LGR5调控TGF-beta信号通路并抑制结直肠癌转移的机制研究

Metastasis is the major cause of death in colorectal cancer (CRC) patients. Further insight into the biology of CRC metastasis is therefore essential in order to gain a greater understanding of this process and ultimately to develop better cancer therapies to prevent metastasis. LGR5 is leucine-rich repeat containing G protein-coupled receptor (GPCR) and was well known as a marker for proliferating adult stem cells in the small intestine and a potential therapeutic target in CRC. However, the function of LGR5 in CRC metastasis is not well studied. Our preliminary studies showed that LGR5 has negative effects in cancer progression both in vitro and in vivo. More importantly, our work underlies a novel mechanism of LGR5 that it activates TGF-beta signaling in CRC cells accompanied by increased stability of TGF-beta receptors. In this proposal, we hypothesize that LGR5 modulates TGF-beta signaling by inhibiting TGF-beta receptors degradation. To test this hypothesis, we will further determine the function of LGR5 in CRC metastasis by orthotopic transplantation mouse model in an inducible system; investigate the molecular mechanism of LGR5 in regulating TGF-beta receptors stability and activation in the process of CRC metastasis. All these findings will be further validated in human patient samples. By completing this study, we would be able to provide an alternative regulatory mechanism of TGF-beta signaling and a novel strategy for the biological therapy of CRC.

肿瘤的转移是结直肠癌患者的主要死因，肿瘤转移机制的研究对制定靶向治疗策略具有重要的意义。LGR5是小肠上皮的干细胞特征蛋白，近年来成为了肿瘤治疗靶点的研究热门，我们的前期工作证明LGR5在体内外均具有抑制肿瘤恶变和转移的生物学功能，并能与TGF-beta信号通路受体相互作用激活下游信号通路（以上工作已发表于Cancer Research）。后续未发表的工作亦提示LGR5可促进TGF-beta受体的蛋白稳定性及细胞定位，通过影响受体的内吞途径调控下游信号通路并抑制肿瘤转移。本项目将在前期工作基础上，利用可诱导表达系统和体内原位移植模型验证LGR5/TGF-beta受体复合物的抑癌功能；在分子水平上明确LGR5和TGF-beta受体的相互作用对下游信号的调控机制；并在人结直肠癌组织中加以验证。为TGF-beta信号通路的调控机制提供更全面的理论依据，也为结直肠肿瘤的靶向治疗提供新的思路。

LGR5是小肠上皮的干细胞特征蛋白，近年来成为了肿瘤治疗靶点的研究热门。本项目通过全面的体内外功能实验研究发现，LGR5可激活结肠癌细胞中的TGF-beta信号通路，增强TGF-beta介导的生长抑制、诱导细胞凋亡和肿瘤转移。我们通过荧光素酶报告、IHC染色等实验发现，LGR5基因敲除后可减弱TGF-beta信号传导、降低下游信号通路的分子激活、从而增加细胞增殖和体外存活率；利用原位移植小鼠模型我们进一步确定LGR5在动物体内具有抑制肿瘤转移的功能，LGR5敲除组小鼠肿瘤生长加速、转移率上升；在分子机制层面，我们利用免疫荧光、Western-blot等基础研究方法发现LGR5与TGF-beta受体可形成复合物，LGR5在其受体RSPO1的激活下可通过增加TGF-beta受体的表达、抑制受体降解来调节TGF-beta信号传导。我们也在临床结直肠癌患者样本中对上述发现进行了基本验证。我们的研究揭示了结肠癌细胞中干细胞标志物LGR5和抑癌信号通路TGF-beta之间的相互作用。由于TGF-beta信号传导在大多数晚期结肠癌中存在缺陷，其抑制功能的丧失是导致肿瘤恶变的关键因素，本项目研究结果可为激活TGF-beta信号传导并抑制结肠癌的发展和进展提供更全面的理论依据。