RESUMEN
Poorly differentiated (PD) chordoma, a rare, aggressive tumor originating from notochordal tissue, shows loss of SMARCB1 expression, a core component of the Switch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complexes. To determine the impact of SMARCB1 re-expression on cell growth and gene expression, two SMARCB1-negative PD chordoma cell lines with an inducible SMARCB1 expression system were generated. After 72 hours of induction of SMARCB1, both SMARCB1-negative PD chordoma cell lines continued to proliferate. This result contrasted with those observed with SMARCB1-negative rhabdoid cell lines in which SMARCB1 re-expression caused the rapid inhibition of growth. We found that the lack of growth inhibition may arise from the loss of CDKN2A (p16INK4A) expression in PD chordoma cell lines. RNA-sequencing of cell lines after SMARCB1 re-expression showed a down-regulation for rRNA and RNA processing as well as metabolic processing and increased expression of genes involved in cell adhesion, cell migration, and development. Taken together, these data establish that SMARCB1 re-expression in PD chordomas alters the repertoire of SWI/SNF complexes, perhaps restoring those associated with cellular differentiation. These novel findings support a model in which SMARCB1 inactivation blocks the conversion of growth-promoting SWI/SNF complexes to differentiation-inducing ones, and they implicate SMARCB1 loss as a late event in tumorigenic progression. Importantly, the absence of growth inhibition after SMARCB1 restoration creates a unique opportunity to identify therapeutic vulnerabilities.
Asunto(s)
Cordoma , Humanos , Cordoma/genética , Cordoma/patología , Factores de Transcripción/metabolismo , Diferenciación Celular/genética , Carcinogénesis , Proteína SMARCB1/genéticaRESUMEN
Nuclear factor erythroid 2-related factor 2 (NFE2L2, also known as NRF2) is a transcription factor and master regulator of cellular antioxidant response. Aberrantly high NRF2-dependent transcription is recurrent in human cancer, but conversely NRF2 activity diminishes with age and in neurodegenerative and metabolic disorders. Although NRF2-activating drugs are clinically beneficial, NRF2 inhibitors do not yet exist. Here, we describe use of a gain-of-function genetic screen of the kinome to identify new druggable regulators of NRF2 signaling. We found that the under-studied protein kinase brain-specific kinase 2 (BRSK2) and the related BRSK1 kinases suppress NRF2-dependent transcription and NRF2 protein levels in an activity-dependent manner. Integrated phosphoproteomics and RNAseq studies revealed that BRSK2 drives 5'-AMP-activated protein kinase α2 (AMPK) signaling and suppresses the mTOR pathway. As a result, BRSK2 kinase activation suppresses ribosome-RNA complexes, global protein synthesis and NRF2 protein levels. Collectively, our data illuminate the BRSK2 and BRSK1 kinases, in part by functionally connecting them to NRF2 signaling and mTOR. This signaling axis might prove useful for therapeutically targeting NRF2 in human disease.This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Factor 2 Relacionado con NF-E2 , Receptor EphA5 , Proteínas Quinasas Activadas por AMP/metabolismo , Mutación con Ganancia de Función , Humanos , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal/genéticaRESUMEN
Activation of the nuclear factor (erythroid-derived 2)-like 2 (NFE2L2 or NRF2) transcription factor is a critical and evolutionarily conserved cellular response to oxidative stress, metabolic stress, and xenobiotic insult. Deficiency of NRF2 results in hypersensitivity to a variety of stressors, whereas its aberrant activation contributes to several cancer types, most commonly squamous cell carcinomas of the esophagus, oral cavity, bladder, and lung. Between 10% and 35% of patients with squamous cell carcinomas display hyperactive NRF2 signaling, harboring activating mutations and copy number amplifications of the NFE2L2 oncogene or inactivating mutations or deletions of KEAP1 or CUL3, the proteins of which co-complex to ubiquitylate and degrade NRF2 protein. To better understand the role of NRF2 in tumorigenesis and more broadly in development, we engineered the endogenous Nfe2l2 genomic locus to create a conditional mutant LSL-Nrf2E79Q mouse model. The E79Q mutation, one of the most commonly observed NRF2-activating mutations in human squamous cancers, codes for a mutant protein that