RESUMO
Malignant tumors exhibit increased dependence on glycolysis, resulting in abundant export of lactic acid, a hypothesized key step in tumorigenesis. Lactic acid is mainly transported by two H(+)/lactate symporters, MCT1/MCT4, that require the ancillary protein CD147/Basigin for their functionality. First, we showed that blocking MCT1/2 in Ras-transformed fibroblasts with AR-C155858 suppressed lactate export, glycolysis, and tumor growth, whereas ectopic expression of MCT4 in these cells conferred resistance to MCT1/2 inhibition and reestablished tumorigenicty. A mutant-derivative, deficient in respiration (res(-)) and exclusively relying on glycolysis for energy, displayed low tumorigenicity. These res(-) cells could develop resistance to MCT1/2 inhibition and became highly tumorigenic by reactivating their endogenous mct4 gene, highlighting that MCT4, the hypoxia-inducible and tumor-associated lactate/H(+) symporter, drives tumorigenicity. Second, in the human colon adenocarcinoma cell line (LS174T), we showed that combined silencing of MCT1/MCT4 via inducible shRNA, or silencing of CD147/Basigin alone, significantly reduced glycolytic flux and tumor growth. However, both silencing approaches, which reduced tumor growth, displayed a low level of CD147/Basigin, a multifunctional protumoral protein. To gain insight into CD147/Basigin function, we designed experiments, via zinc finger nuclease-mediated mct4 and basigin knockouts, to uncouple MCTs from Basigin expression. Inhibition of MCT1 in MCT4-null, Basigin(high) cells suppressed tumor growth. Conversely, in Basigin-null cells, in which MCT activity had been maintained, tumorigenicity was not affected. Collectively, these findings highlight that the major protumoral action of CD147/Basigin is to control the energetics of glycolytic tumors via MCT1/MCT4 activity and that blocking lactic acid export provides an efficient anticancer strategy.
Assuntos
Basigina/metabolismo , Transformação Celular Neoplásica/genética , Glicólise/efeitos dos fármacos , Ácido Láctico/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas Musculares/metabolismo , Subunidades Proteicas/metabolismo , Simportadores/metabolismo , Basigina/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos dos fármacos , Primers do DNA/genética , Citometria de Fluxo , Imunofluorescência , Técnicas de Inativação de Genes , Inativação Gênica , Humanos , Imuno-Histoquímica , Transportadores de Ácidos Monocarboxílicos/antagonistas & inibidores , Transportadores de Ácidos Monocarboxílicos/genética , Proteínas Musculares/genética , Consumo de Oxigênio/fisiologia , Subunidades Proteicas/genética , Simportadores/antagonistas & inibidores , Simportadores/genética , Tiofenos/farmacologia , Uracila/análogos & derivados , Uracila/farmacologiaRESUMO
Accumulation of HIF-1alpha during normoxic conditions at high cell density has previously been shown to occur and can be used to stabilize HIF-1alpha protein in the absence of a specific anaerobic chamber. However, the impact and origin of this pool of HIF-1alpha, obtained under normoxia, has been underestimated. In this study, we have systematically compared the related pools of HIF-1alpha stabilized in normoxia by high cell density to those obtained at low density in hypoxia. At first glance, these two stimuli appear to have similar outcomes: HIF-1alpha stabilization and induction of HIF-1-dependent genes. However, upon careful analysis, we observed that molecular mechanisms involved are different. We clearly demonstrate that density-dependant HIF-1alpha accumulation during normoxia is due to the cells high consumption of oxygen, as demonstrated by using a respiration inhibitor (oligomycin) and respiratory-defective mutant cells (GSK3). Finally and most importantly, our data indicate that a decrease in AKT activity followed by a total decrease in p70(S6K) phosphorylation reflecting a decrease in mTOR activity occurs during high oxygen consumption, resulting from high cell density. In contrast, hypoxia, even at severe low O(2) levels, only slightly impacts upon the mTOR pathway under low cell density conditions. Thus, activation of HIF-1alpha in exponentially growing cells via hypoxic stimulation is independent of the Akt/mTOR pathway whereas HIF-1alpha activation obtained in high confluency is totally dependent on mTOR pathway as rapamycin totally impaired (i) HIF-1alpha stabilization and (ii) mRNA levels of CA9 and BNIP3, two HIF-target genes.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Contagem de Células , Hipóxia Celular/fisiologia , Linhagem Celular , Proliferação de Células , Cricetinae , Cricetulus , Células HeLa , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Consumo de Oxigênio , Interferência de RNA , RNA Interferente Pequeno/genética , Sirolimo/farmacologia , Serina-Treonina Quinases TORRESUMO
