RESUMO
Aberrant activation of GLI transcription factors has been implicated in the pathogenesis of different tumor types including pancreatic ductal adenocarcinoma. However, the mechanistic link with established drivers of this disease remains in part elusive. In this study, using a new genetically engineered mouse model overexpressing constitutively active mouse form of GLI2 and a combination of genome-wide assays, we provide evidence of a novel mechanism underlying the interplay between KRAS, a major driver of pancreatic ductal adenocarcinoma development, and GLI2 to control oncogenic gene expression. These mice, also expressing KrasG12D, show significantly reduced median survival rate and accelerated tumorigenesis compared with the KrasG12D only expressing mice. Analysis of the mechanism using RNA sequencing demonstrate higher levels of GLI2 targets, particularly tumor growth-promoting genes, including Ccnd1, N-Myc, and Bcl2, in KrasG12D mutant cells. Furthermore, chromatin immunoprecipitation sequencing studies showed that in these cells KrasG12D increases the levels of trimethylation of lysine 4 of the histone 3 (H3K4me3) at the promoter of GLI2 targets without affecting significantly the levels of other major active chromatin marks. Importantly, Gli2 knockdown reduces H3K4me3 enrichment and gene expression induced by mutant Kras. In summary, we demonstrate that Gli2 plays a significant role in pancreatic carcinogenesis by acting as a downstream effector of KrasG12D to control gene expression.
Assuntos
Carcinoma Ductal Pancreático , Regulação Neoplásica da Expressão Gênica , Neoplasias Pancreáticas , Proteínas Proto-Oncogênicas p21(ras) , Proteína Gli2 com Dedos de Zinco , Animais , Humanos , Camundongos , Carcinogênese , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Histonas/metabolismo , Histonas/genética , Camundongos Transgênicos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transcrição Gênica , Proteína Gli2 com Dedos de Zinco/genética , Proteína Gli2 com Dedos de Zinco/metabolismoRESUMO
Carcinoma-associated fibroblasts (CAFs) play an important role in the progression of multiple malignancies. Secretion of cytokines and growth factors underlies the pro-tumoral effect of CAFs. Although this paracrine function has been extensively documented, the molecular mechanisms controlling the expression of these factors remain elusive. In this study, we provide evidence of a novel CAF transcriptional axis regulating the expression of SDF1, a major driver of cancer cell migration, involving the transcription factor GLI1 and histone acetyltransferase p300. We demonstrate that conditioned media from CAFs overexpressing GLI1 induce the migration of pancreatic cancer cells, and this effect is impaired by an SDF1-neutralizing antibody. Using a combination of co-immunoprecipitation, proximity ligation assay and chromatin immunoprecipitation assay, we further demonstrate that GLI1 and p300 physically interact in CAFs to co-occupy and drive SDF1 promoter activity. Mapping experiments highlight the requirement of GLI1 N-terminal for the interaction with p300. Importantly, knockdowns of both GLI1 and p300 reduce SDF1 expression. Further analysis shows that knockdown of GLI1 decreases SDF1 promoter activity, p300 recruitment, and levels of its associated histone marks (H4ac, H3K27ac, and H3K14ac). Finally, we show that the integrity of two GLI binding sites in the SDF1 promoter is required for p300 recruitment. Our findings define a new role for the p300-GLI1 complex in the regulation of SDF1, providing new mechanistic insight into the molecular events controlling pancreatic cancer cells migration.
Assuntos
Fibroblastos Associados a Câncer , Neoplasias Pancreáticas , Humanos , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Linhagem Celular Tumoral , Movimento Celular , Imunoprecipitação da Cromatina , Neoplasias Pancreáticas/patologia , Transdução de Sinais , Proteína GLI1 em Dedos de Zinco/genética , Proteína GLI1 em Dedos de Zinco/metabolismo , Quimiocina CXCL12/metabolismo , Neoplasias PancreáticasRESUMO
Germline mutations in CDKN2A, encoding the tumor suppressor p16, are responsible for a large proportion of familial melanoma cases and also increase risk of pancreatic cancer. We identified four families through pancreatic cancer probands that were affected by both cancers. These families bore a germline missense variant of CDKN2A (47T>G), encoding a p16-L16R mutant protein associated with high cancer occurrence. Here, we investigated the biological significance of this variant. When transfected into p16-null pancreatic cancer cells, p16-L16R was expressed at lower levels than wild-type (WT) p16. In addition, p16-L16R was unable to bind CDK4 or CDK6 compared with WT p16, as shown by coimmunoprecipitation assays and also was impaired in its ability to inhibit the cell cycle, as demonstrated by flow cytometry analyses. In silico molecular modeling predicted that the L16R mutation prevents normal protein folding, consistent with the observed reduction in expression/stability and diminished function of this mutant protein. We isolated normal dermal fibroblasts from members of the families expressing WT or L16R proteins to investigate the impact of endogenous p16-L16R mutant protein on cell growth. In culture, p16-L16R fibroblasts grew at a faster rate, and most survived until later passages than p16-WT fibroblasts. Further, western blotting demonstrated that p16 protein was detected at lower levels in p16-L16R than in p16-WT fibroblasts. Together, these results suggest that the presence of a CDKN2A (47T>G) mutant allele contributes to an increased risk of pancreatic cancer as a result of reduced p16 protein levels and diminished p16 tumor suppressor function.
