TCF12-regulated GRB7 facilitates the HER2+ breast cancer progression by activating Notch1 signaling pathway.

BACKGROUND: Human epidermal growth factor receptor 2-positive (HER2+) breast cancer (BC), which accounts for approximately one-fifth of all BCs, are highly invasive with a high rate of recurrence and a poor prognosis. Several studies have shown that growth factor receptor-bound protein 7 (GRB7) might be a potential therapeutic target for tumor diagnosis and prognosis. Nevertheless, the role of GRB7 in HER2+ BC and its underlying mechanisms have not been fully elucidated. The aim of this study was to investigate the biological function and regulatory mechanism of GRB7 in HER2+ BC. METHODS: Bioinformatics analysis was performed using the TCGA, GEO and CancerSEA databases to evaluate the clinical significance of GRB7. RT quantitative PCR, western blot and immunofluorescence were conducted to assess the expression of GRB7 in BC cell lines and tissues. MTT, EdU, colony formation, wound healing, transwell, and xenograft assays were adopted to explore the biological function of GRB7 in HER2+ BC. RNA sequencing was performed to analyze the signaling pathways associated with GRB7 in SK-BR-3 cells after the cells were transfected with GRB7 siRNA. Chromatin immunoprecipitation analysis (ChIP) and luciferase reporter assay were employed to elucidate the potential molecular regulatory mechanisms of GRB7 in HER2+ BC. RESULTS: GRB7 was markedly upregulated and associated with poor prognosis in BC, especially in HER2+ BC. Overexpression of GRB7 increased the proliferation, migration, invasion, and colony formation of HER2+ BC cells, while depletion of GRB7 had the opposite effects in HER2+ BC cells and inhibited xenograft growth. ChIP-PCR and luciferase reporter assay revealed that TCF12 directly bound to the promoter of the GRB7 gene to promote its transcription. GRB7 facilitated HER2+ BC epithelial-mesenchymal transition (EMT) progression by interacting with Notch1 to activate Wnt/ß-catenin pathways and other signaling (i.e., AKT, ERK). Moreover, forced GRB7 overexpression activated Wnt/ß-catenin to promote EMT progression, and partially rescued the inhibition of HER2+ BC proliferation, migration and invasion induced by TCF12 silencing. CONCLUSIONS: Our work elucidates the oncogenic role of GRB7 in HER2+ BC, which could serve as a prognostic indicator and promising therapeutic target.

GRB7 plays a promoting role in the progression of gastric cancer.

BACKGROUND: Gastric cancer is a clinically common tumor, showing an upward trend of both incidence and mortality. GRB7 has been identified as a vital regulator in tumor progression. This study aims to uncover the biological function of GRB7 in gastric cancer process. METHODS: immunohistochemical (IHC) staining using a tissue microarray (TMA), quantitative reverse transcription PCR (qRT-PCR) and Western blotting were performed to detect the expression of genes. Furthermore, gastric cancer cell lines AGS and MGC-803 were transfected with short hairpin RNAs against GRB7. The biological function of GRB7 in gastric cancer cells were examined by CCK-8, flow cytometry, wound healing and Transwell assays. Then, in vivo tumor formation assay was conducted to explore the effects of GRB7 on tumor growth. Finally, expression levels of proteins related to cell functions were determined by Western blotting. Coimmunoprecipitation (CoIP) assay was performed to assess the protein-protein interaction. RESULTS: GRB7 was up-regulated in gastric cancer tissues and cell lines, and its expression was inversely proportional to survival of gastric cancer patients. Moreover, GRB7 knockdown inhibited proliferative, migratory abilities, as well as promoted cell apoptosis in gastric cancer cells. Further study suggested that GRB7 silencing could suppress gastric cancer tumor growth in vivo. Furthermore, our study uncovered an important interaction between GRB7 and MyD88. Silencing MyD88 was observed to alleviate the malignant phenotypes promoted by GRB7 in gastric cancer cells. CONCLUSIONS: Together, this study provided evidence that GRB7 may be an effective molecular targets for the treatment of gastric cancer.

Upregulated GRB7 promotes proliferation and tumorigenesis of Bladder Cancer via Phospho-AKT Pathway.

