RESUMO
Classical homocystinuria (HCU) is a rare inborn error of amino acid metabolism characterized by accumulation of homocysteine, an intermediate product of methionine metabolism, leading to significant systemic toxicities, particularly within the vascular, skeletal, and ocular systems. Most patients require lifelong dietary therapy with severe restriction of natural protein to minimize methionine intake, and many patients still struggle to maintain healthy homocysteine levels. Since eliminating methionine from the diet reduces homocysteine levels, we hypothesized that an enzyme that can degrade methionine within the gastrointestinal (GI) tract could help HCU patients maintain healthy levels while easing natural protein restrictions. We describe the preclinical development of CDX-6512, a methionine gamma lyase (MGL) enzyme that was engineered for stability and activity within the GI tract for oral administration to locally degrade methionine. CDX-6512 is stable to low pH and intestinal proteases, enabling it to survive the harsh GI environment without enteric coating and to degrade methionine freed from dietary protein within the small intestine. Administering CDX-6512 to healthy non-human primates following a high protein meal led to a dose-dependent suppression of plasma methionine. In Tg-I278T Cbs-/- mice, an animal model that recapitulates aspects of HCU disease including highly elevated serum homocysteine levels, oral dosing of CDX-6512 after a high protein meal led to suppression in serum levels of both methionine and homocysteine. When animals received a daily dose of CDX-6512 with a high protein meal for two weeks, the Tg-I278T Cbs-/- mice maintained baseline homocysteine levels, whereas homocysteine levels in untreated animals increased by 39%. These preclinical data demonstrate the potential of CDX-6512 as an oral enzyme therapy for HCU.
Assuntos
Homocistinúria , Humanos , Camundongos , Animais , Homocistinúria/tratamento farmacológico , Homocistinúria/genética , Metionina/metabolismo , Homocisteína , Cistationina beta-Sintase/genética , Cistationina beta-Sintase/metabolismo , Racemetionina , Trato Gastrointestinal/metabolismoRESUMO
Classic homocystinuria is an inborn error of metabolism caused mainly by missense mutations leading to misfolded and/or unstable human cystathionine ß-synthase (CBS) protein, causing the accumulation of excess total homocysteine (tHcy) in tissues. Previously, it has been shown that certain missense containing human CBS proteins can be functionally rescued in mouse models of CBS deficiency by treatment with proteasome inhibitors. The rescue by proteasome inhibitors is thought to work both by inhibiting the degradation of misfolded CBS protein and by inducing the levels of heat-shock chaperone proteins in the liver. Here we examine the effectiveness of two FDA approved protease inhibitors, carfilzomib and bortezomib, on various transgenic mouse models of human CBS deficiency. Our results show that although both drugs are effective in inducing the liver chaperone proteins Hsp70 and Hsp27, and are effective in inhibiting proteasome function, bortezomib was somewhat more robust in restoring the mutant CBS function. Moreover, there was no significant correlation between proteasome inhibition and CBS activity, suggesting that some of bortezomib's effects are via other mechanisms. We also test the use of low-doses of bortezomib and carfilzomib on various mouse models for extended periods of time and find that while low-doses are less toxic, they are also less effective at restoring CBS function. Overall, these results show that while restoration of mutant CBS function is possible with proteasome inhibitors, the exact mechanism is complicated and it will likely be too toxic for long-term patient treatment.
Assuntos
Cistationina beta-Sintase , Homocistinúria , Humanos , Camundongos , Animais , Cistationina beta-Sintase/genética , Cistationina beta-Sintase/metabolismo , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/uso terapêutico , Homocistinúria/tratamento farmacológico , Homocistinúria/genética , Homocistinúria/metabolismo , Bortezomib/farmacologia , Bortezomib/uso terapêutico , Complexo de Endopeptidases do Proteassoma , Camundongos TransgênicosRESUMO
Methylthioadenosine phosphorylase (MTAP) is a key enzyme in the methionine salvage pathway that converts the polyamine synthesis byproduct 5'-deoxy-5'-methylthioadenosine (MTA) into methionine. Inactivation of MTAP, often by homozygous deletion, is found in both solid and hematologic malignancies and is one of the most frequently observed genetic alterations in human cancer. Previous work established that MTAP-deleted cells accumulate MTA and contain decreased amounts of proteins with symmetric dimethylarginine (sDMA). These findings led to the hypothesis that accumulation of intracellular MTA inhibits the protein arginine methylase (PRMT5) responsible for bulk protein sDMAylation. Here, we confirm that MTAP-deleted cells have increased MTA accumulation and reduced protein sDMAylation. However, we also show that addition of extracellular MTA can cause a dramatic reduction of the steady-state levels of sDMA-containing proteins in MTAP+ cells, even though no sustained increase in intracellular MTA is found because of catabolism of MTA by MTAP. We determined that inhibition of protein sDMAylation by MTA occurs within 48 h, is reversible, and is specific. In addition, we have identified two enhancer-binding proteins, FUBP1 and FUBP3, that are differentially sDMAylated in response to MTAP and MTA. These proteins work via the far upstream element site located upstream of Myc and other promoters. Using a transcription reporter construct containing the far upstream element site, we demonstrate that MTA addition can reduce transcription, suggesting that the reduction in FUBP1 and FUBP3 sDMAylation has functional consequences. Overall, our findings show that extracellular MTA can inhibit protein sDMAylation and that this inhibition can affect FUBP function.
