The role of KLF5 in GI epithelial homeostasis and disease

描述（由申请人提供）：Kr¿pel样因子5 （KLF5； IKLF; BTEB2）是一种在体外具有促增殖特性的锌指转录因子，在胃肠道上皮的增殖细胞中表达，包括食管的基底细胞。这些细胞与细胞外基质（ECM）的相互作用对其功能至关重要，我们已经证明Klf5通过表皮生长因子受体（EGFR）调节食管上皮细胞的增殖。在新的初步数据中，我们证明了Klf5通过整合素连接激酶（ILK）在培养的原发性食管角化细胞中增加迁移。鉴于这些发现，Klf5是协调食管上皮细胞在体内增殖和迁移的优秀候选者。然而，尽管有大量证据表明KLF5在非转化上皮细胞中具有促增殖作用，但KLF5抑制增殖，促进变性，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缺失或下调。在上皮癌细胞中，KLF5蛋白比在未转化的上皮细胞中降解得更快。这些发现表明KLF5功能的丧失可能对癌变很重要。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的这种双重作用并不是唯一的，因为其他因素(如TGF？Notch和KLF4。在这里，我们将通过验证以下假设来研究KLF5在正常和转化食管中的作用：(1)KLF5是食管转运扩增细胞增殖、分化和迁移的关键调节剂；(2) KLF5的缺失是食道恶性转化的关键步骤。我们将通过以下相关的具体目标来检验这些假设。在Specific Aim 1中，我们将通过以下方式研究KLF5在食管上皮稳态中的作用：(a)在KLF5过表达或抑制的非转化角化细胞中评估整合素、整合素连接激酶（ILK）和表皮生长因子受体（EGFR）信号传导；(b)研究KLF5对EGFR和ILK的转录调控；(c)检测转基因Klf5在食道中的表达。在Specific Aim 2中，我们将通过以下方式确定KLF5在食管肿瘤发生过程中的功能：(a)检测KLF5在转化的食管鳞状细胞中对整合素、ILK和EGFR通路的调节；(b)评价经致癌物NMBA处理或与ED-L2/cyclin D1转基因小鼠杂交建立的遗传癌模型后，Klf5转基因小鼠食管发育不良和肿瘤形成情况。总之，了解KLF5在食管上皮中调控的通路将为理解食管疾病（包括良性和恶性）的分子事件提供一个框架。公共卫生相关性：食道疾病，如胃食管反流病和食管癌，是美国和全世界最常见的疾病之一，这些疾病是由正常上皮内平衡失调引起的。因此，了解控制正常食管增殖和分化的分子机制，包括特定因素和复杂信号阵列，对于理解食管疾病（无论是良性还是恶性）至关重要。本申请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.