Mitogen-activated protein (MAP) kinase phosphatases (MKPs) are dual-specificity phosphatases that dephosphorylate phosphothreonine and phosphotyrosine residues within MAP kinases. Here, we describe a novel posttranslational mechanism for regulating MKP-3/Pyst1/DUSP6, a member of the MKP family that is highly specific for extracellular signal-regulated kinase 1 and 2 (ERK1/2) inactivation. Using a fibroblast model in which the expression of either MKP-3 or a more stable MKP-3-green fluorescent protein (GFP) chimera was induced by tetracycline, we found that serum induces the phosphorylation of MKP-3 and its subsequent degradation by the proteasome in a MEK1 and MEK2 (MEK1/2)-ERK1/2-dependent manner. In vitro phosphorylation assays using glutathione S-transferase (GST)-MKP-3 fusion proteins indicated that ERK2 could phosphorylate MKP-3 on serines 159 and 197. Tetracycline-inducible cell clones expressing either single or double serine mutants of MKP-3 or MKP-3-GFP confirmed that these two sites are targeted by the MEK1/2-ERK1/2 module in vivo. Double serine mutants of MKP-3 or MKP-3-GFP were more efficiently protected from degradation than single mutants or wild-type MKP-3, indicating that phosphorylation of either serine by ERK1/2 enhances proteasomal degradation of MKP-3. Hence, double mutation caused a threefold increase in the half-life of MKP-3. Finally, we show that the phosphorylation of MKP-3 has no effect on its catalytic activity. Thus, ERK1/2 exert a positive feedback loop on their own activity by promoting the degradation of MKP-3, one of their major inactivators in the cytosol, a situation opposite to that described for the nuclear phosphatase MKP-1.
Assuntos
Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Serina/metabolismo , Animais , Linhagem Celular , Fosfatase 6 de Especificidade Dupla , Estabilidade Enzimática , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Mutagênese Sítio-Dirigida , Fosforilação , Proteínas Tirosina Fosfatases/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/fisiologiaRESUMO
The function of the hypoxia-inducible factor-1 (HIF-1), the key transcription factor involved in cellular adaptation to hypoxia, is restricted to low oxygen tension (pO(2)). As such, this transcription factor is central in modulating the tumor microenvironment, sensing nutrient availability, and controlling anaerobic glycolysis, intracellular pH, and cell survival. Degradation and inhibition of the limiting HIF-1alpha subunit are intimately connected in normoxia. Hydroxylation of two proline residues by prolyl hydroxylase domain (PHD) 2 protein earmarks the protein for degradation, whereas hydroxylation of an asparagine residue by factor-inhibiting HIF-1 (FIH-1 or FIH) reduces its transcriptional activity. Indeed, silencing of either PHD2 or FIH in normoxia partially induced hypoxic genes, whereas combined PHD2/FIH silencing generated a full hypoxic gene response. Given the fact that HIF-1alpha possesses two transcriptional activation domains [TAD; NH(2)-terminal (N-TAD) and COOH-terminal (C-TAD)], we hypothesized on a possible bifunctional activity of HIF-1alpha that could be discriminated by FIH, an inhibitor of the C-TAD. In human cell lines engineered to overexpress or silence FIH in response to tetracycline, we show by quantitative reverse transcription-PCR that a set of hypoxic genes (ca9, phd3, pgk1, and bnip3) respond differently toward FIH expression. This finding, extended to 26 hypoxia-induced genes, indicates differential gene expression by the N-TAD and C-TAD in response to the hypoxic gradient. We propose that the oxygen-sensitive attenuator FIH, together with two distinct TADs, is central in setting the gene expression repertoire dictated by the cell pO(2).
