Differential sensitivities to lactate transport inhibitors of breast cancer cell lines

This is linked to the online version of the paper at: http://dx.doi.org/10.1530/ERC-13-0132.The tumour microenvironment is known to be acidic due to high glycolytic rates of tumour cells. Monocarboxylate transporters (MCTs) play a role in extracellular acidification, which is widely known to be involved in tumour progression. Recently, we have described the upregulation of MCT1 in breast carcinomas and its association with poor prognostic variables. Thus, we aimed to evaluate the effect of lactate transport inhibition in human breast cancer cell lines. The effects of a-cyano-4-hydroxycinnamate, quercetin and lonidamine on cell viability, metabolism, proliferation, apoptosis, migration and invasion were assessed in a panel of different breast cancer cell lines. MCT1, MCT4 and CD147 were differently expressed among the breast cancer cell lines and, as expected, different sensitivities were observed for the three inhibitors. Interestingly, in the most sensitive cell lines, lactate transport inhibition induced a decrease in cell proliferation, migration and invasion, as well as an increase in cell death. Results were validated by silencing MCT1 expression using siRNA. The results obtained here support targeting of lactate transport as a strategy to treat breast cancer, with a special emphasis on the basal-like subtype, which so far does not have a specific molecular therapy.This work was supported by the Fundação para a Ciência e a Tecnologia (FCT) grant ref. PTDC/SAU-FCF/104347/2008, under the scope of ‘Programa Operacional Temático Factores de Competitividade’ (COMPETE) of ‘Quadro Comunitário de Apoio III’ and co-financed by the Fundo Europeu De Desenvolvimento Regional (FEDER)

The cytotoxicity of 3-bromopyruvate in breast cancer cells depends on extracellular pH

Although the anti-cancer properties of 3BP have been described previously, its selectivity for cancer cells still needs to be explained. In the work reported here we characterized the kinetic parameters of radiolabelled [14C]-3BP uptake in three breast cancer cell lines that display different levels of resistance to 3BP: ZR-75-1 < MCF-7 < SK-BR-3. At pH 6.0 the affinity of cancer cells for 3BP transport, correlates with their sensitivity, a pattern that does not occur at pH 7.4. In the three cell lines, the uptake of 3BP is dependent on the proton motive force and is decreased by MCTs inhibitors. In the SK-BR-3 cell line, a sodium-dependent transport also occurs. Butyrate promotes the localization of MCT-1 at the plasma membrane and increases the level of MCT-4 expression, leading to a higher sensitivity for 3BP. Here, we demonstrate that this phenotype is accompanied by an increase in affinity for 3BP uptake. Our results confirm the role of MCTs, especially MCT-1 in 3BP uptake and the importance of CD147 glycosylation in this process. We find that the affinity for 3BP transport is higher when the extracellular milieu is acid. This is a typical phenotype of tumor microenvironment and explains the lack of secondary effects of 3BP already described in in vivo studies.FEDER (Fundo Europeu deDesenvolvimento Regional), through POFC (Programa Operacional Factores de Competitividade) – COMPETE, and by Portuguese National Funds from FCT (Fundac¸˜ao para a Ciˆencia e Tecnologia) in the scope of the project PEst-OE/BIA/U14050/2014. JAS [grant number SFRH/BD/76038/2011] received a fellowship from the Portuguese government from the FCT through FSE (Fundo Social Europeu) and POPH (Programa Operacional Potencial Humano)

Monocarboxylate transporters (MCTs) in gliomas: expression and exploitation as therapeutic targets

Background. Gliomas exhibit high glycolytic rates, and monocarboxylate transporters (MCTs) play a major role in the maintenance of the glycolytic metabolism through the proton-linked transmembrane transport of lactate. However, their role in gliomas is poorly studied. Thus, we aimed to characterize the expression of MCT1, MCT4, and their chaperone CD 147 and to assess the therapeutic impact of MCT inhibition in gliomas. Methods. MCTs and CD 147 expressions were characterized by immunohistochemistry in nonneoplastic brain and glioma samples. The effect of CHC (MCT inhibitor) and MCT1 silencing was assessed in in vitro and in vivo glioblastoma models. Results. MCT1, MCT4, and CD 147 were overexpressed in the plasma membrane of glioblastomas, compared with diffuse astrocytomas and nonneoplastic brain. CHC decreased glycolytic metabolism, migration, and invasion and induced cell death in U251 cells (more glycolytic) but only affected proliferation in SW1088 (more oxidative). The effectiveness of CHC in glioma cells appears to be dependent on MCT membrane expression. MCT1 downregulation showed similar effects on different glioma cells, supporting CHC as an MCT1 inhibitor. There was a synergistic effect when combining CHC with temozolomide treatment in U251 cells. In the CAM in vivo model, CHC decreased the size of tumors and the number of blood vessels formed. Conclusions. This is the most comprehensive study reporting the expression of MCTs and CD 147 in gliomas. The MCT1 inhibitor CHC exhibited anti-tumoral and anti-angiogenic activity in gliomas and, of importance, enhanced the effect of temozolomide. Thus, our results suggest that development of therapeutic approaches targeting MCT1 may be a promising strategy in glioblastoma treatment.Life and Health Sciences Research Institute, University of Minho, Portugal; Fundacao para a Ciencia e Tecnologia [SFRH/BI/33503/2008, SFRH/BPD/69479/2010, SFRH/BD/36463/2007]info:eu-repo/semantics/publishedVersio

