Human Epidermal Growth Factor Receptor Family Research Articles

Potential tripeptides against the tyrosine kinase domain of human epidermal growth factor receptor (HER) 2 through computational and kinase assay approaches

An abnormal activation of human epidermal growth factor receptor (HER) 2 has been found to associate with several types of human cancer, and thus the protein is a prominent target for cancer therapy. Although several small chemical molecules targeting the tyrosine kinase (TK) of HER family have been identified, the development of a new class of inhibitors, i.e., small peptides inhibiting the function of tyrosine kinase is still promising. Here, we screened 8000 tripeptides for candidate potential inhibitors against HER2-TK using molecular docking. Our in vitro kinase assays showed that the candidate tripeptides had more than 50% relative inhibition to HER2-TK. Even though these tripeptides had much lower inhibitory activity than that of the drug Lapatinib, the tripeptides WWW exhibited high inhibitory activity with the IC50 of ≈283 μM, while FYW showed lower activity with the IC50 of ≈1723 μM. The relative binding free energies calculated by MM/PBSA method were comparable to the inhibition experiment in that Lapatinib binding was ≈-139 kJ/mol whereas the binding of WWW and FYW was ≈-112 kJ/mol and ≈-81 kJ/mol, respectively. Energy calculation also indicated that the HER2-TK/inhibitor interactions were dominated by van der Waals over electrostatic contributions. In addition, molecular interaction analyses revealed that several interacting residues with more negative binding free energy could mostly contribute the hydrophobic interaction. Therefore, we suggested preferable interactions for further development of potential tripeptides as a new anticancer peptide targeting HER2-TK.

HER family in cancer progression: From discovery to 2020 and beyond.

The human epidermal growth factor receptor (HER) family of receptor tyrosine kinases (RTKs) are among the first layer of molecules that receive, interpret, and transduce signals leading to distinct cancer cell phenotypes. Since the discovery of the tooth-lid factor-later characterized as the epidermal growth factor (EGF)-and its high-affinity binding EGF receptor, HER kinases have emerged as one of the commonly upregulated or hyperactivated or mutated kinases in epithelial tumors, thus allowing HER1-3 family members to regulate several hallmarks of cancer development and progression. Each member of the HER family exhibits shared and unique structural features to engage multiple receptor activation modes, leading to a range of overlapping and distinct phenotypes. EGFR, the founding HER family member, provided the roadmap for the development of the cell surface RTK-directed targeted cancer therapy by serving as a prototype/precursor for the currently used HER-directed cancer drugs. We herein provide a brief account of the discoveries, defining moments, and historical context of the HER family and guidepost advances in basic, translational, and clinical research that solidified a prominent position of the HER family in cancer research and treatment. We also discuss the significance of HER3 pseudokinase in cancer biology; its unique structural features that drive transregulation among HER1-3, leading to a superior proximal signaling response; and potential role of HER3 as a shared effector of acquired therapeutic resistance against diverse oncology drugs. Finally, we also narrate some of the current drawbacks of HER-directed therapies and provide insights into postulated advances in HER biology with extensive implications of these therapies in cancer research and treatment.

Abstract 2973: Growth response of human liver cancer cell lines to treatment with various agents targeting different members of the HER family and CDKs

Abstract Liver cancer is one of the most lethal types of cancer in the world. The heterogeneous nature of liver cancer, together with primary and secondary resistance to existing drugs are some of the important contributing factors. In the past three decades, abnormal expression and activation of human epidermal growth factor receptor (HER) family members have been reported in a wide range of epithelial cancers and several monoclonal antibody (mAb)-based drugs and small molecule tyrosine kinase inhibitors (TKIs) targeting the HER family members have been approved for the treatment of patients with a wide range of cancers. However, none of the HER inhibitors have yet been approved for the treatment of patients with liver cancer. In some studies, the expression of other members of the HER family or other growth factor receptors has been associated with the resistance to therapy with the HER inhibitors. In this study, we investigated the relative expression of all HER family members, other growth factor receptors (e.g. IGF-IR, C-MET) and the putative cancer stem cell biomarker CD44 in a panel of human liver cancer cell lines (LCCLs). We also investigated the sensitivity of these LCCLs to treatment with various agents including different types of TKIs with specificity to one or more members of the HER family, C-MET or IGFI-IR, as well as inhibitors of CDK4/6 (palbocicilib) and CDK1/2/5/9 (dinacicilib) and compared our data to those obtained with the FDA approved agents sorafenib and regorafenib for the treatment of liver cancer. While the great majority of the LCCLs were positive for the HER family members, overexpression of the EGFR, HER-3, and HER-4 were only present in SNU475, PLC/PRF5 and PLC/PRF5 LCCLs with mean fluorescence intensity (MFI) values of 620, 130 and 84 respectively. In contrast, the expression levels of C-MET and IGF-IR were the highest in SNU449 and HEPG-2 with MFI values of 95 and 99 respectively. Four of the seven LCCLs had overexpression of CD44 with the MFI values ranging from 329 to 704. With the exception of PLC/PRF5 which was found to be more sensitive to treatment with the pan-HER TKI afatinib (IC50 = 86nM) than treatment with sorafenib (IC50= 370nM) and regorafenib (372 nM), most of the other LCCLs were insensitive to treatment with the other types of HER inhibitors. Moreover, out of all the targeted therapies, the CDK inhibitors Dinacicilib and Palbocicilib were the most effective treatment for inhibiting the growth in vitro of these LCCLs. However, using linear regression analysis, we did not find any significant association between the expression level of different growth factor receptors and the response to therapy with various agents. Taken together our results show the heterogeneous nature of human LCCLs and the need for further investigation on the therapeutic potential of afatinib in combination with other agents in liver cancer. Citation Format: Ozlem H. Ozyamaci, Alan M. Seddon, Satvider Mudan, Helmout Modjtahedi. Growth response of human liver cancer cell lines to treatment with various agents targeting different members of the HER family and CDKs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2973.

