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Athletic performance is influenced by many factors such as the environment, diet, training and endurance or speed in physical effort and by genetic predisposition. Just a few studies have analyzed the impact of genotypes on physical performance in rugby. The aim of this study was to verify the modulation of genetic influence on rugby-specific physical performance. Twenty-seven elite rugby union players were involved in the study during the in-season phase. Molecular genotyping was performed for: angiotensin-converting enzyme (ACE rs4646994), alfa-actinin-3 (ACTN3 rs1815739) and monocarboxylate transporter 1 (MCT1 rs1049434) and their variants. Lean mass index (from skinfolds), lower-limb explosive power (countermovement jump), agility (505), speed (20 m), maximal aerobic power (Yo-yo intermittent recovery test level 1) and repeated sprint ability (12 × 20 m) were evaluated. In our rugby union players ACE and ACTN3 variants did not show any influence on athletic performance. MCT1 analysis showed that TT-variant players had the highest peak vertical power (p = 0.037) while the ones with the AA genotype were the fastest in both agility and sprint tests (p = 0.006 and p = 0.012, respectively). Considering the T-dominant model, the AA genotype remains the fastest in both tests (agility: p = 0.013, speed: p = 0.017). Only the MCT1 rs1049434 A allele seems to be advantageous for elite rugby union players, particularly when power and speed are required.

Background We evaluated the therapeutic potential of combining the monocarboxylate transporter 1 (MCT1) inhibitor AZD3965 with the mitochondrial respiratory Complex I inhibitor IACS-010759, for the treatment of diffuse large B-cell lymphoma (DLBCL), a potential clinically actionable strategy to target tumour metabolism. Methods AZD3965 and IACS-010759 sensitivity were determined in DLBCL cell lines and tumour xenograft models. Lactate concentrations, oxygen consumption rate and metabolomics were examined as mechanistic endpoints. In vivo plasma concentrations of IACS-010759 in mice were determined by LC-MS to select a dose that reflected clinically attainable concentrations. Results In vitro, the combination of AZD3965 and IACS-010759 is synergistic and induces DLBCL cell death, whereas monotherapy treatments induce a cytostatic response. Significant anti-tumour activity was evident in Toledo and Farage models when the two inhibitors were administered concurrently despite limited or no effect on the growth of DLBCL xenografts as monotherapies. Conclusions This is the first study to examine a combination of two distinct approaches to targeting tumour metabolism in DLBCL xenografts. Whilst nanomolar concentrations of either AZD3965 or IACS-010759 monotherapy demonstrate anti-proliferative activity against DLBCL cell lines in vitro, appreciable clinical activity in DLBCL patients may only be realised through their combined use.

Background: Studies on functional roles of BACH1 reveal that BACH1 promotes cancer metastasis and regulates metabolic networks for metastatic processes. However, little is known about BACH1 protein expression in breast tumors and its relevance to clinical variables as a biomarker for patients with breast tumors., Methods: Using a tissue microarray (TMA) of breast tumor tissues isolated from a patient cohort (N = 130) expression of BACH1 and its target gene MCT1 (encoded by SLC16A1) were monitored by immunohistochemistry (IHC) assays and scored for further analyses. We examined the association between scores of BACH1 (Allredscoretotal) or MCT1 (Hscoretotal3×2×1x) with clinical variables including: breast cancer subtypes, tissue types, tumor size, patient's racial/ethnic background, and age group. Groups were compared using the Mann-Whitney U test (or the non-parametric Kruskal-Wallis test when appropriate) for numerical data. A proportional odds ordinal logistic model was used to examine multiple covariates. Associations between variables were evaluated with the Spearman's correlation coefficient., Results: BACH1 and MCT1 expression were detected in 90.76% (N = 118/130) and 92.30% (N = 120/130) of patients by IHC, respectively, in our study. After dichotomizing tumor size (small: 3-25 in diameter vs. big: 27-85 mm in diameter), BACH1 expression scores were significantly higher ( p = 0.015) in the bigger tumor group (mean [SD]; 4.20 [1.796]) compared with the smaller tumor group (3.920 [1.693]). Of interest, we also observed significantly higher BACH1 scores ( p = 0.004) in tumors from Black women (3.971 [1.514]; N = 69) compared with those of White women (3.02 [1.942]; N = 49). Consistent with mRNA expression analysis, BACH1 expression is most abundant in the basal-like tumors among all subtypes, specifically in Black women, whereas MCT1 expression scores are considerably higher in the basal-like tumors regardless of race. In addition, there was a positive association between BACH1 and MCT1 IHC scores in tumors from Black women, although a weak association between them in tumors from White women. In general, we did not detect associations between MCT1 IHC scores and race, tumor size, tissue types, or patient's age., Conclusions: We found strong associations of BACH1 expression with tumor size and the basal-like subtype, respectively. Importantly, BACH1 expresses significantly higher in tumors from Black women than White women, as well as in the basal-like subtype of breast tumors from Black women. Our study suggests that BACH1 expression could serve as a potential race-associated biomarker indicating poor prognosis., Competing Interests: Competing interests: All authors declare no competing interests

Dysregulation of cellular pH is frequent in solid tumours and provides potential opportunities for therapeutic intervention. The acidic microenvironment within a tumour can promote migration, invasion and metastasis of cancer cells through a variety of mechanisms. Pathways associated with the control of intracellular pH that are under consideration for intervention include carbonic anhydrase IX (CAIX), the monocarboxylate transporters (MCT1 and MCT4), the vacuolar-type H+-ATPase proton pump (V-ATPase), and the sodium-hydrogen exchanger 1 (NHE1). This review will describe progress in the development of inhibitors to these targets.

