SphK1/S1P信号通路在人结肠癌侵袭转移中对FAK介导的上皮间质转化的影响及其机制研究

Evidences show that epithelial-mesenchymal transition (EMT) plays important roles in cancer metastasis, and focal adhesion kinase (FAK) is thought to be a key regulating factor of EMT. Our recent studies showed that the expression of sphingosine kinase 1 (SphK1), FAK and p-FAK in colon cancer tissues was associated with Dukes' stage, lymph node metastasis and distant metastasis. There was a close correlation between the expression of SphK1 and FAK or p-FAK. Morover, cell invasiveness and expression of FAK were enhanced with the overexpression of SphK1 in colon cancer cells. Studies also showed that the expression of E-cadherin in colon cancer tissues with metastasis was slight than that of colon cancer tissues with metastasis, contrastly, the expression of vimentin was increased, and the expression of E-cadherin and vimentin were closely relative with the expression of SphK1. Thus, it is considerated that SphK1/S1P signaling pathway promotes colon cancer metastasis through regulating EMT mediated by FAK. In this study, the expression of factors which involved in EMT, SphK1/S1P and FAK signal pathways will be investigated in tissues of normal colonic mucosa, colon cancer with or without metastasis. In addition, the effects of SphK1/S1P signal pathway on FAK mediated EMT in colon cancer metastasis will be studied in vivo and in vitro by gene engineering technology and the orthotopic transplantation tumor model. This study will elusidate some mechanisms of colon cancer invasion and metastasis and provide the experimental basis for exploring new therapeutic target on human colon carcinama.

研究表明上皮间质转化（EMT）在肿瘤的侵袭转移中发挥重要作用，而黏着斑激酶（FAK）被认为是调控EMT的关键因素之一。我们前期研究发现SphK1/S1P信号通路通过激活FAK促进结肠癌的侵袭转移；且有转移结肠癌组织中EMT主要标记物E-钙粘蛋白的表达较无转移结肠癌组织明显减弱，而波形蛋白则明显增强，这些标记物表达的变化与SphK1的表达密切相关。因此，我们推测SphK1/S1P信号通路可能是通过调控FAK介导的EMT从而促进结肠癌侵袭转移。本课题拟检测正常结肠粘膜、有转移和无转移结肠癌组织中SphK1/S1P和FAK通路以及EMT相关蛋白的表达；同时，采用基因工程技术及原位移植瘤模型等方法探讨体外及体内结肠癌细胞SphK1/S1P信号通路对FAK介导的EMT及细胞侵袭转移能力的影响，为阐明人结肠癌侵袭转移机制，寻找治疗结肠癌新靶点提供实验依据。

结肠癌是最常见的恶性肿瘤之一。侵袭转移是影响预后的主要原因，上皮间质转化（epithelial–mesenchymal transition，EMT）在结肠癌侵袭转移中的调控机制还远未阐明。黏着斑激酶（Focal adhesion kinase，FAK）参与调节癌细胞侵袭迁移，在EMT中发挥着关键性作用，鞘氨醇激酶-1（sphingosine kinase 1, SphK1）可能是通过调控 FAK 促进侵袭相关因子分泌，从而促进结肠癌的侵袭转移。因此，我们推测 SphK1/S1P信号通路可能是通过调控 FAK 介导的 EMT促进结肠癌侵袭转移。. 研究发现：结肠癌组织中SphK1，FAK，p‑FAK，E‑cadherin 和 vimentin的表达与结肠癌的转移密切相关，而且SphK1的阳性表达与患者的预后负相关。SphK1抑制剂DMS和FAK抑制剂PF-562271抑制人结肠癌细胞的活性和迁移能力，同时抑制p-FAK、上皮相关蛋白和 MMP2蛋白，并上调间质相关蛋白，因此SphK1和FAK通路可能在结肠癌细胞EMT中发挥重要作用。敲低SphK1抑制抗凋亡蛋白ki67和Bcl-2，增加凋亡蛋白caspase-9和caspase-3，增强RKO细胞对DDP的化疗敏感性。抑制SphK1和FAK (p-FAK)后抑制细胞迁移能力，降低Slug，vimentin和N-cadherin并增加E-cadherin，而且抑制SphK1能减少p-FAK。因此SphK1可能通过调节FAK通路介导的EMT促进结肠癌细胞的侵袭转移。敲除FAK和SphK1能抑制RKO细胞p-FAK、AKT和MMP2/9，抑制细胞EMT和迁移能力，而且抑制FAK可显著抑制SphK1过表达所诱导的HT29细胞p-FAK、p-AKT、MMP2/9、EMT和迁移能力。因此，SphK1能通过FAK/AKT/MMPs轴介导的EMT调节结肠癌的侵袭转移。另外，还发现激活SphK1/ERK/p-ERK通路促进结肠癌HT-29细胞的自噬。. 在裸鼠结肠癌原位模型中发现，与空白组相比，抑制SphK1能够抑制结肠癌肿瘤的生长。. 综上，本课题的研究证实了SphK1介导EMT在结肠癌侵袭转移中的重要作用，SphK1和EMT可能成为结肠癌治疗的靶标，同时，为进一步探究SphK1和结肠癌自噬之间的关系提供基