Grb7 Protein Research Articles

Abstract LB009: Unveiling the significance of DARPP-32 and its partners, including GRB7 and DKK1, in breast cancer: Integrative insights from patient data and cell line transcriptomics

Abstract DARPP-32 plays a crucial role in regulating both protein-phosphatase-1 (PP-1) and protein kinase A (PKA), its function being reliant on its phosphorylation status. We have previously demonstrated that low DARPP-32 expression is associated with adverse survival of breast cancer patients, particularly those with oestrogen receptor (ER) positive disease; however, there is limited information on how DARPP-32 may alter breast cancer cell behaviour. We have recently published an integrative approach using patient data and cell line transcriptomics to understand the role of DARPP-32 in breast cancer. We analysed the transcriptome of T47D ER positive breast cancer cells following DARPP-32 knockdown, including treatments in combination with 17β-estradiol or PKA inhibitor fragment (6-22) amide. 202 differentially expressed transcripts were identified in DARPP-32 knock down cells and treatment of these cells with 17β-estradiol or PKA inhibitor fragment (6-22) amide led to the differential expression of 193 and 181 transcripts respectively. Differentially expressed transcripts were compared with genes identified as associated with DARPP-32 expression in 1980 breast cancer patients within the METABRIC cohort using an Artificial Neural Network approach (ANN). Several genes overlapped with those identified from cell line transcriptomics, including PTK7, TRAF5, and KLK6. Both DKK1 and GRB7 were identified as genes associated with DARPP-32 expression in patients using ANN, therefore protein expression was determined in a large cohort of early-stage breast cancer patients (n>1000). We demonstrated that DKK1 and GRB7 protein expression was significantly correlated with DARPP-32 Threonine-34 phosphorylation levels within the same samples (all P<0.05). We have now applied an ANN-based integrative data mining approach to identify genes with the largest effect on other genes resulting in an interactome map, which was based on genes identified within the cell line transcriptomics and original ANN. 1878 genes were identified for inclusion within an interactome map in the total patient cohort, whereas 1003 were identified in ER negative disease alone and 2144 in ER positive disease alone. Pathway enrichment analysis identified the ubiquitin proteosome pathway, the insulin pathway-protein kinase B signalling cascade and the RAS pathway. This study underscores the significance of DARPP-32, a central molecular switch, in breast cancer, elucidating its impact on patient prognosis and highlighting potential molecular targets for therapeutic intervention. The integrative approach combining patient data with cell line transcriptomics provides insights into the complex mechanisms underlying breast cancer progression and opens avenues for further research and targeted interventions. Citation Format: Behnaz Saidy, Richa Vasan, Megan-Rose Greener, Rosie Durrant, Adelynn Immanuel, Andrew Green, Emad Rakha, Ian Ellis, Graham Ball, Stewart Martin, Sarah J. Storr. Unveiling the significance of DARPP-32 and its partners, including GRB7 and DKK1, in breast cancer: Integrative insights from patient data and cell line transcriptomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB009.

Up-regulated GRB7 protein in gastric cancer cells correlates with clinical properties and increases proliferation and stem cell properties.

It has been reported that GRB7 is closely related to a variety of human solid tumors, but its role in gastric cancer has not been reported yet. The purpose of this study was to investigate the expression level and intracellular effects of GRB7 in human gastric cancer. Real-time fluorescent quantitative PCR and Western blot were used to detect the expression of GRB7 in gastric cancer cell lines. The immunohistochemical staining and SPSS analysis verified the GRB7 protein expression. Stable gastric cancer cell lines, MTT experiments, clone formation experiments, cell cycle flow cytometry experiments, sphere formation experiments and lateral subpopulation cell sorting experiments were conducted to investigate the role of GRB7 in gastric cancer cells. We found that the expression of GRB7 in gastric cancer cell lines was higher than that of the corresponding normal gastric epithelial cells, and correspondingly higher in gastric cancer tissues than its paired adjacent tissues. GRB7 protein was expressed more highly in cancer tissues than in adjacent tissues. GRB7 protein expression levels were positively correlated with the clinical stage of gastric cancer patients, and negatively correlated with the survival prognosis of patients. GSEA analysis of GRB7 mRNA levels in gastric cancer tissues and normal gastric epithelial tissues from public databases showed that GRB7 may affect cell proliferation and related processes of intracellular stem cells. GRB7 can promote the proliferation of gastric cancer cells and is positively related to the self-renewal ability of gastric cancer stem cells. This study shows that GRB7 molecules highly expressed in gastric cancer tissues can promote the proliferation of gastric cancer cells and increase the proportion of gastric cancer stem cells, so it is expected to become a diagnostic molecule or potential therapeutic target for gastric cancer.

