RESUMEN
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.
Asunto(s)
Melanoma , Factor de Transcripción Asociado a Microftalmía , Línea Celular Tumoral , Tolerancia a Medicamentos , Regulación Neoplásica de la Expresión Génica , Humanos , Melanoma/tratamiento farmacológico , Melanoma/genética , Factor de Transcripción Asociado a Microftalmía/genética , Factor de Transcripción Asociado a Microftalmía/metabolismoRESUMEN
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.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , ADN Helicasas , Inhibidores de Puntos de Control Inmunológico , Neoplasias Pulmonares , Proteínas Nucleares , Sarcoma , Neoplasias de los Tejidos Blandos , Neoplasias Torácicas , Antígeno B7-H1/antagonistas & inhibidores , Antígeno B7-H1/inmunología , Biomarcadores de Tumor/inmunología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Carcinoma de Pulmón de Células no Pequeñas/patología , ADN Helicasas/deficiencia , ADN Helicasas/inmunología , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Proteínas Nucleares/deficiencia , Proteínas Nucleares/inmunología , Sarcoma/tratamiento farmacológico , Sarcoma/inmunología , Sarcoma/patología , Neoplasias de los Tejidos Blandos/tratamiento farmacológico , Neoplasias de los Tejidos Blandos/inmunología , Neoplasias de los Tejidos Blandos/patología , Neoplasias Torácicas/tratamiento farmacológico , Neoplasias Torácicas/inmunología , Neoplasias Torácicas/patología , Factores de Transcripción/inmunología , Microambiente Tumoral/inmunologíaRESUMEN
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.
Asunto(s)
Diferenciación Celular/genética , Proteínas de Unión al ADN/genética , Proteínas Musculares/genética , Músculos/metabolismo , Mioblastos/metabolismo , Regeneración/genética , Factores de Transcripción/genética , Animales , Línea Celular , Proteínas de Unión al ADN/metabolismo , Elementos de Facilitación Genéticos/genética , Perfilación de la Expresión Génica/métodos , Immunoblotting , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Fluorescente , Desarrollo de Músculos/genética , Proteínas Musculares/metabolismo , Músculos/citología , Músculos/fisiología , Mutación , Mioblastos/citología , Unión Proteica , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genética , Factores de Transcripción de Dominio TEA , Factores de Transcripción/metabolismoRESUMEN
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.
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Proteínas de Drosophila , Células Madre , Ratones , Animales , Diferenciación Celular/genética , Células Madre/metabolismo , Factor de Transcripción TFIID/genética , Mucosa Intestinal/metabolismo , Proteínas de Drosophila/metabolismo , Complejo Represivo Polycomb 2/metabolismo , Epigénesis GenéticaRESUMEN
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.
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Melanoma , Humanos , Línea Celular Tumoral , Melanoma/patología , Secuencias Reguladoras de Ácidos Nucleicos , Inestabilidad Genómica , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , Elementos de Facilitación Genéticos , Proteínas/metabolismoRESUMEN
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.
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Carcinoma Medular , Carcinoma de Células Renales , Ferroptosis , Neoplasias Renales , Rasgo Drepanocítico , Humanos , Neoplasias Renales/patología , Carcinoma Medular/metabolismo , Carcinoma de Células Renales/patología , Proteína SMARCB1/genética , Proteína SMARCB1/metabolismo , Proteínas Represoras , Proteínas de HomeodominioRESUMEN
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.
Asunto(s)
Fibroblastos Asociados al Cáncer , Carcinoma de Células Renales , Neoplasias Renales , Humanos , Fibroblastos Asociados al Cáncer/patología , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/genética , Perfilación de la Expresión Génica , Inmunoterapia , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/genética , Pronóstico , Microambiente Tumoral , Ensayos Clínicos como AsuntoRESUMEN
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.
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Carcinoma de Células Renales , Neoplasias Renales , Humanos , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/patología , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Nivolumab/administración & dosificación , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/genética , Neoplasias Renales/patología , Ipilimumab/administración & dosificación , Metilación de ADN , Fenotipo , Microambiente Tumoral/genética , Oxigenasas de Función Mixta , Proteínas Proto-Oncogénicas/genéticaRESUMEN
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.
Asunto(s)
Melanoma , Inhibidores de Proteínas Quinasas , ARN Largo no Codificante , Proteínas ras , Humanos , Línea Celular Tumoral , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/metabolismo , Dinámicas Mitocondriales , Fosforilación Oxidativa , Inhibidores de Proteínas Quinasas/farmacología , Proteínas ras/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Resistencia a AntineoplásicosRESUMEN
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.