"In-gel" purified ditags direct synthesis of highly efficient SAGE Libraries

BACKGROUND: SAGE (serial analysis of gene expression) is a recently developed technique for systematic analysis of eukaryotic transcriptomes. The most critical step in the SAGE method is large scale amplification of ditags which are then are concatemerized for the construction of representative SAGE libraries. Here, we report a protocol for purifying these ditags via an 'in situ' PAGE purification method. This generates ditags free of linker contaminations, making library construction simpler and more efficient. RESULTS: Ditags used to generate SAGE libraries were demarcated 'in situ' on preparative polyacrylamide gels using XC and BPB dyes, which precisely straddle the ditag band when a 16% PAGE gel (19:1 acrylamide:bis, 5% cross linker) is used to resolve the DNA bands. Here, the ditag DNA was directly excised from gel without visualization via EtBr or fluorescent dye staining, resulting in highly purified ditag DNA free of contaminating linkers. These ditags could be rapidly self ligated even at 4°C to generate concatemers in a controlled manner, which in turn enabled us to generate highly efficient SAGE libraries. This reduced the labor and time necessary, as well as the cost. CONCLUSIONS: This approach greatly simplified the ditag purification procedure for constructing SAGE libraries. Since the traditional post-run staining with EtBr or fluorescent dyes routinely results in cross contamination of a DNA band of interest by other DNA in the gel, the dry gel DNA excision method described here may also be amenable to other molecular biology techniques in which DNA purity is critically important

Lactate transporters in the context of prostate cancer metabolism: what do we know?

Lactate Transporters in the Context of Prostate Cancer Metabolism: What Do We Know?Metabolic changes during malignant transformation have been noted for many years in tumours. Otto Warburg first reported that cancer cells preferentially rely on glycolysis for energy production, even in the presence of oxygen, leading to the production of high levels of lactate. The crucial role of lactate efflux and exchange within the tumour microenvironment drew attention to monocarboxylate transporters (MCTs). MCTs have been recognized as promising targets in cancer therapy, and their expression was described in a large variety of tumours; however, studies showing how these isoforms contribute to the acquisition of the malignant phenotype are scarce and still unclear regarding prostate cancer. In this review, we focus on the role for MCTs in cell metabolism, supporting the development and progression of prostate cancer, and discuss the exploitation of the metabolic nature of prostate cancer for therapeutic and diagnostic purposes.Pertega Gomes N. received a fellowship from the Portuguese Foundation for Science and Technology (FCT), reference SFRH/BD/61027/2009. This work was supported by the FCT grant, reference PTDC/SAUMET/113415/2009, under the scope of "Programa Operacional Tematico Factores de Competitividade" (COMPETE) of "Quadro Comunitario de Apoio III" and co-financed by Fundo Comunitario Europeu FEDER

Significance of glycolytic metabolism-related protein expression in colorectal cancer, lymph node and hepatic metastasis

Background: Colorectal cancer (CRC) is one of the most common malignancies and a leading cause of cancer death worldwide. Most cancer cells display high rates of glycolysis with production of lactic acid, which is then exported to the microenvironment by monocarboxylate transporters (MCTs). The main aim of this study was to evaluate the significance of MCT expression in a comprehensive series of primary CRC cases, lymph node and hepatic metastasis. Methods: Expressions of MCT1, MCT4, CD147 and GLUT1 were studied in human samples of CRC, lymph node and hepatic metastasis, by immunohistochemistry. Results: All proteins were overexpressed in primary CRC, lymph node and hepatic metastasis, when compared with non-neoplastic tissue, with exception of MCT1 in lymph node and hepatic metastasis. MCT1 and MCT4 expressions were associated with CD147 and GLUT1 in primary CRC. These markers were associated with clinical pathological features, reflecting the putative role of these metabolism-related proteins in the CRC setting. Conclusion: These findings provide additional evidence for the pivotal role of MCTs in CRC maintenance and progression, and support the use of MCTs as biomarkers and potential therapeutic targets in primary and metastatic CRC.This work was supported by the Fundação para a Ciência e a Tecnologia (FCT) grant ref. PTDC/SAU-FCF/104347/2008, under the scope of ‘Programa Operacional Temático Factores de Competitividade’ (COMPETE) of ‘Quadro Comunitário de Apoio III’ and co-financed by the Fundo Europeu De Desenvolvimento Regional (FEDER). Ricardo Amorim was recipient of the fellowship SFRH/BD/98002/2013, from Fundação para a Ciência e a Tecnologia (FCT Portugal).info:eu-repo/semantics/publishedVersio