Growth factor receptor-bound protein 7 (GRB7) has been found closely related to the occurrence and development of various tumors, but its function in bladder cancer has not yet been elucidated. The study is aiming at investigating the expression and function of GRB7 in bladder cancer. The Cancer Genome Atlas (TCGA) database was selected to analyze mRNA levels of GRB7 in bladder cancer. RT-qPCR and Western blot were conducted to detect the expression of GRB7 in normal bladder epithelial cells, seven bladder cancer cell lines and eight pairs of malignant/nonmalignant bladder tissues. The role of GRB7 in tumor proliferation and tumorigenesis was explored by establishing stable cells, in vitro cell experiments and in vivo xenograft models. The molecular regulation mechanism of GRB7 in bladder cancer was investigated by treatment with AKT inhibitor. GRB7 mRNA was upregulated in bladder cancer samples compared with that in normal tissue samples. Overexpressing GRB7 significantly promoted the proliferation and tumorigenesis of bladder cancer. However, silencing GRB7 played the retarding part. GRB7 promoted G1/S transition by activating the AKT pathway. Our results indicate that GRB7 plays an important role in promoting proliferation and tumorigenesis of bladder cancer.

Growth factor receptor bound protein-7 regulates proliferation, cell cycle, and mitochondrial apoptosis of thyroid cancer cells via MAPK/ERK signaling.

It is of great significance to explore the molecular mechanism of thyroid cancer (TC) pathogenesis for its improvement and therapy. Growth factor receptor bound protein-7 (GRB7) has been regarded as an important regulatory gene in the developments of various malignant tumors. Our study aimed to illustrate the role of GRB7 in the TC pathology mechanism. Firstly, GRB7 was found to be significantly upregulated in 49 cases of TC tissues and 5 TC cell lines by using real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting. Silencing GRB7 with siRNA dramatically inhibited proliferation and induced cell cycle arrest in TC cells. Besides, GRB7 silence resulted in the decrease of adenosine triphosphate content, glucose uptake, and lactose production in TC cells and attenuated the activity and expression of mitochondrial respiratory complex. We also demonstrated that GRB7 downregulation increased the levels of Bax and caspase 3, and inhibited the expression of Bcl-2, suggesting the induced mitochondrial apoptosis. More importantly, our study proved that mitogen-activated protein kinase/extracellular-regulated protein kinases (MAPK/ERK) signaling played a crucial role in the regulation of GRB7 on TC cell functions. In general, the present research verified that GRB7 was upregulated during TC development and modulated the proliferation, cell cycle, and mitochondrial apoptosis of TC cells by activating MAPK/ERK pathway. This may provide a novel target for the therapeutic strategy of TC.

Partners of wild type Grb7 and a mutant lacking its calmodulin-binding domain.

Growth factor receptor bound protein 7 (Grb7) is a mammalian adaptor protein participating in signaling pathways implicated in cell migration, metastatic invasion, cell proliferation and tumor-associated angiogenesis. We expressed tagged versions of wild type Grb7 and the mutant Grb7Δ, lacking its calmodulin-binding domain (CaM-BD), in human embryonic kidney (HEK) 293 cells and rat glioma C6 cells to identify novel binding partners using shot-gun proteomics. Among the new identified proteins, we validated the ubiquitin-ligase Nedd4 (neural precursor cell expressed developmentally down-regulated protein 4), the heat-shock protein Hsc70/HSPA8 (heat shock cognate protein 70) and the cell cycle regulatory protein caprin-1 (cytoplasmic activation/proliferation-associated protein 1) in rat glioma C6 cells. Our results suggest a role of Grb7 in pathways where these proteins are implicated. These include protein trafficking and degradation, stress-response, chaperone-mediated autophagy, apoptosis and cell proliferation.

Evaluation of Cyclic Peptide Inhibitors of the Grb7 Breast Cancer Target: Small Change in Cargo Results in Large Change in Cellular Activity.