Association of genetic ancestry with HER2, GRB7 AND estrogen receptor expression among Colombian women with breast cancer.

Our previous study reported higher mRNA levels of the human epidermal growth factor receptor 2 (HER2)-amplicon genes ERBB2 and GRB7 in estrogen receptor (ER)-positive breast cancer patients with relatively high Indigenous American (IA) ancestry from Colombia. Even though the protein expression of HER2 and GRB7 is highly correlated, they may also express independently, an event that could change the patients' prognosis. In this study, we aimed to explore the differences in ER, HER2 and GRB7 protein expression according to genetic ancestry, to further assess the clinical implications of this association. We estimated genetic ancestry from non-tumoral breast tissue DNA and assessed tumoral protein expression of ER, HER2, and GRB7 by immunohistochemistry in a cohort of Colombian patients from different health institutions. We used binomial and multinomial logistic regression models to test the association between genetic ancestry and protein expression. Kaplan-Meier and log-rank tests were used to evaluate the effect of HER2/GRB7 co-expression on patients' survival. Our results show that patients with higher IA ancestry have higher odds of having HER2+/GRB7- breast tumors, compared to the HER2-/GRB7- subtype, and this association seems to be stronger among ER-positive tumors (ER+/HER2+/GRB7-: OR=3.04, 95% CI, 1.47-6.37, p<0.05). However, in the multivariate model this association was attenuated (OR=1.80, 95% CI, 0.72-4.44, p=0.19). On the other hand, it was observed that having a higher European ancestry patients presented lower odds of ER+/HER2+/GRB7- breast tumors, this association remained significant in the multivariate model (OR=0.36, 95% CI, 0.13 - 0.93, p= 0.0395). The survival analysis according to HER2/GRB7 co-expression did not show statistically significant differences in the overall survival and recurrence-free survival. Our results suggest that Colombian patients with higher IA ancestry and a lower European fraction have higher odds of ER+/HER2+/GRB7- tumors compared to ER+/HER2-/GRB7- disease. However, this association does not seem to be associated with patients' overall or recurrence-free survival.

Abstract PR-04: Association of HER2/GRB7 co-expression with Indigenous American ancestry and breast cancer clinical-pathological features

Abstract Introduction: Breast cancer is the most prevalent cancer in women worldwide. It is a heterogeneous disease that has been classified into intrinsic subtypes based on gene expression analyses. Previous studies have reported a higher proportion of HER2-positive (HER2+) tumors in Hispanics/Latinas, compared to Non-Hispanic White women, characterized by their aggressive phenotype. A gene expression analysis of breast tumor tissues from Colombian women reported that patients with a higher Indigenous American (IA) ancestry expressed higher mRNA levels of ERBB2, and of other genes in the HER2-amplicon region (17q12) such as GRB7. Although the expression of HER2 and GRB7 is highly correlated, it has been reported that these proteins might also be expressed independently. It is still unclear whether IA ancestry is associated with the co-expression of HER2/GRB7, or only with one of these two proteins. Aim of the study: To explore differences in HER2 and GRB7 protein expression according to genetic ancestry and the presentation of clinical-pathological features in Colombian women with breast cancer. Methods: A total of 517 Colombian breast cancer patients from different health institutions were included. For genetic ancestry estimation, 106 ancestry informative markers were genotyped from non-tumoral DNA. The protein expression of HER2 and GRB7 was evaluated in paraffin blocks with tumoral tissue by immunohistochemistry by a single pathologist. We tested the association between genetic ancestry and protein expression of HER2, GRB7, and the co-expression of both using logistic and multinomial logistic regression models. Results: Higher IA ancestry fractions were observed in patients with HER2+ compared to HER2- tumors (46% vs 41%, respectively, p = 0.013). We found higher odds of having HER2+ (OR=1.65, 95% CI, 1.05-2.63, p=0.0293) and GRB7+ tumors (OR=1.92, 95% CI, 1.03-3.56, p=0.0376) for every 25% increase in the IA ancestry proportion. The association between IA ancestry and HER2/GRB7 co-expression was influenced by estrogen receptor (ER) status. Patients with a higher IA ancestry (&amp;gt;42%) and with ER+ subtypes presented higher odds of having HER2+/GRB7- breast tumors (OR=3.59, 95% CI, 1.11-11.65, p=0.0327), whereas ER- patients with high IA ancestry tended to have HER2+/GRB7+ tumors (OR=2.27, 95% CI, 0.69 - 7.45, p=0.1730). The co-expression of these proteins was found associated with advanced clinical stages (p=0.018), less differentiated tumors (p=0.005), and a higher proliferation index (p&amp;lt;0.001), compared to HER2+/GRB7- and HER2-/GRB7- tumors. Conclusions: Our results confirm the previously reported association between IA ancestry and HER2 expression in breast tumors and suggest that HER2/GRB7 co-expression in patients with high AI ancestry may change according to the ER status. This event also correlates with unfavorable clinical-pathological features. Citation Format: Laura Rey Vargas, Lina Maria Bejarano, Juan Carlos Mejia Henao, Luz Fernanda Sua Villegas, Jhon Faustino Bastidas Andrade, Carlos Andrés Ossa Gómez, Laura Fejerman, Maria Carolina Sanabria Salas, Silvia Juliana Serrano Gomez. Association of HER2/GRB7 co-expression with Indigenous American ancestry and breast cancer clinical-pathological features [abstract]. In: Proceedings of the AACR Virtual Conference: 14th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2021 Oct 6-8. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr PR-04.

