The role of the C. albicans transcriptional repressor NRG1 during filamentation and disseminated candidiasis is strain dependent.

Candida albicans is one of the most common causes of superficial and invasive fungal diseases in humans. Its ability to cause disease is closely linked to its ability to undergo a morphological transition from budding yeast to filamentous forms (hyphae and pseudohyphae). The extent to which C. albicans strains isolated from patients undergo filamentation varies significantly. In addition, the filamentation phenotypes of mutants involving transcription factors that positively regulate hyphal morphogenesis can also vary from strain to strain. Here, we characterized the virulence, in vitro and in vivo filamentation, and in vitro and in vivo hypha-associated gene expression profiles for four poorly filamenting C. albicans isolates and their corresponding deletion mutants of the repressor of filamentation NRG1. The two most virulent strains, 57055 and 78048, show robust in vivo filamentation but are predominately yeast phase under in vitro hypha induction; the two low-virulence strains (94015 and 78042) do not undergo filamentation well under either condition. In vitro, deletion of NRG1 increases hyphae formation in the SC5314 derivative SN250, but only pseudohyphae are formed in the clinical isolates. Deletion of NRG1 modestly increased the virulence of 78042, which was accompanied by increased expression of hypha-associated genes without an increase in filamentation. Strikingly, deletion of NRG1 in 78048 reduced filamentation in vivo, expression of candidalysin (ECE1), and virulence without dramatically altering establishment of infection. Thus, the function of the conserved repressor NRG1 in C. albicans shows strain-based heterogeneity during infection.IMPORTANCEClinical isolates of the human fungal pathogen Candida albicans show significant variation in their ability to undergo in vitro filamentation and in the function of well-characterized transcriptional regulators of filamentation. Here, we show that Nrg1, a key repressor of filamentation and filament specific gene expression in standard reference strains, has strain-dependent functions, particularly during infection. Most strikingly, loss of NRG1 function can reduce filamentation, hypha-specific gene expression such as the toxin candidalysin, and virulence in some strains. Our data emphasize that the functions of seemingly fundamental and well-conserved transcriptional regulators such as Nrg1 are contextual with respect to both environment and genetic backgrounds.

Neuregulin-1 and ALS19 (ERBB4): at the crossroads of amyotrophic lateral sclerosis and cancer.

BACKGROUND: Neuregulin-1 (NRG1) is implicated in both cancer and neurologic diseases such as amyotrophic lateral sclerosis (ALS); however, to date, there has been little cross-field discussion between neurology and oncology in regard to these genes and their functions. MAIN BODY: Approximately 0.15-0.5% of cancers harbor NRG1 fusions that upregulate NRG1 activity and hence that of the cognate ERBB3/ERBB4 (HER3/HER4) receptors; abrogating this activity with small molecule inhibitors/antibodies shows preliminary tissue-agnostic anti-cancer activity. Notably, ERBB/HER pharmacologic suppression is devoid of neurologic toxicity. Even so, in ALS, attenuated ERBB4/HER4 receptor activity (due to loss-of-function germline mutations or other mechanisms in sporadic disease) is implicated; indeed, ERBB4/HER4 is designated ALS19. Further, secreted-type NRG1 isoforms may be upregulated (perhaps via a feedback loop) and could contribute to ALS pathogenesis through aberrant glial cell stimulation via enhanced activity of other (e.g., ERBB1-3/HER1-3) receptors and downstream pathways. Hence, pan-ERBB inhibitors, already in use for cancer, may be agents worthy of testing in ALS. CONCLUSION: Common signaling cascades between cancer and ALS may represent novel therapeutic targets for both diseases.

Diagnostic potential of NRG1 in benign nerve sheath tumors and its influence on the PI3K-Akt signaling and tumor immunity.

