RESUMO
Melanoma cell phenotype switching between differentiated melanocytic and undifferentiated mesenchymal-like states drives metastasis and drug resistance. CDK7 is the serine/threonine kinase of the basal transcription factor TFIIH. We show that dedifferentiation of melanocytic-type melanoma cells into mesenchymal-like cells and acquisition of tolerance to targeted therapies is achieved through chronic inhibition of CDK7. In addition to emergence of a mesenchymal-type signature, we identify a GATA6-dependent gene expression program comprising genes such as AMIGO2 or ABCG2 involved in melanoma survival or targeted drug tolerance, respectively. Mechanistically, we show that CDK7 drives expression of the melanocyte lineage transcription factor MITF that in turn binds to an intronic region of GATA6 to repress its expression in melanocytic-type cells. We show that GATA6 expression is activated in MITF-low melanoma cells of patient-derived xenografts. Taken together, our data show how the poorly characterized repressive function of MITF in melanoma participates in a molecular cascade regulating activation of a transcriptional program involved in survival and drug resistance in melanoma.
Assuntos
Melanoma , Fator de Transcrição Associado à Microftalmia , Linhagem Celular Tumoral , Tolerância a Medicamentos , Regulação Neoplásica da Expressão Gênica , Humanos , Melanoma/tratamento farmacológico , Melanoma/genética , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição Associado à Microftalmia/metabolismoRESUMO
BACKGROUND: Thoracic SMARCA4-deficient undifferentiated tumors (SMARCA4-UT) are aggressive neoplasms. Data linking BAF alterations with tumor microenvironment (TME) and efficacy of immune checkpoint inhibitors (ICI) are contradictory. The TME of SMARCA4-UT and their response to ICI are unknown. MATERIALS AND METHODS: Patients diagnosed with SMARCA4-UT in our institution were included. Immunostainings for tertiary lymphoid structures (TLS), immune cell markers, and checkpoints were assessed. Validation was performed using an independent transcriptome dataset including SMARCA4-UT, non-small cell lung cancers (NSCLC) with/without SMARCA4 mutations, and unclassified thoracic sarcomas (UTS). CXCL9 and PD-L1 expressions were assessed in NSCLC and thoracic fibroblast cell lines, with/without SMARCA4 knockdown, treated with/without interferon gamma. RESULTS: Nine patients were identified. All samples but one showed no TLS, consistent with an immune desert TME phenotype. Four patients received ICI as part of their treatment, but the only one who responded, had a tumor with a TLS and immune-rich TME. Unsupervised clustering of the validation cohort using immune cell scores identified 2 clusters associated with cell ontogeny and immunity (cluster 1 enriched for NSCLC independently of SMARCA4 status (n = 9/10; P = .001); cluster 2 enriched for SMARCA4-UT (n = 11/12; P = .005) and UTS (n = 5/5; P = .0005). SMARCA4 loss-of-function experiments revealed interferon-induced upregulation of CXCL9 and PD-L1 expression in the NSCLC cell line with no effect on the thoracic fibroblast cell line. CONCLUSION: SMARCA4-UT mainly have an immune desert TME with limited efficacy to ICI. TME of SMARCA4-driven tumors varies according to the cell of origin questioning the interplay between BAF alterations, cell ontogeny and immunity.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , DNA Helicases , Inibidores de Checkpoint Imunológico , Neoplasias Pulmonares , Proteínas Nucleares , Sarcoma , Neoplasias de Tecidos Moles , Neoplasias Torácicas , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/imunologia , Biomarcadores Tumorais/imunologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/patologia , DNA Helicases/deficiência , DNA Helicases/imunologia , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Proteínas Nucleares/deficiência , Proteínas Nucleares/imunologia , Sarcoma/tratamento farmacológico , Sarcoma/imunologia , Sarcoma/patologia , Neoplasias de Tecidos Moles/tratamento farmacológico , Neoplasias de Tecidos Moles/imunologia , Neoplasias de Tecidos Moles/patologia , Neoplasias Torácicas/tratamento farmacológico , Neoplasias Torácicas/imunologia , Neoplasias Torácicas/patologia , Fatores de Transcrição/imunologia , Microambiente Tumoral/imunologiaRESUMO
