Human Epidermal Growth Factor Receptor Family Research Articles

The Combination of Afatinib With Dasatinib or Miransertib Results in Synergistic Growth Inhibition of Stomach Cancer Cells.

Of various human epidermal growth factor receptor (HER) inhibitors, only the anti-HER2 monoclonal antibody (mAb) Herceptin/trastuzumab and the antibody-drug conjugate trastuzumab deruxtecan (T-Dxd) has been approved for the treatment of patients with stomach cancer. However, the duration of response may be short in many patients, with tumor heterogeneity being one contributing factor. We investigated the effect of various types of targeted agents on growth in vitro and migration of a panel of human stomach cancer cells (HSCCLs) and the impact of cell proliferation rate on the anti-tumor activities of these agents. We also investigated the association between the cell surface expression of the HER family members, hepatocyte growth factor receptor (c-Met), anaplastic lymphoma kinase (ALK)7 and cancer stem cell markers CD44 and CD133, and the response to the targeted agents. Of the 18 agents examined, the cyclin dependent kinase (CDK) 1/2/5/9 inhibitor dinaciclib was the most effective and inhibited the growth of all human HSCCLs at 50% inhibitory concentration (IC50) values between 9 nM to 23 nM. Of various HER inhibitors, the irreversible pan-HER family inhibitors (e.g., afatinib) were more effective than the reversible dual epidermal growth factor receptor (EGFR)/HER2 tyrosine kinase inhibitor (TKI) lapatinib and the EGFR-specific TKI erlotinib in inhibiting the growth of HSCCLs. Of agents targeting different downstream cell signaling molecules, dasatinib targeting Ab1/Src/C-Kit, trametinib targeting MERK1/2 and miransertib targeting AKT1/2/3 inhibited growth of majority of HSCCLs, with the IC50 values ranging from 2 nM to 7 µM. Many of these agents were more effective in inhibiting the growth of HSCCLs when they were proliferating at a slower rate. Treatment with neratinib, afatinib, dinaciclib, dasatinib, stattic, miransertib and paclitaxel significantly inhibited migration of stomach cancer cells. Interestingly, treatment with a combination of afatinib and dasatinib or afatinib and miransertib resulted in synergistic and additive growth inhibition of stomach cancer cells. These results suggest that treatment with a combination of these agents may be of therapeutic value in stomach cancer and warrants further investigations.

The impact of nitric oxide on HER family post-translational modification and downstream signaling in cancer.

The human epidermal growth factor receptor (HER) family consists of four members, activated by two families of ligands. They are known for mediating cell-cell interactions in organogenesis, and their deregulation has been associated with various cancers, including breast and esophageal cancers. In particular, aberrant epidermal growth factor receptor (EGFR) and HER2 signaling drive disease progression and result in poorer patient outcomes. Nitric oxide (NO) has been proposed as an alternative activator of the HER family and may play a role in this aberrant activation due to its ability to induce s-nitrosation and phosphorylation of the EGFR. This review discusses the potential impact of NO on HER family activation and downstream signaling, along with its role in the efficacy of therapeutics targeting the family.

Abstract 5152: Co-expression and prognostic significance of HER family members and EGFRvIII in patients with hepatocellular carcinoma

Abstract Hepatocellular carcinoma is one of the most common type of liver cancer. Despite advances in its treatment including the approval of several targeted agents in the past few decades, many patients do not respond or have a response of short duration to such therapeutic interventions. As a result, liver cancer is the third leading cause of cancer-related deaths worldwide. Therefore, it is essential to identify additional therapeutic targets and to develop more effective therapeutic interventions. In the past few decades, increased expression, and activation of the human epidermal growth factor receptor (HER) family members have been detected in various cancer types and of these, EGFR and HER2 are important therapeutic targets for the treatment of patients with a wide range of cancers, but currently not in liver cancer. In some studies, cross-talk between different members of the HER family has been associated with tumor progression and resistance to therapy with EGFR inhibitors but there has been no comprehensive study of the expression of all members of the HER family in patients with hepatocellular carcinoma. In this study, we examined the relative expression of all members of the HER family, type-III deletion mutant of EGFR (i.e.EGFRvIII), which is a tumor specific antigen, and two putative liver cancer cell biomarkers (i.e. CD44, EpCAM) in 43 patients with hepatocellular carcinoma by immunohistochemistry. The prognostic significance of such antigens was also investigated. Of these, the expression of EGFR, HER2, HER3, HER4 and EGFRvIII was found in tumor specimens from 35%, 58%, 0%, 19%, and 26% of the cases, respectively. Interestingly, at a cut off value of >5% of tumor cells with staining, 23%, 14% and 9% of the patients had co-expression of EGFR with HER2, EGFRvIII, and HER2/EGFRvIII. In addition, 40% and 33% of the cases were CD44 and EpCAM positive. Of these, the expression of EGFRvIII positivity at >5% ( HR=3.35, p=0.002) and 20% (HR=7.22, p=0.003) of tumour cells and membranous CD44 ( HR=2.12, p=0.04) were associated with a poorer survival in patients with hepatocellular carcinoma. In contrast, the expression of cytoplasmic CD44 was associated with a better overall survival (HR=0.30, p=0.02). These were found to be independent prognostic markers in multivariate analysis. Interestingly, the co-expression of EGFR with EGFRvIII was associated with a poorer overall survival (59.5 vs 21.0 months, p=0.004). Taken together, our results suggest that the expression of EGFRvIII occurs in patients with hepatocellular carcinoma and highlight its importance as biomarker for tumor progression and as a potential target for therapeutic intervention with drugs targeting EGFRvIII and other members of HER family. Citation Format: Ozlem Sherif, Said Khelwatty, Izhar Bagwan, Alan Seddon, Angus Dalgleish, Satvinder Mudan, Helmout Modjtahedi. Co-expression and prognostic significance of HER family members and EGFRvIII in patients with hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5152.