Assuntos
Ciclo Celular , Inibidor p16 de Quinase Dependente de Ciclina/genética , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Heterozigoto , Melanoma/patologia , Neoplasias Pancreáticas/patologia , Adulto , Idoso , Feminino , Humanos , Masculino , Melanoma/genética , Pessoa de Meia-Idade , Neoplasias Pancreáticas/genética , LinhagemRESUMO
The Hedgehog-regulated transcription factors GLI1 and GLI2 play overlapping roles in development and disease; however, the mechanisms underlying their interplay remain elusive. We report for the first time that GLI1 and GLI2 physically and functionally interact in cancer cells. GLI1 and GLI2 were shown to co-immunoprecipitate in PANC1 pancreatic cancer cells and RMS13 rhabdomyosarcoma cells. Mapping analysis demonstrated that the zinc finger domains of both proteins are required for their heteromerization. RNAi knockdown of either GLI1 or GLI2 inhibited expression of many well-characterized GLI target genes (BCL2, MYCN, PTCH2, IL7 and CCND1) in PANC1 cells, whereas PTCH1 expression was only inhibited by GLI1 depletion. qPCR screening of a large set of putative canonical and non-canonical Hedgehog/GLI targets identified further genes (e.g. E2F1, BMP1, CDK2) strongly down-regulated by GLI1 and/or GLI2 depletion in PANC1 cells, and demonstrated that ANO1, AQP1 and SOCS1 are up-regulated by knockdown of either GLI1 or GLI2. Chromatin immunoprecipitation showed that GLI1 and GLI2 occupied the same regions at the BCL2, MYCN and CCND1 promoters. Furthermore, depletion of GLI1 inhibited GLI2 occupancy at these promoters, suggesting that GLI1/GLI2 interaction is required for the recruitment of GLI2 to these sites. Together, these findings indicate that GLI1 and GLI2 co-ordinately regulate the transcription of some genes, and provide mechanistic insight into the roles of GLI proteins in carcinogenesis.
Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas Hedgehog/metabolismo , Proteínas Nucleares/metabolismo , Neoplasias Pancreáticas/metabolismo , Rabdomiossarcoma/metabolismo , Proteína GLI1 em Dedos de Zinco/metabolismo , Proteína Gli2 com Dedos de Zinco/metabolismo , Linhagem Celular Tumoral , Proteínas Hedgehog/genética , Humanos , Proteínas Nucleares/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Multimerização Proteica , Rabdomiossarcoma/genética , Rabdomiossarcoma/patologia , Proteína GLI1 em Dedos de Zinco/genética , Proteína Gli2 com Dedos de Zinco/genéticaRESUMO
The transcription factor GLI1 (GLI family zinc finger 1) plays a key role in the development and progression of multiple malignancies. To date, regulation of transcriptional activity at target gene promoters is the only molecular event known to underlie the oncogenic function of GLI1. Here, we provide evidence that GLI1 controls chromatin accessibility at distal regulatory regions by modulating the recruitment of SMARCA2 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member 2) to these elements. We demonstrate that SMARCA2 endogenously interacts with GLI1 and enhances its transcriptional activity. Mapping experiments indicated that the C-terminal transcriptional activation domain of GLI1 and SMARCA2's central domains, including its ATPase motif, are required for this interaction. Interestingly, similar to SMARCA2, GLI1 overexpression increased chromatin accessibility, as indicated by results of the micrococcal nuclease assay. Further, results of assays for transposase-accessible chromatin with sequencing (ATAC-seq) after GLI1 knockdown supported these findings, revealing that GLI1 regulates chromatin accessibility at several regions distal to gene promoters. Integrated RNA-seq and ATAC-seq data analyses identified a subset of differentially expressed genes located in cis to these regulated chromatin sites. Finally, using the GLI1-regulated gene HHIP (Hedgehog-interacting protein) as a model, we demonstrate that GLI1 and SMARCA2 co-occupy a distal chromatin peak and that SMARCA2 recruitment to this HHIP putative enhancer requires intact GLI1. These findings provide insights into how GLI1 controls gene expression in cancer cells and may inform approaches targeting this oncogenic transcription factor to manage malignancies.