The TEAD family of transcription factors (TEAD1-4) bind the MCAT element in the regulatory elements of both growth promoting and myogenic differentiation genes. Defining TEAD transcription factor function in myogenesis has proved elusive due to overlapping expression of family members and their functional redundancy. We show that silencing of either Tead1, Tead2 or Tead4 did not effect primary myoblast (PM) differentiation, but that their simultaneous knockdown strongly impaired differentiation. In contrast, Tead1 or Tead4 silencing impaired C2C12 differentiation showing their different contributions in PMs and C2C12 cells. Chromatin immunoprecipitation identified enhancers associated with myogenic genes bound by combinations of Tead4, Myod1 or Myog. Tead4 regulated distinct gene sets in C2C12 cells and PMs involving both activation of the myogenic program and repression of growth and signaling pathways. ChIP-seq from mature mouse muscle fibres in vivo identified a set of highly transcribed muscle cell-identity genes and sites bound by Tead1 and Tead4. Although inactivation of Tead4 in mature muscle fibres caused no obvious phenotype under normal conditions, notexin-induced muscle regeneration was delayed in Tead4 mutants suggesting an important role in myogenic differentiation in vivo. By combining knockdown in cell models in vitro with Tead4 inactivation in muscle in vivo, we provide the first comprehensive description of the specific and redundant roles of Tead factors in myogenic differentiation.
Assuntos
Diferenciação Celular/genética , Proteínas de Ligação a DNA/genética , Proteínas Musculares/genética , Músculos/metabolismo , Mioblastos/metabolismo , Regeneração/genética , Fatores de Transcrição/genética , Animais , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos/genética , Perfilação da Expressão Gênica/métodos , Immunoblotting , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Fluorescência , Desenvolvimento Muscular/genética , Proteínas Musculares/metabolismo , Músculos/citologia , Músculos/fisiologia , Mutação , Mioblastos/citologia , Ligação Proteica , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Fatores de Transcrição de Domínio TEA , Fatores de Transcrição/metabolismoRESUMO
Taf4 (TATA-box binding protein-associated factor 4) is a subunit of the general transcription factor TFIID, a component of the RNA polymerase II pre-initiation complex that interacts with tissue-specific transcription factors to regulate gene expression. Properly regulated gene expression is particularly important in the intestinal epithelium that is constantly renewed from stem cells. Tissue-specific inactivation of Taf4 in murine intestinal epithelium during embryogenesis compromised gut morphogenesis and the emergence of adult-type stem cells. In adults, Taf4 loss impacted the stem cell compartment and associated Paneth cells in the stem cell niche, epithelial turnover and differentiation of mature cells, thus exacerbating the response to inflammatory challenge. Taf4 inactivation ex vivo in enteroids prevented budding formation and maintenance and caused broad chromatin remodeling and a strong reduction in the numbers of stem and progenitor cells with a concomitant increase in an undifferentiated cell population that displayed high activity of the Ezh2 and Suz12 components of Polycomb Repressive Complex 2 (PRC2). Treatment of Taf4-mutant enteroids with a specific Ezh2 inhibitor restored buddings, cell proliferation and the stem/progenitor compartment. Taf4 loss also led to increased PRC2 activity in cells of adult crypts associated with modification of the immune/inflammatory microenvironment that potentiated Apc-driven tumorigenesis. Our results reveal a novel function of Taf4 in antagonizing PRC2-mediated repression of the stem cell gene expression program to assure normal development, homeostasis, and immune-microenvironment of the intestinal epithelium.
