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In advanced castration resistant prostate cancer (CRPC), mutations in the DNA damage response (DDR) gene ataxia telangiectasia mutated ( ATM ) are common. While poly(ADP-ribose) polymerase inhibitors are approved in this context, their clinical efficacy remains limited. Thus, there is a compelling need to identify alternative therapeutic avenues for ATM mutant prostate cancer patients. Here, we generated matched ATM-proficient and ATM-deficient CRPC lines to elucidate the impact of ATM loss on DDR in response to DNA damage via irradiation. Through unbiased phosphoproteomic screening, we unveiled that ATM-deficient CRPC lines maintain dependence on downstream ATM targets through activation of ATR and DNA-PKcs kinases. Dual inhibition of ATR and DNA-PKcs effectively inhibited downstream γH2AX foci formation in response to irradiation and radiosensitized ATM-deficient lines to a greater extent than either ATM-proficient controls or single drug treatment. Further, dual inhibition abrogated residual downstream ATM pathway signaling and impaired replication fork dynamics. To circumvent potential toxicity, we leveraged the RUVBL1/2 ATPase inhibitor Compound B, which leads to the degradation of both ATR and DNA-PKcs kinases. Compound B effectively radiosensitized ATM-deficient CRPC in vitro and in vivo , and impacted replication fork dynamics. Overall, dual targeting of both ATR and DNA-PKcs is necessary to block DDR in ATM-deficient CRPC, and Compound B could be utilized as a novel therapy in combination with irradiation in these patients.
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The glucocorticoid receptor (GR) is an important anti-cancer target in lymphoid cancers but has been understudied in solid tumors like lung cancer, although glucocorticoids are often given with chemotherapy regimens to mitigate side effects. Here, we identify a dexamethasone-GR mediated anti-cancer response in a subset of aggressive non-small cell lung cancers (NSCLCs) that harbor Serine/Threonine Kinase 11 (STK11/LKB1) mutations. High tumor expression of carbamoyl phosphate synthase 1 (CPS1) was strongly linked to the presence of LKB1 mutations, was the best predictor of NSCLC dexamethasone (DEX) sensitivity (p < 10-16) but was not mechanistically involved in DEX sensitivity. Subcutaneous, orthotopic and metastatic NSCLC xenografts, biomarker-selected, STK11/LKB1 mutant patient derived xenografts, and genetically engineered mouse models with KRAS/LKB1 mutant lung adenocarcinomas all showed marked in vivo anti-tumor responses with the glucocorticoid dexamethasone as a single agent or in combination with cisplatin. Mechanistically, GR activation triggers G1/S cell cycle arrest in LKB1 mutant NSCLCs by inducing the expression of the cyclin-dependent kinase inhibitor, CDKN1C/p57(Kip2). All findings were confirmed with functional genomic experiments including CRISPR knockouts and exogenous expression. Importantly, DEX-GR mediated cell cycle arrest did not interfere with NSCLC radiotherapy, or platinum response in vitro or with platinum response in vivo. While DEX induced LKB1 mutant NSCLCs in vitro exhibit markers of cellular senescence and demonstrate impaired migration, in vivo DEX treatment of a patient derived xenograft (PDX) STK11/LKB1 mutant model resulted in expression of apoptosis markers. These findings identify a previously unknown GR mediated therapeutic vulnerability in STK11/LKB1 mutant NSCLCs caused by induction of p57(Kip2) expression with both STK11 mutation and high expression of CPS1 as precision medicine biomarkers of this vulnerability.
