HER4 Affects Sensitivity to Tamoxifen and Abemaciclib in Luminal Breast Cancer Cells and Restricts Tumor Growth in MCF-7-Based Humanized Tumor Mice.

The impact of the HER4 receptor on the growth and treatment of estrogen receptor-positive breast cancer is widely uncertain. Using CRISPR/Cas9 technology, we generated stable HER4 knockout variants derived from the HER4-positive MCF-7, T-47D, and ZR-75-1 breast cancer cell lines. We investigated tumor cell proliferation as well as the cellular and molecular mechanisms of tamoxifen, abemaciclib, AMG232, and NRG1 treatments as a function of HER4 in vitro. HER4 differentially affects the cellular response to tamoxifen and abemaciclib treatment. Most conspicuous is the increased sensitivity of MCF-7 in vitro upon HER4 knockout and the inhibition of cell proliferation by NRG1. Additionally, we assessed tumor growth and immunological effects as responses to tamoxifen and abemaciclib therapy in humanized tumor mice (HTM) based on MCF-7 HER4-wildtype and the corresponding HER4-knockout cells. Without any treatment, the enhanced MCF-7 tumor growth in HTM upon HER4 knockout suggests a tumor-suppressive effect of HER4 under preclinical but human-like conditions. This phenomenon is associated with an increased HER2 expression in MCF-7 in vivo. Independent of HER4, abemaciclib and tamoxifen treatment considerably inhibited tumor growth in these mice. However, abemaciclib-treated hormone receptor-positive breast cancer patients with tumor-associated mdm2 gene copy gains or pronounced HER4 expression showed a reduced event-free survival. Evidently, the presence of HER4 affects the efficacy of tamoxifen and abemaciclib treatment in different estrogen receptor-positive breast cancer cells, even to different extents, and is associated with unfavorable outcomes in abemaciclib-treated patients.

The guanine nucleotide exchange factor VAV3 participates in ERBB4-mediated cancer cell migration.

ERBB4 is a member of the epidermal growth factor receptor (EGFR)/ERBB subfamily of receptor tyrosine kinases that regulates cellular processes including proliferation, migration, and survival. ERBB4 signaling is involved in embryogenesis and homeostasis of healthy adult tissues, but also in human pathologies such as cancer, neurological disorders, and cardiovascular diseases. Here, an MS-based analysis revealed the Vav guanine nucleotide exchange factor 3 (VAV3), an activator of Rho family GTPases, as a critical ERBB4-interacting protein in breast cancer cells. We confirmed the ERBB4-VAV3 interaction by targeted MS and coimmunoprecipitation experiments and further defined it by demonstrating that kinase activity and Tyr-1022 and Tyr-1162 of ERBB4, as well as the intact phosphotyrosine-interacting SH2 domain of VAV3, are necessary for this interaction. We found that ERBB4 stimulates tyrosine phosphorylation of the VAV3 activation domain, known to be required for guanine nucleotide exchange factor (GEF) activity of VAV proteins. In addition to VAV3, the other members of the VAV family, VAV1 and VAV2, also coprecipitated with ERBB4. Analyses of the effects of overexpression of dominant-negative VAV3 constructs or shRNA-mediated down-regulation of VAV3 expression in breast cancer cells indicated that active VAV3 is involved in ERBB4-stimulated cell migration. These results define the VAV GEFs as effectors of ERBB4 activity in a signaling pathway relevant for cancer cell migration.

PET and SPECT Imaging of the EGFR Family (RTK Class I) in Oncology.

The human epidermal growth factor receptor family (EGFR-family, other designations: HER family, RTK Class I) is strongly linked to oncogenic transformation. Its members are frequently overexpressed in cancer and have become attractive targets for cancer therapy. To ensure effective patient care, potential responders to HER-targeted therapy need to be identified. Radionuclide molecular imaging can be a key asset for the detection of overexpression of EGFR-family members. It meets the need for repeatable whole-body assessment of the molecular disease profile, solving problems of heterogeneity and expression alterations over time. Tracer development is a multifactorial process. The optimal tracer design depends on the application and the particular challenges of the molecular target (target expression in tumors, endogenous expression in healthy tissue, accessibility). We have herein summarized the recent preclinical and clinical data on agents for Positron Emission Tomography (PET) and Single Photon Emission Tomography (SPECT) imaging of EGFR-family receptors in oncology. Antibody-based tracers are still extensively investigated. However, their dominance starts to be challenged by a number of tracers based on different classes of targeting proteins. Among these, engineered scaffold proteins (ESP) and single domain antibodies (sdAb) show highly encouraging results in clinical studies marking a noticeable trend towards the use of smaller sized agents for HER imaging.