Abstract 5975: Characterization and pharmacokinetics of ligand traps targeting the human epidermal growth factor receptor (HER) family

Abstract Antibodies targeting HER receptors have led to successful therapeutics for multiple human malignancies. Targeting the family of HER ligands provides an alternative strategy to inhibit signaling and subsequent proliferation of cancer cells. We have engineered ligand traps that bind the growth factors activating the HER family and have characterized a series of molecules (EN2000-Fc) using Surface Plasmon Resonance (SPR). Five distinct molecules were further evaluated for improved in vivo pharmacokinetics in mice that express human neonatal Fc receptor (FcRn). Mutations in the IgG Fc known to extend the in-vivo half-life of monoclonal antibodies were incorporated into the parental EN2000-Fc to generate 3 more distinct pan HER ligand traps. In addition, an EN2000-Fc variant with a full-length hinge was engineered, which is 5-amino-acid longer N-terminally than the parental molecule. These five molecules were synthesized (> 300 mg of each), purified (>98%), and characterized for in vitro and in vivo testing. Two variants were compared by evaluating the ligand binding affinity to 11 HER ligands using SPR. Both molecules showed comparable affinity against all ligands tested, confirming reproducibility of the pan HER ligand trap engineering technology. Among the ligands, HB-EGF had the highest affinity. High binding affinities to EGF, NRG1, NRG2, betacellulin and TGFα were also measured. The serum pharmacokinetics of the different ligand traps were compared in 6- to 8-week-old female C57BL/6 mice engineered to express human FcRn. Ligand traps were administered by single tail vein injection, and serum samples collected over a 28-day interval were analyzed by ELISA. Ligand traps with engineered Fc-mutations had 3- to 4-fold greater systemic exposure (area-under-the-curve) compared to ligand traps with the parental sequence, demonstrating improved in vivo characteristics of selected engineered fusion proteins. These results provide useful information for product candidate selection and for further preclinical development of a ligand trap targeting HER family receptors. Citation Format: Daniel C. Maneval, Cindy Yang, Cynthia Trevisiol, H. Michael Shepard, Pei Jin. Characterization and pharmacokinetics of ligand traps targeting the human epidermal growth factor receptor (HER) family [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5975.

Abstract 6132: Impact of treatment with agents targeting different members of HER family, CDKs and downstream cell signaling molecules on growth and migration of stomach cancer cells

Abstract Stomach cancer ranks fifth and fourth for the incidence and mortality rate for cancer worldwide, respectively. In recent years, several inhibitors with specificity to one or more members of human epidermal growth factor receptor (HER) family have been approved for the treatment of patients with different cancer types. Of these, only the anti-HER2 monoclonal antibody (mAb) Herceptin/trastuzumab and the antibody-drug conjugate trastuzumab deruxtecan has been approved for the treatment of patients with stomach cancer. However, the duration of response may be short in many patients and with tumor heterogeneity being one contributing factor. In this study, we investigated the effect of various types of targeted agents on the growth in vitro of a panel of human stomach cancer cells (HSCCLs) and the impact of stomach cancer proliferation rate on the anti-tumor activities of these agents. Moreover, we investigated the cell surface expression of the HER family members, other biomarkers such as C-Met, Alk-7 and cancer stem cell makers CD44 and CD133 by flow cytometry and the effect various targeted agents on tumor migration using Incucyte. Of the 18 agents examined, the CDK 1/2/5/9 inhibitor dinaciclib was the most effective and inhibited the growth of all human HSCCLs at IC50 values between 1nM to 9nM. Of various HER inhibitors, the irreversible pan-HER family inhibitors (e.g., afatinib) were more effective than the reversible dual EGFR/HER2 TKI lapatinib and the EGFR specific TKI erlotinib in inhibiting the growth of HSCCLs. Interestingly, the HER-2 overexpressing cells lines NCI-N87 (Mean Fluorescence Intensity =596) was most sensitive to the HER inhibitors. Of agents targeting different downstream cell signaling molecules, dasatinib targeting Ab1/Src/C-Kit, trametinib targeting MERK1/2 and miransertib targeting AKT1/2/3 inhibited growth of majority of HSCCLs and with the IC50 values ranging from 2nM to 7uM. Interestingly, many of these agents were more effective in inhibiting the growth of HSCCLs when proliferating at slower rate. Of the agent examined, neratinib, afatinib, dinacicilib, dasatinib, stattic and miransertib also inhibited the migration of stomach cancer cells. Finally, treatment with a combination of afatinib with dinaciclib, capmatinib, dasatinib, stattic, ponatinib or miransertib resulted in synergistic and additive growth inhibition of stomach cancer cells. As stomach cancer cells are heterogenous in nature, our results support further research on the therapeutic potential of the CDK dinaciclib in combination with a pan-HER inhibitor in stomach cancer. Citation Format: Tina Al-Janaby, Narmin Nahi, Said A. Khelwatty, Alan Seddon, Izhar Bagwan, Helmout Modjtahedi. Impact of treatment with agents targeting different members of HER family, CDKs and downstream cell signaling molecules on growth and migration of stomach cancer cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6132.