does not undergo KEAP1/CUL3-dependent degradation, resulting in its constitutive activity. Expression of NRF2 E79Q protein in keratin 14 (KRT14)-positive murine tissues resulted in hyperplasia of squamous cell tissues of the tongue, forestomach, and esophagus, a stunted body axis, decreased weight, and decreased visceral adipose depots. RNA-seq profiling and follow-up validation studies of cultured NRF2E79Q murine esophageal epithelial cells revealed known and novel NRF2-regulated transcriptional programs, including genes associated with squamous cell carcinoma (e.g. Myc), lipid and cellular metabolism (Hk2, Ppard), and growth factors (Areg, Bmp6, Vegfa). These data suggest that in addition to decreasing adipogenesis, KRT14-restricted NRF2 activation drives hyperplasia of the esophagus, forestomach, and tongue, but not formation of squamous cell carcinoma. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Asunto(s)
Tejido Adiposo Blanco/patología , Carcinogénesis/genética , Modelos Animales de Enfermedad , Factor 2 Relacionado con NF-E2/genética , Lesiones Precancerosas/genética , Tracto Gastrointestinal Superior/patología , Animales , Carcinoma de Células Escamosas/genética , Esófago/patología , Humanos , Hiperplasia/genética , Ratones , Mutación , Lengua/patologíaRESUMEN
The new paradigm of mutations in chromatin-modifying genes as driver events in the development of cancers has proved challenging to resolve the complex influences over disease phenotypes. In particular, impaired activities of members of the SWI/SNF chromatin remodeling complex have appeared in an increasing variety of tumors. Mutations in SNF5, a member of this ubiquitously expressed complex, arise in almost all cases of malignant rhabdoid tumor in the absence of additional genetic alterations. Therefore, we studied how activation of additional oncogenic pathways might shift the phenotype of disease driven by SNF5 loss. With the use of a genetically engineered mouse model, we examined the effects of a hypomorphic Vhl2B allele on disease phenotype, with a modest up-regulation of the hypoxia response pathway. Snf5+/-;Vhl2B/+ mice did not demonstrate a substantial difference in overall survival or a change in malignant rhabdoid tumor development. However, a high percentage of female mice showed complex hemorrhagic ovarian cysts, a phenotype rarely found in either parental mouse strain. These lesions also showed mosaic expression of SNF5 by immunohistochemistry. Therefore, our studies implicate that modest changes in angiogenic regulation interact with perturbations of SWI/SNF complex activity to modulate disease phenotypes.
Asunto(s)
Hemorragia/patología , Mutación , Quistes Ováricos/patología , Proteína SMARCB1/fisiología , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/fisiología , Animales , Femenino , Hemorragia/etiología , Hemorragia/metabolismo , Ratones , Ratones Noqueados , Quistes Ováricos/etiología , Quistes Ováricos/metabolismo , FenotipoRESUMEN
Small cell carcinoma of the ovary, hypercalcaemic type (SCCOHT) is a rare but aggressive and untreatable malignancy affecting young women. We and others recently discovered that SMARCA4, a gene encoding the ATPase of the SWI/SNF chromatin-remodelling complex, is the only gene recurrently mutated in the majority of SCCOHT. The low somatic complexity of SCCOHT genomes and the prominent role of the SWI/SNF chromatin-remodelling complex in transcriptional control of genes suggest that SCCOHT cells may rely on epigenetic rewiring for oncogenic transformation. Herein, we report that approximately 80% (19/24) of SCCOHT tumour samples have strong expression of the histone methyltransferase EZH2 by immunohistochemistry, with the rest expressing variable amounts of EZH2. Re-expression of SMARCA4 suppressed the expression of EZH2 in SCCOHT cells. In comparison to other ovarian cell lines, SCCOHT cells displayed hypersensitivity to EZH2 shRNAs and two selective EZH2 inhibitors, GSK126 and EPZ-6438. EZH2 inhibitors induced cell cycle arrest, apoptosis, and cell differentiation in SCCOHT cells, along with the induction of genes involved in cell cycle regulation, apoptosis, and neuron-like differentiation. EZH2 inhibitors suppressed tumour growth and improved the survival of mice bearing SCCOHT xenografts. Therefore, our data suggest that loss of SMARCA4 creates a dependency on the catalytic activity of EZH2 in SCCOHT cells and that pharmacological inhibition of EZH2 is a promising therapeutic strategy for treating this disease. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Asunto(s)
