Review Research

Published OnlineFirst August 7, 2012; DOI: 10.1158/1541-7786.MCR-12-0352

Molecular Cancer Review Research

GAB2—a Scaffolding Protein in Cancer

Sarah J. Adams, Iraz T. Aydin, and Julide T. Celebi

Abstract Adaptor or scaffolding proteins mediate protein–protein interactions that drive the formation of protein complexes. Grb2-associated binding protein 2 (GAB2) scaffolding protein is an intermediary molecule that links plasma membrane receptor signaling including receptor tyrosine kinases with the downstream effectors, such as protein tyrosine phosphatase, nonreceptor type 11 (SHP2), p85 subunit of phosphoinositide-3 kinase (PI3-K), phospholipase C-gamma 1 (PLC-g), v-crk sarcoma virus CT10 (CRK), Src homology 2 domain containing transforming protein 1 (SHC), and SH2 containing inositol phosphatase (SHIP). Although, well described in signal transduction, its role in cancer has recently been emerging especially in leukemia, breast and ovarian cancer, and melanoma. GAB2 is essential for two major signal transduction pathways in cancer, the PI3-K-AKT and extracellular signal-regulated kinase (ERK) signaling pathways, and thus regulates a number of key cellular processes. This review focuses on structure and function of GAB2, its regulatory proteins, emerging role in cancer, and potential as a therapeutic target. Mol Cancer Res; 10(10); 1265–70. 2012 AACR.

Introduction reviewed in the literature (2–5). This article will instead Grb2-associated binding protein 2 (GAB2) is a scaf- focusonGAB2withanemphasisonitsemergingrolein folding protein that contains various structural domains human cancer. and docking sites that serve as a platform for the assembly of signaling systems. Although, GAB2 itself lacks enzy- Structural Motifs and Binding Partners of GAB2 matic activity, it acts downstream of receptor tyrosine GAB proteins contain a number of highly conserved kinases (RTK) and non-RTKs, such as cytokine and structural motifs that mediate their interaction with binding G-protein–coupled receptors, to transmit and amplify partners including an N-terminal Pleckstrin homology (PH) signals to downstream effectors. Upon stimulation, GAB2 domain, a central proline-rich domain, and multiple phos- becomes phosphorylated on critical tyrosine residues cre- pho-tyrosine residues (Fig. 1; refs. 3, 6). The PH domain ating binding sites for diverse targets involved in signal binds to cell membrane phospholipids, preferentially phos- transduction. As such, GAB2 serves as a mediator of phatidylinositol phosphates (7, 8) and plays a role in the essential cellular processes including proliferation, surviv- membrane localization of GAB2. al, migration, and differentiation. The proline-rich domain contains numerous PXXP GAB2 belongs to a family of evolutionarily conserved motifs, which serve as docking sites for Src homology 3 proteins consisting of 3 mammalian paralogues, GAB1, (SH3) domain–containing proteins. GAB2 constitutively GAB2, and GAB3, Drosophila melanogaster homolog DOS associates with the Grb2 adaptor protein through a "canon- (daughter of sevenless), and Caenorhabditis elegans homo- ical" (PXXPXR) and "atypical" (PXXXRXXKP) SH3 bind- log SOC1 (suppressor of clear). Family members show a ing motif (9). GRB2 is the main upstream regulator of GAB2 40% to 50% sequence homology but are associated with and indirectly recruits it to the activated plasma membrane unique cellular functions (1). Both GAB1 and GAB2 are receptors. These include RTKs (EGFR, KIT), cytokine expressed ubiquitously, but they are most highly expressed receptors (IL-1, IL-3, IL-15, TPO, EPO, KITL, M-CSF, inbrain,kidney,lung,heart,testis,andovary(2).GAB3 Flt310, gp130), Fc receptors (FceR1, FcgR1), T- and B-cell also has a widespread expression pattern, although it is antigen receptors, and G-protein–coupled receptors (2). most highly expressed in lymphoid tissue (2). The role of GRB2 binds GAB2 via its C-terminal SH3 domain, and GAB proteins in signal transduction has been extensively the complex associates with membrane receptors by binding to phosphorylated tyrosine residues on their intracellular Authors' Afﬁliation: Department of Dermatology, Columbia University domain (10). Certain receptors do not have GRB2 binding Medical Center, New York sites, such as the b-chain of IL-2 and IL-3 receptors. These Corresponding Author: Julide T. Celebi, Columbia University, Russ Berrie signaling cascades require a SHC protein to serve as an Pavillion, 1150 St. Nicholas Avenue, Room 318, New York, NY 10032. additional bridging adaptor between tyrosine-phosphorylat- Phone: 212-851-4546; Fax: 212-851-4540; E-mail: [email protected] ed receptors and the GRB2–GAB2 complex (11, 12). doi: 10.1158/1541-7786.MCR-12-0352 GAB2 harbors multiple tyrosine residues that are phos- 2012 American Association for Cancer Research. phorylated upon activation of signal transduction. These

