靶向KIF23通过失活MYH9/MCM2/PCNA途径抑制宫颈癌症细胞增殖和原发性化疗耐药性

摘要(中文译文)

驱动蛋白家族成员23（KIF23）被认为是参与多种癌症发病机制的重要促癌因子。然而，其在调控宫颈癌（CC）生长和原发性化疗耐药中的作用及潜在分子机制尚未完全阐明。本研究首先通过生物信息学分析评估KIF23的表达及预后意义，随后在临床标本中进行验证。为评估KIF23对宫颈癌细胞增殖和顺铂（DDP）敏感性的影响，采用CRISPR/Cas9敲除、过表达及小鼠异种移植模型进行了体外和体内实验。运用免疫共沉淀、蛋白质半衰期实验和泛素化实验阐明KIF23、肌球蛋白重链9（MYH9）、微小染色体维持蛋白2（MCM2）和增殖细胞核抗原（PCNA）之间的相互作用及调控机制，从而揭示KIF23介导宫颈癌进展和原发性化疗耐药的分子基础。KIF23在宫颈癌组织中高表达，且与患者不良预后和顺铂耐药显著相关。敲除KIF23可抑制细胞增殖、诱导G1期阻滞并增强对顺铂的化疗敏感性。机制上，发现KIF23的C端结构域可直接结合MYH9的肌球蛋白尾部结构域。该相互作用通过招募去泛素化酶7（泛素特异性蛋白酶7 [USP7]）去除K48连接的泛素链，从而稳定MYH9。随之发生的MYH9上调促进了泛素特异性蛋白酶15（USP15）的招募，使MCM2去泛素化，从而防止其降解。MCM2的第469位赖氨酸（K469）被鉴定为MYH9诱导去泛素化的关键位点。此外，MCM2水平升高增强了其与PCNA的结合，从而促进宫颈癌细胞增殖。这些发现表明，KIF23水平升高通过激活MYH9/MCM2/PCNA轴促进细胞增殖和原发性化疗耐药，是宫颈癌的不良预后因素。因此，KIF23可能代表一个改善宫颈癌临床预后的有前景的治疗靶点。顺铂处理以浓度和时间依赖性方式诱导KIF23表达。KIF23招募USP7，去除MYH9的K48连接泛素链，从而稳定MYH9并促进其核转运。MYH9招募USP15，从而稳定MCM2，MCM2进而通过与PCNA结合调控G1/S期转换。靶向KIF23/MYH9/MCM2/PCNA轴可使宫颈癌细胞对顺铂敏感化。

Abstract

Kinesin family member 23 (KIF23) is recognised as an important tumour promoter involved in the pathogenesis of various cancers. However, its role and underlying molecular mechanisms in regulating cervical cancer (CC) growth and primary chemoresistance remain to be fully elucidated. The expression and prognostic significance of KIF23 were initially assessed through bioinformatic analyses and subsequently validated in clinical specimens. To evaluate the effects of KIF23 on cell proliferation and cisplatin (DDP) sensitivity in CC cells, in vitro and in vivo experiments were conducted using CRISPR/Cas9 knockout, overexpression and mouse xenograft models. Co-immunoprecipitation, protein half-life assays and ubiquitination assays were employed to elucidate the interactions and regulatory mechanisms involving KIF23, myosin heavy chain 9 (MYH9), minichromosome maintenance protein 2 (MCM2) and proliferating cell nuclear antigen (PCNA), thereby revealing the molecular basis of KIF23-mediated CC progression and primary chemoresistance. KIF23 is highly expressed in CC tissues and is significantly correlated with poor prognosis and DDP resistance in patients. The knockout of KIF23 inhibited cell proliferation, induced G1-phase arrest and enhanced chemosensitivity to DDP. Mechanistically, the C-terminal domain of KIF23 was found to directly bind to the myosin tail domain of MYH9. This interaction stabilises MYH9 by recruiting deubiquitinase 7 (ubiquitin-specific protease 7 [USP7]), which removes K48-linked ubiquitin chains. The consequent upregulation of MYH9 promoted the recruitment of ubiquitin-specific protease 15 (USP15) to deubiquitinate MCM2, thereby preventing its degradation. Lysine 469 (K469) of MCM2 was identified as the key site for MYH9-induced deubiquitination. Furthermore, elevated MCM2 levels enhanced its binding to PCNA, thereby promoting CC cell proliferation. These findings demonstrated that elevated KIF23 levels act as an unfavorable prognostic factor for CC by promoting cell proliferation and primary chemoresistance via the activation of the MYH9/MCM2/PCNA axis. Thus, KIF23 may represent a promising therapeutic target for improving clinical outcomes in CC. Cisplatin treatment induces KIF23 expression in a concentration- and time-dependent manner. KIF23 recruits USP7, which removes the K48-linked ubiquitin chain of MYH9, thereby stabilising MYH9 and facilitating its nuclear transport. MYH9 recruits USP15, thereby stabilising MCM2, which, in turn, regulates the G1/S phase transition by binding to PCNA. Targeting the KIF23/MYH9/MCM2/PCNA axis sensitises cervical cancer cells to cisplatin.

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