鞘氨醇激酶2(SphK2) 代谢失衡促进慢性结肠炎微环境形成及炎癌转化研究

Colonic tissues characterize with hypoxia, formation of colonic inflammation and growth of adenomatous polyposis. The reason has yet unknown. We recently found that development of colonic tumorigenesis is related to SphK2 overexpression and loss of balance in metabolism of products. Unlike well-studied SphK1, little has yet understood the functions of SphK2. Based on our previous results, we hypothesize that overexpression of SphK2 causes the formation of tumour microenvironment (TME) and promotion of malignant progression in colon. Based on the establishment of transgenic mice models and determination by in vivo visualization probes, including mice models of Sphk2+/+ and APCMin/+Sphk2+/+, we will further analyze the tumor-associated lymphocyte recruited and educated by cancer cells with high SphK2. Expression of PD-L1/PD-1 and M2 and MCSCs cells invaded colon cancer tissues will be analyzed to understand the mechanism that cancer cells with high Sphk2 escape from immunological surveillance through regulation of the PD-1/PD-L1 axis. Further, we will investigate the net work involving the regulation of tumor microenvironment and development of colitis-associated cancer. The experiment will be performed in the APCMin/+/Atg5 and Sphk2+/+/Atg5 mice model as well as HCT-116Sphk2 cells. We will understand the function of molecular switch for Sphk2 through the activation of autophagy function in Atg5 and inhibiting apoptosis in the condition of endoplasmic reticulum (ER) stress. The significance for this project is that SphK2 might play crucial roles in the conversion from chronic inflammation to tumorigenesis and further progression of colon cancer.

结肠组织易缺氧、形成慢性肠炎及炎性腺瘤增生等，原因不明。前期发现，SphK2代谢失衡与结肠癌有关。与SphK1不同，SphK2功能不清。假设SphK2是导致肿瘤微环境主要原因，SphK2表达与代谢异常促进结肠炎癌转化。在建立Sphk2+/+和APCMin/+Sphk2+/+小鼠及分子影像技术确证基础上，分析结肠和淋巴组织肿瘤相关淋巴细胞被招募和驯化状况，分析SphK2上调PD-L1/PD-1及M2、MCSCs浸润，确定Sphk2通过激活免疫逃逸方式促进结肠炎癌转化。以上述小鼠模型及APCMin/+/Atg5和Sphk2+/+/Atg5小鼠等，在基因组测序基础上，研究SphK2具备“分子开关”调控网络机制，确认内质网应激条件下由于SphK2激活所导致的以Atg5为中心形成的凋亡转向自噬调控过程，促进结肠炎癌转化。项目的意义在于明确SphK2是在慢性结肠炎癌转化节点调控关键网络转向的主要分子。

SphK2是鞘脂代谢过程中的重要激酶，迄今对于SphK2功能了解有限。我们旨在阐明SphK2在炎癌转化过程中作用及机制。基于临床调研SphK2与结肠癌发病相关基础上，我们建立SphK2 TG和SphK2-/-小鼠等模型研究发现：1. 高表达SphK2招募结肠局部ATF4等及MDSCs, M2和Th17细胞并激活PD-L1/PD-1促进炎症微环境。SphK2激活内质网GRP78等信号分子激活以Atg5为主自噬。在SphK2机制方面：（1）过表达SphK2通过与核内PKD2竞争方式抑制核β-Catenin磷酸化降解，致持续激活Wnt/β-Catenin下游靶基因转录；（2）发现膜S1PR2在5-FU诱导下移位至内质网，而核内SphK2产生出核并移位至内质网，由于S1PR2与SphK2移位与激活在内质网产生SphK2-S1P-S1PR2-Ca2+信号激活，我们测得HCT116SphK2细胞内质网Ca2+浓度升高，促进炎癌转化。2. 首次发现SphK2与S1PR2同步激活进而联合调控癌细胞对于5-FU耐药，发现源于SphK2 Tg小鼠结肠癌对于5-FU耐药，而源于SphK2-/-小鼠结肠癌对于5-FU敏感。分析显示这与SphK2激活上调癌细胞DPD表达有关，而SphK2通过上调DPD降解胞内5-FU，致使5-FU水平下降，产生耐药性。在SphK2调控耐药机制方面如下发现：以RNA-Seq方法分析HCT116sphk2细胞DPD转录水平，并以RT-qPCR方法确认上调DPD；以H3组蛋白广谱筛选发现SphK2 Tg小鼠H3K79me和H3K56ac乙酰化升高，确认后者为DPD转录位点；以ChIP-seq法分析发现SphK2 TG小鼠结肠上皮细胞内乙酰化H3K56ac在dpd 外显子区结合与野生小鼠明显差异；以Motif、UCSC Genome Browser、CHIP-Secquence法、qRT-PCR法以及双荧光素酶报告法等确定SphK2调控序列在DPYD 外显子区118593600至118595799，以ChIP-seq分析该序列位于800-850bp。结论：（1）SphK2和S1PR2同步激活并联合调控炎癌转化进程；（2）核SphK2通过H3K56ac乙酰化促进转录DPD，导致5-FU耐药性。靶向设计SphK2和S1PR2抑制剂能够抑制DPD进而逆转5-FU耐药性。