Knockdown of growth factor receptor bound protein 7 suppresses angiogenesis by inhibiting the secretion of vascular endothelial growth factor A in ovarian cancer cells

ABSTRACT Growth factor receptor bound protein 7 (GRB7) plays an important role in regulating the growth and metastasis of ovarian cancer. Angiogenesis is the basis for the growth, invasion, and metastasis of malignant tumors. In the current study, we aimed to determine whether GRB7 plays a role in regulating angiogenesis in ovarian cancer. Immunohistochemistry on tissue microarray showed that GRB7 and platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) protein expression were positively correlated in ovarian cancer tissues. GRB7 knockdown suppressed vascular endothelial growth factor A (VEGFA) expression and reduced VEGFA secretion. The effects of GRB7-silenced SKOV-3 cells on human umbilical vein endothelial cells (HUVECs) were evaluated using a transwell cell co-culture model, which showed that knockdown of GRB7 in SKOV-3 cells suppressed HUVEC proliferation, migration, invasion, and tube formation. Moreover, knockdown of GRB7 in SKOV-3 cells downregulated the expression of proteins associated with angiogenesis, including vascular endothelial growth factor receptor-2 (VEGFR2), mitogen-activated protein kinase kinase 1 (MAP2K1/MEK1), extracellular signal-regulated kinases 1 and 2 (ERK1/2), notch receptor 1 (NOTCH1), and delta-like canonical Notch ligand 4 (DLL4) in HUVECs. In conclusion, knockdown of GRB7 in ovarian cancer cells is an attractive potential therapeutic target for the suppression of angiogenesis in ovarian cancer. GRB7 may regulate angiogenesis through VEGFA/VEGFR2 signaling and its downstream pathways.

The prognostic significance of GRB7 protein expression and localization in human breast and ovarian cancers.

Objective: To study GRB7 protein expression in normal human tissues and breast and ovarian cancers, and determine its clinical significance. Results: GRB7 protein was expressed in multiple tissues, including myoepithelial cells of normal breast and fibroadenoma. Cytoplasmic GRB7 expression was seen predominantly in HER-2 positive and, to a lesser extent, triple negative breast cancer. Membrane localization of GRB7 was present in a subset of breast cancers with high cytoplasmic GRB7 expression. Univariate and multivariate analysis found that cytoplasmic GRB7 expression was associated with a negative progesterone receptor status, while membrane GRB7 expression was associated with a negative axillary nodal status. Membrane associated GRB7 expression was present in a subset of ovarian cancers with high cytoplasmic GRB7 expression. Membrane GRB7 expression displayed a trend towards improved recurrence free survival (RFS). Landmark analysis suggested an RFS advantage for ovarian cancers that had GRB7 membrane expression and survived beyond 27 months; GRB7 membrane expression in two or more cores (out of three) predicted an improved RFS. Membrane expression of GRB7 protein was observed in breast cancer cell lines with high GRB7 protein expression in vitro. Conclusion: GRB7 protein membrane expression may be associated with a better prognosis in breast and ovarian cancers. The favorable prognostic value of GRB7 protein membrane expression and its underlying mechanism is worthy of further investigation. Methods: Immunohistochemistry of normal human tissues, breast tissues of various pathologies, and clinically annotated ovarian cancers was performed to correlate the patterns of GRB7 expression with biomarkers or clinical outcome.