Abstract P1-11-10: Reversible versus irreversible tyrosine kinase inhibitors (TKIs) combined with antibody-drug conjugates (ADCs) in HER2-positive (HER2+) breast cancer (BC) cell lines

Abstract Background: Targeting the human epidermal growth factor receptor (HER) family of tyrosine kinase receptors with small molecule TKIs lapatinib, neratinib and tucatinib, monoclonal antibodies like trastuzumab and more recently with ADCs like T-DM1 and T-DXd has resulted in improved survival rates for patients with HER2+ BC. TKIs are primarily utilised in trastuzumab-refractory disease. Neratinib, and the pan-HER TKI afatinib, bind irreversibly to all members of the HER family, while lapatinib binds reversibly to HER2 and EGFR, and tucatinib binds reversibly to HER2 alone. Previous work in our lab has shown that TKIs like lapatinib are capable of modulating tumour surface HER2 levels and increasing trastuzumab load on tumours. Thus, there is a rationale for the use of TKI/ADC combinations in HER2+ BC. Using innately trastuzumab-resistant HER2+ BC cell lines, this in vitro study aims to assess the anti-proliferative potential of HER2-targeting ADCs T-DM1 or T-DXd in combination with the TKIs afatinib, lapatinib, neratinib or tucatinib. Methods: HCC1569 and HCC1954 cells (HER2+, estrogen receptor (ER)-negative; innately trastuzumab-resistant) were grown in RPMI1640/10% FBS at 37°C and 5% CO2. The anti-proliferative effects of afatinib, lapatinib, neratinib and tucatinib; of T-DM1 and T-DXd; and of each TKI/ADC combination thereof were assessed in these cells via 5-day acid phosphatase. ADCs were obtained from Saint Vincent’s University Hospital, Dublin, and TKIs were purchased from commercial sources. Calcusyn software was used to generate IC50 values and combination index (CI) values at ED50. CI values > 1 represent an antagonistic combination, CI values = 1 are additive, and CI values < 1 are synergistic. All assays were carried out in triplicate. Results: The HCC1569 cell line was more sensitive to all four TKIs (IC50 values were 12.8 ± 0.3, 453.8 ± 47.1 nM, 4.7 ± 1.4 and 381.5 ± 37.3 nM for afatinib, lapatinib, neratinib and tucatinib respectively) compared to the HCC1954 cell line (IC50 22.3 ± 2.8, 652.4 ± 36.0, 57.0 ± 6.3 and 2365.1 ± 185.3 nM for afatinib, lapatinib, neratinib and tucatinib respectively). Despite their innate trastuzumab resistance, both cell lines displayed high sensitivity to T-DM1 (IC50 25.2 ± 8.7 ng/mL for HCC1569 and IC50 30.4 ± 3.2 ng/mL for HCC1954) and to T-DXd (IC50 16.0 ± 3.2 ng/mL for HCC1569 and IC50 36.8 ± 8.7 ng/mL for HCC1954). In both cell lines, co-treatment with ADCs and the irreversible TKIs afatinib and neratinib resulted in additive or synergistic responses, while the combination of the ADCs with the reversible TKIs lapatinib and tucatinib resulted in mild to moderate antagonism (Table 1). Conclusions: In this pre-clinical study, T-DM1 and T-DXd consistently exhibited antagonistic interactions with reversible HER2-targeting TKIs, and synergy/additivity in combination with the irreversible TKIs tested. Future work will explore the underlying mechanisms of this observed synergy and antagonism, including impacts of TKIs on HER2 expression and real-time ADC internalisation rates. Table 1. CI values +/- Std Dev for each TKI/ADC treatment combination in HCC1569 and HCC1954 cells Citation Format: Niall Ashfield, Amira F. Mahdi, Neil T. Conlon, John Crown, Denis M. Collins. Reversible versus irreversible tyrosine kinase inhibitors (TKIs) combined with antibody-drug conjugates (ADCs) in HER2-positive (HER2+) breast cancer (BC) cell lines [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-11-10.