Carcinoma de Células Pequeñas/enzimología , Proteína Potenciadora del Homólogo Zeste 2/antagonistas & inhibidores , N-Metiltransferasa de Histona-Lisina/metabolismo , Hipercalcemia/enzimología , Neoplasias Ováricas/enzimología , Animales , Apoptosis/fisiología , Carcinoma Epitelial de Ovario , Puntos de Control del Ciclo Celular/fisiología , Línea Celular Tumoral , Transformación Celular Neoplásica , ADN Helicasas/deficiencia , Regulación hacia Abajo , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Femenino , Histona Metiltransferasas , Humanos , Trasplante de Neoplasias , Neoplasias Glandulares y Epiteliales/enzimología , Proteínas Nucleares/deficiencia , Factores de Transcripción/deficiencia , Trasplante Heterólogo , Células Tumorales Cultivadas , Regulación hacia ArribaRESUMEN
Small cell carcinoma of the ovary, hypercalcaemic type (SCCOHT) is a lethal and sometimes familial ovarian tumour of young women and children. We and others recently discovered that over 90% of SCCOHTs harbour inactivating mutations in the chromatin remodelling gene SMARCA4 with concomitant loss of its encoded protein SMARCA4 (BRG1), one of two mutually exclusive ATPases of the SWI/SNF chromatin remodelling complex. To determine the specificity of SMARCA4 loss for SCCOHT, we examined the expression of SMARCA4 by immunohistochemistry in more than 3000 primary gynaecological tumours. Among ovarian tumours, it was only absent in clear cell carcinoma (15 of 360, 4%). In the uterus, it was absent in endometrial stromal sarcomas (4 of 52, 8%) and high-grade endometrioid carcinomas (2 of 338, 1%). Recent studies have shown that SMARCA2 (BRM), the other mutually exclusive ATPase of the SWI/SNF complex, is necessary for survival of tumour cells lacking SMARCA4. Therefore, we examined SMARCA2 expression and discovered that all SMARCA4-negative SCCOHTs also lacked SMARCA2 protein by IHC, including the SCCOHT cell lines BIN67 and SCCOHT1. Among ovarian tumours, the SMARCA4/SMARCA2 dual loss phenotype appears completely specific for SCCOHT. SMARCA2 loss was not due to mutation but rather from an absence of mRNA expression, which was restored by treatment with the histone deacetylase inhibitor trichostatin A. Re-expression of SMARCA4 or SMARCA2 inhibited the growth of BIN67 and SCCOHT1 cell lines. Our results indicate that SMARCA4 loss, either alone or with SMARCA2, is highly sensitive and specific for SCCOHT and that restoration of either SWI/SNF ATPase can inhibit the growth of SCCOHT cell lines.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Carcinoma de Células Pequeñas/genética , ADN Helicasas/deficiencia , Proteínas Nucleares/deficiencia , Neoplasias Ováricas/diagnóstico , Factores de Transcripción/deficiencia , Adenosina Trifosfatasas/metabolismo , Carcinoma de Células Pequeñas/diagnóstico , Línea Celular Tumoral , Proliferación Celular/fisiología , Transformación Celular Neoplásica/genética , Proteínas Cromosómicas no Histona/deficiencia , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Silenciador del Gen/fisiología , Humanos , Hipercalcemia/genética , Inmunohistoquímica , Mutación/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Neoplasias Ováricas/genética , Proteína SMARCB1 , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The identification of mutations and deletions in the SMARCB1 locus in chromosome band 22q11.2 in pediatric rhabdoid tumors provided the first evidence for the involvement of the SWI/SNF chromatin remodeling complex in cancer. Over the last 15 years, alterations in more than 20 members of the complex have been reported in a variety of human tumors. These include germline mutations and copy number alterations in SMARCB1, SMARCA4, SMARCE1, and PBRM1 that predispose carriers to both benign and malignant neoplasms. Somatic mutations, structural abnormalities, or epigenetic modifications that lead to reduced or aberrant expression of complex members have now been reported in more than 20% of malignancies, including both solid tumors and hematologic disorders in both children and adults. In this review, we will highlight the role of SMARCB1 in cancer as a paradigm for other tumors with alterations in SWI/SNF complex members and demonstrate the broad spectrum of mutations observed in complex members in different tumor types.