Assuntos
Regulação Neoplásica da Expressão Gênica/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Proteínas Repressoras/fisiologia , Fatores de Transcrição/fisiologia , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Inativação Gênica , Células HeLa , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Prolina Dioxigenases do Fator Induzível por Hipóxia , Oxigenases de Função Mista , Pró-Colágeno-Prolina Dioxigenase/antagonistas & inibidores , Pró-Colágeno-Prolina Dioxigenase/genética , Estrutura Terciária de Proteína , RNA Interferente Pequeno/genética , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/biossíntese , Proteínas Repressoras/genética , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Ativação Transcricional , TransfecçãoRESUMO
Lactic acid generated by highly glycolytic tumours is exported by the MonoCarboxylate Transporters, MCT1 and MCT4, to maintain pHi and energy homeostasis. We report that MCT1 inhibition combined with Mct4 gene disruption severely reduced glycolysis and tumour growth without affecting ATP levels. Because of the key role of the 5'-AMP-activated protein kinase (AMPK) in energy homeostasis, we hypothesized that targeting glycolysis (MCT-blockade) in AMPK-null (Ampk(-/-)) cells should kill tumour cells from 'ATP crisis'. We show that Ampk(-/-)-Ras-transformed mouse embryonic fibroblasts (MEFs) maintained ATP levels and viability when glycolysis was inhibited. In MCT-inhibited MEFs treated with OXPHOS inhibitors the ATP level and viability collapsed in both Ampk(+/+) and Ampk(-/-) cells. We therefore propose that the intracellular acidification resulting from lactic acid sequestration mimicks AMPK by blocking mTORC1, a major component of an ATP consuming pathway, thereby preventing 'ATP crisis'. Finally we showed that genetic disruption of Mct4 and/or Ampk dramatically reduced tumourigenicity in a xenograft mouse model suggesting a crucialrolefor these two actors in establishment of tumours in a nutrient-deprived environment. These findings demonstrated that blockade of lactate transport is an efficient anti-cancer strategy that highlights the potential in targeting Mct4 in a context of impaired AMPK activity.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Trifosfato de Adenosina/metabolismo , Transformação Celular Neoplásica/metabolismo , Fibroblastos/patologia , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas Musculares/metabolismo , Animais , Linhagem Celular Tumoral , Técnicas de Inativação de Genes , Glicólise/fisiologia , Xenoenxertos , Humanos , Imuno-Histoquímica , Camundongos , Camundongos NusRESUMO
Rapidly growing glycolytic tumors require energy and intracellular pH (pHi) homeostasis through the activity of two major monocarboxylate transporters, MCT1 and the hypoxia-inducible MCT4, in intimate association with the glycoprotein CD147/BASIGIN (BSG). To further explore and validate the blockade of lactic acid export as an anticancer strategy, we disrupted, via zinc finger nucleases, MCT4 and BASIGIN genes in colon adenocarcinoma (LS174T) and glioblastoma (U87) human cell lines. First, we showed that homozygous loss of MCT4 dramatically sensitized cells to the MCT1 inhibitor AZD3965. Second, we demonstrated that knockout of BSG leads to a decrease in lactate transport activity of MCT1 and MCT4 by 10- and 6-fold, respectively. Consequently, cells accumulated an intracellular pool of lactic and pyruvic acids, magnified by the MCT1 inhibitor decreasing further pHi and glycolysis. As a result, we found that these glycolytic/MCT-deficient cells resumed growth by redirecting their metabolism toward OXPHOS. Third, we showed that in contrast with parental cells, BSG-null cells became highly sensitive to phenformin, an inhibitor of mitochondrial complex I. Phenformin addition to these MCT-disrupted cells in normoxic and hypoxic conditions induced a rapid drop in cellular ATP-inducing cell death by "metabolic catastrophe." Finally, xenograft analysis confirmed the deleterious tumor growth effect of MCT1/MCT4 ablation, an action enhanced by phenformin treatment. Collectively, these findings highlight that inhibition of the MCT/BSG complexes alone or in combination with phenformin provides an acute anticancer strategy to target highly glycolytic tumors. This genetic approach validates the anticancer potential of the MCT1 and MCT4 inhibitors in current development.