Monocarboxylate transporter 4 (MCT4) and CD147 overexpression is associated with poor prognosis in prostate cancer

BACKGROUND. Monocarboxylate transporters (MCTs) are transmembrane proteins involved in the transport of monocarboxylates across the plasma membrane, which appear to play an important role in solid tumours, however the role of MCTs in prostate cancer is largely unknown.The aim of the present work was to evaluate the clinico-pathological value of monocarboxylate transporters (MCTs) expression, namely MCT1, MCT2 and MCT4, together with CD147 and gp70 as MCT1/4 and MCT2 chaperones, respectively, in prostate carcinoma. METHODS. Prostate tissues were obtained from 171 patients, who performed radical prostatectomy and 14 patients who performed cystoprostatectomy. Samples and clinico-pathological data were retrieved and organized into tissue microarray (TMAs) blocks. Protein expression was evaluated by immunohistochemistry in neoplastic (n= 171), adjacent non-neoplastic tissues (n= 135), PIN lesions (n=40) and normal prostatic tissue (n=14). Protein expression was correlated with patients' clinicopathologic characteristics. RESULTS. In the present study, a significant increase of MCT2 and MCT4 expression in the cytoplasm of tumour cells and a significant decrease in both MCT1 and CD147 expression in prostate tumour cells was observed when compared to normal tissue. All MCT isoforms and CD147 were expressed in PIN lesions. Importantly, for MCT2 and MCT4 the expression levels in PIN lesions were between normal and tumour tissue, which might indicate a role for these MCTs in the malignant transformation. Associations were found between MCT1, MCT4 and CD147 expressions and poor prognosis markers; importantly MCT4 and CD147 overexpression correlated with higher PSA levels, Gleason score and pT stage, as well as with perineural invasion and biochemical recurrence. CONCLUSIONS. Our data provides novel evidence for the involvement of MCTs in prostate cancer. According to our results, we consider that MCT2 should be further explored as tumour marker and both MCT4 and CD147 as markers of poor prognosis in prostate cancer.NPG, CP and VMG received fellowships from the Portuguese Foundation for Science and Technology (FCT), refs. SFRH/BD/61027/2009, SFRH/BPD/69479/ 2010 and SFRH/BI/33503/2008, respectively. This work was supported by the FCT grant ref. PTDC/SAU-FCF/104347/2008, under the scope of Programa Operacional Temático Factores de Competitividade” (COMPETE) of Quadro Comunitário de Apoio III and co-financed by Fundo Comunitário Europeu FEDER

Hypoxia-mediated upregulation of MCT1 expression supports the glycolytic phenotype of glioblastomas

Background: Glioblastomas (GBM) present a high cellular heterogeneity with conspicuous necrotic regions associated with hypoxia, which is related to tumor aggressiveness. GBM tumors exhibit high glycolytic metabolism with increased lactate production that is extruded to the tumor microenvironment through monocarboxylate transporters (MCTs). While hypoxia-mediated regulation of MCT4 has been characterized, the role of MCT1 is still controversial. Thus, we aimed to understand the role of hypoxia in the regulation of MCT expression and function in GBM, MCT1 in particular. Methods: Expression of hypoxia-and glycolytic-related markers, as well as MCT1 and MCT4 isoforms was assessed in in vitro and in vivo orthotopic glioma models, and also in human GBM tissues by immunofluorescence/immunohistochemistry and Western blot. Following MCT1 inhibition, either pharmacologically with CHC (a-cyano-4-hydroxynnamic acid) or genetically with siRNAs, we assessed GBM cell viability, proliferation, metabolism, migration and invasion, under normoxia and hypoxia conditions. Results: Hypoxia induced an increase in MCT1 plasma membrane expression in glioma cells, both in in vitro and in vivo models. Additionally, treatment with CHC and downregulation of MCT1 in glioma cells decreased lactate production, cell proliferation and invasion under hypoxia. Moreover, in the in vivo orthotopic model and in human GBM tissues, there was extensive co-expression of MCT1, but not MCT4, with the GBM hypoxia marker CAIX. Conclusion: Hypoxia-induced MCT1 supports GBM glycolytic phenotype, being responsible for lactate efflux and an important mediator of cell survival and aggressiveness. Therefore, MCT1 constitutes a promising therapeutic target in GBM.This study was supported by Projecto Estratégico- LA 26 – 2013-2014 (PEst-C/SAL/LA0026/2013) and ON.2 SR&TD Integrated Program (NORTE-07-0124FEDER-000017)” co-funded by Programa Operacional Regional do Norte (ON.2- O Novo Norte), Quadro de Referência Estratégico Nacional (QREN), through Fundo Europeu de Desenvolvimento Regional (FEDER), as well as MCTI/CNPq Nº 73/2013 (Brazil). VMG received a fellowship from Fundação para a Ciência e Tecnologia (FCT) ref. SFRH/BD/51997/2012.info:eu-repo/semantics/publishedVersio