Abstract 357: Pan-HER, an antibody mixture simultaneously targeting EGFR, HER2, and HER3, is highly effective in triple negative breast cancer patient-derived xenografts

Abstract As a heterogeneous disease, breast cancer is clinically classified taking into account the expression of estrogen receptor alpha (ERα), progesterone (PR), and the presence/amplification status of the oncogenic human epidermal growth factor receptor 2 (HER2). Triple negative breast cancer (TNBC) represents ~10-20% of all cases, and displays poor prognosis and aggressive clinical course. EGFR is more often overexpressed in TNBC than in other breast cancer subtypes, providing cancer cells with compensatory signals that greatly contribute to the development of resistance and survival in response to therapy. This characteristic has made HER family members potentially important therapeutic targets. The present study was designed to investigate the effects of Pan-HER, a novel mixture of six monoclonal antibodies directed against members of the human epidermal growth factor receptor (HER) family EGFR, HER2, and HER3, in a preclinical trial of TNBC patient-derived xenografts (PDXs). Fifteen low passage TNBC PDX tumor samples were transferred and engrafted into the right mammary fat pad of mice for engraftment. When tumors reached an average size of 150-200 mm3, mice were randomized (n ≥ 3 per group), and treated following three, one-week cycles, consisting of 3 times/week intraperitoneally (IP) injection of either formulation buffer (Vehicle control) or Pan-HER (50 mg/kg). At the end of treatment, tumors were collected for western blot, RNA and immunohistochemistry analyses. All 15 TNBC PDXs were responsive to Pan-HER treatment, showing significant reductions in tumor growth consistent with Pan-HER-mediated tumor down-modulation of EGFR and HER3 protein levels and significantly decreased activation of associated HER family signaling pathways including AKT, ERK and NF-κB. Tumor regression was observed in five of the models, which corresponded to those PDX tumor models with the highest level of HER family activation. This observation may provide a plausible explanation for the response of TNBC tumors to Pan-HER as an indication of the potential use of EGFR and associated signaling expression as biomarkers for selecting patients that may benefit of targeting these proteins. In summary, this preclinical TNBC PDX trial with Pan-HER provides strong justification for further biomarker-guided studies in TNBC. The finding that tumors were affected rapidly and effectively, with long-lasting results, offers exciting perspectives to treat this aggressive form of breast cancer, for which available treatment options are currently limited. Citation Format: Roberto R. Rosato, Don S. Choi, Wei Qian, Wen Chen, Johan Lantto, Ivan D. Horak, Michael Kragh, Jenny C. Chang. Pan-HER, an antibody mixture simultaneously targeting EGFR, HER2, and HER3, is highly effective in triple negative breast cancer patient-derived xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 357.

Roles of human epidermal growth factor receptor family in pulmonary lymphangioleiomyomatosis

Lymphangioleiomyomatosis (LAM) is a rare and refractory disease that affects women of reproductive age. Several target therapies are used to manage LAM, but no curative modes of treatment have been reported yet. Therefore, in this study, we focused on targeting human epidermal growth factor receptor (HER) family proteins as a treatment strategy for LAM. In antibody array analysis, HER signaling was detected in the proteins extracted from LAM tissues. We then evaluated the expression of HER family members in 34 pulmonary LAM specimens using both immunohistochemistry and quantitative reverse-transcription polymerase chain reaction. Hierarchical clustering analysis was performed to classify the cases based on the immunohistochemistry results. Both epidermal growth factor receptor (EGFR) and HER4 were expressed in all 34 cases. HER3 was expressed in 25 of 34 cases, but HER2 was not expressed in any case. In addition, results of quantitative reverse-transcription polymerase chain reaction analysis confirmed the expression of EGFR and HER4 expression in LAM cells. Patients with HER3- or HER4-positive tissues were younger and had a history of pneumothorax. The cases were classified into 4 different clusters based on the results of hierarchical cluster analysis. One of these clusters was associated with EGFR, HER3, and HER4; the patients in this cluster were significantly younger and had a history of pneumothorax. These results indicated that HER family could contribute to the progression of pulmonary LAM, and treatments targeted against HER family might be effective for treating pulmonary LAM.

