RESUMEN
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.
Asunto(s)
Arginina , Desoxiadenosinas , Purina-Nucleósido Fosforilasa , Arginina/análogos & derivados , Proteínas de Unión al ADN/metabolismo , Humanos , Metionina/metabolismo , Metilación , Poliaminas , Proteína-Arginina N-Metiltransferasas/genética , Proteína-Arginina N-Metiltransferasas/metabolismo , Purina-Nucleósido Fosforilasa/genética , Purina-Nucleósido Fosforilasa/metabolismo , Proteínas de Unión al ARN/metabolismo , Eliminación de Secuencia , TionucleósidosRESUMEN
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.
Asunto(s)
Homocistinuria , Humanos , Ratones , Animales , Homocistinuria/tratamiento farmacológico , Homocistinuria/genética , Metionina/metabolismo , Homocisteína , Cistationina betasintasa/genética , Cistationina betasintasa/metabolismo , Racemetionina , Tracto Gastrointestinal/metabolismoRESUMEN
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.
Asunto(s)
Cistationina betasintasa/genética , Dependovirus/genética , Terapia Genética , Vectores Genéticos/genética , Homocistinuria/genética , Homocistinuria/terapia , Animales , Cistationina betasintasa/sangre , Cistationina betasintasa/deficiencia , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Técnicas de Transferencia de Gen , Vectores Genéticos/administración & dosificación , Homocistinuria/metabolismo , Hígado/metabolismo , Hígado/patología , Masculino , Ratones , Ratones Noqueados , FenotipoRESUMEN
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.
Asunto(s)
Cistationina betasintasa/genética , Homocistinuria/genética , Alelos , Animales , Bortezomib/farmacología , Análisis Mutacional de ADN , Femenino , Homocisteína/sangre , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Inhibidores de Proteasoma/química , Piridoxina/químicaRESUMEN
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.
Asunto(s)
Regulación de la Expresión Génica , Linfoma de Células T/patología , Linfocitos T/citología , Factores de Transcripción/genética , Animales , Incidencia , Linfoma de Células T/genética , Ratones , Receptores de Antígenos de Linfocitos T/genética , Receptores Notch/metabolismo , Transducción de Señal , TransgenesRESUMEN
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.
Asunto(s)
Biomarcadores de Tumor/sangre , Carcinoma de Células Renales/sangre , Carcinoma de Células Renales/diagnóstico , Histidina/sangre , Neoplasias Renales/sangre , Neoplasias Renales/diagnóstico , Triptófano/sangre , Carcinoma de Células Renales/cirugía , Estudios de Casos y Controles , Humanos , Neoplasias Renales/cirugía , Modelos BiológicosRESUMEN
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.
Asunto(s)
Cistationina betasintasa/genética , Metilación de ADN/genética , Epigénesis Genética/genética , Homocistinuria/genética , Animales , Cistationina betasintasa/metabolismo , ADN/genética , Modelos Animales de Enfermedad , Epigenómica/métodos , Homocisteína/genética , Homocisteína/metabolismo , Homocistinuria/metabolismo , Riñón/metabolismo , Hígado/metabolismo , Ratones , S-Adenosilhomocisteína/metabolismo , S-Adenosilmetionina/metabolismoRESUMEN
T lymphocytes develop into two major lineages characterized by expression of the alphabeta and gammadelta T cell receptor (TCR) heterodimers. Within each major lineage, further specialization occurs, resulting in distinct subsets that differ in TCR specificity, phenotype and functional attributes. Thus, in the murine thymus, two distinct subsets of mature (CD24-) gammadelta cells have been identified, that is NK1.1+ cells, which are enriched for Vgamma1.1 usage and selectively produce IFNgamma on stimulation, and CCR6+ cells, which are enriched for Vgamma2 usage produce IL17. The upstream signals and transcriptional pathways that promote development of these distinct gammadelta subsets remain relatively poorly understood. Here, we show that the Zn-finger transcription factor ThPOK has a critical function in the development of gammadelta thymocytes. Thus, lack of functional ThPOK causes a marked reduction in the percentage and absolute number of mature gammadelta thymocytes, and a particularly severe reduction of NK1.1+ cells. Conversely, constitutive ThPOK expression leads to a striking increase in mature NK1.1+ gammadelta thymocytes. Further, we show that ThPOK induction in gammadelta thymocytes is induced by strong TCR signals mediated by engagement with antibody or high-affinity endogenous ligands, and that an important ThPOK cis-acting element, the distal regulatory element (DRE), is sufficient for this TCR-dependent induction. These results show that ThPOK expression in gammadelta thymocytes is regulated in part by the strength of TCR signalling, identify ThPOK as an important mediator of gammadelta T cell development/maturation, and lend strong support to the view that development of a significant fraction of gammadelta T cells depends on TCR engagement/signalling.
