结肠癌干细胞在结肠癌发生发展中起重要作用，但调控机制不楚。R-spondin3 (RSPO3)是一个新的Wnt/beta-catenin 信号调节基因。我们研究发现，RSPO3在骨髓来源肌成纤维细胞(BMFs)诱导的结肠癌干细胞样细胞(CSC-LC)中的表达显著高于亲本肠癌细胞、且在"干细胞样分型"的人结肠癌组织中的表达显著高于其它分型的结肠癌组织；高表达RSPO3与结肠癌病人不良预后明显相关；BMFs与结肠癌细胞与共培养显著增加了RSPO3在细胞中表达；重组RSPO3能够诱导结肠癌细胞产生CSC-LC。在此基础上，我们拟进一步研究BMFs旁分泌的RSPO3和肿瘤细胞自分泌的RSPO3在结肠癌CSC-LC重编、CSC特性维持和肿瘤形成中的作用，研究LGR4和LGR5在RSPO3诱导CSC-LC中的作用，从而阐明RSPO3调控结肠CSCs的作用及机制，为靶向抑制CSC而治疗结肠癌提供新靶标。

Cancer stem cells (CSCs) play an important role in cancer development and progression. However, the regulation of CSCs and underlying mechanisms remain unclear. R-spondin 3 (RSPO3) is a novel modulator of Wnt/beta-catenin signaling. We recently found that RSPO3 expression was much higher in CSC-like cells (CSC-LCs) induced by bone marrow-derived myofibroblasts (BMFs) than in the parental colorectal cancer (CRC) cells. Furthermore, in human CRC tissues, RSPO3 expression was also higher in stem cell-like subtype than in other subtypes. High expression of RSPO3 was strongly associated with poor prognosis of human CRC patients.Co-culture of BMFs and CRC cells increased RSPO3 expression in both cells. Recombinant RSPO3 induced CSC-LC formation of CRC cells. Based on our new findings, we hypothesized that paracrine and autocrine RSPO3 regulates colon CSCs by enhancing and sustaining Wnt signaling activity. In this proposal, We will: (1) determine the effects of overexpression and knockdown of RSPO3 in BMFs on CSC-LC induction and maintenance, as well as tumorigenesis in a mouse model with co-injection of CRC cells and BMFs; (2) stduy the effects of overexpression and knockdown of RSPO3 in CRC cells on CSC-LC induction and maintenance as well as tumorigenesis; and (3) investigate molecular mechanisms by which RSPO3 induces colon CSC-LCs by determine the effects of overexpression or knockdown of LGR4 and LGR5 in RSPO-activated Wnt/beta-catenin signaling and RSPO3-induced CSC-LCs in CRC cells. Successful completion of the project will: 1) demonstrate the important role of tumor stromal-derived RSPO3 in inducing and maintaining CSCs; 2) reveal a new function of RSPO3 in regulating colon CSCs; 3) delineate new mechanisms by which RSPO3 promotes colon tumor development and progression; and 4) provide new molecular targets for preventing and treating colon cancer.