Assuntos
Arginina , Desoxiadenosinas , Purina-Núcleosídeo Fosforilase , Arginina/análogos & derivados , Proteínas de Ligação a DNA/metabolismo , Humanos , Metionina/metabolismo , Metilação , Poliaminas , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Purina-Núcleosídeo Fosforilase/genética , Purina-Núcleosídeo Fosforilase/metabolismo , Proteínas de Ligação a RNA/metabolismo , Deleção de Sequência , TionucleosídeosRESUMO
Elevated rates of glycolysis in cancer cells support tumor growth, in a process that typically depends on oncogene-induced increases in the expression and/or activity of enzymes in the glycolytic pathway. The NEDD9 scaffolding protein is upregulated in many advanced tumors, with increased NEDD9 promoting the activity of SRC and other effectors that promote invasion and metastasis. We here define a new role for NEDD9 in support of glycolysis. NEDD9 knockdown significantly impaired glycolysis in multiple lung cancer cell lines This was accompanied by post-transcriptional downregulation of steady-state levels of hexokinases (HK1 and HK2), which catalyze early steps in the glycolytic cascade, key rate limiting enzyme phosphofructokinase (PFK1), and downstream glyceraldehyde phosphate dehydrogenase (GAPDH). In mice, protein levels of HK1, HK2, PFK1, and GAPDH were depressed in Krastm4Tyj/J /Trp53tm1Brn/J (KP) non-small cell lung tumors with null versus wild type Nedd9. Reciprocally, depletion of HK1 or HK2 elevated NEDD9 expression, as did the treatment of cells with 2-deoxyglucose (2DG), an inhibitor of glycolysis; whereas overexpression of hexokinases promoted NEDD9 dephosphorylation, associated with reduced NEDD9 activity. Together, these data for the first time suggest a negative feedback circuit involving NEDD9 and glycolytic enzymes that may contribute to NEDD9 action in promoting the aggressive growth of advanced tumors.
RESUMO
Cystathionine ß-synthase (CBS) deficiency is a recessive inborn error of sulfur metabolism characterized by elevated blood levels of total homocysteine (tHcy). Patients diagnosed with CBS deficiency are currently treated by a combination of vitamin supplementation and restriction of foods containing the homocysteine precursor methionine, but the effectiveness of this therapy is limited due to poor compliance. A mouse model for CBS deficiency (Tg-I278T Cbs-/- ) was used to evaluate a potential gene therapy approach to treat CBS deficiency utilizing an AAVrh.10-based vector containing the human CBS cDNA downstream of the constitutive, strong CAG promoter (AAVrh.10hCBS). Mice were administered a single dose of virus and followed for up to 1 year. The data demonstrated a dose-dependent increase in liver CBS activity and a dose-dependent decrease in serum tHcy. Liver CBS enzyme activity at 1 year was similar to Cbs+/- control mice. Mice given the highest dose (5.6 × 1011 genomes/mouse) had mean serum tHcy decrease of 97% 1 week after injection and an 81% reduction 1 year after injection. Treated mice had either full- or substantial correction of alopecia, bone loss, and fat mass phenotypes associated with Cbs deficiency in mice. Our findings show that AAVrh.10-based gene therapy is highly effective in treating CBS deficiency in mice and supports additional pre-clinical testing for eventual use human trials.
