ErbB receptor signaling via small G-proteins in breast cancer

描述(申请人提供)：人表皮生长因子(EGF)酪氨酸激酶受体家族由ErbB1(EGFR)、ErbB2(Neu)、ErbB3和ErbB4组成，在癌症进展中发挥关键作用。由于受体过度激活(如突变的EGFR或ErbB2过度表达)、配体过度表达(如转化生长因子？或Heregulins)，或增强的下游信号(如增强的PI3K信号)在乳腺癌的病因中发挥着重要作用。我们发现，ErbB1和ErbB3受体的配体刺激导致小GTPase rac1的强烈激活，从而促进有丝分裂和运动。在乳腺癌细胞中寻找Rac-GEF介导ErbB配体激活Rac1，揭示了phosphatidylinositol-3，4，5-triphosphate-dependent RAC交换器-1(P-Rex1)的重要作用，它最初在中性粒细胞中被发现，但到目前为止还没有在任何癌症中被研究。值得注意的是，P-REx1在大多数乳腺癌细胞系和肿瘤中都高度表达。P-REx1是一种依赖于PI3K和G？的RAC-gef，巧合的是，ErbB受体介导的rac1的激活被抑制毒素、PI3K抑制剂wortmannin和抑制/耗尽PI3K？所抑制。亚单位。此外，ErbB3诱导的rac1的激活依赖于CXCR4的反式激活，CXCR4是一种广泛参与乳腺癌进展的GI偶联受体，而趋化因子SDF-11直接刺激CXCR4激活rac1也依赖于P-REx1。因此，P-REx1是乳腺癌细胞中ErbB受体和GPCRs激活RAC的共同介质。在特定的目标1中，我们将研究ErbB受体的配体刺激是否能促进乳腺癌细胞中P-REx1的激活，并评估这种激活的功能后果，包括肌动蛋白细胞骨架的重组、迁移和增殖。易位研究以及FRET方法将被用来解决离散的PI3K同工酶和CXCR4-G？P-Rex1和rac1激活的途径。在特定的目标2中，我们将确定P-REx1的RNAi缺失是否影响由ErbB3配体的过表达驱动的乳腺癌细胞的致瘤性和转移扩散。我们还将评估P-REx1在抗雌激素抵抗中的作用。在特定的目标3中，我们将确定通过P-REx1的Rac信号是否参与ErbB2驱动的肿瘤的发生和转移，以及这是否由CXCR4-G？？-PI3K？路径。我们将建立P-REx1过表达的乳腺特异性小鼠模型，并确定它是否会导致肿瘤形成或与ErbB2过表达协同作用。在特定的目标4中，目标是确定P-REx1是否是人类乳腺癌的标志，以及P-REx1在人类肿瘤中的表达与信号参数相关。确定P-REx1是ErbB受体的效应分子，对于了解乳腺癌的分子基础和病因，以及对预后和治疗有重要意义。

DESCRIPTION (provided by applicant): The human epidermal growth factor (EGF) family of tyrosine kinase receptors consists of ErbB1 (EGFR), ErbB2 (Neu), ErbB3, and ErbB4, and plays key roles in cancer progression. Dysregulation of ErbB signaling due to hyperactivation of receptors (such as mutated EGFR or ErbB2 overexpression), overexpression of ligands (such as TGF-? or heregulins), or augmented downstream signaling (such as enhanced PI3K signaling) plays a major role in the etiology of breast cancer. We found that ligand stimulation of ErbB1 and ErbB3 receptors leads to a strong activation of the small GTPase Rac1 to promote mitogenesis and motility. The search for Rac-GEFs that mediate Rac1 activation by ErbB ligands in breast cancer cells revealed a prominent role for phosphatidylinositol-3,4,5-triphosphate-dependent Rac exchanger-1 (P-Rex1), a Rac-GEF originally identified in neutrophils but not studied in any cancer so far. Strikingly, P-Rex1 is highly expressed in most breast cancer cell lines as well as in tumors. P-Rex1 is a PI3K- and G??-dependent Rac-GEF, and coincidentally, ErbB receptor-mediated activation of Rac1 is inhibited by Pertusis toxin, which prevents G23 subunit release from heterotrimeric Gi proteins, by the PI3K inhibitor wortmannin, and by inhibition/depletion of PI3K?, a class Ib PI3K that is activated by G?? subunits. Moreover, ErbB3-induced activation of Rac1 is dependent on the transactivation of CXCR4, a Gi-coupled receptor widely implicated in breast cancer progression, and Rac1 activation by direct stimulation of CXCR4 with the chemokine SDF-11 also depends on P-Rex1. Therefore, P-Rex1 is a common mediator of Rac activation by ErbB receptors and GPCRs in breast cancer cells. In Specific Aim 1 we will investigate whether ligand stimulation of ErbB receptors can promote the activation of P-Rex1 in breast cancer cells, and assess the functional consequences for such activation, including actin cytoskeleton reorganization, migration, and proliferation. Translocation studies as well as FRET approaches will be used to address the relative contribution of discrete PI3K isozymes and the CXCR4-G?? pathway to P-Rex1 and Rac1 activation. In Specific Aim 2 we will determine whether RNAi depletion of P-Rex1 affects tumorigenicity and metastatic dissemination of breast cancer cells driven by overexpression of an ErbB3 ligand. We will also assess the involvement of P-Rex1 in anti-estrogen resistance. In Specific Aim 3 we will determine if Rac signaling via P-Rex1 contributes to ErbB2-driven tumorigenesis and metastasis, and whether this is mediated by the CXCR4-G??-PI3K? pathway. We will generate mammary-specific mouse models for P-Rex1 overexpression and determine whether it can lead to tumor formation or cooperate with ErbB2 overexpression. In Specific Aim 4 the goal is to determine whether P-Rex1 is a hallmark of human breast cancer, and P-Rex1 expression in human tumors correlates with signaling parameters. The identification of P-Rex1 as an effector of ErbB receptors may have major relevance for understanding the molecular basis and etiology of breast cancer as well as significant prognostic and therapeutic implications.