KLF4 regulation of epithelial/mesenchymal transition in breast cancer

描述（由申请人提供）：播散性疾病是乳腺癌死亡的主要原因，确定控制转移形成和生长的分子机制对于开发挽救生命的治疗方法至关重要。转移是一个多步骤的过程，通常涉及肿瘤细胞在上皮和间质状态之间的转变。这种可塑性已经在体外乳腺癌细胞模型和肿瘤中被观察到。此外，表达间充质基因特征的乳腺癌是高度转移的。因此，识别控制上皮/间充质特性和上皮到间充质转化（EMT）的机制应该为转移性疾病的过程提供重要的见解。我们的研究表明KLF4 （Kr¿pel-like Factor 4）是人乳腺上皮细胞上皮/间充质状态的主要调节因子。在未转化的细胞中，KLF4的持续表达是维持上皮表型所必需的，而在乳腺癌细胞中，KLF4的过表达诱导了从间质状态到上皮状态的转换。KLF4刺激E-cadherin基因的转录，E-cadherin基因是一种上皮决定因素，并抑制Snail基因的表达，Snail基因是间充质表型的诱导剂。这些数据表明KLF4是调节乳腺上皮上皮/间充质状态的枢纽。转化生长因子-¿（TGF）诱导的EMT伴随着KLF4表达的抑制，这支持了这种可能性。KLF4加强上皮表型的能力与KLF8进行了对比，KLF8驱动间充质表型，其表达受TGF刺激。因此，Kr - pel样因子决定乳腺上皮细胞的上皮或间充质状态，并可能是转移能力的基础。本研究的重点是描述KLF4在决定乳腺细胞上皮状态中的作用机制。由于KLF4在TGF诱导的EMT中受到抑制，我们将首先确定KLF4是否抑制TGF诱导的EMT，以及这是否涉及KLF4下调Snail。这将揭示KLF4的精确水平是否对确定TGF诱导EMT的程度以及可能的转移至关重要。其次，我们将评估KLF8和KLF4在同一细胞内是否具有拮抗作用。如果是这样，这将表明这两种蛋白的相对浓度决定了乳腺上皮细胞的上皮/间充质状态，这种可能性将通过检测人类肿瘤中KLF4和KLF8的相对表达及其与E-cadherin表达的相关性来进一步评估。在第三个目标中，我们将通过实验转移模型和转基因乳腺癌模型来评估KLF4过表达对转移的影响。最后，我们预计KLF4通过调节一系列基因的转录来维持上皮特征。我们将使用基于基因组的方法来鉴定这些基因并评估它们对上皮表型的贡献。总之，这些研究将揭示由KLF4控制的调节上皮表型的分子电路，并对确定转移潜力具有重要意义。

DESCRIPTION (provided by applicant): Disseminated disease is the primary cause of death in breast cancer and identifying molecular mechanism(s) that control formation and growth of metastases is critical for developing life-saving treatments. Metastasis is a multi-step process that often involves transitions of tumor cells between epithelial and mesenchymal states. This plasticity has been observed in breast cancer cell models in vitro and in tumors. In addition, breast cancers that express a mesenchymal gene signature are highly metastatic. Thus, discerning mechanism(s) that control epithelial/mesenchymal properties and epithelial to mesenchymal transition (EMT) should provide significant insights into the processes that contribute to metastatic disease. Our studies revealed that KLF4 (Kr¿ppel-like Factor 4) is a dominant regulator of the epithelial/mesenchymal status in human breast epithelial cells. Sustained expression of KLF4 is necessary to maintain an epithelial phenotype in non-transformed cells, while KLF4 overexpression induces a switch from a mesenchymal to epithelial state in breast cancer cells. KLF4 stimulates transcription of the E-cadherin gene, an epithelial determinant, and inhibits expression of Snail, an inducer of the mesenchymal phenotype. These data indicate that KLF4 is a hub for regulating the epithelial/mesenchymal states of breast epithelia. Supporting this possibility, EMT induced by Transforming Growth Factor-¿ (TGF¿) is accompanied by a repression of KLF4 expression. The ability of KLF4 to enforce the epithelial phenotype is contrasted by KLF8 which drives a mesenchymal phenotype and whose expression is stimulated by TGF¿. Hence, Kr¿ppel-like factors determine the epithelial or mesenchymal states of breast epithelial cells and may underlie metastatic capacity. This proposal focuses on delineating mechanism(s) of action of KLF4 in dictating the epithelial state of breast cells. Since KLF4 is repressed during TGF¿-induced EMT, we will first determine if KLF4 inhibits TGF¿-induced EMT and if this involves KLF4 downregulation of Snail. This will reveal whether the precise level of KLF4 is critical for determining the extent of EMT induced by TGF¿ and possibly, metastasis. Secondly, we will assess whether KLF8 and KLF4 are antagonistic within the same cells. If so, this would indicate that the relative concentrations of these two proteins defines the epithelial/mesenchymal status of breast epithelial cells and this possibility will be further assessed by examining the relative expression of KLF4 and KLF8 in human tumors and their correlation with E-cadherin expression. In the third aim, we will assess the impact of KLF4 overexpression on metastasis using an experimental metastasis model as well as a transgenic model of autochthonous breast cancer. Lastly, we expect that KLF4 maintains epithelial characteristics by regulating transcription of a spectrum of genes. We will use genome-based approaches to identify these genes and assess their contributions to the epithelial phenotype. Together, these studies will reveal the molecular circuitry controlled by KLF4 that modulates the epithelial phenotype and has significant implications for specifying metastatic potential.