Klf4 in tumor initiation and maintenance of squamous cell carcinoma

描述(申请人提供)：KLF4转录因子在细胞命运和上皮细胞分化中起重要作用。KLF4可迅速引发小鼠皮肤鳞状细胞癌(SCC)，并在人类皮肤肿瘤进展的早期阶段被解除调控。使用条件KLF4-ER融合蛋白，我们确定细胞命运决定因素Notch 1是KLF4的转录靶标。在培养细胞、转基因小鼠和人类肿瘤中，KLF4的表达与Notch1、Notch1配体和Notch1效应因子(如Cyclin D1)的表达有关。RK3E上皮细胞的转化需要Notch1。这些研究确定了KLF4-Notch1通路，该通路似乎在正常上皮细胞和肿瘤细胞的细胞命运指定中具有重要意义。KLF4的诱变鉴定出显性负性(DN)等位基因，可阻断KLF4的体外转化。KLF4-Dns鉴定KLF4在特定类型的人细胞体外生长中的作用，以及通过ErbB2和c-myc等特定癌基因的转化。 为了研究KLF4在皮肤鳞状细胞癌中的作用，我们通过给基因工程小鼠注射致癌物，建立了一种新的高度渗透的皮肤鳞状细胞癌模型。肿瘤在组织学上与人类鳞癌相似，并且表达鳞癌标志物，如P53、角蛋白14和Notch1。在该模型中形成的SCC包括KLF4阳性的肿瘤和一些KLF4阴性的肿瘤。 为了了解正常调控KLF4的机制，我们将研究翻译后修饰在突变KLF4蛋白的DN活性中的作用(目标1)。将在新的SCC小鼠模型中研究内源性KLF4在肿瘤启动和维持中的作用，方法是有条件地、通过CRE介导的KLF4/loxP等位基因的删除，以及通过四环素诱导转基因小鼠的siRNA或KLF4-dN的表达(目标2)。使用类似的功能丧失和功能获得策略，将确定KLF4在乳腺上皮发育和ErbB2转化中的作用(目标3)。在目标4中，将通过识别KLF4 cDNA中的RNA不稳定元件来分析翻译后控制，该控制似乎对KLF4转录产物在小鼠和人类SCC中的上调负责。 这些研究可能为KLF4和Notch1在鳞状细胞癌和乳腺癌等常见人类癌症的发病机制中的作用提供支持。因此，研究的完成将把这一途径确定为预防和治疗的目标，并提供新的动物模型来评估这种疗法。

DESCRIPTION (provided by applicant): The KLF4 transcription factor is important in cell fate and differentiation of epithelial cells. KLF4 rapidly initiates cutaneous squamous cell carcinoma (SCC) in mice, and is deregulated at an early step in human skin tumor progression. Using a conditional KLF4-ER fusion protein, we identified the cell fate determinant Notch 1 as a transcriptional target of KLF4. In cultured cells, in transgenic mice and in human tumors, KLF4 expression is associated with expression of Notch1, Notch1 ligands, and Notch1 effectors such as Cyclin D1. Transformation of RK3E epithelial cells requires Notch1. These studies identify a KLF4-Notch1 pathway that appears important in cell fate specification of normal epithelial cells and tumor cells. Mutagenesis of KLF4 identified dominant negative (DN) alleles that block transformation in vitro by KLF4. KLF4-DNs identify a role of KLF4 in growth of specific human cell types in vitro, and in transformation by specific transforming oncogenes such as ErbB2 and c-MYC. To study the role of Klf4 in cutaneous SCC we developed a novel highly penetrant model of SCC by administering carcinogens to genetically-engineered mice. Tumors were histologically similar to human SCC, and expressed SCC markers such as p53, keratin 14, and Notch1. SCCs that developed in this model included KLF4-positive tumors and some tumors that were KLF4-negative. To understand the mechanisms that normally regulate KLF4, we will study the role of posttranslational modifications in the DN activity of mutant KLF4 proteins (Aim 1). The role of endogenous Klf4 in tumor initiation and maintenance will be studied in the new SCC mouse model using conditional, Cre-mediated deletion of Klf4/LoxP alleles, and by tetracycline-inducible expression of siRNA or KLF4-DN in transgenic mice (Aim 2). Using similar loss- and gain-of-function strategies, the role of Klf4 in mammary epithelial development and in transformation by ErbB2 will be determined (Aim 3). In Aim 4, a posttranslational control that appears responsible for upregulation of Klf4 transcripts in mouse and human SCC will be analyzed by identifying RNA destabilizing elements in the KLF4 cDNA. These studies may provide support of a role for KLF4 and Notch1 in pathogenesis of common human cancers such as SCC and breast cancer. Thus, completion of the studies would identify this pathway as a target for prevention and treatment, and provide new animal models in which to assess such therapies.