酪氨酸激酶受体RON对膀胱肿瘤细胞CXCR4的表达调控及其介导膀胱癌肌层浸润机制

Muscle invasion is an important phenomenon during bladder cancer progression. If muscle invasion happens, the survival rate of the patients will dramatically decrease. Tyrosine kinase receptor RON belongs to MET family, which is usually activated in bladder cancer. During our previous research, we demonstrated that RON is overexpressed in bladder cancer and leads to epithelial to mesenchymal transition by activate erk1/2-rsk signal transduction pathway. CXCR4 is a chemokine receptor which involves in bladder cancer muscle invasion with its ligand CXCL12. CXCL12 is produced by carcinoma-associated fibroblasts or carcinoma-associated mesenchymal cells which locate at subepithelial connective tissue or bladder muscle layer, thus, bladder tumor cells overexpressed CXCR4 may be attracted by CXCL12 and invade into subepithelial connective tissue or bladder muscle layer. Based on: (1) RON and CXCR4 has the same expression characters in bladder cancer. And (2) RON activates erk1/2 and rsk, which also causes downstream molecule CREB binding to CXCR4's promoter, we hypothesis that (1) Coexpression of RON and CXCR4 is a survival prognostic index for muscle invasive bladder cancer; (2) RON mediates bladder cancer muscle invasion by stimulating EMT and increasing expression of CXCR4; (3) Blocking MSP/RON pathway decreases possibility of muscle invasion in bladder cancer. Through multiple experimental methods such as pathological analysis, cell and animal model research, we try to find the mechanisms how RON participates into bladder progression. The results will disclose the role of RON in mediation of bladder cancer muscle invasion and provide a new target for prevention and treatment of muscle invasion in bladder cancer.

膀胱癌肌层浸润是肿瘤发展过程中的重要现象。一旦发生浸润，患者生存率明显下降。我们前期研究发现膀胱癌组织表达酪氨酸激酶受体RON，RON通过ERK1/2-RSK通路介导细胞EMT现象并且明显增强细胞迁移能力。趋化因子受体CXCR4亦是一种与膀胱肿瘤浸润相关的重要因子，在膀胱癌具有与RON类似的肿瘤特征性表达谱。由于RSK下游信号分子CREB是CXCR4转录因子，RON可能通过ERK1/2-RSK-CREB通路上调CXCR4的表达。通过分析膀胱肿瘤组织RON和CXCR4的表达与临床病理及预后关系、膀胱肿瘤细胞中RON介导EMT机制及CXCR4表达上调机制、并进一步研究阻断MSP/RON通路在体外和体内对细胞侵袭力及肌层浸润的影响，以期阐明RON介导膀胱肿瘤肌层浸润机制，并为在膀胱癌进行针对RON的靶向治疗奠定前期基础。

RON是一种在多种肿瘤表达的酪氨酸激酶受体，与肿瘤的生长、浸润及转移相关。CXCR4是一种趋化因子受体，在趋化因子CXC12的作用下，促进细胞迁移。在膀胱肿瘤已发现RON与CXCR4的高表达，均与膀胱肿瘤的预后相关。然而，目前并不清楚RON和CXCR4在膀胱肿瘤浸润性生长中是否呈协同促进作用。本研究提出以下科学假设：“尿液中MSP促进膀胱黏膜层肿瘤细胞高表达的RON激活，RON激活后上调CXCR4表达，进一步在膀胱肌层间质细胞来源的CXCL12趋化下，造成肿瘤细胞向肌层浸润”。RON的激活是膀胱肿瘤由肌层非浸润向肌层浸润的重要因素之一。从以下五方面进行论证。(1).我们的临床病理研究提示RON与CXCR4不但在膀胱肿瘤表达，与肿瘤分期成正相关，而且RON与CXCR4的表达成正相关。我们检测106 例膀胱尿路上皮癌组织及34例癌旁组织中RON蛋白、CXCR4蛋白的表达情况，发现RON与CXCR4在膀胱肿瘤表达成正相关。(2).细胞模型提示RON高表达上调CXCR4表达，抑制RON下调CXCR4表达。在5637细胞株使用siRNA敲低RON表达后CXCR4表达下调，T24细胞株转染RON后CXCR4表达上调。(3).细胞模型提示RON激活后导致MAPK-RSK-CREB信号通路的激活，pCREB与CXCR4启动子结合及CXCR4启动子转录上调。刺激5637细胞后RON活化，进一步引起p-erk，p-RSK, p-CREB的激活。免疫共沉淀提示p-CREB与CXCR4结合，荧光素酶实验提示CXCR4转录子活性增强。(4).RON促进膀胱肿瘤细胞EMT。敲除RON表达，E-cadherin表达上调，vimentin表达下降。提示RON激活引起膀胱肿瘤细胞发生EMT，细胞形变和迁移。(5).联合RON特异性抗体Zt/g4和表柔比星明显抑制膀胱肿瘤细胞增殖、迁移与浸润。以上结果，提示膀胱肿瘤细胞在RON激活后，一方面造成细胞EMT现象与形变，另一方面，在此动态过程中激活MAPK-RSK-CREB信号通路，造成pCREB与CXCR4启动子结合及CXCR4启动子转录上调，CXCR4蛋白表达增强。从而得以在肌层间质细胞产生的CXCL12趋化下，向膀胱肌层浸润。我们的研究为理解膀胱肿瘤由非肌层向肌层浸润的机制提供了新的依据。然而，尚需免疫荧光观察CREB转位情况和动物试验进一步验证。