LRRK2磷酸化LKB1诱导细胞自噬维持膀胱癌高增殖潜能的机制研究

The high recurrence rate and high proliferative capacity of bladder cancer pose a significant clinical problem for urologists. A bulk of recent evidence suggest that a high basal level of autophagic activity is found in human bladder cancer cell lines when compared with normal bladder epithelial cell and other cancer cell lines, and macroautophagy (hereafter referred to as autophagy) is required for the growth and survival of human bladder cancer cells. But the molecular mechanisms that mediated the induction and constitutive activation of autophagy remain largely unknown. In our preliminary study, based on the potential relationship between Parkinson's disease (PD) and cancer and the fact that PD protein LRRK2-induced autophagy in dopaminergic neuron plays a vital role in PD pathogenesis, we firstly identified that LRRK2-induced autophagy in bladder cancer cells positively contributed to its proliferative effects, which was regulated by LKB1. We hypothesize that autophagy induction by LRRK2 via phosphorylation of LKB1 is a novel mechanism for bladder cancer cell to survive under unfavorable conditions. To further elucidate the regulatory mechanisms of autophagy induction by LRRK2 in bladder cancer, we aimed to investigate the role LKB1/AMPK signaling played in LRRK2-induced autophagy based on our preliminary data that LRRK2 increased autophagic flux and phosphorylated LKB1 in vitro. Human bladder cancer cell lines with different expression of LRRK2, nude mice orthotopic bladder cancer and xenografts models will be served as the model systems in vitro and in vivo. Our studies thus will provide a rationale for the better understanding of molecular mechanisms that mediated LRRK2-induced autophagy in the development and progression of bladder cancer.

膀胱癌的高复发特性一直是临床上膀胱癌治疗的难点。而膀胱癌复发的过程势必包含着肿瘤细胞过度增殖的过程，新近研究表明，高自噬活性是维持膀胱癌存活及高增殖能力的重要因素之一，但对于启动并维持膀胱癌自噬的分子机制仍未清楚。我们在前期研究中围绕帕金森病(PD)与肿瘤间的特殊联系，并基于PD致病蛋白LRRK2可诱导神经细胞自噬进而促进PD发生及进展这个现象，发现LRRK2可诱导膀胱癌细胞发生自噬并促进其体外增殖，该过程受LKB1调控。基于此基础，我们提出“LRRK2直接磷酸化LKB1诱导细胞自噬，促进膀胱癌在能量缺乏环境中的存活，进而维持膀胱癌的高增殖潜能”的理论设想。本项目拟采用基因转染/封闭及免疫共沉淀等分子生物学技术，研究LKB1/AMPK信号通路在LRRK2诱导膀胱癌自噬进而影响其增殖活性过程中所扮演的角色，为初步阐明膀胱癌获得高自噬活性并维持其高增殖及高复发潜能的分子机制奠定理论及实验基础。

非肌层浸润性膀胱癌术后的高复发特点及其分子机制一直是泌尿外科基础研究领域的热点和难点之一。而膀胱癌复发的过程势必包含着肿瘤细胞过度增殖的过程。我们的前期研究表明，与正常膀胱上皮细胞相比，膀胱癌细胞显示了更强的分子伴侣介导的自噬（CMA）活性，抑制CMA关键调节蛋白LAMP2A则可显著抑制膀胱癌的体内外增殖。高自噬活性是维持膀胱癌存活及高增殖能力的重要因素之一，但对于启动并维持膀胱癌自噬的分子机制仍未清楚。我们在前期研究中围绕帕金森病(PD)与肿瘤间的特殊“镜像”联系，即是在PD患者中，膀胱肿瘤的患病风险显著下降；并基于PD致病蛋白LRRK2可诱导神经元自噬进而促进PD发生及进展这个现象，发现LRRK2可诱导膀胱癌细胞发生自噬并促进其体外增殖，该过程受LKB1调控。基于此基础，采用膀胱癌细胞系以及体内膀胱癌裸鼠皮下移植瘤模型，我们在本项研究中进一步发现，抑制膀胱癌细胞自噬的发生可显著抑制LRRK2的促增殖活性。在分子机制层面，以LC-3 I/II以及LC-3 puncta作为自噬的标志物，无糖饥饿可显著增加LRRK2的表达，敲低LKB1、抑制AMPK或抑制LRRK2均可显著抑制无糖饥饿所诱导的细胞自噬。更为关键的是，我们发现，LRRK2可直接磷酸化LKB1，磷酸化LKB1可激活AMPK，诱导膀胱癌细胞发生自噬，进而促进膀胱癌的体内外增殖能力。有意思的是，临床组织标本分析结果提示，LRRK2与膀胱癌存在远处转移的患者中表达升高；且患者的临床分期以及病理分级越高，LRRK2的表达越高；但LRRK2与膀胱肿瘤的大小无统计学关联；且复发次数越多的患者LRRK2的表达也显著高于复发次数低的患者。本项目也从分子层面以及临床应用层面初步阐明了膀胱癌获得高自噬活性并维持其高增殖及高复发潜能的分子机制，也为靶向LRRK2作为膀胱癌治疗的新靶点奠定理论及实验基础。

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