Assuntos
Proteínas de Drosophila , Células-Tronco , Camundongos , Animais , Diferenciação Celular/genética , Células-Tronco/metabolismo , Fator de Transcrição TFIID/genética , Mucosa Intestinal/metabolismo , Proteínas de Drosophila/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Epigênese GenéticaRESUMO
Super-enhancers (SEs) are stretches of enhancers ensuring a high level of expression of key genes associated with cell function. The identification of cancer-specific SE-driven genes is a powerful means for the development of innovative therapeutic strategies. Here, we identify a MITF/SOX10/TFIIH-dependent SE promoting the expression of BAHCC1 in a broad panel of melanoma cells. BAHCC1 is highly expressed in metastatic melanoma and is required for tumor engraftment, growth, and dissemination. Integrative genomics analyses reveal that BAHCC1 is a transcriptional regulator controlling expression of E2F/KLF-dependent cell-cycle and DNA-repair genes. BAHCC1 associates with BRG1-containing remodeling complexes at the promoters of these genes. BAHCC1 silencing leads to decreased cell proliferation and delayed DNA repair. Consequently, BAHCC1 deficiency cooperates with PARP inhibition to induce melanoma cell death. Our study identifies BAHCC1 as an SE-driven gene expressed in melanoma and demonstrates how its inhibition can be exploited as a therapeutic target.
Assuntos
Melanoma , Humanos , Linhagem Celular Tumoral , Melanoma/patologia , Sequências Reguladoras de Ácido Nucleico , Instabilidade Genômica , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Elementos Facilitadores Genéticos , Proteínas/metabolismoRESUMO
Renal medullary carcinoma (RMC) is an aggressive tumour driven by bi-allelic loss of SMARCB1 and tightly associated with sickle cell trait. However, the cell-of-origin and oncogenic mechanism remain poorly understood. Using single-cell sequencing of human RMC, we defined transformation of thick ascending limb (TAL) cells into an epithelial-mesenchymal gradient of RMC cells associated with loss of renal epithelial transcription factors TFCP2L1, HOXB9 and MITF and gain of MYC and NFE2L2-associated oncogenic and ferroptosis resistance programs. We describe the molecular basis for this transcriptional switch that is reversed by SMARCB1 re-expression repressing the oncogenic and ferroptosis resistance programs leading to ferroptotic cell death. Ferroptosis resistance links TAL cell survival with the high extracellular medullar iron concentrations associated with sickle cell trait, an environment propitious to the mutagenic events associated with RMC development. This unique environment may explain why RMC is the only SMARCB1-deficient tumour arising from epithelial cells, differentiating RMC from rhabdoid tumours arising from neural crest cells.
Assuntos
Carcinoma Medular , Carcinoma de Células Renais , Ferroptose , Neoplasias Renais , Traço Falciforme , Humanos , Neoplasias Renais/patologia , Carcinoma Medular/metabolismo , Carcinoma de Células Renais/patologia , Proteína SMARCB1/genética , Proteína SMARCB1/metabolismo , Proteínas Repressoras , Proteínas de HomeodomínioRESUMO
Immune checkpoint inhibitors (ICI) represent the cornerstone for the treatment of patients with metastatic clear cell renal cell carcinoma (ccRCC). Despite a favorable response for a subset of patients, others experience primary progressive disease, highlighting the need to precisely understand the plasticity of cancer cells and their cross-talk with the microenvironment to better predict therapeutic response and personalize treatment. Single-cell RNA sequencing of ccRCC at different disease stages and normal adjacent tissue (NAT) from patients identified 46 cell populations, including 5 tumor subpopulations, characterized by distinct transcriptional signatures representing an epithelial-to-mesenchymal transition gradient and a novel inflamed state. Deconvolution of the tumor and microenvironment signatures in public data sets and data from the BIONIKK clinical trial (NCT02960906) revealed a strong correlation between mesenchymal-like ccRCC cells and myofibroblastic cancer-associated fibroblasts (myCAF), which are both enriched in metastases and correlate with poor patient survival. Spatial transcriptomics and multiplex immune staining uncovered the spatial proximity of mesenchymal-like ccRCC cells and myCAFs at the tumor-NAT interface. Moreover, enrichment in myCAFs was associated with primary resistance to ICI therapy in the BIONIKK clinical trial. These data highlight the epithelial-mesenchymal plasticity of ccRCC cancer cells and their relationship with myCAFs, a critical component of the microenvironment associated with poor outcome and ICI resistance. SIGNIFICANCE: Single-cell and spatial transcriptomics reveal the proximity of mesenchymal tumor cells to myofibroblastic cancer-associated fibroblasts and their association with disease outcome and immune checkpoint inhibitor response in clear cell renal cell carcinoma.