Abstract B68: EGFR signaling mediated modulation of transcription and probable crosstalk with components of Wnt signaling

Abstract Introduction: The purpose of the study is to examine the crosstalk between HER2/EGFR and Wnt signaling in HER2-positive (HER2+) breast cancer cells. Human epidermal growth factor receptors (HER) constitute a family of four transmembrane receptors (HER1-HER4). Ligand binding to HER1 (EGFR), HER3, and HER4 results in heterodimerization with other HER family members, including HER2. HER2+ breast cancer is characterized by an amplification of the HER2 gene, resulting in an increase in HER2 protein presence on the surface of cells, magnification of downstream intracellular signaling, and enhanced responsiveness to ligand stimulation (e.g., EGF). Approximately 20-30% of breast cancers have HER2 amplifications. The Wnt pathway is highly conserved in mammals and overexpression of some Wnt family members results in cancer. Wnts are ligands that bind to Frizzled/LRP receptors to initiate downstream signaling that results in the stabilization and nuclear translocation of β-catenin. Once in the nucleus, β-catenin associates with activators such as TCF/LEF, SMADs, ATF2, and KLF4 to promote the transcription of many target genes. EGFR and Wnt crosstalk has been observed in various cell types following EGF treatment. As an example, EGF treatment of human epidermoid carcinoma cells results in β-catenin nuclear translocation and activation of TCF/LEF dependent reporters. Mitogen-Activated Protein Kinases (MAPKs), which are activated following EGF stimulation, have been demonstrated to inhibit GSK3β, a negative regulator of β-catenin. However, no genome-wide analysis has been conducted to determine what genes are regulated by β-catenin following EGF treatment in HER2+ breast cancer cells. We sought to determine the modulation of gene expression following the stimulation of HER2+ breast cancer cells with EGF and to investigate the mechanisms that underlie the changes observed in gene expression. Our studies have revealed exciting and novel findings that elucidate the effects of EGFR signaling on the epigenetic landscape. Specifically, we have identified putative β-catenin targets that become activated following EGFR stimulation. We hypothesize that EGFR signaling promotes the activation of specific β-catenin genes in order to alter cellular identity. Methods: RNA-seq and ChIP-seq for H3K18ac and H3K27ac was conducted following an EGF treatment time course in SKBR3 cells. The levels of several proteins of interest were determined by Western blot analysis. The cellular localization of proteins of interest was examined using biochemically fractionated lysates followed by Western blot analysis. Results: RNA-seq analysis following an EGF treatment time course revealed that approximately 2,200 genes are either upregulated or downregulated compared to untreated cells. Moreover, the expression profiles clearly demonstrated waves of transcription. Next, we determined the status of H3K18ac and H3K27ac using ChIP-seq following an EGF time course. We found that H3K18ac and H3K27ac increased globally within 1h post-EGF treatment compared to untreated cells. We conducted a motif discovery search for transcription factor binding sites contained from -1000bp to +200bp for all activated genes and determined that each wave of transcription had some unique putative regulators. As expected, the genes activated at 1h and 2h post-EGF treatment contained c-Jun and JunD binding sites. Surprisingly, TCF3, TCF5, and LEF1 motifs were enriched in some genes that peaked in expression at 6h, 16h, and 24h post EGF treatment. Lastly, we biochemically fractionated the cellular compartments and detected an increase in chromatin associated β-catenin following EGF treatment, suggesting a crosstalk between EGFR and Wnt signaling components. We plan to determine the genome-wide localization of β-catenin following EGF treatment. Conclusions: Our data suggest that a crosstalk between EGFR and Wnt signaling components may regulate β-catenin target genes and lead HER2+ cells' resistance to therapeutic treatment. Citation Format: Miguel Nava, Nwamaka Amobi, Nathan Zemke, Arnold Berk, Robin Farias-Eisner, Jay Vadgama, Yanyuan Wu. EGFR signaling mediated modulation of transcription and probable crosstalk with components of Wnt signaling [abstract]. In: Proceedings of the Tenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2017 Sep 25-28; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2018;27(7 Suppl):Abstract nr B68.

