Monocarboxylate transporter 1 is up‐regulated in basal‐like breast carcinoma

Pinheiro C, Albergaria A, Paredes J, Sousa B, Dufloth R, Vieira D, Schmitt F & Baltazar F
(2010) Histopathology 56, 860–867
Monocarboxylate transporter 1 is up‐regulated in basal‐like breast carcinoma Aims: Monocarboxylate transporters (MCTs) have been considered promising targets for cancer therapy, since they facilitate lactate efflux in glycolytic tumours. However, their role in solid tumours is still poorly understood. Thus, the present work aimed to contribute to understanding the involvement of MCT1 and MCT4 in breast cancer progression as well as MCT regulation by CD147. Methods and results: The expression of the membrane transporters MCT1 and MCT4 was analysed in a series of breast carcinomas (249 cases) and their clinicopathological significance investigated. Additionally, we analysed the significance of CD147 co‐expression, as an important regulator of MCT expression and activity. MCT1 was significantly increased in breast carcinomas when compared with normal breast tissue and, importantly, both MCT1 and CD147 were associated with poor prognostic variables such as basal‐like subtype and high grade tumours. Conclusions: These results provide evidence for a prognostic value of MCT1 in breast carcinoma and support the exploitation of the complex MCT1/CD147 as a promising target for cancer therapy, especially in basal‐like breast carcinoma.     

Current aspects of lactate exchange: lactate/H+ transport in human skeletal muscle

Skeletal muscle is capable of producing and releasing large amounts of lactate and at the same time taking up lactate and using it as a respiratory fuel. The release and uptake of lactate both involve transmembrane transport, which is mediated mainly by a membrane protein called the monocarboxylate transporter (MCT). MCTs mediate membrane transport with an obligatory 1:1 coupling between lactate and H⁺ fluxes, and is therefore of great importance for pH regulation, especially during intense muscle activity. The total lactate and H⁺ transport capacity is higher in membranes from oxidative fibers than in membranes from more glycolytic fibers. There are two isoforms of MCT present in skeletal muscle, MCT1 and MCT4. In human muscle samples, there is a positive correlation between the proportion of type I fibers and MCT1 density. In contrast, the MCT4 density in human muscle is independent of fiber type and displays a large interindividual variation. Although the two isoforms have identical transport kinetics (K _m), they may have different roles in muscle. MCT1 and MCT4 respond differently to a high-intensity training session, which suggests that these two isoforms are regulated differently. Electronic Publication     

HIC1 loss promotes prostate cancer metastasis by triggering epithelial–mesenchymal transition

Metastatic disease is the leading cause of death due to prostate cancer (PCa). Although the hypermethylated in cancer 1 (HIC1) gene has been observed to be epigenetically modified in PCa, its intrinsic role and mechanism in PCa metastasis still remain uncertain. Here, we show that hypermethylation of the HIC1 promoter markedly reduces its suppressive function in metastatic PCa tissues as compared with primary and adjacent normal prostate tissues, and is associated with poor patient survival. PCas in cancer‐prone mice homozygous for a prostate‐targeted Hic1 conditional knockout showed stronger metastatic behaviour than those in heterozygous mice, as a result of epithelial–mesenchymal transition (EMT). Moreover, impairment of HIC1 expression in PCa cells induced their migration and metastasis through EMT, by enhancing expression of Slug and CXCR4, both of which are critical to PCa metastasis; the CXCL12–CXCR4 axis promotes EMT by activating the extracellular signal‐regulated kinase (ERK) 1/2 pathway. Taken together, our results suggest that evaluation of HIC1–CXCR4–Slug signalling may provide a potential predictor for PCa aggressiveness. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Co-expression of CD147/EMMPRIN with monocarboxylate transporters and multiple drug resistance proteins is associated with epithelial ovarian cancer progression