Grb7 is an adapter protein, overexpressed in HER2+ve breast and other cancers, and identified as a therapeutic target. Grb7 promotes both proliferative and migratory cellular pathways through interaction of its SH2 domain with upstream binding partners including HER2, SHC, and FAK. Here we present the evaluation of a series of monocyclic and bicyclic peptide inhibitors that have been developed to specifically and potently target the Grb7 SH2-domain. All peptides tested were found to inhibit signaling in both ERK and AKT pathways in SKBR-3 and MDA-MB-231 cell lines. Proliferation, migration, and invasion assays revealed, however, that the second-generation bicyclic peptides were not more bioactive than the first generation G7-18NATE peptide, despite their higher in vitro affinity for the target. This was found not to be due to steric hindrance by the cell-permeability tag, as ascertained by ITC, but to differences in the ability of the bicyclic peptides to interact with and penetrate cellular membranes, as determined using SPR and mass spectrometry. These studies reveal that just small differences to amino acid composition can greatly impact the effectiveness of peptide inhibitors to their intracellular target and demonstrate that G7-18NATE remains the most effective peptide inhibitor of Grb7 developed to date.

Key candidate genes associated with BRAFV600E in papillary thyroid carcinoma on microarray analysis.

The mechanisms of B-type Raf kinase (BRAF) V600E mutation in papillary thyroid cancer (PTC) remain to be elucidated. With the aim to investigate the key candidate genes distinctive to BRAFV600E -PTC, we analyzed the transcriptomics data from three microarray datasets (GSE27155, GSE54958, and GSE58545) and identified 491 differentially expressed genes (DEGs) between BRAFV600E -PTC and BRAFwild type -PTC. Functional enrichment analysis of DEGs revealed that negative regulation of wound healing may be involved in the BRAFV600E -related pathogenesis in PTC. Weighted gene coexpression network analysis revealed BRAFV600E -related coexpressed genes in PTC, from which hub genes were selected. The intersection of DEGs and hub genes revealed 31 candidates, wherein GRB7, SNAP25, SLC35F2, FAM155B, HGD, and ITPR1 were rendered the key candidate genes via receiver operating characteristic curve analysis. On further characterization, the six key genes displayed significantly different expression patterns at different cytomorphology, however, with no significant difference in overall survival. These results provide novel insights into the key genes distinctive to of BRAFV600E in PTC and might be suggestive as therapeutic targets for further application.

Epigenetically altered miR­193a­3p promotes HER2 positive breast cancer aggressiveness by targeting GRB7.

Emerging evidence has demonstrated that microRNAs (miRNAs/miRs) have various biological functions in the development of human epidermal growth factor receptor 2 (HER2) positive breast cancer. The aim of the present study is to reveal the mechanism of miR­193a­3p inhibiting the progress of HER2 positive breast cancer. The expression of miR­193a­3p was evaluated by quantitative polymerase chain reaction (PCR). The methylation status of miR­193a­3p was evaluated by PCR and pyrosequencing analysis. Overexpression of miR­193a­3p and growth factor receptor bound protein 7 (GRB7) combined with in vitro tumorigenic assays were conducted to determine the carcinostatic capacities of miR­193a­3p in HER2 positive breast cancer cells. The association between miR­193a­3p and GRB7 was determined by luciferase reporter assay. Protein level was evaluated using western blot analysis. miR­193a­3p was downregulated in HER2 positive breast cancer cells and clinical tissues. Methylation­mediated silencing led to decreased expression of miR­193a­3p in HER2 positive breast cancer. Overexpression of miR­193a­3p could inhibit proliferation, migration and invasion of breast cancer cells. Overexpression of GRB7 could abolish this effect. miR­193a­3p could directly target the 3' untranslated region of GRB7. miR­193a­3p could directly or indirectly target extracellular signal­regulated kinase 1/2 (ERK1/2) and forkhead box M1 (FOXM1) signaling. In conclusion, it was identified that silencing of miR­193a­3p through hypermethylation can promote HER2 positive breast cancer progress by targeting GRB7, ERK1/2 and FOXM1 signaling. The function of miR­193a­3p in HER2 positive breast cancer implicates its potential application in therapy.

Methylation-associated silencing of miR-193a-3p promotes ovarian cancer aggressiveness by targeting GRB7 and MAPK/ERK pathways.