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PURPOSE: Anaplastic lymphoma kinase (ALK) aberrations have been identified in pediatric-type infant gliomas, but their occurrence across age groups, functional effects, and treatment response has not been broadly established. EXPERIMENTAL DESIGN: We performed a comprehensive analysis of ALK expression and genomic aberrations in both newly generated and retrospective data from 371 glioblastomas (156 adult, 205 infant/pediatric, and 10 congenital) with in vitro and in vivo validation of aberrations. RESULTS: ALK aberrations at the protein or genomic level were detected in 12% of gliomas (45/371) in a wide age range (0-80 years). Recurrent as well as novel ALK fusions (LRRFIP1-ALK, DCTN1-ALK, PRKD3-ALK) were present in 50% (5/10) of congenital/infant, 1.4% (3/205) of pediatric, and 1.9% (3/156) of adult GBMs. ALK fusions were present as the only candidate driver in congenital/infant GBMs and were sometimes focally amplified. In contrast, adult ALK fusions co-occurred with other oncogenic drivers. No activating ALK mutations were identified in any age group. Novel and recurrent ALK rearrangements promoted STAT3 and ERK1/2 pathways and transformation in vitro and in vivo. ALK-fused GBM cellular and mouse models were responsive to ALK inhibitors, including in patient cells derived from a congenital GBM. Relevant to the treatment of infant gliomas, we showed that ALK protein appears minimally expressed in the forebrain at perinatal stages, and no gross effects on perinatal brain development were seen in pregnant mice treated with the ALK inhibitor ceritinib. CONCLUSIONS: These findings support use of brain-penetrant ALK inhibitors in clinical trials across infant, pediatric, and adult GBMs. See related commentary by Mack and Bertrand, p. 2567.
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Glioblastoma , Glioma , Ratones , Animales , Quinasa de Linfoma Anaplásico/genética , Glioblastoma/tratamiento farmacológico , Glioblastoma/genética , Estudios Retrospectivos , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Glioma/tratamiento farmacológicoRESUMEN
Comprehensive cis-regulatory landscapes are essential for accurate enhancer prediction and disease variant mapping. Although cis-regulatory element (CRE) resources exist for most tissues and organs, many rare - yet functionally important - cell types remain overlooked. Despite representing only a small fraction of the heart's cellular biomass, the cardiac conduction system (CCS) unfailingly coordinates every life-sustaining heartbeat. To globally profile the mouse CCS cis-regulatory landscape, we genetically tagged CCS component-specific nuclei for comprehensive assay for transposase-accessible chromatin-sequencing (ATAC-Seq) analysis. Thus, we established a global CCS-enriched CRE database, referred to as CCS-ATAC, as a key resource for studying CCS-wide and component-specific regulatory functions. Using transcription factor (TF) motifs to construct CCS component-specific gene regulatory networks (GRNs), we identified and independently confirmed several specific TF sub-networks. Highlighting the functional importance of CCS-ATAC, we also validated numerous CCS-enriched enhancer elements and suggested gene targets based on CCS single-cell RNA-Seq data. Furthermore, we leveraged CCS-ATAC to improve annotation of existing human variants related to cardiac rhythm and nominated a potential enhancer-target pair that was dysregulated by a specific SNP. Collectively, our results established a CCS-regulatory compendium, identified novel CCS enhancer elements, and illuminated potential functional associations between human genomic variants and CCS component-specific CREs.
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Núcleo Celular , Cromatina , Sistema de Conducción Cardíaco , Contracción Miocárdica , Animales , Humanos , Ratones , Núcleo Celular/genética , Cromatina/genética , Redes Reguladoras de Genes , Contracción Miocárdica/genética , Contracción Miocárdica/fisiología , Factores de Transcripción/genética , Sistema de Conducción Cardíaco/fisiologíaRESUMEN
The neomorphic transcription factor EWS-FLI1 is a key driver of Ewing sarcoma. Ablation of EWS-FLI1 may present a promising therapeutic strategy for this malignancy. Here we found that the deubiquitinase, ubiquitin specific peptidase 9 X-linked (USP9X) stabilizes EWS-FLI1 protein expression in Ewing sarcoma. We show that USP9X binds the ETS domain of EWS-FLI1 in Ewing sarcoma cells and deubiquitinates EWS-FLI1 and that USP9X and EWS-FLI1 protein expression is correlated in clinical Ewing sarcoma specimens. We found that treatment of Ewing sarcoma cells with the USP9X inhibitor WP1130 mediates rapid EWS-FLI1 degradation in vitro and in vivo which coincides with reduced growth of Ewing sarcoma cells and tumors. Our results suggest that USP9X might be a potential therapeutic target to mediate EWS-FLI1 depletion in Ewing sarcoma.
