The role of Twist in inducing breast cancer initiating cells and metastasis

描述(由申请人提供)：这项研究提案的总体目标是描述Twist在促进乳腺癌发病机制中的癌症起始细胞和转移方面的生物学功能。为了支持这一目标，我们早些时候已经证明，乳腺细胞(MCF-7)中的一种基本螺旋-环-螺旋转录因子Twist的过表达促进了异种原位移植瘤上皮-间充质转化，增强了肿瘤的发生，增加了遗传不稳定性，并增加了肿瘤微环境中的血管形成和通透性。我们还证明了Twist在乳腺细胞中的过表达可以促进乳腺癌起始细胞的表型，其特征是CD44高表达，CD24低表达或不表达，ALDH活性增加。此外，TWIST过表达细胞表现出高外流的Hoechst 33342和罗丹明123，这是由于ABCC1(MRP1)转运体表达增加的结果，ABCC1(MRP1)转运体是癌症起始细胞的一种特性。重要的是，我们发现，只有20个高表达CD44？/低亚群的Twist细胞接种能够在SCID小鼠的乳房脂肪垫中形成肿瘤。在我们最近的研究中，在乳腺癌细胞中，Twist的表达与粘蛋白1(MUC1)的异常调节直接相关。有趣的是，我们的研究还发现，Twist的表达调节p27和胆碱激酶的表达，这两个基因是侵袭性癌症表型的公认标志。在这项修订的应用中，我们旨在为Twist在乳腺癌发病机制中的作用以及在乳腺癌个体发育过程中改变代谢组学的作用建立机制基础。我们的假设是，Twist的表达改变了正常的细胞通路，促进了向肌上皮祖细胞的分化，并增加了转移潜能。为了检验这一假设，我们提出了三个具体目标。在目标1中，我们将鉴定Twist诱导的致癌细胞，并从功能上证明它们的成瘤潜能。在目标2中，我们将通过其对p27和胆碱激酶的调节来确定Twist诱导乳腺癌进展和转移的分子机制。此外，我们将在临床前模型中纵向确定Twist表达引起的代谢变化。在目标3中，我们将在纯DCIS样本中识别由Twist表达诱导的侵袭性表型的早期标记和致癌标记，并利用快速尸检程序中的样本来确定Twist表达是否与器官特异性乳腺癌转移相关。重要的是，我们将在患者的乳腺肿瘤中鉴定乳腺癌起始细胞，并确定它们在乳腺肿瘤发生中的功能意义。综上所述，这些研究将确定Twist在诱导乳腺癌、启动细胞和转移方面的作用，以及它作为乳腺癌早期标志物的实用性。

DESCRIPTION (provided by applicant): The overall objective of this research proposal is to delineate the biological functions of Twist in promoting cancer initiating cells and metastasis in the pathogenesis of breast cancer. In support for this objective, we had earlier demonstrated that over-expression of Twist, a basic helix-loop-helix transcription factor, in breast cells (MCF-7) promotes epithelial-mesenchymal transition, enhanced tumorigenesis, increased genetic instability, and increased vasculature and permeability within the tumor microenvironment of orthotopic xenografts. We also demonstrated that the over-expression of Twist in breast cells can promote breast cancer initiating cell phenotype characterized by high CD44 expression, little or no CD24 expression and increased ALDH activity. In addition, Twist over-expressing cells exhibit high efflux of Hoechst 33342 and Rhodamine 123 as a result of increased expression of ABCC1 (MRP1) transporters, a property of cancer initiating cells. Importantly, we show that inoculums of only twenty cells of the Twist over-expressing CD44?/low sub-population are capable of forming tumors in the mammary fat pad of SCID mice. In our recent studies, a direct association between Twist expression and the dysregulation of Mucin 1 (MUC1) was demonstrated in breast cancer cells. Interestingly, our studies have also revealed that Twist expression regulates the expression of p27 and choline kinase, which are well-established markers of aggressive cancer phenotypes. In this revised application, we aim to establish the mechanistic basis for the role of Twist in pathogenesis of breast cancer and in altering metabolomics during the ontogeny of breast cancer. Our hypothesis is that Twist expression alters normal cellular pathways, facilitating the differentiation towards myoepithelial progenitor cells and increasing metastatic potential. To test this hypothesis, we propose three specific aims. In Aim 1, we will identify cancer initiating cells induced by Twist and functionally demonstrate their tumor forming potential. In Aim 2, we will determine the molecular mechanisms of Twist induced breast cancer progression and metastasis through its regulation of p27 and choline kinase. In addition, we will longitudinally determine the metabolic changes induced by Twist expression in preclinical models. In Aim 3, we will identify early markers of aggressive phenotypes and cancer initiating markers induced by Twist expression in pure DCIS samples and utilize the samples from the rapid autopsy program to determine if Twist expression can be associated with organ specific breast cancer metastasis. Importantly, we will identify breast cancer initiating cells in patient breast tumors and determine their functional implications in breast tumorigenesis. Taken together these studies will establish the role of Twist in inducing breast cancer initiating cells and metastasis as well as its utility as an early marker of breast cancer.