Assuntos
Basigina/genética , Neoplasias do Colo/terapia , Hipoglicemiantes/farmacologia , Transportadores de Ácidos Monocarboxílicos/genética , Fenformin/farmacologia , Animais , Basigina/metabolismo , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Glicólise , Humanos , Masculino , Camundongos , Camundongos Nus , Transportadores de Ácidos Monocarboxílicos/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
While hypoxia-inducible factor (HIF) is a major actor in the cell survival response to hypoxia, HIF also is associated with cell death. Several studies implicate the HIF-induced putative BH3-only proapoptotic genes bnip3 and bnip3l in hypoxia-mediated cell death. We, like others, do not support this assertion. Here, we clearly demonstrate that the hypoxic microenvironment contributes to survival rather than cell death by inducing autophagy. The ablation of Beclin1, a major actor of autophagy, enhances cell death under hypoxic conditions. In addition, the ablation of BNIP3 and/or BNIP3L triggers cell death, and BNIP3 and BNIP3L are crucial for hypoxia-induced autophagy. First, while the small interfering RNA-mediated ablation of either BNIP3 or BNIP3L has little effect on autophagy, the combined silencing of these two HIF targets suppresses hypoxia-mediated autophagy. Second, the ectopic expression of both BNIP3 and BNIP3L in normoxia activates autophagy. Third, 20-mer BH3 peptides of BNIP3 or BNIP3L are sufficient in initiating autophagy in normoxia. Herein, we propose a model in which the atypical BH3 domains of hypoxia-induced BNIP3/BNIP3L have been designed to induce autophagy by disrupting the Bcl-2-Beclin1 complex without inducing cell death. Hypoxia-induced autophagy via BNIP3 and BNIP3L is clearly a survival mechanism that promotes tumor progression.
Assuntos
Autofagia/fisiologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Hipóxia Celular , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteínas de Membrana/metabolismo , Fragmentos de Peptídeos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteína Beclina-1 , Linhagem Celular Tumoral , Fibroblastos/citologia , Fibroblastos/fisiologia , Regulação da Expressão Gênica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Alinhamento de Sequência , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genéticaRESUMO
Hypoxia-inducible factor (HIF), a transcriptional complex conserved from Caenorhabditis elegans to vertebrates, plays a pivotal role in cellular adaptation to low oxygen availability. In normoxia, the HIF-alpha subunits are targeted for destruction by prolyl hydroxylation, a specific modification that provides recognition for the E3 ubiquitin ligase complex containing the von Hippel-Lindau tumour suppressor protein (pVHL). Three HIF prolyl-hydroxylases (PHD1, 2 and 3) were identified recently in mammals and shown to hydroxylate HIF-alpha subunits. Here we show that specific 'silencing' of PHD2 with short interfering RNAs is sufficient to stabilize and activate HIF-1alpha in normoxia in all the human cells investigated. 'Silencing' of PHD1 and PHD3 has no effect on the stability of HIF-1alpha either in normoxia or upon re-oxygenation of cells briefly exposed to hypoxia. We therefore conclude that, in vivo, PHDs have distinct assigned functions, PHD2 being the critical oxygen sensor setting the low steady-state levels of HIF-1alpha in normoxia. Interestingly, PHD2 is upregulated by hypoxia, providing an HIF-1-dependent auto-regulatory mechanism driven by the oxygen tension.