PO.ET03.04 · 实验与分子治疗
界定核仁应激在介导 DDB2 诱导的结直肠癌奥沙利铂耐药中的作用
Defining the role of nucleolar stress in mediating DDB2 induced oxaliplatin resistance in colorectal cancer
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：结直肠癌（CRC）是美国癌症死亡的第二大常见原因。含奥沙利铂的方案通常被视为身体状况良好患者的一线治疗，形式为 FOLFIRINOX 或 NALIRIFOX。然而，获得性耐药不可避免，且相当比例接受辅助性 FOLFOX（5-FU/亚叶酸钙/奥沙利铂）治疗的患者未能应答。此外，奥沙利铂治疗具有毒性副作用。近期研究表明，奥沙利铂通过诱导核仁应激杀伤细胞。我们和其他团队已证明，肿瘤细胞对奥沙利铂（而非顺铂）的耐药依赖于 DDB2 的水平——DDB2 是一种在全基因组核苷酸切除修复（GG-NER）过程中参与染色质损伤识别的 DNA 修复蛋白。然而，DDB2 在抑制奥沙利铂诱导的核仁应激中的作用尚不清楚。因此，界定 DDB2 在奥沙利铂反应中的分子作用、评估 CRC 应答率是否与低 DDB2 水平相关，以及阐明 DDB2 清除这些 DNA 损伤的机制，将填补我们知识中的一项紧迫空白。
方法：通过 Western blot 和 IHC 检测在 CRC 组织微阵列中测定 DDB2 蛋白水平。通过 cell titer glo（CTG）细胞活力实验和克隆形成实验获得奥沙利铂和顺铂的 IC50。通过共聚焦显微镜成像奥沙利铂处理后 DDB2 在核仁中的积累。我们采用新开发的 SMADNE 实验计算 DDB2 的结合动力学和平衡解离常数（KD）。
结果：我们发现，高表达 DDB2 的 CRC 细胞系对奥沙利铂的 IC50 较高，而 DDB2 敲除细胞对奥沙利铂比对顺铂治疗更敏感。我们的 IHC 数据提示 DDB2 在 CRC 患者肿瘤中差异表达。有趣的是，通过 C-trap 进行的单分子分析揭示，DDB2 以相似的亲和力结合奥沙利铂和顺铂加合物，表明尽管 DDB2 识别顺铂损伤，但 DDB2 缺失并不增加顺铂敏感性。共聚焦显微成像显示，奥沙利铂处理显著促进了 DDB2 在核仁中的积累，并与 NPM1（核仁应激标志物）相互共定位。DDB2 抑制剂（NAEi 或 CSN5i-3）与奥沙利铂的联用协同杀伤 HCT116 CRC 细胞。
结论：我们的研究表明，DDB2 是奥沙利铂反应的潜在生物标志物，也是 CRC 治疗中一个新的临床可靶向蛋白。这些研究将为 Pevonedistat 或 CSN5i 与奥沙利铂及 5FU 联合治疗的临床试验奠定基础。
查看英文原文 English abstract
Background: Colorectal cancer (CRC) is the second most common cause of cancer deaths in the USA. Oxaliplatin containing regimens are generally considered first line therapy for fit patients, either in the form of FOLFIRINOX or NALIRIFOX. However, acquired resistance is inevitable and a significant percentage of patients treated with adjuvant FOLFOX (5-FU/leucovorin/oxaliplatin) fail to respond. Furthermore, oxaliplatin treatment has toxic side effects. Recent studies have shown that oxaliplatin kill cells by inducing stress to nucleolus. We and others have shown that oxaliplatin, but not cisplatin resistance in tumor cells is dependent upon the levels of DDB2, a DNA repair protein involved in damage recognition in chromatin during global genome nucleotide excision repair (GG-NER). However, the role of DDB2 in suppressing oxaliplatin induced nucleolar stress is not known. Therefore, defining the molecular roles of DDB2 in oxaliplatin response, to assess CRC response rates correlate with low DDB2 levels and the mechanisms of how DDB2 acts to remove these DNA lesions will fill an urgent gap in our knowledge.
Methods: DDB2 protein levels were measured by western blot and IHC assay in CRC tissue microarrays. The IC50s for oxaliplatin and cisplatin was obtained by performing cell titer glo (CTG) cell viability and clonogenic assay. The accumulation of DDB2 in nucleolus after oxaliplatin treatment was imaged by confocal microscopy. We used our newly developed SMADNE assay to calculate DDB2 binding kinetics and equilibrium dissociation constant (KD).
Results: We found that high DDB2 expressing CRC cell lines have higher IC50 for oxaliplatin and DDB2 KO cells are more sensitive to oxaliplatin than cisplatin therapy. Our IHC data suggests that DDB2 is differentially expressed in CRC patient's tumor. Interestingly, single molecule analysis by C-trap, reveals that DDB2 binds to oxaliplatin and cisplatin adducts with similar affinities, indicating that while DDB2 recognizes cisplatin lesions loss of DDB2 does not increase cisplatin sensitivity. Confocal microscopic imaging demonstrates that oxaliplatin treatment dramatically facilitated DDB2 accumulation in nucleolus with mutual colocalization of NPM1 (a marker for nucleolar stress). The combination of DDB2 inhibitors (NAEi or CSN5i-3) and oxaliplatin kills HCT116 CRC cells synergistically.
Conclusions: Our studies demonstrate that DDB2 is a potential biomarker for response to oxaliplatin and a new clinically targetable protein in the treatment of CRC. These studies will lay the foundation for clinical trials of combination therapy of Pevonedistat or CSN5i with oxaliplatin and 5FU.
利益披露 Disclosure
V. Kumar, None..
D. Hsu, None..
O. D. Schärer, None..
M. A. Schaich, None..
K. E. Helfrich, None..
S. C. Watkins, None..
B. Van Houten, None.