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Rapidly proliferative processes in mammalian tissues including tumorigenesis and embryogenesis rely on the glycolytic pathway for energy and biosynthetic precursors. The enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) plays an important regulatory role in glycolysis by activating the key rate-limiting glycolytic enzyme, 6-phosphofructo-1-kinase (PFK-1). We have previously determined that decreased PFKFB3 expression reduced glycolysis and growth in transformed cells in vitro and suppressed xenograft growth in vivo. In earlier studies, we created a constitutive knockout mouse to interrogate the function of PFKFB3 in vivo but failed to generate homozygous offspring due to the requirement for PFKFB3 for embryogenesis. We have now developed a novel transgenic mouse model that exhibits inducible homozygous pan-tissue Pfkfb3 gene deletion (Pfkfb3fl/fl). We have induced Pfkfb3 genomic deletion in these mice and found that it effectively decreased PFKFB3 expression and activity. To evaluate the functional consequences of Pfkfb3 deletion in vivo, we crossed Cre-bearing Pfkfb3fl/fl mice with oncogene-driven tumor models and found that Pfkfb3 deletion markedly decreased their glucose uptake and growth. In summary, our studies reveal a critical regulatory function for PFKFB3 in glycolysis and tumorigenesis in vivo and characterize an effective and powerful model for further investigation of its role in multiple biological processes.
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Recurrence of metastatic breast cancer stemming from acquired endocrine and chemotherapy resistance remains a health burden for women with luminal (ER+) breast cancer. Disseminated ER+ tumor cells can remain viable but quiescent for years to decades. Contributing factors to metastatic spread include the maintenance and expansion of breast cancer stem cells (CSCs). Breast CSCs frequently exist as a minority population in therapy resistant tumors. In this study, we show that cytoplasmic complexes composed of steroid receptor (SR) co-activators, PELP1 and SRC-3, modulate breast CSC expansion through upregulation of the HIF-activated metabolic target genes PFKFB3 and PFKFB4. Seahorse metabolic assays demonstrated that cytoplasmic PELP1 influences cellular metabolism by increasing both glycolysis and mitochondrial respiration. PELP1 interacts with PFKFB3 and PFKFB4 proteins, and inhibition of PFKFB3 and PFKFB4 kinase activity blocks PELP1-induced tumorspheres and protein-protein interactions with SRC-3. PFKFB4 knockdown inhibited in vivo emergence of circulating tumor cell (CTC) populations in mammary intraductal (MIND) models. Application of PFKFB inhibitors in combination with ER targeted therapies blocked tumorsphere formation in multiple models of advanced breast cancer including tamoxifen (TamR) and paclitaxel (TaxR) resistant models, murine tumor cells, and ER+ patient-derived organoids (PDxO). Together, our data suggest that PELP1, SRC-3, and PFKFBs cooperate to drive ER+ tumor cell populations that include CSCs and CTCs. Identifying non-ER pharmacological targets offers a useful approach to blocking metastatic escape from standard of care ER/estrogen (E2)-targeted strategies to overcome endocrine and chemotherapy resistance.
Assuntos
Neoplasias da Mama/genética , Proteínas Correpressoras/genética , Resistencia a Medicamentos Antineoplásicos/genética , Coativador 3 de Receptor Nuclear/genética , Fosfofrutoquinase-2/genética , Receptores de Estrogênio/genética , Fatores de Transcrição/genética , Animais , Mama/patologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Estrogênios/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Células MCF-7 , Camundongos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Paclitaxel/farmacologia , Fosforilação/genética , Tamoxifeno/farmacologia , Regulação para Cima/genéticaRESUMO
Constitutive activation of the epidermal growth factor receptor (EGFR) because of somatic mutations of the EGFR gene is commonly observed in tumors of non-small cell lung cancer (NSCLC) patients. Consequently, tyrosine kinase inhibitors (TKI) targeting the EGFR are among the most effective therapies for patients with sensitizing EGFR mutations. Clinical responses to the EGFR-targeting TKIs are evaluated through 2-[18F]fluoro-2-deoxy-glucose (18FDG)-PET uptake, which is decreased in patients responding favorably to therapy and is positively correlated with survival. Recent studies have reported that EGFR signaling drives glucose metabolism in NSCLC cells; however, the precise downstream effectors required for this EGFR-driven metabolic effect are largely unknown. 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3) is an essential glycolytic regulator that is consistently overexpressed in lung cancer. Here, we found that PFKFB3 is an essential target of EGFR signaling and that PFKFB3 activation is required for glycolysis stimulation upon EGFR activation. We demonstrate that exposing NSCLC cells harboring either WT or mutated EGFR to EGF rapidly increases PFKFB3 phosphorylation, expression, and activity and that PFKFB3 inhibition markedly reduces the EGF-mediated increase in glycolysis. Furthermore, we found that prolonged NSCLC cell exposure to the TKI erlotinib drives PFKFB3 expression and that chemical PFKFB3 inhibition synergizes with erlotinib in increasing erlotinib's anti-proliferative activity in NSCLC cells. We conclude that PFKFB3 has a key role in mediating glucose metabolism and survival of NSCLC cells in response to EGFR signaling. These results support the potential clinical utility of using PFKFB3 inhibitors in combination with EGFR-TKIs to manage NSCLC.