Asunto(s)
Ensamble y Desensamble de Cromatina , Proteínas Cromosómicas no Histona/genética , ADN Helicasas/genética , Proteínas de Unión al ADN/genética , Neoplasias/genética , Proteínas Nucleares/genética , Factores de Transcripción/genética , Animales , Carcinoma de Células Renales/genética , Modelos Animales de Enfermedad , Predisposición Genética a la Enfermedad , Humanos , Neoplasias Renales/genética , Ratones Noqueados , Mutación , Neurilemoma/genética , Neurofibromatosis/genética , Tumor Rabdoide/genética , Proteína SMARCB1 , Neoplasias Cutáneas/genéticaRESUMEN
Prior to the next generation sequencing and characterization of the tumor genome landscape, mutations in the SWI/SNF chromatin remodeling complex and the KEAP1-NRF2 signaling pathway were underappreciated. While these two classes of mutations appeared to independently contribute to tumor development, recent reports have demonstrated a mechanistic link between these two regulatory mechanisms in specific cancer types and cell models. In this work, we expand upon these data by exploring the relationship between mutations in BAF and PBAF subunits of the SWI/SNF complex and activation of NRF2 signal transduction across many cancer types. ARID1A/B mutations were strongly associated with NRF2 transcriptional activity in head and neck squamous carcinomas (HNSC). Many additional tumor types showed significant association between NRF2 signaling and mutation of specific components of the SWI/SNF complex. Different effects of BAF and PBAF mutations on the polarity of NRF2 signaling were observed. Overall, our results support a context-dependent functional link between SWI/SNF and NRF2 mutations across human cancers and implicate ARID1A inactivation in HPV-negative HNSC in promoting tumor progression and survival through activation of the KEAP1-NRF2 signaling pathway. The tumor-specific effects of these mutations open a new area of study for how mutations in the KEAP1-NRF2 pathway and the SWI/SNF complex contribute to cancer.
Asunto(s)
Proteínas de Unión al ADN , Neoplasias de Cabeza y Cuello , Factor 2 Relacionado con NF-E2 , Factores de Transcripción , Humanos , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Neoplasias de Cabeza y Cuello/genética , Proteína 1 Asociada A ECH Tipo Kelch/genética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Mutación , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transducción de Señal/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Squamous cell carcinomas (SCCs), including lung, head & neck, bladder, and skin SCCs often display constitutive activation of the KEAP1-NRF2 pathway. Constitutive activation is achieved through multiple mechanisms, including activating mutations in NFE2L2 (NRF2). To determine the functional consequences of Nrf2 activation on skin SCC development, we assessed the effects of mutant Nrf2E79Q expression, one of the most common activating mutations in human SCCs, on tumor promotion and progression in the mouse skin multistage carcinogenesis model using a DMBA-initiation/TPA-promotion protocol where the Hras A->T mutation (Q61L) is the canonical driver mutation. Nrf2E79Q expression was temporally and conditionally activated in the epidermis at two stages of tumor development: 1) after DMBA initiation in the epidermis but before cutaneous tumor development and 2) in pre-existing DMBA-initiated/TPA-promoted squamous papillomas. Expression of Nrf2E79Q in the epidermis after DMBA initiation but before tumor occurrence inhibited the development/promotion of 70% of squamous papillomas. However, the remaining papillomas often displayed non-canonical Hras and Kras mutations and enhanced progression to SCCs compared to control mice expressing wildtype Nrf2. Nrf2E79Q expression in pre-existing tumors caused rapid regression of 60% of papillomas. The remaining papillomas displayed the expected canonical Hras A->T mutation (Q61L) and enhanced progression to SCCs. These results demonstrate that mutant Nrf2E79Q enhances the promotion and progression of a subset of skin tumors and alters the frequency and diversity of oncogenic Ras mutations when expressed early after initiation.
RESUMEN
Aberrant activation of the NRF2/NFE2L2 transcription factor commonly occurs in head and neck squamous cell carcinomas (HNSCC). Mouse model studies have shown that NRF2 activation alone does not result in cancer. When combined with classic oncogenes and at the right dose, NRF2 activation promotes tumor initiation and progression. Here we deleted the tumor suppressor genes p16INK4A and p53 (referred to as CP mice), which are commonly lost in human HNSCC, in the presence of a constitutively active NRF2E79Q mutant (CPN mice). NRF2E79Q expression in CPN mice resulted in squamous cell hyperplasia or dysplasia with hyperkeratosis in the esophagus, oropharynx, and forestomach. In addition, CPN mice displayed oral cavity squamous cell carcinoma (OSCC); CP mice bearing wild-type NRF2 expression did not develop oral cavity hyperplasia, dysplasia or OSCC. In both CP and CPN mice, we also observed predominantly abdominal sarcomas and carcinomas. Our data show that in the context of p53 and p16 tumor suppressor loss, NRF2 activation serves oncogenic functions to drive OSCC. CPN mice represent a new model for OSCC that closely reflects the genetics of human HNSCC. SIGNIFICANCE: Human squamous cancers frequently show constitutive NRF2 activation, associated with poorer outcomes and resistance to multiple therapies. Here, we report the first activated NRF2-driven and human-relevant mouse model of squamous cell carcinoma that develops in the background of p16 and p53 loss. The availability of this model will lead to a clearer understanding of how NRF2 contributes to the initiation, progression, and therapeutic response of OSCC.
