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

描述（由申请人提供）：本研究计划的总体目标是描述Twist在乳腺癌发病机制中促进癌起始细胞和转移的生物学功能。为了支持这一目标，我们之前已经证明，乳腺细胞（MCF-7）中Twist（一种基本的螺旋-环-螺旋转录因子）的过度表达促进了上皮-间质转化，增强了肿瘤发生，增加了遗传不稳定性，增加了原位异种移植物肿瘤微环境中的血管和通透性。我们还证实，Twist在乳腺细胞中的过表达可以促进乳腺癌启动细胞表型，其特征是CD44高表达，CD24少表达或不表达，ALDH活性增加。此外，Twist过表达的细胞由于ABCC1 （MRP1）转运体的表达增加，表现出Hoechst 33342和Rhodamine 123的高外排，这是癌症启动细胞的一个特性。重要的是，我们发现只有20个Twist细胞的接种体过表达CD44？/低亚群能够在SCID小鼠乳腺脂肪垫形成肿瘤。在我们最近的研究中，Twist的表达与Mucin 1 （MUC1）的失调直接相关。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，而p27和胆碱激酶是侵袭性癌症表型的公认标志物。在这次修订的申请中，我们旨在建立Twist在乳腺癌发病机制中的作用以及在乳腺癌个体发生过程中改变代谢组学的机制基础。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，增加了转移潜力。为了验证这一假设，我们提出了三个具体目标。在Aim 1中，我们将鉴定Twist诱导的癌症启动细胞，并从功能上证明它们的肿瘤形成潜力。在Aim 2中，我们将通过Twist对p27和胆碱激酶的调控来确定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测定Twist表达引起的代谢变化。在Aim 3中，我们将在纯DCIS样本中确定Twist表达诱导的侵袭性表型的早期标记和癌症启动标记，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有关。重要的是，我们将在患者乳腺肿瘤中识别乳腺癌起始细胞，并确定其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在诱导乳腺癌起始细胞和转移中的作用，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. PUBLIC HEALTH RELEVANCE: The project will help define the role of Twist in inducing cancer initiating cells and in metastasis during the pathogenesis of breast cancer. The clinical translatability of these findings will be confirmed in human breast cancer tissue samples such as pure DCIS and that obtained from the rapid autopsy program at Johns Hopkins. We anticipate that the clinical applications derived from the insights and knowledge provided by the successful completion of this research project will lead to molecular approaches to identify and target this lethal subpopulation of cells, which will improve treatment outcomes. In the long term, these studies will help characterize breast cancer initiating cells and provide insights that will contribute to the development of novel imaging markers and chemotherapeutic drugs for breast cancer.