Assuntos
Fosfofrutoquinase-2/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Cloridrato de Erlotinib/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glicólise , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Mutação , Fosfofrutoquinase-2/antagonistas & inibidores , Fosfofrutoquinase-2/genética , Fosforilação , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Metabolic alterations are increasingly recognized as important novel anti-cancer targets. Among several regulators of metabolic alterations, fructose 2,6 bisphosphate (F2,6BP) is a critical glycolytic regulator. Inhibition of the active form of PFKFB3ser461 using a novel inhibitor, PFK158 resulted in reduced glucose uptake, ATP production, lactate release as well as induction of apoptosis in gynecologic cancer cells. Moreover, we found that PFK158 synergizes with carboplatin (CBPt) and paclitaxel (PTX) in the chemoresistant cell lines, C13 and HeyA8MDR but not in their chemosensitive counterparts, OV2008 and HeyA8, respectively. We determined that PFK158-induced autophagic flux leads to lipophagy resulting in the downregulation of cPLA2, a lipid droplet (LD) associated protein. Immunofluorescence and co-immunoprecipitation revealed colocalization of p62/SQSTM1 with cPLA2 in HeyA8MDR cells uncovering a novel pathway for the breakdown of LDs promoted by PFK158. Interestingly, treating the cells with the autophagic inhibitor bafilomycin A reversed the PFK158-mediated synergy and lipophagy in chemoresistant cells. Finally, in a highly metastatic PTX-resistant in vivo ovarian mouse model, a combination of PFK158 with CBPt significantly reduced tumor weight and ascites and reduced LDs in tumor tissue as seen by immunofluorescence and transmission electron microscopy compared to untreated mice. Since the majority of cancer patients will eventually recur and develop chemoresistance, our results suggest that PFK158 in combination with standard chemotherapy may have a direct clinical role in the treatment of recurrent cancer.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Autofagia/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Fosfofrutoquinase-2/antagonistas & inibidores , Piridinas/farmacologia , Quinolinas/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose/efeitos dos fármacos , Carboplatina/administração & dosagem , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Inibidores Enzimáticos/uso terapêutico , Feminino , Glicólise/efeitos dos fármacos , Humanos , Gotículas Lipídicas/efeitos dos fármacos , Gotículas Lipídicas/metabolismo , Camundongos Nus , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Paclitaxel/administração & dosagem , Fosfofrutoquinase-2/metabolismo , Piridinas/uso terapêutico , Quinolinas/uso terapêuticoRESUMO
Talimogene laherparepvec is a genetically modified herpes simplex virus type 1-based oncolytic immunotherapy for the local treatment of unresectable subcutaneous and nodal tumors in patients with melanoma recurrent after initial surgery. We report on two patients with melanoma who, after progression on numerous systemic therapies, derived clinical benefit from talimogene laherparepvec in an expanded-access protocol (ClinicalTrials.gov, NCT02147951). Intralesional talimogene laherparepvec (day 1, ≤4 ml 10 PFU/ml; after 3 weeks, ≤4 ml 10 PFU/ml every 2 weeks) was administered until complete response, no injectable tumors, progressive disease, or intolerance occurred. Patient 1 was 71 years old, had stage IIIB disease, and had previously received granulocyte-macrophage colony-stimulating factor, vemurafenib, metformin, ipilimumab, dabrafenib, trametinib, and pembrolizumab. Patient 2 was 45 years old, had stage IIIC disease, and had previously received nivolumab/ipilimumab combination therapy. There were marked reductions in the number and size of melanoma lesions during treatment with talimogene laherparepvec. Both patients experienced mild-to-moderate nausea and vomiting, which were managed using ondansetron, metoclopramide, and pantoprazole. Both patients completed treatment with talimogene laherparepvec in the expanded-access protocol on 24 November 2015, but received talimogene laherparepvec in clinical practice. Patient 1 continues to receive therapy (>60 weeks); patient 2 experienced a complete response at 23 weeks. Immunohistochemistry of a biopsied dermal metastasis from patient 1 showed a marked infiltration of CD4 and CD8 T cells after 1 year of treatment. Talimogene laherparepvec was active in patients with advanced melanoma with disease progression following multiple previous systemic therapies; no new safety signals were identified.
