适应反应基因ATF3调控细胞骨架重构抑制膀胱癌转移的作用机制

Invasion and metastasis are the leading cause of unfavorable prognosis and death for bladder cancer patients. However, the mechanism of bladder cancer metastasis remain to be determined. Our preliminary study revealed that ATF3, adaptive response gene, is a new tumor metastasis suppressor gene of bladder cancer by the method of tumor tissue microarray, functional assay and in vivo mouse metastasis model. To our knowledge, this is the first report on the role of ATF3 in bladder cancer. Simultaneously, our preliminary data also have suggested that ATF3 can modulate the depolymerization and polymerization of actin cytoskeleton. It has been shown that the remodeling of actin cytoskeleton involved in tumor metastasis, and Rho GTPase was the key regulator for the remolding of cytoskeleton. Therefore, we propose that ATF3 may suppress bladder cancer metastasis through the Rho GTPase regulation of cytoskeleton remodeling..On the base of our preliminary studies, this project will further intend to investigate the mechanism of ATF3 in bladder cancer metastasis suppression. We will evaluate the expression of cytoskeleton regulatory proteins in bladder cancer and analyze their correlation with ATF3 using the large bladder cancer tissue microarray. To identify the target protein of ATF3, the methods of co-immunoprecipitation and immunofluorescence will be applied to explore the interaction of ATF3 with the proteins involved in Rho GTPase signaling pathway and characterize the role of ATF3 in the modulate cytoskeleton polymerization with the different metastatic potential of bladder cancer cells and WT,ATF3-/- MEF cells. Using in vivo imaging technology combined with the Rho GTPase pathway inhibitors, we will dynamically observe the tumor development and metastasis in the bladder cancer tumor-bearing mice. This project will characterize the detailed molecular mechanism of ATF3 in bladder cancer metastasis suppression through the remodeling of actin cytoskeleton at the molecular, cellular, tissue and body level, which can provide a new target and new idea to benefit the prognosis and treatment of bladder cancer.

侵袭和转移是影响膀胱癌预后和导致死亡的主要原因，但机制未明确。我们前期通过表型、体内外功能研究首次证实适应反应基因ATF3发挥膀胱癌转移抑制基因的作用,而且ATF3可影响细胞骨架的解聚和重组。已知细胞骨架重构是肿瘤细胞转移的基础,主要受Rho GTPase信号调节。因此我们提出假说：ATF3可能通过Rho GTPase调节细胞骨架重构抑制膀胱癌转移。本项目拟在前期基础上，利用膀胱癌组织芯片分析细胞骨架调控蛋白表达及与ATF3的相关性；利用不同转移潜能膀胱癌细胞和WT、ATF3-/-MEF细胞通过免疫共沉淀、免疫荧光等方法鉴定Rho GTPase通路中ATF3的作用靶蛋白及对细胞骨架重构的调控作用；利用活体成像技术结合Rho通路抑制剂动态观察膀胱癌荷瘤小鼠的肿瘤转移情况。本项目将在分子、细胞、组织及整体等多水平探讨ATF3抑制膀胱癌转移的机制，为膀胱癌转移的预后判断和治疗提供新靶点和思路。

膀胱癌是泌尿系统最常见的恶性肿瘤，侵袭转移是膀胱癌最主要的致死原因，因此研究膀胱癌侵袭转移的分子机制具有重要的理论意义和临床应用价值。肿瘤转移抑制基因在肿瘤侵袭–转移级联反应的多个步骤中发挥调控作用。本研究项目鉴定发现适应反应基因ATF3在膀胱癌转移中的作用及其分子机制。.我们首先通过临床膀胱癌组织芯片检测发现ATF3 在膀胱癌中低表达，且与膀胱癌的侵袭转移负相关。临床生存期资料显示低表达ATF3的膀胱癌患者生存期相对较短。体外功能实验和动物体内模型证实ATF3可抑制膀胱癌细胞的侵袭和转移。为了探讨ATF3抑制膀胱癌转移的分子机制，我们发现ATF3可影响细胞骨架重构，基因敲除ATF3小鼠成纤维细胞和沉默ATF3膀胱癌细胞株的细胞骨架F-actin强度明显增强。进一步检测结果显示ATF3可正向调控细胞骨架结合蛋白—凝溶胶蛋白（Gelsolin，GSN）的表达。通过CoIP技术结合生物信息学方法分析发现在GSN上游启动子区域存在六个ATF3的结合位点。临床样本也证实GSN在膀胱癌中的表达降低，而且ATF3与GSN的表达呈正相关。ATF3与GSN同时低表达的膀胱癌患者生存期明显缩短，而且两者同时低表达较单一指标对患者生存期和转移的预测价值更大。深入分析GSN调控的下游信号通路证实ATF3-GSN调控网络可影响p38信号通路参与的肿瘤细胞上皮间质转换（EMT）进而抑制肿瘤细胞转移。这一发现在膀胱癌、胃癌等肿瘤细胞中均得到证实。.该研究鉴定发现ATF3是一种新的膀胱癌转移抑制基因，并阐明了ATF3通过调控Gelsolin介导的细胞骨架蛋白重构抑制膀胱癌细胞转移的新机制。在该研究项目的基础上，对ATF3-GSN调控的下游信号通路一步步阐明不仅揭示了膀胱癌新的转移机制，而且在胃癌等其他肿瘤细胞上的验证进一步拓展了这一调控网络在其他实体瘤中的临床应用广泛性，为临床提供了转移及预后判断的新指标和将来可能的治疗靶目标。