Asunto(s)
Carcinoma de Células Escamosas , Neoplasias de Cabeza y Cuello , Neoplasias de la Boca , Animales , Humanos , Ratones , Carcinoma de Células Escamosas/genética , Modelos Animales de Enfermedad , Neoplasias de Cabeza y Cuello/genética , Hiperplasia/genética , Neoplasias de la Boca/genética , Factor 2 Relacionado con NF-E2/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Proteína p53 Supresora de Tumor/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismoRESUMEN
PURPOSE: Large cell neuroendocrine carcinoma (LCNEC) is a high-grade neuroendocrine malignancy that, like small cell lung cancer (SCLC), is associated with an absence of druggable oncogenic drivers and dismal prognosis. In contrast to SCLC, however, there is little evidence to guide optimal treatment strategies which are often adapted from SCLC and non-small cell lung cancer (NSCLC) approaches. EXPERIMENTAL DESIGN: To better define the biology of LCNEC, we analyzed cell line and patient genomic data and performed immunohistochemistry and single-cell (sc)RNAseq of core needle biopsies from LCNEC patients and preclinical models. RESULTS: Here, we demonstrate that the presence or absence of YAP1 distinguishes two subsets of LCNEC. The YAP1-high subset is mesenchymal and inflamed and characterized, alongside TP53 mutations, by co-occurring alterations in CDKN2A/B and SMARCA4. Therapeutically, the YAP1-high subset demonstrates vulnerability to MEK and AXL targeting strategies, including a novel preclinical AXL CAR-T cell. Meanwhile, the YAP1-low subset is epithelial and immune-cold and more commonly features TP53 and RB1 co-mutations, similar to those observed in pure SCLC. Notably, the YAP1-low subset is also characterized by expression of SCLC subtype-defining transcription factors - especially ASCL1 and NEUROD1 - and, as expected given its transcriptional similarities to SCLC, exhibits putative vulnerabilities reminiscent of SCLC, including Delta-like ligand 3 (DLL3) and CD56 targeting, as with novel preclinical DLL3 and CD56 CAR T-cells, and DNA damage repair (DDR) inhibition. CONCLUSION: YAP1 defines distinct subsets of LCNEC with unique biology. These findings highlight the potential for YAP1 to guide personalized treatment strategies for LCNEC.
RESUMEN
Malignant rhabdoid tumors (MRTs) are rare, aggressive cancers occuring in young children primarily through inactivation of the SNF5(INI1, SMARCB1) tumor suppressor gene. We and others have demonstrated that mice heterozygous for a Snf5 null allele develop MRTs with partial penetrance. We have also shown that Snf5(+/-) mice that lack expression of the pRb family, due to TgT121 transgene expression, develop MRTs with increased penetrance and decreased latency. Here, we report that altering the genetic background has substantial effects upon MRT development in Snf5(+/--) and TgT121 ;Snf5(+/-) mice, with a mixed F1 background resulting in increased latency and the appearance of brain tumors. We also report the establishment of the first mouse MRT cell lines that recapitulate many features of their human counterparts. Our studies provide further insight into the genetic influences on MRT development as well as provide valuable new cell culture and genetically engineered mouse models for the study of CNS-MRT etiology.
Asunto(s)
Línea Celular Tumoral , Tumor Rabdoide/genética , Animales , Proliferación Celular , Transformación Celular Neoplásica/genética , Neoplasias del Plexo Coroideo/genética , Neoplasias del Plexo Coroideo/patología , Proteínas Cromosómicas no Histona/genética , Modelos Animales de Enfermedad , Genotipo , Humanos , Cariotipo , Ratones , Ratones Transgénicos , Fenotipo , Tumor Rabdoide/patología , Proteína SMARCB1RESUMEN
SMARCA4 (BRG1) and SMARCA2 (BRM) are the two paralogous ATPases of the SWI/SNF chromatin remodeling complexes frequently inactivated in cancers. Cells deficient in either ATPase have been shown to depend on the remaining counterpart for survival. Contrary to this paralog synthetic lethality, concomitant loss of SMARCA4/2 occurs in a subset of cancers associated with very poor outcomes. Here, we uncover that SMARCA4/2-loss represses expression of the glucose transporter GLUT1, causing reduced glucose uptake and glycolysis accompanied with increased dependency on oxidative phosphorylation (OXPHOS); adapting to this, these SMARCA4/2-deficient cells rely on elevated SLC38A2, an amino acid transporter, to increase glutamine import for fueling OXPHOS. Consequently, SMARCA4/2-deficient cells and tumors are highly sensitive to inhibitors targeting OXPHOS or glutamine metabolism. Furthermore, supplementation of alanine, also imported by SLC38A2, restricts glutamine uptake through competition and selectively induces death in SMARCA4/2-deficient cancer cells. At a clinically relevant dose, alanine supplementation synergizes with OXPHOS inhibition or conventional chemotherapy eliciting marked antitumor activity in patient-derived xenografts. Our findings reveal multiple druggable vulnerabilities of SMARCA4/2-loss exploiting a GLUT1/SLC38A2-mediated metabolic shift. Particularly, unlike dietary deprivation approaches, alanine supplementation can be readily applied to current regimens for better treatment of these aggressive cancers.