Cancer metastasis and anti-cancer drug resistance are the major reason for the failure of clinical cancer treatment. We evaluated CD147, monocarboxylate transporters (MCT1 and MCT4), and multidrug resistance (MDR) markers (MDR1 and MRP2) in 4 epithelial ovarian cancer (EOC) cell lines and primary tumors (n = 120) along with the matched metastatic lesions (n = 40) with immunofluorescence labeling. We correlated CD147 with MCT1, MCT4, MDR1 and MRP2 markers in primary and metastatic cells in cell lines and tissues using confocal microscopy. We also investigated the relationship of expression of CD147, MCT1 and MCT4 with various progression parameters. Our results indicate that the co-expression of CD147 with MCTs or MDR markers was found in primary and metastatic EOC cells and stromal cells; the over-expression of CD147, MCT1 and MCT4 was found in most primary and the matched metastatic lesions of EOC, and was significantly associated with tumor stage, grade, residual disease status and presence of ascites (P < 0.05) but not with histology type (P > 0.05). These results suggest that over-expression of CD147, MCT1 and MCT4 is correlated with EOC progression, and co-expression of CD147 and MCT1/MCT4 is related to drug resistance during EOC metastasis and could be useful therapeutic targets to prevent the development of incurable, recurrent and drug resistance EOC.     

Is lactate food for neurons? Comparison of monocarboxylate transporter subtypes in brain and muscle

Intercellular monocarboxylate transport is important, particularly in tissues with high energy demands, such as brain and muscle. In skeletal muscle, it is well established that glycolytic fast twitch muscle fibers produce lactate, which is transported out of the cell through the monocarboxylate transporter (MCT) 4. Lactate is then taken up and oxidized by the oxidative slow twitch muscle fibers, which express MCT1. In the brain it is still questioned whether lactate produced in astrocytes is taken up and oxidized by neurons upon activation. Several studies have reported that astrocytes express MCT4, whereas neurons express MCT2. By comparing the localizations of MCTs in oxidative and glycolytic compartments I here give support to the idea that there is a lactate shuttle in the brain similar to that in muscle. This conclusion is based on studies in rodents using high resolution immunocytochemical methods at the light and electron microscopical levels.     

HDAC inhibition in glioblastoma monitored by hyperpolarized 13C MRSI

Vorinostat is a histone deacetylase (HDAC) inhibitor that inhibits cell proliferation and induces apoptosis in solid tumors, and is in clinical trials for the treatment of glioblastoma (GBM). The goal of this study was to assess whether hyperpolarized ¹³C MRS and magnetic resonance spectroscopic imaging (MRSI) can detect HDAC inhibition in GBM models. First, we confirmed HDAC inhibition in U87 GBM cells and evaluated real‐time dynamic metabolic changes using a bioreactor system with live vorinostat‐treated or control cells. We found a significant 40% decrease in the ¹³C MRS‐detectable ratio of hyperpolarized [1‐¹³C]lactate to hyperpolarized [1‐¹³C]pyruvate, [1‐¹³C]Lac/Pyr, and a 37% decrease in the pseudo‐rate constant, k_PL, for hyperpolarized [1‐¹³C]lactate production, in vorinostat‐treated cells compared with controls. To understand the underlying mechanism for this finding, we assessed the expression and activity of lactate dehydrogenase (LDH) (which catalyzes the pyruvate to lactate conversion), its associated cofactor nicotinamide adenine dinucleotide, the expression of monocarboxylate transporters (MCTs) MCT1 and MCT4 (which shuttle pyruvate and lactate in and out of the cell) and intracellular lactate levels. We found that the most likely explanation for our finding that hyperpolarized lactate is reduced in treated cells is a 30% reduction in intracellular lactate levels that occurs as a result of increased expression of both MCT1 and MCT4 in vorinostat‐treated cells. In vivo ¹³C MRSI studies of orthotopic tumors in mice also showed a significant 52% decrease in hyperpolarized [1‐¹³C]Lac/Pyr when comparing vorinostat‐treated U87 GBM tumors with controls, and, as in the cell studies, this metabolic finding was associated with increased MCT1 and MCT4 expression in HDAC‐inhibited tumors. Thus, the ¹³C MRSI‐detectable decrease in hyperpolarized [1‐¹³C]lactate production could serve as a biomarker of response to HDAC inhibitors.     