BackgroundAkt signalling regulates glycolysis and drives the Warburg effect in cancer, thus decreased glucose utilisation is a pharmacodynamic marker of Akt inhibition. However, cancer cells can utilise alternative nutrients to glucose for energy such as lactate, which is often elevated in tumours together with increased acidity. We therefore hypothesised that lactic acidosis may confer resistance to Akt inhibition.MethodsThe effect of the pan-Akt inhibitor uprosertib (GSK2141795), on HCT116 and LS174T colon cancer cells was evaluated in the presence and absence of lactic acid in vitro. Expression of downstream Akt signalling proteins was determined using a phosphokinase array and immunoblotting. Metabolism was assessed using1H nuclear magnetic resonance spectroscopy, stable isotope labelling and gas chromatography-mass spectrometry.ResultsLactic acid-induced resistance to uprosertib was characterised by increased cell survival and reduced apoptosis. Uprosertib treatment reduced Akt signalling and glucose uptake irrespective of lactic acid supplementation. However, incorporation of lactate carbon and enhanced respiration was maintained in the presence of uprosertib and lactic acid. Inhibiting lactate transport or oxidative phosphorylation was sufficient to potentiate apoptosis in the presence of uprosertib.ConclusionsLactic acidosis confers resistance to uprosertib, which can be reversed by inhibiting lactate transport or oxidative metabolism.

The oral antidiabetic, metformin, has been documented to have antineoplastic activity in various cancer cell lines, associated with the inhibition of mitochondrial complex I. However, pre-clinical studies have struggled to demonstrate such antineoplastic activity utilising metformin concentrations attainable with standard antidiabetic dosing. Research has revealed that metformin combined with the anti-hypertensive drug, syrosingopine, lowers metformin’s therapeutic threshold and sensitizes cancer cells to killing, by inhibiting monocarboxylate transporters (MCT)1/MCT4. The potent interaction between these drugs elicits a synthetic lethality, specific to transformed cells. This preliminary study aimed to investigate metformin’s antineoplastic effects and its synergistic relationship with syrosingopine by measuring cell viability and extracellular lactate in PC3 (adherent) and THP-1 (suspension) cancer cell lines. Overall, the PC3 cell line responded better to treatment with combined doses of metformin and syrosingopine, or either drug alone; however, synthetic lethality was not observed in either cell line. In both cell lines, the interaction between metformin and syrosingopine was not statistically significant in the cell viability assays (p>0.05). Analysis of MCT1/MCT4 inhibition through the measurement of extracellular lactate was not statistically significant (p>0.05) and proved inconclusive. Additionally, the nature of the cell line, adherent or suspension, was statistically significant (p

Monocarboxylate transporters (MCTs) are cell membrane proteins transporting lactate, pyruvate, and ketone bodies across the plasma membrane. The prognostic role of MCTs in neuroendocrine tumors is unknown. We aimed to analyze MCT1 and MCT4 expression in small bowel neuroendocrine tumors (SB-NETs). The cohort included 109 SB-NETs and 61 SB-NET lymph node metastases from two Finnish hospitals. Tumor samples were immunohistochemically stained with MCT1 and MCT4 monoclonal antibodies. The staining intensity, percentage of positive cells, and stromal staining were assessed. MCT1 and MCT4 scores (0, 1 or 2) were composed based on the staining intensity and the percentage of positive cells. Survival analyses were performed with the Kaplan–Meier method and Cox regression, adjusted for confounders. The primary outcome was disease-specific survival (DSS). A high MCT4 intensity in SB-NETs was associated with better DSS when compared to low intensity (85.7 vs. 56.6%, p = 0.020). A high MCT4 percentage of positive cells resulted in better DSS when compared to a low percentage (77.4 vs. 49.1%, p = 0.059). MCT4 scores 0, 1, and 2 showed DSS of 52.8 vs. 58.8 vs. 100% (p = 0.025), respectively. After adjusting for confounders, the mortality hazard was lowest in the patients with a high MCT4 score. MCT1 showed no association with survival. According to our study, a high MCT4 expression is associated with an improved prognosis in SB-NETs.

Several genes are involved in sport performance, especially in injuries incidence. The aim of this study was to investigate the association of ACE, ACTN3, COL1A1, and MCT1 genotypes and injuries in rugby players in order to find a genotype/phenotype correlation and provide useful information improving athletic performance. One-hundred male professional and semiprofessional rugby players were selected. Analysis was performed genotyping the genes ACE, ACTN3, COL1A1, and MCT1 as candidate gene of interest involved in athletic performance. A control group of non-athletic Italian male participants was analyzed to compare the results. We found statistical significance of MCT1 rs1049434 AA for total injuries (χ2 = 0.115; p = 0.003) and bone injuries (χ2 = 0.603; p = 0.007) in the rugby athlete population. No statistical significance was found between injury incidence and ACE, ACTN3, COL1A1 genotypes. The MCT1 AA genotype is associated with the incidence of total and bone injuries in the rugby player population. Although environmental factors such as lifestyle, diet, training, and stress can influence athletic performance, our data demonstrated the importance of genetic study in sport aimed at developing personalized training and achieving the best possible athletic excellence.

