IQGAP1 protein binds human epidermal growth factor receptor 2 (HER2) and modulates trastuzumab resistance

Human epidermal growth factor receptor 2 (HER2) is overexpressed in 20-25% of breast cancers. Increased HER2 expression is an adverse prognostic factor and correlates with decreased patient survival. HER2-positive (HER2(+)) breast cancer is treated with trastuzumab. Unfortunately, some patients are intrinsically refractory to therapy, and many who do respond initially become resistant within 1 year. Understanding the molecular mechanisms underlying HER2 signaling and trastuzumab resistance is essential to reduce breast cancer mortality. IQGAP1 is a ubiquitously expressed scaffold protein that contains multiple protein interaction domains. By regulating its binding partners IQGAP1 integrates signaling pathways, several of which contribute to breast tumorigenesis. We show here that IQGAP1 is overexpressed in HER2(+) breast cancer tissue and binds directly to HER2. Knockdown of IQGAP1 decreases HER2 expression, phosphorylation, signaling, and HER2-stimulated cell proliferation, effects that are all reversed by reconstituting cells with IQGAP1. Reducing IQGAP1 up-regulates p27, and blocking this increase attenuates the growth inhibitory effects of IQGAP1 knockdown. Importantly, IQGAP1 is overexpressed in trastuzumab-resistant breast epithelial cells, and reducing IQGAP1 both augments the inhibitory effects of trastuzumab and restores trastuzumab sensitivity to trastuzumab-resistant SkBR3 cells. These data suggest that inhibiting IQGAP1 function may represent a rational strategy for treating HER2(+) breast carcinoma.     

Functional expression of a single-chain antibody fragment against human epidermal growth factor receptor 2 (HER2) in Escherichia coli

The human epidermal growth factor receptor (HER) family plays an important role in cell growth and signaling and alteration of its function has been demonstrated in many different kinds of cancer. Receptor dimerization is necessary for the HER signal transduction pathway and tyrosine kinase activity. Recently, several monoclonal antibodies have been developed to directly interfere with ligand–HER receptor binding and receptor dimerization. A single chain variable fragment (ScFv) is a valuable alternative to an intact antibody. This report describes the production and purification of an ScFv specific for domain II of the HER2 receptor in Escherichia coli BL21 (DE3) cytoplasm. The majority of expressed of anti-her2his-ScFv protein was produced as inclusion bodies. A Ni-NTA affinity column was used to purify the anti-her2his-ScFv protein. The molecular weight of anti-her2his-ScFv protein was estimated to be approximately 27 kDa, as confirmed by SDS-PAGE and Western blotting assay. The anti-her2his-ScFv showed near 95 % purity and reached a yield of approximately 29 mg/l in flask fermentation. The purified anti-her2his-ScFv showed its biological activity by binding to HER2 receptor on the surface of BT-474 cells. This ScFv may be a potential pharmaceutical candidate for targeting tumour cells overexpressing HER2 receptor.     

Impact of protein tyrosine kinase 6 (PTK6) on human epidermal growth factor receptor (HER) signalling in breast cancer

PTK6, also known as Brk, is highly expressed in over 80% of breast cancers. In the last decade several substrates and interaction partners were identified localising PTK6 downstream of HER receptors. PTK6 seems to be involved in progression of breast tumours, in particular in HER receptor signalling. Here, we show the down-regulation effects of PTK6 in the T47D, BT474 and JIMT-1 breast cancer cell lines. PTK6 knockdown leads to a decreased phosphorylation of HER2, PTEN, MAPK (ERK), p38 MAPK, STAT3 and to a reduced expression of cyclin E. Our findings show that silencing PTK6 impairs the downstream targets of HER receptors and consequently the activation of signalling molecules. Furthermore, lower levels of PTK6 result in reduced migration of T47D and JIMT-1 breast cancer cells. Due to decreased migration, the PTK6 RNA interference might contribute to reduced metastasis and malignant potential of breast cancer cells. Since PTK6 plays an important role in HER receptor signal transduction, its down-regulation might be suitable for future therapy approaches in breast cancer.     

