Table1_Novel MDM2 Inhibitor XR-2 Exerts Potent Anti-Tumor Efficacy and Overcomes Enzalutamide Resistance in Prostate Cancer.DOC

Background: The inactivation of tumor-suppressor p53 plays an important role in second generation anti-androgens (SGAs) drug resistance and neuroendocrine differentiation in castration-resistant prostate cancer (CRPC). The reactivation of p53 by blocking the MDM2–p53 interaction represents an attractive therapeutic remedy in cancers with wild-type or functional p53. Whether MDM2-p53 inhibitor could overcome SGAs drug resistance in CRPC is still needed further research. Here, we investigated the anti-tumor efficacy and mechanisms of a novel MDM2-p53 inhibitor XR-2 in CRPC.
Methods: To investigate the functions and mechanisms of XR-2 in prostate cancer, in vitro and in vivo biofunctional assays were performed. Western blot and qRT-PCR assay were performed to detect the protein and mRNA expression levels of indicated genes. CCK8, colony formation, flow cytometry and senescence assays were performed for cell function identifications. RNA-sequencing and bioinformatics analysis were mainly used to identify the influence of XR-2 on prostate cancer cells transcriptome. Subcutaneous 22Rv1 derived xenografts mice model was used to investigate the in vivo anti-tumor activity of XR-2. In addition, the broad-spectrum anti-tumor activities in vivo of XR-2 were evaluated by different xenografts mice models.
Results: XR-2 could directly bind to MDM2, potently reactivate the p53 pathway and thus induce cell cycle arrest and apoptosis in wild-type p53 CRPC cell lines. XR-2 also suppresses the AR pathway as p53 regulates AR transcription inhibition and MDM2 participates in AR degradation. As a result, XR-2 efficiently inhibited CRPC cell viability, showed a synergistic effect with enzalutamide and overcame enzalutamide resistance both in vitro and in vivo. Moreover, results illustrated that XR-2 possesses broad-spectrum anti-tumor activities in vivo with favourable safety.
Conclusion: MDM2-p53 inhibitor (XR-2) possesses potently prostate cancer progresses inhibition activity both in vitro and in vivo. XR-2 shows a synergistic effect with enzalutamide and overcomes enzalutamide resistance.

【背景】肿瘤抑制蛋白p53（tumor-suppressor p53）的失活，在去势抵抗性前列腺癌（castration-resistant prostate cancer, CRPC）的第二代抗雄激素药物（second generation anti-androgens, SGAs）耐药及神经内分泌分化（neuroendocrine differentiation）过程中发挥关键作用。通过阻断MDM2-p53相互作用以重激活p53，是针对野生型或功能完整p53肿瘤的极具潜力的治疗策略。然而MDM2-p53抑制剂能否克服CRPC中SGAs耐药性，仍有待进一步研究阐明。本研究针对新型MDM2-p53抑制剂XR-2在CRPC中的抗肿瘤功效及作用机制展开了探究。
【方法】为探究XR-2在前列腺癌中的功能与作用机制，本研究开展了体外（in vitro）与体内（in vivo）生物功能实验。采用蛋白质免疫印迹（Western blot）与实时定量逆转录聚合酶链反应（quantitative reverse transcription PCR, qRT-PCR），检测目标基因的蛋白与mRNA表达水平；通过CCK8实验、集落形成实验、流式细胞术（flow cytometry）及细胞衰老检测，完成细胞功能鉴定；主要借助RNA测序（RNA-sequencing）与生物信息学分析，明确XR-2对前列腺癌细胞转录组（transcriptome）的影响；构建皮下22Rv1细胞来源的异种移植瘤小鼠模型，以评估XR-2的体内抗肿瘤活性；此外，通过多种异种移植瘤小鼠模型，评价XR-2的体内广谱抗肿瘤活性。
【结果】XR-2可直接结合MDM2，强效重激活p53通路，进而在野生型p53 CRPC细胞系中诱导细胞周期阻滞与细胞凋亡（apoptosis）。同时，XR-2可抑制雄激素受体（androgen receptor, AR）通路：一方面p53可调控AR的转录抑制，另一方面MDM2参与AR的降解。综上，XR-2可有效抑制CRPC细胞活力，与恩扎卢胺（enzalutamide）展现出协同效应，并在体外与体内均成功克服了恩扎卢胺耐药性。此外，实验结果证实，XR-2在体内具备广谱抗肿瘤活性，且安全性良好。
【结论】MDM2-p53抑制剂XR-2在体外与体内均具备强效抑制前列腺癌进展的活性，可与恩扎卢胺发挥协同效应并克服其耐药性。