TLR-9/NF-κB/ODAM信号通路在前列腺癌转移中的作用及其机制研究

Tumor metastasis is the main cause of death in patients with prostate cancer. Many studies displayed that activation of TLR-9 enhanced the metastasis of prostate cancer cells, but the mechanism is unclear. In the previous study, we found that activation of TLR-9 decreased expression of ODAM, overexpression of which inhibited TLR-9-induced metastasis of prostate cancer cells. Based on these results, our further researches revealed that activation of TLR-9 could activate NF-κB and down-regulate ODAM expression. Moreover, inhibition of NF-κB blocked TLR-9-induced down-regulation of ODAM. We also found that NF-κB could directly bind to the promoter region of ODAM gene. Therefore, we speculate that TLR-9 promotes the invasion and metastasis of prostate cancer via negative regulation of ODAM by NF-κB. In this project, we will use multiple approaches, including CRISPER/Cas9, site-directed mutation, confocal laser scanning microscopy and animal model of prostate cancer in situ to carry out this study, in order to clarify the mechanism of ODAM down-regulated by TLR-9. Combined with needle biopsy, surgical specimens and follow-up data of prostate cancer patients, the project would systematically clarify the effects of TLR-9/ NF-κB /ODAM signaling pathway in the invasion and metastasis of prostate cancer and the potential mechanisms, which will provide new biomarkers to predict the metastasis of prostate cancer and therapeutic targets, a novel scientific theory and tools for diagnosis and treatment of prostate cancer metastasis.

肿瘤转移是导致前列腺癌患者死亡最主要原因。研究表明激活TLR-9可促进前列腺癌转移，但机制未明。我们前期研究发现激活TLR-9可下调ODAM表达、过表达ODAM可抑制TLR-9引起的前列腺癌转移。进一步研究发现：TLR-9激活NF-κB的同时可导致ODAM表达下调，且抑制NF-κB可阻断TLR-9引起的ODAM表达下调；我们还发现NF-κB可以与ODAM启动子直接结合，据此我们推测TLR-9通过激活NF-κB而下调ODAM表达来促进前列腺癌转移。本项目拟通过CRISPER/Cas9、启动子定点突变与复原、共聚焦显微镜及原位前列腺癌动物模型等手段，明确TLR-9下调ODAM表达的机制；结合术前前列腺穿刺活检及术后标本、临床随访数据，系统阐明TLR-9/NF-κB/ODAM信号通路在前列腺癌转移中的作用及机制，以期发现预测前列腺癌转移标志物及治疗靶点，为防治前列腺癌转移提供新的理论和诊治手段。

前列腺癌患者死亡最常见的原因为肿瘤转移，现有的治疗手段仍难以有效地遏制转移事件的发生，因此，迫切需要新型标志物及治疗干预靶点来改善患者预后。本课题组通过体外细胞实验、原位前列腺癌动物模型及前列腺癌病人标本等前期研究已明确Toll 样受体-9（Toll Like Receptors-9，TLR-9）的表达和激活其可促进前列腺癌的转移。为了探求 TLR-9 促进前列腺癌转移的机制，我们通过全基因组微阵列筛选出了 TLR-9 下游关键的调控分子-- 颌骨成釉细胞相关蛋白（Odontogenic ameloblast-associated protein，ODAM）。ODAM 的肿瘤转移抑制效应在体外细胞实验、动物模型及前列腺癌病人标本上均得到了验证。在此基础上，我们进一步研究发现过表达 ODAM 可明显使得 TLR-9 增强前列腺癌转移能力的效应受到抑制。在探究 TLR-9 调控 ODAM 表达机制的研究中，我们发现 TLR-9 可激活核转录因子κB（nuclear factor kappa-B，NF-κB）并发生核转位。此外，生物信息软件分析、ChIP、EMSA、双荧光素酶报告基因实验显示NF-κB 可以与 ODAM 基因的启动子的结合，阻遏了 ODAM 基因的转录，从而下调该基因的表达。至此，我们系统阐明了TLR-9/NF-κB/ODAM信号通路在前列腺癌转移中的作用及机制，结合患者术前前列腺穿刺活检及术后标本、临床随访数据，揭示了TLR-9/NF-κB/ODAM信号通路可作为预测前列腺癌转移新标志物及治疗靶点，为防治前列腺癌转移提供新的理论和诊治手段。