Human growth factor receptor-bound protein-7 (GRB7) is a pivotal mediator involved in receptor tyrosine kinase signaling and governing diverse cellular processes. Aberrant upregulation of GRB7 is frequently associated with the progression of human cancers. However, the molecular mechanisms leading to the upregulation of GRB7 remain largely unknown. Here, we propose that the epigenetic modification of GRB7 at the post-transcriptional level may be a crucial factor leading to GRB7 upregulation in ovarian cancers. Methods: The upstream miRNA regulators were predicted by in silico analysis. Expression of GRB7 was examined by qPCR, immunoblotting and immunohistochemical analyses, while miR-193a-3p levels were evaluated by qPCR and in situ hybridization in ovarian cancer cell lines and clinical tissue arrays. MS-PCR and pyrosequencing analyses were used to assess the methylation status of miR-193a-3p. Stable overexpression or gene knockdown and Tet-on inducible approaches, in combination with in vitro and in vivo tumorigenic assays, were employed to investigate the functions of GRB7 and miR-193a-3p in ovarian cancer cells. Results: Both miR-193a-3p and its isoform, miR-193b-3p, directly targeted the 3' UTR of GRB7. However, only miR-193a-3p showed a significantly inverse correlation with GRB7-upregulated ovarian cancers. Epigenetic studies revealed that methylation-mediated silencing of miR-193a-3p led to a stepwise decrease in miR-193a-3p expression from low to high-grade ovarian cancers. Intriguingly, miR-193a-3p not only modulated GRB7 but also ERBB4, SOS2 and KRAS in the MAPK/ERK signaling pathway to enhance the oncogenic properties of ovarian cancer cells in vitro and in vivo. Conclusion: These findings suggest that epigenetic silencing of miR-193a-3p by DNA hypermethylation is a dynamic process in ovarian cancer progression, and miR-193a-3p may be explored as a promising miRNA replacement therapy in this disease.

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.

Rational design of novel N-alkyl-N capped biostable RNA nanostructures for efficient long-term inhibition of gene expression.

Computational techniques have been used to design a novel class of RNA architecture with expected improved resistance to nuclease degradation, while showing interference RNA activity. The in silico designed structure consists of a 24-29 bp duplex RNA region linked on both ends by N-alkyl-N dimeric nucleotides (BCn dimers; n = number of carbon atoms of the alkyl chain). A series of N-alkyl-N capped dumbbell-shaped structures were efficiently synthesized by double ligation of BCn-loop hairpins. The resulting BCn-loop dumbbells displayed experimentally higher biostability than their 3'-N-alkyl-N linear version, and were active against a range of mRNA targets. We studied first the effect of the alkyl chain and stem lengths on RNAi activity in a screen involving two series of dumbbell analogues targeting Renilla and Firefly luciferase genes. The best dumbbell design (containing BC6 loops and 29 bp) was successfully used to silence GRB7 expression in HER2+ breast cancer cells for longer periods of time than natural siRNAs and known biostable dumbbells. This BC6-loop dumbbell-shaped structure displayed greater anti-proliferative activity than natural siRNAs.

[Knockdown of Grb7 inhibits growth of oral squamous cell carcinoma, cell proliferation and promoted apoptosis through ERK/FOXM1 pathway].

PURPOSE: To investigate the effect of growth factor receptor-bound 7 (Grb7) on oral squamous cell carcinoma growth and tumor xenografts. METHODS: Cal27 and hNOK cells were cultivated, real-time PCR and Western blotting were used to detect the expression of Grb7 in hNOK and Cal27. Cal27 was transfected with Grb7 siRNA for 48 h, cell proliferation was assayed using MTT. Flow cytometry was used to determine the cell cycle and apoptosis. Western blot was used to evaluate the expression of caspase3, Bax, bcl-2, Cyclin D1, Rb, E2F1, ERK and FOXM1. Grb7 siRNA and negative control were designed and injected subcutaneously into the mice, tumor volume and weight were measured. Statistical analysis was performed using SPSS 11.0 software package. RESULTS: Grb7 was highly expressed in Cal27 compared with hNOK. Depletion of Grb7 significantly inhibited cell proliferation, blocked G1/S phase transition, promoted cell apoptosis. Knockdown of Grb7 suppressed the expression of Cyclin D1 and Rb, upregulated E2F1 expression. Moreover, c-caspase 3 and Bax was also reduced after inhibition of Grb7. ERK/FOXM1 signaling pathway was also inhibited by Grb7. In addition, the volume and weight of tumor xenografts were reduced by siGrb7. CONCLUSIONS: Depletion of Grb7 inhibits cell proliferation, promotes apoptosis and reduces tumor xenografts through ERK/FOXM1 pathway.