Asunto(s)
Antígeno CD24/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T/inmunología , Timo/citología , Factores de Transcripción/inmunología , Animales , Antígeno CD24/genética , Activación de Linfocitos/inmunología , Ratones , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Transducción de Señal/inmunología , Timo/inmunología , Factores de Transcripción/genéticaRESUMEN
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.
Asunto(s)
Gelatinasas/antagonistas & inhibidores , Gelatinasas/inmunología , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/inmunología , Serina Endopeptidasas/inmunología , Anticuerpos de Cadena Única/inmunología , Animales , Afinidad de Anticuerpos , Endopeptidasas , Citometría de Flujo , Gelatinasas/genética , Gelatinasas/metabolismo , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Terapia Molecular Dirigida , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/inmunología , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Biblioteca de Péptidos , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/metabolismo , Resonancia por Plasmón de SuperficieRESUMEN
Hirschsprung disease (HSCR) is a multigenic, congenital disorder that affects 1 in 5,000 newborns and is characterized by the absence of neural crest-derived enteric ganglia in the colon. One of the primary genes affected in HSCR encodes the G protein-coupled endothelin receptor-B (EDNRB). The expression of Ednrb is required at a defined time period during the migration of the precursors of the enteric nervous system (ENS) into the colon. In this study, we describe a conserved spatiotemporal ENS enhancer of Ednrb. This 1-kb enhancer is activated as the ENS precursors approach the colon, and partial deletion of this enhancer at the endogenous Ednrb locus results in pigmented mice that die postnatally from megacolon. We identified binding sites for SOX10, an SRY-related transcription factor associated with HSCR, in the Ednrb ENS enhancer, and mutational analyses of these sites suggested that SOX10 may have multiple roles in regulating Ednrb in the ENS.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Proteínas del Grupo de Alta Movilidad/fisiología , Proteínas de Neoplasias/fisiología , Receptores de Endotelina/genética , Animales , Secuencia de Bases , Elementos de Facilitación Genéticos , Sistema Nervioso Entérico/fisiología , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Factores de Transcripción SOXE , Homología de Secuencia de Ácido Nucleico , Factores de TranscripciónRESUMEN
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.
Asunto(s)
Cistationina betasintasa , Homocistinuria , Humanos , Ratones , Animales , Cistationina betasintasa/genética , Cistationina betasintasa/metabolismo , Inhibidores de Proteasoma/farmacología , Inhibidores de Proteasoma/uso terapéutico , Homocistinuria/tratamiento farmacológico , Homocistinuria/genética , Homocistinuria/metabolismo , Bortezomib/farmacología , Bortezomib/uso terapéutico , Complejo de la Endopetidasa Proteasomal , Ratones TransgénicosRESUMEN
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.