Assuntos
Cistationina beta-Sintase/genética , Dependovirus/genética , Terapia Genética , Vetores Genéticos/genética , Homocistinúria/genética , Homocistinúria/terapia , Animais , Cistationina beta-Sintase/sangue , Cistationina beta-Sintase/deficiência , Modelos Animais de Doenças , Feminino , Expressão Gênica , Técnicas de Transferência de Genes , Vetores Genéticos/administração & dosagem , Homocistinúria/metabolismo , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Knockout , FenótipoRESUMO
BACKGROUND: CIMP (CpG island methylator phenotype) is an epigenetic molecular subtype, observed in multiple malignancies and associated with the epigenetic silencing of tumor suppressors. Currently, for most cancers including gastric cancer (GC), mechanisms underlying CIMP remain poorly understood. We sought to discover molecular contributors to CIMP in GC, by performing global DNA methylation, gene expression, and proteomics profiling across 14 gastric cell lines, followed by similar integrative analysis in 50 GC cell lines and 467 primary GCs. RESULTS: We identify the cystathionine beta-synthase enzyme (CBS) as a highly recurrent target of epigenetic silencing in CIMP GC. Likewise, we show that CBS epimutations are significantly associated with CIMP in various other cancers, occurring even in premalignant gastroesophageal conditions and longitudinally linked to clinical persistence. Of note, CRISPR deletion of CBS in normal gastric epithelial cells induces widespread DNA methylation changes that overlap with primary GC CIMP patterns. Reflecting its metabolic role as a gatekeeper interlinking the methionine and homocysteine cycles, CBS loss in vitro also causes reductions in the anti-inflammatory gasotransmitter hydrogen sulfide (H2S), with concomitant increase in NF-κB activity. In a murine genetic model of CBS deficiency, preliminary data indicate upregulated immune-mediated transcriptional signatures in the stomach. CONCLUSIONS: Our results implicate CBS as a bi-faceted modifier of aberrant DNA methylation and inflammation in GC and highlights H2S donors as a potential new therapy for CBS-silenced lesions.
Assuntos
Ilhas de CpG/genética , Cistationina beta-Sintase/genética , Metilação de DNA/genética , Inflamação/genética , Mutação/genética , Neoplasias Gástricas/genética , Animais , Linhagem Celular Tumoral , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Deleção de Genes , Humanos , Intestinos/patologia , Metaplasia , Camundongos Transgênicos , Fenótipo , Proteoma/metabolismo , Transcriptoma/genéticaRESUMO
Classical homocystinuria is a recessive inborn error of metabolism caused by mutations in the cystathionine beta-synthase (CBS) gene. The highest incidence of CBS deficiency in the world is found in the country of Qatar due to the combination of high rates of consanguinity and the presence of a founder mutation, c.1006C>T (p.R336C). This mutation does not respond to pyridoxine and is considered severe. Here we describe the creation of a mouse that is null for the mouse Cbs gene and expresses human p.R336C CBS from a zinc-inducible transgene (Tg-R336C Cbs -/- ). Zinc-treated Tg-R336C Cbs -/- mice have extreme elevation in both serum total homocysteine (tHcy) and liver tHcy compared with control transgenic mice. Both the steady-state protein levels and CBS enzyme activity levels in liver lysates from Tg-R336C Cbs -/- mice are significantly reduced compared to that found in Tg-hCBS Cbs -/- mice expressing wild-type human CBS. Treatment of Tg-R336C Cbs -/- mice with the proteasome inhibitor bortezomib results in stabilization of liver CBS protein and an increase in activity to levels found in corresponding Tg-hCBS Cbs -/- wild type mice. Surprisingly, serum tHcy did not fully correct even though liver enzyme activity was as high as control animals. This discrepancy is explained by in vitro enzymatic studies of mouse liver extracts showing that p.R336C causes reduced binding affinity for the substrate serine by almost 7-fold and significantly increased dependence on pyridoxal phosphate in the reaction buffer. These studies demonstrate that the p.R336C alteration effects both protein stability and substrate/cofactor binding.