Assuntos
Melanoma/terapia , Terapia Viral Oncolítica/métodos , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Idoso , Terapia Combinada , Progressão da Doença , Herpesvirus Humano 1/fisiologia , Humanos , Masculino , Melanoma/tratamento farmacológico , Melanoma/enzimologia , Melanoma/patologia , Pessoa de Meia-IdadeRESUMO
PURPOSE: Human epidermal growth factor receptor-2 (HER2) has been implicated in the progression of multiple tumor types, including breast cancer, and many downstream effectors of HER2 signaling are primary regulators of cellular metabolism, including Ras and Akt. A key downstream metabolic target of Ras and Akt is the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 isozyme (PFKFB3), whose product, fructose-2,6-bisphosphate (F26BP), is a potent allosteric activator of a rate-limiting enzyme in glycolysis, 6-phosphofructo-1-kinase (PFK-1). We postulate that PFKFB3 may be regulated by HER2 and contribute to HER2-driven tumorigenicity. METHODS: Immunohistochemistry and Kaplan-Meier analysis of HER2+ patient samples investigated the relevance of PFKFB3 in HER2+ breast cancer. In vitro genetic and pharmacological inhibition of PFKFB3 was utilized to determine effects on HER2+ breast cancer cells, while HER2 antagonist treatment assessed the mechanistic regulation on PFKFB3 expression and glucose metabolism. Administration of a PFKFB3 inhibitor in a HER2-driven transgenic breast cancer model evaluated this potential therapeutic approach in vivo. RESULTS: PFKFB3 is elevated in human HER2+ breast cancer and high PFKFB3 transcript correlated with poorer progression-free (PFS) and distant metastatic-free (DFMS) survival. Constitutive HER2 expression led to elevated PFKFB3 expression and increased glucose metabolism, while inhibition of PFKFB3 suppressed glucose uptake, F26BP, glycolysis, and selectively decreased the growth of HER2-expressing breast cancer cells. In addition, treatment with lapatinib, an FDA-approved HER2 inhibitor, decreased PFKFB3 expression and glucose metabolism in HER2+ cells. In vivo administration of a PFKFB3 antagonist significantly suppressed the growth of HER2-driven breast tumors and decreased 18F-2-deoxy-glucose uptake. CONCLUSIONS: Taken together, these data support the potential clinical utility of PFKFB3 inhibitors as chemotherapeutic agents against HER2+ breast cancer.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Glucose/metabolismo , Fosfofrutoquinase-2/antagonistas & inibidores , Receptor ErbB-2/genética , Animais , Antineoplásicos/farmacologia , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Expressão Gênica , Glicólise , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Camundongos , Camundongos Transgênicos , Fosfofrutoquinase-2/genética , Fosfofrutoquinase-2/metabolismo , Prognóstico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor ErbB-2/metabolismo , Carga TumoralRESUMO
Human tumors exhibit increased glucose uptake and metabolism as a result of high demand for ATP and anabolic substrates and this metabolotype is a negative prognostic indicator for survival. Recent studies have demonstrated that cancer cells from several tissue origins and genetic backgrounds require the expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4 (PFKFB4), a regulatory enzyme that synthesizes an allosteric activator of glycolysis, fructose-2,6-bisphosphate. We report the discovery of a first-in-class PFKFB4 inhibitor, 5-(n-(8-methoxy-4-quinolyl)amino)pentyl nitrate (5MPN), using structure-based virtual computational screening. We find that 5MPN is a selective inhibitor of PFKFB4 that suppresses the glycolysis and proliferation of multiple human cancer cell lines but not non-transformed epithelial cells in vitro. Importantly, 5MPN has high oral bioavailability and per os administration of a non-toxic dose of 5MPN suppresses the glucose metabolism and growth of tumors in mice.