Asunto(s)
Embrión de Mamíferos/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Línea Celular , Proliferación Celular , Embrión de Mamíferos/citología , Matriz Extracelular/genética , Fibroblastos/citología , Fibronectinas/genética , Fibronectinas/metabolismo , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Proteína de Unión al GTP cdc42/genética , Quinasas p21 Activadas/genética , Quinasas p21 Activadas/metabolismoRESUMEN
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.
RESUMEN
MED13L syndrome is a haploinsufficiency developmental disorder characterized by intellectual disability, heart malformation, and hypotonia. MED13L controls transcription by tethering the cyclin C-Cdk8 kinase module (CKM) to the Mediator complex. In addition, cyclin C has CKM-independent roles in the cytoplasm directing stress-induced mitochondrial fragmentation and regulated cell death. Unstressed MED13L S1497 F/fs patient fibroblasts exhibited aberrant cytoplasmic cyclin C localization, mitochondrial fragmentation, and a 6-fold reduction in respiration. In addition, the fibroblasts exhibited reduced mtDNA copy number, reduction in mitochondrial membrane integrity, and hypersensitivity to oxidative stress. Finally, transcriptional analysis of MED13L mutant fibroblasts revealed reduced mRNA levels for several genes necessary for normal mitochondrial function. Pharmacological or genetic approaches preventing cyclin C-mitochondrial localization corrected the fragmented mitochondrial phenotype and partially restored organelle function. In conclusion, this study found that mitochondrial dysfunction is an underlying defect in cells harboring the MED13L S1497 F/fs allele and identified cyclin C mis-localization as the likely cause. These results provide a new avenue for understanding this disorder.
RESUMEN
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.
Asunto(s)
Adenocarcinoma/patología , Proteínas de la Matriz Extracelular/metabolismo , Matriz Extracelular/fisiología , Fibroblastos/enzimología , Gelatinasas/fisiología , Proteínas de la Membrana/fisiología , Invasividad Neoplásica/patología , Proteínas de Neoplasias/fisiología , Neoplasias Pancreáticas/patología , Serina Endopeptidasas/fisiología , Microambiente Tumoral/fisiología , Adenocarcinoma/enzimología , Animales , Western Blotting , Neoplasias de la Mama/patología , Técnicas de Cultivo de Célula , Línea Celular Tumoral/enzimología , Línea Celular Tumoral/patología , Movimiento Celular , Colágeno Tipo I/metabolismo , Endopeptidasas , Matriz Extracelular/ultraestructura , Fibronectinas/metabolismo , Fibronectinas/ultraestructura , Quinasa 1 de Adhesión Focal/fisiología , Gelatinasas/genética , Humanos , Integrina beta1/fisiología , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos ICR , Ratones SCID , Células 3T3 NIH/enzimología , Neoplasias Pancreáticas/enzimología , Proteínas Recombinantes de Fusión/fisiología , Serina Endopeptidasas/genética , Imagen de Lapso de Tiempo , Trasplante HeterólogoRESUMEN
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.
Asunto(s)
Islas de CpG/genética , Cistationina betasintasa/genética , Metilación de ADN/genética , Inflamación/genética , Mutación/genética , Neoplasias Gástricas/genética , Animales , Línea Celular Tumoral , Células Epiteliales/metabolismo , Células Epiteliales/patología , Eliminación de Gen , Humanos , Intestinos/patología , Metaplasia , Ratones Transgénicos , Fenotipo , Proteoma/metabolismo , Transcriptoma/genéticaRESUMEN
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.