The marine natural product coibamide targets expression of HER family receptors

Natural products have historically been a significant source of inspiration for drug design and development, particularly in the areas of cancer and infectious disease. The utility of natural products as drug leads is attributed to the fact that these molecules have evolved to bind specific biological targets and have the potential to reveal new aspects of cell signaling. We previously reported the discovery of coibamide, a cytotoxic N‐methyl‐stabilized depsipeptide isolated from a marine cyanobacterium growing within Coiba National Park, Panama. Initial testing of coibamide in the National Cancer Institute in vitro 60 (NCI60) cancer cell line panel revealed pM to nM potency as a growth inhibitor, an unmatched selectivity profile and histological selectivity for breast, CNS‐derived tumors, ovarian and colon cancer cells.We have undertaken studies to determine the mechanism of action of coibamide and have determined that coibamide selectively targets the ER secretory pathway. To date, these relatively rare natural products are known for their ability to selectively inhibit biosynthesis receptor tyrosine kinases, growth factors and cell adhesion proteins. We studied the relative expression of human epidermal growth factor receptor (HER) proteins in human HER2‐amplified and triple negative breast cancer cells following exposure to coibamide. Coibamide inhibited expression of HER family members with differential sensitivity (EGFR=HER3<<HER2). Time‐ and concentration‐dependent decreases in HER protein expression were accompanied by a dampening in AKT/MAPK signaling. Coibamide promoted proteasomal degradation of HER family proteins consistent with the action of an ER secretory pathway inhibitor. These results suggest that coibamide preferentially inhibits the biosynthesis of a subset of ER secretory pathway proteins to induce cancer cell death rather than a total inhibition of cellular secretory function. The HER family of proteins are associated with drug resistance and treatment failure in response to many modern, targeted anti‐cancer medicines and thus this work has the potential to reveal a new way to target HER signaling.Support or Funding InformationThis work was supported by the Oregon State University (OSU) College of Pharmacy, the American Brain Tumor Association (JEI) and an American Foundation for Pharmaceutical Education (AFPE) Pre‐Doctoral Fellowship in the Pharmaceutical Sciences (JDS)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Analysis of HER Family (HER1-4) Expression as a Biomarker in Combination Therapy with Pertuzumab, Trastuzumab and Docetaxel for Advanced HER2-positive Breast Cancer.

Chemotherapy with trastuzumab, pertuzumab and docetaxel (TPD regimen) is now strongly recommended as a treatment option for first-line therapy for advanced human epidermal growth factor receptor (HER)2-positive breast cancer. In this study, we analyzed the expression of HER 1-4 proteins, and investigated whether or not their expression was predictive of the response of advanced HER2-positive breast cancer to chemotherapy with the TPD regimen. The study consisted of 29 cases in which TPD regimen chemotherapy was carried out from September 2013 to November 2015. The expression levels of estrogen receptor (ER), progesterone receptor (PgR), Ki67, HER1, HER2, HER3 and HER4 were evaluated using immunostaining employing needle biopsy specimens. The overall response rate (ORR) was significantly higher in the HER3-positive group than in the HER3-negative group (p=0.002). In prognostic analysis, the HER3-positive group showed a significant progression-free survival extension over the HER3-negative group (p=0.042, log-rank). In univariate analysis, objective response (p=0.004, hazard ratio(HR)=0.123) and positive HER3 expression (p=0.023, HR=0.279) significantly contributed to extension of progression-free survival interval. HER3 expression may be a useful factor for predicting the response of HER2-positive breast cancer to chemotherapy with the TPD regimen.

Resistance to receptor-blocking therapies primes tumors as targets for HER3-homing nanobiologics

Resistance to anti-tumor therapeutics is an important clinical problem. Tumor-targeted therapies currently used in the clinic are derived from antibodies or small molecules that mitigate growth factor activity. These have improved therapeutic efficacy and safety compared to traditional treatment modalities but resistance arises in the majority of clinical cases. Targeting such resistance could improve tumor abatement and patient survival. A growing number of such tumors are characterized by prominent expression of the human epidermal growth factor receptor 3 (HER3) on the cell surface. This study presents a “Trojan-Horse” approach to combating these tumors by using a receptor-targeted biocarrier that exploits the HER3 cell surface protein as a portal to sneak therapeutics into tumor cells by mimicking an essential ligand. The biocarrier used here combines several functions within a single fusion protein for mediating targeted cell penetration and non-covalent self-assembly with therapeutic cargo, forming HER3-homing nanobiologics. Importantly, we demonstrate here that these nanobiologics are therapeutically effective in several scenarios of resistance to clinically approved targeted inhibitors of the human EGF receptor family. We also show that such inhibitors heighten efficacy of our nanobiologics on naïve tumors by augmenting HER3 expression. This approach takes advantage of a current clinical problem (i.e. resistance to growth factor inhibition) and uses it to make tumors more susceptible to HER3 nanobiologic treatment. Moreover, we demonstrate a novel approach in addressing drug resistance by taking inhibitors against which resistance arises and re-introducing these as adjuvants, sensitizing tumors to the HER3 nanobiologics described here.