Assuntos
Aminoquinolinas/farmacologia , Antineoplásicos/farmacologia , Carcinoma Pulmonar de Lewis/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Glicólise/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Nitratos/farmacologia , Fosfofrutoquinase-2/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Administração Oral , Aminoquinolinas/administração & dosagem , Aminoquinolinas/química , Aminoquinolinas/farmacocinética , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Antineoplásicos/farmacocinética , Disponibilidade Biológica , Carcinoma Pulmonar de Lewis/enzimologia , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/patologia , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Proliferação de Células/efeitos dos fármacos , Desenho Assistido por Computador , Relação Dose-Resposta a Droga , Feminino , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Células HCT116 , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estrutura Molecular , Terapia de Alvo Molecular , Nitratos/administração & dosagem , Nitratos/química , Nitratos/farmacocinética , Fosfofrutoquinase-2/genética , Fosfofrutoquinase-2/metabolismo , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacocinética , Interferência de RNA , Relação Estrutura-Atividade , Transfecção , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Ras pathway mutation is frequent in carcinomas where it induces expression of the transcriptional repressor ZEB1. Although ZEB1 is classically linked to epithelial-mesenchymal transition and tumour metastasis, it has an emerging second role in generation of cancer-initiating cells. Here we show that Ras induction of ZEB1 is required for tumour initiation in a lung cancer model, and we link this function to repression Pten, whose loss is critical for emergence of cancer-initiating cells. These two roles for ZEB1 in tumour progression can be distinguished by their requirement for different levels of ZEB1. A lower threshold of ZEB1 is sufficient for cancer initiation, whereas further induction is necessary for tumour metastasis.
Assuntos
Adenocarcinoma/genética , Adenoma/genética , Transformação Celular Neoplásica/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/genética , Fatores de Transcrição Kruppel-Like/genética , Neoplasias Pulmonares/genética , PTEN Fosfo-Hidrolase/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , RNA Mensageiro/metabolismo , Adenocarcinoma/patologia , Adenoma/patologia , Animais , Modelos Animais de Doenças , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Metástase Neoplásica , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de ZincoRESUMO
Fructose-2,6-bisphosphate (F2,6BP) is a shunt product of glycolysis that allosterically activates 6-phosphofructo-1-kinase (PFK-1) resulting in increased glucose uptake and glycolytic flux to lactate. The F2,6BP concentration is dictated by four bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB1-4) with distinct kinase:phosphatase activities. PFKFB4 is over-expressed in human cancers, induced by hypoxia and required for survival and growth of several cancer cell lines. Although PFKFB4 appears to be a rational target for anti-neoplastic drug development, it is not clear whether its kinase or phosphatase activity is required for cancer cell survival. In this study, we demonstrate that recombinant human PFKFB4 kinase activity is 4.3-fold greater than its phosphatase activity, siRNA and genomic deletion of PFKFB4 decrease F2,6BP, PFKFB4 over-expression increases F2,6BP and selective PFKFB4 inhibition in vivo markedly reduces F2,6BP, glucose uptake and ATP. Last, we find that PFKFB4 is required for cancer cell survival during the metabolic response to hypoxia, presumably to enable glycolytic production of ATP when the electron transport chain is not fully operational. Taken together, our data indicate that the PFKFB4 expressed in multiple transformed cells and tumors functions to synthesize F2,6BP. We predict that pharmacological disruption of the PFKFB4 kinase domain may have clinical utility for the treatment of human cancers.
Assuntos
Adenocarcinoma/metabolismo , Frutosedifosfatos/biossíntese , Glucose/metabolismo , Neoplasias Pulmonares/metabolismo , Fosfofrutoquinase-2/metabolismo , Adenocarcinoma/enzimologia , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Animais , Hipóxia Celular/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Feminino , Glicólise , Células HCT116 , Xenoenxertos , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/patologia , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Oxirredução , FosforilaçãoRESUMO
Estradiol (E2) administered to estrogen receptor-positive (ER(+)) breast cancer patients stimulates glucose uptake by tumors. Importantly, this E2-induced metabolic flare is predictive of the clinical effectiveness of anti-estrogens and, as a result, downstream metabolic regulators of E2 are expected to have utility as targets for the development of anti-breast cancer agents. The family of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB1-4) control glycolytic flux via their product, fructose-2,6-bisphosphate (F26BP), which activates 6-phosphofructo-1-kinase (PFK-1). We postulated that E2 might promote PFKFB3 expression, resulting in increased F26BP and glucose uptake. We demonstrate that PFKFB3 expression is highest in stage III lymph node metastases relative to normal breast tissues and that exposure of human MCF-7 breast cancer cells to E2 causes a rapid increase in [(14)C]glucose uptake and glycolysis that is coincident with an induction of PFKFB3 mRNA (via ER binding to its promoter), protein expression and the intracellular concentration of its product, F26BP. Importantly, selective inhibition of PFKFB3 expression and activity using siRNA or a PFKFB3 inhibitor markedly reduces the E2-mediated increase in F26BP, [(14)C]glucose uptake, and glycolysis. Furthermore, co-treatment of MCF-7 cells with the PFKFB3 inhibitor and the anti-estrogen ICI 182,780 synergistically induces apoptotic cell death. These findings demonstrate for the first time that the estrogen receptor directly promotes PFKFB3 mRNA transcription which, in turn, is required for the glucose metabolism and survival of breast cancer cells. Importantly, these results provide essential preclinical information that may allow for the ultimate design of combinatorial trials of PFKFB3 antagonists with anti-estrogen therapies in ER(+) stage IV breast cancer patients.
