RESUMEN
BRCA1 mutations are associated with increased breast and ovarian cancer risk. BRCA1-mutant tumors are high-grade, recurrent, and often become resistant to standard therapies. Herein, we performed a targeted CRISPR-Cas9 screen and identified MEPCE, a methylphosphate capping enzyme, as a synthetic lethal interactor of BRCA1. Mechanistically, we demonstrate that depletion of MEPCE in a BRCA1-deficient setting led to dysregulated RNA polymerase II (RNAPII) promoter-proximal pausing, R-loop accumulation, and replication stress, contributing to transcription-replication collisions. These collisions compromise genomic integrity resulting in loss of viability of BRCA1-deficient cells. We also extend these findings to another RNAPII-regulating factor, PAF1. This study identifies a new class of synthetic lethal partners of BRCA1 that exploit the RNAPII pausing regulation and highlight the untapped potential of transcription-replication collision-inducing factors as unique potential therapeutic targets for treating cancers associated with BRCA1 mutations.
Asunto(s)
Proteína BRCA1 , Replicación del ADN , Síndrome de Cáncer de Mama y Ovario Hereditario , Mutación , Transcripción Genética , Humanos , Proteína BRCA1/deficiencia , Proteína BRCA1/genética , Replicación del ADN/genética , Síndrome de Cáncer de Mama y Ovario Hereditario/genética , Síndrome de Cáncer de Mama y Ovario Hereditario/patología , Síndrome de Cáncer de Mama y Ovario Hereditario/fisiopatología , ARN Polimerasa II/metabolismo , Transcripción Genética/genética , Regiones Promotoras Genéticas , Metiltransferasas/deficiencia , Metiltransferasas/genética , Estructuras R-Loop , Muerte CelularRESUMEN
BACKGROUND & AIMS: N6-methyladenosine (m6A) governs the fate of RNAs through m6A readers. Colorectal cancer (CRC) exhibits aberrant m6A modifications and expression of m6A regulators. However, how m6A readers interpret oncogenic m6A methylome to promote malignant transformation remains to be illustrated. METHODS: YTH N6-methyladenosine RNA binding protein 1 (Ythdf1) knockout mouse was generated to determine the effect of Ythdf1 in CRC tumorigenesis in vivo. Multiomic analysis of RNA-sequencing, m6A methylated RNA immunoprecipitation sequencing, YTHDF1 RNA immunoprecipitation sequencing, and proteomics were performed to unravel targets of YTHDF1 in CRC. The therapeutic potential of targeting YTHDF1-m6A-Rho/Rac guanine nucleotide exchange factor 2 (ARHGEF2) was evaluated using small interfering RNA (siRNA) encapsulated by lipid nanoparticles (LNP). RESULTS: DNA copy number gain of YTHDF1 is a frequent event in CRC and contributes to its overexpression. High expression of YTHDF1 is significantly associated with metastatic gene signature in patient tumors. Ythdf1 knockout in mice dampened tumor growth in an inflammatory CRC model. YTHDF1 promotes cell growth in CRC cell lines and primary organoids and lung and liver metastasis in vivo. Integrative multiomics analysis identified RhoA activator ARHGEF2 as a key downstream target of YTHDF1. YTHDF1 binds to m6A sites of ARHGEF2 messenger RNA, resulting in enhanced translation of ARHGEF2. Ectopic expression of ARHGEF2 restored impaired RhoA signaling, cell growth, and metastatic ability both in vitro and in vivo caused by YTHDF1 loss, verifying that ARHGEF2 is a key target of YTHDF1. Finally, ARHGEF2 siRNA delivered by LNP significantly suppressed tumor growth and metastasis in vivo. CONCLUSIONS: We identify a novel oncogenic epitranscriptome axis of YTHDF1-m6A-ARHGEF2, which regulates CRC tumorigenesis and metastasis. siRNA-delivering LNP drug validated the therapeutic potential of targeting this axis in CRC.
