RESUMEN
Here, we present a functional assay to detect the repair switch to the alternative PARP1-dependent end joining (PARP1-EJ) pathway and the associated susceptibility to PARPi-mediated radiosensitization in freshly collected tumor samples from prostate cancer (PCa) patients, thereby facilitating the selection of patients who should benefit from combined PARPi plus radiotherapy (RT) treatment. Our optimized ex-vivo approach sustains tumor slices for up to 15 days under culture conditions that maintain proliferation and oxygenation rates, as measured by EdU incorporation and pimonidazole staining, respectively. We present a robust system to analyze DSB repair using, for the first time in an ex vivo tumor slice setting, two DSB-markers simultaneously i.e. γH2AX and 53BP1. A computer-based processing method (i) controls variations in DNA content and slicing on the number of repair foci and (ii) measures the PARPi-mediated enhancement ratio on DSB foci numbers to ensure inter-patient-comparability. We validated this approach using a PC3 xenograft model with its previously described repair switch to PARP1-EJ. More importantly, we show that approximately 30% of the analyzed tumor tissue samples collected from PCa patients display a switch to PARP1-EJ, as indicated by the enhanced number of residual γH2AX/53BP1 foci exclusively after PARPi+RT. Furthermore, normal prostatic tissues show no repair switch to PARP1-EJ, indicating that this repair switch and its associated radiosensitizing effect is tumor-specific. Collectively, we present here a predictive assay for the switch to PARP1-EJ that enables individualization of anti-cancer treatment using a combination of RT and radiosensitizing anticancer agents such as PARPi in PCa.
Asunto(s)
Reparación del ADN por Unión de Extremidades/efectos de los fármacos , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/administración & dosificación , Neoplasias de la Próstata/terapia , Fármacos Sensibilizantes a Radiaciones/administración & dosificación , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades/efectos de la radiación , Histonas/metabolismo , Humanos , Masculino , Ratones , Clasificación del Tumor , Poli(ADP-Ribosa) Polimerasa-1/antagonistas & inhibidores , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Fármacos Sensibilizantes a Radiaciones/farmacología , Técnicas de Cultivo de Tejidos , Proteína 1 de Unión al Supresor Tumoral P53/metabolismoRESUMEN
Here we report that BCL2 blocks DNA double strand break (DSB) repair via nonhomologous end-joining (NHEJ), through sequestration of KU80 protein outside the nucleus. We find that this effect is associated with a repair switch to the error-prone PARP1-dependent end-joining (PARP1-EJ). We present in-vitro proof-of-concept for therapeutic targeting of this switch using PARP inhibitor to specifically enhance the radiosensitivity of BCL2-overexpressing cells. Given its erroneous behavior, PARP1-EJ might allow for the accumulation of genetic alterations and tumor progression. Consistently, we report an inverse correlation between BCL2 expression and biochemical recurrence-free survival of 10.259 prostate cancer (PCa) patients who underwent primary radical-prostatectomy for localized disease. Further, we evaluated retrospectively the impact of BCL2 expression on clinical outcome of 1.426 PCa patients, who had been given salvage radiotherapy at relapse after radical prostatectomy. In line with its role in blocking NHEJ, BCL2 over-expressers showed significantly better response to salvage radiotherapy compared to low-expressers. Collectively, our findings identify BCL2 status in PCa as a putative predictor of (i) radiotherapy response and (ii) response to treatment with PARP inhibitor olaparib as a radiosensitizing agent.
Asunto(s)
Ftalazinas/uso terapéutico , Piperazinas/uso terapéutico , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Neoplasias de la Próstata/terapia , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Regulación hacia Arriba , Línea Celular Tumoral , Reparación del ADN por Unión de Extremidades , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Humanos , Autoantígeno Ku/metabolismo , Masculino , Poli(ADP-Ribosa) Polimerasa-1/genética , Prostatectomía , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Radioterapia , Terapia Recuperativa , Análisis de SupervivenciaRESUMEN
End processing at DNA double strand breaks (DSB) is a decisive step in repair pathway selection. Here, we investigated the role of 53BP1/RIF1 in limiting BRCA1/CtIP-mediated end resection to control DSB repair pathway choice. ATM orchestrates this process through 53BP1 phosphorylation to promote RIF1 recruitment. As cells enter S/G2-phase, end resection is activated, which displaces pATM from DSB sites and diminishes 53BP1 phosphorylation and RIF1 recruitment. Consistently, the kinetics of ATM and 53BP1 phosphorylation in S/G2-phase concur. We show that defective 53BP1/RIF1-mediated DSB end-protection in G1-phase stimulates CtIP/MRE11-dependent end-resection, which requires Polo-like kinase 3. This end resection activity in G1 was shown to produce only short tracks of ssDNA overhangs, as evidenced by the findings that in 53BP1 depleted cells, (i) RPA focus intensity was significantly lower in G1 compared to that in S/G2 phase, and (ii) EXO1 knockdown did not alter either number or intensity of RPA foci in G1 but significantly decreased the RPA focus intensity in S/G2 phase. Importantly, we report that the observed DSB end resection in G1 phase inhibits DNA-PK-dependent nonhomologous end joining but is not sufficient to stimulate HR. Instead, it switches the repair to the alternative PARP1-dependent end joining pathway.
Asunto(s)
Proteínas Portadoras/genética , Roturas del ADN de Doble Cadena , Reparación del ADN , Proteínas Nucleares/genética , Poli(ADP-Ribosa) Polimerasa-1/genética , Proteínas de Unión a Telómeros/genética , Proteína 1 de Unión al Supresor Tumoral P53/genética , Proteína BRCA1/genética , Línea Celular Tumoral , Reparación del ADN por Unión de Extremidades , ADN de Cadena Simple/genética , Endodesoxirribonucleasas , Fase G1 , Células HeLa , Humanos , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Interferente Pequeño/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Proteínas Supresoras de TumorRESUMEN
PURPOSE: The aim of this study was to elucidate the impact of DNA damage response (DDR) proteins 53BP1 and BRCA1 on the double-strand break (DSB)-repair choice. This is important not only in order to understand the underlying mechanisms of DSB-repair pathway regulation but also to determine the therapeutic implications for BRCA1-associated tumors. MATERIALS AND METHODS: Human tumor cell lines A549 and HeLa were used. Non-homologous end-joining (NHEJ) and homologous recombination (HR) were assessed using NHEJ and HR reporter constructs. Colocalization of HR-proteins RPA and RAD51 with 53BP1 was evaluated by confocal microscopy and 3D-analysis. RESULTS: We demonstrate a specific crosstalk between 53BP1 and BRCA1. While 53BP1 does not colocalize with RPA or RAD51 and prohibits the recruitment of BRCA1 to DSBs to stimulate NHEJ, BRCA1 promotes the 53BP1 displacement specifically in S/G2-phase to allow end-resection, initiating HR. HR-efficiency was restored in BRCA1-depleted cells upon additional 53BP1-knockdown. Further, we found that 53BP1-mediated end protection precedes BRCA1-dependent end-resection. CONCLUSION: These results demonstrate that the interplay between 53BP1/NHEJ and BRCA1/HR is of great relevance for tumor treatment, as the 53BP1 status would be highly important for the treatment response of BRCA1-associated tumors.