Grb7 Protein Research Articles

Novel Nonphosphorylated Peptides with Conserved Sequences Selectively Bind to Grb7 SH2 Domain with Affinity Comparable to Its Phosphorylated Ligand

The Grb7 (growth factor receptor-bound 7) protein, a member of the Grb7 protein family, is found to be highly expressed in such metastatic tumors as breast cancer, esophageal cancer, liver cancer, etc. The src-homology 2 (SH2) domain in the C-terminus is reported to be mainly involved in Grb7 signaling pathways. Using the random peptide library, we identified a series of Grb7 SH2 domain-binding nonphosphorylated peptides in the yeast two-hybrid system. These peptides have a conserved GIPT/K/N sequence at the N-terminus and G/WD/IP at the C-terminus, and the region between the N-and C-terminus contains fifteen amino acids enriched with serines, threonines and prolines. The association between the nonphosphorylated peptides and the Grb7 SH2 domain occurred in vitro and ex vivo. When competing for binding to the Grb7 SH2 domain in a complex, one synthesized nonphosphorylated ligand, containing the twenty-two amino acid-motif sequence, showed at least comparable affinity to the phosphorylated ligand of ErbB3 in vitro, and its overexpression inhibited the proliferation of SK-BR-3 cells. Such nonphosphorylated peptides may be useful for rational design of drugs targeted against cancers that express high levels of Grb7 protein.

New unphosphorylated peptide motif interacts with Grb7 SH2 domain

The Grb7 protein is found to be highly expressed in metastatic tumors. And the SH2 domain in Grb7 C‐terminal is reported to be mainly involved in Grb7 signal pathways. Currently, most reported motifs interacting with Grb7 SH2 domain are phosphotyrosine motifs. This domain can also bind dephosphorylated motifs steadily from degradation of phosphatases. By yeast two hybrid screening random peptide library, we have found some nonphosphorylated consensus sequences interacting with Grb7 SH2 domain. All positive peptides comprised a 22 amino acids sequence, containing 7 conserved ones at the two terminals and 15 random ones in the interval including S, T and P residues. This new way of interaction was not a common phenomenon, for we did not detect the interaction between these peptides and other SH2 domain proteins such as ABL, Grb2 and Grb14. Deleting assays in yeast two hybrid system showed that 7 conserved amino acids were necessary for interaction between Grb7 SH2 domain and nonphosphotyrosine peptides. To further confirm the interaction, we selected two peptides and a mutation which was short of 7 conserved sequences and co‐transfected into 293T cells with Grb7 SH2 domain. We found 7 conserved residues played essential roles in interacting with Grb7 SH2 domain in co‐IP assay. In further experiments, it is interested to see if those dephosphorylated peptides as drugs against cancer with high expression of Grb7 protein.

Grb7 binds to Hax‐1 and undergoes an intramolecular domain association that offers a model for Grb7 regulation

Adaptor proteins mediate signal transduction from cell surface receptors to downstream signaling pathways. The Grb7 protein family of adaptor proteins is constituted by Grb7, Grb10, and Grb14. This protein family has been shown to be overexpressed in certain cancers and cancer cell lines. Grb7-mediated cell migration has been shown to proceed through a focal adhesion kinase (FAK)/Grb7 pathway, although the specific participants downstream of Grb7 in cell migration signaling have not been fully determined. In this study, we report that Grb7 interacts with Hax-1, a cytoskeletal-associated protein found overexpressed in metastatic tumors and cancer cell lines. Additionally, in yeast 2-hybrid assays, we show that the interaction is specific to the Grb7-RA and -PH domains. We have also demonstrated that full-length Grb7 and Hax-1 interact in mammalian cells and that Grb7 is tyrosine phosphorylated. Isothermal titration calorimetry measurements demonstrate the Grb7-RA-PH domains bind to the Grb7-SH2 domain with micromolar affinity, suggesting full-length Grb7 can exist in a head-to-tail conformational state that could serve a self-regulatory function.