A Review of HER4 (ErbB4) Kinase, Its Impact on Cancer, and Its Inhibitors.

HER4 is a receptor tyrosine kinase that is required for the evolution of normal body systems such as cardiovascular, nervous, and endocrine systems, especially the mammary glands. It is activated through ligand binding and activates MAPKs and PI3K/AKT pathways. HER4 is commonly expressed in many human tissues, both adult and fetal. It is important to understand the role of HER4 in the treatment of many disorders. Many studies were also conducted on the role of HER4 in tumors and its tumor suppressor function. Mostly, overexpression of HER4 kinase results in cancer development. In the present article, we reviewed the structure, location, ligands, physiological functions of HER4, and its relationship to different cancer types. HER4 inhibitors reported mainly from 2016 to the present were reviewed as well.

Biasing human epidermal growth factor receptor 4 (HER4) tyrosine kinase signaling with antibodies: Induction of cell death by antibody-dependent HER4 intracellular domain trafficking.

Human epidermal growth factor receptor 4 (HER4) isoforms have oncogenic or tumor suppressor functions depending on their susceptibility to proteolytic cleavage and HER4 intracellular domain (4ICD) translocation. Here, we report that the neuregulin 1 (NRG1) tumor suppressor mechanism through the HER4 JMa/CYT1 isoform can be mimicked by the agonist anti-HER4 Ab C6. Neuregulin 1 induced cleavage of poly(ADP-ribose) polymerase (PARP) and sub-G1 DNA fragmentation, and also reduced the metabolic activity of HER3- /HER4+ cervical (C-33A) and ovarian (COV318) cancer cells. This effect was confirmed in HER4 JMa/CYT1-, but not JMa/CYT2-transfected BT549 triple-negative breast cancer cells. Neuregulin 1 favored 4ICD cleavage and retention in mitochondria in JMa/CYT1-transfected BT549 cells, leading to reactive oxygen species (ROS) production through mitochondrial depolarization. Similarly, the anti-HER4 Ab C6, which binds to a conformational epitope located on a.a. 575-592 and 605-620 of HER4 domain IV, induced 4ICD cleavage and retention in mitochondria, and mimicked NRG1-mediated effects on PARP cleavage, ROS production, and mitochondrial membrane depolarization in cancer cells. In vivo, C6 reduced growth of COV434 and HCC1187 tumor cell xenografts in nude mice. Biasing 4ICD trafficking to mitochondria with anti-HER4 Abs to mimic NRG1 suppressor functions could be an alternative anticancer strategy.

Poziotinib suppresses ovarian cancer stem cell growth via inhibition of HER4-mediated STAT5 pathway.

Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy, with an overall 5-year survival rate of only 30%. EOC is associated with drug resistance, frequent recurrence, and poor prognosis. A major contributor toward drug resistance might be cancer stem cells (CSCs), which may remain after chemotherapy. Here, we aimed to find therapeutic agents that target ovarian CSCs. We performed a high-throughput screening using the Clinical Compound Library with a sphere culture of A2780 EOCs. Poziotinib, a pan-human epidermal growth factor receptor (HER) inhibitor, decreased sphere formation, viability, and proliferation, and induced G1 cell cycle arrest and apoptosis in ovarian CSCs. In addition, poziotinib suppressed stemness and disrupted downstream signaling of Wnt/ß-catenin, Notch, and Hedgehog pathways, which contribute to many characteristics of CSCs. Interestingly, HER4 was overexpressed in ovarian CSCs and Poziotinib reduced the phosphorylation of STAT5, AKT, and ERK, which are regulated by HER4. Our results suggest that HER4 may be a promising therapeutic target for ovarian CSCs, and that poziotinib may be an effective therapeutic option for the prevention of ovarian cancer recurrence.

Potent dual EGFR/Her4 tyrosine kinase inhibitors containing novel (1,2-dithiolan-4-yl)acetamides.