Characterization of the full‐length human Grb7 protein and a phosphorylation representative mutant

It is well known the dimerization state of receptor tyrosine kinases (RTKs), in conjunction with binding partners such as the growth factor receptor bound protein 7 (Grb7) protein, plays an important role in cell signaling regulation. Previously, we proposed, downstream of RTKs, that the phosphorylation state of Grb7SH2 domain tyrosine residues could control Grb7 dimerization, and dimerization may be an important regulatory step in Grb7 binding to RTKs. In this manner, additional dimerization-dependent regulation could occur downstream of the membrane-bound kinase in RTK-mediated signaling pathways. Extrapolation to the full-length (FL) Grb7 protein, and the ability to test this hypothesis further, has been hampered by the availability of large quantities of pure and stable FL protein. Here, we report the biophysical characterization of the FL Grb7 protein and also a mutant representing a tyrosine-phosphorylated Grb7 protein form. Through size exclusion chromatography and analytical ultracentrifugation, we show the phosphorylated-tyrosine-mimic Y492E-FL-Grb7 protein (Y492E-FL-Grb7) is essentially monomeric at expected physiological concentrations. It has been shown previously the wild-type FL Grb7(WT-FLGrb7) protein is dimeric with a dissociation constant (Kd) of approximately 11μM. Our studies here measure a FL protein dimerization Kd of WT-FL-Grb7 within one order of magnitude at approximately 1μM. The approximate size and shape of the WT-FL-Grb7 in comparison the tyrosine-phosphorylation mimic Y492E-FL-Grb7 protein was determined by dynamic light scattering methods. In vitro phosphorylation of the Grb7SH2 domain indicates only one of the available tyrosine residues is phosphorylated, suggesting the same phosphorylation pattern could be relevant in the FL protein. The biophysical characterization studies in total are interpreted with a view towards understanding the functionally active Grb7 protein conformation.

Mutational analysis of interdomain binding interface of Grb7

Grb7 is a member of the Grb7 adaptor protein family (Grb7, Grb10 and Grb14) and is a suspected promoter of tumor progression. The multi‐domain Grb7 protein is composed of the Proline‐Rich (PR), Ras‐associating (RA), Pleckstrin Homology (PH), and Src Homology 2 (SH2) domain[Office1] s. Grb7 is observed over‐expressed, along with the receptor tyrosine kinase ErbB2, in 20–30% of breast cancer cells, and is involved in increased cell migration and formation of metastases [1]. It is believed the RA, PH and SH2 domains interact with various downstream signaling partners to potentiate Grb7's role in cell migration [1]. Grb7 undergoes intermolecular homo‐dimerization through its SH2 domain [2] while also undergoing an intramolecular domain interaction via its SH2 and RA‐PH domains [3]. These interactions are believed to affect the Grb7 protein functional state. Based upon our previous results substantiating the Grb7 intramolecular domain interaction, we now seek to characterize the binding interface between the Grb7 RA‐PH, and Grb7 SH2 domains. Our approach is to use mutational analysis to target the RA‐PH and SH2 domain interfaces we hypothesize may be involved in the intramolecular interaction. At present we have successfully produced the SH2 domain mutants V45A, K46R, D47E and S48A by overexpression in a bacterial system and GST fusion‐protein based purification. We have also expressed and purified the Grb7 RA‐PH two‐domain construct by a method previously developed in our laboratory. With these protein domains in hand we will perform biophysical studies to probe the interaction between the Grb7 RA‐PH, and SH2 domains. Proposed experimental approaches for this study include isothermal titration calorimetry, surface plasmon resonance, nuclear magnetic resonance, circular dichroism, and dynamic light scattering. These studies will help us to better understand how Grb7 functions in tumor progression, and further our ultimate goal of designing Grb7‐based therapeutics for treating aggressive breast cancers.[Office1]You can't use acronyms in an abstract without first defining them. Your reader will get lost.Support or Funding InformationNIH5‐SC3‐GM116728This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Toward Understanding the Functional State of the Grb7 Protein

Human growth factor receptor bound protein 7 (Grb7) proteins are the family of adaptor proteins that include Grb7, Grb10 and Grb14. These proteins have a conserved topology that includes a proline rich N‐terminal domain, an RA domain, a PH domain and a C‐terminal SH2 domain. Grb7 proteins bind with activated receptor tyrosine kinase (RTK) receptors through their SH2 domain to mediate downstream signal transduction events. Previous studies in our laboratory have shown the Grb7SH2 domain exists in a dimeric form and this may control the dimerization of the full‐length protein (FLGrb7). In our current studies, we show a FLGrb7 phosphorylation mimic, Y492E‐FLGrb7, results in a monomeric form of the protein, by in vitro biophysical measurements. Specifically, size exclusion and analytical ultracentrifugation results demonstrate Y492E‐FLGrb7 is essentially monomeric, while under the same conditions wild‐type Grb7 (WTFLGrb7) exists as a dimer. Further, the size and shape of these proteins were determined through dynamic light scattering measurements. These studies suggest the phosphorylation mimic Y492E‐FLGrb7 may exist in a more open, as opposed to compact globular, conformational form. We interpret the above results with a view towards understanding the functional state of Grb7 protein, and its role in controlling cell signaling pathways via interaction with its binding partners.Support or Funding InformationNIH5‐SC3‐6M116728This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

GRB7 dependent proliferation of basal-like, HER-2 positive human breast cancer cell lines is mediated in part by HER-1 signaling.