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Sarcoma de Ewing , Humanos , Sarcoma de Ewing/tratamiento farmacológico , Sarcoma de Ewing/genética , Sarcoma de Ewing/patología , Línea Celular Tumoral , Proteína EWS de Unión a ARN/genética , Proteína EWS de Unión a ARN/metabolismo , Proteína Proto-Oncogénica c-fli-1/genética , Proteína Proto-Oncogénica c-fli-1/metabolismo , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Transformación Celular Neoplásica , Regulación Neoplásica de la Expresión Génica , Ubiquitina Tiolesterasa/genética , Ubiquitina Tiolesterasa/metabolismoRESUMEN
EGFR oncogenic mutations predict sensitivity to EGFR inhibitors in NSCLC, but less is known about EGFR "variants of unknown significance." Using preclinical models, 3D structure analyses, and patient response data, Robichaux et al. show in Nature that mutations in structural regions of EGFR predict responses to different EGFR inhibitors.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Receptores ErbB/genética , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , MutaciónRESUMEN
Glioblastomas (GBM) are routinely treated with ionizing radiation (IR) but inevitably recur and develop therapy resistance. During treatment, the tissue surrounding tumors is also irradiated. IR potently induces senescence, and senescent stromal cells can promote the growth of neighboring tumor cells by secreting factors that create a senescence-associated secretory phenotype (SASP). Here, we carried out transcriptomic and tumorigenicity analyses in irradiated mouse brains to elucidate how radiotherapy-induced senescence of non-neoplastic brain cells promotes tumor growth. Following cranial irradiation, widespread senescence in the brain occurred, with the astrocytic population being particularly susceptible. Irradiated brains showed an altered transcriptomic profile characterized by upregulation of CDKN1A (p21), a key enforcer of senescence, and several SASP factors, including HGF, the ligand of the receptor tyrosine kinase (RTK) Met. Preirradiation of mouse brains increased Met-driven growth and invasiveness of orthotopically implanted glioma cells. Importantly, irradiated p21-/- mouse brains did not exhibit senescence and consequently failed to promote tumor growth. Senescent astrocytes secreted HGF to activate Met in glioma cells and to promote their migration and invasion in vitro, which could be blocked by HGF-neutralizing antibodies or the Met inhibitor crizotinib. Crizotinib also slowed the growth of glioma cells implanted in preirradiated brains. Treatment with the senolytic drug ABT-263 (navitoclax) selectively killed senescent astrocytes in vivo, significantly attenuating growth of glioma cells implanted in preirradiated brains. These results indicate that SASP factors in the irradiated tumor microenvironment drive GBM growth via RTK activation, underscoring the potential utility of adjuvant senolytic therapy for preventing GBM recurrence after radiotherapy. SIGNIFICANCE: This study uncovers mechanisms by which radiotherapy can promote GBM recurrence by inducing senescence in non-neoplastic brain cells, suggesting that senolytic therapy can blunt recurrent GBM growth and aggressiveness.
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Encéfalo/patología , Senescencia Celular , Rayos gamma/efectos adversos , Glioblastoma/patología , Recurrencia Local de Neoplasia/patología , Fenotipo Secretor Asociado a la Senescencia , Microambiente Tumoral , Compuestos de Anilina/farmacología , Animales , Antineoplásicos/farmacología , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Astrocitos/patología , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Glioblastoma/tratamiento farmacológico , Glioblastoma/etiología , Glioblastoma/metabolismo , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Recurrencia Local de Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/etiología , Recurrencia Local de Neoplasia/metabolismo , Sulfonamidas/farmacologíaRESUMEN
Identifying resistance mutations in a drug target provides crucial information. Lentiviral transduction creates multiple types of mutations due to the error-prone nature of the HIV-1 reverse transcriptase (RT). Here we optimized and leveraged this property to identify drug resistance mutations, developing a technique we term LentiMutate. This technique was validated by identifying clinically relevant EGFR resistance mutations, then applied to two additional clinical anticancer drugs: imatinib, a BCR-ABL inhibitor, and AMG 510, a KRAS G12C inhibitor. Novel deletions in BCR-ABL1 conferred resistance to imatinib. In KRAS-G12C or wild-type KRAS, point mutations in the AMG 510 binding pocket or oncogenic non-G12C mutations conferred resistance to AMG 510. LentiMutate should prove highly valuable for clinical and preclinical cancer-drug development. SIGNIFICANCE: LentiMutate can evaluate a drug's on-target activity and can nominate resistance mutations before they occur in patients, which could accelerate and refine drug development to increase the survival of patients with cancer.