Assuntos
Fibroblastos Associados a Câncer , Carcinoma de Células Renais , Neoplasias Renais , Humanos , Fibroblastos Associados a Câncer/patologia , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/genética , Perfilação da Expressão Gênica , Imunoterapia , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/genética , Prognóstico , Microambiente Tumoral , Ensaios Clínicos como AssuntoRESUMO
PURPOSE: Immune checkpoint inhibitors (ICI) have revolutionized the treatment of patients with clear-cell renal cell carcinomas (ccRCC). Although analyses of transcriptome, genetic alterations, and the tumor microenvironment (TME) have shed light into mechanisms of response and resistance to these agents, the role of epigenetic alterations in this process remains fully unknown. EXPERIMENTAL DESIGN: We investigated the methylome of six ccRCC cohorts as well as one cell line dataset. Of note, we took advantage of the BIONIKK trial aiming to tailor treatments according to Paris Descartes 4-gene expression subgroups, and performed Illumina EPIC profiling for 46 samples related to patients treated with ipilimumab plus nivolumab, and 17 samples related to patients treated with sunitinib. RESULTS: A group of tumors associated with enhancer demethylation was discovered, namely TED. TED was associated with tumors with sarcomatoid differentiation and poor clinical outcome. TED harbored TET1 promoter demethylation, activated the gene expression signature of epithelial-mesenchymal transition and IL6/JAK/STAT3 pathways, and displayed a TME characterized by both immune activation and suppressive populations, fibroblast infiltration, and endothelial depletion. In addition, TED was a predictive factor of resistance to the combination of first-line ipilimumab-nivolumab in the BIONIKK clinical trial. Finally, TED was associated with activation of specific regulons, which we also found to be predictive of resistance to immunotherapy in an independent cohort. CONCLUSIONS: We report on the discovery of a novel epigenetic phenotype associated with resistance to ICIs that may pave the way to better personalizing patients' treatments. See related commentary by Zhou and Kim, p. 1170.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Humanos , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Nivolumabe/administração & dosagem , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/genética , Neoplasias Renais/patologia , Ipilimumab/administração & dosagem , Metilação de DNA , Fenótipo , Microambiente Tumoral/genética , Oxigenases de Função Mista , Proteínas Proto-Oncogênicas/genéticaRESUMO
Tumor heterogeneity is a key feature of melanomas that hinders development of effective treatments. Aiming to overcome this, we identified LINC00518 (LENOX; lincRNA-enhancer of oxidative phosphorylation) as a melanoma-specific lncRNA expressed in all known melanoma cell states and essential for melanoma survival in vitro and in vivo. Mechanistically, LENOX promoted association of the RAP2C GTPase with mitochondrial fission regulator DRP1, increasing DRP1 S637 phosphorylation, mitochondrial fusion, and oxidative phosphorylation. LENOX expression was upregulated following treatment with MAPK inhibitors, facilitating a metabolic switch from glycolysis to oxidative phosphorylation and conferring resistance to MAPK inhibition. Consequently, combined silencing of LENOX and RAP2C synergized with MAPK inhibitors to eradicate melanoma cells. Melanomas are thus addicted to the lncRNA LENOX, which acts to optimize mitochondrial function during melanoma development and progression. SIGNIFICANCE: The lncRNA LENOX is a novel regulator of melanoma metabolism, which can be targeted in conjunction with MAPK inhibitors to eradicate melanoma cells.