GRB7 gene encodes a multi-domain signal transduction molecule and is part of the core of the HER-2 amplicon. GRB7 is commonly co-amplified and overexpressed with HER-2 in human breast cancer. This study addresses the role of GRB7 in HER-2 positive human breast cancers resistant to HER-2 targeted therapy. HCC1954, 21MT1, and JIMT1 are basal like HER-2 positive breast cancer cell lines based on expression profiling. These three cell lines are resistant to trastuzumab and lapatinib treatment. Knockdown of GRB7 protein expression with siRNA transfection as well as lentiviral vector mediated shRNA over-expression decreased the growth of HCC1954, 21MT1, and JIMT1 cells in vitro and the growth of tumor xenografts these cells formed in animal models. When assayed by ki-67 staining and TUNEL assay, the mechanism of reduced tumor xenograft growth appeared to be distinct. Reduced proliferation and increased apoptosis were seen in 21MT1 cells, while reduced proliferation was seen in HCC1954 cells and increased apoptosis in JIMT1 cells. Phospho-proteome profiling found HER-1 tyrosine phosphorylation was reduced with GRB7 knock down in JIMT1 cells. Immuno-blotting and immuno-precipitation experiments found HER-1 phosphorylation was reduced with GRB7 knock down in all three cell lines. HER-1 knock down via siRNA transient transfection as well as blocking HER-1 function with panitumumab decreased proliferation of all three cell lines in vitro. Our study finds that GRB7 has an essential growth promoting function which is mediated in part by HER-1 activation. The potential of HER-1 targeting in therapy resistant HER-2 positive breast cancer merits further study.

127P - Studies on the carcinogenic mechanisms of Growth Receptor Bound Protein 7 in breast cancer cells and its clinical therapeutic applications

Background: Growth factor receptor bound protein 7 (GRB7) is a member of Grb family, and it is an adaptor protein. GRB7 has five functional domains, participated in many different signaling pathway.Human epidermal growth factor receptor 2 (HER2) is classified as a subtype in breast cancer. Studies until now have indicated that GRB7 and HER2 are highly correlated. Methods: To further confirm the relationship between GRB7 and HER2, GRB7 mRNA and protein levels were analyzed in different breast cancer cell lines and clinical patients tissue (N = 213). Results: We found that GRB7 highly expressed in HER2 positive breast cancer cell lines and HER2 positive patients’ tissues. GRB7 definitely existed in cytosol and nucleus in SKBR3 breast cancer cell, but GRB7 could not regulate HER2 gene expression directly. In addition, we constructed the knocked down GRB7 plasmid and overexpression GRB7 plasmid. We proved that HER2 would decrease when we knocked down GRB7; HER2 protein level would not change when we overexpressed GRB7.Utilizing a nature compound named curcumin to treat SKBR3 cells decreased GRB7 protein expression. Further analysis using cycloheximide to identify the possible degradation mechanism of GRB7, and the consequence indicated that GRB7 protein was directed to the path of hydrolysis, rather than being inhibited during the synthesis. Conclusions: By inhibiting GRB7 and can decrease the excessive expression of HER2. These results imply that GRB7 could be a potential molecule for therapy and prognosis prediction in breast cancer patients. Legal entity responsible for the study: N/A Funding: This study was supported by the Ministry of Science and Technology, Taiwan (MOST 106-2320-B-038-061 -MY3). Disclosure: The author has declared no conflicts of interest.

Abstract P2-08-03: An essential role of GRB7 in promoting the growth of therapy resistant HER-2 positive human breast cancer cells in culture and animal models