Modifications at C6 and C7 positions of 3-cyanoquinolines 6 and 7 led to potent inhibitors of the ErbB family of kinases particularly against EGFRWT and Her4 enzymes in the radioisotope filter binding assay. The lead (4, SAB402) displayed potent dual biochemical activities with EGFRWT/Her4 IC50 ratio of 80 due to its potent inhibition of Her4 activity (IC50 0.03 nM), however, the selectivity towards activating mutations (EGFRL858R, EGFRex19del) was decreased. Inhibitor 4 also exhibited excellent growth inhibition in seven different cancer types and reduced cell viability in female NMRI nude mice in the intraperitoneally implanted hollow fibers which have been loaded with MOLT-4 (leukemia) and NCI-H460 (NSCLC) cells in a statistically significant manner.

HER4 promotes the growth and metastasis of osteosarcoma via the PI3K/AKT pathway.

Osteosarcoma is the most common primary malignant bone tumor, which occurs in adolescents. As reported by our previous studies, HER4 indicates a poor prognosis of primary osteosarcoma. However, its mechanisms in the pathogenesis of osteosarcoma have not yet been studied. The purpose of this study was to investigate the role of HER4 in osteosarcoma and whether the PI3K/AKT pathway is involved. In this study, western blot analysis was used to investigate the expression of HER4 protein in osteosarcoma tissues and cell lines. CCK8 and transwell assays were used to detect the effects of HER4 on the proliferation, migration, and invasion of osteosarcoma cells in vitro. The effects of HER4 on the growth and metastasis of osteosarcoma in vivo were detected by tumor formation and immunofluorescence in nude mice. The role of the PI3K/AKT pathway in HER4 regulation of the growth and metastasis of osteosarcoma was examined by western blot analysis and immunofluorescence assay. We found that HER4 protein was highly expressed in clinical osteosarcoma specimens and osteosarcoma cells. HER4 markedly promoted the proliferation, migration, and invasion of osteosarcoma cells in vitro as well as the growth and metastasis of osteosarcoma in vivo. HER4 overexpression upregulated the expression of phosphorylated protein kinase B (pAKT), proliferation marker antigen Ki67, and metastasis cell marker matrix metalloproteinase 9 (MMP9). Notably, PI3K/AKT inhibitor LY294002 significantly inhibited the effects of HER4 via the downregulation of pAKT, Ki67, and MMP9. Moreover, LY294002 markedly blocked the effects of HER4-induced upregulation of tumor malignancy. The present study suggests that HER4 may promote the growth and metastasis of osteosarcoma via the PI3K/AKT pathway. The HER4/PI3K/AKT pathway could serve as a potential target for the treatment of osteosarcoma.

ErbB4 3'-UTR Variant (c.*3622A>G) is Associated with ER/PR Negativity and Advanced Breast Cancer.

A genetic variant may alter a gene expression level and as a result be associated with pathological characteristics in breast cancer. In this research, the frequency and association of the ErbB4 3'-untranslated region (3'-UTR) variant, rs12471583 (c.*3622A>G) was studied in an Iranian breast cancer patients. In silico assessment was performed to predict the function of the rs12471583 variant located on the 3'-UTR of ErbB4. Furthermore, as a case-control study, this polymorphism was genotyped in 243 breast cancer patients and non-cancerous controls using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. The Armitage's trend test and regular association tests were performed to analyze a possible association between the rs12471583 and risk of breast cancer and its relevant pathological traits. The bioinformatics analysis predicted that the rs12471583 SNP is located on the four miRNA binding sites, including miR-511-5p, miR-4659a-5p, miR-4659b-5p, and miR-6830-3p. According to logistic regression tests, the G allele is negatively associated with ER- (OR = 0.20, 95% C.I. = 0.04-0.93, p = 0.026), PR- (OR = 0.31, 95% C.I. = 0.10-0.98, p = 0.039), ER-/PR- (OR = 0.20, 95% C.I. = 0.04-0.93, p = 0.026), and advanced breast cancer (OR = 0.40, 95% C.I. = 0.18-0.85, p = 0.016). It has been found that ErbB4 expression may be linked to unfavorable outcomes in breast cancer. Likewise, our results suggest that the G allele may strengthen miRNA-ErbB4 binding efficiency and as a result reduce expression of ErbB4. This is a possible explanation for the observed association.

SUMOylation regulates nuclear accumulation and signaling activity of the soluble intracellular domain of the ErbB4 receptor tyrosine kinase.

