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

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。

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.