Asunto(s)
Cistationina betasintasa/genética , ADN Circular/genética , Terapia Genética , Vectores Genéticos/genética , Homocistinuria/genética , Homocistinuria/terapia , Animales , Biomarcadores , Cistationina betasintasa/sangre , Cistationina betasintasa/deficiencia , ADN Circular/administración & dosificación , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Técnicas de Transferencia de Gen , Vectores Genéticos/administración & dosificación , Homocistinuria/metabolismo , Hígado/metabolismo , Hígado/patología , Masculino , Ratones , Ratones Noqueados , Fenotipo , Transfección/métodos , Resultado del TratamientoRESUMEN
PURPOSE: Human fibroblast activation protein (FAP)/seprase is a 97-kDa surface glycoprotein expressed on tumor associated fibroblasts in the majority of epithelial cancers including colon adenocarcinomas. FAP overexpression in human tumor cells has been shown to promote tumor growth in animal models, and clinical trials targeting FAP enzymatic activity have been initiated. The primary objective of this study was to evaluate the clinical significance of stromal FAP in human colon cancers by immunohistochemisty. EXPERIMENTAL DESIGN: Sections of paraffin-embedded resected primary human colon cancer specimens from 1996 through 2001 within the Fox Chase Cancer Center tumor bank were stained with D8 antibody directed against FAP/seprase. Xenotransplanted human colorectal tumors in mice were examined similarly for stromal FAP in tumors of different sizes. Overall percentage of stromal FAP staining of the primary tumor was assessed semiquantitatively (0, 1+, 2+, 3+) and staining intensity was also graded (none, weak, intermediate, strong). Survival time and time to recurrence data were analyzed using Kaplan-Meier plots, log-rank tests, and Cox proportional hazards models. RESULTS: One hundred thirty-eight patients with resected specimens were available for study (mean follow-up, 1,050 days) with 6 (4%) stage I, 52 (38%) stage II, 43 (31%) stage III, and 37 (27%) stage IV patients. FAP was detected in >93% of specimens. Semiquantitative staining was scored as 1+ in 28 (20%), 2+ in 52 (38%), and 3+ in 49 (35%). FAP staining intensity was graded as weak in 45 (33%), intermediate in 48 (35%), and dark in 36 (26%). Stromal FAP was found to correlate inversely with tumor stage (semiquantitative, P = 0.01; intensity, P = 0.009) and with tumor size of the tumor xenograft model (correlation coefficient, -0.61; P = 0.047), suggesting that stromal FAP may have a greater role in the early development of tumors. Furthermore, greater stromal FAP for patients with known metastatic disease was associated with a decreased survival. CONCLUSION: Our data indicate that patients whose colon tumors have high levels of stromal FAP are more likely to have aggressive disease progression and potential development of metastases or recurrence. This study affirms the rationale for ongoing clinical investigations using FAP as a therapeutic target to disrupt FAP-driven tumor progression in patients with metastatic disease. It also suggests that the effects of FAP inhibition should be investigated in earlier-stage tumors, given its high levels and potential effect earlier in the course of the disease.
Asunto(s)
Adenocarcinoma/genética , Antígenos de Neoplasias/genética , Biomarcadores de Tumor/genética , Neoplasias del Colon/genética , Regulación Neoplásica de la Expresión Génica , Serina Endopeptidasas/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/mortalidad , Adenocarcinoma/patología , Adulto , Anciano , Anciano de 80 o más Años , Animales , Antígenos de Neoplasias/metabolismo , Biomarcadores de Tumor/metabolismo , Neoplasias del Colon/metabolismo , Neoplasias del Colon/mortalidad , Neoplasias del Colon/patología , Endopeptidasas , Femenino , Gelatinasas , Células HT29 , Humanos , Inmunohistoquímica , Masculino , Proteínas de la Membrana , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Persona de Mediana Edad , Serina Endopeptidasas/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
Asunto(s)
Adenina/análogos & derivados , Desoxiadenosinas/farmacología , Proteínas Asociadas a Microtúbulos/antagonistas & inhibidores , Tionucleósidos/farmacología , Adenina/metabolismo , Adenina/farmacología , Animales , Línea Celular , Línea Celular Tumoral , Homocigoto , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Células 3T3 NIH , Purina-Nucleósido Fosforilasa/metabolismo , Eliminación de Secuencia/efectos de los fármacos , Tioguanina/farmacologíaRESUMEN
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.