Abstract P2-19-01: Characterization of fully human internalizing anti-HER2 monoclonal antibodies that compete with Trastuzumab for binding to HER2

Abstract The human epidermal growth factor receptor (HER) family of receptors plays a significant role in the pathogenesis of several human cancers. Among them, HER2 is overexpressed in several cancers. In particular, overexpression with or without gene amplification occurs in 15-30% of breast cancers and this has both prognostic and predictive implications. HER2 overexpression and amplification is associated with shorter disease-free and overall survivals and with resistance to certain hormonal agents as well as increased risk of metastasis to the brain. Two types of treatment targeting HER2 biological activity have been developed: anti-Her2 monoclonal antibodies and small molecule tyrosine kinase inhibitors such as lapatinib and neratinib targeting HER2 and EGFR and Afatinib targeting all HER family members. Trastuzumab is a humanized anti-HER2 monoclonal antibody that binds to domain IV of HER2 extracellular segment and blocks its signaling. It is the first therapeutic antibody targeting Her2 overexpression approved by the FDA. It is administered in combination with standard of care chemotherapy. More recently, since it was shown that Trastuzumab was an internalizing antibody, two antibody drug conjugates using trastuzumab have been approved and used in the standard of care: Ado-Trastuzumab-Emtansine (Kadcyla) consisting of trastuzumab conjugated to the drug mertansine DM1 and fam-trastuzumab-deruxtecan-nhki (Enhertu). Enhertu is another antibody drug conjugate using Trastuzumab to deliver a topoisomerase inhibitor deruxtecan. Our laboratory has been focused in developing fully human internalizing anti-Her2 antibodies that compete or not with trastuzumab for binding to Her2. These antibodies have been developed by immunizing fully human mice with recombinant Her2 protein. Human Ab producing Tc mice (TC-mAb mice) stably maintain a mouse-derived engineered chromosome containing the entire human Ig heavy and kappa chain loci in a mouse Ig knockout background. After production of hybridoma secreting fully human immunoglobulins, the screening process included inhibition of binding of trastuzumab to Her2 by enzyme linked immunoassay and by Octet epitope binning as well as internalization assay. Several internalizing antibodies with Kd ranging from 10-9 M to 10-12 M were selected. They were stratified by their ability to compete or not with trastuzumab. They were further characterized for their ability to deliver a cytotoxic payload in Her2 overexpressing cells as well as for their efficacy to inhibit proliferation, signaling and migration of Her2 overexpressing breast cancer cells. Data related to these antibodies will be presented here. In conclusion, the use of fully human TC mice in our laboratory represents an attractive approach to develop fully human monoclonal antibodies to high value cancer targets that can by-pass the need for humanization and affinity maturation of antibodies. Citation Format: Ginette Serrero, Jianping Dong, Binbin Yue, Udaya Yerramalla, Jun Hayashi. Characterization of fully human internalizing anti-HER2 monoclonal antibodies that compete with Trastuzumab for binding to HER2 [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-19-01.

493TiP A CRUK phase I/IIA, first in human dose-escalation and expansion trial of HMBD-001 (an anti-HER-3 antibody) in patients with advanced HER3-positive solid tumours

Human epidermal growth factor receptor (HER)3 is a member of the HER family of transmembrane proteins. HER3 lacks kinase activity and must be transactivated by dimerisation with a kinase-active partner for signal transduction. In ligand-dependent activation, the NRG1 ligand can stabilise HER3 in the open conformation. However, ligand-independent heterodimerization can also occur. Overexpression of HER3 is observed in multiple tumour types and associated with poor clinical outcome. HMBD-001 is an IgG1 humanized monoclonal antibody specifically targeting HER3 with a novel mechanism of action.

Antibody-Drug Conjugates Targeting the Human Epidermal Growth Factor Receptor Family in Cancers.

Members of the human epidermal growth factor receptor (HER) family, which includes HER1 (also known as EGFR), HER2, HER3 and HER4, have played a central role in regulating cell proliferation, survival, differentiation and migration. The overexpression of the HER family has been recognized as one of the most common cellular dysregulation associated with a wide variety of tumor types. Antibody-drug conjugates (ADCs) represent a new and promising class of anticancer therapeutics that combine the cancer specificity of antibodies with cytotoxicity of chemotherapeutic drugs. Two HER2-directed ADCs, trastuzumane-emtansine (T-DM1) and trastuzumab-deruxtecan (DS-8201a), have been approved for HER2-positive metastatic breast cancer by the U.S. Food and Drug Administration (FDA) in 2013 and 2019, respectively. A third HER2-directed ADC, disitamab vedotin (RC48), has been approved for locally advanced or metastatic gastric or gastroesophageal junction cancer by the NMPA (National Medical Products Administration) of China in 2021. A total of 11 ADCs that target HER family receptors (EGFR, HER2 or HER3) are currently under clinical trials. In this review article, we summarize the three approved ADCs (T-DM1, DS-8201a and RC48), together with the investigational EGFR-directed ADCs (ABT-414, MRG003 and M1231), HER2-directed ADCs (SYD985, ARX-788, A166, MRG002, ALT-P7, GQ1001 and SBT6050) and HER3-directed ADC (U3-1402). Lastly, we discuss the major challenges associated with the development of ADCs, and highlight the possible future directions to tackle these challenges.