Oral squamous cell carcinoma (OSCC) is among the leading causes of cancer-associated death worldwide. miR-31 is an oncogenic miRNA in OSCC. NUMB is an adaptor protein capable of suppressing malignant transformation. Disruption of the miR-31-NUMB regulatory axis has been demonstrated in malignancies. Mitochondrial dysfunction and adaptation to glycolytic respiration are frequent events in malignancies. Monocarboxylate transporters (MCTs) function to facilitate lactate flux in highly glycolytic cells. Upregulation of MCT1 and MCT4 has been shown to be a prognostic factor of OSCC. Here, we reported that miR-31-NUMB can modulate glycolysis in OSCC. Using the CRISPR/Cas9 gene editing strategy, we identified increases in oncogenic phenotypes, MCT1 and MCT4 expression, lactate production, and glycolytic respiration in NUMB-deleted OSCC subclones. Transfection of the Numb1 or Numb4 isoform reversed the oncogenic induction elicited by NUMB deletion. This study also showed, for the first time, that NUMB4 binds MCT1 and MCT4 and that this binding increases their ubiquitination, which may decrease their abundance in cell lysates. The disruptions in oncogenicity and metabolism associated with miR-31 deletion and NUMB deletion were partially rescued by MCT1/MCT4 expression or knockdown. This study demonstrated that NUMB is a novel binding partner of MCT1 and MCT4 and that the miR-31-NUMB-MCT1/MCT4 regulatory cascade is present in oral carcinoma.

Gene delivery vehicles currently in the clinic for treatment of monogenic disorders lack sufficient carrying capacity to efficiently address complex polygenic diseases. Thus, to engineer multifaceted genetic circuits for bioengineering human cells as a therapeutic option for polygenic diseases, we require new tools that are currently in their infancy. Mammalian artificial chromosomes, or synthetic chromosomes, represent a viable approach for delivery of large genetic payloads that are mitotically stable and remain independent of the host genome. Previously, we described a mammalian synthetic chromosome platform, termed the ACE system, that requires a single unidirectional integrase for the introduction of multiple genes onto the ACE platform chromosome. In this report, we provide a proof of concept that the ACE synthetic chromosome bioengineering platform is amenable to sequential delivery of off-the-shelf large genomic fragments. Specifically, large genomic clones spanning the human solute carrier family 2, facilitated glucose transporter member 1 (SLC2A1 or GLUT1, 169 kbp), and human monocarboxylate transporter 1 (SLC16A1 or MCT1, 144 kbp) genetic loci were engineered onto the ACE platform and demonstrated to express and correctly splice both gene transcripts. Thus, the ACE system provides a facile and tractable engineering platform for the development of gene-based therapeutic agents targeting polygenic diseases., Graphical Abstract

Malignant glioma represents a fatal disease with a poor prognosis and development of resistance mechanisms against conventional therapeutic approaches. The distinct tumor zones of this heterogeneous neoplasm develop their own microenvironment, in which subpopulations of cancer cells communicate. Adaptation to hypoxia in the center of the expanding tumor mass leads to the glycolytic and angiogenic switch, accompanied by upregulation of different glycolytic enzymes, transporters, and other metabolites. These processes render the tumor microenvironment more acidic, remodel the extracellular matrix, and create energy gradients for the metabolic communication between different cancer cells in distinct tumor zones. Escape mechanisms from hypoxia-induced cell death and energy deprivation are the result. The functional consequences are more aggressive and malignant behavior with enhanced proliferation and survival, migration and invasiveness, and the induction of angiogenesis. In this review, we go from the biochemical principles of aerobic and anaerobic glycolysis over the glycolytic switch, regulated by the key transcription factor hypoxia-inducible factor (HIF)-1α, to other important metabolic players like the monocarboxylate transporters (MCTs)1 and 4. We discuss the metabolic symbiosis model via lactate shuttling in the acidic tumor microenvironment and highlight the functional consequences of the glycolytic switch on glioma malignancy. Furthermore, we illustrate regulation by micro ribonucleic acids (miRNAs) and the connection between isocitrate dehydrogenase (IDH) mutation status and glycolytic metabolism. Finally, we give an outlook about the diagnostic and therapeutic implications of the glycolytic switch and the relation to tumor immunity in malignant glioma.

ObjectivesThis study aims to investigate the underlying mechanisms of KAT2A/MCT1 axis in renal cell carcinoma (RCC), providing potential therapeutic targets.MethodsWe obtained the expression data of KAT2A and MCT1 from The Cancer Genome Atlas Kidney Clear Cell Carcinoma (TCGA-KIRC) and International Cancer Genome Consortium (ICGC) databases. Differential analysis was conducted via the limma package. The CCK8 assay, soft agar assay, clone formation assay, and patients-derived organoid models were used to detect cell growth. The transwell and wound-healing assays were utilized to detect cell migration. The in vitro and in vivo assays were further conducted to assess the oncogenic roles of KAT2A. The transcriptome sequencing and chromatin immunoprecipitation (ChIP) sequencing were conducted to screen KAT2A downstream targets. The dose-effect curves were used to detect the 50% inhibiting concentration (IC50) of AZD3965. Data analysis was performed in the Graphpad Prism (Version 8.3.0) and R software (Version 3.6.1).ResultsOur study found that KAT2A was highly expressed in RCC versus normal samples. Prognostic analysis indicated that a high KAT2A was an independent biomarker and associated with poor survival outcomes. KAT2A could promote RCC proliferation and distal metastasis in vitro and in vivo. Transcriptome analysis and ChIP-seq were combined to find that KAT2A mainly regulated the glycolytic process. Validation and rescue assays revealed that MCT1 was the downstream target of KAT2A, and KAT2A depended on MCT1 to promote RCC malignant phenotypes. Lastly, MCT1 inhibitor (AZD3965) was effective to treat KAT2A-induced RCC progression.ConclusionOur study indicated that KAT2A was an oncogenic chromatin modifier that promotes RCC progression by inducing MCT1 expression. We proposed that MCT1 inhibitor (AZD3965) was useful for suppressing RCC.

