Elucidating the Role of Src and JNK signaling in Net1A-dependent Breast Cancer Metastasis

项目摘要 每年有40,000多名妇女死于乳腺癌转移，而且很少有有效的治疗方法 以此过程为目标。乳腺癌转移的一个标志是癌细胞迁移和侵袭增加， 这部分是由小的GTP酶RhoA驱动的。不幸的是，RhoA的治疗靶向尚未得到 成功是因为它在全身普遍表达，而且没有有针对性的接口。这个 神经上皮细胞转化基因1(NET1)是一种高表达的Rho鸟嘌呤交换因子 在乳腺癌中，是乳腺癌细胞运动所必需的。Net1异构体是由核唯一调控的 自动减支。然而，对静止细胞的配体刺激允许Net1A亚型重新定位到 细胞质，在那里它可以激活RhoA。我们实验室最近的工作已经建立了c-jun-N末端激酶 (JNK)和Src信号是配体刺激的Net1A胞浆定位所必需的，而这些 激酶使Net1a内不同的位点磷酸化，从而导致这种效应。基于这些发现，我们 假设Src与JNK协同调控Net1a胞质积累促进 乳腺癌的运动和转移。在目标1中，我们将确定Src依赖的磷酸化是如何 Y373上的Net1A控制其亚细胞定位。我们将确定直接磷酸化的激酶 Net1a在这个网站上，并阐明了这一驱动Net1a核出口的机制。在《目标2》中我们将 确定JNK和Src信号如何协同促进Net1A的胞质积累以刺激 RhoA激活与乳腺癌细胞体外运动在目标3中，我们将确定JNK-和SRC-的作用。 Net1a依赖磷酸化在乳腺原位肿瘤发生和转移中的作用 乳腺肿瘤模型。我们假设这些信号事件是Net1A介导的运动的关键驱动因素 和入侵。识别这些调节机制可能会提供新的治疗方法 减少乳腺癌转移。

Project Summary Breast cancer metastasis kills over 40,000 women annually and there are very few therapies that efficiently target this process. A hallmark of breast cancer metastasis is an increase in cancer cell migration and invasion, which is driven in part by the small GTPase RhoA. Unfortunately, therapeutic targeting of RhoA has not been successful because it is expressed ubiquitously throughout the body and has no targetable interfaces. The neuroepithelial transforming gene 1 (Net1), is a Rho guanine exchange factor (RhoGEF) that is overexpressed in breast cancer and is required for breast cancer cell motility. Net1 isoforms are uniquely regulated by nuclear sequestration. However, ligand stimulation of quiescent cells allows the Net1A isoform to relocalize to the cytoplasm, where it can activate RhoA. Recent work from our lab has established c-Jun-N-terminal kinase (JNK) and Src signaling are required for ligand stimulated cytosolic localization of Net1A, and that these kinases phosphorylate distinct sites within Net1A to cause this effect. Based on these findings, we hypothesize that Src cooperates with JNK to regulate Net1A cytoplasmic accumulation to promote breast cancer motility and metastasis. In Aim 1, we will determine how Src-dependent phosphorylation of Net1A on Y373 controls its subcellular localization. We will identify the kinase that directly phosphorylates Net1A on this site, and elucidate the mechanism by which this drives Net1A nuclear export. In Aim 2 we will determine how JNK and Src signaling synergize to promote cytoplasmic accumulation of Net1A to stimulate RhoA activation and breast cancer cell motility in vitro. In Aim 3, we will establish the role of JNK- and Src- dependent phosphorylation of Net1A in mammary gland tumorigenesis and metastasis using orthotopic mammary tumor models. We hypothesize these signaling events to be key drivers of Net1A-mediated motility and invasion. Identification of these regulatory mechanisms may provide novel therapeutic approaches to mitigate breast cancer metastasis.