad, CA, USA): MCT1 siRNA: siMCT1 #3, 5′-CCAAGGCAGGGAAAGAUAAGUCUAA-3′ (sense) and 5′-UUAGACUUAUCUUUCCCUGCCUUGG-3′ (antisense); siMCT1 #4, 5′-CACCACCAGCGAAGUGUCAUGGAUA-3′ (sense) and 5′-UAUCCAUGACACUUCGCUGGUGGUG-3′ (antisense); MCT4 siRNA: siMCT4 #1, 5′-CGACCCACGUCUACAUGUACGUGUU-3′ (sense) and 5′-AACACGUACAUGUAGACGUGGGUCG-3′ (antisense); siMCT4 #2, 5′-UCUG-CAGUGUGUGCGUGAACCGCUU-3′ (sense) and 5′-AAGCG-GUUCACGCACACACUGCAGA-3′ (antisense); BSG siRNA: siBSG #2, 5′-AGUGAAGGCUGUGAAGUCGUCAGAA-3′ (sense) and 5′-UUCUGACGACUUCACAGCCUUCACU-3′ (antisense); siBSG #3, 5′-UCCGAGAGCAGGUUCUUCGUGAGUU-3′ (sense) and 5′-AACUCACGAAGAACCUGCUCUC-GGA-3′ (antisense). Two hundred picomoles of siRNA were mixed with 10 μL Lipofectamine 2000 (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. After 20 min, 1 × 106 A110L cells were gently added, mixed and incubated for a further 20 min. Transfected cells were used for western blotting, cell proliferation assays, migration assays and invasion assays.
Migration and invasion assay. Cancer cells (2.5 × 104) were seeded into each control or invasion cell culture insert (control: #3097, pore size 8 μm; Becton Dickinson Labware, Bedford, MA, USA; invasion: BD BioCoat Matrigel Invasion Chamber, #354480; Becton Dickinson Labware) according to the manufacturer’s instructions. After 22 h, non-migrating and non-invading cells were removed and migrating and invading cells were fixed with methanol and stained with 1 μg/mL DAPI. Digital photographs were taken with an ECLIPSE E600 fluorescence microscope (Nikon, Tokyo, Japan) and DS-5M, -L1 (Nikon), and the migrating and invading cells were counted. The invasion ratios were calculated using: (the mean number of cells invading through the Matrigel insert membrane)/(the mean number of cells migrating through the control insert membrane). For the siRNA experiments, siRNA-transfected A110L cells were collected after 48 h and seeded into the chamber. For addition of inhibitor and lactate, the indicated concentration of inhibitor and sodium lactate was added at the same time as the A110L cells were seeded.
Western blotting. The preparation of cytoplasmic proteins and western blotting has been described previously.(16) Briefly, cell lysates were prepared in 10 mM Tris-HCl (pH 7.9), 150 mM NaCl, 0.5% NP40, 1 mM PMSF, and the nuclei were removed by centrifugation at 800g for 5 min at 4°C. Cytoplasmic proteins (50 μg) were separated on a 10% SDS-PAGE gel and transferred to a filter with a semidry blotter. Detection was performed using enhanced chemiluminescence (Amersham, Piscataway, NJ, USA). The protein expression levels were quantitated using a Multi Gauge Version 3.0 (Fujifilm, Tokyo, Japan). For the siRNA experiments, siRNA-transfected A110L cells were collected after 48 h. For inhibitor treatment, the indicated concentration of inhibitor was added at the same time as the A110L cells were seeded, and cells were collected after 22 h.
Cell proliferation. The cell proliferation assay has been previously described.(17) Briefly, siRNA-transfected A110L cells were seeded into 12-well plates at a density of 1 × 104 cells per well. Twenty-four hours after transfection was set as time zero. The cells were harvested by trypsinization and counted every 24 h with a Coulter-type cell size analyzer (CDA-500; Sysmex Corp., Kobe, Japan).
Gelatin-zymography. Gelatin-zymography assay was performed with a Gelatin-zymography kit (AK36) (Primary Cell, Hokkaido, Japan) following the instruction manual. Briefly, 11 lung cancer cells (1 × 105) or siRNA-transfected cells were seeded into six-well plates for 48 h. The following day, the cells were washed twice with PBS and once with serum-free medium and were cultured with serum-free medium (1 mL) for 24 h. The culture medium was collected and centrifuged at 800g for 5 min. Supernatant was mixed with an equal volume of dye provided in the kit. Each 20 μL was used for the Gelatin-zymography assay.
Cell viability assay by water-soluble tetrazolium salt (WST)-8 assays. A110L cells (1 × 103) were seeded into 96-well plates. The following day, the indicated concentrations of the inhibitors were applied. After 72 h, the surviving cells were stained with TetraColor ONE (Seikagaku Corporation, Tokyo, Japan) for 90 min at 37°C according to the manufacturer’s instructions. The absorbance was then measured at 450 nm.