Abstract Background- GRB7 gene encodes a multi-domain signal transduction molecule and is part of the core of the HER-2 amplicon. GRB7 is commonly co-amplified and over-expressed with HER-2 in human breast cancer. Earlier studies found a functional role of GRB7 in breast cancer. The role of GRB7 in HER-2 positive human breast cancer resistant to HER-2 targeted therapy remains unexplored however. Materials and Methods- HCC-1954, 21MT1 and JimT1 are human HER-2 positive breast cancer cell lines that are resistant to trastuzumab and lapatinib treatment. Transient knock down of GRB7 protein expression was achieved with siRNA transfection and stable knock down with lentiviral vector mediated shRNA over-expression. Cell lines transfected with non-targeting siRNA or shRNA serve as negative controls. Knock down of GRB7 protein expression is verified by Western blotting. The growth of human breast cancer cell lines after GRB7 knock down in vitro is measured with the CellTiter Glo assay as well as the Incucyte live cell imaging. Activation status of specific signaling pathways was examined with phospho-specific antibody by immune-blotting and immune-precipitation. To assess the growth promoting function of GRB7 in human breast cancer cell lines in vivo, polyclonal HCC-1954, 21MT1 and JimT1 cells, with GRB7 knock down or their corresponding negative control, were orthotopically injected into the mammary fat pads of female immune-deficient NSG mice. The growth rates of these tumors, measured serially with caliper, and final tumor weights were compared between GRB7 knock down and the negative control. The proliferation rate and apoptosis of these tumors were studied with ki-67 staining and Tunel assay.The effects of GRB7 knock down on signaling were investigated with a proteome profiler receptor tyrosine kinase kit (R&amp;D). The role of signaling molecules differentially activated in the growth of breast cancer cells by GRB7 knock down was examined utilizing siRNA mediated knock down, and antibody and small molecule inhibitors. Results- GRB7 knock down decreased the growth of HCC-1954, 21MT1 and JimT1 cells in vitro and the growth of tumor xenograft these cells formed in animal models. When assayed by ki67 staining and Tunel assay, the mechanism of reduced tumor xenograft growth appeared to be distinct. Reduced proliferation and increased apoptosis were seen in 21MT1 cells, while reduced proliferation was seen in HCC-1954 cells and increased apoptosis in JimT1 cells. Protein profiling found that tyrosine phosphorylation of candidate signaling molecules was reduced with GRB7 knock down in JimT1 cells. Immuno-blotting and immuno-precipitation experiments were performed to evaluate these effects in other cell lines. The effect of targeting these molecules in breast cancer cell growth by siRNA and inhibitors is being examined. Discussion- GRB7 has essential growth promoting function in therapy resistant HER-2 positive human breast cancer cells. GRB7 knock down has pleiotropic effects on signaling in various cellular contexts. The potential of targeting GRB7 signaling in treating therapy resistant HER-2 positive breast cancer merits further study. Citation Format: Luoh S-W, Wagoner W, Lai X, Hu Z, Chin K, Ramsey B. An essential role of GRB7 in promoting the growth of therapy resistant HER-2 positive human breast cancer cells in culture and animal models [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-08-03.

Grb7 is over-expressed in cervical cancer and facilitate invasion and inhibit apoptosis in cervical cancer cells

Growth factor receptor bound protein-7 (Grb7) is a multi-domain adaptor protein that is co-opted by numerous tyrosine kinases involved in various cellular signaling. The objective of this study was to investigate the expression of Grb7 and its clinicopathological significance in cervical cancer. Utilising immunohistochemical staining, we examined the expression of Grb7 in 120 cases of human cervical cancer tissue and 10 cases of adjacent non-cancerous cervical tissue. The positive rate of Grb7 protein expression was 34.2%, which was significantly higher than that in adjacent non-cancerous cervical tissue (0%, p<0.05). The expression of Grb7 was found to be correlated with age, tumor size, serosal invasion, differentiated degree, tumor stage, early or advanced stage and lymph node metastasis. Kaplan-Meier survival analysis showed that patients with positive Grb7 protein expression had a lower overall survival rate than patients without Grb7 expression. In addition, Grb7 plays an important role in promoting tumor progression, including invasion and anti-apoptosis, in cervical cancer cell line. Down-regulation of Grb7 repressed the expression of MMP-9 and Bcl-2, and increased the expression of Bax in Grb7 knockdown Hela cells. Cell invasion assay showed decreased number of Grb7 knockdown Hela cells (18.7±2.1) compared to Hela cells (65.3±2.5, P<0.05). Our results indicated that Grb7 over-expression may facilitate invasion and inhibit apoptosis in cervical cancer and Grb7 is a potentially molecular target of cervical cancer chemotherapy.

Grb7 protein RA domain oligomerization.