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Melanoma , Neoplasias Cutáneas , Humanos , Ratones , Animales , Microambiente Tumoral , Melanoma/genética , Melanoma/metabolismo , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo , Citocinas/metabolismo , Queratinocitos/metabolismo , Linfopoyetina del Estroma Tímico , Melanoma Cutáneo MalignoRESUMEN
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.
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Transdiferenciación Celular/genética , Perfilación de la Expresión Génica , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Subunidades de Proteína/metabolismo , Análisis de la Célula Individual , Factor de Transcripción TFIID/metabolismo , Envejecimiento/metabolismo , Animales , Peso Corporal/efectos de los fármacos , Transdiferenciación Celular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Células Secretoras de Glucagón/efectos de los fármacos , Células Secretoras de Glucagón/metabolismo , Glucosa/farmacología , Insulina/metabolismo , Secreción de Insulina/efectos de los fármacos , Células Secretoras de Insulina/efectos de los fármacos , Ratones , Mutación/genética , Factor de Transcripción TFIID/deficiencia , Factores de Transcripción/metabolismoRESUMEN
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.
Asunto(s)
Proliferación Celular/fisiología , Citrulinación/fisiología , Glucólisis/fisiología , Neoplasias/metabolismo , Arginina Deiminasa Proteína-Tipo 1/metabolismo , Arginina Deiminasa Proteína-Tipo 3/metabolismo , Piruvato Quinasa/metabolismo , Regulación Alostérica , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Humanos , Melanoma , Proteínas de la Membrana , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2 , Neoplasias/genética , Arginina Deiminasa Proteína-Tipo 1/genética , Arginina Deiminasa Proteína-Tipo 3/genética , Hormonas Tiroideas , Regulación hacia Arriba , Proteínas de Unión a Hormona TiroideRESUMEN
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.
RESUMEN
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.
Asunto(s)
Antígenos Nucleares/fisiología , ADN Helicasas/fisiología , Regulación Neoplásica de la Expresión Génica , Melanoma Experimental/genética , Proteínas del Tejido Nervioso/fisiología , Proteínas Nucleares/fisiología , Proteínas Proto-Oncogénicas B-raf/genética , Neoplasias Cutáneas/genética , Factores de Transcripción/fisiología , Animales , Antígenos Nucleares/genética , ADN Helicasas/genética , Progresión de la Enfermedad , Epigénesis Genética , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Ratones , Factor de Transcripción Asociado a Microftalmía/genética , Factor de Transcripción Asociado a Microftalmía/metabolismo , Mutación , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Fosfohidrolasa PTEN/genética , Factores de Transcripción SOXE/genética , Factores de Transcripción SOXE/metabolismo , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Factores de Transcripción/genética , Células Tumorales CultivadasRESUMEN
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.
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Antígenos de Neoplasias/metabolismo , Carcinogénesis/genética , Melanoma/genética , Recurrencia Local de Neoplasia/genética , Neoplasias Cutáneas/genética , Proteínas de Anclaje a la Quinasa A/genética , Adulto , Antígenos de Neoplasias/inmunología , Biopsia , Carcinogénesis/inmunología , Evolución Clonal , Proteínas del Citoesqueleto/genética , Análisis Mutacional de ADN , Células HEK293 , Humanos , MAP Quinasa Quinasa 1/genética , Masculino , Melanoma/inmunología , Melanoma/patología , Mutación , Mutación Missense , Recurrencia Local de Neoplasia/inmunología , Recurrencia Local de Neoplasia/patología , Proteínas de Fusión Oncogénica/genética , Proteínas Proto-Oncogénicas B-raf/genética , Piel/patología , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/patología , Secuenciación del ExomaRESUMEN
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.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Melanoma/patología , Timina ADN Glicosilasa/genética , Animales , Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular/genética , Citosina/análogos & derivados , Citosina/metabolismo , Metilación de ADN , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma Experimental/genética , Melanoma Experimental/patología , Ratones Noqueados , Ratones SCID , Ratones Transgénicos , Terapia Molecular Dirigida/métodos , Timina ADN Glicosilasa/antagonistas & inhibidores , Timina ADN Glicosilasa/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Heterogeneidad Genética , Melanoma/genética , Melanoma/patología , Factor de Transcripción Asociado a Microftalmía/genética , Adulto , Anciano de 80 o más Años , Biomarcadores de Tumor , Línea Celular Tumoral , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Melanoma/metabolismo , Factor de Transcripción Asociado a Microftalmía/metabolismo , Mutación , Análisis de la Célula IndividualRESUMEN
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.