Heregulin signaling via small GTPases in mitogenesis and tumorigenesis

描述（由申请人提供）：大约30%的乳腺肿瘤表达高水平的heregulins (HRG)，这是一组表皮生长因子（EGF）样配体肽，可诱导肿瘤向侵袭性和转移性表型发展。HRG与ErbB3和ErbB4受体结合，ErbB3和ErbB4受体与ErbB受体家族成员异二聚体结合，并信号激活有丝分裂和生存途径。我们最近在乳腺癌细胞中发现，小g蛋白Rac1通过HRG介导G1/S的细胞周期进程，这一作用涉及细胞周期蛋白D1的诱导和Rb的过度磷酸化。HRG在乳腺癌细胞中引起强烈的Rac活化；然而，与通过ErbB1 （EGFR）发出信号的EGF相比，它在动力学上存在显著差异。令人惊讶的是，我们发现HRG对Rac的激活不仅是由ErbB3和ErbB2介导的，还可以通过EGFR的反激活介导，而且它不依赖于ErbB4。hrg诱导的Rac激活依赖于pi3k。hrg诱导的Rac反应也有类似的要求，包括肌动蛋白细胞骨架重组、运动和通过ERK和cyclin D1进行的细胞周期进展。PCR阵列筛选和RNAi研究显示PI3K-和G？依赖性Rac- gef P-Rex1介导HRG在乳腺癌细胞中的Rac活化。有趣的是，初步研究表明，P-Rex1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与人类乳腺癌的相关性。在Specific Aim 1中，我们将研究P-Rex1r Rac通路在有丝分裂、非锚定生长和运动中的相关性。我们预测HRG通过EGFR和PI3K途径引起P-Rex1的激活。我们还将研究P-Rex1过表达是否会带来生长优势和运动表型。在Specific Aim 2中，我们将在小鼠模型中确定Rac信号和P-Rex1在HRG肿瘤发生和转移中的相关性。异种移植、转基因和敲除方法将用于确定P-Rex1rRac1通路在乳腺癌进展中的相关性。在Specific Aim 3中，我们将开展一项严谨的研究来表征P-Rex1在人类乳腺癌标本中的表达，并确定其是否与乳腺癌的分级及其他相关临床病理参数存在相关性。在Specific Aim 4中，目标是剖析EGFR参与Rac HRG激活的分子基础。一个关键的目标是确定单个EGFR酪氨酸残基在将信号传递给P- Rex1rRac中的作用。当P-Rex1被PI3K和G？亚基，我们将探索gpcr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。我们的提案涵盖分子、细胞、动物和人类标本研究，因此具有重要的翻译价值。

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.