The growth factor receptor bound protein 7 (Grb7) is an adaptor protein that is often coamplified with the erythroblastosis oncogene B 2 receptor in 20% to 30% of breast cancer patients. Grb7 overexpression has been linked to increased cell migration and cancer metastasis. The ras associating and pleckstrin homology domain region of Grb7 has been reported to interact with various other downstream signaling proteins such as four and half Lin11, Isl-1, Mec-3 (LIM) domains isoform 2 and filamin α. These interactions are believed to play a role in regulating Grb7-mediated cell migration function. The full-length Grb7 protein has been shown to dimerize, and the oligomeric state of the Grb7SH2 domain has been extensively studied; however, the oligomerization state of the ras associating and pleckstrin homology domains, and the importance of this oligomerization in Grb7 function, is yet to be fully known. In this study, we characterize the oligomeric state of the Grb7RA domain using size exclusion chromatography, nuclear magnetic resonance, nuclear relaxation studies, glutaraldehyde cross linking, and dynamic light scattering. We report the Grb7RA domain can exist in transient multimeric forms and, based upon modeling results, postulate the potential role of Grb7RA domain oligomerization in Grb7 function.

Grb7 Protein Stability Modulated by Pin1 in Association with Cell Cycle Progression.

Growth factor receptor bound protein-7 (Grb7) is a multi-domain adaptor protein that is co-opted by numerous tyrosine kinases involved in various cellular signaling and functions. The molecular mechanisms underlying the regulation of Grb7 remain unclear. Here, we revealed a novel negative post-translational regulation of Grb7 by the peptidyl-prolyl cis/trans isomerase, Pin1. Our data show that phosphorylation of Grb7 protein on the Ser194-Pro motif by c-Jun N-terminal kinase facilitates its binding with the WW domain of Pin1. Subsequently, Grb7 is degraded by the ubiquitin- and proteasome-dependent proteolytic pathway. Indeed, we found that Pin1 exerts its peptidyl-prolyl cis/trans isomerase activity in the modulation of Grb7 protein stability in regulation of cell cycle progression at the G2-M phase. This study illustrates a novel regulatory mechanism in modulating Grb7-mediated signaling, which may take part in pathophysiological consequences.

Grb7 and Hax1 may colocalize partially to mitochondria in EGF-treated SKBR3 cells and their interaction can affect Caspase3 cleavage of Hax1.

Growth factor receptor bound protein 7 (Grb7) is a signal-transducing adaptor protein that mediates specific protein-protein interactions in multiple signaling pathways. Grb7, with Grb10 and Grb14, is members of the Grb7 protein family. The topology of the Grb7 family members contains several protein-binding domains that facilitate the formation of protein complexes, and high signal transduction efficiency. Grb7 has been found overexpressed in several types of cancers and cancer cell lines and is presumed involved in cancer progression through promotion of cell proliferation and migration via interactions with the erythroblastosis oncogene B 2 (human epidermal growth factor receptor 2) receptor, focal adhesion kinase, Ras-GTPases, and other signaling partners. We previously reported Grb7 binds to Hax1 (HS1 associated protein X1) isoform 1, an anti-apoptotic protein also involved in cell proliferation and calcium homeostasis. In this study, we confirm that the in vitro Grb7/Hax1 interaction is exclusive to these two proteins and their interaction does not depend on Grb7 dimerization state. In addition, we report Grb7 and Hax1 isoform 1 may colocalize partially to mitochondria in epidermal growth factor-treated SKBR3 cells and growth conditions can affect this colocalization. Moreover, Grb7 can affect Caspase3 cleavage of Hax1 isoform 1 in vitro, and Grb7 expression may slow Caspase3 cleavage of Hax1 isoform 1 in apoptotic HeLa cells. Finally, Grb7 is shown to increase cell viability in apoptotic HeLa cells in a time-dependent manner. Taken together, these discoveries provide clues for the role of a Grb7/Hax1 protein interaction in apoptosis pathways involving Hax1. Copyright © 2016 John Wiley & Sons, Ltd.

Abstract LB-026: Pin1 negatively regulated Grb7 protein stability via the ubiquitin-proteasome cascade requires the peptidyl-prolyl cis/trans isomerase activity of Pin1

Abstract Growth factor receptor bound protein-7 (Grb7) is a multi-domain adaptor protein in cooperation with numerous tyrosine kinases to regulate various cellular signaling and functions. Although the regulatory mechanisms on the activation of Grb7 have been documented, the molecular mechanism governing Grb7 stability and its functional consequence is still not clearly understood. Here, we observed a novel negative regulatory mechanism of Grb7 by peptidyl-prolyl cis/trans isomerases Pin1 at the post-translational level. The Grb7 phosphorylation on the Ser194-Pro motif facilitated its binding to the WW domain of Pin1 and subsequently ubiquitination on Grb7, which, in turn, enhanced the process of proteosome-dependent proteolysis. Moreover, the interaction between Grb7 and Pin1 is dependent on the phosphorylation that is mediated by c-Jun N-terminal kinases MAPK. Both the phosphorylated Ser/Thr-Pro motif binding module and the peptidyl-prolyl cis/trans isomerase activity of Pin1 is essentail for Pin1-mediated Grb7 ubiquitin-proteasome proteolysis. By contrast, inhibition of Pin by lentiviral-mediated gene silencing resulted in accumulation of Grb7 protein as well as prolonged Grb7 protein stability. A stable Grb7S194A mutant that cannot be bound to and modulated by Pin1 utilize its potential to influence cell cycle progression. Our finding revealed that the Pin1/Grb7 complex formation enables negatively regulating Grb7-mediated cell proliferation due to the influence of Grb7 protein stability. Note: This abstract was not presented at the meeting. Citation Format: Yu-Ling Tai, Tang-Long Shen. Pin1 negatively regulated Grb7 protein stability via the ubiquitin-proteasome cascade requires the peptidyl-prolyl cis/trans isomerase activity of Pin1. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-026. doi:10.1158/1538-7445.AM2015-LB-026