Grb7 gene amplification and protein expression by FISH and IHC in ovarian cancer.

OBJECTIVE: Overexpression of growth factor receptor-bound protein 7 (Grb7) has been found in numerous human cancers. The aim of this study was to evaluate the correlation between Grb7 gene amplification and protein expression in ovarian cancer (OC). METHODS: We use Tissue Microarray (TMA) respectively to detect the gene amplification and protein expression of Grb7 in 90 cases OC and 10 control specimens of normal ovarian tissues by IHC and FISH. RESULTS: The Grb7 protein expression by IHC analysis was observed in 52/90 (57.8%) OC with 3 cases (3.3%) scored 3(+) and 9 cases (10%) scored 2(+) Grb7 gene amplification by FISH analysis was successfully detectable in 6 specimens with a positive rate of 6.8% (6/88) in which immunostaining 3(+), 2(+) and negative (1(+)/0) expressions of Grb7 were 100.0% (3/3), 11.1% (1/9) and 2.6% (2/76), respectively. Our data exhibited that the IHC and FISH results had a good consistency between Grb7 gene amplification and Grb7 protein expression (Kappa = 0.651, P < 0.001). Both the results of IHC and FISH revealed that Grb7 did not seem to have a role in OC clinicopathology. CONCLUSION: There is a close relationship between Grb7 gene amplification and GRB7 protein overexpression in human OC. IHC might have limited diagnostic value especially in these tumors and especially in characterizing genetically diverse borderline cases, FISH could be superior to IHC.

An Integrative Analysis to Identify Driver Genes in Esophageal Squamous Cell Carcinoma.

BACKGROUND: Few driver genes have been well established in esophageal squamous cell carcinoma (ESCC). Identification of the genomic aberrations that contribute to changes in gene expression profiles can be used to predict driver genes. METHODS: We searched for driver genes in ESCC by integrative analysis of gene expression microarray profiles and copy number data. To narrow down candidate genes, we performed survival analysis on expression data and tested the genetic vulnerability of each genes using public RNAi screening data. We confirmed the results by performing RNAi experiments and evaluating the clinical relevance of candidate genes in an independent ESCC cohort. RESULTS: We found 10 significantly recurrent copy number alterations accompanying gene expression changes, including loci 11q13.2, 7p11.2, 3q26.33, and 17q12, which harbored CCND1, EGFR, SOX2, and ERBB2, respectively. Analysis of survival data and RNAi screening data suggested that GRB7, located on 17q12, was a driver gene in ESCC. In ESCC cell lines harboring 17q12 amplification, knockdown of GRB7 reduced the proliferation, migration, and invasion capacities of cells. Moreover, siRNA targeting GRB7 had a synergistic inhibitory effect when combined with trastuzumab, an anti-ERBB2 antibody. Survival analysis of the independent cohort also showed that high GRB7 expression was associated with poor prognosis in ESCC. CONCLUSION: Our integrative analysis provided important insights into ESCC pathogenesis. We identified GRB7 as a novel ESCC driver gene and potential new therapeutic target.

Validation of analytical breast cancer microarray analysis in medical laboratory.

A previously reported microarray data analysis by RISS algorithm on breast cancer showed over-expression of the growth factor receptor (Grb7) and it also highlighted Tweety (TTYH1) gene to be under expressed in breast cancer for the first time. Our aim was to validate the results obtained from the microarray analysis with respect to these genes. Also, the relationship between their expression and the different prognostic indicators was addressed. RNA was extracted from the breast tissue of 30 patients with primary malignant breast cancer. Control samples from the same patients were harvested at a distance of ≥5 cm from the tumour. Semi-quantitative RT-PCR analysis was done on all samples. There was a significant difference between the malignant and control tissues as regards Grb7 expression. It was significantly related to the presence of lymph node metastasis, stage and histological grade of the malignant tumours. There was a significant inverse relation between expression of Grb7 and expression of both oestrogen and progesterone receptors. Grb7 was found to be significantly related to the biological classification of breast cancer. TTYH1 was not expressed in either the malignant or the control samples. The RISS by our group algorithm developed was laboratory validated for Grb7, but not for TTYH1. The newly developed software tool needs to be improved.