Abstract P4-08-03: False Positive HER-2 Testing by Fluorescence In Situ Hybridization in Human Breast Cancer

Abstract Background: Testing for human epidermal growth factor receptor-2 (HER-2) is routinely performed after breast cancer diagnosis by either immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH). FISH is more accurate and reproducible, often considered the gold standard, and a more reliable predictor of a patient's response to HER-2 directed therapies in clinical practice. The growth factor receptor-bound protein-7 gene (GRB7) encodes a multi-domain signal transduction molecule located in close proximity to HER-2 on chromo some 17q11-12 and has been shown to be an independent adverse prognostic marker in breast cancer. Methods: We performed western blotting analysis of protein extracts from 563 annotated frozen breast tumors, collected from 1988 - 1998. GRB7 and HER-2 bands were assigned low or high values compared to specific protein controls, and tubulin bands. HER-2 FISH was performed on a subset of these tumors using an FDA approved Path Vysion kit on 4 μm frozen sections. HER-2 and chromosome 17 centromere (CEP17) signals were enumerated according to ASCO/CAP guidelines. Results: Sixty-six tumors (11.5%) over-expressed HER-2 and GRB7 proteins by Western analysis, 66 (11.5%) over-expressed HER-2 but not GRB7, and 30 (5.5%) over-expressed GRB7 but not HER-2. All 32 tumors (32/32) submitted to FISH analysis that over-expressed both HER-2 and GRB7 proteins demonstrated HER-2 gene amplification, while only one out of thirty-five (1/35) tumors that over-expressed HER-2 without GRB7 protein over-expression were HER-2 amplified. Interestingly, 7 of 28 tumors over-expressing GRB7 but not HER-2 protein were amplified for the HER-2 gene. Clinical laboratory HER-2 IHC testing confirmed the absence of HER-2 protein over-expression in all 7 of these tumors. Conclusions: Our study demonstrates that about ten percent of human breast cancers that test positive with FISH for HER-2 gene amplification do not over-express HER-2 protein, which may influence sensitivity to HER-2 directed therapy. All tumors that show false positive HER-2 gene amplification over-express GRB7 protein, suggesting the presence of GRB7 driven gene amplification in these tumors. Given that the HER-2 FISH probe contains GRB7 sequences, it is possible that HER-2 FISH false positivity may reflect GRB7 gene amplification. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-08-03.

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

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.

GRB7 protein over-expression and clinical outcome in breast cancer

The growth factor receptor-bound protein-7 gene (GRB7) encodes a multi-domain signal transduction molecule. The purpose of this study was to examine the clinical significance of GRB7 protein expression in human breast cancer. Western blotting analysis of protein extracts from 563 annotated frozen breast tumors was performed. Expression status of GRB7 and HER-2 was correlated with clinical covariates and outcomes. Cox proportional hazards were used to identify factors associated with breast cancer-free interval. The median follow-up was 71 months. P values <0.05 were considered statistically significant (two-sided). A discrepancy between HER-2 and GRB7 protein over-expression was observed. GRB7 protein over-expression was associated with negative estrogen and progesterone receptor status, higher tumor grade, larger primary tumor size, (more) axillary lymph node involvement, higher clinical stage, and shortened breast cancer-free interval. HER-2 protein over-expression was associated only with higher tumor grade. Multi-variate analysis revealed that GRB7 protein over-expression was an independent adverse prognostic factor for breast cancer-free interval (hazard ratio 1.69, 95% confidence interval 1.07-2.67; P = 0.024). The same was true of the subset of patients who did not receive any adjuvant systemic therapy (hazard ratio 1.68, 95% confidence interval 1.16-2.31; P = 0.0055). Using FISH analysis, 32/32 (100%; 95% CI 89-100%) tumors which over-expressed both HER-2 and GRB7 proteins and 1/35 (3%; 95% CI 0-15%) tumors with HER-2 but no GRB7 protein over-expression with Western blotting analysis demonstrated HER-2 gene amplification. GRB7 protein over-expression is an independent adverse prognostic factor in human breast cancer.