Clinical significance of epidermal growth factor receptor content in gastric cancer

The objective of this work was to evaluate the epidermal growth factor receptor (EGFR) content in gastric cancer, its possible relationship with clinicopathological parameters of tumors and its prognostic significance. Membranous EGFR levels were examined by radioligand binding assays in 110 patients with gastric cancer. The mean follow-up period was 30.7 months. EGFR levels of tumors ranged widely, from 0.3 to 510 fmol/mg protein. EGFR levels were significantly higher (p<0.0005) in neoplastic tissue than in paired adjacent mucosa samples (median) (n= 84; 8.7 vs. 3.9 fmol/mg protein). Intratumoral EGFR levels were significantly correlated with tumor stage (p<0.05), and were higher in patients with stage III tumors (median) (7.6, 6.4, 12.3 and 7.5 fmol/mg protein for stages I, II, III and IV, respectively). In addition, the tumor/mucosa ratios of the EGFR content were significantly higher (p<0.05) in patients with stage III tumors (1, 1.8, 3.9, and 0.92, respectively). Although there was no significant relationship between EGFR levels of tumors and overall survival, the results suggest a role for EGFR in tumor progression of gastric cancer.     

Amplification of epidermal growth factor receptor (EGFR) gene and oncogenes in human gastric carcinomas

DNAs from 37 human gastric carcinomas and seven lymph node metastases were analyzed for alterations of the epidermal growth factor receptor (EGFR) gene and oncogenes by the Southern blot hybridization method. The probes used were EGFR gene, c-Ha-ras, v-Ki-ras, N-ras, c-myc, v-myb, v-fos, c-erbB-2, v-erbA, v-abl and v-fes. Amplification of the EGFR gene was detected in only one poorly differentiated adenocarcinoma. Amplifications of c-myc gene and c-erbB-2 gene were each observed in two well differentiated adenocarcinomas. One of these tumors had coamplification of c-erbB-2 and c-erbA genes but there were no amplifications nor rearrangements of other oncogenes. The poorly differentiated adenocarcinom with amplified EGFR gene also showed enhanced expression of EGFR gene by Northern blot analysis and additionally had strong synchronous immunoreactivity for EGFR and EGF.     

Amplification of epidermal growth factor receptor (EGFR) gene and oncogenes in human gastric carcinomas

DNAs from 37 human gastric carcinomas and seven lymph node metastases were analyzed for alterations of the epidermal growth factor receptor (EGFR) gene and oncogenes by the Southern blot hybridization method. The probes used were EGFR gene, c-Ha-ras, v-Ki-ras, N-ras, c-myc, v-myb, v-fos, c-erbB-2, v-erbA, v-abl and v-fes. Amplification of the EGFR gene was detected in only one poorly differentiated adenocarcinoma. Amplifications of c-myc gene and c-erbB-2 gene were each observed in two well differentiated adenocarcinomas. One of these tumors had coamplification of c-erbB-2 and c-erbA genes but there were no amplifications nor rearrangements of other oncogenes. The poorly differentiated adenocarcinom with amplified EGFR gene also showed enhanced expression of EGFR gene by Northern blot analysis and additionally had strong synchronous immunoreactivity for EGFR and EGF. Supported in part by Grants-in Aid for Cancer Research from the Ministry of Education, Science and Culture of Japan and for Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health and Welfare of Japan     

Sphingosine 1-phosphate transactivates c-Met as well as epidermal growth factor receptor (EGFR) in human gastric cancer cells

Receptor tyrosine kinases (RTKs) are transactivated by the stimulation of G protein-coupled receptors (GPCRs). Sphingosine 1-phosphate (S1P), a ligand of GPCR, is known as a tumor-promoting lipid, but its signaling pathways are not fully understood. We here demonstrated that S1P induces rapid and transient tyrosine phosphorylation of epidermal growth factor receptor (EGFR) and c-Met in gastric cancer cells, both of which have been proposed as prognostic markers of gastric cancers. The pathway of S1P-induced c-Met transactivation is Gi-independent and matrix metalloproteinase-independent, which differs from that of EGFR transactivation. Our results indicate that S1P acts upstream of various RTKs and thus may act as a potent stimulator of gastric cancer.     