Erb-B2 receptor tyrosine kinase 4 (ErbB4) is a kinase that can signal via a proteolytically released intracellular domain (ICD) in addition to classical receptor tyrosine kinase-activated signaling cascades. Previously, we have demonstrated that ErbB4 ICD is posttranslationally modified by the small ubiquitin-like modifier (SUMO) and functionally interacts with the PIAS3 SUMO E3 ligase. However, direct evidence of SUMO modification in ErbB4 signaling has remained elusive. Here, we report that the conserved lysine residue 714 in the ErbB4 ICD undergoes SUMO modification, which was reversed by sentrin-specific proteases (SENPs) 1, 2, and 5. Although ErbB4 kinase activity was not necessary for the SUMOylation, the SUMOylated ErbB4 ICD was tyrosine phosphorylated to a higher extent than unmodified ErbB4 ICD. Mutation of the SUMOylation site compromised neither ErbB4-induced phosphorylation of the canonical signaling pathway effectors Erk1/2, Akt, or STAT5 nor ErbB4 stability. In contrast, SUMOylation was required for nuclear accumulation of the ErbB4 ICD. We also found that Lys-714 was located within a leucine-rich stretch, which resembles a nuclear export signal, and could be inactivated by site-directed mutagenesis. Furthermore, SUMOylation modulated the interaction of ErbB4 with chromosomal region maintenance 1 (CRM1), the major nuclear export receptor for proteins. Finally, the SUMO acceptor lysine was functionally required for ErbB4 ICD-mediated inhibition of mammary epithelial cell differentiation in a three-dimensional cell culture model. Our findings indicate that a SUMOylation-mediated mechanism regulates nuclear localization and function of the ICD of ErbB4 receptor tyrosine kinase.

HER4 expression in estrogen receptor-positive breast cancer is associated with decreased sensitivity to tamoxifen treatment and reduced overall survival of postmenopausal women.

BACKGROUND: The sensitivity of estrogen receptor-positive breast cancers to tamoxifen treatment varies considerably, and the molecular mechanisms affecting the response rates are manifold. The human epidermal growth factor receptor-related receptor HER2 is known to trigger intracellular signaling cascades that modulate the activity of coregulators of the estrogen receptor which, in turn, reduces the cell sensitivity to tamoxifen treatment. However, the impact of HER2-related receptor tyrosine kinases HER1, HER3, and, in particular, HER4 on endocrine treatment is largely unknown. METHODS: Here, we retrospectively evaluated the importance of HER4 expression on the outcome of tamoxifen- and aromatase inhibitor-treated estrogen receptor-positive breast cancer patients (n = 258). In addition, we experimentally analyzed the efficiency of tamoxifen treatment as a function of HER4 co-expression in vitro. RESULTS: We found a significantly improved survival in tamoxifen-treated postmenopausal breast cancer patients in the absence of HER4 compared with those with pronounced HER4 expression. In accordance with this finding, the sensitivity to tamoxifen treatment of estrogen and HER4 receptor-positive ZR-75-1 breast cancer cells can be significantly enhanced by HER4 knockdown. CONCLUSION: We suggest an HER4/estrogen receptor interaction that impedes tamoxifen binding to the estrogen receptor and reduces treatment efficiency. Whether the sensitivity to tamoxifen treatment can be enhanced by anti-HER4 targeting needs to be prospectively evaluated.

HER4 promotes cell survival and chemoresistance in osteosarcoma via interaction with NDRG1.

Osteosarcoma (OS) is the most common primary malignant bone tumor in children and adolescents. The abilities of chemotherapy resistance are major roadblock in the successful treatment of OS. The clarification of mechanism regarding cell survival during OS chemotherapy are important. Here, we examined HER4 expression by immunohistochemistry in a large series of OS tissues, and found HER4 expression correlated with tumor characteristics and patient survival rates. HER4 knockdown by shRNA inhibited OS cell growth and tumorigenesis, and induced cell senescence and apoptosis in vitro and in vivo. We demonstrated that HER4 expression upregulated in the adverse conditions, such as serum starvation and sphere culture. Moreover, HER4 knockdown cells became more sensitive in stressful conditions such as loss of attachment, cytotoxic agents or nutrition insufficiency. Mechanism studies revealed that HER4 interacted with NDRG1, and NDRG1 overexpression could antagonize HER4 knockdown-mediated cell growth and apoptosis in stressed conditions. There was a positive correlation between HER4 and NDRG1 immunoreactivity in OS patients. Together, our present study shows that HER4 and/or NDRG1 might play a critical role for the cell survival and chemo-resistance of OS, and could be used as potential therapeutic targets in OS.