Context-dependent role of Grb7 in HER2+ve and triple-negative breast cancer cell lines.

Grb7 is an adapter protein, aberrantly co-overexpressed with HER2 and identified as an independent prognostic marker in breast cancer. It has been established that Grb7 exacerbates the cellular growth and migratory behaviour of HER2+ve breast cancer cells. Less is known about Grb7's role in the context of HER2-ve cells. Here we directly compare the effect of stable Grb7 knockdown in oestrogen sensitive (T47D), HER2+ve (SKBR3) and triple-negative (MDA-MB-468 and MDA-MB-231) breast cancer cell lines on anchorage dependent and independent cell growth, wound healing and chemotaxis. All cell lines showed reduced ability to migrate upon Grb7 knockdown, despite their greatly varied endogenous levels of Grb7. Decreased cell proliferation was not observed in any of the cell lines upon Grb7 knockdown; however, decreased ability to form colonies was observed for all but the oestrogen sensitive cell line, depending upon the stringency of the growth conditions. The data reveal that Grb7 plays an important role in breast cancer progression, beyond the context of HER2+ve cell types.

Deletion of the calmodulin-binding domain of Grb7 impairs cell attachment to the extracellular matrix and migration.

The adaptor Grb7 is a calmodulin (CaM)-binding protein that participates in signaling pathways involved in cell migration, proliferation and the control of angiogenesis, and plays a significant role in tumor growth, its metastatic spread and tumor-associated neo-vasculature formation. In this report we show that deletion of the CaM-binding site of Grb7, located in the proximal region of its pleckstrin homology (PH) domain, impairs cell migration, cell attachment to the extracellular matrix, and the reorganization of the actin cytoskeleton occurring during this process. Moreover, we show that the cell-permeable CaM antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) and N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide (W-13) both retard the migration of cells expressing wild type Grb7, but not the migration of cells expressing the mutant protein lacking the CaM-binding site (Grb7Δ), underscoring the proactive role of CaM binding to Grb7 during this process.

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.

Expression screening of 17q12-21 amplicon reveals GRB7 as an ERBB2-dependent oncogene.

Gene amplification is a major genetic alteration in human cancers. Amplicons, amplified genomic regions, are believed to contain "driver" genes responsible for tumorigenesis. However, the significance of co-amplified genes has not been extensively studied. We have established an integrated analysis system of amplicons using retrovirus-mediated gene transfer coupled with a human full-length cDNA set. Applying this system to 17q12-21 amplicon observed in breast cancer, we identified GRB7 as a context-dependent oncogene, which modulates the ERBB2 signaling pathway through enhanced phosphorylation of ERBB2 and Akt. Our work provides an insight into the biological significance of gene amplification in human cancers.

Expression levels of HER2/neu and those of collocated genes at 17q12-21, in breast cancer.

HER2/neu is associated with poorer clinical outcome in breast cancer. Expression patterns of co-localised cancer-associated genes at 17q12-21 were examined using RT-PCR. The study group consisted of a 96-patient cohort. Relative quantity of mRNA expression was calculated using the comparative cycle threshold method and Qbase software. Results were analysed to detect expression patterns among the genes, and to identify associations between expression levels and clinical data. Levels of HER2/neu correlated with those of GRB7 (r=0.551, p<0.001), RARA (r=0.391, p<0.001), RPL19 (r=0.549, p<0.001) and LASP1 (r=0.399, p<0.001). GRB7 was significantly inversely associated with improved DFS at 60 months (p=0.036). RARA levels were greater in HER2/neu-positive as opposed to HER2/neu-negative patients (p=0.021); levels were significantly higher in ER-positive patients, relative to those who were ER-negative (p=0.003). Levels of RPL19 were significantly higher in the HER2/neu-overexpressing (p=0.010) and luminal B subtypes (p=0.007). LASP1 levels were higher in those patients who had been classified clinically as HER2/neu-positive (p=0.004). This study reaffirms the correlation between HER2/neu and the co-localised LASP1 and GRB7; the latter target may hold additional significance in addition to being a surrogate marker for HER2/neu expression. The relationship identified between RARA and ER-positivity may herald an avenue for targeted therapy of these tumours.