A gene-protein assay for human epidermal growth factor receptor 2 (HER2): brightfield tricolor visualization of HER2 protein, the HER2 gene, and chromosome 17 centromere (CEN17) in formalin-fixed, paraffin-embedded breast cancer tissue sections

ABSTRACT: BACKGROUND: The eligibility of breast cancer patients for human epidermal growth factor receptor 2 (HER2)-directed therapies is determined by the HER2 gene amplification and/or HER2 protein overexpression status of the breast tumor as determined by in situ hybridization (ISH) or immunohistochemistry (IHC), respectively. Our objective was to combine the US Food and Drug Administration (FDA)-approved HER2 & chromosome 17 centromere (CEN17) brightfield ISH (BISH) and HER2 IHC assays into a single automated HER2 gene-protein assay allowing simultaneous detection of all three targets in a single tissue section. METHODS: The HER2 gene-protein assay was optimized using formalin-fixed, paraffin-embedded (FFPE) samples of the xenograft tumors MCF7 [HER2 negative (non-amplified gene, protein negative)] and Calu-3 [HER2 positive (amplified gene, protein positive)]. HER2 IHC was performed using a rabbit monoclonal anti-HER2 antibody (clone 4B5) and a conventional 3,3' -diaminobenzidine IHC detection. The HER2 & CEN17 BISH signals were visualized using horseradish peroxidase-based silver and alkaline phosphatase-based red detection systems, respectively with a cocktail of 2,4-dinitrophenyl-labeled HER2 and digoxigeninlabeled CEN17 probes. The performance of the gene-protein assay on tissue microarray slides containing 189 randomly selected FFPE clinical breast cancer tissue cores was compared to that of the separate HER2 IHC and HER2 & CEN17 BISH assays. RESULTS: HER2 protein detection was optimal when the HER2 IHC protocol was used before (rather than after) the BISH protocol. The sequential use of HER2 IHC and HER2 & CEN17 BISH detection steps on FFPE xenograft tumor sections appropriately co-localized the HER2 protein, HER2 gene, and CEN17 signals after mitigating the silver background staining by using a naphthol phosphate-containing hybridization buffer for the hybridization step. The HER2 protein and HER2 gene status obtained using the multiplex HER2 gene-protein assay demonstrated high concordance with those obtained using the separate HER2 IHC and HER2 & CEN17 BISH assays, respectively. CONCLUSIONS: We have developed a protocol that allows simultaneous visualization of the HER2 IHC and HER2 & CEN17 BISH targets. This automated protocol facilitated the determination of HER2 protein and HER2 gene status in randomly selected breast cancer samples, particularly in cases that were equivocal or exhibited tumor heterogeneity. The HER2 gene-protein assay produced results virtually equivalent to those of the single FDA-approved HER2 IHC and HER2 & CEN17 BISH assays. Virtual slides The virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2041964038705297.     