Asunto(s)
Glutamina , Neoplasias , Humanos , Transportador de Glucosa de Tipo 1 , Adenosina Trifosfatasas/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Suplementos Dietéticos , ADN Helicasas/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
For DNA replication to occur, chromatin must be remodeled. Yet, we know very little about which proteins alter nucleosome occupancy at origins and replication forks and for what aspects of replication they are required. Here, we demonstrate that the BRG1 catalytic subunit of mammalian SWI/SNF-related complexes co-localizes with origin recognition complexes, GINS complexes, and proliferating cell nuclear antigen at sites of DNA replication on extended chromatin fibers. The specific pattern of BRG1 occupancy suggests it does not participate in origin selection but is involved in the firing of origins and the process of replication elongation. This latter function is confirmed by the fact that Brg1 mutant mouse embryos and RNAi knockdown cells exhibit a 50% reduction in replication fork progression rates, which is associated with decreased cell proliferation. This novel function of BRG1 is consistent with its requirement during embryogenesis and its role as a tumor suppressor to maintain genome stability and prevent cancer.
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ADN Helicasas/fisiología , Replicación del ADN , Proteínas Nucleares/fisiología , Factores de Transcripción/fisiología , Animales , Proliferación Celular , Cromatina/química , ADN Helicasas/análisis , ADN Helicasas/genética , Proteínas de Unión al ADN/análisis , Desarrollo Embrionario , Células Eritroides/metabolismo , Células HeLa , Humanos , Ratones , Mutación , Proteínas Nucleares/análisis , Proteínas Nucleares/genética , Fenotipo , Factores de Transcripción/análisis , Factores de Transcripción/genéticaRESUMEN
Studies have shown that Nrf2E79Q/+ is one of the most common mutations found in human tumors. To elucidate how this genetic change contributes to lung cancer, we compared lung tumor development in a genetically-engineered mouse model (GEMM) with dual Trp53/p16 loss, the most common mutations found in human lung tumors, in the presence or absence of Nrf2E79Q/+. Trp53/p16-deficient mice developed combined-small cell lung cancer (C-SCLC), a mixture of pure-SCLC (P-SCLC) and large cell neuroendocrine carcinoma. Mice possessing the LSL-Nrf2E79Q mutation showed no difference in the incidence or latency of C-SCLC compared with Nrf2+/+ mice. However, these tumors did not express NRF2 despite Cre-induced recombination of the LSL-Nrf2E79Q allele. Trp53/p16-deficient mice also developed P-SCLC, where activation of the NRF2E79Q mutation associated with a higher incidence of this tumor type. All C-SCLCs and P-SCLCs were positive for NE-markers, NKX1-2 (a lung cancer marker) and negative for P63 (a squamous cell marker), while only P-SCLC expressed NRF2 by immunohistochemistry. Analysis of a consensus NRF2 pathway signature in human NE+-lung tumors showed variable activation of NRF2 signaling. Our study characterizes the first GEMM that develops C-SCLC, a poorly-studied human cancer and implicates a role for NRF2 activation in SCLC development.
