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，肠道炎症会增加结直肠癌的风险，特别是当患者缺乏硒时——硒蛋白的关键成分，参与抗氧化防御。谷胱甘肽过氧化物酶-2 （GPx2）是一种胃肠道硒蛋白，是Wnt靶基因之一，它是在大肠腺瘤性息肉病（APC）等基因缺失时诱导的，APC是结直肠癌中常见的突变基因。此外，GPx2被认为介导肠上皮的自我更新，其在肠道微生物定植反应中的上调表明其在上皮保护中起作用。最近的研究表明，GPx2在肿瘤环境中高度表达，并被认为根据癌症分期发挥抗炎或抗凋亡作用。然而，GPx2在肠隐窝生长和apc驱动的肿瘤发生中的作用尚不清楚。本研究的核心假设是，GPx2可能在APC驱动的结肠肿瘤发生过程中促进上皮癌性转化的进展，并将通过以下目的进行验证：1)GPx2的功能将在小鼠模型中进行表征，其中可追溯的Lrig1+干细胞中诱导APC功能的丧失主要导致结肠腺瘤。GPx2在肿瘤发生中的作用将通过三种基因型（GPx2 +/+; Lrig1cre/+; ApcΔ14/+, GPx2 +/-; Lrig1cre/+; ApcΔ14/+, GPx2 -/-; Lrig1cre/+; ApcΔ14/+）小鼠进行肿瘤发生来评估。此外，小鼠将在肿瘤诱导前接受不同的饮食硒浓度。除了使用流式细胞术对固有层免疫细胞进行免疫表型分析外，还将通过连续结肠镜检查来跟踪肿瘤的发展。已知影响细胞存活的氧化还原敏感信号通路将通过RNA-seq和免疫印迹在肿瘤样本中进行检测。除了小鼠类肿瘤（Gpx2-/-; Lrigcre/+; ApcΔ14/+）外，Gpx2在人类肿瘤中的表达也将被修饰，以确定Gpx2是否在上皮癌性转化的进展中发挥作用。此外，16S rRNA测序将用于表征上述基因型的肠道微生物组，以确定GPx2的缺失是否与癌症进展和微生物组中的氧化还原依赖性转移相关。2) GPx2在上皮隐窝中的功能将通过对GPx2 -/-肠样中GPx2突变体构建体进行详细的结构-功能研究来确定。

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.