OBJECTIVE: Benign nerve sheath tumors (BNSTs) present diagnostic challenges due to their heterogeneous nature. This study aimed to determine the significance of NRG1 as a novel diagnostic biomarker in BNST, emphasizing its involvement in the PI3K-Akt pathway and tumor immune regulation. METHODS: Differential genes related to BNST were identified from the GEO database. Gene co-expression networks, protein-protein interaction networks, and LASSO regression were utilized to pinpoint key genes. The CIBERSORT algorithm assessed immune cell infiltration differences, and functional enrichment analyses explored BNST signaling pathways. Clinical samples helped establish PDX models, and in vitro cell lines to validate NRG1's role via the PI3K-Akt pathway. RESULTS: Nine hundred eighty-two genes were upregulated, and 375 downregulated in BNST samples. WGCNA revealed the brown module with the most significant difference. Top hub genes included NRG1, which was also determined as a pivotal gene in disease characterization. Immune infiltration showed significant variances in neutrophils and M2 macrophages, with NRG1 playing a central role. Functional analyses confirmed NRG1's involvement in key pathways. Validation experiments using PDX models and cell lines further solidified NRG1's role in BNST. CONCLUSION: NRG1 emerges as a potential diagnostic biomarker for BNST, influencing the PI3K-Akt pathway, and shaping the tumor immune microenvironment.

nNOS in Erbb4-positive neurons regulates GABAergic transmission in mouse hippocampus.

Neuronal nitric oxide synthase (nNOS, gene name Nos1) orchestrates the synthesis of nitric oxide (NO) within neurons, pivotal for diverse neural processes encompassing synaptic transmission, plasticity, neuronal excitability, learning, memory, and neurogenesis. Despite its significance, the precise regulation of nNOS activity across distinct neuronal types remains incompletely understood. Erb-b2 receptor tyrosine kinase 4 (ErbB4), selectively expressed in GABAergic interneurons and activated by its ligand neuregulin 1 (NRG1), modulates GABA release in the brain. Our investigation reveals the presence of nNOS in a subset of GABAergic interneurons expressing ErbB4. Notably, NRG1 activates nNOS via ErbB4 and its downstream phosphatidylinositol 3-kinase (PI3K), critical for NRG1-induced GABA release. Genetic removal of nNos from Erbb4-positive neurons impairs GABAergic transmission, partially rescued by the NO donor sodium nitroprusside (SNP). Intriguingly, the genetic deletion of nNos from Erbb4-positive neurons induces schizophrenia-relevant behavioral deficits, including hyperactivity, impaired sensorimotor gating, and deficient working memory and social interaction. These deficits are ameliorated by the atypical antipsychotic clozapine. This study underscores the role and regulation of nNOS within a specific subset of GABAergic interneurons, offering insights into the pathophysiological mechanisms of schizophrenia, given the association of Nrg1, Erbb4, Pi3k, and Nos1 genes with this mental disorder.

Real-world outcomes associated with afatinib use in patients with solid tumors harboring NRG1 gene fusions.

OBJECTIVES: Neuregulin-1 (NRG1) fusions may drive oncogenesis via constitutive activation of ErbB signaling. Hence, NRG1 fusion-driven tumors may be susceptible to ErbB-targeted therapy. Afatinib (irreversible pan-ErbB inhibitor) has demonstrated activity in individual patients with NRG1 fusion-positive solid tumors. This study collected real-world data on demographics, clinical characteristics, and clinical outcomes in this patient population. MATERIALS AND METHODS: In this retrospective, multicenter, non-comparative cohort study, physicians in the US-based Cardinal Health Oncology Provider Extended Network collected data from medical records of patients with NRG1 fusion-positive solid tumors who received afatinib (afatinib cohort) or other systemic therapies (non-afatinib cohort) in any therapy line. Objectives included demographics, clinical characteristics, and outcomes (overall response rate [ORR], progression-free survival [PFS], and overall survival [OS]). RESULTS: Patients (N = 110) with a variety of solid tumor types were included; 72 received afatinib, 38 other therapies. In the afatinib cohort, 70.8 % of patients received afatinib as second-line treatment and Eastern Cooperative Oncology Group performance status (ECOG PS) was 2-4 in 69.4 % at baseline. In the non-afatinib cohort, 94.7 % of patients received systemic therapy as first-line treatment and ECOG PS was 2-4 in 31.6 % at baseline. In the afatinib cohort, ORR was 37.5 % overall (43.8 % when received as first-line therapy); median PFS and OS were 5.5 and 7.2 months, respectively. In the non-afatinib cohort, ORR was 76.3 %; median PFS and OS were 12.9 and 22.6 months, respectively. CONCLUSION: This study provides real-world data on the characteristics of patients with NRG1 fusion-positive solid tumors treated with afatinib or other therapies; durable responses were observed in both groups. However, there were imbalances between the cohorts, and the study was not designed to compare outcomes. Further prospective/retrospective trials are required.