Assuntos
Cistationina beta-Sintase/genética , Homocistinúria/genética , Alelos , Animais , Bortezomib/farmacologia , Análise Mutacional de DNA , Feminino , Homocisteína/sangue , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Inibidores de Proteassoma/química , Piridoxina/químicaRESUMO
Cystathionine ß-synthase (CBS) deficiency is a recessive inborn error of metabolism characterized by extremely elevated total homocysteine (tHcy) in the blood. Patients diagnosed with CBS deficiency have a variety of clinical problems, including dislocated lenses, osteoporosis, cognitive and behavioral issues, and a significantly increased risk of thrombosis. Current treatment strategies involve a combination of vitamin supplementation and restriction of foods containing the homocysteine precursor methionine. Here, a mouse model for CBS deficiency (Tg-I278T Cbs-/-) was used to evaluate the potential of minicircle-based naked DNA gene therapy to treat CBS deficiency. A 2.3 kb DNA-minicircle containing the liver-specific P3 promoter driving the human CBS cDNA (MC.P3-hCBS) was delivered into Tg-I278T Cbs-/- mice via a single hydrodynamic tail vein injection. Mean serum tHcy decreased from 351 µM before injection to 176 µM 7 days after injection (p = 0.0005), and remained decreased for at least 42 days. Western blot analysis reveals significant minicircle-directed CBS expression in the liver tissue. Liver CBS activity increased 34-fold (12.8 vs. 432 units; p = 0.0004) in MC.P3-hCBS-injected animals. Injection of MC.P3-hCBS in young mice, subsequently followed for 202 days, showed that the vector can ameliorate the mouse homocystinuria alopecia phenotype. The present findings show that minicircle-based gene therapy can lower tHcy in a mouse model of CBS deficiency.
Assuntos
Cistationina beta-Sintase/genética , DNA Circular/genética , Terapia Genética , Vetores Genéticos/genética , Homocistinúria/genética , Homocistinúria/terapia , Animais , Biomarcadores , Cistationina beta-Sintase/sangue , Cistationina beta-Sintase/deficiência , DNA Circular/administração & dosagem , Modelos Animais de Doenças , Feminino , Expressão Gênica , Técnicas de Transferência de Genes , Vetores Genéticos/administração & dosagem , Homocistinúria/metabolismo , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Knockout , Fenótipo , Transfecção/métodos , Resultado do TratamentoRESUMO
Elevated plasma total homocysteine (tHcy) is associated with a number of human diseases including coronary artery disease, stroke, osteoporosis and dementia. It is highly correlated with intracellular S-adenosylhomocysteine (SAH). Since SAH is a strong inhibitor of methyl-transfer reactions involving the methyl-donor S-adenosylmethionine (SAM), elevation in SAH could be an explanation for the wide association of tHcy and human disease. Here, we have created a transgenic mouse (Tg-hAHCY) that expresses human S-adenosylhomocysteine hydrolase (AHCY) from a zinc-inducible promoter in the liver and kidney. Protein analysis shows that human AHCY is expressed well in both liver and kidney, but elevated AHCY enzyme activity (131% increase) is only detected in the kidney due to the high levels of endogenous mouse AHCY expression in liver. Tg-hAHCY mice were crossed with mice lacking cystathionine ß-synthase activity (Tg-I278T Cbs-/- ) to explore the effect to AHCY overexpression in the context of elevated serum tHcy and elevated tissue SAM and SAH. Overexpression of AHCY had no significant effect on the phenotypes of Tg-I278T Cbs-/- mice or any effect on the steady state concentrations of methionine, total homocysteine, SAM, SAH, and SAM/SAH ratio in the liver and kidney. Furthermore, enhanced AHCY activity did not lower serum and tissue tHcy or methionine levels. Our data suggests that enhancing AHCY activity does not alter the distribution of methionine recycling metabolites, even when they are greatly elevated by Cbs mutations.
RESUMO
Homozygous deletion of the methylthioadenosine phosphorylase (MTAP) gene is a frequent event in a wide variety of human cancers and is a possible molecular target for therapy. One potential therapeutic strategy to target MTAP-deleted tumors involves combining toxic purine analogues such as 6'-thioguanine (6TG) or 2'-fluoroadenine (2FA) with the MTAP substrate 5'-deoxy-5'-methylthioadenosine (MTA). The rationale is that excess MTA will protect normal MTAP+ cells from purine analogue toxicity because MTAP catalyzes the conversion of MTA to adenine, which then inhibits the conversion of purine base analogues into nucleotides. However, in MTAP- tumor cells, no protection takes place because adenine is not formed. Here, we examine the effects of 6TG and 2FA in combination with MTA in vitro and in vivoIn vitro, MTA protected against both 6TG and 2FA toxicity in an MTAP-dependent manner, shifting the IC50 concentration by one to three orders of magnitude. However, in mice, MTA protected against toxicity from 2FA but failed to protect against 6TG. Addition of 100 mg/kg MTA to 20 mg/kg 2FA entirely reversed the toxicity of 2FA in a variety of tissues and the treatment was well tolerated by mice. The 2FA+MTA combination inhibited tumor growth of four different MTAP- human tumor cell lines in mouse xenograft models. Our results suggest that 2FA+MTA may be a promising combination for treating MTAP-deleted tumors.Significance: Loss of MTAP occurs in about 15% of all human cancers; the MTAP protection strategy presented in this study could be very effective in treating these cancers. Cancer Res; 78(15); 4386-95. ©2018 AACR.