Assuntos
Cromatina/genética , Elementos Reguladores de Transcrição , Fatores de Transcrição/metabolismo , Sítio de Iniciação de Transcrição , Proteína GLI1 em Dedos de Zinco/metabolismo , Linhagem Celular Tumoral , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , DNA/genética , DNA/metabolismo , Células HEK293 , Humanos , Domínios Proteicos , Mapas de Interação de Proteínas , Fatores de Transcrição/química , Ativação Transcricional , Proteína GLI1 em Dedos de Zinco/químicaRESUMO
PURPOSE: Genomic testing has increased the quantity of information available to oncologists. Unfortunately, many identified sequence alterations are variants of unknown significance (VUSs), which thus limit the clinician's ability to use these findings to inform treatment. We applied a combination of in silico prediction and molecular modeling tools and laboratory techniques to rapidly define actionable VUSs. MATERIALS AND METHODS: Exome sequencing was conducted on 308 tumors from various origins. Most single nucleotide alterations within gene coding regions were VUSs. These VUSs were filtered to identify a subset of therapeutically targetable genes that were predicted with in silico tools to be altered in function by their variant sequence. A subset of receptor tyrosine kinase VUSs was characterized by laboratory comparison of each VUS versus its wild-type counterpart in terms of expression and signaling activity. RESULTS: The study identified 4,327 point mutations of which 3,833 were VUSs. Filtering for mutations in genes that were therapeutically targetable and predicted to affect protein function reduced these to 522VUSs of interest, including a large number of kinases. Ten receptortyrosine kinase VUSs were selected to explore in the laboratory. Of these, seven were found to be functionally altered. Three VUSs (FGFR2 F276C, FGFR4 R78H, and KDR G539R) showed increased basal or ligand-stimulated ERK phosphorylation compared with their wild-type counterparts, which suggests that they support transformation. Treatment of a patient who carried FGFR2 F276C with an FGFR inhibitor resulted in significant and sustained tumor response with clinical benefit. CONCLUSION: The findings demonstrate the feasibility of rapid identification of the biologic relevance of somatic mutations, which thus advances clinicians' ability to make informed treatment decisions.
RESUMO
Ig secretion by terminally differentiated B cells is an important component of the immune response to foreign pathogens. Its overproduction is a defining characteristic of several B cell malignancies, including Waldenström macroglobulinemia (WM), where elevated IgM is associated with significant morbidity and poor prognosis. Therefore, the identification and characterization of the mechanisms controlling Ig secretion are of great importance for the development of future therapeutic approaches for this disease. In this study, we define a novel pathway involving the oncogenic transcription factor GLI2 modulating IgM secretion by WM malignant cells. Pharmacological and genetic inhibition of GLI2 in WM malignant cells resulted in a reduction in IgM secretion. Screening for a mechanism identified the IL-6Rα (gp80) subunit as a downstream target of GLI2 mediating the regulation of IgM secretion. Using a combination of expression, luciferase, and chromatin immunoprecipitation assays we demonstrate that GLI2 binds to the IL-6Rα promoter and regulates its activity as well as the expression of this receptor. Additionally, we were able to rescue the reduction in IgM secretion in the GLI2 knockdown group by overexpressing IL-6Rα, thus defining the functional significance of this receptor in GLI2-mediated regulation of IgM secretion. Interestingly, this occurred independent of Hedgehog signaling, a known regulator of GLI2, as manipulation of Hedgehog had no effect on IgM secretion. Given the poor prognosis associated with elevated IgM in WM patients, components of this new signaling axis could be important therapeutic targets.
