Klf4 in tumor initiation and maintenance of squamous cell carcinoma

描述（由申请人提供）：KLF4转录因子在上皮细胞的细胞命运和分化中很重要。KLF4在小鼠中迅速引发皮肤鳞状细胞癌（SCC），并在人类皮肤肿瘤进展的早期阶段失调。使用条件KLF4-ER融合蛋白，我们确定了细胞命运决定因子Notch 1作为KLF4的转录靶点。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效应物（如Cyclin D1）的表达相关。RK3E上皮细胞的转化需要Notch1。这些研究确定了KLF4-Notch1通路，该通路在正常上皮细胞和肿瘤细胞的细胞命运规范中显得重要。KLF4的诱变鉴定了在体外阻断KLF4转化的显性阴性（DN）等位基因。KLF4-DNs鉴定了KLF4在体外特定人类细胞类型生长中的作用，以及在特定转化癌基因（如ErbB2和c-MYC）转化中的作用。 为了研究Klf4在皮肤SCC中的作用，我们通过向基因工程小鼠施用致癌物开发了一种新型的高度渗透的SCC模型。肿瘤在组织学上与人类SCC相似，并表达SCC标志物，如p53、角蛋白14和Notch1。在该模型中形成的SCC包括KLF4阳性肿瘤和一些KLF4阴性肿瘤。 为了了解通常调节KLF4的机制，我们将研究突变KLF4蛋白的DN活性中翻译后修饰的作用（目的1）。将在新的SCC小鼠模型中使用条件性Cre介导的Klf4/LoxP等位基因缺失，并通过转基因小鼠中四环素诱导的siRNA或KLF4-DN表达，研究内源性Klf4在肿瘤发生和维持中的作用（目的2）。使用类似的功能丧失和获得策略，Klf4在乳腺上皮发育和ErbB2转化中的作用将被确定（目的3）。在目标4中，将通过鉴定KLF4 cDNA中的RNA去稳定元件来分析似乎负责小鼠和人SCC中Klf4转录物上调的翻译后控制。 这些研究可能为KLF4和Notch1在SCC和乳腺癌等常见人类癌症发病机制中的作用提供支持。因此，研究的完成将确定这一途径作为预防和治疗的靶点，并提供新的动物模型来评估这些疗法。

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.