Statistical analysis. Pearson’s correlation was used for statistical analysis, and significance was set at the 5% level.
MCT4 expression correlates with the concentration of lactate in the culture medium. To assess the cellular expression of MCT family proteins, western blotting was performed with 11 lung cancer cell lines. All cells expressed MCT1, MCT5 and BSG, but MCT4 was not expressed in B1203L cells (Fig. 1A). Because MCT family proteins are associated with the transport of lactate,(1) the lactate concentration in each medium was measured. As shown in Figure 1B, lactate concentration correlated with the expression of MCT4 but not MCT1 (Fig. 1B), MCT5 or BSG (data not shown).
MCT1 and MCT4 expression correlates with the invasion ratio. Inhibition of BSG expression reduces tumor cell invasion(18,19) and BSG is tightly associated with MCT1 and MCT4.(12) Therefore, we assessed migration and invasion activities by comparing the numbers of cells invading through a Matrigel insert membrane versus those migrating through a control insert membrane (Table 1). We found that the invasion ratio was strongly positively correlated with both MCT1 and MCT4 expression, but was not correlated with MCT5 expression (Fig. 2). Although both MCT1 and MCT4 expression were correlated with invasion activity, there is no statistical correlation between MCT1 and MCT4 expressions (data not shown). To assess the effect of MCT1 and MCT4 expression on invasion activity, we used specific siRNA targeting MCT1 and MCT4 in A110L cells that strongly express both genes. As shown in Figure 3A, each siRNA reduced the expression of its target protein. Interestingly, knockdown of MCT1 and MCT4 reduced the expression of BSG, and conversely, knockdown of BSG effectively reduced the expression of MCT4 and slightly reduced that of MCT1.
Table 1. Migration and invasion of 11 lung cancer cell lines
Invasion ration = Average of invading cells/Average of migrating cells.
We then performed cell proliferation assays in the presence of the siRNA. As shown in Figure 3B, and consistent with a previous report,(19) transfection of siRNA directed against BSG, MCT1 or MCT4 reduced cell proliferation. In the invasion assay, BSG siRNA reduced invasion activity but not migration activity (Fig. 3C); MCT1 and MCT4 siRNA both inhibited invasion activity more strongly than did BSG siRNA (Fig. 3D).
Both MCT1 and MCT4 expressions do not associate with MMP2 and 9 expression and activity. To assess the effect of MCT expression on MMP expression and activity, we used the gelatin-zymography assay. As shown in Figure 4A, three cell lines including A110L cells had strong MMP9 activity in the culture medium. This result was consistent with western blot with anti-MMP9 antibody. Strong MMP2 activity was only observed in one cell line. Next, we investigated whether MCT1, MCT4 and BSG expressions were associated with MMP9 expression and activity with the culture medium of A110L cells. Knockdown of each gene did not reduce MMP9 expression and activity (Fig. 4B). Similarly, cellular expression of MMP9 was not decreased by transfection of MCT1 and MCT4 siRNA (Fig. 4C).
Lactate does not increase migration and invasion activities. The correlation between MCT4 expression and lactate concentration is shown in Figure 1B. We then investigated whether lactate induced migration and invasion activity using A110L cells. First, we measured the concentration of lactate in the medium when cells were treated with siRNA. As expected, lactate concentration of the medium was increased by MCT1 siRNA and decreased by MCT4 siRNA (Fig. 5A) significantly, but there was little fluctuation. On the other hand, the concentration of intracellular lactate was almost constant when both MCT1 and MCT4 expressions were downregulated (Fig. 5A). Furthermore, addition of three different kinds of sodium lactate did not induce migration and invasion activity (Fig. 5B).
MCT inhibitors reduce both migration and invasion activity. To assess the effect of MCT function on invasion activity, we used the anion transporter inhibitor DIDS to inhibit all MCT proteins, quercetin to inhibit MCT1 and simvastatin to inhibit MCT4.(20) At first, we assessed cell viability using a WST-8 assay. As shown in Figure 6A, DIDS did not affect cell viability. We then assessed the effect of the inhibitors of MCT family proteins on cell migration. Lower 5 μM quercetin and 0.3 μM simvastatin reduced cell viability by 5% (Fig. 6A) at the maximum. These drugs also effectively reduced migration activity (Fig. 6B). Treatment with these drugs at the indicated concentrations did not affect the expression of either MCT1 or MCT4 (data not shown). One-hundred nanomolar DIDS, 50 nM quercetin and 30 nM simvastatin reduced invasion activity, but not migration activity (Fig. 6C).
To develop new therapeutic strategies for cancer, it is important to clarify the mechanisms of both invasion and metastasis of cancer cells. Tumor invasion has several steps, including: (i) detachment from other tumor cells; (ii) adhesion to the extracellular matrix (ECM); (iii) proteolytic degradation of the extracellular matrix (ECM); and (iv) motility and migration into the ECM. It has been reported that MMP family proteins,(21,22) plasminogen activator,(23,24) integrin,(25,26) E-cadherin and the catenin complex(27,28) and the small GTPase Rho(29,30) are key molecules for tumor invasion. In addition, tumor metabolism and the microenvironment, including the extracellular pH, are known to be involved in tumor growth and invasion activity. However, MCT family proteins have not yet been directly implicated in these processes.