CD147 immunohistochemistry discriminates between reactive mesothelial cells and malignant mesothelioma

Malignant mesothelioma (MM) is a rare form of cancer. Its histopathological diagnosis is very difficult, as it exhibits a number of different appearances that can be misinterpreted as metastatic invasion or atypical hyperplasia. Thus, there is an urgent need to identify adequate markers to distinguish between benign and malignant cells, allowing the implementation of appropriate therapies and, possibly, specific directed therapies. MM, like other tumors, show an increase in glucose uptake, due to high rates of glycolysis, inducing an intracellular overload of acids. In this context, monocarboxylate transporters (MCTs) emerge as important players, by mediating the transmembranar co-transport of lactate with a proton, thereby, regulating pH and allowing continuous glycolysis. Importantly, proper MCT expression and activity depend on its co-expression with a chaperone, CD147, which is associated with poor prognosis in cancer. Twenty-two samples including reactive mesothelial cells, MM, and atypical mesothelial hyperplasias were evaluated for immunoexpression of MCT1, MCT4, and CD147. Expression of these proteins was compared with GLUT1 as a new promising marker for MM. Although MCT isoforms were not differentially expressed in the two types of cytological specimens, CD147, as GLUT1, was almost exclusively expressed in MM. Both MCT1 and MCT4 are not able to discriminate between mesothelial reactive cells and mesothelial malignant cells, while CD147 was able to distinguish these two proliferations. If confirmed, besides being a good marker for identification of MM, CD147 may also be a target for therapeutical strategies in this rare type of tumor.     

Monocarboxylate Transporters 1 and 4 Are Associated with CD147 in Cervical Carcinoma

Due to the highly glycolytic metabolism of solid tumours, there is an increased acid production, however, cells are able to maintain physiological pH through plasma membrane efflux of the accumulating protons. Acid efflux through MCTs (monocarboxylate transporters) constitutes one of the most important mechanisms involved in tumour intracellular pH maintenance. Still, the molecular mechanisms underlying the regulation of these proteins are not fully understood. We aimed to evaluate the association between CD147 (MCT1 and MCT4 chaperone) and MCT expression in cervical cancer lesions and the clinico-pathological significance of CD147 expression, alone and in combination with MCTs. The series included 83 biopsy samples of precursor lesions and surgical specimens of 126 invasive carcinomas. Analysis of CD147 expression was performed by immunohistochemistry. CD147 expression was higher in squamous and adenocarcinoma tissues than in the non-neoplastic counterparts and, importantly, both MCT1 and MCT4 were more frequently expressed in CD147 positive cases. Additionally, co-expression of CD147 with MCT1 was associated with lymph-node and/or distant metastases in adenocarcinomas. Our results show a close association between CD147 and MCT1 and MCT4 expressions in human cervical cancer and provided evidence for a prognostic value of CD147 and MCT1 co-expression.     

Somatic copy number alterations by whole‐exome sequencing implicates YWHAZ and PTK2 in castration‐resistant prostate cancer

Castration‐resistant prostate cancer (CRPC) is the most aggressive form of prostate cancer (PCa) and remains a significant therapeutic challenge. The key to the development of novel therapeutic targets for CRPC is to decipher the molecular alterations underlying this lethal disease. The aim of our study was to identify therapeutic targets for CRPC by assessing somatic copy number alterations (SCNAs) by whole‐exome sequencing on five CRPC/normal paired formalin‐fixed paraffin‐embedded (FFPE) samples, using the SOLiD4 next‐generation sequencing (NGS) platform. Data were validated using fluorescence in situ hybridization (FISH) on a PCa progression cohort. PTK2 and YWHAZ amplification, mRNA and protein expression were determined in selected PCa cell lines. Effects of PTK2 inhibition using TAE226 inhibitor and YWHAZ knock‐down on cell proliferation and migration were tested in PC3 cells in vitro. In a larger validation cohort, the amplification frequency of YWHAZ was 3% in localized PCa and 48% in CRPC, whereas PTK2 was amplified in 1% of localized PCa and 35% in CRPC. YWHAZ knock‐down and PTK2 inhibition significantly affected cell proliferation and migration in the PC3 cells. Our findings suggest that inhibition of YWHAZ and PTK2 could delay the progression of the disease in CRPC patients harbouring amplification of the latter genes. Furthermore, our validated whole‐exome sequencing data show that FFPE tissue could be a promising alternative for SCNA screening using next‐generation sequencing technologies. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Highly differential expression of the monocarboxylate transporters MCT2 and MCT4 in the developing rat brain

