鞘氨醇激酶2（SphK2)促进结肠炎癌转化的机制研究与N-芳基-2-氨基噻唑类化合物的活性筛选

The malignant progression from inflammation to tumorigenesis is the critical point in the development of colon cancer. However, the cause and mechanism for colon cancer have still been unknown. Based on the establishment of mice models with high expression of SphK2, we hypothesize that SphK2 cause the formation of tumour microenvironment (TME) and the promotion of malignant progression in colon. The project will be mainly designed in mice models of Sphk2+/+ and APCMin/+Sphk2+/+ as well as cell culture. The experiments will be performed to analyze the number of tumor-associated lymphocyte recruited and educated by cancer cells with high expression of SphK2 in colon cancer tissues and lymphonodus. We will analyze the expression of PD-L1/PD-1 on cancer cells and M2 and MCSCs cells invade in colon cancer tissues. Based on these data, we will conclude that cancer cells with high level of Sphk2 may escape from immunological surveillance through up regulation of the PD-1/PD-L1 axis. Using APCMin/+/Atg5 and Sphk2+/+/Atg5 mice model as well as HCT-116Sphk2 cells, we will investigate the role of molecular switch for Sphk2 through activating the function of autophagy in Atg5 and inhibiting the function of apoptosis in the condition of endoplasmic reticulum (ER) stress. The compound targeting SphK2 might inhibit the malignant progression of colon cancer. The significance for this project is that SphK2 might play crucial roles in the progression of colon cancer. These data will be provided to clinical study for improving the chemoprevention results for colon cancers.

炎癌转化是结肠癌发病过程中的关键节点，但原因与机制不清。在建立结肠炎癌转化模型基础上，假设SphK2是导致肿瘤微环境的原因、其异常表达促进了炎癌转化进程。拟以高表达Sphk2+/+和杂合APCMin/+Sphk2+/+小鼠结合细胞方法，分析结肠和淋巴组织等肿瘤相关淋巴细胞被招募和驯化状况，分析SphK2上调癌细胞表面PD-L1/PD-1表达及M2、MCSCs等免疫细胞浸润程度，确认Sphk2通过肿瘤免疫逃逸机制促进了炎癌转化。再以APCMin/+/Atg5和Sphk2+/+/Atg5小鼠及裸鼠接种HCT-116Sphk2方法，通过研究SphK2的“分子开关”机制，确认在内质网应激条件下激活Atg5调控的凋亡转向自噬活动促进了结肠炎癌转化进程。靶向Sphk2的化合物可能抑制上述炎癌转化过程。本项目的意义在于明确了SphK2是导致结肠炎癌转化的关键因子，将为临床制定更加有效的防治方案奠定基础。

通过研究鞘氨醇激酶-2 (SphK2)作为炎癌转化关键分子功能，阐释了SphK2-S1P-S1PR2信号轴介导慢性结肠炎癌转化及结肠癌对5-FU类药物产生耐药性机制，确定SphK2或S1PR2为新靶点，藉此靶向设计全新结构SphK2或S1PR2抑制剂。我们在以下方面获得进展：.（1）明确了激活SphK2在介导慢性结肠炎癌转化中的功能：通过比较转基因小鼠SphK2 Tg和SphK2-/-模型发现：激活SphK2能聚集结肠粘膜下层免疫抑制细胞MDSCs、M2和Th17等促进形成炎症微环境；在研究SphK2激活机制方面，我们发现：1）激活SphK2通过与核内PKD2竞争方式抑制核β-Catenin磷酸化降解，进而导致持续激活Wnt/β-Catenin下游靶基因转录；2）我们发现膜S1PR2能在5-FU诱导下移位至内质网，此时的核内SphK2出核移位内质网，由于在内质网聚集的S1PR2与SphK2导致SphK2-S1P-S1PR2-Ca2+信号轴激活，我们据此测得HCT116SphK2细胞内质网Ca2+浓度急剧升高，促进慢性炎症及炎癌转化过程。.（2）发现SphK2与S1PR2同步激活联合调控结肠癌细胞产生5-FU耐药性，源于SphK2 Tg小鼠结肠癌对5-FU耐药，源于SphK2-/-小鼠结肠癌对5-FU敏感，进一步研究显示这与SphK2激活上调癌细胞二氢嘧啶脱氢酶DPD表达有关，也即SphK2通过上调DPD降解癌细胞内5-FU，进而产生5-FU耐药性。SphK2调控DPD机制如下：以H3组蛋白广谱筛选法发现SphK2 Tg小鼠的H3K56ac乙酰化水平明显上调，据此确定DPD转录位点；进一步以ChIP-seq法分析发现SphK2 TG小鼠结肠癌上皮细胞内乙酰化H3K56ac在dpd 外显子区结合与野生小鼠具有明显差异性；再以Motif、UCSC Genome Browser、CHIP-Secquence法、qRT-PCR法以及双荧光素酶报告实验法等确定了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。.（3）靶向S1PR2设计合成了全新结构吡咯烷吡唑衍生物100余个，筛选出10个候选化合物，其中化合物10c活性最高，确立其为候选药物，拟进入临床前研究阶段。