Assuntos
Adenina/análogos & derivados , Desoxiadenosinas/farmacologia , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Tionucleosídeos/farmacologia , Adenina/metabolismo , Adenina/farmacologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Homozigoto , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Células NIH 3T3 , Purina-Núcleosídeo Fosforilase/metabolismo , Deleção de Sequência/efeitos dos fármacos , Tioguanina/farmacologiaRESUMO
BACKGROUND: In previous work, we showed that serum-free amino acid (SFAA) profiles were different between kidney cancer patients and age and sex matched controls. The goals of the current study are to: (1) confirm our initial observation on an independent sample set; (2) examine if there were similar differences in plasma-free amino acids (PFAA); and (3) determine if removal of tumors changed SFAA and PFAA profiles. METHODS: SFAA and PFAA profiles were measured in 484 samples taken from 124 healthy controls and 56 clear cell renal cell carcinoma (ccRCC) patients both before and after resection of renal tumors. RESULTS: SFAA and PFAA profiles taken from identical blood samples were remarkably different, with the mean individual amino acid concentrations being 40% less in plasma compared to serum. Both SFAA and PFAA profiles differed significantly between ccRCC patients and controls, but the individual amino acids that differed the most, and the direction of the changes, were quite different between the two blood components. Removal of the tumor had almost no effect on either the SFAA or PFAA profiles. A logistic regression model using serum histidine and plasma tryptophan correctly classified 85.5% of control and 84.7% of case samples. CONCLUSIONS: Our findings show that that tumor mass is not directly linked to alterations in blood amino acid levels, and that a combination of serum histidine and plasma tryptophan may be useful as a biomarker to detect ccRCC.
Assuntos
Biomarcadores Tumorais/sangue , Carcinoma de Células Renais/sangue , Carcinoma de Células Renais/diagnóstico , Histidina/sangue , Neoplasias Renais/sangue , Neoplasias Renais/diagnóstico , Triptofano/sangue , Carcinoma de Células Renais/cirurgia , Estudos de Casos e Controles , Humanos , Neoplasias Renais/cirurgia , Modelos BiológicosRESUMO
Cystathionine ß-synthase (CBS) deficiency is a recessive inborn error of metabolism in which patients have extremely elevated plasma total homocysteine and have clinical manifestations in the vascular, visual, skeletal, and nervous systems. Homocysteine is an intermediary metabolite produced from the hydrolysis of S-adenosylhomocysteine (SAH), which is a by-product of methylation reactions involving the methyl-donor S-adenosylmethionine (SAM). Here, we have measured SAM, SAH, DNA and histone methylation status in an inducible mouse model of CBS deficiency to test the hypothesis that homocysteine-related phenotypes are caused by inhibition of methylation due to elevated SAH and reduced SAM/SAH ratio. We found that mice lacking CBS have elevated cellular SAH and reduced SAM/SAH ratios in both liver and kidney, but this was not associated with alterations in the level of 5-methylcytosine or various histone modifications. Using methylated DNA immunoprecipitation in combination with microarray, we found that of the 241 most differentially methylated promoter probes, 89 % were actually hypermethylated in CBS deficient mice. In addition, we did not find that changes in DNA methylation correlated well with changes in RNA expression in the livers of induced and uninduced CBS mice. Our data indicates that reduction in the SAM/SAH ratio, due to loss of CBS activity, does not result in overall hypomethylation of either DNA or histones.