Asunto(s)
Neoplasias Colorrectales , Regulación Neoplásica de la Expresión Génica , Adenosina/análogos & derivados , Adenosina/metabolismo , Animales , Carcinogénesis/genética , Neoplasias Colorrectales/patología , Humanos , Liposomas , Ratones , Nanopartículas , ARN Interferente Pequeño , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/genética , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Prostate cancer (PCa) risk-associated SNPs are enriched in noncoding cis-regulatory elements (rCREs), yet their modi operandi and clinical impact remain elusive. Here, we perform CRISPRi screens of 260 rCREs in PCa cell lines. We find that rCREs harboring high risk SNPs are more essential for cell proliferation and H3K27ac occupancy is a strong indicator of essentiality. We also show that cell-line-specific essential rCREs are enriched in the 8q24.21 region, with the rs11986220-containing rCRE regulating MYC and PVT1 expression, cell proliferation and tumorigenesis in a cell-line-specific manner, depending on DNA methylation-orchestrated occupancy of a CTCF binding site in between this rCRE and the MYC promoter. We demonstrate that CTCF deposition at this site as measured by DNA methylation level is highly variable in prostate specimens, and observe the MYC eQTL in the 8q24.21 locus in individuals with low CTCF binding. Together our findings highlight a causal mechanism synergistically driven by a risk SNP and DNA methylation-mediated 3D genome architecture, advocating for the integration of genetics and epigenetics in assessing risks conferred by genetic predispositions.
Asunto(s)
Sistemas CRISPR-Cas , Metilación de ADN , Edición Génica/métodos , Predisposición Genética a la Enfermedad/genética , Estudio de Asociación del Genoma Completo/métodos , Neoplasias de la Próstata/genética , Animales , Factor de Unión a CCCTC/genética , Factor de Unión a CCCTC/metabolismo , Carcinogénesis/genética , Línea Celular Tumoral , Humanos , Masculino , Ratones Endogámicos NOD , Ratones SCID , Polimorfismo de Nucleótido Simple , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas c-myc/genética , Sitios de Carácter Cuantitativo/genética , Elementos Reguladores de la Transcripción/genética , Factores de RiesgoRESUMEN
Neuroendocrine prostate cancer (NEPC), a lethal form of the disease, is characterized by loss of androgen receptor (AR) signaling during neuroendocrine transdifferentiation, which results in resistance to AR-targeted therapy. Clinically, genomically and epigenetically, NEPC resembles other types of poorly differentiated neuroendocrine tumors (NETs). Through pan-NET analyses, we identified ONECUT2 as a candidate master transcriptional regulator of poorly differentiated NETs. ONECUT2 ectopic expression in prostate adenocarcinoma synergizes with hypoxia to suppress androgen signaling and induce neuroendocrine plasticity. ONEUCT2 drives tumor aggressiveness in NEPC, partially through regulating hypoxia signaling and tumor hypoxia. Specifically, ONECUT2 activates SMAD3, which regulates hypoxia signaling through modulating HIF1α chromatin-binding, leading NEPC to exhibit higher degrees of hypoxia compared to prostate adenocarcinomas. Treatment with hypoxia-activated prodrug TH-302 potently reduces NEPC tumor growth. Collectively, these results highlight the synergy between ONECUT2 and hypoxia in driving NEPC, and emphasize the potential of hypoxia-directed therapy for NEPC patients.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Tumores Neuroendocrinos/genética , Neoplasias de la Próstata/genética , Proteína smad3/genética , Factores de Transcripción/metabolismo , Animales , Carcinogénesis/genética , Carcinogénesis/patología , Hipoxia de la Célula/efectos de los fármacos , Hipoxia de la Célula/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Conjuntos de Datos como Asunto , Progresión de la Enfermedad , Perfilación de la Expresión Génica , Proteínas de Homeodominio/genética , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Tumores Neuroendocrinos/patología , Nitroimidazoles/farmacología , Mostazas de Fosforamida/farmacología , Próstata/patología , Neoplasias de la Próstata/patología , ARN Interferente Pequeño/metabolismo , Transducción de Señal/genética , Proteína smad3/metabolismo , Factores de Transcripción/genética , Regulación hacia Arriba , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Optical roughness was introduced into the bidirectional reflectance distribution function (BRDF) model to simulate the reflectance characteristics of thermal radiation. The optical roughness BRDF model stemmed from the influence of surface roughness and wavelength on the ray reflectance calculation. This model was adopted to simulate real metal emissivity. The reverse Monte Carlo method was used to display the distribution of reflectance rays. The numerical simulations showed that the optical roughness BRDF model can calculate the wavelength effect on emissivity and simulate the real metal emissivity variance with incidence angles.