A multiplica??o celular descontrolada constitui a ess?ncia do comportamento biol?gico das neoplasias malignas e envolve mecanismos altamente complexos. Eventos celulares relacionados ? respira??o s?o respons?veis pela produ??o de energia e, por isso, essenciais ? sobreviv?ncia celular. A compreens?o dos processos neopl?sicos ? um desafio, e o conhecimento de biomarcadores espec?ficos envolvidos na capta??o energ?tica das c?lulas malignas ? crucial para o melhor entendimento do c?ncer. O presente estudo teve por objetivo investigar a express?o dos transportadores de monocarboxilato 1 (MCT1) e 4 (MCT4), bem como sua rela??o com a taxa de prolifera??o celular (Ki67) e caracter?sticas cl?nicas em diferentes tipos histol?gicos de tumores da cavidade oral. Prontu?rios e esp?cimes de bi?psia de tumores orais foram distribu?dos nos seguintes grupos: (1) 14 linfomas; (2) 5 melanomas; (3) 11 adenocarcinomas (prim?rios e met?stases); (4) 15 carcinomas espinocelulares (CEC); e (5) grupo-controle: 15 hiperplasias fibroepiteliais. Os prontu?rios foram revisados considerando idade e sexo dos pacientes; uso de ?lcool e tabaco; sintomas; e s?tio anat?mico, tamanho e tempo de evolu??o das les?es. Os esp?cimes foram submetidos a processamento imunoistoqu?mico para avaliar a express?o dos marcadores MCT1, MCT4 e Ki-67. De modo geral, os tumores orais avaliados tiveram coexpress?o de MCT1 e MCT4, especialmente o CEC e o melanoma. MCT1 e MCT4 exibiram correla??o positiva entre si; MCT1 tamb?m teve correla??o positiva com Ki67, enquanto o MCT4 n?o teve. Os tr?s marcadores exibiram correla??o positiva com tamanho do tumor, enquanto somente o MCT4 teve correla??o negativa com o tempo de evolu??o. Conclus?o: O perfil de express?o dos marcadores MCT1 e MCT4 corrobora sua rela??o com fatores progn?sticos, bem como a participa??o do efeito Warburg reverso no perfil energ?tico do c?ncer oral. S?o necess?rios novos estudos para subsidiar evid?ncias desses fatores como um novo alvo na terap?utica oncol?gica. Uncontrolled cell proliferation is the essence of biological behavior of malignancies, comprising highly complex mechanisms. Cellular processes related to respiration are responsible for the energy production and, therefore, essential for cell surviving. Understanding tumor biology is a challenging task, and the knowledge of biomarkers involved in the energy processes of cancer cells is extremely important to better understand the cancer itself. The aim of the present study was to analyze the expression of monocarboxylate transporter 1 (MCT1) and 4 (MCT4) and the relationship of these transporters to cell proliferation rate (Ki67) and clinical features in malignant tumors of the oral cavity. Paraffin-embedded oral biopsy specimens were allocated into the following groups: (1) 14 lymphomas; (2) 5 melanomas; (3) 11 adenocarcinomas (primary and metastases); (4) 15 squamous cell carcinomas (SCCs); and (5) 15 fibroepithelial hyperplasias (control group). Medical records were reviewed considering patients? age and sex, alcohol and tobacco use, symptoms, and the anatomical site, evolution time and size of the tumor. The immunohistochemical expression of Ki67, MCT1 and MCT4 was analyzed in the sample. Overall, MCT1 and MCT4 were co-expressed in the oral tumors analyzed, especially in SCC and melanoma. MCT1 and MCT4 were positively correlated to each other; MCT1 was also correlated to Ki67, whereas MCT4 was not. These three markers were positively correlated to tumor size, whereas only MCT4 was negatively correlated to the evolution time of the tumor. Conclusion: MCT1 and MCT4 expression supports reverse Warburg effect as an energy profile in oral cancer and its relationship to prognostic factors. Further studies analyzing the specificities of this relationship are needed to provide evidence of a new target in oral oncology therapeutics. Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES

Rationale: CAV3 (caveolin3) variants associated with arrhythmogenic cardiomyopathy and muscular dystrophy can disrupt post-Golgi surface trafficking. As CAV1 (caveolin1) was recently identified in cardiomyocytes, we hypothesize that conserved isoform-specific protein/protein interactions orchestrate unique cardiomyocyte microdomain functions. To analyze the CAV1 versus CAV3 interactome, we employed unbiased live-cell proximity proteomic, isoform-specific affinity, and complexome profiling mass spectrometry techniques. We demonstrate the physiological relevance and loss-of-function mechanism of a novel CAV3 interactor in gene-edited human induced pluripotent stem cell cardiomyocytes. Objective: To identify differential CAV1 versus CAV3 protein interactions and to define the molecular basis of cardiac CAV3 loss-of-function. Methods and Results: Combining stable isotope labeling with proximity proteomics, we applied mass spectrometry to screen for putative CAV3 interactors in living cardiomyocytes. Isoform-specific affinity proteomic and co-immunoprecipitation experiments confirmed the monocarboxylate transporter McT1 (monocarboxylate transporter type 1) versus aquaporin1, respectively, as CAV3 or CAV1 specific interactors in cardiomyocytes. Superresolution stimulated emission depletion microscopy showed distinct CAV1 versus CAV3 cluster distributions in cardiomyocyte transverse tubules. CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/Cas9 nuclease)-mediated CAV3 knockout uncovered a stabilizing role for McT1 surface expression, proton-coupled lactate shuttling, increased late Na + currents, and early afterdepolarizations in human induced pluripotent stem cell-derived cardiomyocytes. Complexome profiling confirmed that McT1 and the Na,K-ATPase form labile protein assemblies with the multimeric CAV3 complex. Conclusions: Combining the strengths of proximity and affinity proteomics, we identified isoform-specific CAV1 versus CAV3 binding partners in cardiomyocytes. McT1 represents a novel class of metabolically relevant CAV3-specific interactors close to mitochondria in cardiomyocyte transverse tubules. CAV3 knockout uncovered a previously unknown role for functional stabilization of McT1 in the surface membrane of human cardiomyocytes. Strikingly, CAV3 deficient cardiomyocytes exhibit action potential prolongation and instability, reproducing human reentry arrhythmias in silico. Given that lactate is a major substrate for stress adaption both in the healthy and the diseased human heart, future studies of conserved McT1/CAV3 interactions may provide rationales to target this muscle-specific assembly function therapeutically.

Enlarged, hypertrophic adipocytes are less responsive to insulin and are a hallmark feature of obesity, contributing to many of the negative metabolic consequences of excess adipose tissue. Although the mechanisms remain unclear, the adipocyte size appears to be inversely correlated with insulin sensitivity and glucose tolerance, wherein smaller adipocytes are insulin-sensitive and larger adipocytes develop insulin resistance and exhibit an impaired glucose uptake. Thus, pharmacological strategies aimed at regulating adipocyte hypertrophy (increase in adipocyte size) in favor of promoting hyperplasia (increase in adipocyte number) have the potential to improve adipocyte insulin sensitivity and provide therapeutic benefits in the context of metabolic disorders. As white adipose tissue can metabolize large amounts of glucose to lactate, using transcriptomics and in vitro characterization we explore the functional consequences of inhibiting monocarboxylate transporter 1 (MCT1) activity in fully differentiated adipocytes. Our studies show that the pharmacological inhibition of MCT1, a key regulator of the cellular metabolism and proliferation, promotes the re-entry of mature adipocytes into the cell cycle. Furthermore, we demonstrate that inhibitor-treated adipocytes exhibit an enhanced insulin-stimulated glucose uptake as compared with untreated adipocytes, and that this outcome is dependent on the cyclin-dependent kinase 1 (CDK1) activity. In summary, we identify a mechanism though which MCT1 inhibition improves the insulin sensitivity of mature adipocytes by inducing cell cycle re-entry. These results provide the foundation for future studies investigating the role MCT1 plays in adipocyte hyperplasia, and its therapeutic potential as a drug target for obesity and metabolic disease.

Most cancer cells rely on glycolysis to sustain their high proliferation rates with the production of lactate. For many years, lactate was seen as a metabolic waste of glycolytic metabolism in the tumor microenvironment, however, lactate has been recently associated as a key metabolic fuel and as an important signaling molecule 1,2. This substrate is responsible for extracellular acidification, which, is a feature of the tumor environment, and favors tumor invasion. The transport of lactate across the plasma membrane is mediated by a family of proton coupled monocarboxylate transporters (MCTs), which comprises 14 members 3. MCT1 and MCT4 serve as metabolic links between cancer cells via lactate exchange within tumors. This form of metabolic symbiosis illustrates how the apparent waste product from hypoxic tumor cells may be exploited by oxidative tumor cells to sustain their energy production under nutrient deprived conditions 4. MCTs are not only gatekeepers of intercellular metabolic cooperation, but also important regulators of angiogenesis and tumor migration, invasion and metastasis 5 . However, the role of MCTs in tumors is far from being well understood and their potential as therapeutic targets is poorly explored. Given the relationships between MCT1 and MCT4 in cancer cells, they offer a unique opportunity for novel treatment strategies. In this work, a set of molecular tools was generated for the expression and trafficking analyses of MCT1 and MCT4. Plasmids were designed harboring MCT1 or MCT4 with GFP or mCherry at the C- or N- terminal following the classical DNA cloning method. These molecular tools will be essential to study the expression and localization of MCT1 and MCT4 and to study the conditions and mechanisms underlying the endocytic trafficking of both transporters to further elucidate the significance of MCTs expression in tumor cells.