Defucosylated Anti-Epidermal Growth Factor Receptor Monoclonal Antibody 134-mG2a-f Exerts Antitumor Activities in Mouse Xenograft Models of Dog Epidermal Growth Factor Receptor-Overexpressed Cells

The epidermal growth factor receptor (EGFR) is a type I transmembrane protein, which is a member of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases. EGFR is a crucial mediator of cell growth and differentiation and forms homodimers or heterodimers with other HER family members to activate downstream signaling cascades. We previously established an anti-human EGFR (hEGFR) monoclonal antibody (mAb), clone EMab-134 (mouse IgG1), by immunizing mice with the ectodomain of hEGFR. In this study, the subclass of EMab-134 was converted from IgG1 to IgG2a (134-mG2a) and further defucosylated (134-mG2a-f) to facilitate antibody-dependent cellular cytotoxicity (ADCC). Although 134-mG2a-f was developed against hEGFR, it was shown to cross-react with dog EGFR (dEGFR) using flow cytometry. The dissociation constant (KD) of 134-mG2a-f against dEGFR-overexpressed CHO-K1 (CHO/dEGFR) cells was determined by flow cytometry to be 3.3 × 10-9 M, indicating that 134-mG2a-f possesses a high binding affinity to dEGFR. Analysis in vitro revealed that 134-mG2a-f contributed to high levels of ADCC and complement-dependent cytotoxicity (CDC) in experiments targeting CHO/dEGFR cells. Furthermore, the in vivo administration of 134-mG2a-f significantly inhibited the development of CHO/dEGFR in comparison with the results observed in response to control mouse IgG. Taken together, the findings of this study demonstrate that 134-mG2a-f could be useful as part of a therapeutic regimen for dEGFR-expressing canine cancers.

Mig6 not only inhibits EGFR and HER2 but also targets HER3 and HER4 in a differential specificity: Implications for targeted esophageal cancer therapy

The human EGF receptor family plays pivotal roles in physiology and cancer, which contains four closely-related members: HER1/EGFR, HER2, HER3 and HER4. Previously, it was found that the mitogen-inducible gene 6 (Mig6) protein is a negative regulator of EGFR and HER2 by using its S1 segment to bind at the kinase dimerization interface. However, it is still unclear whether the S1 segment can also effectively target HER3 and HER4? Here, we performed a systematic investigation to address this issue. The segment can bind to all the four HER kinases with a varying affinity and moderate selectivity; breaking of the segment into shorter hotspot peptides would largely impair the affinity and selectivity, indicating that the full-length sequence is required for the effective binding of S1 to these kinases. The hs2 peptide, which corresponds to the middle hotspot region of S1 segment, can partially retain the affinity to HER kinases, can moderately compete with S1 segment at the dimerization interfaces, and can mimic the biological function of Mig6 protein to suppress HER4+ esophageal cancer at cellular level. In addition, we also analyzed the binding potency of S1 segment and hs2 peptide to the kinase domains of other five widely documented growth factor receptors (GFRs). It was showed that both the S1 and hs2 cannot effectively interact with these receptors. Overall, the Mig6 is suggested as a specific pan-HER inhibitor, which can target and suppress HER family members with a broad selectivity, but exhibits weak or no activity towards other GFRs.

Quantification of HER family dimers by proximity ligation assay and its clinical evaluation in non–small cell lung cancer patients treated with osimertinib

ObjectivesThe epidermal growth factor receptor (EGFR, also known as Her1) is a member of the human epidermal growth factor receptor (HER) family of proteins and a target of tyrosine kinase inhibitors (TKIs) in the treatment of non–small cell lung cancer (NSCLC) positive for activating mutations ofEGFR. Signal transduction by HER family proteins is dependent on their homo- or heterodimerization, but little is known of the relation between the relative proportions of such dimers of Her1 and sensitivity to EGFR-TKIs. We here investigated the feasibility of assessing this relation with the in situ proximity ligation assay (PLA) technique, which is able to detect the interaction of two proteins of interest when they are in close proximity. Materials and methodsIn situ PLA was applied to detect Her1 homodimers and Her1 heterodimers in NSCLC cell lines and tissue specimens positive for EGFR activating mutations. ResultsIn situ PLA allowed visualization and quantitative assessment of Her1 homodimers as well as of Her1 heterodimers with Her2, Her3, or Her4 not only in NSCLC cell lines but also in NSCLC tissue specimens obtained from various anatomic sites and by different collection methods. Treatment of NSCLC cell lines with EGFR-TKIs resulted in a decrease in the number of Her1 dimers, with the effect on homodimers being greater than that on heterodimers. A high ratio of Her1 heterodimers to homodimers was associated with poor progression-free survival in NSCLC patients treated with osimertinib. ConclusionIn situ PLA allows the detection of HER family dimers in NSCLC tissue, and quantitative assessment of Her1 homo- and heterodimers may prove informative for prediction of the response of NSCLC patients to EGFR-TKI treatment.