Neuregulin 1 as a potential biomarker for disease progression in moyamoya disease: A case-control study in Chinese population.

OBJECTIVE: Moyamoya disease (MMD) is a rare and progressive stenosis of cerebral arteries characterized by abnormally proliferative vasculopathy. Current studies have demonstrated that Neuregulin 1 (NRG1) plays a key role in angiogenesis-related disorders. Thus, the aim of our study is to investigate the serum NRG1 levels and their clinical correlations in MMD patients. METHODS: In this study, thirty adult patients with MMD and age-gender matched healthy controls were enrolled from our hospital between July 2020 and April 2022. Peripheral blood samples were collected at baseline, and clinical data were obtained from the electronic medical record system. Serum NRG1 concentrations were measured by enzyme-linked immunosorbent assay. Sanger sequencing was applied to detect the RNF213 p.R4810K mutation. RESULTS: The serum NRG1 levels were significantly higher in MMD patients compared to controls (14.48 ± 10.81 vs.7.54 ± 6.35mmol/L, p < 0.001). No statistical difference in baseline clinical characteristics was found between both groups. Correlation analyses showed that NRG1 levels were positively associated with Suzuki staging (r = 0.4137, p = 0.023) while not related to other clinical features (reduced cerebral blood flow, posterior cerebral artery involvement, bilateral or unilateral steno-occlusive changes). Furthermore, subgroup analysis revealed that MMD patients with the RNF213 p.R4810K mutation presented with significantly higher NRG1 levels than those without the mutation (9.60 ± 0.929 vs. 25.89 ± 4.338 mmol/L, p = 0.001). CONCLUSIONS: Our study suggests that increased serum NRG1 levels may constitute a characteristic feature of MMD, indicating a potential positive correlation with disease progression and the presence of the RNF213 mutation. This positions NRG1 as a potentially crucial target for further studies aimed at comprehending the pathogenesis of MMD.

Fluorescent in situ hybridization has limitations in screening NRG1 gene rearrangements.

BACKGROUND: NRG1 fusion is a promising therapeutic target for various tumors but its prevalence is extremely low, and there are no standardized testing algorithms for genetic assessment. MOTHODS: In this study, we analyzed 3008 tumors using Fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) to screen for NRG1 translocation and p-HER3 expression. RESULTS: Our results demonstrated no cases with p-HER3 positivity through IHC. Nonetheless, 29 cases (0.96%) were identified positive for NRG1 translocation through FISH, with three different signal types. FISH-positive cases were subsequently subjected to next-generation sequencing (NGS) testing. However, only eight of these cases were confirmed with NRG1 fusion through NGS. Notably, we divided FISH into three types and FISH type C group was consistent with NGS results. All NGS NRG1 fusion tumors were adenocarcinomas, with a higher prevalence in females. Our findings indicate that although FISH has limitations in screening NRG1 gene rearrangements, NRG1 fusions can be reliably detected with signals exhibiting low copy numbers of the 5'-end of the gene and no fusion signals. CONCLUSION: Considering the high cost of NGS, FISH remains a useful method for screening NRG1 fusions in various types of tumors. This study provides valuable insights into the molecular mechanisms of NRG1 fusion and identifies potential treatment targets for patients suffering from this disease.

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.