Assuntos
Cistationina beta-Sintase/genética , Metilação de DNA/genética , Epigênese Genética/genética , Homocistinúria/genética , Animais , Cistationina beta-Sintase/metabolismo , DNA/genética , Modelos Animais de Doenças , Epigenômica/métodos , Homocisteína/genética , Homocisteína/metabolismo , Homocistinúria/metabolismo , Rim/metabolismo , Fígado/metabolismo , Camundongos , S-Adenosil-Homocisteína/metabolismo , S-Adenosilmetionina/metabolismoRESUMO
mTOR inhibitors are used to treat metastatic renal cell cancer (RCC), but most patients eventually become resistant. One possible mechanism for resistance is upregulation of autophagy, a pathway that helps recycle intracellular proteins and promotes cell survival. Hydroxychloroquine (HCQ), a potent autophagy inhibitor used to treat malaria and autoimmune disorders, is currently being studied in the context of cancer treatment. Here, we have investigated the effects of HCQ on three different renal carcinoma derived cell lines. We found that HCQ treatment inhibits RCC cell growth, promotes apoptosis, inhibits mitochondrial oxygen consumption, and increases rates of glycolysis. To understand the molecular mechanism behind these effects, we examined various nodes in the mTOR pathway and compared the effects of HCQ with the effects of the mTOR inhibitor RAD001. A key downstream readout of the pathway, phospho-S6 protein, was inhibited by both HCQ and RAD001. However, the upstream kinase, P70S6K was only inhibited by RAD001 and not HCQ, suggesting that the block by HCQ was downstream of P70S6K. Treatment with the proteasome inhibitor bortezomib restored phospho-S6 levels, suggesting that the reduction of phospho-S6 is caused by increased degradation of phospho-S6, but not total S6. Surprisingly, treatment with other autophagy inhibitors did not exhibit the same effects. Our findings suggest that HCQ causes the down-regulation of phospho-S6 in RCC cell lines via a novel mechanism that is not shared with other autophagy inhibitors.
Assuntos
Células Epiteliais/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Hidroxicloroquina/farmacologia , Proteínas Quinases S6 Ribossômicas/antagonistas & inibidores , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Bortezomib/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Everolimo/farmacologia , Glicólise/efeitos dos fármacos , Glicólise/genética , Humanos , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Quinases S6 Ribossômicas/genética , Proteínas Quinases S6 Ribossômicas/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
The transcription factor T-helper-inducing POZ/Krueppel-like factor (ThPOK, encoded by the Zbtb7b gene) plays widespread and critical roles in T-cell development, particularly as the master regulator of CD4 commitment. Here we show that mice expressing a constitutive T-cell-specific ThPOK transgene (ThPOK(const) mice) develop thymic lymphomas. These tumors resemble human T-cell acute lymphoblastic leukemia (T-ALL), in that they predominantly exhibit activating Notch1 mutations. Lymphomagenesis is prevented if thymocyte development is arrested at the DN3 stage by recombination-activating gene (RAG) deficiency, but restored by introduction of a T-cell receptor (TCR) transgene or by a single injection of anti-αßTCR antibody into ThPOK(const) RAG-deficient mice, which promotes development to the CD4(+)8(+) (DP) stage. Hence, TCR signals and/or traversal of the DN (double negative) > DP (double positive) checkpoint are required for ThPOK-mediated lymphomagenesis. These results demonstrate a novel link between ThPOK, TCR signaling, and lymphomagenesis. Finally, we present evidence that ectopic ThPOK expression gives rise to a preleukemic and self-perpetuating DN4 lymphoma precursor population. Our results collectively define a novel role for ThPOK as an oncogene and precisely map the stage in thymopoiesis susceptible to ThPOK-dependent tumor initiation.
Assuntos
Regulação da Expressão Gênica , Linfoma de Células T/patologia , Linfócitos T/citologia , Fatores de Transcrição/genética , Animais , Incidência , Linfoma de Células T/genética , Camundongos , Receptores de Antígenos de Linfócitos T/genética , Receptores Notch/metabolismo , Transdução de Sinais , TransgenesRESUMO
Fibroblast activation protein (FAP) is a serine protease selectively expressed on tumor stromal fibroblasts in epithelial carcinomas and is important in cancer growth, adhesion, and metastases. As FAP enzymatic activity is a potent therapeutic target, we aimed to identify inhibitory antibodies. Using a competitive inhibition strategy, we used phage display techniques to identify 53 single-chain variable fragments (scFvs) after three rounds of panning against FAP. These scFvs were expressed and characterized for binding to FAP by surface plasmon resonance and flow cytometry. Functional assessment of these antibodies yielded an inhibitory scFv antibody, named E3, which could attenuate 35% of FAP cleavage of the fluorescent substrate Ala-Pro-7-amido-4-trifluoromethylcoumarin compared with nonfunctional scFv control. Furthermore, a mutant E3 scFv was identified by yeast affinity maturation. It had higher affinity (4-fold) and enhanced inhibitory effect on FAP enzyme activity (3-fold) than E3. The application of both inhibitory anti-FAP scFvs significantly affected the formation of 3-dimensional FAP-positive cell matrix, as demonstrated by reducing the fibronectin fiber orientation from 41.18% (negative antibody control) to 34.06% (E3) and 36.15% (mutant E3), respectively. Thus, we have identified and affinity-maturated the first scFv antibody capable of inhibiting FAP function. This scFv antibody has the potential to disrupt the role of FAP in tumor invasion and metastasis.
