Colorectal Cancer Prevention Through Thyroid Hormone Targets

描述(申请人提供)：结直肠癌是美国人口中第三种最常见的癌症，是与癌症相关的死亡的主要原因，排名第二。被诊断为结直肠癌的平均年龄为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方面发挥作用。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和甲状腺激素敏感性降低。KR样因子9(KLF9)是一种甲状腺激素诱导的转录因子，可能是人类结肠直肠的肿瘤抑制因子。这项建议的目的是研究KLF9预防小鼠结直肠癌发生的机制，并评估衰老对这种关系的影响。我的初步数据表明，KLF9在体外是脂肪酸合成酶(FASN)的负调节因子，脂肪酸合成酶是结肠腺癌中普遍上调的基因，也是癌症的潜在治疗靶点。这项申请提出了两个目的来检验这一假设，即衰老通过循环中甲状腺激素的急剧减少和甲状腺激素敏感性的降低，导致KLF9在结肠粘膜的表达减少，从而导致FASN的表达增加。此外，我们认为KLF9作为结肠的肿瘤抑制因子发挥作用，并且KLF9等位基因的缺失将增加ApcMin/小鼠肠道肿瘤的数量和大小。目的1探讨衰老是否会通过降低甲状腺激素敏感性，降低KLF9在结肠的表达，从而增加FASN在结肠的表达。将收集6、12和18月龄小鼠的结肠，并在mRNA和蛋白质水平上评估KLF9和FASN的粘膜表达水平。目的2通过将Klf9基因敲除小鼠与ApcMin/小鼠杂交，并测量肿瘤的发生和发展，以确定Klf9在结肠中的表达是否影响肠道肿瘤的发生。带有两个、一个或没有Klf9等位基因的ApcMin/小鼠的小肠和大肠将被切除，肿瘤数量和大小将被记录下来。最大的腺瘤将接受免疫组织化学检查，以监测磷组蛋白H3、TUNEL、KLF9和Tr？1的表达。从腺瘤和未受累的粘膜中提取RNA和蛋白质，用Western印迹和qPCR方法检测KLF9、TR1、FASN和端粒酶逆转录酶(TERT)的表达水平。最后，对微腺瘤进行H&E染色和计数。这项研究的成功完成将提供对潜在的年龄相关靶点的洞察，这些靶点可能导致开发新的疗法来预防或治疗与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.