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Biomarcadores de Tumor , Descubrimiento de Drogas/métodos , Resistencia a Antineoplásicos/genética , Vectores Genéticos/genética , Lentivirus/genética , Mutación , Neoplasias/genética , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Modelos Moleculares , Neoplasias/tratamiento farmacológico , Relación Estructura-ActividadRESUMEN
PURPOSE: FGFR1 overexpression has been associated with endocrine resistance in ER+ breast cancer. We found FGFR1 localized in the nucleus of breast cancer cells in primary tumors resistant to estrogen suppression. We investigated a role of nuclear FGFR1 on gene transcription and antiestrogen resistance. EXPERIMENTAL DESIGN: Tumors from patients treated with letrozole were subjected to Ki67 and FGFR1 IHC. MCF7 cells were transduced with FGFR1(SP-)(NLS) to promote nuclear FGFR1 overexpression. FGFR1 genomic activity in ER+/FGFR1-amplified breast cancer cells ± FOXA1 siRNA or ± the FGFR tyrosine kinase inhibitor (TKI) erdafitinib was examined by chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq). The nuclear and chromatin-bound FGFR1 interactome was investigated by mass spectrometry (MS). RESULTS: High nuclear FGFR1 expression in ER+ primary tumors positively correlated with post-letrozole Ki67 values. Nuclear FGFR1 overexpression influenced gene transcription and promoted resistance to estrogen suppression and to fulvestrant in vivo. A gene expression signature induced by nuclear FGFR1 correlated with shorter survival in the METABRIC cohort of patients treated with antiestrogens. ChIP-Seq revealed FGFR1 occupancy at transcription start sites, overlapping with active transcription histone marks. MS analysis of the nuclear FGFR1 interactome identified phosphorylated RNA-Polymerase II and FOXA1, with FOXA1 RNAi impairing FGFR1 recruitment to chromatin. Treatment with erdafitinib did not impair nuclear FGFR1 translocation and genomic activity. CONCLUSIONS: These data suggest nuclear FGFR1 contributes to endocrine resistance by modulating gene transcription in ER+ breast cancer. Nuclear FGFR1 activity was unaffected by FGFR TKIs, thus supporting the development of treatment strategies to inhibit nuclear FGFR1 in ER+/FGFR1 overexpressing breast cancer.
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Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Resistencia a Antineoplásicos/genética , Moduladores de los Receptores de Estrógeno/uso terapéutico , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/fisiología , Transcripción Genética/fisiología , Neoplasias de la Mama/química , Núcleo Celular , Femenino , Humanos , Receptores de Estrógenos/análisis , Células Tumorales CultivadasRESUMEN
Estrogen receptor alpha gene (ESR1) mutations occur frequently in ER-positive metastatic breast cancer, and confer clinical resistance to aromatase inhibitors. Expression of the ESR1 Y537S mutation induced an epithelial-mesenchymal transition (EMT) with cells exhibiting enhanced migration and invasion potential in vitro. When small subpopulations of Y537S ESR1 mutant cells were injected along with WT parental cells, tumor growth was enhanced with mutant cells becoming the predominant population in distant metastases. Y537S mutant primary xenograft tumors were resistant to the antiestrogen tamoxifen (Tam) as well as to estradiol (E2) withdrawal. Y537S ESR1 mutant primary tumors metastasized efficiently in the absence of E2; however, Tam treatment significantly inhibited metastasis to distant sites. We identified a nine-gene expression signature, which predicted clinical outcomes of ER-positive breast cancer patients, as well as breast cancer metastasis to the lung. Androgen receptor (AR) protein levels were increased in mutant models, and the AR agonist dihydrotestosterone significantly inhibited estrogen-regulated gene expression, EMT, and distant metastasis in vivo, suggesting that AR may play a role in distant metastatic progression of ESR1 mutant tumors.