A pan-cancer analysis of the HER family gene and their association with prognosis, tumor microenvironment, and therapeutic targets

AimsThe human epidermal growth factor receptor (HER) family gene is involved in a wide range of biological functions in human cancers. Nevertheless, there is little research that comprehensively analysis the correlation between HER family members and prognosis, tumor microenvironment (TME) in different cancers. Materials and methodsBased on updated public databases and integrated several bioinformatics analysis methods, we evaluated expression level, prognostic values of HER family gene and explore the association between expression of HER family gene and TME, Stemness score, immune subtype, drug sensitivity in pan-cancer. Key findingsEGFR, ERBB2, ERBB3, and ERBB4 were higher expressed in four cancers, five cancers, ten cancers, and two cancers, respectively. HER family gene expression is related to the prognosis in several cancers from TCGA and has a significant correlation with stromal and immune scores in pan-cancer also has a significant correlation with RNA stemness score and DNA stemness score in pan-cancer. Expression level of HER family gene is associated with immune subtype in head and neck squamous cell carcinoma and kidney renal clear cell carcinoma. EGFR expression was negatively associated with drug sensitivity of Pipamperone, Tamoxifen, Bafetinib and positively related to drug sensitivity of Dasatinib and Staurosporine. ERBB2 expression was negatively related to drug sensitivity of Ifosfamide, Imexon, and Oxaliplatin. ERBB4 expression was positively related to drug sensitivity of E−7820. SignificanceThese findings may elucidate the roles played by HER family gene in cancer progression and providing insights for further investigation of the HER family gene as potential targets in pan-cancer.

HER2 Expression Is Predictive of Survival in Cetuximab Treated Patients with RAS Wild Type Metastatic Colorectal Cancer.

Simple SummaryTreatment with anti-epidermal growth factor receptor (EGFR) monoclonal antibodies (mAbs)—cetuximab and panitumumab—produced clinical benefits in a subset of patients with metastatic colorectal cancer (mCRC). Here, the authors investigated the relative expression and predictive value of HER family members in 144 patients with CRC. They found high levels of expression of HER2 in patients treated with cetuximab; these were associated with shorter progression free survival (PFS). The results provide support for the emergence of HER2 as a therapeutic target in patients with mCRC.The overexpressed HER2 is an important target for treatment with monoclonal antibody (mAb) trastuzumab, only in patients with breast and gastric cancers, and is an emerging therapeutic biomarker in metastatic colorectal cancer (mCRC) treated with anti-epidermal growth factor receptor (EGFR) mAbs cetuximab and panitumumab. In this study, we investigated the relative expression and predictive value of all human epidermal growth factor receptor (HER) family members in 144 cetuximab-treated patients with wild type RAS mCRC. The relative expression of EGFR and HER2 have also been examined in 21-paired primary tumours and their metastatic sites by immunohistochemistry. Of the 144 cases examined, 25%, 97%, 79%, 48%, and 10% were positive for EGFR, HER2, HER3, and HER4 and all four HER family members, respectively. The expression of EGFR was an indicator of poorer overall survival and the membranous expression of HER2 and HER3 3+ intensity was associated with a shorter progression free survival (PFS). In contrast, the cytoplasmic expression of HER2 was associated with better PFS. In 48% and 71% of the cases, there were discordance in the expression of EGFR or one or more HER family members in paired primary and related metastatic tumours, respectively. Our results implicate the importance of a large prospective investigation of the expression level and predictive value of not only the therapeutic target (i.e., EGFR protein) but also HER2 and other HER family members as therapeutic targets, or for response to therapy with anti-EGFR mAbs and other forms of HER inhibitors, in both the primary tumours and metastatic sites in mCRC.

Coordinated dysregulation of cancer progression by the HER family and p21-activated kinases.