Assuntos
Melanoma , Inibidores de Proteínas Quinases , RNA Longo não Codificante , Proteínas ras , Humanos , Linhagem Celular Tumoral , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/metabolismo , Dinâmica Mitocondrial , Fosforilação Oxidativa , Inibidores de Proteínas Quinases/farmacologia , Proteínas ras/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Resistencia a Medicamentos AntineoplásicosRESUMO
Malignant melanoma is a major public health issue displaying frequent resistance to targeted therapy and immunotherapy. A major challenge lies in better understanding how melanoma cells evade immune elimination and how tumor growth and metastasis is facilitated by the tumor microenvironment. Here, we show that expression of the cytokine thymic stromal lymphopoietin (TSLP) by epidermal keratinocytes is induced by cutaneous melanoma in both mice and humans. Using genetically engineered models of melanoma and tumor cell grafting combined with TSLP-KO or overexpression, we defined a crosstalk between melanoma cells, keratinocytes, and immune cells in establishing a tumor-promoting microenvironment. Keratinocyte-derived TSLP is induced by signals derived from melanoma cells and subsequently acts via immune cells to promote melanoma progression and metastasis. Furthermore, we show that TSLP signals through TSLP receptor-expressing (TSLPR-expressing) DCs to play an unrecognized role in promoting GATA3+ Tregs expressing a gene signature including ST2, CCR8, ICOS, PD-1, CTLA-4, and OX40 and exhibiting a potent suppressive activity on CD8+ T cell proliferation and IFN-γ production. An analogous population of GATA3-expressing Tregs was also identified in human melanoma tumors. Our study provides insights into the role of TSLP in programming a protumoral immune microenvironment in cutaneous melanoma.
Assuntos
Melanoma , Neoplasias Cutâneas , Humanos , Camundongos , Animais , Microambiente Tumoral , Melanoma/genética , Melanoma/metabolismo , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Citocinas/metabolismo , Queratinócitos/metabolismo , Linfopoietina do Estroma do Timo , Melanoma Maligno CutâneoRESUMO
Regulation of gene expression involves a complex and dynamic dialogue between transcription factors, chromatin remodelling and modification complexes and the basal transcription machinery. To address the function of the Taf4 subunit of general transcription factor TFIID in the regulation of insulin signalling, it was inactivated in adult murine pancreatic beta cells. Taf4 inactivation impacted the expression of critical genes involved in beta-cell function leading to increased glycaemia, lowered plasma insulin levels and defective glucose-stimulated insulin secretion. One week after Taf4-loss, single-cell RNA-seq revealed cells with mixed beta cell, alpha and/or delta cell identities as well as a beta cell population trans-differentiating into alpha-like cells. Computational analysis of single-cell RNA-seq defines how known critical beta cell and alpha cell determinants may act in combination with additional transcription factors and the NuRF chromatin remodelling complex to promote beta cell trans-differentiation.
Assuntos
Transdiferenciação Celular/genética , Perfilação da Expressão Gênica , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Subunidades Proteicas/metabolismo , Análise de Célula Única , Fator de Transcrição TFIID/metabolismo , Envelhecimento/metabolismo , Animais , Peso Corporal/efeitos dos fármacos , Transdiferenciação Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Células Secretoras de Glucagon/efeitos dos fármacos , Células Secretoras de Glucagon/metabolismo , Glucose/farmacologia , Insulina/metabolismo , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Camundongos , Mutação/genética , Fator de Transcrição TFIID/deficiência , Fatores de Transcrição/metabolismoRESUMO
Chromodomain helicase DNA binding protein 4 (CHD4) is an ATPase subunit of the Nucleosome Remodelling and Deacetylation (NuRD) complex that regulates gene expression. CHD4 is essential for growth of multiple patient derived melanoma xenografts and for breast cancer. Here we show that CHD4 regulates expression of PADI1 (Protein Arginine Deiminase 1) and PADI3 in multiple cancer cell types modulating citrullination of arginine residues of the allosterically-regulated glycolytic enzyme pyruvate kinase M2 (PKM2). Citrullination of PKM2 R106 reprogrammes cross-talk between PKM2 ligands lowering its sensitivity to the inhibitors Tryptophan, Alanine and Phenylalanine and promoting activation by Serine. Citrullination thus bypasses normal physiological regulation by low Serine levels to promote excessive glycolysis and reduced cell proliferation. We further show that PADI1 and PADI3 expression is up-regulated by hypoxia where PKM2 citrullination contributes to increased glycolysis. We provide insight as to how conversion of arginines to citrulline impacts key interactions within PKM2 that act in concert to reprogramme its activity as an additional mechanism regulating this important enzyme.
