Using Cultured Human Mammary Epithelial Cells to Explore the Role of the Yap Oncogene in Early Breast Cancer Progression

雅普是一种与许多癌症进展有关的癌基因。然而，大多数关于雅普的研究 致癌功能已经在已经经历了许多的永生化细胞系中进行， 恶性进展的步骤。本研究将使用HMEC细胞培养系统， 乳腺癌体内进展的早期步骤，以了解雅普在早期乳腺癌中的作用。 乳腺癌进展。特别强调将放在审查雅普的作用，在关键的 重新激活端粒酶以克服复制衰老障碍的步骤， 显示了两个步骤的进展。首先，雅普导致了一个变化，允许收购 条件不朽（Conditional Immortality）然后，HMEC然后需要经历变量 过程（称为转换）转换其端粒维护机制，以支持短 在癌细胞中观察到的稳定构型。初步数据得出以下假设：雅普 早期乳腺癌进展过程中的失调改变了基因表达，并导致细胞 容易获得癌症特性，包括条件性永生和癌症干细胞 特色本提案将严格检验这一假设，目的有三。(1)雅普人的目标 促进国家允许获得有条件的永生将由以下因素决定： 进行RNAseq和差异基因表达分析。(2)肿瘤干细胞的雅普诱导 将用三种测定法检查永生化早期的性质： 干细胞表型、耐药性和3D培养中乳腺球的形成。的基因 还将确定促进这些干细胞表型的雅普失调下游。 (3)其中雅普诱导乳腺癌进展的乳腺癌阶段将被定义为 从正常、DCIS和增加的乳腺癌阶段获得组织， 使用抗体的免疫荧光检测雅普和在目的1和2中鉴定的基因， 促进永生化和癌症干细胞表型。此外，已发表的转录组和 微阵列数据集将用于确定雅普和永生化以及癌症干细胞 雅普下游的诱导基因在乳腺癌的特定阶段增加。的 本提案中概述的实验将确定雅普失调在早期免疫反应中的作用。 乳腺癌进展，并可能确定新的治疗靶点，以预防乳腺癌， 阻止永生化步骤。雅普癌基因与许多癌症有关， 永生化是大多数人类癌症早期进展的障碍;因此这些新的 治疗靶点也可能与大多数人类癌症的预防有关。

Yap is an oncogene implicated in the progression of many cancers. However, most research on Yap oncogenic function has been performed in immortalized cell lines that have already undergone many steps of malignant progression. This study will use a HMEC cell culture system that closely models the early steps of breast cancer progression in vivo to understand the function of Yap during early breast cancer progression. Particular emphasis will be place on examining Yap’s role in the crucial step of reactivating telomerase to overcome the replicative senescence barrier, a process that we have shown proceeds in two steps. First Yap causes a change permissive for the acquisition of immortality (termed conditional immortality). Then, the HMEC then need to undergo a variable process (termed conversion) to convert their telomere maintenance mechanisms to support the short stable configuration seen in cancer cells. Preliminary data has led to the following hypothesis: Yap dysregulation during early breast cancer progression alters gene expression and causes cells to be susceptible to acquire cancer properties including conditional immortality and cancer stem cell characteristics. This proposal will rigorously test this hypothesis with three aims. (1) The Yap targets that promote a state permissive for the acquisition of conditional immortality will be determined by performing RNAseq and differential gene expression analysis. (2) Yap induction of cancer stem cell properties early in immortalization will be examined with three assays: qPCR of RNA’s involved in stem cell phenotypes, drug resistance, and formation of mammospheres in 3D culture. The genes downstream of Yap dysregulation that promote these stem cell phenotypes will also be determined. (3) the breast cancer stages in which Yap induces breast cancer progression will be defined by obtaining tissue from normal, DCIS, and increasing breast cancer stages and performing immunofluorescence using antibodies that detect Yap and genes identified in aims 1 and 2 that promote immortalization and cancer stem cell phenotypes. Furthermore, published transcriptome and microarray datasets will be used to determine if Yap and immortalization and cancer stem cell inducing genes downstream of Yap are increased in specific stages of breast cancer. The experiments outlined in this proposal will determine the function of Yap dysregulation during early breast cancer progression and may identify novel therapeutic targets to prevent breast cancer by preventing the immortalization step. The Yap oncogene is implicated in many cancers and immortalization is a barrier in the early progression of most human carcinomas; therefore these novel therapeutic targets could also be relevant to the prevention of most human carcinomas.