Most epithelial cancer types are polygenic in nature and are driven by coordinated dysregulation of multiple regulatory pathways, genes, and protein modifications. The process of coordinated regulation of cancer promoting pathways in response to extrinsic and intrinsic signals facilitates the dysregulation of several pathways with complementary functions, contributing to the hallmarks of cancer. Dysregulation and hyperactivation of cell surface human epidermal growth factor receptors (HERs) and cytoskeleton remodeling by p21-activated kinases (PAKs) are two prominent interconnected aspects of oncogenesis. We briefly discuss the discoveries and significant advances in the area of coordinated regulation of HERs and PAKs in the development and progression of breast and other epithelial cancers. We also discuss how initial studies involving heregulin signaling via HER3-HER2 axis and HER2-overexpressing breast cancer cells not only discovered a mechanistic role of PAK1 in breast cancer pathobiology but also acted as a bridge in generating a broader cancer research interest in other PAK family members and cancer types and catalyzed establishing the role of PAKs in human cancer, at-large. In addition, growth factor stimulation of the PAK pathway also helped to recognize new facets of PAKs, connecting the PAK pathway to oncogenesis, nuclear signaling, gene expression, mitotic progression, DNA damage response, among other phenotypic responses, and shaped the field of PAK cancer research. Finally, we recount some of the current limitations of HER- and PAK-directed therapeutics in counteracting acquired therapeutic resistance and discuss how cancer's as a polygenic disease may be best targeted with a polygenic approach.

Abstract 1424: Her3 up regulation in response to AKT and PI3K inhibition causes differential responses dependent on the presence of neuregulin isoforms in the microenvironmental milieu

Abstract Introduction: Triple Negative Breast Cancer (TNBC) is an aggressive form of Breast Cancer (BC) affecting ~20% of BC patients. TNBC is a highly heterogenous disease and numerous tyrosine kinase inhibitors (TKI) targeting different pathways including the PI3K, AKT and MAPK pathway have shown limited benefit in the clinical setting. Intrinsic and extrinsic resistance mechanisms to TKI are the main reason of therapeutic failure. Signalling from the human epidermal growth factor receptor (HER) family of proteins is dependent on a well-orchestrated series of interactions between family members to form either homo- or heterodimers. The heterodimeric complex formed with HER3 is a particularly potent oncogenic signalling unit that can act as a driver of cancer growth. HER3 upregulation in response to small molecule inhibitors has been shown to rescue a large number of cancers including TNBC. In this study we aim to characterize the extent of HER3 reliance in TNBC and to define the effect of neuregulin isoforms in TNBC cancers. Method and Materials: Basal and Claudin type Breast cancer cell lines were treated with a range of small molecule inhibitors, known to exhibit limited efficacy as single agents, including the AKT inhibitor (GDC0068), the PI3K inhibitor (GDC0077) and the EGFR inhibitors (Gefitinib and Lapatinib) or a combination of these drugs in the presence or absence of the HER3 ligand neuregulin. The effect these inhibitors on Akt/ MAPK signalling was characterized using specific biomarkers of the respective pathways. Combination therapy was evaluated in human cancer cell lines through Annexin V staining. Results: Basal (HCC-70 and MDA-MB-468) and Caudin type (MDA-MB-231) TNBC cell lines displayed differential reliance on the HER family of receptors. Dynamic HER3 upregulation was predominant in the TNBC cells that show HER signaling reliance coinciding with the basal subtype. Furthermore, the presence of the natural ligand neuregulin showed potent signaling through the HER3-AKT pathway and significantly diminished the efficacy of the inhibitors tested. We report that neuregulin augments the HER3 feedback mechanism for continued proliferation in TNBC. Interestingly, we show that neuregulin mediated resistance is dependent on the type and dose of the neuregulin isoform. Neuregulin 1β showed potent proliferative effects and overcoming this effect required a 10 fold increase in the drug concentration. In contrast, the presence of neuregulin 1α isoform may paradoxically confer sensitivity to small molecule inhibitors. We show that cellular proliferation is maintained through both the PI3K and MAPK pathway and single targeting is inefficient in overcoming resistance. We demonstrate that combination strategies that block compensatory mechanisms are effective strategies to neuregulin dependent and independent resistance mechanisms. Conclusion: The highly dynamic nature of HER3 expression and signalling combined with redundant HER signalling and downstream mechanisms allow for continued signalling in a subtype of TNBC patients. Targeted therapy to ensure complete abrogation of HER signalling is essential for effective therapeutic response. In conclusion, TNBC cancers display differential mechanism of resistance that should be adequately addressed for therapeutic strategies. Citation Format: Nicoleta Sinevici, Pedram Heidari, Umar Mahmood. Her3 up regulation in response to AKT and PI3K inhibition causes differential responses dependent on the presence of neuregulin isoforms in the microenvironmental milieu [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1424.

Anti‑EGFR monoclonal antibody 134‑mG2a exerts antitumor effects in mouse xenograft models of oral squamous cell carcinoma.