Assuntos
Proliferação de Células/fisiologia , Citrulinação/fisiologia , Glicólise/fisiologia , Neoplasias/metabolismo , Proteína-Arginina Desiminase do Tipo 1/metabolismo , Proteína-Arginina Desiminase do Tipo 3/metabolismo , Piruvato Quinase/metabolismo , Regulação Alostérica , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Humanos , Melanoma , Proteínas de Membrana , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase , Neoplasias/genética , Proteína-Arginina Desiminase do Tipo 1/genética , Proteína-Arginina Desiminase do Tipo 3/genética , Hormônios Tireóideos , Regulação para Cima , Proteínas de Ligação a Hormônio da TireoideRESUMO
Well-differentiated pancreatic neuroendocrine tumors (pNET) have an unpredictable natural history. The identification of both blood and tumor immune features associated with patients' outcomes remains limited. Herein, we evaluated the best prognostic value of the neutrophils-to-lymphocyte ratio (NLR) in a cohort of 144 pNETs. The NLR ≥ 4 was associated with worse overall survival in both univariate analysis (HR = 3.53, CI95% = 1.50-8.31, p = 0.004) and multivariate analysis (HR = 2.57, CI95% = 1.061-6.216, p = 0.036). The presence of synchronous liver metastasis was identified as a prognostic factor in multivariate analysis (HR = 3.35, CI95% = 1.411-7.973, p = 0.006). Interestingly, the absolute tumor-associated neutrophils count was higher in liver metastasis as compared to their paired primary tumor (p = 0.048). Deconvolution of immune cells from the transcriptome of 83 primary tumors and 30 liver metastases reveals enrichment for neutrophils in metastasis relative to primary tumors (p = 0.005), and this was associated with upregulation of the complement pathway (NES = 1.84, p < 0.0001). Combining neutrophils signature and complement pathway genes, unsupervised clustering identified two pNETs subgroups, namely Neu-Comp1 and Neu-Comp2. Characterized by neutrophils infiltration and activation of the complement pathway, Neu-Comp1 was highly enriched for metastatic liver samples as compared to Neu-Comp2 (p < 0.0001). These data suggest the possible link between liver metastasis, complement pathway activation, and neutrophils infiltration in well-differentiated pNET and open avenues for targeting complement pathways in these tumors.
RESUMO
Somatic oncogenic mutation of BRAF coupled with inactivation of PTEN constitute a frequent combination of genomic alterations driving the development of human melanoma. Mice genetically engineered to conditionally express oncogenic BrafV600E and inactivate Pten in melanocytes following tamoxifen treatment rapidly develop melanoma. While early-stage melanomas comprised melanin-pigmented Mitf and Dct-expressing cells, expression of these and other melanocyte identity genes was lost in later stage tumours that showed histological and molecular characteristics of de-differentiated neural crest type cells. Melanocyte identity genes displayed loss of active chromatin marks and RNA polymerase II and gain of heterochromatin marks, indicating epigenetic reprogramming during tumour progression. Nevertheless, late-stage tumour cells grown in culture re-expressed Mitf, and melanocyte markers and Mitf together with Sox10 coregulated a large number of genes essential for their growth. In this melanoma model, somatic inactivation that the catalytic Brg1 (Smarca4) subunit of the SWI/SNF complex and the scaffolding Bptf subunit of the NuRF complex delayed tumour formation and deregulated large and overlapping gene expression programs essential for normal tumour cell growth. Moreover, we show that Brg1 and Bptf coregulated many genes together with Mitf and Sox10. Together these transcription factors and chromatin remodelling complexes orchestrate essential gene expression programs in mouse melanoma cells.