Molecular dynamics simulations of asymmetric heterodimers of HER1/HER2 complexes

The family of human epidermal growth factor receptors (HER) is involved in tumor cell growth. Homodimerization and heterodimerization of the HER family are important for activation of these receptors. The structures of homodimer conformation are well characterized, while the structures of heterodimer conformations, especially between HER1 and HER2, are not completely understood. In this study, two models of possible asymmetric HER1/HER2 kinase domains were built. Molecular dynamics simulations and molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) methods were applied to examine the possibility of these two-heterodimer interactions. From our results, it could be concluded that the HER2 kinase domain prefers to serve as the receiver rather than the activator. Key binding residues of this dimer complex at N lobe of HER2 is ALA683 and at C lobe of HER1 are GLU914, GLU917, and ASP930. This study will be useful in allowing us to predict and be able to control activity of this enzyme in disease in the future. Graphical abstract A model of the asymmetric dimer of HER1-HER2 heterodimer with key intereaction residues in (a) HER1A and (b) HER2R by molecular dynamic simulation.

SiRNA-mediated inactivation of HER3 improves the antitumour activity and sensitivity of gefitinib in gastric cancer cells.

The human EGFR family consists of four type-1 transmembrane tyrosine kinase receptors: HER1 (EGFR, ErbB1), HER2 (Neu, ErbB2), HER3 (ErbB3), and HER4 (ErbB4). HER3 can dimerize with EGFR, HER2 and even c-Met and likely plays a central role in the response to EGFR-targeted therapy. Because HER3 lacks significant kinase activity and cannot be inhibited by tyrosine kinase inhibitors, neutralizing antibodies and alternative inhibitors of HER3 have been sought as cancer therapeutics. Here, we describe the stable suppression of HER3 mRNA and protein using siRNA. The inhibition of HER3 expression decreased cell proliferation, suppressed cell cycle progression, induced apoptosis and inhibited cell motility, migration, invasiveness, and soft agar growth. In addition, we found that gefitinib treatment increased the HER3 and HER2 mRNA levels. The administration of various concentrations of gefitinib to HER3-knockdown cells enhanced antitumour activity and sensitivity due to the downregulation of protein phosphorylation via PI3K/AKT and ERK signalling. Our results support the use of combined treatments targeting multiple EGFR receptors, particularly the use of HER3 inhibitors combined with EGFR inhibitors, such as gefitinib.

Natural Products as Chemopreventive Agents by Potential Inhibition of the Kinase Domain in ErbB Receptors.

Small molecules found in natural products provide therapeutic benefits due to their pharmacological or biological activity, which may increase or decrease the expression of human epidermal growth factor receptor (HER), a promising target in the modification of signaling cascades involved in excessive cellular growth. In this study, in silico molecular protein-ligand docking protocols were performed with AutoDock Vina in order to evaluate the interaction of 800 natural compounds (NPs) from the NatProd Collection (http://www.msdiscovery.com/natprod.html), with four human HER family members: HER1 (PDB: 2ITW), HER2 (PDB: 3PP0), HER3 (PDB: 3LMG) and HER4 (PDB: 2R4B). The best binding affinity values (kcal/mol) for docking pairs were obtained for HER1-podototarin (−10.7), HER2-hecogenin acetate (−11.2), HER3-hesperidin (−11.5) and HER4-theaflavin (−10.7). The reliability of the theoretical calculations was evaluated employing published data on HER inhibition correlated with in silico binding calculations. IC50 values followed a significant linear relationship with the theoretical binding Affinity data for HER1 (R = 0.656, p < 0.0001) and HER2 (R = 0.543, p < 0.0001), but not for HER4 (R = 0.364, p > 0.05). In short, this methodology allowed the identification of several NPs as HER inhibitors, being useful in the discovery and design of more potent and selective anticancer drugs.

Research status quo and progression in targeted therapy for advanced gastric cancer

With the deeper research of the proliferation, invasion and metastasis mechanisms of the gastric cancer, targeted therapy has become a hot spot in this field. The exploration of targeted agents for gastric cancer is mainly concentrated upon the drugs that target human epidermal growth factor receptor (HER) family, the vascular endothelial growth factor (VEGF), the phosphatidylinositol 3-kinase-protein kinase/mammalian target of rapamycin (PI3K/mTOR) and the NF-κB signaling pathways. The targeted drugs relevant to HER family include the Cetuximab, Nimotuzumab, Matuzumab, Panitumumab and Erlotinib which are aimed at HER-1, the Trastuzumab, Pertuzumab and T-DM1 (trastuzumab emtansine) which are aimed at HER-2, and the Lapatinib and Afatinib which are the multi-target agents of HER. The agents which target VEGF signaling pathway include the anti-VEGF monoclonal antibody (Bevacizumab), the anti-VEGFR drugs (Ramucirumab, Apatinib, Sorafenib, Sunitinib and cediranib), and the recombinant fusion protein (Aflibercept). The LY294002, BEZ235 and Everolimus which are aimed at PI3K/mTOR signaling pathway have shown great promise. Bortezomib has been researched more as a new agent which targets NF-κB signaling pathway. Currently, only Trastuzumab, Ramucirumab and Apatinib have been completed the stage 3 clinical trials and succeeded. In future, researches should focus on multi-target agents or applications in combination treatment. This review collects the recent researches and the clinical trials to summarize the current state and progress of research on gastric cancer targeted therapy.