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Neoplasias de la Mama/tratamiento farmacológico , Receptor alfa de Estrógeno/genética , Receptores Androgénicos/genética , Tamoxifeno/farmacología , Animales , Inhibidores de la Aromatasa/farmacología , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Dihidrotestosterona/farmacología , Estradiol/metabolismo , Estrógenos/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Mutación/genética , Metástasis de la Neoplasia , Receptores Androgénicos/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The study of DNA damage repair response (DDR) in prostate cancer is restricted by the limited number of prostate cancer cell lines and lack of surrogates for heterogeneity in clinical samples. Here, we sought to leverage our experience with patient derived explants (PDEs) cultured ex vivo to study dynamics of DDR in primary tumors following application of clinically relevant doses of ionizing radiation (IR) to tumor cells in their native 3-dimensional microenvironment. We compared DDR dynamics between prostate cancer cell lines, PDEs and xenograft derived explants (XDEs) following treatment with IR (2Gy) either alone or in combination with pharmacological modulators of DDR. We have shown that following treatment with 2Gy, DDR can be consistently detected in PDEs from multiple solid tumors, including prostate, kidney, testes, lung and breast, as evidenced by γ-H2AX, 53BP1, phospho-ATM and phospho-DNA-PKcs foci. By examining kinetics of resolution of IR-induced foci, we have shown that DDR in prostate PDEs (complete resolution in 8â¯h) is much faster than in prostate cancer cell lines (<50% resolution in 8â¯h). The transcriptional profile of DDR genes following 2Gy IR appears to be distinct between PDEs and cell lines. Pre-treatment with drugs targeting DDR pathways differentially alter the kinetics of DDR in the PDEs and cell lines, as evidenced by altered kinetics of foci resolution. This study highlights the utility of PDEs as a robust model system for short-term evaluation of DDR in primary solid tumors in clinically relevant microenvironment.
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Using a mini-library of 1062 lentiviral shRNAs targeting 40 nuclear hormone receptors and 70 of their co-regulators, we searched for potential therapeutic targets that would be important during in vivo tumor growth using a parallel in vitro and in vivo shRNA screening strategy in the non-small cell lung cancer (NSCLC) line NCI-H1819. We identified 21 genes essential for in vitro growth, and nine genes specifically required for tumor survival in vivo, but not in vitro: NCOR2, FOXA1, HDAC1, RXRA, RORB, RARB, MTA2, ETV4, and NR1H2. We focused on FOXA1, since it lies within the most frequently amplified genomic region in lung adenocarcinomas. We found that 14q-amplification in NSCLC cell lines was a biomarker for FOXA1 dependency for both in vivo xenograft growth and colony formation, but not mass culture growth in vitro. FOXA1 knockdown identified genes involved in electron transport among the most differentially regulated, indicating FOXA1 loss may lead to a decrease in cellular respiration. In support of this, FOXA1 amplification was correlated with increased sensitivity to the complex I inhibitor phenformin. Integrative ChipSeq analyses reveal that FOXA1 functions in this genetic context may be at least partially independent of NKX2-1. Our findings are consistent with a neomorphic function for amplified FOXA1, driving an oncogenic transcriptional program. These data provide new insight into the functional consequences of FOXA1 amplification in lung adenocarcinomas, and identify new transcriptional networks for exploration of therapeutic vulnerabilities in this patient population.
