胰腺癌细胞与成纤维细胞直接接触调控其基因组甲基化修饰的细胞膜受体筛选及鉴定

Cancer-associated fibroblasts (CAF) are very important components of pancreatic cancer, and a major obstacle to effective treatments of pancreatic cancer. Abnormal genomic methylation was observed in several kinds of tumor, but the specific mechanism is unknown. Previous studies showed that SOCS1 was frequently methylated in CAF of pancreatic cancer tissues and primary cultures. Gene array showed that DNA methylation was presented in a global panel of genes and caused their down-regulation of CAF. DNA methyltransferase 1(DNMT1) was overexpressed in pancreatic cancer CAF induced by interacting with pancreatic tumor cells, and that CAF were epigenetically reprogrammed by promoter methylation induced by DNMT1. Moreover, the methylation of SOCS1 in CAF was specifically induced upon direct contact with tumor cells. However, the membrane receptor on CAF and the specific signaling pathway is unknown, but was supposed to be associated with phosphorylation activation. In this project, we will utilize multiple molecular biological techniques including, among others, receptor shRNA knock down, quantitative real time PCR, western blotting, as well as quantitative phosphoproteomic mass spectrometry in CAF and tumor cell co-culture system, to screen and identify the critical receptor(s) and elucidate the DNMT1 inducing signaling pathway. It is hoped that the findings from this project will help clarify the mechanism of genomic methylation of CAF induced by pancreatic cancer cells and provide new targets for reversing CAF methylation by small molecule inhibitors.

肿瘤相关成纤维细胞（CAF）是胰腺癌组织的重要成分，是制约胰腺癌有效化疗和靶向治疗的重要障碍。研究发现多种肿瘤组织CAF基因组存在异常甲基化修饰，但具体调控机制不明。我们前期也发现胰腺癌组织及原代培养的CAF中包括SOCS1基因在内的基因组存在甲基化修饰，由胰腺癌细胞诱导CAF过表达甲基转移酶1（DNMT1）导致；进一步研究发现SOCS1甲基化只能由胰腺癌细胞与CAF的直接接触而诱导，介导此过程的CAF细胞膜受体及其分子机制尚不明确，但与磷酸化激活的膜受体信号通路相关。本项目将应用定量磷酸化蛋白质组学技术筛选磷酸化激活的CAF细胞膜受体，并应用基因敲降技术抑制膜受体表达，以DNMT1表达和SOCS1甲基化为指标，鉴定出关键的细胞膜受体，并初步阐明其下游活化DNMT1的信号通路。本研究旨在一定程度上明确胰腺癌调控CAF细胞基因组的分子机制，为发掘小分子抑制剂逆转CAF细胞甲基化提供靶点。

前期研究发现胰腺癌组织及原代培养的CAF中包括SOCS1在内的基因组存在甲基化修饰，由胰腺癌细胞诱导CAF过表达甲基转移酶1（DNMT1）导致；进一步研究发现SOCS1甲基化只能由胰腺癌细胞与CAF的直接接触而诱导，介导此过程的CAF细胞信号通路尚不明确，但与磷酸化激活的膜受体信号通路相关。本项目的研究目标是筛选鉴定CAF细胞受胰腺癌细胞调控发生基因组甲基化过程中，CAF细胞与肿瘤细胞相互作用的关键细胞膜受体及该受体磷酸化激活后诱导DNMT1表达的信号通路。本项目完成了CAF与肿瘤细胞共培养后的磷酸化蛋白质质谱分析，共鉴定出10363个肽段，其中磷酸化修饰肽段8619个，其中2884个蛋白上的8609个位点具有定量信息。将FC> 1.3的蛋白质磷酸化位点进行生物信息学分析，包括GO富集，KEGG通路富集，聚类分析等信息学处理，结合前期表达谱基因芯片结果，发现HRAS，P53及TGFβR1是细胞内磷酸化上调最为显著的蛋白质分子，其中HRAS-P38信号通路是潜在最为可能的信号通路，MST1R是潜在的细胞膜信号受体。采用基因敲降技术获得稳定敲降细胞系进行细胞共培养试验，通过实时荧光PCR及蛋白质免疫印迹等分子检测发现HRAS-P38-Mnk1信号通路是诱导DNMT1过表达的重要信号通路分子。动物实验进一步确认CAF中HRAS-P38-Mnk1调控的DNMT1表达对于其促进肿瘤细胞增殖有显著意义。此外，通过研究不同肿瘤细胞诱导CAF甲基化修饰的水平发现，肿瘤细胞诱导CAF甲基化修饰具有普遍性，但不同肿瘤细胞诱导CAF甲基化能力存在差异，且CAF细胞促肿瘤细胞增殖能力与其SOCS1基因甲基化水平正相关。综上，本研究进一步明确了CAF细胞基因组被诱导甲基化修饰在各种肿瘤中具有普遍性，CAF细胞中HRAS-P38-Mnk1信号通路活化是肿瘤细胞诱导DNMT1过表达的关键分子机制。