The role of KLF5 in GI epithelial homeostasis and disease

描述(申请人提供)：KR？ppel-like factor5(KLF5；IKLF；BTEB2)是一种体外具有促增殖特性的锌指转录因子，表达于胃肠道上皮细胞的增殖细胞，包括食道的基底细胞。这些细胞与细胞外基质(ECM)的相互作用对它们的功能至关重要，我们已经证明KLF5通过表皮生长因子受体(EGFR)调节食道上皮细胞的增殖。在新的初步数据中，我们证明KLF5通过整合素连接的激酶(ILK)促进原代培养的食道角质形成细胞的迁移。鉴于这些发现，KLF5是在体内协调食道上皮细胞增殖和迁移的极佳候选者。然而，尽管有重要证据表明KLF5在未转化的上皮细胞中具有促增殖作用，但KLF5抑制增殖，促进失巢凋亡，并减少食管鳞癌细胞的侵袭。KLF5在人类乳腺癌和前列腺癌以及小鼠和人类的肠腺瘤中也被缺失或下调。在上皮癌细胞中，KLF5蛋白的降解速度比未转化的上皮细胞更快。这些发现表明，KLF5功能的消失可能在肿瘤的发生中起重要作用。事实上，KLF5已被认为在人类乳腺癌、前列腺癌、食道癌和结肠癌中具有肿瘤抑制功能。KLF5的这种二分作用并不是独一无二的，因为已经报道了其他因子的不同功能，如转化生长因子β、Notch和KLF4。在这里，我们将通过检验以下假设来研究KLF5在正常和转化的食道中的作用：(1)KLF5是食道运输放大细胞增殖、分化和迁移的关键调节因子；(2)KLF5的缺失是食道恶性转化的关键步骤。我们将通过以下相互关联的具体目标来检验这些假设。在具体目标1中，我们将通过：(A)评估KLF5过表达或抑制的未转化角质形成细胞中的整合素、整合素连接激酶(ILK)和表皮生长因子受体(EGFR)信号；(B)研究KLF5对EGFR和ILK的转录调控；(C)检测KLF5在食道中转基因表达的小鼠，以探讨KLF5在食道上皮细胞动态平衡中的作用。在特定的目标2中，我们将通过：(A)检测KLF5对转化的食道鳞状细胞中整合素、ILK和EGFR通路的调节；以及(B)评估KLF5在食道中转基因表达的小鼠的异型增生和肿瘤形成，这些小鼠已经用致癌物NMBA处理过或与ED-L2/Cyclin D1转基因小鼠杂交，建立了一个已建立的遗传癌症模型。总体而言，了解KLF5在食道上皮细胞中调控的通路将为理解食道疾病的分子事件提供一个框架，无论是良性疾病还是恶性疾病。公共卫生相关性：食道疾病，如胃食道反流病和食道癌，是美国和世界各地最常见的疾病之一，这些疾病是由于正常的上皮细胞动态平衡失调引起的。因此，了解控制正常食道增殖和分化的分子机制，包括特定的因素和复杂的信号阵列，对于理解食管良恶性疾病至关重要。本申请中提出的研究旨在通过对关键调控蛋白KLF5的研究来阐明这些因素和信号通路。

DESCRIPTION (provided by applicant): Kr¿ppel-like factor 5 (KLF5; IKLF; BTEB2), a zinc-finger transcription factor with pro-proliferative properties in vitro, is expressed in proliferating cells of gastrointestinal tract epithelia, including the basal cells of the esophagus. The interplay of these cells with the extracellular matrix (ECM) is critical to their function, and we have shown that Klf5 regulates proliferation in esophageal epithelial cells through the epidermal growth factor receptor (EGFR). In novel preliminary data, we demonstrate that Klf5 increases migration via the integrin- linked kinase (ILK) in primary esophageal keratinocytes in culture. Given these findings, Klf5 is an excellent candidate to orchestrate esophageal epithelial cell proliferation and migration in vivo. However, despite significant evidence of a pro-proliferative role for KLF5 in non-transformed epithelial cells, KLF5 inhibits proliferation, promotes anoikis, and decreases invasion of esophageal squamous cancer cells. KLF5 is also deleted or down-regulated in human breast and prostate cancers, as well as intestinal adenomas from mice and humans. In epithelial cancer cells, KLF5 protein undergoes more rapid degradation than in non- transformed epithelial cells. These findings suggest that extinction of KLF5 function may be important for carcinogenesis. In fact, KLF5 has been suggested to have tumor suppressive functions in human breast, prostate, esophageal, and colon cancers. This dichotomous role for KLF5 is not unique, as divergent functions have been reported for other factors, such as TGF?, Notch, and KLF4. Here, we will investigate the role of KLF5 in normal and transformed esophagus by testing the following hypotheses: (1) KLF5 is a critical regulator of proliferation, differentiation, and migration in transit amplifying cells of the esophagus; and (2) loss of KLF5 is a key step in malignant transformation in the esophagus. We will test these hypotheses through the following interrelated Specific Aims. In Specific Aim 1, we will investigate the role of KLF5 in esophageal epithelial homeostasis by: (a) evaluating integrin, integrin-linked kinase (ILK), and epidermal growth factor receptor (EGFR) signaling in non-transformed keratinocytes with overexpression or suppression of KLF5; (b) studying the transcriptional regulation of EGFR and ILK by KLF5; (c) examining mice with transgenic expression of Klf5 in the esophagus. In Specific Aim 2, we will determine the function of KLF5 during esophageal tumorigenesis by: (a) examining the regulation of the integrin, ILK, and EGFR pathways by KLF5 in transformed esophageal squamous cells; and (b) evaluating dysplasia and tumor formation in mice with transgenic expression of Klf5 in esophagus which have been treated with the carcinogen NMBA or crossed with ED-L2/cyclin D1 transgenic mice, an established genetic cancer model. Overall, an understanding of the pathways regulated by KLF5 in esophageal epithelia will provide a framework to understand the molecular events underlying esophageal diseases, both benign and malignant. PUBLIC HEALTH RELEVANCE: Diseases of the esophagus, such as gastroesophageal reflux disease and esophageal cancer, are among the most common ailments in the United States and throughout the world, and these diseases result from dysregulation of normal epithelial homeostasis. Thus, an appreciation of the molecular mechanisms, including the specific factors and complex signaling arrays, which govern normal esophageal proliferation and differentiation is critical to the understanding of esophageal diseases, both benign and malignant. The research proposed in this application aims to elucidate these factors and signaling pathways through studies of the key regulatory protein KLF5.