Assuntos
Estradiol/farmacologia , Glucose/metabolismo , Fosfofrutoquinase-2/metabolismo , Apoptose/efeitos dos fármacos , Transporte Biológico/efeitos dos fármacos , Neoplasias da Mama/patologia , Sobrevivência Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Estradiol/análogos & derivados , Receptor alfa de Estrogênio/metabolismo , Frutosedifosfatos/metabolismo , Fulvestranto , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Humanos , Metástase Linfática , Células MCF-7 , Fosfofrutoquinase-2/antagonistas & inibidores , Fosfofrutoquinase-2/genética , Elementos de Resposta/efeitos dos fármacos , Elementos de Resposta/genéticaRESUMO
BACKGROUND: Unlike glycolytic enzymes that directly catabolize glucose to pyruvate, the family of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFBs) control the conversion of fructose-6-phosphate to and from fructose-2,6-bisphosphate, a key regulator of the glycolytic enzyme phosphofructokinase-1 (PFK-1). One family member, PFKFB3, has been shown to be highly expressed and activated in human cancer cells, and derivatives of a PFKFB3 inhibitor, 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), are currently being developed in clinical trials. However, the effectiveness of drugs such as 3PO that target energetic pathways is limited by survival pathways that can be activated by reduced ATP and nutrient uptake. One such pathway is the process of cellular self-catabolism termed autophagy. We hypothesized that the functional glucose starvation induced by inhibition of PFKFB3 in tumor cells would induce autophagy as a pro-survival mechanism and that inhibitors of autophagy could increase the anti-tumor effects of PFKFB3 inhibitors. RESULTS: We found that selective inhibition of PFKFB3 with either siRNA transfection or 3PO in HCT-116 colon adenocarcinoma cells caused a marked decrease in glucose uptake simultaneously with an increase in autophagy based on LC3-II and p62 protein expression, acridine orange fluorescence of acidic vacuoles and electron microscopic detection of autophagosomes. The induction of autophagy caused by PFKFB3 inhibition required an increase in reactive oxygen species since N-acetyl-cysteine blocked both the conversion of LC3-I to LC3-II and the increase in acridine orange fluorescence in acidic vesicles after exposure of HCT-116 cells to 3PO. We speculated that the induction of autophagy might protect cells from the pro-apoptotic effects of 3PO and found that agents that disrupt autophagy, including chloroquine, increased 3PO-induced apoptosis as measured by double staining with Annexin V and propidium iodide in both HCT-116 cells and Lewis lung carcinoma (LLC) cells. Chloroquine also increased the anti-growth effect of 3PO against LLCs in vivo and resulted in an increase in apoptotic cells within the tumors. CONCLUSIONS: We conclude that PFKFB3 inhibitors suppress glucose uptake, which in turn causes an increase in autophagy. The addition of selective inhibitors of autophagy to 3PO and its more potent derivatives may prove useful as rational combinations for the treatment of cancer.
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Strategies targeting pathological angiogenesis have focused primarily on blocking vascular endothelial growth factor (VEGF), but resistance and insufficient efficacy limit their success, mandating alternative antiangiogenic strategies. We recently provided genetic evidence that the glycolytic activator phosphofructokinase-2/fructose-2,6-bisphosphatase 3 (PFKFB3) promotes vessel formation but did not explore the antiangiogenic therapeutic potential of PFKFB3 blockade. Here, we show that blockade of PFKFB3 by the small molecule 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) reduced vessel sprouting in endothelial cell (EC) spheroids, zebrafish embryos, and the postnatal mouse retina by inhibiting EC proliferation and migration. 3PO also suppressed vascular hyperbranching induced by inhibition of Notch or VEGF receptor 1 (VEGFR1) and amplified the antiangiogenic effect of VEGF blockade. Although 3PO reduced glycolysis only partially and transiently in vivo, this sufficed to decrease pathological neovascularization in ocular and inflammatory models. These insights may offer therapeutic antiangiogenic opportunities.
