Leptin/Cofilin通路促进前列腺癌侵袭性伪足形成的机制研究

Obesity is regarded as a risk factor for invasion and metastasis of prostate cancer. The excessive Leptin that secreted by abundant adipose tissue is proved to induce cancer progression, while the mechanisms remain unclear. We previously discovered that Leptin would dose-dependently upregulate the expression of active Cofilin-1, and it would promote the formation of invadopodia in cancer cells in a 3D culture model. It is reported that Cofilin-1 will be activated by dephosphorylation and consequently promotes the reform of actin and cytoskeleton. Indicated by bioinformatics analysis, we speculate that the combination of Leptin and its membrane receptor would activate the downstream signaling, upregulate the expression of active Cofilin-1 at transcription and phosphorylation levels, and induce the formation of invadopodia of prostate cancer. We intend to discover: (1) the associations between the leptin level, clinicopathological characteristics and prognosis of prostate cancer patients; (2) the impact on invadopodia by adding leptin in the micro-environment or regulating the expression of Leptin receptor; (3) the downstream transcriptor of Cofilin-1 and phosphorylation network in Leptin-associated signaling and regulation via ChIP-seq and phosphorylation proteomics. The present study aim to explore mechanisms for invasion and metastasis of prostate cancer, as well as provide novel strategy about treatment targets.

肥胖是前列腺癌侵袭转移的高危因素。研究表明，脂肪沉积通过超量分泌脂肪因子Leptin参与肿瘤进展，其机制不明。我们前期发现，Leptin剂量依赖性上调活性Cofilin-1表达，并在三维细胞培养中诱导肿瘤细胞侵袭性伪足形成。文献报道，Cofilin-1通过去磷酸化激活，促进肌动蛋白剪切与重组。结合生物信息学分析，我们推测，Leptin与膜受体结合后激活胞内信号转导，通过转录与磷酸化调控使活性Cofilin-1表达上调，促进前列腺癌侵袭性伪足形成。拟研究：（1）临床层面，分析Leptin表达与临床病理特征及预后关系；（2）细胞及动物层面，通过外源性Leptin或干预其受体表达，评价对侵袭性伪足的影响；（3）机制层面，利用ChIP-seq、磷酸化蛋白质组学等技术，寻找Leptin下游Cofilin-1的转录因子，并探索磷酸化调控网络。本研究将完善前列腺癌侵袭转移机制，为探索治疗靶点提供新策略。

既往研究提示肥胖是前列腺癌侵袭性增强的高危因素。我们的前期工作提示，脂肪沉积通过超量分泌脂肪因子Leptin参与肿瘤进展，本研究拟探索其具体分子机制。实验中现有体外细胞培养模型不足以用来观察和表征前列腺癌的侵袭性特征变化，为此我们研发了适合前列腺癌体外实验的三维细胞培养模型进行后续研究。. 本研究获得以下结果：（1）体外实验发现，短期（24-48小时）高浓度Leptin对于前列腺癌细胞侵袭存在抑制，长期（28天）中等浓度Leptin显著增加雄激素非依赖性前列腺癌细胞DU145的迁移与侵袭。（2）对Leptin下游关键蛋白进行研究，前列腺癌患者肿瘤组织Cofilin表达较高。Leptin长期作用下，前列腺癌细胞DU145的Cofilin表达上调，同时磷酸化后失活的p-Cofilin表达下调，提示Leptin通过转录调控上调了Cofilin表达，并通过磷酸化调控使Cofilin/p-Cofilin去磷酸化激活，促进细胞骨架重构和细胞侵袭。同时通过shRNA在DU145细胞基础上成功建立LEPR与CFL1干扰稳定株，两种干扰稳定株均表现出明显的侵袭能力下降，提示Leptin受体与Cofilin均是细胞侵袭的关键通路分子。（3）对Leptin调控前列腺癌细胞基因表达的转录组进行生物信息学分析，发现Leptin作用与中性粒细胞激活、RNA与mRNA分解、细胞外结构组成、细胞-底物连接、胞质囊腔、粘着斑和细胞因子介导信号通路相关。重点对细胞外基质-受体互作通路进行研究，发现位于细胞膜上的整合素αv和syndecan均出现表达上调，提示两者参与Leptin促进前列腺癌细胞侵袭性增强的过程。（4）制备并表征新型复合细胞支架，构建与之共培养的前列腺癌体外三维培养体系，用以最大程度模拟体内肿瘤生长微环境，适用于体外肿瘤细胞侵袭性表征、动态分子生物学研究、药物靶点筛选等多种用途。