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Biomarcadores de Tumor/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Genómica/métodos , Factor Nuclear 3-alfa del Hepatocito/metabolismo , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Neoplasias Pulmonares/patología , Trombospondina 1/metabolismo , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/metabolismo , Adenocarcinoma del Pulmón/patología , Animales , Apoptosis , Biomarcadores de Tumor/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Proliferación Celular , Femenino , Regulación Neoplásica de la Expresión Génica , Estudio de Asociación del Genoma Completo , Factor Nuclear 3-alfa del Hepatocito/genética , Humanos , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Receptores Citoplasmáticos y Nucleares , Trombospondina 1/genética , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The dependency of prostate cancer (PCa) growth on androgen receptor (AR) signaling has been harnessed to develop first-line therapies for high-risk localized and metastatic PCa treatment. However, the occurrence of aberrant expression, mutated or splice variants of AR confers resistance to androgen ablation therapy (ADT), radiotherapy or chemotherapy in AR-positive PCa. Therapeutic strategies that effectively inhibit the expression and/or transcriptional activity of full-length AR, mutated AR and AR splice variants have remained elusive. In this study, we report that mithramycin (MTM), an antineoplastic antibiotic, suppresses cell proliferation and exhibits dual inhibitory effects on expression and transcriptional activity of AR and AR splice variants. MTM blocks AR recruitment to its genomic targets by occupying AR enhancers and causes downregulation of AR target genes, which includes key DNA repair factors in DNA damage repair (DDR). We show that MTM significantly impairs DDR and enhances the effectiveness of ionizing radiation or the radiomimetic agent Bleomycin in PCa. Thus, the combination of MTM treatment with RT or radiomimetic agents, such as bleomycin, may present a novel effective therapeutic strategy for patients with high-risk, clinically localized PCa.
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Antibióticos Antineoplásicos/farmacología , Daño del ADN/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Plicamicina/farmacología , Neoplasias de la Próstata Resistentes a la Castración/patología , Receptores Androgénicos/metabolismo , Línea Celular Tumoral , Humanos , Masculino , Receptores Androgénicos/efectos de los fármacosRESUMEN
RUVBL1 and RUVBL2 (collectively RUVBL1/2) are essential AAA+ ATPases that function as co-chaperones and have been implicated in cancer. Here we investigated the molecular and phenotypic role of RUVBL1/2 ATPase activity in non-small cell lung cancer (NSCLC). We find that RUVBL1/2 are overexpressed in NSCLC patient tumors, with high expression associated with poor survival. Utilizing a specific inhibitor of RUVBL1/2 ATPase activity, we show that RUVBL1/2 ATPase activity is necessary for the maturation or dissociation of the PAQosome, a large RUVBL1/2-dependent multiprotein complex. We also show that RUVBL1/2 have roles in DNA replication, as inhibition of its ATPase activity can cause S-phase arrest, which culminates in cancer cell death via replication catastrophe. While in vivo pharmacological inhibition of RUVBL1/2 results in modest antitumor activity, it synergizes with radiation in NSCLC, but not normal cells, an attractive property for future preclinical development.
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ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Proteínas Portadoras/metabolismo , ADN Helicasas/metabolismo , Replicación del ADN , Neoplasias Pulmonares/metabolismo , Complejos Multiproteicos/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas/antagonistas & inhibidores , ATPasas Asociadas con Actividades Celulares Diversas/genética , Antineoplásicos/química , Antineoplásicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , ADN Helicasas/antagonistas & inhibidores , ADN Helicasas/genética , Replicación del ADN/efectos de los fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/tratamiento farmacológico , Estructura Molecular , Complejos Multiproteicos/antagonistas & inhibidores , Complejos Multiproteicos/genética , Tolerancia a RadiaciónRESUMEN
The propagation of species depends on the ability of germ cells to protect their genome from numerous exogenous and endogenous threats. While these cells employ ubiquitous repair pathways, specialized mechanisms that ensure high-fidelity replication, chromosome segregation, and repair of germ cell genomes remain incompletely understood. We identified Germ Cell Nuclear Acidic Peptidase (GCNA) as a conserved regulator of genome stability in flies, worms, zebrafish, and human germ cell tumors. GCNA contains an acidic intrinsically disordered region (IDR) and a protease-like SprT domain. In addition to chromosomal instability and replication stress, Gcna mutants accumulate DNA-protein crosslinks (DPCs). GCNA acts in parallel with the SprT domain protein Spartan. Structural analysis reveals that while the SprT domain is needed to limit DNA damage, the IDR imparts significant function. This work shows that GCNA protects germ cells from various sources of damage, providing insights into conserved mechanisms that promote genome integrity across generations.