The effect of a cellular prion protein (PrP c ) deficiency on neuroenergetics was primarily analyzed via surveying the expression of genes specifically involved in lactate/pyruvate metabolism, such as monocarboxylate transporters (MCT1, MCT2, MCT4). The aim of the present study was to elucidate a potential involvement of PrP c in the regulation of energy metabolism in different brain regions. By using quantitative real-time polymerase chain reaction (qRT-PCR), we observed a marked reduction in MCT1 mRNA expression in the cortex of symptomatic Zürich I Prnp -/- mice, as compared to their wild-type (WT) counterparts. MCT1 downregulation in the cortex was accompanied with significantly decreased expression of the MCT1 functional interplayer, the Na + /K + ATPase α2 subunit. Conversely, the MCT1 mRNA level was significantly raised in the cerebellum of Prnp -/- vs. WT control group, without a substantial change in the Na + /K + ATPase α2 subunit expression. To validate the observed mRNA findings, we confirmed the observed change in MCT1 mRNA expression level in the cortex at the protein level. MCT4, highly expressed in tissues that rely on glycolysis as an energy source, exhibited a significant reduction in the hippocampus of Prnp -/- vs. WT mice. The present study demonstrates that a lack of PrP c leads to altered MCT1 and MCT4 mRNA/protein expression in different brain regions of Prnp -/- vs. WT mice. Our findings provide evidence that PrP c might affect the monocarboxylate intercellular transport, which needs to be confirmed in further studies.

Dissertação de mestrado em Bioquímica Aplicada (área de especialização em Biomedicina), Cancer is a complex disease that represents a vast problem worldwide due to its high incidence and mortality, being the second leading cause of death globally. Even with the decrease of mortality in many countries, the incidence of this disease will increase, causing serious problems even in countries where health systems are more advanced. So, efforts have been made in order to identify new-targeted therapies. In the past decade, it has been reported that the monocarboxylate transporter MCT1 is highly expressed in multiple cancer types. MCT1 is a symporter that is responsible for the influx of lactate across the cell membrane and, in cancer cells, it supplies their metabolism and their rapid proliferation and expansion. This makes it a potential target molecule for therapy as well as a useful prognostic factor. This project aimed at evaluating the expression and localization of the EGFP-MCT1 transporter, by confocal microscopy, in U2OS cells, edited by the CRISPR-Cas9 technology, and grown under different metabolic conditions or in the presence of specific compounds. In this work we show that the EGFP-MCT1 transporter remains stable at the plasma membrane, in cells incubated in a medium without glucose or glutamine. These results highlight the relevance of expressing nutrient transporters at an endogenous level to study their intracellular trafficking. Additionally, our preliminary studies suggest the involvement of the Wnt signaling pathway in MCT1 internalization. We found that the presence of lithium chloride (LiCl), an activator of the Wnt pathway, also seems to lead to an increased MCT1 internalization, after 24 h of incubation. The results obtained in this study will contribute towards a better understanding of the molecular mechanisms involved in the expression and endocytic regulation of MCT1, and open novel therapeutic possibilities in the fight against tumors., O cancro é uma doença complexa que representa um vasto problema mundial devido ao seu nível de incidência e mortalidade, sendo a segunda causa de morte no mundo. Mesmo com a diminuição da mortalidade em muitos países, a incidência desta doença na população continuará a aumentar, o que irá causar sérios problemas, mesmo em países com sistemas de saúde mais avançados. Assim, vários esforços têm sido feitos a fim de identificar novas terapias direcionadas. Na última década, foi descrito que o transportador de monocarboxilatos MCT1 é altamente expresso em vários tipos de cancro. O MCT1 é um transportador responsável pelo influxo de lactato através da membrana plasmática e, em células cancerígenas, promove o seu metabolismo e a sua rápida proliferação e expansão. Assim, o MCT1 é potencialmente uma molécula alvo que poderá ser usada para fins terapêuticos, bem como um fator de prognóstico útil. Este projeto teve como objetivo avaliar a expressão e localização do transportador EGFP-MCT1, por microscopia confocal, em células U2OS, editas pela tecnologia CRISPR-Cas9, e crescidas em diferentes condições metabólicas ou na presença de compostos específicos. Demonstrámos que o transportador EGFP-MCT1 permanece estável na membrana plasmática, em células incubadas num meio sem glucose ou glutamina. Estes resultados evidenciam a relevância de expressar transportadores de nutrientes a um nível endógeno para estudar o seu tráfego intracelular. Adicionalmente, os nossos estudos preliminares sugerem o envolvimento da via de sinalização Wnt na internalização do MCT1. Verificámos que a presença de cloreto de lítio (LiCl), um ativador da via Wnt, parece levar ao aumento da internalização do MCT1, após 24 h de incubação. Os resultados obtidos neste estudo pretendem contribuir para um melhor entendimento dos mecanismos moleculares envolvidos na expressão e regulação endocítiva de MCT1, e, futuramente, abrir novas possibilidades terapêuticas no combate tumoral.