A radioimmunoassay for human epidermal growth factor receptor

The development of a radioimmunoassay (RIA) for the human epidermal growth factor receptor solubilized with nonionic detergents which employs iodinated epidermal growth factor (125I-EGF) as the specific ligand is described. A monoclonal antibody (R1) that binds specifically to human EGF receptors [Waterfield, M. D., et al. (1982) J. Cell Biochem. 20, 149-161] was used to separate solubilized receptors saturated with 125I-EGF from free ligand by absorption to protein A-Sepharose, and the bound radioactivity was determined. The RIA was linear when increasing amounts of solubilized membrane protein were added and, when compared to the standard polyethylene glycol assay, was more reproducible. In addition, the background nonspecific binding obtained in the presence of a hundred-fold excess of unlabeled EGF was less in the RIA. Substitution of normal mouse serum for the monoclonal antibody gave very low nonspecific background ligand binding and avoided the use of large amounts of unlabeled EGF in the assay. Two major classes of binding sites for EGF were observed in membrane preparations from the cervical carcinoma cell line A431 or from normal human placental tissue. These were present in approximately equal amounts, with apparent dissociation constants of 4 X 10(-10) and 4 X 10(-9) M. Upon solubilization with the nonionic detergent Triton X-100, only one class of EGF binding sites was detected in both cases, with a dissociation constant of 3 X 10(-8) M. The RIA can be used to monitor receptor purification and for quantitation of receptor number and affinity in various cell types.     

Size heterogeneity of epidermal growth factor in human body fluids

We measured the concentration of immunoreactive (IR) hEGF in various body fluids by radioimmunoassay (RIA) and evaluated its size heterogeneity by size exclusion high performance liquid chromatography combined with RIA or with time-resolved immunoflurometric assay (TR-IFMA). Mean concentration was 80 ng/ml in urine, 65 ng/ml in milk, 50 ng/ml in seminal plasma, 25 ng/ml in armpit sweat, 1 ng/ml in breast sweat, 0.3 ng/ml in third-trimester amniotic fluid, 3 ng/ml in saliva, 1.5 ng/ml in tears and 0.3 ng/ml in gastric juice.

All the fluids except armpit sweat and gastric juice contained two to five molecular sizes of IR-hEGF. As well as the 6200-dalton (6.2kDa) hEGF we found at least four other different molecular sizes with approximate weights of 300, 150, 70 and 20kDa. The authentic 6.2kDa form made up >90% of the total IR-hEGF in all except the amniotic fluid where its proportion was 71%, and the seminal plasma where the proportion could not be determined.     

Imaging epidermal growth factor receptor phosphorylation in human colorectal cancer cells and human tissues

In tumor cells, high phosphorylation levels of receptor tyrosine kinases may occur in the absence of exogenous ligands due to autocrine signaling or enhanced tyrosine kinase activity. Here we show that the phosphorylation state of the endogenous epidermal growth factor receptor (EGFR) can be quantitatively imaged in tumor cells and tissues by detecting fluorescence resonance energy transfer between fluorophores conjugated to antibodies against the receptor and phosphotyrosine, respectively. Five different human colorectal cell lines were analyzed for activity and expression of EGFR. All cell lines exhibited basal EGFR phosphorylation under serum starvation conditions. Phosphorylation levels increased after stimulation with EGF or pervanadate, dependent on the level of basal EGFR phosphorylation in the respective cell lines. This basal activity correlated inversely with receptor expression. Using the acceptor photobleaching fluorescence resonance energy transfer imaging approach, a significantly higher phosphorylation state of EGFR was also found in resected human colorectal tumor samples as compared with adjacent healthy tissue. Imaging of EGFR phosphorylation may thus serve as a valuable tool to investigate the role of receptor tyrosine kinase activity in malignant cell growth.     

A caspase-6 and anti-human epidermal growth factor receptor-2 (HER2) antibody chimeric molecule suppresses the growth of HER2-overexpressing tumors

Clinical studies have suggested that human epidermal growth factor receptor-2 (HER2) provide a useful target for antitumor therapy. We previously described the generation of a chimeric HER2-targeted immunocasp-3 protein. In this study, we extend the repertoire of chimeric proapoptotic proteins with immunocasp-6, a construct that comprises a HER2-specific single-chain Ab, a single-chain Pseudomonas exotoxin A, and an active caspase-6, which can directly cleave lamin A leading to nucleus damage and inducing programmed cell death. We demonstrate that the secreted immunocasp-6 molecule selectively recognizes and induces apoptosis in HER2-overexpressing tumor cells in vitro, but not in cells with undetectable HER2. The immunocasp-6 gene was next transferred into BALB/c athymic mice bearing human breast SK-BR-3 tumors by i.m. injection of liposome-encapsulated vectors, by intratumor injection of adenoviral vectors, or by i.v. injection of PBMC modified by retroviral infection. Regardless of the method used, expression of immunocasp-6 suppressed tumor growth and prolonged animal survival significantly. Our data show that the chimeric immunocasp-6 molecule can recognize HER2-positive tumor cells, promptly attack their nucleus, and induce their apoptotic death, suggesting the potential of this strategy for the treatment of human cancers that overexpress HER2.     