Assuntos
Linfócitos B/imunologia , Imunoglobulina M/imunologia , Fatores de Transcrição Kruppel-Like/imunologia , Receptores de Interleucina-6/imunologia , Macroglobulinemia de Waldenstrom/patologia , Animais , Linhagem Celular , Imunoprecipitação da Cromatina , Feminino , Proteínas Hedgehog/genética , Humanos , Receptores de Hialuronatos/imunologia , Imunoglobulina M/biossíntese , Fatores de Transcrição Kruppel-Like/antagonistas & inibidores , Fatores de Transcrição Kruppel-Like/genética , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Regiões Promotoras Genéticas/genética , Ligação Proteica/imunologia , Receptores de Interleucina-6/biossíntese , Transdução de Sinais/imunologia , Macroglobulinemia de Waldenstrom/metabolismo , Proteína Gli2 com Dedos de ZincoRESUMO
The role of the epithelial-to-mesenchymal transition (EMT) during hepatocellular carcinoma (HCC) progression is well established, however the regulatory mechanisms modulating this phenomenon remain unclear. Here, we demonstrate that transcription factor glioma-associated oncogene 1 (GLI1) modulates EMT through direct up-regulation of SNAI1 and serves as a downstream effector of the transforming growth factor-ß1 (TGFß1) pathway, a well-known regulator of EMT in cancer cells. Overexpression of GLI1 increased proliferation, viability, migration, invasion, and colony formation by HCC cells. Conversely, GLI1 knockdown led to a decrease in all the above-mentioned cancer-associated phenotypes in HCC cells. Further analysis of GLI1 regulated cellular functions showed that this transcription factor is able to induce EMT and identified SNAI1 as a transcriptional target of GLI1 mediating this cellular effect in HCC cells. Moreover, we demonstrated that an intact GLI1-SNAI1 axis is required by TGFß1 to induce EMT in these cells. Together, these findings define a novel cellular mechanism regulated by GLI1, which controls the growth and EMT phenotype in HCC.
Assuntos
Carcinoma Hepatocelular/metabolismo , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/metabolismo , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Humanos , Microscopia de Fluorescência/métodos , Invasividade Neoplásica , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Fenótipo , Plasmídeos/metabolismo , RNA Mensageiro/metabolismo , Fatores de Transcrição da Família Snail , Transfecção , Proteína GLI1 em Dedos de ZincoRESUMO
The Hedgehog (HH) pathway has been identified as an important deregulated signal transduction pathway in pancreatic ductal adenocarcinoma (PDAC), a cancer type characterized by a highly metastatic phenotype. In PDAC, the canonical HH pathway activity is restricted to the stromal compartment while HH signaling in the tumor cells is reduced as a consequence of constitutive KRAS activation. Here, we report that in the tumor compartment of PDAC the HH pathway effector transcription factor GLI1 regulates epithelial differentiation. RNAi-mediated knockdown of GLI1 abolished characteristics of epithelial differentiation, increased cell motility, and synergized with TGFß to induce an epithelial-to-mesenchymal transition (EMT). Notably, EMT conversion in PDAC cells occurred in the absence of induction of SNAIL or SLUG, two canonical inducers of EMT in many other settings. Further mechanistic analysis revealed that GLI1 directly regulated the transcription of E-cadherin, a key determinant of epithelial tissue organization. Collectively, our findings identify GLI1 as an important positive regulator of epithelial differentiation, and they offer an explanation for how decreased levels of GLI1 are likely to contribute to the highly metastatic phenotype of PDAC.
Assuntos
Carcinoma Ductal Pancreático/patologia , Transição Epitelial-Mesenquimal , Neoplasias Pancreáticas/patologia , Fatores de Transcrição/antagonistas & inibidores , Junções Aderentes/patologia , Western Blotting , Linhagem Celular Tumoral , Imunofluorescência , Inativação Gênica , Humanos , Fatores de Transcrição/genética , Proteína GLI1 em Dedos de ZincoRESUMO
PURPOSE: To determine the concentration of myocilin in primary open-angle glaucoma (POAG) and pseudoexfoliation glaucoma (PEXG) aqueous humor. METHODS: Aqueous humor was collected during surgery from patients with POAG, PEXG, and elective cataract removal (control). Volume-equivalent aqueous samples were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gradient gels. Quantification of myocilin levels was performed using Western blots probed with 2 independent N-terminal polyclonal anti-myocilin antibodies (AB1 and AB2) followed by densitometry. Myocilin levels in aqueous humor were quantified by plotting the densitometry readings of the aqueous samples against a recombinant myocilin standard curve. Total protein concentration was determined by Bradford protein assay. Transforming growth factor ß 2 levels were assessed by enzyme-linked immunosorbent assay. RESULTS: Myocilin levels are significantly elevated in human POAG aqueous humor when compared with control aqueous humor (AB1: 0.66±0.53 ng/µL vs. 0.23±0.20 ng/µL, P<0.001; AB2: 0.98±0.59 ng/µL vs. 0.65±0.5 ng/µL, P<0.03; mean±SD). Myocilin makes up a larger percent of the total protein in POAG aqueous humor compared with control aqueous (AB1: 0.26±0.20% vs. 0.10±0.20%, P<0.001; AB2: 0.43±0.32% vs. 0.28±0.18%, P<0.05). In contrast to POAG, myocilin levels were not elevated in PEXG aqueous humor when compared with control aqueous humor. No correlation between myocilin and transforming growth factor ß 2 levels was observed. CONCLUSIONS: Myocilin is elevated in POAG, but not in PEXG aqueous humor.