Monocarboxylate transporters (MCTs) play an important role in the metabolism of all cells. They mediate the transport of lactate and pyruvate but also some other substrates such as ketone bodies. It has been proposed that glial cells release monocarboxylates to fuel neighbouring neurons. A key element in this hypothesis is the existence of neuronal MCTs. Amongst the three MCTs known to be expressed in the brain (MCT1, 2 and 4) only MCT2 has been found in neurons. Here we have studied the expression pattern of MCT2 during postnatal development. By use of immunoperoxidase and double immunofluorescence microscopy we report that neuronal MCT2 occurs in most brain areas, including the hippocampus and cerebellum, from birth to adult. MCT2 is also expressed in specific subpopulations of astrocytes. Neuronal MCT2 is most abundant in the first 3 postnatal weeks and thereafter decreases toward adulthood. In contrast to MCT2, MCT4 is exclusively present in astroglia during all stages of development. Furthermore, MCT4 expression is very low at birth and reaches adult level by P14. Our results are consistent with previous data suggesting that in the immature brain much of the energy demand is met by monocarboxylates and ketone bodies.     

Monocarboxylate Transporter 1 (MCT1) is an independent prognostic biomarker in endometrial cancer

Background

Endometrial cancer (EC) is a major health concern due to its rising incidence. Whilst early stage disease is generally cured by surgery, advanced EC has a poor prognosis with limited treatment options. Altered energy metabolism is a hallmark of malignancy. Cancer cells drive tumour growth through aerobic glycolysis and must export lactate to maintain intracellular pH. The aim of this study was to evaluate the expression of the lactate/proton monocarboxylate transporters MCT1 and MCT4 and their chaperone CD147 in EC, with the ultimate aim of directing future drug development.

Methods

MCT1, MCT4 and CD147 expression was examined using immunohistochemical analysis in 90 endometrial tumours and correlated with clinico-pathological characteristics and survival outcomes.

Results

MCT1 and MCT4 expression was observed in the cytoplasm, the plasma membrane or both locations. CD147 was detected in the plasma membrane and associated with MCT1 (p =?0.003) but not with MCT4 (p =?0.207) expression. High MCT1 expression was associated with reduced overall survival (p =?0.029) and remained statistically significant after adjustment for survival covariates (p =?0.017).

Conclusion

Our data suggest that MCT1 expression is an important marker of poor prognosis in EC. MCT1 inhibition may have potential as a treatment for advanced or recurrent EC.

     

Cullin-1 promotes cell proliferation via cell cycle regulation and is a novel in prostate cancer

Background: There is no reliable marker available for early detection, diagnostic confirmation, or disease prognosis available of prostate cancer (PCa). We aimed to evaluate the function of Cullin-1 and unravel its underlying molecular mechanism to develop novel treatment options equivalent to PCa. Method: We used immunohistochemistry to analyze the correlation between Cullin-1 expression and clinicopathologic variables and patient survival. The Cullin-1 level was tested in PCa cells. The role of regulation of Cullin-1 in PCa was applied in vitro and vivo. In addition, we further investigated the signaling pathway of Cullin-1 in prostate cancer cell proliferation. Result: We first discovered that Cullin-1 expression was upregulated in human PCa tissues and inversely related with PCa differentiation. We then found that high expression of Cullin-1 protein suggested a poor prognosis in PCa patients. Also, Cullin-1 promotes PCa cell proliferation in vitro and tumor growth in vivo. We then found that the mechanism of Cullin-1 regulation on cell-cycle progression is due to increased expression of p21 and p27, and decreased expression of cyclin D1 and cyclin E after Cullin-1 knockdown. Conclusion: Cullin-1 exerts multiple biological effects in the PCa cell line. Through promoting proliferation and by countering cisplatin-induced apoptosis, Cullin-1 has been deeply implicated in the pathogenesis and development of PCa.     

