Heregulin signaling via small GTPases in mitogenesis and tumorigenesis

描述（由申请人提供）：约30%的乳腺肿瘤表达高水平的调蛋白（HRG），这是一组表皮生长因子（EGF）样配体肽，可诱导进展为侵袭性和转移性表型。HRG与ErbB 3和ErbB 4受体结合，ErbB 3和ErbB 4受体与ErbB受体家族成员异二聚化，并发出激活促有丝分裂和存活途径的信号。我们最近在乳腺癌细胞中发现，小G蛋白Rac 1通过HRG介导细胞周期进程通过G1/S，这种作用涉及细胞周期蛋白D1诱导和Rb过度磷酸化。HRG在乳腺癌细胞中引起强烈的Rac激活;然而，与通过ErbB 1（EGFR）发出信号的EGF相比，它在动力学上存在显着差异。令人惊讶的是，我们发现HRG对Rac的激活不仅由ErbB 3和ErbB 2介导，而且还由EGFR的反式激活介导，并且它不依赖于ErbB 4。HRG诱导的Rac激活是PI 3 K依赖性的。HRG诱导的Rac反应也观察到类似的要求，包括肌动蛋白细胞骨架重组、运动性以及通过ERK和细胞周期蛋白D1的细胞周期进展。PCR阵列筛选后的RNAi研究显示，PI 3 K-和G？？-依赖Rac-GEF P-Rex 1介导乳腺癌细胞中HRG对Rac的激活。有趣的是，初步研究显示P-Rex 1在人类乳腺癌细胞系和患者肿瘤中显著上调。现在的目标是确定参与这些复杂信号相互作用的分子机制及其在人类乳腺癌中的相关性。在具体目标1中，我们将研究P-Rex 1 r Rac通路在有丝分裂、锚定非依赖性生长和运动中的相关性。我们预测HRG通过EGFR和PI 3 K途径激活P-Rex 1。我们还将研究P-Rex 1过表达是否赋予生长优势和运动表型。在具体目标2中，我们将确定Rac信号传导和P-Rex 1在小鼠模型中HRG肿瘤发生和转移中的相关性。异种移植、转基因和基因敲除方法将用于确定P-Rex 1 rRac 1通路在乳腺癌进展中的相关性。在具体目标3中，我们将进行严格的研究，以表征P-Rex 1在人类乳腺癌标本中的表达，并确定是否与乳腺癌的分级和其他相关临床病理参数存在相关性。在具体目标4中，目标是剖析EGFR参与Rac的HRG激活的分子基础。一个关键目标是确定单个EGFR酪氨酸残基在将信号传递给P-Rex 1 rRac中的作用。由于P-Rex 1被PI 3 K和G？亚基，我们将探讨通过G？？到Rac激活。总之，我们的研究将为ErbB受体和下游效应物在乳腺癌中的作用提供新的机制和功能见解。我们的提案涵盖了分子、细胞、动物和人类标本研究，因此具有重要的转化价值。 公共卫生相关性：在这项提案中，我们将表征一种新的信号通路激活的生长因子heregulin（HRG），涉及小G蛋白Rac 1和它的激活剂P-Rex 1。Rac 1和P-Rex 1介导HRG诱导的促有丝分裂和运动信号。我们的目标是使用细胞和动物模型来确定这一途径在乳腺癌中的意义，并阐明HRG通过P-Rex 1向Rac发出信号的分子机制。

DESCRIPTION (provided by applicant): Approximately 30% of breast tumors express high levels of heregulins (HRG), a group of epidermal growth factor (EGF)-like ligand peptides that induce the progression towards an aggressive and metastatic phenotype. HRG binds to ErbB3 and ErbB4 receptors which heterodimerize with members of the ErbB receptor family and signal for the activation of mitogenic and survival pathways. We have recently established in breast cancer cells that the small G-protein Rac1 mediates cell cycle progression through G1/S by HRG, an effect that involves cyclin D1 induction and Rb hyperphosphorylation. HRG causes strong Rac activation in breast cancer cells; however, it does so with striking differences in kinetics compared to EGF, which signals through ErbB1 (EGFR). Surprisingly, we found that activation of Rac by HRG is mediated not only by ErbB3 and ErbB2 but also by transactivation of EGFR, and it is independent of ErbB4. HRG-induced Rac activation was PI3K-dependent. Similar requirements were observed for HRG-induced Rac responses, including actin cytoskeleton reorganization, motility, and cell cycle progression via ERK and cyclin D1. A PCR array screening followed by RNAi studies revealed that the PI3K- and G??-dependent Rac-GEF P-Rex1 mediates Rac activation by HRG in breast cancer cells. Interestingly, preliminary studies show a striking up-regulation of P-Rex1 in human breast cancer cell lines and tumors from patients. The goal now is to define the molecular mechanisms involved in these complex signaling interactions and their relevance in human breast cancer. In Specific Aim 1 we will examine the relevance of the P-Rex1r Rac pathway in mitogenesis, anchorage-independent growth and motility. We predict that HRG causes the activation of P-Rex1 via the EGFR and PI3K pathways. We will also examine if P-Rex1 overexpression confers growth advantage and a motile phenotype. In Specific Aim 2 we will determine the relevance of Rac signaling and P-Rex1 in HRG tumorigenesis and metastasis in mouse models. Xenograft, transgenic, and knock-out approaches will be used to establish the relevance of the P-Rex1rRac1 pathway in breast cancer progression. In Specific Aim 3 we will carry out a rigorous study to characterize the expression of P-Rex1 in human breast cancer specimens and establish whether a correlation exists with grade and other relevant clinicopathological parameters in breast cancer. In Specific Aim 4 the goal is to dissect the molecular basis for the involvement of EGFR in HRG activation of Rac. A key objective is to determine the contribution of individual EGFR tyrosine residues in relaying the signal to P- Rex1rRac. As P-Rex1 is activated by PI3K and G?? subunits, we will explore a potential contribution from GPCRs via G?? to Rac activation. In summary, our studies will provide novel mechanistic and functional insights into the role of ErbB receptors and downstream effectors in breast cancer. Our proposal encompasses molecular, cellular, animal, and human specimen studies, and therefore it has significant translational value. PUBLIC HEALTH RELEVANCE: In this proposal we will characterize a novel signaling pathway activated by the growth factor heregulin (HRG) that involves the small G-protein Rac1 and its activator P-Rex1. Rac1 and P-Rex1 mediate mitogenic and motile signals induced by HRG. The goal is to determine the implications of this pathway in breast cancer using cellular and animal models, and to elucidate molecular mechanisms by which HRG signals to Rac via P-Rex1.