RESUMEN
Evidence that long non-coding RNAs (lncRNAs) participate in DNA repair is accumulating, however, whether they can control DNA repair pathway choice is unknown. Here we show that the small Cajal body-specific RNA 2 (scaRNA2) can promote HR by inhibiting DNA-dependent protein kinase (DNA-PK) and, thereby, NHEJ. By binding to the catalytic subunit of DNA-PK (DNA-PKcs), scaRNA2 weakens its interaction with the Ku70/80 subunits, as well as with the LINP1 lncRNA, thereby preventing catalytic activation of the enzyme. Inhibition of DNA-PK by scaRNA2 stimulates DNA end resection by the MRN/CtIP complex, activation of ATM at DNA lesions and subsequent repair by HR. ScaRNA2 is regulated in turn by WRAP53ß, which binds this RNA, sequestering it away from DNA-PKcs and allowing NHEJ to proceed. These findings reveal that RNA-dependent control of DNA-PK catalytic activity is involved in regulating whether the cell utilizes NHEJ or HR.
Asunto(s)
Proteínas Quinasas , ARN , ADN/genética , ADN/metabolismo , Reparación del ADN por Unión de Extremidades , Reparación del ADN , Proteína Quinasa Activada por ADN/genética , Proteína Quinasa Activada por ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Autoantígeno Ku/genética , Autoantígeno Ku/metabolismo , Proteínas Quinasas/metabolismoRESUMEN
The clinically aggressive alveolar rhabdomyosarcoma (RMS) subtype is characterized by expression of the oncogenic fusion protein PAX3-FOXO1, which is critical for tumorigenesis and cell survival. Here, we studied the mechanism of cell death induced by loss of PAX3-FOXO1 expression and identified a novel pharmacologic combination therapy that interferes with PAX3-FOXO1 biology at different levels. Depletion of PAX3-FOXO1 in fusion-positive (FP)-RMS cells induced intrinsic apoptosis in a NOXA-dependent manner. This was pharmacologically mimicked by the BH3 mimetic navitoclax, identified as top compound in a screen from 208 targeted compounds. In a parallel approach, and to identify drugs that alter the stability of PAX3-FOXO1 protein, the same drug library was screened and fusion protein levels were directly measured as a read-out. This revealed that inhibition of Aurora kinase A most efficiently negatively affected PAX3-FOXO1 protein levels. Interestingly, this occurred through a novel specific phosphorylation event in and binding to the fusion protein. Aurora kinase A inhibition also destabilized MYCN, which is both a functionally important oncogene and transcriptional target of PAX3-FOXO1. Combined treatment with an Aurora kinase A inhibitor and navitoclax in FP-RMS cell lines and patient-derived xenografts synergistically induced cell death and significantly slowed tumor growth. These studies identify a novel functional interaction of Aurora kinase A with both PAX3-FOXO1 and its effector MYCN, and reveal new opportunities for targeted combination treatment of FP-RMS. SIGNIFICANCE: These findings show that Aurora kinase A and Bcl-2 family proteins are potential targets for FP-RMS.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Aurora Quinasa A/metabolismo , Proteína Proto-Oncogénica N-Myc/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Factores de Transcripción Paired Box/metabolismo , Rabdomiosarcoma Alveolar/tratamiento farmacológico , Compuestos de Anilina/farmacología , Compuestos de Anilina/uso terapéutico , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Apoptosis/efectos de los fármacos , Aurora Quinasa A/antagonistas & inhibidores , Sinergismo Farmacológico , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Ratones , Mutagénesis Sitio-Dirigida , Proteínas de Fusión Oncogénica/genética , Factores de Transcripción Paired Box/genética , Fosforilación/efectos de los fármacos , Cultivo Primario de Células , Estabilidad Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Interferente Pequeño/metabolismo , Rabdomiosarcoma Alveolar/genética , Rabdomiosarcoma Alveolar/patología , Sulfonamidas/farmacología , Sulfonamidas/uso terapéutico , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Proper repair of DNA double-strand breaks is critical for maintaining genome integrity and avoiding disease. Modification of damaged chromatin has profound consequences for the initial signaling and regulation of repair. One such modification involves ubiquitination by E3 ligases RNF8 and RNF168 within minutes after DNA double-strand break formation, altering chromatin structure and recruiting factors such as 53BP1 and BRCA1 for repair via non-homologous end-joining (NHEJ) and homologous recombination (HR), respectively. The WD40 protein WRAP53ß plays an essential role in localizing RNF8 to DNA breaks by scaffolding its interaction with the upstream factor MDC1. Loss of WRAP53ß impairs ubiquitination at DNA lesions and reduces downstream repair by both NHEJ and HR. Intriguingly, WRAP53ß depletion attenuates repair of DNA double-strand breaks more than depletion of RNF8, indicating functions other than RNF8-mediated ubiquitination. WRAP53ß plays key roles with respect to the nuclear organelles Cajal bodies, including organizing the genome to promote associated transcription and collecting factors involved in maturation of the spliceosome and telomere elongation within these organelles. It is possible that similar functions may aid also in DNA repair. Here we describe the involvement of WRAP53ß in Cajal bodies and DNA double-strand break repair in detail and explore whether and how these processes may be linked. We also discuss the possibility that the overexpression of WRAP53ß detected in several cancer types may reflect its normal participation in the DNA damage response rather than oncogenic properties.
RESUMEN
BACKGROUND: MYCN is amplified in small cell lung cancers and several pediatric tumors, including alveolar rhabdomyosarcomas and neuroblastomas. MYCN protein is known to play a key oncogenic role in both alveolar rhabdomyosarcomas and neuroblastomas. MYCN opposite strand (MYCNOS) is a gene located on the antisense strand to MYCN that encodes alternatively spliced transcripts, two of which (MYCNOS-01 and MYCNOS-02) are known to be expressed in neuroblastoma and small cell lung cancer with reciprocal regulation between MYCNOS-02 and MYCN reported for neuroblastomas. We sought to determine a functional role for MYCNOS-01 in alveolar rhabdomyosarcoma and neuroblastoma cells and identify any associated regulatory effects between MYCN and MYCNOS-01. METHODS: MYCNOS-01, MYCNOS-02 and MYCN expression levels were assessed in alveolar rhabdomyosarcoma and neuroblastoma cell lines and tumor samples from patients using Affymetrix microarray data and quantitative RT-PCR. Following MYCNOS-01 or MYCN siRNA knockdown and MYCNOS-01 overexpression, transcript levels were assayed by quantitative RT-PCR and MYCN protein expression assessed by Western blot and immunofluorescence. Additionally, effects on cell growth, apoptosis and cell cycle profiles were determined by a metabolic assay, caspase activity and flow cytometry, respectively. RESULTS: MYCNOS-01 transcript levels were generally higher in NB and RMS tumor samples and cell lines with MYCN genomic amplification. RNA interference of MYCNOS-01 expression did not alter MYCN transcript levels but decreased MYCN protein levels. Conversely, MYCN reduction increased MYCNOS-01 transcript levels, creating a negative feedback loop on MYCN protein levels. Reduction of MYCNOS-01 or MYCN expression decreased cell growth in MYCN-amplified alveolar rhabdomyosarcoma and neuroblastoma cell lines. This is consistent with MYCNOS-01-mediated regulation of MYCN contributing to the phenotype observed. CONCLUSIONS: An alternative transcript of MYCNOS, MYCNOS-01, post-transcriptionally regulates MYCN levels and affects growth in MYCN-amplified rhabdomyosarcoma and neuroblastoma cells.