Assuntos
Gelatinases/antagonistas & inibidores , Gelatinases/imunologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/imunologia , Serina Endopeptidases/imunologia , Anticorpos de Cadeia Única/imunologia , Animais , Afinidade de Anticorpos , Endopeptidases , Citometria de Fluxo , Gelatinases/genética , Gelatinases/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Terapia de Alvo Molecular , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/imunologia , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Biblioteca de Peptídeos , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/metabolismo , Ressonância de Plasmônio de SuperfícieRESUMO
Extracellular matrix (ECM) actively participates in normal cell regulation and in the process of tumor progression. The Rho GTPase Cdc42 has been shown to regulate cell-ECM interaction in conventional two-dimensional culture conditions by using dominant mutants of Cdc42 in immortalized cell lines that may introduce nonspecific effects. Here, we employ three-dimensional culture systems for conditional gene targeted primary mouse embryonic fibroblasts that better simulate the reciprocal and adaptive interactions between cells and surrounding matrix to define the role of Cdc42 signaling pathways in ECM organization. Cdc42 deficiency leads to a defect in global cell-matrix interactions reflected by a decrease in collagen gel contraction. The defect is associated with an altered cell-matrix interaction that is evident by morphologic changes and reduced focal adhesion complex formation. The matrix defect is also associated with a reduction in synthesis and activation of matrix metalloproteinase 9 (MMP9) and altered fibronectin deposition patterning. A Cdc42 mutant rescue experiment found that downstream of Cdc42, p21-activated kinase (PAK), but not Par6 or WASP, may be involved in regulating collagen gel contraction and fibronectin organization. Thus, in addition to the previously implicated roles in intracellular regulation of actin organization, proliferation, and vesicle trafficking, Cdc42 is essential in ECM remodeling in three dimensions.
Assuntos
Embrião de Mamíferos/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Linhagem Celular , Proliferação de Células , Embrião de Mamíferos/citologia , Matriz Extracelular/genética , Fibroblastos/citologia , Fibronectinas/genética , Fibronectinas/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Proteína cdc42 de Ligação ao GTP/genética , Quinases Ativadas por p21/genética , Quinases Ativadas por p21/metabolismoRESUMO
Interactions between cancer cells and their microenvironment are crucial for promoting tumor growth and invasiveness. In the tumor adaptive landscape model, hypoxic and acidic microenvironmental conditions reduce the fitness of cancer cells and significantly restrict their proliferation. This selects for enhanced motility as cancer cells may evolve an invasive phenotype if the consequent cell movement is rewarded by proliferation. Here, we used an integrative approach combining a mathematical tumor adaptive landscape model with experimental studies to examine the evolutionary dynamics that promote an invasive cancer phenotype. Computer simulation results hypothesized an explicit coupling of motility and proliferation in cancer cells. The mathematical modeling results were also experimentally examined by selecting Panc-1 cells with enhanced motility on a fibroblast-derived 3-dimensional matrix for cells that move away from the unfavorable metabolic constraints. After multiple rounds of selection, the cells that adapted through increased motility were characterized for their phenotypic properties compared with stationary cells. Microarray and gene depletion studies showed the role of Rho-GDI2 in regulating both cell movement and proliferation. Together, this work illustrates the partnership between evolutionary mathematical modeling and experimental validation as a potentially useful approach to study the complex dynamics of the tumor microenvironment.