Asunto(s)
Carcinoma Neuroendocrino , Neoplasias Pulmonares , Carcinoma Pulmonar de Células Pequeñas , Animales , Carcinoma Neuroendocrino/patología , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Proteínas de Homeodominio/metabolismo , Humanos , Incidencia , Neoplasias Pulmonares/patología , Ratones , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Proteínas Nucleares/metabolismo , Carcinoma Pulmonar de Células Pequeñas/patología , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Eukaryotic organisms package DNA into chromatin for compact storage in the cell nucleus. However, this process promotes transcriptional repression of genes. To overcome the transcriptional repression, chromatin remodeling complexes have evolved that alter the configuration of chromatin packaging of DNA into nucleosomes by histones. The SWI/SNF chromatin remodeling complex uses energy from ATP hydrolysis to reposition nucleosomes and make DNA accessible to transcription factors. Recent studies showing mutations of BRG1, one of two mutually exclusive ATPase subunits, in human tumor cell lines and primary tissue samples have implicated a role for its loss in cancer development. While most of the mutations lead to complete loss of BRG1 protein expression, others result in single amino acid substitutions. To better understand the role of these BRG1 point mutations in cancer development, we characterized SWI/SNF function in human tumor cell lines with these mutations in the absence of BRM expression, the other ATPase component. We found that the mutant BRG1 proteins still interacted with the core complex members and appeared at the promoters of target genes. However, while these mutations did not affect CD44 and CDH1 expression, known targets of the SWI/SNF complex, they did abrogate Rb-mediated cell-cycle arrest. Therefore, our results implicate that these mutations disrupt the de novo chromatin remodeling activity of the complex without affecting the status of existing nucleosome positioning.
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Ciclo Celular/genética , ADN Helicasas/genética , Proteínas Nucleares/genética , Mutación Puntual , Proteína de Retinoblastoma/metabolismo , Factores de Transcripción/genética , Adenosina Trifosfatasas/genética , Antígenos CD , Cadherinas/metabolismo , Línea Celular Tumoral , Proteínas Cromosómicas no Histona/metabolismo , ADN Helicasas/metabolismo , Humanos , Receptores de Hialuranos/metabolismo , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismoRESUMEN
Epigenetic effectors "read" marks "written" on chromatin to regulate function and fidelity of the genome. Here, we show that this coordinated read-write activity of the epigenetic machinery extends to the cytoskeleton, with PBRM1 in the PBAF chromatin remodeling complex reading microtubule methyl marks written by the SETD2 histone methyltransferase. PBRM1 binds SETD2 methyl marks via BAH domains, recruiting PBAF components to the mitotic spindle. This read-write activity was required for normal mitosis: Loss of SETD2 methylation or pathogenic BAH domain mutations disrupt PBRM1 microtubule binding and PBAF recruitment and cause genomic instability. These data reveal PBRM1 functions beyond chromatin remodeling with domains that allow it to integrate chromatin and cytoskeletal activity via its acetyl-binding BD and methyl-binding BAH domains, respectively. Conserved coordinated activity of the epigenetic machinery on the cytoskeleton opens a previously unknown window into how chromatin remodeler defects can drive disease via both epigenetic and cytoskeletal dysfunction.
Asunto(s)
Microtúbulos , Lectura , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina , Citoesqueleto/metabolismo , Microtúbulos/metabolismoRESUMEN
The NF-E2-related factor 2 (referred to as NRF2) transcription factor binds antioxidant responsive elements within the promoters of cytoprotective genes to induce their expression. Next-generation sequencing studies in lung cancer have shown a significant number of activating mutations within the NRF2 signaling pathway. Mutations in components of the SWI/SNF chromatin-remodeling complex, a general regulator of transcription using either BRG1 or BRM as the catalytic subunit, also frequently occur in lung cancers. Importantly, low BRG1 expression levels in primary human NSCLC correlated with increased NRF2-target gene expression. Here, we show that loss of SWI/SNF complex function activated a subset of NRF2-mediated transcriptional targets. Using a series of isogenic NSCLC lines with reduced or depleted BRG1 and/or BRM expression, we observed significantly increased expression of the NRF2-target genes HMOX1 and GSTM4. In contrast, expression of the NRF2 target genes NQO1 and GCLM modestly increased following BRM reduction. Chromatin immunoprecipitation showed that BRG1 knockdown led to increased NRF2 binding at its respective ARE sites in the HMOX1 promoter but not in NQO1 and GCLM. Our data demonstrate that loss of BRG1 or BRM in lung cancer results in activation of the NRF2/KEAP1 pathway and HMOX1 expression. Therefore, we provide an additional molecular explanation for why patients harboring BRG1 or BRM mutations show poor prognoses. A better understanding of this mechanism may yield novel insights into the design of targeted treatment modalities. IMPLICATIONS: Our study identifies a novel mechanism for how mutations in the SMARCA4 gene may drive progression of human lung adenocarcinomas.