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Receptor

Shc Figure 1. Schematic diagram of Gab2 Grb2 GAB2 signaling and the GAB2 protein structure. The two main P85 Shp2 effector arms of GAB2 signaling, P110 SHP2-ERK and PI3K-AKT, are shown. Structural domains of the GAB2 protein including an N- Ras terminal PH domain critical for Akt membrane localization and central Grb2* Grb2† proline-rich regions mediating interactions with SH2- and SH3- Raf Gab2 676 domain containing proteins are indicated. Interacting partners of Crk Shp2 GAB2 such as GRB2, p85, SHP2, γ p85 Mek PLC CRK, PLCg, and 14-3-3 are shown. 14-3-3 14-3-3 Survival, growth

Erk

Proliferation, migration, differentiation

critical phospho-tyrosine moieties are capable of interacting and SHIP-2 are SH2 domain–containing inositol 50-phos- – with Src homology 2 (SH2) domain containing proteins phatases that hydrolyze PIP(3,4,5)3 to PIP(3,4)2 and have and mediate the interaction of GAB2 with downstream been shown to associate with GAB2 (16, 17). Both SHIP-1 effectors, such as SHP2, p85, PLC-g, CRK, SHC, and and -2 act as negative regulators of mast cell activation SHIP (6). GAB2 plays a central role in the propagation of upon high affinity IgE receptor (FceRI) stimulation (17, mitogen-activated protein kinase (MAPK) and PI3-K-AKT 18). GAB2 is also involved in propagating the type I IFN signaling pathways. Binding of GAB2 to SHP2 activates response and likely competes with interferon (alpha, beta and extracellular signal-regulated kinase (ERK; GAB2–SHP2– omega) receptor (IFNAR 2) for binding to IFNAR 1 (19). ERK) and MAPK signaling. This well-defined relationship is dependent on tyrosine residues Y614 and Y643 in GAB2, Genomic Studies in Cancer which engage the SH2 domain of SHP2 (6). Conversely, The DNA amplification is a common mechanism leading docking of the P85 subunit of phosphoinositide-3 kinase to oncogenic activation in human cancer. GAB2 is located on (PI3-K) onto Y452, Y476, and Y584 of GAB2 leads to the chromosomal band 11q14.1. Amplification of 11q13-14.1 activation of AKT (GAB2–p85–AKT; ref. 6). is frequently observed in human malignancies (20). CCND1 GAB2 forms complexes with a variety of proteins in which located on 11q13.2 has long been considered the key gene functional significance has not been fully characterized. In driving this amplicon (21, 22). However, the large size of osteoclasts, PLC-g2 binds GAB2, is required for GAB2 11q13-14.1 (9–10 Mb), the presence of other potential phosphorylation, and modulates GAB2 recruitment to proto-oncogenes in this region, and the identification of receptor activator of NF-kB (RANK), thereby regulating narrow amplicons telomeric and distinct from CCND1 osteoclastogenesis (13). 14-3-3 Proteins have an inhibitory suggest the possibility of multiple drivers underlying these effect on GAB2 signaling. They bind to S210 and T391 in a amplifications. Studies have identified several candidates phosphorylation-dependent manner and attenuate GAB2- including EMS1, EMSY, PAK1, RSF1, and GAB2 (23). mediated signal transduction. This may be due to the The identification of GAB2 as a potential oncogene comes disassembly of GAB2–GRB2 complexes or shifting of the from studies in breast (24, 25) and ovarian cancer (23), equilibrium between GAB2–GRB2 and GAB2–14-3-3 leukemia (26), and melanoma (27). 11q13-14.1 is amplified complexes favoring the latter (14). A GTP-ase activating in approximately 10% to 15% of breast cancers (28), and protein for the Rho family, named Rho GTPase activating GAB2 is overexpressed in human primary breast cancer protein 32 (GC-GAP), interacts with both GAB1 and tumors and cell lines as compared with normal ductal GAB2. GC-GAP is highly expressed in the brain, colocalizes epithelium (29). Eight percent of human breast carcinomas with GAB2 at the cell membrane, and stimulates GTPase show upregulation of GAB2 by gene expression analysis, activity of the Rho family of small GTPases (15). SHIP-1 and a subset of these have concomitant genomic