Zinc Finger Nuclease mediated knockout of ADP dependent Glucokinase in Cancer cell lines: Effects on cell survival and Mitochondrial Oxidative Metabolism

<div><p>Zinc finger nucleases (ZFN) are powerful tools for editing genes in cells. Here we use ZFNs to interrogate the biological function of <i>ADPGK</i>, which encodes an ADP-dependent glucokinase (ADPGK), in human tumour cell lines. The hypothesis we tested is that ADPGK utilises ADP to phosphorylate glucose under conditions where ATP becomes limiting, such as hypoxia. We characterised two ZFN knockout clones in each of two lines (H460 and HCT116). All four clones had frameshift mutations in all alleles at the target site in exon 1 of <i>ADPGK,</i> and were ADPGK-null by immunoblotting. <i>ADPGK</i> knockout had little or no effect on cell proliferation, but compromised the ability of H460 cells to survive siRNA silencing of hexokinase-2 under oxic conditions, with clonogenic survival falling from 21±3% for the parental line to 6.4±0.8% (p = 0.002) and 4.3±0.8% (p = 0.001) for the two knockouts. A similar increased sensitivity to clonogenic cell killing was observed under anoxia. No such changes were found when <i>ADPGK</i> was knocked out in HCT116 cells, for which the parental line was less sensitive than H460 to anoxia and to hexokinase-2 silencing. While knockout of <i>ADPGK</i> in HCT116 cells caused few changes in global gene expression, knockout of <i>ADPGK</i> in H460 cells caused notable up-regulation of mRNAs encoding cell adhesion proteins. Surprisingly, we could discern no consistent effect on glycolysis as measured by glucose consumption or lactate formation under anoxia, or extracellular acidification rate (Seahorse XF analyser) under oxic conditions in a variety of media. However, oxygen consumption rates were generally lower in the <i>ADPGK</i> knockouts, in some cases markedly so. Collectively, the results demonstrate that <i>ADPGK</i> can contribute to tumour cell survival under conditions of high glycolytic dependence, but the phenotype resulting from knockout of <i>ADPGK</i> is cell line dependent and appears to be unrelated to priming of glycolysis in these lines.</p></div

The creatine kinase pathway is a metabolic vulnerability in EVI1-positive acute myeloid leukemia

Expression of the MECOM (also known as EVI1) proto-oncogene is deregulated by chromosomal translocations in some cases of acute myeloid leukemia (AML) and is associated with poor clinical outcome. Here, through transcriptomic and metabolomic profiling of hematopoietic cells, we reveal that EVI1 overexpression alters cellular metabolism. A screen using pooled short hairpin RNAs (shRNAs) identified the ATP-buffering, mitochondrial creatine kinase CKMT1 as necessary for survival of EVI1-expressing cells in subjects with EVI1-positive AML. EVI1 promotes CKMT1 expression by repressing the myeloid differentiation regulator RUNX1. Suppression of arginine-creatine metabolism by CKMT1-directed shRNAs or by the small molecule cyclocreatine selectively decreased the viability, promoted the cell cycle arrest and apoptosis of human EVI1-positive cell lines, and prolonged survival in both orthotopic xenograft models and mouse models of primary AML. CKMT1 inhibition altered mitochondrial respiration and ATP production, an effect that was abrogated by phosphocreatine-mediated reactivation of the arginine-creatine pathway. Targeting CKMT1 is thus a promising therapeutic strategy for this EVI1-driven AML subtype that is highly resistant to current treatment regimens. Keywords: AML; RUNX1; CKMT1; cyclocreatine; arginine metabolismNational Cancer Institute (U.S.) (NIH 1R35 CA210030-01)Stand Up To CancerBridge ProjectNational Cancer Institute (U.S.) (David H. Koch Institute for Integrative Cancer Research at MIT. Grant P30-CA14051