Increased expression of monocarboxylate transporters 1, 2, and 4 in colorectal carcinomas

Tumour cells are known to be highly glycolytic, thus producing high amounts of lactic acid. Monocarboxylate transporters (MCTs), by promoting the efflux of the accumulating acids, constitute one of the most important mechanisms in the maintenance of tumour intracellular pH. Since data concerning MCT expression in colorectal carcinomas (CRC) are scarce and controversial, the present study aimed to assess the expressions of MCT1, 2, and 4 in a well characterized series of CRC and assess their role in CRC carcinogenesis. CRC samples (126 cases) were analyzed for MCT1, MCT2, and MCT4 immunoexpression and findings correlated with clinico-pathological parameters. Expression of all MCT isoforms in tumour cells was significantly increased when compared to adjacent normal epithelium. Remarkably, there was a significant gain of membrane expression for MCT1 and MCT4 and loss of plasma membrane expression for MCT2 in tumour cells. Plasma membrane expression of MCT1 was directly related to the presence of vascular invasion. This is the larger study on MCT expression in CRC and evaluates for the first time its clinico-pathological significance. The increased expression of these transporters suggests an important role in CRC, which might justify their use, especially MCT1 and MCT4, as targets in CRC drug therapy.     

Treatment with the MEK inhibitor U0126 induces decreased hyperpolarized pyruvate to lactate conversion in breast,but not prostate,cancer cells

Alterations in cell metabolism are increasingly being recognized as a hallmark of cancer and are being exploited for the development of diagnostic tools and targeted therapeutics. Recently, ¹³C MRS‐detectable hyperpolarized pyruvate to lactate conversion has been validated in models as a noninvasive imaging method for the detection of tumors and treatment response, and has successfully passed phase I clinical trials. To date, response to treatment has been associated with a decrease in hyperpolarized lactate production. In this study, we monitored the effect of treatment with the mitogen‐activated protein kinase (MEK) inhibitor U0126 in prostate and breast cancer cells. Following treatment, we observed a 31% decrease in the flux of hyperpolarized ¹³C label in treated MCF‐7 breast cancer cells relative to controls. In contrast, and unexpectedly, the flux increased to 167% in treated PC3 prostate cancer cells. To mechanistically explain these observations, we investigated treatment‐induced changes in the different factors known to affect the pyruvate to lactate conversion. NADH (nicotinamide adenine dinucleotide, reduced form) levels remained unchanged, whereas lactate dehydrogenase expression and activity, as well as intracellular lactate, increased in both cell lines, providing an explanation for the elevated hyperpolarized lactate observed in PC3 cells. The expression of MCT1, which mediates pyruvate transport, decreased in treated MCF‐7, but not PC3, cells. This identifies pyruvate transport as rate limiting in U0126‐treated MCF‐7 cells and explains the decrease in hyperpolarized lactate observed in these cells following treatment. Our findings highlight the complexity of interactions between MEK and metabolism, and the need for mechanistic validation before hyperpolarized ¹³C MRS can be used to monitor treatment‐induced molecular responses. Copyright © 2012 John Wiley & Sons, Ltd.     

Cytotoxic necrotizing factor 1 promotes prostate cancer progression through activating the Cdc42–PAK1 axis

Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections and plays a role in prostatic carcinogenesis and prostate cancer (PCa) progression. However, the mechanisms through which UPEC promotes PCa development and progression are unclear. Cytotoxic necrotizing factor 1 (CNF1) is one of the most important UPEC toxins and its role in PCa progression has never been studied. We found that UPEC‐secreted CNF1 promoted the migration and invasion of PCa cells and PCa metastasis. In vitro studies showed that CNF1 promotes pro‐migratory and pro‐invasive activity through entering PCa cells and activating Cdc42, which subsequently induced PAK1 phosphorylation and up‐regulation of MMP‐9 expression. CNF1 also promoted pulmonary metastasis in a xenograft mouse model through these mechanisms. PAK1 phosphorylation correlated with advanced grades of PCa in human clinical PCa tissues. These results suggest that CNF1 derived from UPEC plays an important role in PCa progression through activating a Cdc42–PAK1 signal axis and up‐regulating the expression of MMP‐9. Therefore, surveillance for and treatment of cnf1‐carrying UPEC strains may diminish PCa progression and thus have an important clinical therapeutic impact. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Hyperpolarized 13C NMR studies of glucose metabolism in living breast cancer cell cultures

