Rspo2和Rspo3基因融合突变促进结直肠癌发生、转移及预测wnt抑制剂疗效的机制研究

Latest cancer genome study finds that 4-10% of colorectal cancer patients have fusion mutation of Rspo2 or Rspo3, which is known to activate Wnt signaling pathway and promote proliferation of CRC cells. We performed nanostring assay to screen Rspo2 and Rspo3 fusion in 24 CRC PDXs, of which 16 were known having such fusions. The reliability of the assay were verified by the results. Then 7 out of 28 samples of CRCLM were found Rspo2/Rspo3 fusions by nanostring assay, which was far more frequent than it was reported in common CRC population. Furthermore, we found Wnt inhibitor CGX1231 could strongly inhibit the tumor growth in two PDXs with Rspo2/Rspo3 fusion mutations. Therefore, We raise the hypothesis that Rspo2/Rspo3 fusion mutations could drive the tumorigenesis and liver metastasis of CRC, and could be a potential predict biomarker for Wnt inhibitor. In this study, we intend to test Rspo2 and Rspo3 fusion mutations in 200 colorectal cancer samples by nanostring assay and find its prevalence in Chinese population as well as its relation with the colorectal cancer liver metastasis.Secondly we will investigate whether RSPO2 and RSPO3 fusion mutations can cause the malignant transformation of NIH/3T3 cells. The effect of RSPO2 and RSPO3 fusion mutation on the tumorigenesis and metastasis of colorectal cancer and its mechanism will be investigated, as well as its relationship with drug resistance. Finally, the possibility of RSPO2 and RSPO3 fusion mutations as predict biomarkers for Wnt inhibitor and its mechanism will be further verified in more PDXs.

最新测序研究发现，4-10%结直肠癌具有Rspo2或Rspo3融合突变。机制研究认为该融合激活Wnt通路，并驱动依赖于该通路的肿瘤增殖。申请人采用Nanostring法在24例已知基因型人源性肿瘤移植瘤（PDX）中证实了该方法的可靠性；并在结直肠癌肝转移临床标本发现25%（7/28）出现融合，显著高于文献报道水平；药效研究发现Wnt抑制剂显著抑制具有该融合的PDX。因此，该融合突变可能与结直肠癌发生、转移相关，并可能是Wnt抑制剂的疗效预测标志。本研究拟采用Nanostring方法检测200例结直肠癌（肝转移/无肝转移）的RSPO2/3融合突变，明确中国人群发生该融合的频率、新的融合方式及与结肠癌肝转移的相关性。明确该融合突变能否导致NIH/3T3细胞发生恶性转化及其机制；研究该融合突变对结直肠癌发生、转移和耐药的影响及其分子机制；并扩大PDX样本明确其作为Wnt抑制剂疗效预测标志的可能。

多数的结直肠癌患者的患病原因与APC基因以及CTNNB1基因发生突变导致的WNT/β-catenin异常活化有关，但是也有部分患者并不存在以上两种突变，存在RSPO2或者RSPO3的融合突变，该融合突变也与结肠癌的发生密切相关，可能能成为一个新的治疗靶点。.本研究主要围绕寻找RSPO3融合突变的新的治疗药物开展研究，首先我们通过临床标本的检测确定了RSPO3融合突变在中国人群中的发生比例以及融合突变类型。结果显示RSPO3的融合突变在中国结肠癌患者中的发生比例为6%，并且融合突变的类型有两种，第一种是PTPRKexon1-RSPO3exon2，第二种是PTPRKexon7-RSPO3exon2，这两种类型的融合突变与国外发现的RSPO3的融合突变一致。为了寻找RSPO3的新的治疗药物，本研究建立了可以用于药物筛选的新的PDO平台，可以用于寻找新的RSPO3融合突变的治疗药物以及相关的细胞生物学实验;并且本研究成功的构建了带有RSPO3融合突变的PDX模型，通过该模型我们发现RSPO3融合突变患者的肿瘤组织对WNT通路抑制剂敏感，提示WNT通路的抑制剂可以用来治疗该类患者。本研究也成功构建了这两种融合突变的过表达质粒进行后续的分子生物学功能的研究，通过研究我们发现PTPRKexon1-RSPO3 exon2融合突变能够明显的促进结肠癌细胞系的生长，后续我们将对其促进结肠癌细胞生长的分子机制展开更为深入的研究。于此同时，我们也针对WNT通路上的受体FZD5开发了新的可以用于抑制WNT通路的药物，获得了可以抑制该蛋白活性的单克隆抗体，后续将利用带有RSPO3融合突变的稳定过表达细胞系以及我们构建的PDO平台以及PDX模型来对其治疗效果进行评估，为该类患者的治疗提供新的参考。