Assuntos
Antígenos Nucleares/fisiologia , DNA Helicases/fisiologia , Regulação Neoplásica da Expressão Gênica , Melanoma Experimental/genética , Proteínas do Tecido Nervoso/fisiologia , Proteínas Nucleares/fisiologia , Proteínas Proto-Oncogênicas B-raf/genética , Neoplasias Cutâneas/genética , Fatores de Transcrição/fisiologia , Animais , Antígenos Nucleares/genética , DNA Helicases/genética , Progressão da Doença , Epigênese Genética , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição Associado à Microftalmia/metabolismo , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , PTEN Fosfo-Hidrolase/genética , Fatores de Transcrição SOXE/genética , Fatores de Transcrição SOXE/metabolismo , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Fatores de Transcrição/genética , Células Tumorais CultivadasRESUMO
In melanoma, initiating oncogenic mutations in BRAF or NRAS are detected in premalignant lesions that accumulate additional mutations and genomic instability as the tumor evolves to the metastatic state. Here we investigate evolution of clonal composition and neoantigen landscape in an atypical melanoma displaying recurrent cutaneous lesions over a 6-year period without development of extracutaneous metastases. Whole exome sequencing of four cutaneous lesions taken during the 6-year period identified a collection of single nucleotide variants and small insertions and deletions shared among all tumors, along with progressive selection of subclones displaying fewer single nucleotide variants. Later tumors also displayed lower neoantigen burden compared to early tumors, suggesting that clonal evolution was driven, at least in part, by counter selection of subclones with high neoantigen burdens. Among the selected mutations are a missense mutation in MAP2K1 (F53Y) and an inversion on chromosome 7 generating a AKAP9-BRAF fusion. The mutant proteins cooperatively activate the MAPK signaling pathway confirming they are potential driver mutations of this tumor. We therefore describe the long-term genetic evolution of cutaneous metastatic melanoma characterized by an unexpected phenotypic stability and neoantigen-driven clonal selection.
Assuntos
Antígenos de Neoplasias/metabolismo , Carcinogênese/genética , Melanoma/genética , Recidiva Local de Neoplasia/genética , Neoplasias Cutâneas/genética , Proteínas de Ancoragem à Quinase A/genética , Adulto , Antígenos de Neoplasias/imunologia , Biópsia , Carcinogênese/imunologia , Evolução Clonal , Proteínas do Citoesqueleto/genética , Análise Mutacional de DNA , Células HEK293 , Humanos , MAP Quinase Quinase 1/genética , Masculino , Melanoma/imunologia , Melanoma/patologia , Mutação , Mutação de Sentido Incorreto , Recidiva Local de Neoplasia/imunologia , Recidiva Local de Neoplasia/patologia , Proteínas de Fusão Oncogênica/genética , Proteínas Proto-Oncogênicas B-raf/genética , Pele/patologia , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologia , Sequenciamento do ExomaRESUMO
Melanoma is an aggressive neoplasm with increasing incidence that is classified by the NCI as a recalcitrant cancer, i.e., a cancer with poor prognosis, lacking progress in diagnosis and treatment. In addition to conventional therapy, melanoma treatment is currently based on targeting the BRAF/MEK/ERK signaling pathway and immune checkpoints. As drug resistance remains a major obstacle to treatment success, advanced therapeutic approaches based on novel targets are still urgently needed. We reasoned that the base excision repair enzyme thymine DNA glycosylase (TDG) could be such a target for its dual role in safeguarding the genome and the epigenome, by performing the last of the multiple steps in DNA demethylation. Here we show that TDG knockdown in melanoma cell lines causes cell cycle arrest, senescence, and death by mitotic alterations; alters the transcriptome and methylome; and impairs xenograft tumor formation. Importantly, untransformed melanocytes are minimally affected by TDG knockdown, and adult mice with conditional knockout of Tdg are viable. Candidate TDG inhibitors, identified through a high-throughput fluorescence-based screen, reduced viability and clonogenic capacity of melanoma cell lines and increased cellular levels of 5-carboxylcytosine, the last intermediate in DNA demethylation, indicating successful on-target activity. These findings suggest that TDG may provide critical functions specific to cancer cells that make it a highly suitable anti-melanoma drug target. By potentially disrupting both DNA repair and the epigenetic state, targeting TDG may represent a completely new approach to melanoma therapy.