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amplification (24). Evaluation of DNA copy number was shown to be tyrosine-phosphorylated by c-SRC in changes in 172 primary breast cancers by comparative response to EGF treatment, leading to downstream activa- genomic hybridization shows a proximal amplicon at tion of PI3-K (33). Both GAB2 and c-SRC are overexpressed 11q13.2-13.3 spanning CCND1 in 28% of cases and a in breast cancer, and their cotransfection in MCF10A cells distal amplicon at 11q13.5-14.1 with a 15% frequency (25). promotes a growth advantage and EGF-independence (34). The distal amplicon occurs without coamplification at In addition, expression of GAB2 together with the viral CCND1 in 3% of cases, suggesting a distinct driver event. oncoprotein v-SRC (35) or the activated c-SRC-Y527F The smallest common region spans ALG8, KCTD21, mutant (36) brings about the dissociation of 3-dimensional USP35, and GAB2. spheroids in culture, creating a highly dispersed and dis- Similar copy number profiles are observed in melanoma. rupted acinar morphology. This is thought to be mediated GAB2 was amplified in 11% of 64 human metastatic via localized GAB2–SRC–PI3-K signaling at the cell–cell melanoma samples and 20 cell lines examined by array- junction, which disrupts the integrity and binding strength comparative genomic hybridization, and a subset occurred of E-cadherin with resultant loss of cellular adhesion (34). without coamplification of CCND1 (27). When taken Furthermore, these cells show increased migration and together, these genomic studies validate the presence of a invasion, but these effects are independent of PI3-K activa- distinct amplicon in human cancer at 11q13.5-14.1 that is tion (34). Finally, overexpression of GAB2 in MCF10A cells independent of the CCND1 locus. Functional studies promotes anchorage-independent growth, leading to larger implicate GAB2 as a driving event (24, 27). and more numerous colonies on soft agar. These effects are dependent on SRC signaling and the downstream activation GAB2 and Breast Cancer of the oncogenic transcription factor, STAT3 (37). The mechanisms by which GAB2 contributes to breast Additional insight into the role of GAB2 on cell motility, cancer pathogenesis are beginning to be elucidated (29, 30). invasiveness, and thus metastatic spread is obtained through GAB2 contributes to an invasive and metastatic phenotype studies examining its effects on Rho family of GTPases. in breast carcinogenesis. Overexpression of GAB2 in RhoA promotes the formation of contractile actomyosin MCF10A cells, an immortalized human mammary epithelial stress fibers and focal adhesions. GAB2, when overexpressed cell line, results in increased proliferation and altered depen- in MCF10A mammary epithelial cells, was found to decrease dency on EGF and other growth factors (31). Conversely, activation of RhoA, lead to delayed cell spreading, a decrease silencing of GAB2 in several breast cancer cell lines harboring in stress fibers and mature focal adhesions, and enhanced cell genomic amplifications leads to a decrease in proliferation, migration (38). Modulation of focal adhesions and stress attributing to slowed cell-cycle progression and increased fibers by GAB2 was dependent on the SHP2 binding sites. apoptosis, and a reduction in their invasive potential (25). These studies shed light onto mechanisms of GAB2-medi- Studies show that GAB2 alone is insufficient to transform ated cancer progression. primary mammary epithelial cells. However, it can cooperate GAB2 signaling in breast cancer results in the activation of with other proto-oncogenes, such as ErbB2 (Neu or HER2) numerous downstream effectors that contributes to cancer and Src, to potentiate tumorigenic signaling. GAB2 acts development. A genomic "signature" of 205 transcripts was downstream of ErbB2 and is tyrosyl-phosphorylated upon obtained from GAB2 overexpressing, anchorage-indepen- activation of signal transduction (24). GAB2 and ErbB2 are dent MCF10A cells and was found to correlate with met- coamplified in a subset of breast cancers, and coexpression of astatic relapse in patients, independent of existing clinical GAB2 with ErbB2 leads to an invasive multiacinar pheno- and genomic classifiers (37). This suggests a novel role for type in a 3-dimensional culture system. This effect is GAB2 and its transcriptional targets as a biomarker for the mediated via downstream GAB2–SHP2–ERK signaling and prediction of metastatic disease. is independent of PI3-K-AKT activation (24). Transgenic miRNAs posttranscriptionally repress gene expression mice with mammary-specific overexpression of GAB2 do mostly by recognizing complementary target sites in the not show a significant tumor phenotype. However, when 30-untranslated region of target mRNAs. The human let-7 crossed with mice overexpressing ErbB2, an accelerated rate miRNA family consists of 13 members located in 8 genomic of tumor onset is observed (24). Notably, in ErbB2-driven regions frequently deleted in human cancers. Among the let- transgenic mouse models, the deletion of GAB2 has only a 7 family of miRNAs, let-7g was found to have reduced minor effect on the initiation and growth of mammary expression that correlated significantly with lymph node tumors but strongly suppresses the development of pulmo- metastasis and poor survival in patients with breast cancer nary metastases. In this model, GAB2-deficient tumor cells (39). Repression of let-7g expression led to increased GAB2 proliferate normally but display impaired motility in vitro, and fibronectin I expression, which, in turn, cooperatively which can be rescued by the introduction of wild-type increased the activity of ERK, matrix metalloproteinase GAB2. These effects are mediated by impaired activation (MMP-2), and MMP-4. This exemplifies yet another mech- of ERK/MAPK signaling and occur independently of the anism of GAB2 regulation in breast carcinogenesis. AKT pathway (32). GAB2 not only cooperates with RTK but also with GAB2 and Leukemia receptor-associated kinases of the SRC family. This relation- GAB2 regulates hematopoietic cell survival and prolifer- ship was first defined in primary hepatocytes in which GAB2 ation. Although, GAB2 knockout mice are viable and grossly