Most cancer cells produce ATP by aerobic glycolysis and synthesize large quantities of lactate. We measured the lactate concentration in the extracellular medium of 11 lung cancer cell lines and found that lactate concentration correlated with the expression of MCT4, which excretes lactate from cells, but did not correlate with the expression of MCT1, which enables lactate entry into cells (Fig. 1). When MCT4 expression was repressed using a specific MCT4 siRNA, the lactate concentration in the medium was significantly decreased (Fig. 5A), but there was very little fluctuation. These results suggest that MCT4 may predominate over MCT1 to provide the extracellular lactate concentration.
It has been reported that accumulation of lactate within tumors is associated with a poor clinical outcome.(13,31) We investigated the association between MCT expression and invasion activity. The invasion ratios in 11 lung cancer cell lines were significantly correlated with the expression levels of MCT1 and MCT4, but not MCT5 (Fig. 2) or lactate concentration (data not shown). Basigin modulates MMP activity and cancer progression, and is tightly associated with MCT1 and MCT4. We therefore compared the effect of BSG knockdown with knockdown of MCT1 or MCT4 in in vitro invasion assays. We found that knockdown of MCT1 and MCT4 reduced the cellular expression of BSG. Conversely, knockdown of BSG slightly reduced the expression of MCT4 but not that of MCT1 (Fig. 3A). The numbers of invading cells were potently decreased when cells were transfected with MCT1 or MCT4 siRNA compared with BSG siRNA (Fig. 3D). Next, we made the expression plasmids of MCT1, MCT4 and BSG. Unexpectedly, we found that the MCT1 expression was downregulated by the overexpression of MCT4, and inversely, the MCT4 expression was downregulated by the overexpression of MCT1, indicating that both MCT1 and MCT4 expressions might be mutually regulated in a post-transcriptional manner. We also investigated whether MMP2 and MMP9 expressions were associated with cancer invasion. A few cancer cells expressed MMP2 or MMP9 (Fig. 4A). MMP9 was not reduced by the knockdown of MCT1, MCT4 or BSG expression in A110L cells, which has high invasion activity (Fig. 4B). These results suggest that the enzymatic activity and expression of both MMP2 and MMP9 were not associated with invasion activity in lung cancer cell lines. Furthermore, addition of lactate did not increase the number of migrating and invading cells (Fig. 5B). These results suggest that invasion activity is mainly associated with MCT1 or MCT4 expression but not with lactate. It is also possible that lactate concentration in the medium may be saturated to induce invasion through MCT expression regardless of the further addition of lactate.
Many inhibitors of the MCT family have been reported.(20) DIDS is an anion transporter inhibitor and suppresses the function of all proteins in the MCT family. Quercetin and simvastatin inhibit human MCT1 and human MCT4, respectively. First, we assessed the cytotoxicity of these three inhibitors using a WST-8 assay (Fig. 6A). Quercetin (10 μM) and simvastatin (0.5 μM) had a cytotoxic activity of <10% in A110L cells, whereas DIDS at concentrations <100 μM was not cytotoxic at all. When A110L cells were treated with DIDS (10 μM), Quercetin (5 μM) and simvastatin (0.3 μM), cell migration was strongly repressed (Fig. 6B) and there were no invading cells (data not shown) without influencing the expression of MCT1 and MCT4 (data not shown). Furthermore, when A110L cells were treated with DIDS (100 nM), Quercetin (50 nM) and simvastatin (30 nM), cell migration was not repressed (Fig. 6B), but invasion activity still strongly repressed (Fig. 6C). These results are consistent with results using MCT1 and MCT4 siRNA (Fig. 3C,D). It has been reported that quercetin and simvastatin suppress invasion by inhibition of MMP2 and MMP9 activity.(32–35) This is also possible that DIDS, quercetin and simvastatin might decrease the invasion activity not only by direct suppression of MMP activities but also by inhibition of monocarboxylate transporter activity or inhibitory activity against interacting molecules involving the function of MCT1 or MCT4. Further study is required to elucidate the mechanism of invasion involving the MCT proteins, although MCT1 and MCT4 are of general importance in both glucose metabolism and tumor growth. Our work suggests that inhibitors of the MCT may provide a novel strategy to prevent cancer metastasis.
This work was supported by a Grant-in-Aid for Scientific Research from the Ministry for Education, Culture, Sports, Science and Technology of Japan (17016075), a University of Occupational and Environmental Health (UOEH) Japan Grant for Advanced Research and the Vehicle Racing Commemorative Foundation.
The authors have no conflicts of interest to declare.