GRB7 and HER2 protein overexpression and breast cancer outcome

11102 Background: The growth factor receptor-bound protein-7 gene (GRB7) encodes a multi-domain signal transduction molecule and is located in close proximity to human epidermal growth factor receptor 2 (HER2) on chromosome 17q11–12. This study examines the roles of GRB7 protein in human breast cancer. Methods: We performed western blotting analysis of protein extracts from 563 annotated frozen breast tumors, collected from 1988 - 1998. GRB7 and HER2 bands were assigned low or high values compared to specific protein controls, and tubulin bands. Chi-square tests were used to test the hypothesis that there was no association between expression status of GRB7 or HER2 with clinical covariates and outcomes. Univariate Cox regression was performed to identify risk factors and Cox proportional hazards backward step model for variable selection to identify independent predictors of progression free survival. All P values were two sided. P values less than 0.05 were considered statistically significant. HER 2 gene amplification status was determined by fluorescence in situ hybridization (FISH). Results: GRB7 protein over-expression was associated with negative estrogen (&lt; 5 fmole/mg protein) and progesterone receptor (&lt; 10fmole/mg protein) status, higher grade (1, 2, or 3), larger primary tumor size (&lt; 2.0 vs. &gt; 2.0 cm), (more) axillary lymph node involvement (continuous), higher clinical stage (1, 2, 3, or 4) and shortened progression free survival (months). We found a discrepancy in HER2 and GRB7 protein over-expression. Isolated GRB7 protein over-expression correlated with inferior progression free survival, while HER2 protein over-expression without GRB7 protein over-expression did not. Multivariate analysis revealed that GRB7 over-expression was an independent predictor of progression free survival (hazard ratio 1.69: 95% confidence interval, 1.073 - 2.672; P = 0.0236). All 18 tumors submitted to FISH analysis that over-expressed both HER2 and GRB7 proteins and no tumors (0 / 10) with HER2 but no GRB7 protein over-expression demonstrated HER2 gene amplification. Conclusions: GRB7 protein over-expression is an independent adverse prognostic factor in breast cancer. No significant financial relationships to disclose.

The cell migration protein Grb7 associates with transcriptional regulator FHL2 in a Grb7 phosphorylation‐dependent manner

Grb7 is an adaptor molecule that can mediate signal transduction from multiple cell surface receptors to various downstream signaling pathways. Grb7, along with Grb10 and Grb14, make up the Grb7 protein family. This protein family has been shown to be overexpressed in certain cancers and cancer cell lines. Grb7 and a receptor tyrosine kinase (RTK), erbB2, are overexpressed in 20-30% of breast cancers. Grb7 overexpression has been linked to enhanced cell migration and metastasis, though the participants in these pathways have not been determined. In this study, we report that Grb7 interacts with four and half lim domains isoform 2 (FHL2), a transcription regulator with an important role in oncogenesis, including breast cancer. Additionally, in yeast 2-hybrid (Y2H) assays, we show that the interaction is specific to the Grb7 RA and PH domains. We have also demonstrated that full-length (FL) Grb7 and FHL2 interact in mammalian cells and that Grb7 must be tyrosine phosphorylated for this interaction to occur. Immunofluorescent microscopy demonstrates possible co-localization of Grb7 and FHL2. A model with supporting NMR evidence of Grb7 autoinhibition is proposed.

Genomic Organization and Control of the Grb7 Gene Family

Grb7 and their related family members Grb10 and Grb14 are adaptor proteins, which participate in the functionality of multiple signal transduction pathways under the control of a variety of activated tyrosine kinase receptors and other tyrosine-phosphorylated proteins. They are involved in the modulation of important cellular and organismal functions such as cell migration, cell proliferation, apoptosis, gene expression, protein degradation, protein phosphorylation, angiogenesis, embryonic development and metabolic control. In this short review we shall describe the organization of the genes encoding the Grb7 protein family, their transcriptional products and the regulatory mechanisms implicated in the control of their expression. Finally, the alterations found in these genes and the mechanisms affecting their expression under pathological conditions such as cancer, diabetes and some congenital disorders will be highlighted.

Role of Growth Factor Receptor–Bound Protein 7 in Hepatocellular Carcinoma

The human growth factor receptor-bound protein 7 (Grb7) is an adaptor molecule and is related to cell invasion. In this present study, we investigated the clinical and biological significance of Grb7 expression in human hepatocellular carcinoma (HCC). We reviewed 64 consecutive patients who had undergone liver resection for HCC, and we investigated the correlation between Grb7 expression and clinical outcome. To analyze the biological behavior of Grb7 in vitro and in vivo, we established Grb7 stable knockdown HCC cells using RNA interference technology. The positive staining of Grb7 protein was correlated with portal venous invasion (P < 0.01), hepatic venous invasion (P < 0.01), and intrahepatic metastasis (P < 0.05). Positive expression of Grb7 was significantly correlated with focal adhesion kinase (FAK) protein levels in HCC (P < 0.01). The Grb7- and FAK-positive group showed a significantly poorer prognosis as compared with the Grb7- and FAK-negative group (P < 0.05). Grb7 knockdown HCC cells exhibited significantly lower levels of invasion potential (P < 0.05) and motility (P < 0.05) than the control cells in vitro; moreover, Grb7 knockdown HCC cells showed delayed onset of the tumors compared with the control cells in vivo. Grb7 expression can modulate the invasive phenotype of HCC. Grb7 plays an important role in HCC progression and is strongly associated with expression of FAK. Grb7 could be a therapeutic target in HCC.

