Reduced Proficiency in Homologous Recombination Underlies the High Sensitivity of Embryonal Carcinoma Testicular Germ Cell Tumors to Cisplatin and Poly (ADP-Ribose) Polymerase Inhibition

Testicular Germ Cell Tumors (TGCT) and patient-derived cell lines are extremely sensitive to cisplatin and other interstrand cross-link (ICL) inducing agents. Nevertheless, a subset of TGCTs are either innately resistant or acquire resistance to cisplatin during treatment. Understanding the mechanisms underlying TGCT sensitivity/resistance to cisplatin as well as the identification of novel strategies to target cisplatin-resistant TGCTs have major clinical implications. Herein, we have examined the proficiency of five embryonal carcinoma (EC) cell lines to repair cisplatin-induced ICLs. Using γH2AX staining as a marker of double strand break formation, we found that EC cell lines were either incapable of or had a reduced ability to repair ICL-induced damage. The defect correlated with reduced Homologous Recombination (HR) repair, as demonstrated by the reduction of RAD51 foci formation and by direct evaluation of HR efficiency using a GFP-reporter substrate. HR-defective tumors cells are known to be sensitive to the treatment with poly(ADP-ribose) polymerase (PARP) inhibitor. In line with this observation, we found that EC cell lines were also sensitive to PARP inhibitor monotherapy. The magnitude of sensitivity correlated with HR-repair reduced proficiency and with the expression levels and activity of PARP1 protein. In addition, we found that PARP inhibition strongly enhanced the response of the most resistant EC cells to cisplatin, by reducing their ability to overcome the damage. These results point to a reduced proficiency of HR repair as a source of sensitivity of ECs to ICL-inducing agents and PARP inhibitor monotherapy, and suggest that pharmacological inhibition of PARP can be exploited to target the stem cell component of the TGCTs (namely ECs) and to enhance the sensitivity of cisplatin-resistant TGCTs to standard treatments.

睾丸生殖细胞肿瘤（Testicular Germ Cell Tumors, TGCT）及患者来源细胞系对顺铂与其他链间交联（interstrand cross-link, ICL）诱导剂均具有极高敏感性。然而，部分TGCT要么天生对顺铂耐药，要么在治疗过程中获得顺铂耐药性。阐明TGCT对顺铂敏感或耐药的潜在机制，并开发靶向顺铂耐药TGCT的全新治疗策略，具有重大临床价值。本研究检测了5株胚胎癌（embryonal carcinoma, EC）细胞系修复顺铂诱导的ICL损伤的能力。以γH2AX染色（γH2AX staining）作为双链断裂形成的标志物，我们发现这些EC细胞系要么无法修复ICL诱导的损伤，要么修复能力显著受损。该缺陷与同源重组（Homologous Recombination, HR）修复能力降低密切相关，这一点可通过RAD51焦点形成减少，以及利用GFP报告底物直接评估HR效率得以验证。已知同源重组缺陷的肿瘤细胞对聚ADP核糖聚合酶（poly(ADP-ribose) polymerase, PARP）抑制剂治疗敏感。基于这一研究结论，我们观察到EC细胞系同样对PARP抑制剂单药治疗敏感，其敏感程度与HR修复能力降低、PARP1蛋白的表达水平及活性呈正相关。此外，我们发现PARP抑制可通过降低最耐药的EC细胞克服损伤的能力，显著增强其对顺铂的应答。上述结果表明，HR修复能力缺陷是EC细胞对ICL诱导剂及PARP抑制剂单药治疗敏感的核心原因之一，并提示可通过PARP的药理学抑制靶向TGCT的干细胞组分（即EC细胞），同时增强顺铂耐药TGCT对标准治疗的敏感性。