Heregulin signaling via small GTPases in mitogenesis and tumorigenesis

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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.