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normal in appearance, they harbor certain select defects as GAB2 is a direct binding partner of SHP2 and is required a result. These mice lack a proper allergic response, which is for the transformation of primary murine myeloid cells the result of impaired FceRI stimulation in mast cells secondary to its ability to activate ERK, AKT, and STAT5 and is PI3-K dependent (40). Their bone marrow is osteo- signaling (56). A knockin mouse model with a SHP2D61G petrotic due to the decreased RANK-mediated osteoclast -activating mutation leads to hyperactive stem cell function- differentiation (41) and, while their bone marrow cellularity ing with accelerated stem cycling, expansion of the stem cell and peripheral blood counts are normal, there is defective pool, and improved repopulating capacities, which can bring hematopoiesis as a result of decreased proliferation and about the myeloproliferative disease. This aberrant hemato- attenuation of PI3-K-AKT and ERK/MAPK signaling in poietic signaling is rescued by the concomitant deletion of response to early-acting cytokines (42). In addition, GAB2 GAB2, which highlights this adaptors centrality in promot- ablation results in defective expansion of colony stimulating ing oncogenesis (57). factor 1 receptor–dependent mononuclear phagocyte pro- genitors in the bone marrow through decreased proliferation GAB2 and Melanoma and survival (43). The development of melanoma is inextricably linked to GAB2 mediates a critical role in leukemogenic signaling oncogenic activation of ERK and PI3-K-AKT signaling driven by oncogenic fusion tyrosine kinases. This has been (58, 59). GAB2 scaffolding protein is central to the prop- best studied in chronic myelogenous leukemia (CML), agation of these signaling cascades and has been implicated which is caused by a translocation between chromosome as a driver of melanomagenesis. GAB2 is expressed at 9 and 22; t(9;22)(q34;q11), creating BCR-ABL, a consti- significantly higher levels in metastatic melanomas as com- tutively active fusion tyrosine kinase capable of transforming pared with primary melanomas and melanocytic nevi, and hematopoietic stem cells (44). The autophosphorylated can thus be seen as a marker of neoplastic progression. Tyr177 residue on BCR-ABL recruits the GRB2–GAB2 Silencing of GAB2 in metastatic melanoma cell lines leads complex, which can then activate several divergent pathways to a decrease in their invasive potential, whereas overexpres- involved in promoting leukemogenesis, including RAS– – – sion in primary melanomas promotes migration and inva- RAF MEK ERK, PI3-K-AKT, and JAK-STAT (45). sion. In addition, in vivo studies show that overexpression of GAB2 is required for myeloid cell, but not lymphoid cell, GAB2 leads to enhanced tumor growth and development of transformation by BCR-ABL. Cells expressing mutant – – metastases (27). BCR ABL T177F show impaired ERK and AKT activation Although GAB2 cannot independently transform mela- with diminished proliferation and spontaneous migration nocytes, it can cooperate with other oncogenes to promote (45). In addition, silencing of GAB2 or its downstream tumor development. GAB2 is coexpressed with mutant effectors, SHP2 and STAT5, leads to a decrease in cell NRAS in a subset of melanomas, and these proteins act in proliferation and colony formation in BCR-ABL–mediated concert to promote a more aggressive phenotype. GAB2 CML (46). Notably, GAB2 has a similar function in prop- overexpression leads to increased metastatic potential with agating oncogenic signaling derived from other fusion onco- anchorage independence in soft agar, and xenograft