The Role of SRC-1 in Breast Cancer

描述（由申请人提供）：转移（Meta）是几乎所有乳腺癌（BC）死亡率的原因;然而，对其病理生理机制了解甚少。本项目的长期目标是了解BC Meta的分子机制，并开发新的思路/策略来控制BC Meta。类固醇受体辅激活因子1（SRC-1）是一个新发现的Meta基因，它通过促进Twist的表达而促进BC细胞上皮间质转化（EMT）和肺Meta。Twist是一种在侵袭性BC中过表达的DNA结合转录因子，是EMT和BC Meta的主要调节因子。BC中Twist表达也与化疗耐药性的获得相关。此外，BC细胞可塑性之间的EMT和间充质上皮转化（MET）已被提出是可转换的，并发挥关键作用，在Meta，但直接的体内证据，这样的顺序事件及其直接联系到Meta仍然缺乏。在目前的项目中，研究人员假设BC细胞EMT-MET可塑性存在于局部和转移性肿瘤中。Twist对EMT有促进作用，对MET有抑制作用。Twist的灭活将增强BC化疗的疗效，而不会导致成人严重的健康问题。Twist蛋白的稳定性受磷酸化控制，抑制负责这种磷酸化的激酶并增加相应的磷酸酶活性将降低BC细胞中Twist蛋白的水平及其功能。提出了三个具体目标（SA）来检验这些假设。第一个SA是定义Twist在MG肿瘤细胞可塑性中的作用和MG肿瘤细胞可塑性在BC Meta中的作用。将使用EMT依赖性启动子-报告子传感器系统来追踪BC细胞EMT-MET可塑性和肺Meta过程中的EMT细胞谱系。将使用慢病毒诱导Twist内源性表达、过表达或敲除（KO）的小鼠乳腺肿瘤，以确定Twist在肿瘤细胞可塑性和Meta中的作用。第二个SA是评估Twist失活对一般健康的副作用，并测试靶向Twist是否增强BC化疗疗效。将使用诱导型KO系统在成年小鼠中敲除Twist。研究人员将检查Twist KO在成年小鼠中是否会引起任何严重的健康问题，以及Twist KO是否会增强Paclitaxel化疗的疗效。这将提供有关Twist是否可用作具有可耐受副作用的癌症治疗靶点的信息。使用诱导型KO模型，将确定Twist失活对BC化疗的影响。第三个SA是确定Twist磷酸化在调节其蛋白稳定性中的作用和分子机制，并测试通过控制其磷酸化程度使Twist不稳定是否会抑制BC细胞侵袭和Meta。这些研究的成功结果不仅将为理解Twist如何调节BC细胞可塑性和癌症Meta提供见解，而且还将验证Twist作为控制BC Meta的靶点，并提供直接的体内证据来证明或反驳BC Meta期间EMT和MET之间的顺序联系。

DESCRIPTION (provided by applicant): Metastasis (Meta) is responsible for almost all breast cancer (BC) mortality; however, its pathophysiological mechanism is poorly understood. The long-term goal of this project is to understand the molecular mechanisms of BC Meta and develop new ideas/strategies to control BC Meta. The steroid receptor coactivator 1 (SRC-1) is a newly identified Meta gene and it promotes Twist expression to enhance BC cell epithelial-mesenchymal transition (EMT) and lung Meta. Twist is a DNA-binding transcription factor overexpressed in invasive BCs and a master regulator of EMT and BC Meta. Twist expression in BC is also associated with acquisition of chemotherapy resistance. In addition, the BC cell plasticity between EMT and mesenchymal epithelial transition (MET) has been proposed to be convertible and play a crucial role in Meta, but direct in vivo evidences for such sequential events and their direct link to Meta are still absent. In the current project, the investigators hypothesize that BC cell EMT-MET plasticity exists in both local and metastatic tumors. Twist plays a role in promoting EMT and suppressing MET. Inactivation of Twist will enhance the efficacy of BC chemotherapy without causing severe health problems in adults. Twist protein stability is controlled by phosphorylation, and inhibition of kinase responsible for such phosphorylation combined with an increase in corresponding phosphotase activity will reduce Twist protein level and its function in BC cells. Three specific aims (SAs) are proposed to test these hypotheses. The first SA is to define the role of Twist in MG tumor cell plasticity and the role of MG tumor cell plasticity in BC Meta. An EMT- dependent promoter-reporter senor system will be used to trace the EMT cell lineage during the processes of BC cell EMT-MET plasticity and lung Meta. Lentivirus will be used to induce mouse mammary tumors with endogenous expression, overexpression or knockout (KO) of Twist to determine the role of Twist in tumor cell plasticity and Meta. The second SA is to evaluate the side effects of Twist inactivation on general health and test whether targeting Twist enhances BC chemotherapy efficacy. Twist will be knocked out in adult mice using an inducible KO system. Investigators will examine whether Twist KO in adult mice causes any severe health problems and whether Twist KO will enhance the efficacy of Paclitaxel chemotherapy. This will provide information about whether Twist can be used as a cancer therapy target with tolerable side effects. With the inducible KO model, the effect of Twist inactivation on BC chemotherapy will be determined. The third SA is to define the role and molecular mechanisms of Twist phosphorylation in regulation of its protein stability and test whether destabilization of Twist by controlling its phosphorylation degree will inhibit BC cel invasion and Meta. Successful results from these studies will not only provide insights into understanding how Twist regulates BC cell plasticity and cancer Meta, but also validate Twist as a target for controlling BC Meta and provide direct in vivo evidence to prove or disprove the sequential connection between EMT and MET during BC Meta.