Increased expression of EHF via gene amplification contributes to the activation of HER family signaling and associates with poor survival in gastric cancer.

The biological function of E26 transformation-specific (ETS) transcription factor EHF/ESE-3 in human cancers remains largely unknown, particularly gastric cancer. The aim of this study was to explore the role of EHF in tumorigenesis and its potential as a therapeutic target in gastric cancer. By using quantitative RT-PCR (qRT-PCR), immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) assays, we investigated the expression and copy number of EHF in a cohort of gastric cancers and control subjects. Specific EHF siRNAs was used to determine the biologic impacts and mechanisms of altered EHF expression in vitro and in vivo. Dual-luciferase reporter, chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA) assays were performed to identify its downstream targets. Our results demonstrated that EHF was significantly upregulated and frequently amplified in gastric cancer tissues as compared with control subjects. Moreover, EHF amplification was positively correlated with its overexpression and significantly associated with poor clinical outcomes of gastric cancer patients. We also found that EHF knockdown notably inhibited gastric cancer cell proliferation, colony formation, migration, invasion and tumorigenic potential in nude mice and induced cell cycle arrest and apoptosis. Importantly, we identified EHF as a new HER2 transcription factor and the modulator of HER3 and HER4 in gastric cancer. Collectively, our findings suggest that EHF is a novel functional oncogene in gastric cancer by regulating the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases and may represent a potential prognostic marker and therapeutic target for this cancer.

Prognostic value of ERBB4 expression in patients with triple negative breast cancer.

BackgroundTriple-negative breast cancer (TNBC) is known for aggressive biologic features and poor prognosis. Epidermal growth factor receptor (EGFR) overexpression in TNBC indicates poor prognosis. However, there is no previous study of the relationship between expression of the entire human epidermal growth factor receptor (HER) family genes and patient prognosis in TNBC. Accordingly, we investigated the expression profiles of HER family genes in patients with TNBC to determine the prognostic value and clinical implications of HER family expression.MethodsWe used the nCounter expression assay (NanoString®) to measure the expression of EGFR, erb-B2 receptor tyrosine kinase 2 (ERBB2), ERBB3, ERBB4, and estrogen receptor 1 (ESR1) genes using mRNA extracted from paraffin-embedded tumor tissues from 203 patients diagnosed with TNBC. Our data were validated using a separate cohort of 84 TNBC patients.ResultsA total of 203 TNBC patients who received adjuvant chemotherapy after curative surgery from 2000 to 2004 formed the training set. The 84 TNBC patients in the validation consort were selected from breast cancer patients who received curative surgery since 2005 to 2010. Analysis of the expression profiles of the HER family genes in TNBC tissue specimens revealed that increased expression of ERBB4 was associated with poor prognosis according to survival analysis (5-year distant relapse free survival [5Y DRFS], low vs. high expression [cut-off: median]: 90.1 % vs. 80.2 %; p = 0.022). This trend was also observed in the validation set of TNBC patients (5Y DRFS, low vs. high: 69.4 % vs. 44.7 %; p = 0.053). In a multivariate Cox regression model, ERBB4 expression was identified as a indicator of long-term prognosis in patients with TNBC.ConclusionsThe expression profile of ERBB4, a member of the HER family, might serve as a prognostic marker in patients with TNBC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2195-3) contains supplementary material, which is available to authorized users.

Abstract P6-15-01: Triple negative breast cancer is vulnerable to Pan-HER, an antibody mixture simultaneously targeting EGFR, HER2 and HER3