studies proteins including TEL-ABL (47), TEL-JAK2 (48), NPM- show angiogenic switch as a result of upregulation of HIF-1a ALK (49), and BCR-FGFR1 (50) as well as in SF-STK and VEGF (60). activation by Friend virus in murine erythroleukemia (51). Treatment of CML with imatinib, a small-molecule inhibitor of BCR-ABL, has led to significant clinical success. GAB2 and Ovarian Cancer However, resistance mechanisms have evolved including Genomic amplifications of GAB2 have been described in continuous activation of v-yes-1 Yamaguchi sarcoma viral approximately 15% of ovarian cancers (23). GAB2 over- related oncogene homolog (LYN) kinase, which complexes expression is also seen at the mRNA level in serous cysta- with GAB2 and results in persistent phosphorylation of denocarcinoma tumors and at the protein level in several GAB2 and BCR–ABL. Downstream leukemogenic signal- ovarian cancer cell lines (61). Exogenous overexpression of ing persists in the presence or absence of imatinib, and it is GAB2 in cell lines with low baseline expression promotes only in silencing of LYN that drug sensitivity is restored (52). characteristics of the epithelial-to-mesenchymal transition. Persistent activation of the transcription factor STAT5 is These cells have increased migratory and invasive potential, frequently found in hematologic malignancies. GAB2 sig- assessed by wound healing and transwell migration assays, nals upstream of JAK-STAT and is an essential mediator of and show downregulation of E-cadherin. Conversely, silenc- hematopoietic stem cell homeostasis (6). STAT5 and GAB2 ing of GAB2 in cell lines with high expression inhibits double mutant mice show a significant reduction in stem cell migration and invasion and causes upregulation of E-cad- number, reduced cell survival, and a dramatic loss of self- herin. GAB2 mediates these effects by activating the PI3-K renewing potential (53). On the other hand, GAB2-medi- pathway, which results in increased expression of Zeb1, a ated PI3-K-AKT activation potentiates cell growth and transcription factor involved in epithelial-to-mesenchymal survival in STAT5-induced leukemia (54). transition (61). This novel GAB2–PI3-K–ZEB1 pathway GAB2 is a key mediator of leukemogenic signaling driven can be targeted by PI3-K and mTOR inhibitors and can by SHP2 mutations. Somatic gain of function mutations in perhaps serve as a therapeutic target in the treatment of SHP2 causes juvenile myelomonocytic leukemia (55). GAB2-driven ovarian cancer.

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Disclosure of Potential Conﬂicts of Interest Acknowledgments No potential conﬂicts of interest were disclosed. The authors thank Dr. Bin Zheng for his critical review of the manuscript. Because of space constraints, it is impossible to reference every article in this review. We apologize for any oversights. Authors' Contributions Conception and design: J.T. Celebi, I.T. Aydin Grant Support Analysis and interpretation of data (e.g., statistical analysis, biostatistics, compu- This work was supported by a grant from the NIH, R01 CA138678. tational analysis): J.T. Celebi Writing, review, and/or revision of the manuscript: J.T. Celebi, S.J. Adams, I.T. Aydin Received June 14, 2012; revised July 30, 2012; accepted July 31, 2012; Study supervision: J.T. Celebi published OnlineFirst August 7, 2012.

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1270 Mol Cancer Res; 10(10) October 2012 Molecular Cancer Research

GAB2−−a Scaffolding Protein in Cancer

Sarah J. Adams, Iraz T. Aydin and Julide T. Celebi

Mol Cancer Res 2012;10:1265-1270. Published OnlineFirst August 7, 2012.

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