Cell-autonomous redox-sensitive role of selenoprotein glutathione peroxidase-2 (Gpx2) in APC-driven colorectal carcinogenesis and stem cell function

结直肠癌（CRC）是西方国家癌症相关死亡的主要原因之一。研究表明，肠道炎症会增加CRC的风险，特别是当患者缺乏硒时-硒是参与抗氧化防御的硒蛋白的关键成分。谷胱甘肽过氧化物酶-2（GPx 2）是一种胃肠道硒蛋白，是在大肠腺瘤性息肉病（APC）等基因缺失时诱导的几种Wnt靶基因之一，APC是CRC中经常突变的基因。此外，GPx 2被认为介导肠上皮的自我更新，并且其响应于肠道微生物定植的上调表明在上皮保护中的作用。最近的研究表明，GPx 2在肿瘤环境中高度表达，并被认为根据癌症阶段发挥抗炎或抗凋亡作用。然而，GPx 2在肠隐窝生长和APC驱动的肿瘤发生中的作用仍然难以捉摸。该提议的中心假设是，GPx 2可能有助于APC驱动的结肠肿瘤发生中上皮致癌转化的进展，并将通过以下目的进行测试：1）GPx 2的功能将在小鼠模型中表征，其中可追踪的Lrig 1+干细胞内APC功能的可诱导丧失主要导致结肠腺瘤。将通过使三种基因型（Gpx 2 +/+; Lrig 1cre/+; ApcΔ14/+，Gpx 2 +/-; Lrig 1cre/+; Apc Δ14/+，Gpx 2-/-; Lrigcre/+; ApcΔ14/+）小鼠发生肿瘤来评价GPx 2在肿瘤发生过程中的作用。此外，在肿瘤诱导之前，小鼠将经受不同的膳食硒浓度。除了使用流式细胞术对固有层中的免疫细胞进行免疫表型分析外，还将通过连续结肠镜检查跟踪肿瘤发展。将通过RNA-seq和免疫印迹在肿瘤样品中检查已知影响细胞存活的氧化还原敏感性信号传导途径。除小鼠类肿瘤（Gpx 2-/-; Lrigcre/+; ApcΔ14/+）外，还将在人类肿瘤中修饰GPx 2表达，以确定GPx 2是否在上皮致癌转化的进展中发挥作用。此外，16 S rRNA测序将用于表征上述基因型的肠道微生物组，以确定GPx 2的缺失是否与癌症进展和微生物组的氧化还原依赖性转变相关。2)GPx 2在上皮隐窝中的功能将通过在Gpx 2-/-类肠中进行Gpx 2突变体构建体的详细结构-功能研究来定义。

Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths in western countries. Studies have shown that gut inflammation increases the risk of CRC, especially when patients are deficient in Selenium - a key component of selenoproteins involved in antioxidant defenses. Glutathione peroxidase-2 (GPx2), a gastrointestinal selenoprotein, is one of several Wnt target genes that are induced upon the loss of genes like the adenomatous polyposis coli (APC) - a frequently mutated gene in CRC. Additionally, GPx2 is thought to mediate self-renewal of the intestinal epithelium, and its upregulation in response to microbial colonization of the gut suggests a role in epithelium protection. Recent studies have shown that GPx2 is highly expressed in the tumor environment and is thought to either play an anti-inflammatory or anti-apoptotic role, depending on the cancer stage. However, the role of GPx2 in intestinal crypt growth and APC-driven tumorigenesis remains elusive. The central hypothesis of this proposal is that, GPx2 may contribute to the progression of epithelial oncogenic transformation in APC-driven colon tumorigenesis, and will be tested by the following aims: 1) The function of GPx2 will be characterized in a mouse model wherein the inducible loss of APC function within traceable Lrig1+ stem cells, predominantly leads to colonic adenomas. The role of GPx2 during tumorigenesis will be evaluated by subjecting mice of three genotypes (Gpx2+/+; Lrig1cre/+; ApcΔ14/+, Gpx2+/-; Lrig1cre/+; ApcΔ14/+, Gpx2-/-; Lrigcre/+; ApcΔ14/+) to tumorigenesis. Furthermore, the mice will be subjected to different dietary Selenium concentrations before tumor induction. Tumor development will be tracked by serial colonoscopy, in addition to immunophenotyping of immune cells in the lamina propria using flow cytometry. Redox-sensitive signaling pathways known to influence cell survival will be examined in tumor samples via RNA-seq and immunoblotting. In addition to mouse tumoroids (Gpx2-/-; Lrigcre/+; ApcΔ14/+), GPx2 expression will be modified in human tumoroids, to determine if GPx2 plays a role in progression of epithelial oncogenic transformation. Additionally, 16S rRNA sequencing will be used to characterize the gut microbiomes of the aforementioned genotypes to determine if the absence of GPx2 correlates with cancer progression and a redox-dependent shift in the microbiome. 2) The function of GPx2 in epithelial crypts will be defined by performing detailed structure-function studies of Gpx2 mutants constructs in Gpx2-/- enteroids.