The epidermal growth factor receptor (EGFR), a transmembrane receptor and member of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases, is a critical mediator of cell growth and differentiation. EGFR forms homo- or heterodimers with other HER family members to activate downstream signaling cascades in a number of cancer cells. In a previous study, the authors established an anti-EGFR monoclonal antibody (mAb), EMab-134, by immunizing mice with the ectodomain of human EGFR. EMab-134 binds specifically to endogenous EGFR and can be used to detect receptor on oral cancer cell lines by flow cytometry and western blot analysis; this antibody is also effective for the immunohistochemical evaluation of oral cancer tissues. In the present study, the subclass of EMab-134 was converted from IgG1 to IgG2a (134-mG2a) to facilitate antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The dissociation constants (KDs) of EMab-134 and 134-mG2a against EGFR-expressing CHO-K1 (CHO/EGFR) cells were deter-mined by flow cytometry to be 3.2×10−9 M and 2.1×10−9 M, respectively; these results indicate that 134-mG2a has a higher binding affinity than EMab-134. The 134-mG2a antibody was more sensitive than EMab-134 with respect to antigen detection in oral cancer cells in both western blot analysis and immunohistochemistry applications. Analysis in vitro revealed that 134-mG2a contributed to high levels of ADCC and CDC in experiments targeting CHO/EGFR, HSC-2, and SAS cells. Moreover, the in vivo administration of 134-mG2a significantly inhibited the development of CHO/EGFR, HSC-2, and SAS mouse xenografts in comparison to the results observed in response to EMab-134. Taken together, the findings of the present study demonstrate that the newly-formulated 134-mG2a is useful for detecting EGFR by flow cytometry, western blot analysis and immunohistochemistry. Furthermore, the in vivo results suggested that it may also be useful as part of a therapeutic regimen for patients with EGFR-expressing oral cancer.

Simultaneous targeting of HER family pro-survival signaling with Pan-HER antibody mixture is highly effective in TNBC: a preclinical trial with PDXs

BackgroundThe human epidermal growth factor receptor (HER) family, notably EGFR, is overexpressed in most triple-negative breast cancer (TNBC) cases and provides cancer cells with compensatory signals that greatly contribute to the survival and development of resistance in response to therapy. This study investigated the effects of Pan-HER (Symphogen, Ballerup, Denmark), a novel mixture of six monoclonal antibodies directed against members of the HER family EGFR, HER2, and HER3, in a preclinical trial of TNBC patient-derived xenografts (PDXs).MethodsFifteen low passage TNBC PDX tumor samples were transferred into the right mammary fat pad of mice for engraftment. When tumors reached an average size of 100–200 mm3, mice were randomized (n ≥ 6 per group) and treated following three 1-week cycles consisting of three times/week intraperitoneal (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.ResultsAll 15 TNBC PDXs were responsive to Pan-HER treatment, showing significant reductions in tumor growth consistent with Pan-HER-mediated tumor downmodulation of EGFR and HER3 protein levels and significantly decreased activation of associated HER family signaling pathways AKT and ERK. Tumor regression was observed in five of the models, which corresponded to those PDX tumor models with the highest level of HER family activation.ConclusionsThe marked effect of Pan-HER in numerous HER family-dependent TNBC PDX models justifies further studies of Pan-HER in TNBC clinical trials as a potential therapeutic option.

Heregulin controls ERα and HER2 signaling in mammospheres of ERα-positive breast cancer cells and interferes with the efficacy of molecular targeted therapy

Estrogen receptor (ER)α and the human epidermal growth factor receptor (HER) family are inversely expressed in ERα-positive cancer in association with resistance to hormonal therapy, but the mechanism underlying their relationship remains unknown. We analyzed the effect of HER family ligands on the expression of ER and the HER family in ERα-positive MCF-7 and T47D breast cancer cell lines in 3D spheroid culture. Here, we demonstrated for the first time that heregulin-1β (HRG), a HER3 and HER4 ligand, most effectively regulated ER/HER family expression by decreasing ERα mRNA expression and increasing HER family mRNA expression. HRG treatment attenuated fulvestrant-mediated growth inhibition, and promoted the migration of MCF-7 cells. Moreover, HRG increased the CD44+/CD24− cell fraction and side population cells, both of which are recognized as prospective breast cancer stem cell markers. HRG activated both phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin (PI3K/AKT/mTOR) and mitogen-activated protein kinase (MAPK) pathways. Inhibitors of these pathways reduced the growth of MCF-7 cells, but the addition of HRG has different effects on these pathways. HRG blocked the inhibitory effect of mTOR inhibitors, such as rapamycin and everolimus, on cell growth but not that of a PI3K inhibitor. Furthermore, HRG slightly decreased the inhibitory effect of an AKT inhibitor on cell growth. In contrast, HRG enhanced the MEK inhibitor-induced inhibition of cell growth. These findings suggest that HRG-stimulated signaling pathways allow ERα-positive breast cancer cells to escape from growth inhibition caused by everolimus, via MAPK signaling and/or other signaling pathways. Everolimus improves progression-free survival in combination with exemestane as second-line therapy for metastatic hormone receptor-positive breast cancer. Our study suggests that HRG is a novel target for ERα-positive breast cancer therapy.

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.