Lysophospholipids transactivate HER2/neu (erbB-2) in human gastric cancer cells

The ligand-less receptor HER2/neu (erbB-2) has been proposed as a prognostic marker of gastric cancer that correlates with poor clinical outcome, indicating that HER2 signals play an important role in gastric cancer progression. This study demonstrated that two major natural lysophospholipids, lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P), induce rapid and transient phosphorylation of HER2 in two human gastric cancer cell lines, MKN28 and MKN74 cells. We also revealed that tyrosine phosphorylation of HER2 induced by both lysophospholipids was significantly attenuated by two inhibitors, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, AG1478, and a broad-spectrum matrix metalloproteinase inhibitor, GM6001. This suggests that the pathway of HER2 transactivation induced by these lysophospholipids is dependent on the proteolytically released EGFR ligands. Our results indicate that LPA and S1P act upstream of HER2 in gastric cancer cells, and thus may act as potent stimulators of gastric cancer.     

Localization of the epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) in the bovine testis

In the last few decades, several growth factors were identified in the testis of various mammalian species. Growth factors are shown to promote cell proliferation, regulate tissue differentiation, and modulate organogenesis. In the present investigation we have studied the localization of EGF and EGFR in the adult bovine testis by means of immunohistochemical method. Our results demonstrated that EGF and EGFR were localized solely to the bovine testicular germ cells (spermatogonia, spermatocytes, and round spermatids). In contrast, the somatic testicular cells (i.e., Sertoli, Leydig, and myofibroblast cells) exhibited no staining affinity. EGF and EGFR were additionally detected in the epithelial lining of straight tubules and rete testis. Interestingly, the distribution of EGF and EGFR in the germ cells was mainly dependent upon the cycle of the seminiferous epithelium since their localization appeared to be preponderant during the spermatogonia proliferation and during the meiotic and spermiogenic processes. In conclusion, such findings may suggest that EGF and EGFR are important paracrine and/or autocrine regulators of spermatogenesis in bovine.     

Parsing ERK activation reveals quantitatively equivalent contributions from epidermal growth factor receptor and HER2 in human mammary epithelial cells

HER2, a member of the epidermal growth factor receptor (EGFR) tyrosine kinase family, functions as an accessory EGFR signaling component and alters EGFR trafficking by heterodimerization. HER2 overexpression leads to aberrant cell behavior including enhanced proliferation and motility. Here we applied a combination of computational modeling and quantitative experimental studies of the dynamic interactions between EGFR and HER2 and their downstream activation of ERK to understand this complex signaling system. Using cells expressing different levels of HER2 relative to the EGFR, we could separate relative contributions of EGFR and HER2 to signaling amplitude and duration. Based on our model calculations, we demonstrated that, in contrast with previous suggestions in the literature, the intrinsic capabilities of EGFR and HER2 to activate ERK were quantitatively equivalent. We found that HER2-mediated effects on EGFR dimerization and trafficking were sufficient to explain the observed HER2-mediated amplification of epidermal growth factor-induced ERK signaling. Our model suggests that transient amplification of ERK activity by HER2 arises predominantly from the 2-to-1 stoichiometry of receptor kinase to bound ligand in EGFR/HER2 heterodimers compared with the 1-to-1 stoichiometry of the EGFR homodimer, but alterations in receptor trafficking yielding increased EGFR sparing cause the sustained HER2-mediated enhancement of ERK signaling.     