Calorimetric investigation of phosphorylated and non‐phosphorylated peptide ligand binding to the human Grb7‐SH2 domain

Grb7 is a member of the Grb7 family of proteins, which also includes Grb10 and Grb14. All three proteins have been found to be overexpressed in certain cancers and cancer cell lines. In particular, Grb7 (along with the receptor tyrosine kinase erbB2) is overexpressed in 20-30% of breast cancers. In general, growth factor receptor bound (Grb) proteins bind to activated membrane-bound receptor tyrosine kinases (RTKs; e.g., the epidermal growth factor receptor, EGFR) through their Src homology 2 (SH2) domains. In particular, Grb7 binds to erbB2 (a.k.a. EGFR2) and may be involved in cell signaling pathways that promote the formation of metastases and inflammatory responses. In previous studies, we reported the solution structure and the backbone relaxation behavior of the Grb7-SH2/erbB2 peptide complex. In this study, isothermal titration calorimetry studies have been completed by measuring the thermodynamic binding parameters of several phosphorylated and non-phosphorylated peptides representative of natural Grb7 receptor ligands as well as ligands developed through combinatorial peptide screening methods. The entirety of these calorimetric studies is interpreted in an effort to describe the specific ligand binding characteristics of the Grb7 protein.

Transduced PEP-1-Grb7 Fusion Protein Suppressed LPS-induced COX-2 Expression

Although the incidence and severity of atopic dermatitis (AD) is steadily increasing at an alarming rate, its pathogenic mechanisms remain poorly understood yet. Recently, we found that the expression of Grb7 protein was markedly decreased in AD patients using proteomic analysis. In the present study, human Grb7 gene was fused with PEP-1 peptide in a bacterial expression vector to produce a genetic in-frame PEP-1-Grb7 fusion protein. The expressed and purified PEP-1-Grb7 fusion proteins transduced efficiently into skin cells in a time- and dose-dependent manner when added exogenously in culture media. Once inside the cells, the transduced PEP-1-Grb7 protein was stable for 48 h. In addition, transduced PEP-1-Grb7 fusion protein markedly increased cell viability in macrophage RAW 264.7 cells treated with LPS by inhibition of the COX-2 expression level. These results suggest that the PEP-1-Grb7 fusion protein can be used in protein therapy for inflammatory skin disorders, including AD.

The adaptor Grb7 links netrin-1 signaling to regulation of mRNA translation

We previously reported a novel biological activity of Netrin-1 in translational stimulation of kappa opioid receptor (KOR). We now identify Grb7 as a new RNA-binding protein that serves as the molecular adaptor for transmitting Netrin-1 signals, through focal adhesion kinase (FAK), to the translation machinery. Grb7 binds specifically to the first stem loop of kor mRNA 5'-UTR through a new RNA-binding domain located in its amino terminus. Upon binding to its capped, target mRNA, Grb7 blocks the recruitment of eIF4E, rendering mRNA untranslatable. The RNA-binding and translation-repressive activity is reduced by FAK-mediated hyperphosphorylation on two tyrosine residues of its carboxyl terminus. This study reports an adaptor protein Grb7 that transmits the stimulating signals of Netrin-1 to the translational machinery to rapidly regulate mRNA translation.

Specific Peptide Ligand for Grb7 Signal Transduction Protein and Pancreatic Cancer Metastasis

