Colorectal Cancer Prevention Through Thyroid Hormone Targets

描述（由申请人提供）：结直肠癌是第三大最常见的癌症类型，在美国人群中排名第二，是癌症相关死亡的主要原因。结直肠癌诊断的中位年龄为71岁，这表明它主要是一种老年疾病。研究表明，甲状腺激素可能在预防结肠癌中发挥作用。其他研究表明，许多老年患者表现出亚临床甲状腺功能减退症和甲状腺激素敏感性下降。KLF 9是一种甲状腺激素诱导的转录因子，也是一种公认的人类结肠直肠肿瘤抑制因子。该提案的目标是研究KLF 9预防小鼠结直肠肿瘤发生的拟议机制，并评估衰老对这种关系的影响。我的初步数据表明，KLF 9是体外脂肪酸合成酶（Fatty acid synthase，FATCH）的负调节因子，FATCH是结肠腺癌中常见的上调基因，也是癌症的潜在治疗靶点。本申请提出了两个目的来检验以下假设：衰老通过循环甲状腺激素的急性减少和甲状腺激素敏感性的降低导致KLF 9的结肠粘膜表达的减少，这导致FLF 9的表达增加。此外，我们提出KLF 9作为结肠的肿瘤抑制因子，并且Klf 9等位基因的缺失将增加ApcMin/+小鼠中肠肿瘤的数量和大小。目的1试图确定衰老是否会通过降低甲状腺激素敏感性降低Klf 9的结肠表达，从而增加老年小鼠中Fasn的结肠表达。收集6、12和18月龄小鼠的结肠，并在mRNA和蛋白质水平上评价Klf 9和Fasn的粘膜表达水平。目的2将通过Klf 9基因敲除小鼠与ApcMin/+小鼠杂交并测量肿瘤发生和发展来确定Klf 9的结肠表达是否影响肠道肿瘤发生。将从具有两个、一个或无Klf 9等位基因的ApcMin/+小鼠中取出小肠和大肠，并记录肿瘤数量和大小。最大的腺瘤将进行免疫组化以监测磷酸化组蛋白H3、TUNEL、KLF 9和Tr <$1表达。将从腺瘤和未受累粘膜中提取RNA和蛋白质，并通过Western印迹和qPCR评价KLF 9、TR 1、FXR和端粒酶逆转录酶（TERT）的表达水平。最后，将通过H&E染色和计数来量化微腺瘤。该研究的成功完成将为潜在的年龄依赖性靶点提供深入了解，这些靶点可能导致开发新的治疗方法来预防或治疗年龄相关的结直肠癌。

DESCRIPTION (provided by applicant): Colorectal cancer is the third most prevalent type of cancer and ranks second as the major cause of cancer-related deaths in the US population. The median age of diagnosis for colorectal cancer is 71, demonstrating that it is primarily a disease of old age. Studies have suggested that thyroid hormone may have a role in colon cancer prevention. Other studies have shown that many elderly patients exhibit sub-clinical hypothyroidism and decreased thyroid hormone sensitivity. Kr¿ppel-like factor 9 (KLF9) is a thyroid hormone- induced transcription factor, and a putative tumor suppressor of the human colo-rectum. The goal of this proposal is to investigate a proposed mechanism by which KLF9 prevents colorectal tumorigenesis in mice, and to evaluate the effects of aging on this relationship. My preliminary data suggest that KLF9 is a negative regulator of fatty acid synthase (FASN) in vitro, which is a gene commonly up-regulated in colon adenocarcinomas, and a potential therapeutic target for cancer. This application proposes two aims to test the hypothesis that aging, via acute reduction in circulating thyroid hormone and decreased thyroid hormone sensitivity, results in a reduction in colon mucosal expression of KLF9, which leads to increased expression of FASN. Further, we propose that KLF9 functions as a tumor suppressor of the colon, and that loss of Klf9 alleles will increase number and size of intestinal tumors in ApcMin/+ mice. Aim 1 seeks to determine if aging will, via decreased thyroid hormone sensitivity, decrease colonic expression of Klf9 and thereby increase colonic expression of Fasn in aged mice. Colons of mice at ages 6, 12, and 18 months will be collected and mucosal expression levels of Klf9 and Fasn will be evaluated at the mRNA and protein levels. Aim 2 will determine whether colonic expression of Klf9 affects intestinal tumorigenesis by crossing Klf9 knockout mice with ApcMin/+ mice and measuring tumor incidence and development. Small and large intestines will be removed from ApcMin/+ mice with two, one, or no Klf9 alleles and tumor number and sizes will be recorded. The largest adenomas will be subjected to immunohistochemistry to monitor phosphohistone H3, TUNEL, KLF9, and Tr¿1 expression. RNA and protein will be extracted from adenomas and uninvolved mucosa and expression levels of KLF9, TR¿1, FASN, and telomerase reverse transcriptase (TERT) will be evaluated by Western blot and qPCR. Finally, micro-adenomas will be quantified by H&E staining and counting. The successful completion of the study will provide insight into potential age-dependent targets that may lead to the development of new therapies to prevent or treat age-related colorectal cancer.