Assuntos
Modelos Biológicos , Invasividade Neoplásica , Microambiente Tumoral , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Simulação por Computador , Deleção de Genes , Perfilação da Expressão Gênica , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Humanos , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Inibidores da Dissociação do Nucleotídeo Guanina rho-EspecíficoRESUMO
BACKGROUND: Alterations towards a permissive stromal microenvironment provide important cues for tumor growth, invasion, and metastasis. In this study, Fibroblast activation protein (FAP), a serine protease selectively produced by tumor-associated fibroblasts in over 90% of epithelial tumors, was used as a platform for studying tumor-stromal interactions. We tested the hypothesis that FAP enzymatic activity locally modifies stromal ECM (extracellular matrix) components thus facilitating the formation of a permissive microenvironment promoting tumor invasion in human pancreatic cancer. METHODS: We generated a tetracycline-inducible FAP overexpressing fibroblastic cell line to synthesize an in vivo-like 3-dimensional (3D) matrix system which was utilized as a stromal landscape for studying matrix-induced cancer cell behaviors. A FAP-dependent topographical and compositional alteration of the ECM was characterized by measuring the relative orientation angles of fibronectin fibers and by Western blot analyses. The role of FAP in the matrix-induced permissive tumor behavior was assessed in Panc-1 cells in assorted matrices by time-lapse acquisition assays. Also, FAP+ matrix-induced regulatory molecules in cancer cells were determined by Western blot analyses. RESULTS: We observed that FAP remodels the ECM through modulating protein levels, as well as through increasing levels of fibronectin and collagen fiber organization. FAP-dependent architectural/compositional alterations of the ECM promote tumor invasion along characteristic parallel fiber orientations, as demonstrated by enhanced directionality and velocity of pancreatic cancer cells on FAP+ matrices. This phenotype can be reversed by inhibition of FAP enzymatic activity during matrix production resulting in the disorganization of the ECM and impeded tumor invasion. We also report that the FAP+ matrix-induced tumor invasion phenotype is ß1-integrin/FAK mediated. CONCLUSION: Cancer cell invasiveness can be affected by alterations in the tumor microenvironment. Disruption of FAP activity and ß1-integrins may abrogate the invasive capabilities of pancreatic and other tumors by disrupting the FAP-directed organization of stromal ECM and blocking ß1-integrin dependent cell-matrix interactions. This provides a novel preclinical rationale for therapeutics aimed at interfering with the architectural organization of tumor-associated ECM. Better understanding of the stromal influences that fuel progressive tumorigenic behaviors may allow the effective future use of targeted therapeutics aimed at disrupting specific tumor-stromal interactions.
Assuntos
Adenocarcinoma/patologia , Proteínas da Matriz Extracelular/metabolismo , Matriz Extracelular/fisiologia , Fibroblastos/enzimologia , Gelatinases/fisiologia , Proteínas de Membrana/fisiologia , Invasividade Neoplásica/patologia , Proteínas de Neoplasias/fisiologia , Neoplasias Pancreáticas/patologia , Serina Endopeptidases/fisiologia , Microambiente Tumoral/fisiologia , Adenocarcinoma/enzimologia , Animais , Western Blotting , Neoplasias da Mama/patologia , Técnicas de Cultura de Células , Linhagem Celular Tumoral/enzimologia , Linhagem Celular Tumoral/patologia , Movimento Celular , Colágeno Tipo I/metabolismo , Endopeptidases , Matriz Extracelular/ultraestrutura , Fibronectinas/metabolismo , Fibronectinas/ultraestrutura , Quinase 1 de Adesão Focal/fisiologia , Gelatinases/genética , Humanos , Integrina beta1/fisiologia , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos ICR , Camundongos SCID , Células NIH 3T3/enzimologia , Neoplasias Pancreáticas/enzimologia , Proteínas Recombinantes de Fusão/fisiologia , Serina Endopeptidases/genética , Imagem com Lapso de Tempo , Transplante HeterólogoRESUMO
PURPOSE: Fibroblast Activation Protein (FAP) is a tumor fibroblast protease that has been shown to potentiate colorectal cancer growth. The clinical impact of FAP inhibition was tested using Val-boroPro (Talabostat), the first clinical inhibitor of FAP enzymatic activity, in a phase II study of patients with metastatic colorectal cancer. METHODS: Patients with metastatic colorectal cancer who had previously received systemic chemotherapies were treated with single agent Val-boroPro 200 microg p.o. BID continuously. Eligibility included measurable disease, performance status of 0 to 2, and adequate organ function. Laboratory correlates evaluated the pharmacodynamic effects of Val-boroPro on FAP enzymatic function in the peripheral blood. RESULTS: Twenty-eight patients (median age 62; 12 males, 16 females) were enrolled in this study. There were no objective responses. Six of 28 (21%) patients had stable disease for a median of 25 weeks (range 11-38 weeks). Laboratory analysis demonstrated significant, although incomplete inhibition of FAP enzymatic activity in the peripheral blood. CONCLUSION: This phase II trial of Val-boroPro demonstrated minimal clinical activity in patients with previously treated metastatic colorectal cancer. However it provides the initial proof-of-concept that physiologic inhibition of FAP activity can be accomplished in patients with colorectal cancer, and lays the groundwork for future studies targeting the tumor stroma.