4-Phenybutyric acid promotes gastric cancer cell migration via histone deacetylase inhibition-mediated HER3/HER4 up-regulation.

Dysregulation of histone acetylation plays an important role in tumor development. Histone acetylation regulates gene transcription and expression, which is reversibly regulated by histone acetyltransferase (HAT) and histone deacetylase (HDAC). As an HDAC inhibitor, 4-phenylbutyric acid (4-PBA) can increase histone acetylation levels by inhibiting HDAC activity. While 4-PBA inhibits proliferation of tumor cells in vitro, clinical trials have failed to show benefits of 4-PBA for refractory solid tumors. Here, we found that 4-PBA could enhance the migration capacity of gastric cancer cells. Upregulation of HER3/HER4 and activation of HER3/HER4-ERK pathway was shown to be involved in 4-PBA-induced gastric cancer cell migration. Knockdown of HER3/HER4 blocked HER3/HER4-ERK activation and partially prevented 4-PBA-induced cell migration. Consistently, the ERK inhibitor PD98059 also partially prevented 4-PBA-induced cell migration. Moreover, enhanced levels of acetyl-histones were detected following 4-PBA-treatment, and histone3 acetylation in promoter regions of HER3 and HER4 were confirmed by ChIP. These results demonstrate that 4-PBA promotes gastric cancer cells migration through upregulation of HER3/HER4 subsequent to increased levels of acetyl-histone and activation of ERK signaling. These novel findings provide important considerations for the use of 4-PBA in cancer therapeutics.

Tracking the fate of her4 expressing cells in the regenerating retina using her4:Kaede zebrafish.

The Basic-Helix-Loop-Helix-Orange (bHLH-O) transcription factor Hairy-related 4 (her4) is a downstream effector of Notch-Delta signaling that represses expression of typically pro-neural genes in proliferative domains of the central nervous system. Notch-Delta signaling in the retina has been shown to increase in response to injury and influences neuroprotective properties of Müller glia. In contrast to mammals, teleost fish are able to regenerate retinal neurons in response to injury. In zebrafish, her4 is upregulated in the regenerating neural retina in response to both acute and chronic photoreceptor damage, but the contribution of her4 expressing cells to neurogenesis following acute or chronic retinal damage has remained unexplored. Here we investigate the role of her4 in the regenerating retina in a background of chronic, rod-specific degeneration as well as following acute light damage. We demonstrate that her4 is expressed in the persistently neurogenic ciliary marginal zone (CMZ), as well as in small subsets of slowly proliferating Müller glia in the inner nuclear layer (INL) of the central retina. We generated a transgenic line of zebrafish that expresses the photoconvertible Kaede reporter driven by a her4 promoter and validated that expression of the transgene faithfully recapitulates endogenous her4 expression. Lineage tracing analysis revealed that her4-expressing cells in the INL contribute to the rod lineage, and her4 expressing cells in the CMZ are capable of generating any retinal cell type except rod photoreceptors. Our results indicate that her4 is involved in a replenishing pathway that maintains populations of stem cells in the central retina, and that the magnitude of the her4-associated proliferative response mirrors the extent of retinal damage.

Co-expression of HER3 and MUC1 is associated with a favourable prognosis in patients with bladder cancer.

OBJECTIVES: To investigate the functional impact of the interaction of MUC1 with the epidermal growth factor receptors HER3 and HER4 in patients with bladder cancer. PATIENTS AND METHODS: Using reverse transcription quantitative polymerase chain reaction, we examined MUC1 expression in 82 bladder cancer biopsies previously examined for the expression of HER3. RESULTS: Patients expressing high MUC1 had a favourable survival when the expression of HER3 was also high compared with when the expression of HER3 was low (P = 0.004). When MUC1 expression was low, HER3 co-expression did not influence the prognostic value of MUC1 (P = 0.488). MUC1 expression had no correlation with survival, tumour stage or grade, or to the prognostic value of HER4. CONCLUSIONS: A high MUC1 expression was associated with a favourable prognosis in patients with bladder cancer when the expression of HER3 was also high. This suggests an involvement of HER3 in MUC1 function in bladder cancer.