Assuntos
Glicólise , Neovascularização Patológica/enzimologia , Fosfofrutoquinase-2/antagonistas & inibidores , Inibidores da Angiogênese/farmacologia , Animais , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/enzimologia , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica/genética , Neovascularização Fisiológica/efeitos dos fármacos , Neovascularização Fisiológica/genética , Fosfofrutoquinase-2/metabolismo , Piridinas/farmacologia , Vasos Retinianos/efeitos dos fármacos , Vasos Retinianos/crescimento & desenvolvimento , Vasos Retinianos/patologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Peixe-ZebraRESUMO
Ras mutations are frequent in cancer cells where they drive proliferation and resistance to apoptosis. However in primary cells, mutant Ras instead can cause oncogene-induced senescence, a tumor suppressor function linked to repression of the polycomb factor Bmi1, which normally regulates cell cycle inhibitory cyclin-dependent kinase inhibitors (cdki). It is unclear how Ras causes repression of Bmi1 in primary cells to suppress tumor formation while inducing the gene in cancer cells to drive tumor progression. Ras also induces the EMT transcription factor ZEB1 to trigger tumor invasion and metastasis. Beyond its well-documented role in EMT, ZEB1 is important for maintaining repression of cdki. Indeed, heterozygous mutation of ZEB1 is sufficient for elevated cdki expression, leading to premature senescence of primary cells. A similar phenotype is evident with Bmi1 mutation. We show that activation of Rb1 in response to mutant Ras causes dominant repression of ZEB1 in primary cells, but loss of the Rb1 pathway is a hallmark of cancer cells and in the absence of such Rb1 repression Ras induces ZEB1 in cancer cells. ZEB1 represses miR-200 in the context of a mutual repression loop. Because miR-200 represses Bmi1, induction of ZEB1 leads to induction of Bmi1. Rb1 pathway status then dictates the opposing effects of mutant Ras on the ZEB1-miR-200 loop in primary versus cancer cells. This loop not only triggers EMT, surprisingly we show it acts downstream of Ras to regulate Bmi1 expression and thus the critical decision between oncogene-induced senescence and tumor initiation.
Assuntos
Transformação Celular Neoplásica/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , MicroRNAs/metabolismo , Neoplasias/metabolismo , Proteína Oncogênica p21(ras)/biossíntese , Complexo Repressor Polycomb 1/biossíntese , Proteínas Proto-Oncogênicas/biossíntese , RNA Neoplásico/metabolismo , Proteína do Retinoblastoma/biossíntese , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Senescência Celular/genética , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Nus , MicroRNAs/genética , Mutação , Neoplasias/genética , Neoplasias/patologia , Proteína Oncogênica p21(ras)/genética , Complexo Repressor Polycomb 1/genética , Proteínas Proto-Oncogênicas/genética , RNA Neoplásico/genética , Proteína do Retinoblastoma/genética , Fatores de Transcrição/genética , Homeobox 1 de Ligação a E-box em Dedo de ZincoRESUMO
It is thought that genomic instability precipitated by Rb1 pathway loss rapidly triggers additional cancer gene mutations, accounting for rapid tumour onset following Rb1 mutation. However, recent whole-genome sequencing of retinoblastomas demonstrated little genomic instability, but instead suggested rapid epigenetic activation of cancer genes. These results raise the possibility that loss of the Rb1 pathway, which is a hallmark of cancers, might be sufficient for cancer initiation. Yet, mutation of the Rb1 family or inactivation of the Rb1 pathway in primary cells has proven insufficient for tumour initiation. Here we demonstrate that traditional nude mouse assays impose an artificial anoikis and proliferation barrier that prevents Rb1 family mutant fibroblasts from initiating tumours. By circumventing this barrier, we show that primary fibroblasts with only an Rb1 family mutation efficiently form sarcomas in nude mice, and a Ras-ZEB1-Akt pathway then causes transition of these tumours to an invasive phenotype.