The recent development of dissolution dynamic nuclear polarization (DNP) gives NMR the sensitivity to follow metabolic processes in living systems with high temporal resolution. In this article, we apply dissolution DNP to study the metabolism of hyperpolarized U‐¹³C,²H₇‐glucose in living, perfused human breast cancer cells. Spectrally selective pulses were used to maximize the signal of the main product, lactate, whilst preserving the glucose polarization; in this way, both C₁‐lactate and C₃‐lactate could be observed with high temporal resolution. The production of lactate by T47D breast cancer cells can be characterized by Michaelis–Menten‐like kinetics, with K_m = 3.5 ± 1.5 mm and V_max = 34 ± 4 fmol/cell/min. The high sensitivity of this method also allowed us to observe and quantify the glycolytic intermediates dihydroxyacetone phosphate and 3‐phosphoglycerate. Even with the enhanced DNP signal, many other glycolytic intermediates could not be detected directly. Nevertheless, by applying saturation transfer methods, the glycolytic intermediates glucose‐6‐phosphate, fructose‐6‐phosphate, fructose‐1,6‐bisphosphate, glyceraldehyde‐3‐phosphate, phosphoenolpyruvate and pyruvate could be observed indirectly. This method shows great promise for the elucidation of the distinctive metabolism and metabolic control of cancer cells, suggesting multiple ways whereby hyperpolarized U‐¹³C,²H₇‐glucose NMR could aid in the diagnosis and characterization of cancer in vivo. Copyright © 2013 John Wiley & Sons, Ltd.     

Effect of training intensity on muscle lactate transporters and lactate threshold of cross-country skiers.

The training intensity may affect the monocarboxylate transporters MCT1 and MCT4 in skeletal muscle. Therefore, 20 elite cross-country skiers (11 men and nine women) trained hard for 5 months at either moderate (MIG, 60-70% of VO2max) or high intensity (HIG, 80-90%). The lactate threshold, several performance parameters, and the blood lactate concentration (cLa) after exhausting treadmill running were also determined. Muscle biopsies taken from the vastus lateralis muscle before and after the training period were analysed for the two MCTs and for muscle fibre types and six enzymes. The concentration of MCT1 did not change for HIG (P=0.3) but fell for MIG (-12 +/- 3%, P=0.01); the training response differed between the two groups (P=0.05). The concentration of MCT4 did not change during the training period (P > 0.10). The concentration of the two MCTs did not differ between the two sexes (P=0.9). The running speed at the lactate threshold rose for HIG (+3.2 +/- 0.9%, P=0.003), while no change was seen for MIG (P=0.54); the training response differed between the two groups (P=0.04). The cLa after long-lasting exhausting treadmill running correlated with the concentration of MCT1 (rs=0.69, P=0.002), but not with that of MCT4 (rs=0.2, P=0.2). There were no other significant correlations between the concentrations of the two MCTs and the performance parameters, muscle fibre types, or enzymes (r < or = 0.36, P > 0.10). Thus, the training response differed between MIG and HIG both in terms of performance and of the effect on MCT1. Training at high intensity may be more effective for cross-country skiers. Finally, MCT1 may be important for releasing lactate to the blood during long-lasting exercise.     

Expression of monocarboxylate transporter (MCT) 1 and MCT4 in overloaded mice plantaris muscle

A number of studies have shown that changes in muscle contractile activity regulate the expression of monocarboxylate transporters (MCTs) in the skeletal muscle. The aim of this study was to investigate the effect of functional overload on MCT1 and MCT4 protein expression. Plantaris muscles were functionally overloaded for 15 days by ablation of the synergistic muscles. MCT1 and MCT4 mRNA abundance increased by 160–161% (p < 0.01) and 265–325% (p < 0.05), respectively, after 1–3 days of functional overload. MCT1 and MCT4 protein expression increased by 92 and 61%, respectively, after 12 days of functional overload (p < 0.05). AMP-activated protein kinase (AMPK) phosphorylation status [phospho-AMPK (Thr172)/total AMPK] was significantly elevated after 3–9 days of functional overload. Plasma testosterone concentration was elevated after 12 days of functional overload, while blood lactate concentration was not altered. Thus, the current study demonstrated that heavy mechanical loading induces increase in MCT1 and MCT4 protein expression in the muscles with increase in AMPK phosphorylation status and plasma testosterone concentration.