Activated PI3K/AKT and MAPK pathways are potential good prognostic markers in node-positive, triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) patients are a poor prognostic subgroup, and currently, there is no biomarker for targeted therapy. PATIENTS AND METHODS: Tissue samples were obtained from 75 TNBC patients with lymph-node metastases who had received adjuvant chemotherapy. We examined 11 biomarkers, including PIK3CA and AKT1mutation, with regard to event-free survival (EFS) and overall survival (OS) of patients. RESULTS: In the tumor tissues, phospho-AKT (pAKT) expression was significantly related to HER4 expression. Expression of each of these biomarkers was significantly related to longer EFS (P = 0.024 and 0.03, respectively). pERK expression was also a good prognostic factor regarding EFS and OS in TNBC (P = 0.002 and 0.006, respectively). We also identified a correlation between epidermal growth factor receptor positivity and insulin-like growth factor receptor type 1 positivity (P = 0.001). pERK and T-stage (1-3 versus >3) were independent good prognostic factors by multivariate analysis. CONCLUSIONS: We determined that tumors expressing pAKT or pERK are a good prognostic subtype in node-positive TNBC. Different targeted therapies may be necessary for TNBC that involves activation of PI3K/AKT or MAPK pathways.

HER4 selectively coregulates estrogen stimulated genes associated with breast tumor cell proliferation.

The EGFR-family member HER4 undergoes regulated intramembrane proteolysis (RIP) to generate an intracellular domain (4ICD) that functions as a transcriptional coactivator. Accordingly, 4ICD coactivates the estrogen receptor (ER) and associates with ER at target gene promoters in breast tumor cells. However, the extent of 4ICD coactivation of ER and the functional significance of the 4ICD/ER transcriptional complex is unclear. To identify 4ICD coactivated genes we performed a microarray gene expression analysis of ß-estradiol treated cells comparing control MCF-7 breast cancer cells to MCF-7 cells where HER4 expression was stably suppressed using a shRNA. In the MCF-7 cell line, ß-estradiol significantly stimulated or repressed by 2-fold or more 726 or 53 genes, respectively. Significantly, HER4/4ICD was an obligate coactivator for 277 or 38% of the ß-estradiol stimulated genes. Ingenuity Pathway Analysis of ß-estradiol regulated genes identified significant associations with multiple cellular functions regulating cellular growth and proliferation, cell cycle progression, cancer metastasis, decreased hypoplasia, tumor cell migration, apoptotic resistance of tumor cells, and increased transcription. Genes coactivated by 4ICD displayed functional specificity by only significantly contributing to cellular growth and proliferation, cell cycle progression, and decreased hypoplasia. In direct concordance with these in situ results we show that HER4 knockdown in MCF-7 cells results in a loss of estrogen stimulated tumor cell proliferation and cell cycle progression, whereas, estrogen stimulated tumor cell migration was unaffected by loss of HER4 expression. In summary, we demonstrate for the first time that a cell surface receptor functions as an obligate ER coactivator with functional specificity associated with breast tumor cell proliferation and cell cycle progression. Nearly 90% of ER positive tumors coexpress HER4, therefore we predict that the majority of breast cancer patients would benefit from a strategy to therapeutic disengage ER/4ICD coregulated tumor cell proliferation.

A variant in the 5'UTR of ERBB4 is associated with lifespan in Golden Retrievers.

Genome-wide association studies (GWAS) in long-lived human populations have led to identification of variants associated with Alzheimer's disease and cardiovascular disease, the latter being the most common cause of mortality in people worldwide. In contrast, naturally occurring cancer represents the leading cause of death in pet dogs, and specific breeds like the Golden Retriever (GR) carry up to a 65% cancer-related death rate. We hypothesized that GWAS of long-lived GRs might lead to the identification of genetic variants capable of modifying longevity within this cancer-predisposed breed. A GWAS was performed comparing GR dogs ≥ 14 years to dogs dying prior to age 12 which revealed a significant association to ERBB4, the only member of the epidermal growth factor receptor family capable of serving as both a tumor suppressor gene and an oncogene. No coding variants were identified, however, distinct haplotypes in the 5'UTR were associated with reduced lifespan in two separate populations of GR dogs. When all GR dogs were analyzed together (n = 304), the presence of haplotype 3 was associated with shorter survival (11.8 years vs. 12.8 years, p = 0.024). GRs homozygous for haplotype 3 had the shortest survival, and GRs homozygous for haplotype 1 had the longest survival (11.6 years vs. 13.5 years, p = 0.0008). Sub-analyses revealed that the difference in lifespan for GRs carrying at least 1 copy of haplotype 3 was specific to female dogs (p = 0.009), whereas survival remained significantly different in both male and female GRs homozygous for haplotype 1 or haplotype 3 (p = 0.026 and p = 0.009, respectively). Taken together, these findings implicate a potential role for ERBB4 in GR longevity and provide evidence that within-breed canine lifespan studies could serve as a mechanism to identify favorable or disease-modifying variants important to the axis of aging and cancer.