Assuntos
Transformação Celular Neoplásica/genética , Regulação Neoplásica da Expressão Gênica , Mutação , Proteína do Retinoblastoma/genética , Sarcoma Experimental/genética , Neoplasias Cutâneas/genética , Animais , Anoikis , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos Nus , Invasividade Neoplásica , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína do Retinoblastoma/metabolismo , Sarcoma Experimental/metabolismo , Sarcoma Experimental/patologia , Transdução de Sinais , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Homeobox 1 de Ligação a E-box em Dedo de Zinco , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
Vessel sprouting by migrating tip and proliferating stalk endothelial cells (ECs) is controlled by genetic signals (such as Notch), but it is unknown whether metabolism also regulates this process. Here, we show that ECs relied on glycolysis rather than on oxidative phosphorylation for ATP production and that loss of the glycolytic activator PFKFB3 in ECs impaired vessel formation. Mechanistically, PFKFB3 not only regulated EC proliferation but also controlled the formation of filopodia/lamellipodia and directional migration, in part by compartmentalizing with F-actin in motile protrusions. Mosaic in vitro and in vivo sprouting assays further revealed that PFKFB3 overexpression overruled the pro-stalk activity of Notch, whereas PFKFB3 deficiency impaired tip cell formation upon Notch blockade, implying that glycolysis regulates vessel branching.
Assuntos
Células Endoteliais/metabolismo , Glicólise , Neovascularização Fisiológica , Fosfofrutoquinase-2/metabolismo , Animais , Linhagem Celular Tumoral , Células Cultivadas , Células Endoteliais/citologia , Feminino , Deleção de Genes , Inativação Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosfofrutoquinase-2/genética , Pseudópodes/metabolismo , Peixe-ZebraRESUMO
In human cancers, loss of PTEN, stabilization of hypoxia inducible factor-1α, and activation of Ras and AKT converge to increase the activity of a key regulator of glycolysis, 6-phosphofructo-2-kinase (PFKFB3). This enzyme synthesizes fructose 2,6-bisphosphate (F26BP), which is an activator of 6-phosphofructo-1-kinase, a key step of glycolysis. Previously, a weak competitive inhibitor of PFKFB3, 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), was found to reduce the glucose metabolism and proliferation of cancer cells. We have synthesized 73 derivatives of 3PO and screened each compound for activity against recombinant PFKFB3. One small molecule, 1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one (PFK15), was selected for further preclinical evaluation of its pharmacokinetic, antimetabolic, and antineoplastic properties in vitro and in vivo. We found that PFK15 causes a rapid induction of apoptosis in transformed cells, has adequate pharmacokinetic properties, suppresses the glucose uptake and growth of Lewis lung carcinomas in syngeneic mice, and yields antitumor effects in three human xenograft models of cancer in athymic mice that are comparable to U.S. Food and Drug Administration-approved chemotherapeutic agents. As a result of this study, a synthetic derivative and formulation of PFK15 has undergone investigational new drug (IND)-enabling toxicology and safety studies. A phase I clinical trial of its efficacy in advanced cancer patients will initiate in 2013 and we anticipate that this new class of antimetabolic agents will yield acceptable therapeutic indices and prove to be synergistic with agents that disrupt neoplastic signaling.
Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Neoplasias/metabolismo , Fosfofrutoquinase-2/antagonistas & inibidores , Animais , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/química , Feminino , Glucose/metabolismo , Humanos , Células Jurkat , Camundongos , Modelos Moleculares , Conformação Molecular , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Fosfofrutoquinase-2/química , Fosfofrutoquinase-2/metabolismo , Ligação Proteica , Bibliotecas de Moléculas Pequenas , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Rb1 restricts cell cycle progression, and it imposes cell contact inhibition to suppress tumor outgrowth. It also triggers oncogene-induced senescence to block Ras mutation. Loss of the Rb1 pathway, which is a hallmark of cancer cells, then provides a permissive environment for Ras mutation, and Ras is sufficient for invasive tumor formation in Rb1 family mutant mouse embryo fibroblasts (MEFs). These results demonstrate that sequential mutation of the Rb1 and Ras pathways comprises a tumor initiation axis. Both Rb1 and Ras regulate expression of the transcription factor ZEB1, thereby linking tumor initiation to the subsequent invasion and metastasis, which is induced by ZEB1. ZEB1 acts in a negative feedback loop to block expression of miR-200, which is thought to facilitate tumor invasion and metastasis. However, ZEB1 also represses cyclin-dependent kinase (cdk) inhibitors to control the cell cycle; its mutation in MEFs leads to induction of these inhibitors and premature senescence. Here, we provide evidence for two sequential inductions of ZEB1 during Ras transformation of MEFs. Rb1 constitutively represses cdk inhibitors, and induction of ZEB1 when the Rb1 pathway is lost is required to maintain this repression, allowing for the classic immortalization and loss of cell contact inhibition seen when the Rb1 pathway is lost. In vivo, we show that this induction of ZEB1 is required for Ras-initiated tumor formation. ZEB1 is then further induced by Ras, beyond the level seen with Rb1 mutation, and this Ras superinduction is required to reach a threshold of ZEB1 sufficient for repression of miR-200 and tumor invasion.