Pancreatic cancer is one of the most aggressive malignancies, with high rates of invasion and metastasis and with generally poor prognosis. We previously found that metastasis was strongly associated with the expression of growth factor receptor-bound protein 7 (Grb7), which contains a Src homology 2 (SH2) domain. In this study, we evaluated Grb7 protein as a molecular target of therapy for metastatic pancreatic cancer. Grb7 protein expression was measured by immunohistochemistry in 36 human pancreatic cancer specimens and adjacent normal pancreatic tissue. We synthesized a nonphosphorylated peptide inhibitor that binds specifically to the SH2 domain of Grb7. Intracellular signaling was assessed by immunoprecipitation and immunoblot assays in cultured human pancreatic cancer cells. Cell migration was measured with a modified Boyden chamber method. Peritoneal metastasis of the pancreatic cancer cells was measured with a mouse model. All statistical tests were two-sided. We found that 22 (61%) of 36 pancreatic cancer specimens had higher levels of Grb7 protein than their corresponding normal pancreatic tissue specimens. Grb7 expression was statistically significantly different between specimens from patients without lymph node metastasis (stage N0; two of the 10 patients) and patients with lymph node metastasis (stages N1 + N2; 20 of the 26 patients) (P = .006). The Grb7 peptide inhibitor selectively blocked the interaction between Grb7 and focal adhesion kinase and blocked the phosphorylation of Grb7 protein. In vivo Grb7 peptide inhibitor statistically significantly attenuated cell migration (for control peptide, 87.5 cells migrated, 95% confidence interval [CI] = 82.6 to 92.4 cells; for Grb7 peptide, 5.7 cells migrated, 95% CI = 2.3 to 9.0 cells; P < .001) and peritoneal metastasis of the pancreatic cancer cells in a mouse model, as assessed by the number of nodules (control = 72.6 nodules, 95% CI = 55.8 to 89.4 nodules; and for Grb7 peptide = 3.2 nodules, 95% CI = 1.6 to 4.8 nodules; P < .001, t test) and their weight (control = 4.13 g, 95% CI = 3.40 to 4.86 g; Grb7 peptide = 0.19 g, 95% CI = 0.06 to 0.32 g; P < .001, t test). The Grb7 peptide inhibitor appears to be a promising molecularly targeted therapeutic agent against metastatic pancreatic cancer.

Adapter protein connections: The MRL and Grb7 protein families

Adapter protein connections: The MRL and Grb7 protein families

Grb7 signal transduction protein mediates metastatic progression of esophageal carcinoma.

We have previously reported the association of tumor cell invasion with expression of growth factor receptor-bound protein 7 (Grb7). This molecule contains a Src homology 2 (SH2) domain and shares structural homology with a cell migration molecule designated Mig-10 found in Caenorhabditis elegans. In the present study, Grb7 expression was analyzed in human esophageal carcinomas with or without metastatic spread. The Grb7 protein was overexpressed in 14 of 31 esophageal carcinomas as compared to the adjacent normal mucosa (45%) and this finding was significantly correlated with the presence of lymph node metastases. We also identified that Grb7 protein in esophageal carcinoma cells was phosphorylated on tyrosine by epidermal growth factor as well as attachment to extracellular matrix proteins including fibronectin. Such fibronectin-dependent phosphorylation of Grb7 was regulated by integrin signaling that leads to the interaction with focal adhesion kinase protein. Furthermore, ectopic expression of a Grb7-SH2 dominant-negative fragment inhibited the fibronectin-dependent phosphorylation of endogenous Grb7, and reduced migration of esophageal carcinoma cells into fibronectin. Our results suggest a role of Grb7 mediated signal transduction in generation of an invasive cell phenotype against extracellular matrix, and thus contributes to metastatic progression of human esophageal carcinoma.

Expression of Grb7 growth factor receptor signaling protein in kidney development and in adult kidney.

Grb7, a signaling protein whose physiological function is unknown, binds receptor tyrosine kinases important for normal kidney development. By investigating and correlating Grb7 gene expression with that reported for Grb7-binding receptors, we provide clues to Grb7 function(s). RT-PCR and immunoblot were used to demonstrate Grb7 gene and protein expression in the mature kidney. Additional RT-PCR studies detected gene expression in all microdissected adult nephron segments examined, except glomeruli, and in the mouse metanephric kidney from embryonic day 11 (E11) through to day 17 (E17). In situ hybridization at E14 demonstrated the following cellular pattern of localization: Grb7 mRNA in metanephric epithelia of mesenchymal and ureteric bud origin; no expression in the undifferentiated mesenchyme; and little expression in podocyte-destined cells or primitive glomeruli. Grb7 mRNA was also present in the epithelia of the lung and gut at E14. Thus Grb7 may have a basic function in growth factor signaling in terminally differentiated epithelia along the nephron and in developing epithelia in the kidney, lung, and gut. It is localized in a pattern permissive for a role in Her2 and Ret receptor signaling.