HER4 and EGFR Activate Cell Signaling in NRG1 Fusion-Driven Cancers: Implications for HER2-HER3-specific Versus Pan-HER Targeting Strategies.

INTRODUCTION: NRG1 gene fusions are clinically actionable alterations identified in NSCLC and other tumors. Previous studies have reported that NRG1 fusions signal through HER2 and HER3 but, thus far, strategies targeting HER3 specifically or HER2-HER3 signaling have exhibited modest activity in patients with NSCLC bearing NRG1 fusions. Although NRG1 fusion proteins can bind HER4 in addition to HER3, the contribution of HER4 and other HER family members in NRG1 fusion-positive cancers is not fully understood. METHODS: We investigated the role of HER4 and EGFR-HER3 signaling in NRG1 fusion-positive cancers using Ba/F3 models engineered to express various HER family members in combination with NRG1 fusions and in vitro and in vivo models of NRG1 fusion-positive cancer. RESULTS: We determined that NRG1 fusions can stimulate downstream signaling and tumor cell growth through HER4, independent of other HER family members. Moreover, EGFR-HER3 signaling is also activated in cells expressing NRG1 fusions, and inhibition of these receptors is also necessary to effectively inhibit tumor cell growth. We observed that cetuximab, an anti-EGFR antibody, in combination with anti-HER2 antibodies, trastuzumab and pertuzumab, yielded a synergistic effect. Furthermore, pan-HER tyrosine kinase inhibitors were more effective than tyrosine kinase inhibitors with greater specificity for EGFR, EGFR-HER2, or HER2-HER4, although the relative degree of dependence on EGFR or HER4 signaling varied between different NRG1 fusion-positive cancers. CONCLUSIONS: Our findings indicate that pan-HER inhibition including HER4 and EGFR blockade is more effective than selectively targeting HER3 or HER2-HER3 in NRG1 fusion-positive cancers.

Targeting the EGF receptor family in non-small cell lung cancer-increased complexity and future perspectives.

Lung cancer remains the leading cause of cancer-associated mortality worldwide, but with the emergence of oncogene targeted therapies, treatment options have tremendously improved. Owing to their biological relevance, members of the ERBB receptor family, including the EGF receptor (EGFR), HER2, HER3 and HER4, are among the best studied oncogenic drivers. Activating EGFR mutations are frequently observed in non-small cell lung cancer (NSCLC), and small molecule tyrosine kinase inhibitors (TKIs) are the established first line treatment option for patients whose tumors bear "typical/classical" EGFR mutations (exon 19 deletions, L858R point mutations). Additionally, new TKIs are rapidly evolving with better efficacy to overcome primary and secondary treatment resistance (e.g., that due to T790M or C797S resistance mutations). Some atypical EGFR mutations, such as the most frequent exon 20 insertions, exhibit relative resistance to earlier generation TKIs through steric hindrance. In this subgroup, newer TKIs, such as mobocertinib and the bi-specific antibody amivantamab have recently been approved, whereas less frequent atypical EGFR mutations remain understudied. In contrast to EGFR, HER2 has long remained a challenging target, but better structural understanding has led to the development of newer generations of TKIs. The recent FDA approval of the antibody-drug conjugate trastuzumab-deruxtecan for pretreated patients with HER2 mutant NSCLC has been an important therapeutic breakthrough. HER3 and HER4 also exert oncogenic potential, and targeted treatment approaches are being developed, particularly for HER3. Overall, strategies to inhibit the oncogenic function of ERBB receptors in NSCLC are currently evolving at an unprecedented pace; therefore, this review summarizes current treatment standards and discusses the outlook for future developments.