Assuntos
Inibidores Enzimáticos/farmacologia , Melanoma/patologia , Timina DNA Glicosilase/genética , Animais , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Citosina/análogos & derivados , Citosina/metabolismo , Metilação de DNA , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma Experimental/genética , Melanoma Experimental/patologia , Camundongos Knockout , Camundongos SCID , Camundongos Transgênicos , Terapia de Alvo Molecular/métodos , Timina DNA Glicosilase/antagonistas & inibidores , Timina DNA Glicosilase/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Purpose: Understanding tumor heterogeneity is an important challenge in current cancer research. Transcription and epigenetic profiling of cultured melanoma cells have defined at least two distinct cell phenotypes characterized by distinctive gene expression signatures associated with high or low/absent expression of microphthalmia-associated transcription factor (MITF). Nevertheless, heterogeneity of cell populations and gene expression in primary human tumors is much less well characterized.Experimental Design: We performed single-cell gene expression analyses on 472 cells isolated from needle biopsies of 5 primary human melanomas, 4 superficial spreading, and one acral melanoma. The expression of MITF-high and MITF-low signature genes was assessed and compared to investigate intra- and intertumoral heterogeneity and correlated gene expression profiles.Results: Single-cell gene expression analyses revealed varying degrees of intra- and intertumor heterogeneity conferred by the variable expression of distinct sets of genes in different tumors. Expression of MITF partially correlated with that of its known target genes, while SOX10 expression correlated best with PAX3 and ZEB2 Nevertheless, cells simultaneously expressing MITF-high and MITF-low signature genes were observed both by single-cell analyses and RNAscope.Conclusions: Single-cell analyses can be performed on limiting numbers of cells from primary human melanomas revealing their heterogeneity. Although tumors comprised variable proportions of cells with the MITF-high and MITF-low gene expression signatures characteristic of melanoma cultures, primary tumors also comprised cells expressing markers of both signatures defining a novel cell state in tumors in vivoClin Cancer Res; 23(22); 7097-107. ©2017 AACR.
Assuntos
Regulação Neoplásica da Expressão Gênica , Heterogeneidade Genética , Melanoma/genética , Melanoma/patologia , Fator de Transcrição Associado à Microftalmia/genética , Adulto , Idoso de 80 Anos ou mais , Biomarcadores Tumorais , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Melanoma/metabolismo , Fator de Transcrição Associado à Microftalmia/metabolismo , Mutação , Análise de Célula ÚnicaRESUMO
Mammalian genomes encode two genes related to the TATA-box binding protein (TBP), TBP-related factors 2 and 3 (TRF2 and TRF3). Male Trf2(-/-) mice are sterile and characterized by arrested spermatogenesis at the transition from late haploid spermatids to early elongating spermatids. Despite this characterization, the molecular function of murine Trf2 remains poorly characterized and no direct evidence exists to show that it acts as a bona fide chromatin-bound transcription factor. We show here that Trf2 forms a stable complex with TFIIA or the testis expressed paralogue ALF chaperoned in the cytoplasm by heat shock proteins. We demonstrate for the first time that Trf2 is recruited to active haploid cell promoters together with Tbp, Taf7l and RNA polymerase II. RNA-seq analysis identifies a set of genes activated in haploid spermatids during the first wave of spermatogenesis whose expression is down-regulated by Trf2 inactivation. We therefore propose that Trf2 is recruited to the preinitiation complex as a testis-specific subunit of TFIIA/ALF that cooperates with Tbp and Taf7l to promote haploid cell gene expression.