Dissertação de mestrado em Bioquímica Aplicada (área de especialização em Biomedicina), Most cancer cells rely on glycolysis to sustain their high proliferation rates with the production of lactate. For many years, lactate was seen as a metabolic waste of glycolytic metabolism in the tumor microenvironment, however, lactate has been recently associated as a key metabolic fuel and as an important signaling molecule. This substrate is responsible for extracellular acidification, which, is a feature of the tumor environment, and favors tumor invasion. The transport of lactate across the plasma membrane is mediated by a family of proton coupled monocarboxylate transporters (MCTs), which comprises 14 members. MCT1 and MCT4 serve as metabolic links between cancer cells via lactate exchange within tumors. This form of metabolic symbiosis illustrates how the apparent waste product from hypoxic tumor cells may be exploited by oxidative tumor cells to sustain their energy production under nutrient deprived conditions. MCTs are not only gatekeepers of intercellular metabolic cooperation, but also important regulators of angiogenesis and tumor migration, invasion, and metastasis. However, the role of MCTs in tumors is far from being well understood and their potential as therapeutic targets is poorly explored. Given the relationships between MCT1 and MCT4 in cancer cells, they offer a unique opportunity for novel treatment strategies. In this work, a set of molecular tools was generated for the expression and trafficking analyses of MCT1 and MCT4 transporters. Plasmids were designed harboring MCT1 or MCT4 genes with GFP or mCherry at their 3’- or 5’- ends, following the classical DNA cloning method. These molecular tools will be essential to study the expression and localization of MCT1 and MCT4 and to investigate the conditions and mechanisms underlying the endocytic trafficking of both transporters in tumor cells. Additionally, these constructs can be used for colocalization or interaction studies, with advanced techniques like FRET., A maioria das células cancerígenas depende da glicólise para sustentar altas taxas de proliferação através da produção de lactato. Durante muito tempo, o lactato foi visto como um resíduo metabólico da glicólise no microambiente tumoral, no entanto, recentemente, esta molécula foi destacada como um combustível metabólico chave e uma molécula sinalizadora crucial. O lactato é responsável pela acidificação extracelular, característica do ambiente tumoral e que favorece o crescimento e a invasão das células tumorais. O transporte de lactato através da membrana plasmática, é mediado por uma família de transportadores de monocarboxilatos (MCTs), acoplados a protões que inclui 14 membros. MCT1 e MCT4 estabelecem ligações metabólicas entre as células cancerígenas por meio da troca de lactato. Esta forma de simbiose metabólica ilustra como o produto residual aparente de células tumorais hipóxicas pode ser explorado por células tumorais oxidativas para sustentar a produção de energia sob condições de privação de nutrientes. Os MCTs não são apenas guardiões da cooperação metabólica intercelular, mas também reguladores importantes da angiogénese e da migração, invasão e da metastização. No entanto, o papel dos MCTs no cancro está longe de ser bem compreendido e o seu potencial como alvos terapêuticos é pouco explorado. A relação entre MCT1 e MCT4 em células cancerígenas, oferece, portanto, uma oportunidade única para novas estratégias terapêuticas para o cancro. Deste trabalho, resultou um conjunto de ferramentas moleculares relevantes para o estudo da expressão e análise do tráfego dos transportadores, MCT1 e MCT4. Foram construídos 8 plasmídeos com as sequências dos genes MCT1 ou MCT4 fundidas com os genes que codificam as proteínas fluorescentes, GFP ou mCherry, nas extremidades 3’ ou 5’ dos MCTs, seguindo o método clássico de clonagem de DNA. Essas ferramentas moleculares serão essenciais para estudar a expressão e localização desses transportadores e para avaliar as condições e mecanismos subjacentes ao tráfego endocítico dos transportadores, MCT1 e MCT4, em células tumorais. Além disso, estes plasmídeos podem ser usados para estudos de co-localização ou estudos de interação, com técnicas avançadas como FRET.

Background & aims: Hepatocellular carcinoma (HCC) is a cancer with multiple aetiologies and widespread prevalence. Largely refractory to current treatments, HCC is the fourth leading cause of cancer-related deaths worldwide. MicroRNAs (miRNAs) are important regulators in HCCs. We aimed to identify tumour suppressor miRNAs during tumour regression in a conditional c-MYC-driven mouse model (LT2/MYC) of HCC, and to evaluate their therapeutic potential for HCC treatment. Methods: We performed miRNA expression profiling of developed and regressing LT2/MYC tumours and in-depth in vitro gain- and loss-of-function analyses. The effect of adeno-associated virus (AAV) vector-mediated miR-342-3p treatment was evaluated in 3 HCC mouse models. Results: We identified miR-342-3p as a tumour suppressor miRNA in HCC, with increased expression in regressing tumours. Forced miR-342-3p expression in hepatoma cells showed significantly decreased cell proliferation, migration, and colony formation. In vivo administration of AAV-miR-342-3p led to significant attenuation of tumour development and increased overall survival. We identified monocarboxylic acid transporter 1 (MCT1) as a bona fide target of miR-342-3p in HCC. We show that the tumour suppressor role of miR-342-3p is executed partly by modulating the lactate transport function of MCT1. Importantly, we find miR-342-3p downregulated in tumours from patients with HCC compared with matched non-tumour tissues, inversely correlating with MCT1 expression. We observed similar findings in TCGA-LIHC data. Conclusions: In our study, we identified and validated miR-342-3p as a tumour suppressor miRNA in HCC. We demonstrated its therapeutic efficacy in significantly attenuating tumour development, and prolonging survival, in different HCC mouse models. Identification of miR-342-3p as an effective tumour suppressor opens a therapeutic avenue for miRNA-mediated attenuation of HCC development. Lay summary: Hepatocellular carcinoma (HCC), the most common type of liver cancer, affects diverse populations and has a global impact, being the fourth leading cause of cancer deaths worldwide. There are currently no systemic therapies for HCC that can significantly prolong long-term survival. Thus, novel effective treatment options are urgently required. To understand the molecular basis of tumour regression, we compared tumours and regressing liver tumours in mice. We show that a small non-coding miRNA, miR-342-3p, is a tumour suppressor in HCC. Expression of miR-342-3p is low in tumours and high in regressing tumours. When miR-342-3p is delivered to mouse livers with HCC, it can significantly slow down liver tumour development and improve survival. Our study highlights the promising therapeutic potential of miR-342-3p intervention in HCC. Deutsche Krebshilfe