Cellular localisation of human epidermal growth factor receptor

We show here, using immunohistological techniques and a monoclonal antibody to the receptor for epidermal growth factor (EGFR) (Waterfield et al. 1982) that EGFR is present on a wide range of normal epithelial tissues and tumours arising from those sites. The distribution of the receptor suggests that EGF may be involved in the control of proliferation and possibly differentiation of surface epithelia. The strong tumour cell staining suggests an increased expression of the receptor in certain carcinomas.     

Prognostic value of epidermal growth factor receptor in patients with gastric cancer: A meta-analysis

Background

The epidermal growth factor receptor (EGFR) plays important roles in the development of gastric cancer. This study aims to analyze the prognostic value of EGFR in patients with gastric cancer.

Methods

A meta-analysis is performed by searching Cochrane Library, PubMed, EMBASE and Science Direct databases from Jan 1970 to May 2013. Data are extracted from studies evaluating the survival of gastric cancer patients with either positive or negative EGFR expression. Pooled hazard ratios (HRs) and 95% confidence intervals (CIs) are calculated.

Results

Totally 1600 cases of gastric cancer patients from five studies are subjected to final analysis. The HR of post-operational survival of patients with positive EGFR expression is 1.16 (95% CI: 0.94–1.43) as compared with those with negative expression, indicating that positive EGFR expression does not significantly predict the poor survival of gastric cancer.

Conclusions

EGFR expression is not an independent predictor for the survival of gastric cancer patients.     

Immunohistochemical localization of epidermal growth factor receptor (EGF-r) and transforming growth factor alpha (TGF-alpha) in developing human ovarian follicles

In this study, we aimed to detect the distribution of epidermal growth factor receptor (EGF-r) and transforming growth factor alpha in ovarian follicles at different stages. Indirect immunohistochemical methods and EGF-r polyclonal and TGF-alpha monoclonal antibodies were used; tissues were examined with light microscope. While dense collection of both growth factors were observed in primordial follicles, there was a strong reaction especially for EGF-r in follicles. Strong reactivity for EGF-r and moderate reactivity for TGF-alpha were observed in the nearby connective tissue. In examinations of primary follicles for EGF-r presence only, dye uptake was moderate in oocytes and dense in apical and basal cytoplasm of follicle cells. Reactivity was moderate in the nearby connective tissue. In the corpus luteum, there was weak reaction for both growth factors. But in stromal cells, reaction was strong. In degenerated follicle cells and in stroma of atretic follicles, reaction was positive for both growth factors; but EGF-r reactivity was more obvious. While strong staining was observed for both factors especially in granulosa cells surrounding the oocyte in Graafian follicle, moderate TGF-alpha reactivity was determined in oocyte cytoplasm. In conclusion, it is possible that EGF-r and TGF-alpha have ortocrine and paracrine effects on development and regression of human ovarian follicles.     

In silico proteomic characterization of human epidermal growth factor receptor 2 (HER-2) for the mapping of high affinity antigenic determinants against breast cancer

Multiple different approaches are being used to activate the immune system against breast cancer. Vaccine therapy in general follows the principle that injections of various substances ultimately result in the presentation of tumor peptides to the patient’s immune system. We proposed a potential in silico DNA vaccine against breast cancer by integrating high affinity T cell (MHC-I and MHC-II) and B cell (continuous and discontinuous) epitopes. The matching of the HLA haplotype and antigen was performed to provide the appropriate peptide epitope suitable for majority of the patients. The immunogenic nature of the antigenic construct was also enhanced by the administration of consensus epitopes. The potency of DNA vaccines depends on the efficient expression and presentation of the encoded antigen of interest and the chances of efficient expression of our antigenic construct in host organism was also verified by in silico approaches. An attempt was made to overcome the limited potency of the DNA vaccine by targeting DNA to professional antigen-presenting cells (APCs). A higher immune response theoretically corresponds to a higher survival rate of patients. Therefore, optimization studies were also employed to enhance the immunogenicity of proposed in silico DNA vaccine.