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/genética , ADN Helicasas/genética , Neoplasias Pulmonares/genética , Proteínas Nucleares/genética , Análisis de Secuencia de ADN/métodos , Transducción de Señal , Factores de Transcripción/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Ensamble y Desensamble de Cromatina , Regulación Neoplásica de la Expresión Génica , Glutamato-Cisteína Ligasa/genética , Glutatión Transferasa/genética , Hemo-Oxigenasa 1/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/genética , Mutación , NAD(P)H Deshidrogenasa (Quinona)/genética , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismoRESUMEN
Small-cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare and highly aggressive ovarian malignancy. In almost all cases, it is associated with somatic and often germline pathogenic variants in SMARCA4, which encodes for the SMARCA4 protein (BRG1), a subunit of the SWI/SNF chromatin remodeling complex. Approximately 20% of human cancers possess pathogenic variants in at least one SWI/SNF subunit. Because of their role in regulating many important cellular processes including transcriptional control, DNA repair, differentiation, cell division, and DNA replication, SWI/SNF complexes with mutant subunits are thought to contribute to cancer initiation and progression. Fewer than 500 cases of SCCOHT have been reported in the literature and approximately 60% are associated with hypercalcemia. SCCOHT primarily affects females under 40 years of age who usually present with symptoms related to a pelvic mass. SCCOHT is an aggressive cancer, with long-term survival rates of 30% in early-stage cases. Although various treatment approaches have been proposed, there is no consensus on surveillance and therapeutic strategy. An international group of multidisciplinary clinicians and researchers recently formed the International SCCOHT Consortium to evaluate current knowledge and propose consensus surveillance and therapeutic recommendations, with the aim of improving outcomes. Here, we present an overview of the genetics of this cancer, provide updates on new treatment targets, and propose management guidelines for this challenging cancer.
Asunto(s)
Carcinoma de Células Pequeñas/genética , ADN Helicasas/genética , Hipercalcemia/genética , Proteínas Nucleares/genética , Neoplasias Ováricas/genética , Factores de Transcripción/genética , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma de Células Pequeñas/sangre , Carcinoma de Células Pequeñas/mortalidad , Carcinoma de Células Pequeñas/terapia , Quimioterapia Adyuvante/métodos , Quimioterapia Adyuvante/normas , Ensamble y Desensamble de Cromatina/genética , Femenino , Ginecología/normas , Humanos , Hipercalcemia/sangre , Hipercalcemia/patología , Hipercalcemia/terapia , Oncología Médica/normas , Mutación , Neoplasias Ováricas/sangre , Neoplasias Ováricas/mortalidad , Neoplasias Ováricas/terapia , Ovariectomía/normas , Ovario/patología , Ovario/cirugía , Guías de Práctica Clínica como Asunto , Radioterapia Adyuvante/normas , Trasplante de Células Madre/normas , Tasa de Supervivencia , Resultado del TratamientoRESUMEN
PURPOSE: Many rare ovarian cancer subtypes, such as small-cell carcinoma of the ovary, hypercalcemic type (SCCOHT), have poor prognosis due to their aggressive nature and resistance to standard platinum- and taxane-based chemotherapy. The development of effective therapeutics has been hindered by the rarity of such tumors. We sought to identify targetable vulnerabilities in rare ovarian cancer subtypes. EXPERIMENTAL DESIGN: We compared the global proteomic landscape of six cases each of endometrioid ovarian cancer (ENOC), clear cell ovarian cancer (CCOC), and SCCOHT to the most common subtype, high-grade serous ovarian cancer (HGSC), to identify potential therapeutic targets. IHC of tissue microarrays was used as validation of arginosuccinate synthase (ASS1) deficiency. The efficacy of arginine-depriving therapeutic ADI-PEG20 was assessed in vitro using cell lines and patient-derived xenograft mouse models representing SCCOHT. RESULTS: Global proteomic analysis identified low ASS1 expression in ENOC, CCOC, and SCCOHT compared with HGSC. Low ASS1 levels were validated through IHC in large patient cohorts. The lowest levels of ASS1 were observed in SCCOHT, where ASS1 was absent in 12 of 31 cases, and expressed in less than 5% of the tumor cells in 9 of 31 cases. ASS1-deficient ovarian cancer cells were sensitive to ADI-PEG20 treatment regardless of subtype in vitro. Furthermore, in two cell line mouse xenograft models and one patient-derived mouse xenograft model of SCCOHT, once-a-week treatment with ADI-PEG20 (30 mg/kg and 15 mg/kg) inhibited tumor growth in vivo. CONCLUSIONS: Preclinical in vitro and in vivo studies identified ADI-PEG20 as a potential therapy for patients with rare ovarian cancers, including SCCOHT.