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Daño del ADN , Reparación del ADN , Replicación del ADN , Fertilidad , Inestabilidad Genómica , Proteínas Nucleares/metabolismo , Péptido Hidrolasas/metabolismo , Animales , Caenorhabditis elegans , Variaciones en el Número de Copia de ADN , Drosophila melanogaster , Femenino , Genoma , Células Germinativas/citología , Células Germinativas/metabolismo , Humanos , Masculino , Neoplasias de Células Germinales y Embrionarias/genética , Neoplasias de Células Germinales y Embrionarias/metabolismo , Neoplasias de Células Germinales y Embrionarias/patología , Proteínas Nucleares/genética , Péptido Hidrolasas/genética , Dominios Proteicos , Especificidad de la Especie , Pez CebraRESUMEN
Glioblastomas are lethal brain tumors that are treated with conventional radiation (X-rays and gamma rays) or particle radiation (protons and carbon ions). Paradoxically, radiation is also a risk factor for GBM development, raising the possibility that radiotherapy of brain tumors could promote tumor recurrence or trigger secondary gliomas. In this study, we determined whether tumor suppressor losses commonly displayed by patients with GBM confer susceptibility to radiation-induced glioma. Mice with Nestin-Cre-driven deletions of Trp53 and Pten alleles were intracranially irradiated with X-rays or charged particles of increasing atomic number and linear energy transfer (LET). Mice with loss of one allele each of Trp53 and Pten did not develop spontaneous gliomas, but were highly susceptible to radiation-induced gliomagenesis. Tumor development frequency after exposure to high-LET particle radiation was significantly higher compared with X-rays, in accordance with the irreparability of DNA double-strand breaks (DSB) induced by high-LET radiation. All resultant gliomas, regardless of radiation quality, presented histopathologic features of grade IV lesions and harbored populations of cancer stem-like cells with tumor-propagating properties. Furthermore, all tumors displayed concomitant loss of heterozygosity of Trp53 and Pten along with frequent amplification of the Met receptor tyrosine kinase, which conferred a stem cell phenotype to tumor cells. Our results demonstrate that radiation-induced DSBs cooperate with preexisting tumor suppressor losses to generate high-grade gliomas. Moreover, our mouse model can be used for studies on radiation-induced development of GBM and therapeutic strategies. SIGNIFICANCE: This study uncovers mechanisms by which ionizing radiation, especially particle radiation, promote GBM development or recurrence.
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Neoplasias Encefálicas/genética , Roturas del ADN de Doble Cadena , Glioblastoma/genética , Glioma/genética , Neoplasias Inducidas por Radiación/genética , Fosfohidrolasa PTEN/genética , Proteína p53 Supresora de Tumor/genética , Animales , Neoplasias Encefálicas/patología , Femenino , Glioblastoma/patología , Glioma/patología , Humanos , Transferencia Lineal de Energía , Pérdida de Heterocigocidad , Masculino , Ratones , Ratones Endogámicos C57BL , Clasificación del Tumor , Células Madre Neoplásicas/patología , Células Madre Neoplásicas/efectos de la radiaciónRESUMEN
The nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) is a master regulator of adipocyte differentiation and is the target for the insulin-sensitizing thiazolidinedione (TZD) drugs used to treat type 2 diabetes. In cell-based in vitro studies, the transcriptional activity of PPARγ is inhibited by covalent attachment of small ubiquitin-related modifier (SUMOylation) at K107 in its N terminus. However, whether this posttranslational modification is relevant in vivo remains unclear. Here, using mice homozygous for a mutation (K107R) that prevents SUMOylation at this position, we demonstrate that PPARγ is SUMOylated at K107 in white adipose tissue. We further show that in the context of diet-induced obesity PPARγ-K107R-mutant mice have enhanced insulin sensitivity without the corresponding increase in adiposity that typically accompanies PPARγ activation by TZDs. Accordingly, the PPARγ-K107R mutation was weaker than TZD treatment in stimulating adipocyte differentiation in vitro. Moreover, we found that both the basal and TZD-dependent transcriptomes of inguinal and epididymal white adipose tissue depots were markedly altered in the K107R-mutant mice. We conclude that PPARγ SUMOylation at K107 is physiologically relevant and may serve as a pharmacologic target for uncoupling PPARγ's beneficial insulin-sensitizing effect from its adverse effect of weight gain.
