PI3K/AKT信号通过ANLN对膀胱癌侵袭进展的调控作用及机制研究

Aberrant activation of PI3K/AKT signaling pathway plays a key role in the carcinogenesis of bladder cancer. Our previous studies confirmed that this pathway was an important therapeutic target for bladder cancer. However, the molecular mechanism of this pathway in promoting bladder cancer invasion remains unclear. Our previous RNA-sequencing revealed that the ANLN was higher in muscle invasive bladder cancer than non-muscle invasive bladder cancer, and we proved that ANLN played an important role in regulating the invasion and progression of bladder cancer, which was positively correlated with the pathological stage and negatively correlated with the prognosis of bladder cancer. Functional studies revealed that ANLN was closely related to invasion and migration of bladder cancer cells, and immunofluorescence suggested co-localization between ANLN and RhoA in cells. We also found the PI3K/AKT signaling pathway could regulate RhoA through phosphorylation. Therefore, we put forward the hypothesis that PI3K / AKT signaling pathway could phosphorylate RhoA and regulate RhoA / ANLN complex, which could promote the invasion of bladder cancer by regulating the cytoskeleton and tension fiber. This project will reveal the new molecular mechanism of bladder cancer invasion, and help to explore the molecular markers for predicting the progression and prognosis of bladder cancer. Meanwhile, this study will provide theoretical and experimental basis for discovering new targets to interrupt the progression process of bladder cancer.

PI3K/AKT信号通路异常激活在膀胱癌发生发展中起关键作用，我们前期研究证实该通路是膀胱癌重要的治疗靶点，但该通路促进膀胱癌侵袭进展分子机制仍未阐明。前期我们通过全转录组测序发现调控膀胱癌侵袭进展的关键基因ANLN，其高表达与膀胱癌浸润程度呈正相关，和膀胱癌患者预后呈负相关，且ANLN与膀胱癌细胞侵袭、迁移能力密切相关。进一步研究发现了ANLN与RhoA存在共定位现象，且PI3K/AKT信号通路可磷酸化调节RhoA。由此我们提出科学假设：PI3K/AKT信号通路通过磷酸化RhoA，调控RhoA/ANLN复合物在膀胱癌细胞侵袭过程中细胞骨架和张力纤维丝重塑，最终促进膀胱癌侵袭进展。该课题将揭示膀胱癌侵袭进展新的分子机制，有助于探索膀胱癌进展及预后评估分子标记物，为发现干预膀胱癌侵袭进展新靶点提供理论依据及实验基础。

膀胱癌的发病是一个多因素混合、多基因参与、多步骤形成的过程。因此，对于膀胱癌恶性进展密切相关基因的分子机制及其临床意义的深入研究，有助于认识膀胱癌发生及进展过程中关键基因所起的调控机制，这对于实现膀胱癌的分子分型及个体化治疗，开发膀胱癌的靶向治疗药物至关重要。我们前期通过全转录组测序发现调控膀胱癌侵袭进展的关键基因ANLN，其高表达与膀胱癌浸润程度呈正相关，和膀胱癌患者预后呈负相关，且ANLN与膀胱癌细胞侵袭、迁移能力密切相关。进一步研究发现ANLN与CCNB1，BUB1B以及CENPF呈共表达，参与到膀胱癌细胞的周期调节及有丝分裂中。通过免疫荧光实验提示，ANLN编码的Anillin蛋白主要在细胞周期中分裂期起到分裂间沟的形成，敲减ANLN基因可引起有丝分裂期分裂间沟形成障碍导致细胞核分离而细胞质未分裂，从而形成多核细胞。研究发现RhoA抑制剂及PI3K抑制剂均能有效抑制RhoA活性，RhoA表达量较对照组明显减少，但是Anillin蛋白无显著改变，推测PI3K/AKT/RhoA信号通路对Anillin的表达量无直接的调节作用。探索发现YAP是调节ANLN的重要上游信号通路。我们利用YAP的干扰RNA敲减RNA表达后，发现ANLN mRNA出现相同的低表达趋势。进一步行western-blot检测发现siRNA能显著抑制YAP蛋白的表达，并且同时出现ANLN编码蛋白的表达下调。我们在ANLN过表达的膀胱癌细胞系5637中使用YAP干扰RNA后发现，抑制YAP基因后可引起其下游的ANKDR1及TEAD1基因的表达下调，同时ANLN基因的表达呈现出相同的趋势。ANLN基因则通过影响F-actin蛋白的功能，改变细胞骨架及对细胞外应力的改变来负反馈调节YAP通路。ANLN敲减引起F-actin表达下降，进而引起YAP去磷酸化减少，YAP入核减少。该研究发现了YAP/TEAD/ANLN/F-actin负反馈调节环路对膀胱癌细胞功能的调节，该基础研究结果有望为探索膀胱癌分子分层标记物及寻找靶向治疗药物提供新思路。