Klf4 in tumor initiation and maintenance of squamous cell carcinoma

描述（由申请人提供）：KLF4转录因子对于上皮细胞的细胞命运和分化很重要。 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，我们将细胞命运决定因子 Notch 1 确定为 KLF4 的转录靶标。在培养细胞、转基因小鼠和人类肿瘤中，KLF4 表达与 Notch1、Notch1 配体和 Notch1 效应子（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很重要。 KLF4 的诱变鉴定出显性失活 (DN) 等位基因，可阻止 KLF4 体外转化。 KLF4-DN 鉴定了 KLF4 在特定人类细胞类型体外生长以及特定转化癌基因（例如 ErbB2 和 c-MYC）转化中的作用。 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施用致癌物质，开发了一种新型高渗透性鳞状细胞癌模型。肿瘤在组织学上与人类鳞状细胞癌相似，并表达鳞状细胞癌标志物，如 p53、角蛋白 14 和 Notch1。该模型中形成的 SCC 包括 KLF4 阳性肿瘤和一些 KLF4 阴性肿瘤。 为了了解正常调节 KLF4 的机制，我们将研究翻译后修饰在突变 KLF4 蛋白 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 在肿瘤发生和维持中的作用，使用条件性 Cre 介导的 Klf4/LoxP 等位基因删除，并通过转基因小鼠中四环素诱导的 siRNA 或 KLF4-DN 表达（目标 2）。使用类似的功能丧失和获得策略，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，将通过鉴定 KLF4 cDNA 中的 RNA 不稳定元件来分析似乎负责小鼠和人类 SCC 中 Klf4 转录本上调的翻译后对照。 这些研究可能支持 KLF4 和 Notch1 在常见人类癌症（如鳞状细胞癌和乳腺癌）发病机制中的作用。因此，研究的完成将确定该途径作为预防和治疗的目标，并提供新的动物模型来评估此类疗法。

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.