Abstract Background: Triple negative breast cancer (TNBC) is a highly heterogeneous and aggressive subtype of cancer, lacking expression of estrogen and progesterone receptors as well as human epidermal growth factor receptor (HER) 2 protein. Limited standard therapeutic options, absence of effective targeted therapies, and early metastatic spread have contributed to poor prognosis and outcomes associated with this disease. Although overexpression of EGFR has been reported in nearly 80% TNBC, EGFR-targeted therapy has yielded little clinical benefit, and the outcome is still under debate. In conjunction, we also found mixed effects of EGFR-targeted therapy on TNBC xenograft tumors despite significant target engagement, suggesting that tumor heterogeneity and compensating mechanisms may contribute to the variable drug responses to the EGFR-targeted therapy. Recently, we reported superior anti-cancer effects of Pan-HER, a mixture of antibodies targeting the HER family members EGFR, HER2 and HER3 on various types of cancer by overcoming drug resistance and tumor heterogeneity. To this end, we hypothesized that Pan-HER can effectively inhibit tumor growth in TNBC by inhibiting tumor heterogeneity and drug resistance. Objective: The goal of this study is to test the effect of Pan-HER antibody mixture (Sym013) on tumor growth and recurrence of 14 patient-derived (PDX) TNBC orthotopic xenograft tumor models and to investigate molecular biomarkers which can predict drug response to Pan-HER. Methods: We evaluated in-vivo anti-tumor effects of Pan-HER (50 mg/kg, i.p. three times/week, 10 doses in total for 3 weeks) over vehicle on tumor growth and tumor recurrence on 14 PDX TNBC models with known expression levels of EGFR and HER3 (n=3/group). HER family proteins and related downstream molecules (Akt, Erk, Stat3, FAK) in the tumor tissues were evaluated by Western blot assay and immunohistochemistry analysis. Additionally, using dCHIP and ingenuity pathway analysis, we compared microarray data from the tested cohorts and other TNBC PDX models with known HER family receptor status. Results: We found that Pan-HER alone effectively inhibited tumor growth in all 14 PDX models and showed statistical significance (p=0.0103) when compared to the vehicle groups. Among these, one PDX model, BCM-3186, showed substantial tumor reduction and additional two (MC1 and BCM-4913) showed complete response with no recurrence after the last treatment of Pan-HER. The significant anti-tumor effects of Pan-HER were positively correlated with inhibition of phosphorylation and expression of EGFR, HER3, Akt, Erk, and FAK, but not Stat3, and this was consistent in all PDX models tested. Additionally, the microarray and the pathway enrichment analyses suggest that loss of PTEN expression and up-regulation of FAK and RAS pathways may be the predictive markers for the Pan-HER drug response in TNBC. Conclusion: Our in-vivo data suggest that simultaneous targeting of the three HER family receptors is a potential new approach for treatment of TNBC. Further confirmation of our in-vivo results will warrant a phase I clinical trial and lend support to single agent Pan-HER as a viable treatment strategy for TNBC patients in the clinic. Citation Format: Choi DS, Qian W, Davila-Gonzalez D, Ensor JE, Lantto J, Kragh M, Horak ID, Chang JC. Triple negative breast cancer is vulnerable to Pan-HER, an antibody mixture simultaneously targeting EGFR, HER2 and HER3. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-15-01.

Genetic alterations of HER genes in chromophobe renal cell carcinoma

Chromophobe (ch) renal cell carcinoma (RCC) is the 3rd most common subtype of RCC and occurs in 5% of all RCCs. Although chRCC generally demonstrates more favorable outcomes compared with other subtypes of RCC, there is a 6-7% probability of tumor progression and metastasis in this disease. The subclassification of a more aggressive subtype of chRCC may be useful for the management of this cancer. The Erb-B2 receptor tyrosine kinase 2 [also known as human epidermal growth factor receptor (HER) 2] gene has been reported to be important in chRCC. The present study aimed to further investigate the abnormalities of the HER family genes and their potential association with chRCC. Fluorescence in situ hybridization was performed on 11 chRCC tissue specimens, and the Spearman's rank correlation coefficient analysis was used to assess the results. The loss of one copy of the HER2 and HER4 genes was observed to be the major alteration of the tumor cells in all chRCC cases. Statistical data indicated that loss of the HER2 gene was strongly correlated with loss of the HER4 gene (P=0.019). The findings of previous studies were also combined for analysis, and were consistent with those of the present study. In addition, the amplification of HER1 was also strongly correlated with the amplification of HER4 (P=0.004). Furthermore, a high percentage of genetic structural rearrangements was observed in HER3 genes, which was significantly associated with amplification of HER2 (P=0.005). Certain alterations in the HER gene family were also noted as a phenomenom in chRCC. Therefore, the characterization of the underlying aberrant functions of HER genes may be of interest for additional studies in the context of using HER genes to distinguish between RCC subtypes in order to establish improved treatment guidelines.

