EGF-induced Grb7 Recruits and Promotes Ras Activity Essential for the Tumorigenicity of Sk-Br3 Breast Cancer Cells

Abstract

Co-amplification and co-overexpression of ErbB2 and Grb7 are frequently found in various cancers, including breast cancer. Biochemical and functional correlations of the two molecules have identified Grb7 to be a pivotal mediator downstream of ErbB2-mediated oncogenesis. However, it remains largely unknown how Grb7 is involve in the ErbB2-mediated tumorigenesis. In this study, we show that Grb7-mediated cell proliferation and growth are essential for the tumorigenesis that occurs in ErbB2-Grb7-overexpressing breast cancer cells. Intrinsically, EGF-induced de novo Grb7 tyrosine phosphorylation/activation recruits and activates Ras-GTPases and subsequently promotes the phosphorylation of ERK1/2, thereby stimulating tumor growth. Furthermore, we also found the anti-tumor effect could be synergized by co-treatment with Herceptin plus Grb7 knockdown in Sk-Br3 breast cancer cells. Our findings illustrate an underlying mechanism by which Grb7 promotes tumorigenesis through the formation of a novel EGFR-Grb7-Ras signaling complex, thereby highlighting the potential strategy of targeting Grb7 as an anti-breast cancer therapy.Growth factor receptor-bound protein-7 (Grb7) 2 is the prototype of an emerging adaptor protein family that includes Grb10 and Grb14 and contains an N-terminal proline-rich region followed by a putative RA (Ras-associating) domain, a central PH (pleckstrin homology) domain, a BPS (Between the PH and SH2 domains), motif and a C-terminal SH2 domain (1, 2). Most of these domains are known to interact with a variegated spectrum of receptors and/or regulators, through which Grb7 is capable of activating versatile signal transduction pathways to modulate various cellular functions (1, 2). For instance, the SH2 domain of Grb7 is capable of binding to the phosphotyrosine sites of receptor tyrosine kinases such as ErbB family receptors, PDGF receptor, Tek/Tie2, c-Kit, and EphB1, as well as cytoplasmic proteins such as SHPTP2, Shc, and FAK (focal adhesion kinase) (2). The PH domain is the calmodulin-or phosphoinositide-binding domain that is responsible for plasma membrane localization and is also accessible for phosphorylation by FAK (3, 4). Structural prediction and biochemical validation both support the direct interaction of the RA domain with Ras-GTPases, similar to the canonical ubiquitin-like folded Ras-binding domain of c-Raf and RalGDS (5). Using yeast two-hybrid screening, Tankyrase 2, an ankyrin repeat-containing poly(ADP-ribose) polymerase, was identified to interact with the N-terminal prolinerich region of Grb14 (6). However, the downstream signaling targets of the identified interactions and their corresponding functional consequences remain largely unknown.Originally identified as an activated EGF receptor-binding partner, Grb7 has often been found to be co-amplified with ErbB2 in several cancers because both genes are located in the chromosome 17q12 (7). In fact, Grb7 is concurrently overexpressed with ErbB2 in about 30% of human breast cancers, and in these cases, the patients present a poor ...

Expression screening of 17q12–21 amplicon reveals GRB7 as an ERBB2‐dependent oncogene

“…Previous studies showed that GRB7 was required for SK-BR-3 cell proliferation and ERK1/2 and Akt phosphorylation [22,23]. Conversely, overexpression of GRB7 and ERBB2 in MCF-7 breast cancer cells enhance ERBB2 and Akt phosphorylation and tumor xenograft growth [24].…”

Kindlin-2 regulates renal tubular cell plasticity by activation of Ras and its downstream signaling

“…A GST-(Raf)-Ras binding domain (RBD) pulldown assay was performed according to the method described previously (14). The RBD of Raf glutathione S-transferase (GST) fusion protein (GST-Raf-RBD) was precoupled with glutathione-Sepharose 4B according to the manufacturer's instructions and then incubated with HKC lysates for 2 h at 4°C to detect GTP-bound Ras-GTPases as described previously.…”

Mmu-miR-193 Is Involved in Embryo Implantation in Mouse Uterus by Regulating GRB7 Gene Expression

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