Asunto(s)
Adiposidad , Insulina/metabolismo , Lisina/metabolismo , Obesidad/metabolismo , PPAR gamma/metabolismo , Tejido Adiposo/metabolismo , Secuencias de Aminoácidos , Animales , Femenino , Humanos , Lisina/genética , Masculino , Ratones , Mutación Missense , Obesidad/genética , Obesidad/fisiopatología , PPAR gamma/química , PPAR gamma/genética , Proteína SUMO-1 , SumoilaciónRESUMEN
The current therapeutic armamentarium for castration-resistant prostate cancer (CRPC) includes second-generation agents such as the Androgen Receptor (AR) inhibitor enzalutamide and the androgen synthesis inhibitor abiraterone acetate, immunotherapies like sipuleucel-T, chemotherapies including docetaxel and cabazitaxel and the radiopharmaceutical radium 223 dichloride. However, relapse of CRPC resistant to these therapeutic modalities occur rapidly. The mechanisms of resistance to these treatments are complex, including specific mutations or alternative splicing of oncogenic proteins. An alternative approach to treating CRPC may be to target the turnover of these molecular drivers of CRPC. In this review, the mechanisms by which protein stability of several oncoproteins such as AR, ERG, GR, CYP17A1 and MYC, will be discussed, as well as how these findings could be translated into novel therapeutic agents.
Asunto(s)
Antineoplásicos/uso terapéutico , Resistencia a Antineoplásicos/efectos de los fármacos , Recurrencia Local de Neoplasia/prevención & control , Proteínas Oncogénicas/antagonistas & inhibidores , Neoplasias de la Próstata Resistentes a la Castración/terapia , Antagonistas de Receptores Androgénicos/uso terapéutico , Resistencia a Antineoplásicos/genética , Humanos , Masculino , Mutación , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/genética , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismoRESUMEN
Chromosome rearrangements are large-scale structural variants that are recognized drivers of oncogenic events in cancers of all types. Cytogenetics allows for their rapid, genome-wide detection, but does not provide gene-level resolution. Massively parallel sequencing (MPS) promises DNA sequence-level characterization of the specific breakpoints involved, but is strongly influenced by bioinformatics filters that affect detection efficiency. We sought to characterize the breakpoint junctions of chromosomal translocations and inversions in the clonal derivatives of human cells exposed to ionizing radiation. Here, we describe the first successful use of DNA paired-end analysis to locate and sequence across the breakpoint junctions of a radiation-induced reciprocal translocation. The analyses employed, with varying degrees of success, several well-known bioinformatics algorithms, a task made difficult by the involvement of repetitive DNA sequences. As for underlying mechanisms, the results of Sanger sequencing suggested that the translocation in question was likely formed via microhomology-mediated non-homologous end joining (mmNHEJ). To our knowledge, this represents the first use of MPS to characterize the breakpoint junctions of a radiation-induced chromosomal translocation in human cells. Curiously, these same approaches were unsuccessful when applied to the analysis of inversions previously identified by directional genomic hybridization (dGH). We conclude that molecular cytogenetics continues to provide critical guidance for structural variant discovery, validation and in "tuning" analysis filters to enable robust breakpoint identification at the base pair level.