R-spondin3调控结肠癌干细胞及促进肿瘤生长的作用及机制

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.

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。

结肠癌是最常见的恶性肿瘤，发生机制仍不清楚。先前研究已证明，RSPO3在结肠癌中存在突变，RSPO3突变体和BMFs来源的RSPO3能够调控结肠癌的干细胞生成，与结肠癌发生相关。但野生型RSPO3在结肠癌发生中确切功能尚不清楚。本研究首先构建了野生型RSPO3过表达的3个结肠癌细胞株，结果意外发现RSPO3过表达抑制了结肠癌裸鼠移植瘤生长。对29例结肠癌患者的癌组织和癌旁组织RSPO3的表达进行检测分析，发现癌组织中的RSPO3表达显著低于癌旁组织。ELISA检测结果显示结肠癌患者血清中的RSPO3水平低于非肿瘤患者和正常人。免疫组化检测了一个300例结肠癌组织芯片中的RSPO3蛋白表达，也证明RSPO3在结肠癌组织中表达显著低于癌旁正常组织。GEO数据库和TCGA数据库的4个人结肠癌患者样本库分析显示，RSPO3在癌组织中表达显著低于正常组织。而且，CCLE数据库中所有结肠癌细胞株、仁济医院结肠癌临床样本和TCGA数据库样本分析显示，结肠癌细胞和组织中RSPO3的启动子存在高甲基化；应用5-AZA促进结肠癌细胞，能够恢复RSPO3的表达。这些研究结果，提示RSPO3可能具有肿瘤抑制基因的功能。为进一步证实RSPO3是一个肿瘤抑制基因，我们构建了RSPO3肠道上皮特异性敲除小鼠（RSPO3-/-;Villin-Cre小鼠）。对RSPO3-/-;Villin-Cre小鼠及对照小鼠给予灌注AOM/DSS肠处理，诱导炎性相关的结肠癌小鼠模型。结果发现，与野生型小鼠（RSPO3+/+;Villin-Cre ）相比， RSPO3在结肠上完全敲除的RSPO3-/-;Villin-Cre和RSPO3部分敲除的RSPO3+/-;Villin-Cre，其结直肠组织中长出了数量更多、体积更大的肿瘤；特别是在RSPO3-/-;Villin-Cre小鼠发生浸润性癌中。这些研究结果，首次证明野生型RSPO3是一个新的肿瘤抑制基因,从而为结肠癌治疗提供了新靶点。