Assuntos
Transformação Celular Neoplásica/metabolismo , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Mutação , Neoplasias/metabolismo , Proteína do Retinoblastoma/metabolismo , Proteínas ras/metabolismo , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Senescência Celular/genética , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Regulação Neoplásica da Expressão Gênica/genética , Proteínas de Homeodomínio/genética , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Nus , MicroRNAs/biossíntese , MicroRNAs/genética , Invasividade Neoplásica , Metástase Neoplásica , Neoplasias/genética , Neoplasias/patologia , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Proteína do Retinoblastoma/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco , Proteínas ras/genéticaRESUMO
BACKGROUND: Constitutive activation of Ras in immortalized bronchial epithelial cells increases electron transport chain activity, oxygen consumption and tricarboxylic acid cycling through unknown mechanisms. We hypothesized that members of the Ras family may stimulate respiration by enhancing the expression of the Vb regulatory subunit of cytochrome c oxidase (COX). RESULTS: We found that the introduction of activated H-Ras(V12) into immortalized human bronchial epithelial cells increased eIF4E-dependent COX Vb protein expression simultaneously with an increase in COX activity and oxygen consumption. In support of the regulation of COX Vb expression by the Ras family, we also found that selective siRNA-mediated inhibition of K-Ras expression in A549 lung adenocarcinoma cells reduced COX Vb protein expression, COX activity, oxygen consumption and the steady-state concentration of ATP. We postulated that COX Vb-mediated activation of COX activity may be required for the anchorage-independent growth of A549 cells as soft agar colonies or as lung xenografts. We transfected the A549 cells with COX Vb small interfering or shRNA and observed a significant reduction of their COX activity, oxygen consumption, ATP and ability to grow in soft agar and as poorly differentiated tumors in athymic mice. CONCLUSION: Taken together, our findings indicate that the activation of Ras increases COX activity and mitochondrial respiration in part via up-regulation of COX Vb and that this regulatory subunit of COX may have utility as a Ras effector target for the development of anti-neoplastic agents.
Assuntos
Adenocarcinoma/enzimologia , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Neoplasias Pulmonares/enzimologia , Proteínas ras/metabolismo , Adenocarcinoma/química , Adenocarcinoma/genética , Adenocarcinoma de Pulmão , Trifosfato de Adenosina/metabolismo , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Complexo IV da Cadeia de Transporte de Elétrons/química , Complexo IV da Cadeia de Transporte de Elétrons/genética , Ativação Enzimática , Fator de Iniciação 4E em Eucariotos/metabolismo , Feminino , Humanos , Neoplasias Pulmonares/química , Neoplasias Pulmonares/genética , Camundongos , Camundongos Nus , Consumo de Oxigênio , RNA Interferente Pequeno/genética , Transplante HeterólogoRESUMO
BACKGROUND: We previously found that administration of an interleukin 2/diphtheria toxin conjugate (DAB/IL2; Denileukin Diftitox; ONTAK) to stage IV melanoma patients depleted CD4(+)CD25(HI)Foxp3(+) regulatory T cells and expanded melanoma-specific CD8(+) T cells. The goal of this study was to assess the clinical efficacy of DAB/IL2 in an expanded cohort of stage IV melanoma patients. METHODS: In a single-center, phase II trial, DAB/IL2 (12 µg/kg; 4 daily doses; 21 day cycles) was administered to 60 unresectable stage IV melanoma patients and response rates were assessed using a combination of 2-[(18)F]-fluoro-2-deoxy-glucose (FDG)-positron emission tomography (PET) and computed tomography (CT) imaging. RESULTS: After DAB/IL2 administration, 16.7% of the 60 patients had partial responses, 5% stable disease and 15% mixed responses. Importantly, 45.5% of the chemo/immuno-naïve sub-population (11/60 patients) experienced partial responses. One year survival was markedly higher in partial responders (80 ± 11.9%) relative to patients with progressive disease (23.7 ± 6.5%; p value < 0.001) and 40 ± 6.2% of the total DAB/IL2-treated population were alive at 1 year. CONCLUSIONS: These data support the development of multi-center, randomized trials of DAB/IL2 as a monotherapy and in combination with other immunotherapeutic agents for the treatment of stage IV melanoma. TRIAL REGISTRATION: NCT00299689.