(1) Background: Metabolic reprogramming has been postulated to be one of the hallmarks of cancer, thus representing a promising therapeutic target also in glioblastoma multiforme (GBM). Hypoxic tumor cells produce lactate, and monocarboxylate transporters (MCTs) play an important role in its distribution, (2) Methods: We examined the distribution of lactate by multi voxel magnetic resonance spectroscopic imaging and ELISA in glioblastoma multiforme (GBM) patients. In addition, we investigated the expression and cellular localization of MCT1, MCT4, and of several markers connected to tumor progression by quantitative PCR and immunofluorescence double-staining in human GBM ex vivo tissues, (3) Results: The highest lactate concentration was found at the center of the vital parts of the tumor. Three main GBM groups could be distinguished according to their regional gene expression differences of the investigated genes. MCT1 and MCT4 were found on cells undergoing epithelial to mesenchymal transition and on tumor stem-like cells. GBM cells revealing an expression of cellular dormancy markers, showed positive staining for MCT4, (4) Conclusion: Our findings indicate the existence of individual differences in the regional distribution of MCT1 and MCT4 and suggest that both transporters have distinct connections to GBM progression processes, which could contribute to the drug resistance of MCT-inhibitors.

Metabolism in acute myeloid leukemia (AML) cells is dependent primarily on oxidative phosphorylation. However, in order to sustain their high proliferation rate and metabolic demand, leukemic blasts use a number of metabolic strategies, including glycolytic metabolism. Understanding whether monocarboxylate transporters MCT1 and MCT4, which remove the excess of lactate produced by cancer cells, represent new hematological targets, and whether their respective inhibitors, AR-C155858 and syrosingopine, can be useful in leukemia therapy, may reveal a novel treatment strategy for patients with AML. We analyzed MCT1 and MCT4 expression and function in hematopoietic progenitor cells from healthy cord blood, in several leukemic cell lines and in primary leukemic blasts from patients with AML, and investigated the effects of AR-C155858 and syrosingopine, used alone or in combination with arabinosylcytosine, on leukemic cell proliferation. We found an inverse correlation between MCT1 and MCT4 expression levels in leukemic cells, and showed that MCT4 overexpression is associated with poor prognosis in AML patients. We also found that AR-C155858 and syrosingopine inhibit leukemic cell proliferation by activating two different cell-death related pathways, i.e., necrosis for AR-C155858 treatment and autophagy for syrosingopine, and showed that AR-C155858 and syrosingopine exert an anti-proliferative effect, additive to chemotherapy, by enhancing leukemic cells sensitivity to chemotherapeutic agents. Altogether, our study shows that inhibition of MCT1 or MCT4 impairs leukemic cell proliferation, suggesting that targeting lactate metabolism may be a new therapeutic strategy for AML, and points to MCT4 as a potential therapeutic target in AML patients and to syrosingopine as a new anti-proliferative drug and inducer of autophagy to be used in combination with conventional chemotherapeutic agents in AML treatment.

Summary Endothelial cell (EC)-derived signals contribute to organ regeneration, but angiocrine metabolic communication is not described. We found that EC-specific loss of the glycolytic regulator pfkfb3 reduced ischemic hindlimb revascularization and impaired muscle regeneration. This was caused by the reduced ability of macrophages to adopt a proangiogenic and proregenerative M2-like phenotype. Mechanistically, loss of pfkfb3 reduced lactate secretion by ECs and lowered lactate levels in the ischemic muscle. Addition of lactate to pfkfb3-deficient ECs restored M2-like polarization in an MCT1-dependent fashion. Lactate shuttling by ECs enabled macrophages to promote proliferation and fusion of muscle progenitors. Moreover, VEGF production by lactate-polarized macrophages was increased, resulting in a positive feedback loop that further stimulated angiogenesis. Finally, increasing lactate levels during ischemia rescued macrophage polarization and improved muscle reperfusion and regeneration, whereas macrophage-specific mct1 deletion prevented M2-like polarization. In summary, ECs exploit glycolysis for angiocrine lactate shuttling to steer muscle regeneration from ischemia., Graphical Abstract, Highlights • Endothelial loss of pfkfb3 impairs ischemic muscle revascularization and regeneration • EC-derived lactate instructs MCT1-dependent macrophage functional polarization • Lactate-polarized macrophages promote muscle revascularization and regeneration • Restoring lactate levels improves macrophage polarization and recovery from ischemia, Endothelial cells (ECs) critically control muscle recovery from ischemia by secreting lactate. Angiocrine lactate is taken up and oxidized by macrophages in an MCT1-dependent fashion. Lactate-mediated macrophage polarization promotes revascularization and muscle regeneration. Consequently, EC-specific loss of pfkfb3 lowers muscle lactate levels and impairs muscle recovery from ischemia.