Defining the Molecular Mechanisms of KLF17 Regulation and Function in EMT

描述（由申请人提供）：我们最近在小鼠中使用全基因组RNAi筛选发现Kr|苹果样因子7 （KLF17）是上皮-间质转化（EMT）的负调节因子。人类KLF17基因位于染色体1p34，其中等位基因杂合性缺失（LOH）与乳腺癌预后不良相关。本课题的总体目标是确定转录因子KLF17在肿瘤维持和EMT中的分子机制和遗传调控，确定KLF17调控在乳腺癌中的治疗价值和预后价值。我们将利用分子分析、细胞分析和动物模型来阐明KLF17的功能和调控机制。我们将从以下目标出发：目标1。确定KLF17调控肿瘤生长和EMT的分子机制及功能。我们将在体外和小鼠模型中分别确定由KLF17调控的Id2和ARHGAP29对肿瘤细胞生长和EMT的影响，以及这两个基因对肿瘤细胞生长和EMT的影响。我们将使用体外实验和体内原位移植小鼠模型来测试它们在肿瘤侵袭和转移中的功能。将进行上位分析，以确定这些基因是否是KLF17在肿瘤生长和EMT中的主要介质。我们还将研究已知EMT调节因子SNAI1 （Snail）转录和microRNA miR-129转录后调控KLF17的分子机制。我们将确定KLF17是否是SNAI1的直接靶点，以及miR-129是否直接靶向KLF17的3'UTR。SNAI1和miR-129在EMT和肿瘤侵袭中的功能将通过体外实验和肿瘤异种移植小鼠模型来确定。将进行上位分析，以确定KLF17是否是SNAI1和miR-129在EMT和肿瘤侵袭中的主要介质。目标2。表征KLF17在肿瘤维持中的功能。我们将使用异种移植模型和在诱导载体中表达KLF17短发夹RNA （shRNA）和Ras的细胞来测试单独调节KLF17表达或联合抑制Ras是否能引起乳腺癌的肿瘤消退。我们将利用异种移植动物模型研究KLF17敲低是否能拮抗ras诱导的肿瘤衰老，并确定KLF17在ras诱导的衰老中干扰的途径。这些实验将确定KLF17单独或与Ras联合是否可以作为治疗乳腺癌的潜在治疗靶点。目标3。确定临床乳腺癌样本中KLF17、Id1、SNAI1、miR-129的预后价值。我们将测定KLF17和Id1以及Snail和miR-129在已知临床结局的临床原发性乳腺癌样本中的表达，以评估它们是否可以用作乳腺癌复发的预后标志物。

DESCRIPTION (provided by applicant): We recently identified Kr|pple-like factor 7 (KLF17) as a negative regulator of epithelial-mesenchymal transition (EMT) using a genome-wide RNAi screen in mice. The human KLF17 gene is located at chromosome 1p34 where allelic loss of heterozygosity (LOH) correlates with poor prognosis in breast cancer. The overall goal of this proposal is to determine the molecular mechanisms and genetic regulation of transcription factor KLF17 in tumor maintenance and EMT, and determine the therapeutic value of KLF17 modulation and prognostic value of KLF17 in breast cancer. We will use molecular profiling, cellular assays and animal models to elucidate the mechanisms of KLF17 functions and regulation. We will proceed from the following aims: Aim 1. Determine the molecular mechanisms of KLF17 regulation and function in tumor growth and EMT. We will determine the effect of Id2 and ARHGAP29, regulated by KLF17, individually and the combined effect of these two genes on tumor cell growth and EMT both in vitro and in mouse models. We will use in vitro assays and in vivo orthotopic transplantation mouse models to test their functions in tumor invasion and metastasis. Epistasis analysis will be performed to determine whether these genes are major mediators of KLF17 in tumor growth and EMT. We will also investigate the molecular mechanisms of KLF17 regulation by SNAI1 (Snail), a known EMT regulator, transcriptionally and by microRNA miR-129 post-transcriptionally. We will determine whether KLF17 is a direct target of SNAI1 and whether miR-129 directly targets KLF17 at its 3'UTR. The functions of SNAI1 and miR-129 in EMT and tumor invasion will be determined by in vitro assays and tumor xenograft mouse models. Epistasis analysis will be performed to determine whether KLF17 is a major mediator of SNAI1 and miR-129 in EMT and tumor invasion. Aim 2. Characterize the function of KLF17 in tumor maintenance. We will use xenograft models and cells expressing KLF17 short hairpin RNA (shRNA) and Ras in an inducible vector to test whether modulation of KLF17 expression alone or in combination with Ras inhibition can cause tumor regression in breast cancer. We will investigate whether KLF17 knockdown antagonizes Ras-induced senescence in tumors using xenograft animal models and determine the pathways that KLF17 interferes with in Ras-induced senescence. These experiments will determine whether KLF17 alone or in combination with Ras can serve as a potential therapeutic target in the treatment of breast cancer. Aim 3. Determine the prognostic value of KLF17, Id1, SNAI1 and miR-129 in clinical breast cancer samples. We will determine the expression of KLF17 and Id1, along with Snail and miR-129 in clinical primary breast cancer samples with known clinical outcomes to assess whether they can be used as prognostic markers for relapse in breast cancer. PUBLIC HEALTH RELEVANCE: This proposal studies transcription factor KLF17 in tumor maintenance, epithelial- mesenchymal transition (EMT) and metastasis. Tumor spread from a primary site to distant sites is responsible for most cancer death. EMT plays a critical role in tumor spread. Currently there are few prognostic markers and therapeutics for metastatic disease. KLF17 was recently discovered in my laboratory to be involved in EMT by a genome-wide RNAi screen. Elucidation of the molecular mechanisms of KLF17 in EMT and metastasis will provide potential prognostic marker and therapeutics for the treatment of this deadly disease.