Grb7 and Filamin‐a associate and are colocalized to cell membrane ruffles upon EGF stimulation

Grb7 is an adaptor molecule mediating signal transduction from multiple cell surface receptors to diverse downstream 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, ErbB2, are overexpressed in 20-30% of breast cancers. Grb7 overexpression has been linked to enhanced cell migration and metastasis, although the participants in these pathways have not been fully determined. In this study, we report the Grb7 protein interacts with Filamin-a, an actin-crosslinking component of the cell cytoskeleton. Additionally, we have demonstrated the interaction between Grb7 and Flna is specific to the RA-PH domains of Grb7, and the immunoglobulin-like repeat 16-19 domains of Flna. We demonstrate that full-length Grb7 and Flna interact in the mammalian cellular environment, as well as in vitro. Immunofluorescent microscopy shows potential co-localization of Grb7 and Flna in membrane ruffles upon epidermal growth factor stimulation. These studies are amongst the first to establish a clear connection between Grb7 signaling and cytoskeletal remodeling.

Growth factor receptor-bound protein 14: a potential new gene associated with oocyte competence

The Grb14 protein is a member of the Grb7 protein family. This protein family acts by binding to tyrosine kinase receptors, promoting cell proliferation and differentiation. There is evidence of the involvement of tyrosine kinase factors in the bovine oocyte maturation process. However, Grb14 has not been studied for bovine cumulus-oocyte complexes (COCs). The aim of the present study was to characterize Grb14 mRNA expression in bovine COCs during follicular development. Furthermore, we demonstrated that the expression of Grb14 mRNA is not regulated by estradiol. mRNA expression of Grb14 was assessed in 480 COCs from follicles of different sizes (1-3, 4-6, 6-8 or >8 mm) by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Grb14 mRNA expression decreased in COCs throughout follicular growth (P < 0.05). The role of estradiol in the expression of Grb14 mRNA in COCs was studied. Grb14 mRNA abundance did not differ in COCs cultured in the presence or absence of 17β-estradiol or fulvestrant. In conclusion, we showed that Grb14 mRNA is downregulated in COCs during antral follicle development, a finding that suggests a role for Grb14 in oocyte competence.

Dimerization in the Grb7 Protein

In previous studies, we showed that the tyrosine phosphorylation state of growth factor receptor-bound protein 7 (Grb7) affects its ability to bind to the transcription regulator FHL2 and the cortactin-interacting protein, human HS-1-associated protein-1. Here, we present results describing the importance of dimerization in the Grb7-Src homology 2 (SH2) domain in terms of its structural integrity and the ability to bind phosphorylated tyrosine peptide ligands. A tyrosine phosphorylation-mimic mutant (Y80E-Grb7-SH2) is largely dimerization deficient and binds a tyrosine-phosphorylated peptide representative of the receptor tyrosine kinase (RTK) erbB2 with differing thermodynamic characteristics than the wild-type SH2 domain. Another dimerization-deficient mutant (F99R-Grb7-SH2) binds the phosphorylated erbB2 peptide with similarly changed thermodynamic characteristics. Both Y80E-Grb7-SH2 and F99R-Grb7-SH2 are structured by circular dichroism measurements but show reduced thermal stability relative to the wild type-Grb7-SH2 domain as measured by circular dichroism and nuclear magnetic resonance. It is well known that the dimerization state of RTKs (as binding partners to adaptor proteins such as Grb7) plays an important role in their regulation. Here, we propose the phosphorylation state of Grb7-SH2 domain tyrosine residues could control Grb7 dimerization, and dimerization may be an important regulatory step in Grb7 binding to RTKs such as erbB2. In this manner, additional dimerization-dependent regulation could occur downstream of the membrane-bound kinase in RTK-mediated signaling pathways.