USP7稳定MDC1的分子机制及其在基因组稳定性和乳腺癌中生物学意义的研究

Breast cancer is the most common malignancy in women, and deregulation of key players involved in DNA double-strand break (DSB) repair is believed to play an important role in the development and progression of breast cancer. Particularly, deficiency or mutation in BRCA1 (Breast cancer 1), BRCA2 (Breast cancer 2) or BLM (Bloom syndrome RecQ like helicase) has been tightly linked to breast carcinogenesis. Meanwhile, cancerous cells often carry abnormalities in DSB repair machinery, which render these cells resistant to DNA damage-based therapy and gain of aberrant chromosome structures including translocations, deletions, inversions, as well as duplications. Protein deubiquitinase USP7 (Ubiquitin specific protease 7) is reported to stabilize a number of proteins thus regulate multiple biological processes including DNA damage response and cancer development and progression. However, whether USP7 is directly involved in DSB repair and the mechanistic insights into the role of USP7 in DNA repair and tumorigenesis remain to be investigated. Our preliminary data suggested that USP7 is physically associated with MRN complex (MRE11–RAD50–NBS1) and is recruited to DSBs in an MRN complex dependent manner. In addition, we revealed that USP7 depletion was associated with decreased expression of MDC1 (Mediator of DNA damage checkpoint 1) at the protein level, the effect of which could be reverted by proteasome inhibitor MG132, suggesting that MDC1 is a candidate substrate of USP7. Moreover, we found that USP7 is highly expressed in breast carcinomas and USP7-promoted MDC1 stabilization in breast cancer cell confers cellular resistance to DNA damage. These preliminary results indicate that USP7 plays a role of importance in DSB repair and dysregulation of USP7 is associated with DNA damage tolerance in breast cancer cells. In the future, we will further explore the underlying mechanisms of how USP7 regulates MDC1 deubiquitination and how USP7 influences on DSB repair as well as genome instability in breast cancer. Our study provides a mechanistic link of USP7 to DSB repair, and supports the pursuit of USP7 as potential targets for breast cancer intervention when combined with chemo- or radio-therapies.

乳腺癌是危害妇女健康最常见的恶性肿瘤之一。近来研究表明，DNA损伤异常修复可使肿瘤细胞基因组更加趋于不稳定，并赋予肿瘤细胞面对压力选择时逃避死亡的能力。蛋白质去泛素化酶USP7在肿瘤发生发展中起着重要作用，但是其是否直接参与DNA双链损伤修复进而影响肿瘤基因组的不稳定性尚需要进一步探索。我们的初步研究表明，USP7可以被MRN复合物直接招募到DNA双链损伤区域；并依赖于其酶活性通过泛素蛋白酶体途径稳定MDC1；同时，我们发现USP7在乳腺癌中高表达，其功能缺失显著的增加了细胞对损伤处理的敏感性,并且这一效应可被过表达MDC1逆转。这些结果初步提示USP7在DNA损伤修复、基因组稳定性维持和乳腺癌细胞应答损伤刺激的过程中起着重要的作用。我们将在今后的工作中进一步探索USP7在该过程中的作用以及相关分子机制。该研究，为联合使用靶向USP7的小分子抑制剂和放化疗治疗乳腺癌提供了理论基础。

近来研究表明，DNA损伤异常修复可使肿瘤细胞基因组更加趋于不稳定，并赋予肿瘤细胞面对压力选择时逃避死亡的能力。蛋白质去泛素化酶USP7在肿瘤发生发展中起着重要作用，但是其是否直接参与DNA双链损伤修复进而影响基因组稳定性尚需要进一步探索。在该基金项目的资助下，我们发现MRN/MDC1复合体与蛋白质去泛素化酶USP7在物理上存在相互作用，并且MRN/MDC1复合体为USP7更有效的去泛素化和稳定MDC1提供了平台，从而促进DNA损伤修复应答。进一步研究揭示了USP7在宫颈癌中过度表达，其表达水平与MDC1的表达水平呈正相关，与宫颈癌患者的生存水平呈负相关。此外，该研究发现在DNA损伤条件下，USP7 与MRN/MDC1复合体的相互作用增强，MDC1的稳定性升高，从而更加有利于招募BRCA1和53BP1，进而促进细胞同源重组 (Homologous recombination)修复和DNA末端链接(Non-homologous end joining) 修复。USP7介导的MRN/MDC1复合体的稳定性赋予了细胞抵抗DNA损伤的能力，使宫颈癌细胞更容易存活。该研究不仅探索了USP7与MRN/MDC1复合体在宫颈癌发生发展中的作用和分子机制，而且为宫颈癌的治疗提供了可能的分子靶点；进一步研究提示，在USP7和MDC1高表达的宫颈癌病人中，使用靶向USP7的小分子抑制剂和放化疗手段，可以更有效的杀伤宫颈癌细胞。该研究不仅为认识MRN/MDC1复合体如何高效调控损伤应答起始效率提供了新的认识，而且为宫颈癌的治疗提供了可能的分子靶点。该工作于2018年发表在J Clin Invest杂志。在此研究基础上，我们进一步揭示了USP7多聚化和自身稳定性的调节机制，发现上皮细胞转化因子ECT2介导的这一效应在肿瘤发展中起着重要作用（Theranostics， 2020）。在该项目的资助下，我们还发现了去泛素化酶USP9X和USP52可以在染色体分离、组装等角度调节基因组稳定性，且异常调控可以促进乳腺癌的发生发展（Nature Communications，2017；2018）。