Assuntos
Humor Aquoso/metabolismo , Proteínas do Citoesqueleto/metabolismo , Síndrome de Exfoliação/metabolismo , Proteínas do Olho/metabolismo , Glaucoma de Ângulo Aberto/metabolismo , Glicoproteínas/metabolismo , Idoso , Idoso de 80 Anos ou mais , Western Blotting , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fator de Crescimento Transformador beta2/metabolismoRESUMO
The domestic cat (Felix domesticus) is a useful model for understanding the implications of long-term expression and function of normal and mutant myocilin. To better understand the role myocilin has in the cat eye, we isolated and characterized cat myocilin. Oligonucleotides designed against conserved nucleotide regions of myocilin mammalian orthologs were used to PCR amplify a partial cat myocilin cDNA clone. Rapid amplification of cDNA ends (5' and 3' RACE) was used to obtain full-length cat myocilin. The 2125 nucleotide cat myocilin cDNA contains a 490 amino acid open reading frame. Comparison of cat myocilin to human myocilin shows a 87% similarity, including conservation of the N-terminal leucine zipper, N-linked glycosylation site, C-terminal olfactomedin domain, and all five cysteine residues thought to be involved in disulfide bond formation. Expression in a transformed human trabecular cell line or in Crandall feline kidney cells showed cat myocilin was secreted from these cells, similar to human myocilin, suggesting cat myocilin contains a functional signal peptide sequence. In contrast, expression of cat myocilin containing a known human glaucoma-associated mutation (Y423H in cat; Y437H in human) was not secreted. Characterization of cat myocilin will enable long-term studies be performed in Felix domesticus to analyze changes to intraocular pressure and the aqueous outflow pathway following expression of myocilin and glaucoma causing mutations.
Assuntos
Gatos/genética , Proteínas do Citoesqueleto/genética , Proteínas do Olho/genética , Glicoproteínas/genética , Sequência de Aminoácidos/genética , Aminoácidos/genética , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular/métodos , Cisteína/genética , DNA Circular/genética , Proteínas da Matriz Extracelular/genética , Expressão Gênica/genética , Glaucoma/genética , Humanos , Rim/citologia , Zíper de Leucina/genética , Modelos Animais , Modelos Genéticos , Proteínas Mutantes/genética , Mutação/genética , Filogenia , Malha Trabecular/citologiaRESUMO
PURPOSE: Myocilin forms large complexes in aqueous humor. Part of this complex formation is due to myocilin-myocilin protein non-covalent interactions within the leucine zipper. However, additional covalent interactions also exist. We investigated the role of these covalent interactions in disulfide bond formation within myocilin. METHODS: Human aqueous humor was separated by denatured/non-reduced SDS-PAGE followed by Western blot analysis with myocilin specific antibodies. In part two of the study, site-directed mutagenesis was used to selectively mutate one, two, three, four, and all five cysteine residues in the mature myocilin protein expressed in an in vitro system. Products were immunoprecipitated with a hemagglutinin polyclonal antibody following in vitro transcription/translation and analyzed by SDS-PAGE. In part three of the study, glaucoma associated myocilin mutations Arg82Cys and Cys433Arg were created and complex formation analyzed in trabecular meshwork cells. RESULTS: Human aqueous humor showed myocilin in several distinct large complexes in non-reduced SDS-PAGE gels, indicating disulfide bonds occur. Similarly, in vitro expressed myocilin also produced large complexes. Mutation of all five cysteines (within the mature myocilin protein) eliminated this large complex formation. A combination of cysteine to alanine substitutions at amino acids 185, 245, and 433 had the most influence on myocilin complex formation under non-reducing conditions, however individual substitutions at each of the five cysteine amino acids had little influence on myocilin complexes. In trabecular meshwork cells, Arg82Cys was secreted but formed different sized complexes than wild type myocilin. Cys433Arg was not secreted and remained intracellular in a pattern that differed from wild type myocilin and Arg82Cys. CONCLUSIONS: Myocilin complexes present in human aqueous humor are in part due to disulfide bond formation between cysteine amino acids. Glaucoma associated mutations that affect the number of cysteine residues may alter covalent interactions.