In vivoimaging of therapy response to a novel Pan-HER antibody mixture using FDG and FLT positron emission tomography

Overexpression of the human epidermal growth factor receptor (HER) family and their ligands plays an important role in many cancers. Targeting multiple members of the HER family simultaneously may increase the therapeutic efficacy. Here, we report the ability to image the therapeutic response obtained by targeting HER family members individually or simultaneously using the novel monoclonal antibody (mAb) mixture Pan-HER. Mice with subcutaneous BxPC-3 pancreatic adenocarcinomas were divided into five groups receiving vehicle or mAb mixtures directed against either EGFR (HER1), HER2, HER3 or all three receptors combined by Pan-HER. Small animal positron emission tomography/computed tomography (PET/CT) with 2'-deoxy-2'-[(18)F]fluoro-D-glucose (FDG) and 3'-deoxy-3'-[(18)F]fluorothymidine (FLT) was performed at baseline and at day 1 or 2 after initiation of therapy. Changes in tumor uptake of tracers were quantified and compared to reduction in tumor size. Imaging results were further validated by immunohistochemistry and qPCR. Mean FDG and FLT uptake in the Pan-HER treated group decreased by 19 ± 4.3% and 24 ± 3.1%, respectively. The early change in FDG and FLT uptake correlated with tumor growth at day 23 relative to day 0. Ex vivo molecular analyses of markers associated with the mechanisms of FDG and FLT uptake confirmed the in vivo imaging results. Taken together, the study supports the use of FDG and FLT as imaging biomarkers of early response to Pan-HER therapy. FDG and FLT PET/CT imaging should be considered as imaging biomarkers in clinical evaluation of the Pan-HER mAb mixture.

Pan-HER, an Antibody Mixture Simultaneously Targeting EGFR, HER2, and HER3, Effectively Overcomes Tumor Heterogeneity and Plasticity.

Accumulating evidence indicates a high degree of plasticity and compensatory signaling within the human epidermal growth factor receptor (HER) family, leading to resistance upon therapeutic intervention with HER family members. We have generated Pan-HER, a mixture of six antibodies targeting each of the HER family members EGFR, HER2, and HER3 with synergistic pairs of antibodies, which simultaneously remove all three targets, thereby preventing compensatory tumor promoting mechanisms within the HER family. Pan-HER induces potent growth inhibition in a range of cancer cell lines and xenograft models, including cell lines with acquired resistance to therapeutic antibodies. Pan-HER is also highly efficacious in the presence of HER family ligands, indicating that it is capable of overcoming acquired resistance due to increased ligand production. All three target specificities contribute to the enhanced efficacy, demonstrating a distinct benefit of combined HER family targeting when compared with single-receptor targeting. Our data show that simultaneous targeting of three receptors provides broader efficacy than targeting a single receptor or any combination of two receptors in the HER family, especially in the presence of HER family ligands. Pan-HER represents a novel strategy to deal with primary and acquired resistance due to tumor heterogeneity and plasticity in terms of HER family dependency and as such may be a viable alternative in the clinic.

Abstract P5-04-02: Hyaluronan (HA) depletion sensitizes HAhigh tumors to antibody-dependent cell-mediated cytotoxicity

Abstract Therapeutic efficacy of monoclonal antibodies (MAbs) against solid tumor targets, like trastuzumab (anti-HER2) and cetuximab (anti-EGFR), have been used successfully to treat cancer, despite the many physical barriers impeding their access to the malignant cell surface1. For example, only about 50% of HER23+ patients have a durable response to therapy with trastuzumab2. Extracellular matrix (ECM)-mediated inhibition may be among the mechanisms of resistance to MAb therapy of solid tumors. Aberrant accumulation of hyaluronan (HA), a major component of the ECM in many tumors, is associated with poor prognosis and treatment-resistance in multiple malignancies3-5. We investigated HA-dependent pericellular matrix-mediated inhibition to ADCC in HAhigh human cancer cells in vitro and in vivo. We observed high levels of tumor associated HA (HA3+) in &amp;gt;50% of HER23+ breast adenocarcinoma and ∼40% of EGFR+ head and neck squamous cell carcinoma (HNSCC) primary tumors. Human hyaluronan synthase 2 (HAS2)-overexpressing breast cancer cells formed an HAhigh pericellular matrix, which inhibited both natural killer (NK) cell access to tumor cells and ADCC in vitro. Hyaluronan depletion by PEGPH20, a pegylated recombinant human PH20 hyaluronidase currently in clinical study for pancreatic cancer, increased NK cell access to HAS2-overexpressing breast cancer cells and greatly enhanced trastuzumab- or cetuximab-dependent ADCC. Trastuzumab and NK cell accessibility to HAS2-overexpressing tumors was enhanced following HA-depletion by PEGPH20. In an in vivo ADCC-based efficacy study, PEGPH20 treatment in combination with trastuzumab and NK cells enhanced tumor growth inhibition. This work describes a novel tumor microenvironment (TME)-dependent mechanism of inherent resistance to therapeutic antibody-mediated ADCC in vitro and in vivo, and furthermore shows that ADCC can be enhanced by hyaluronan depletion. These results may help to explain as to why tumors with high levels of HA are more aggressive, and suggest potential benefits of PEGPH20-mediated HA depletion in combination with therapeutic antibodies like trastuzumab or cetuximab in the treatment of HAhigh solid tumors.