A Novel Form of Neuregulin 1 Type III Caused by N-Terminal Processing.

Nrg1 (Neuregulin 1) type III, a susceptible gene of schizophrenia, exhibits a critical role in the central nervous system and is essential at each stage of Schwann's cell development. Nrg1 type III comprises double-pass transmembrane domains, with the N-terminal and C-terminal localizing inside the cells. The N-terminal transmembrane helix partially overlaps with the cysteine-rich domain (CRD). In this study, Nrg1 type III constructs with different tags were transformed into cultured cells to verify whether CRD destroyed the transmembrane helix formation. We took advantage of immunofluorescent and immunoprecipitation assays on whole cells and analyzed the N-terminal distribution. Astonishingly, we found that a novel form of Nrg1 type III, about 10% of Nrg1 type III, omitted the N-terminal transmembrane helix, with the N-terminal positioning outside the membrane. The results indicated that the novel single-pass transmembrane status was a minor form of Nrg1 type III caused by N-terminal processing, while the major form was a double-pass transmembrane status.

Parvalbumin interneurons dysfunction is potentially associated with FαMNs decrease and NRG1-ErbB4 signaling inhibition in spinal cord in amyotrophic lateral sclerosis.

OBJECTIVE: To investigate the alteration of PV interneurons in ALS mainly focusing its dynamic changes and its relationship with motor neurons and ErbB4 signaling. METHODS: SOD1G93A mice were used as ALS model. ALS animals were divided into different groups according to birth age: symptomatic prophase (50~60 days), symptomatic phase (90~100 days), and symptomatic progression (130~140 days). Immunofluorescence was performed for measurement of PV-positive interneurons, MMP-9, ChAT, NeuN and ErbB4. RT-qPCR and western blot were used to determine the expression of PV and MMP-9. RESULTS: PV expression was remarkably higher in the anterior horn of gray matter compared with posterior horn and area in the middle of gray matter in control mice. In ALS mice, PV, MMP-9 and ErbB4 levels were gradually decreased along with onset. PV, MMP-9 and ErbB4 levels in ALS mice were significantly down-regulated than control mice after onset, indicating the alteration of PV interneurons, FαMNs and ErbB4. SαMNs levels only decreased remarkably at symptomatic progression in ALS mice compared with control mice, while γMNs levels showed no significant change during whole period in all mice. MMP-9 and ErbB4 were positively correlated with PV. NRG1 treatment significantly enhanced the expression of ErBb4, PV and MMP-9 in ALS mice. CONCLUSION: PV interneurons decrease is along with FαMNs and ErbB4 decrease in ALS mice.

NRG1 promotes tumorigenesis and metastasis and afatinib treatment efficiency is enhanced by NRG1 inhibition in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a highly aggressive tumor with significant heterogeneity in incidence and outcomes. The role of Neuregulin 1 (NRG1) in ESCC and its contribution to aggressiveness remain unknown. This study aims to investigate the functions and molecular mechanisms of NRG1 in ESCC as well as the treatment strategy for ESCC with overexpression of NRG1. We firstly demonstrated the upregulation of NRG1 and a negative correlation trend between patients' overall survival (OS) and the expression level of NRG1 in esophageal cancer. And then we found NRG1 promoted cell proliferation, migration, inhibited apoptosis, and accelerated tumorigenesis and metastasis in ESCC using cell lines and xenograft models. Furthermore, we discovered that NRG1 activated the NF-κB/MMP9 signaling pathway, contributing to the metastatic phenotype in ESCC. Finally, we show that afatinib (FDA approved cancer growth blocker) could inhibit ESCC with overexpressed NRG1 and down-regulation of NRG1 along with afatinib treatment provides higher efficient strategy. This study uncovers the critical role and molecular mechanism of NRG1 in ESCC tumorigenesis and metastasis, suggesting its potential as a novel biomarker for ESCC treatment.

Nrg1 Regulates Cardiomyocyte Migration and Cell Cycle in Ventricular Development.

BACKGROUND: Cardiac ventricles provide the contractile force of the beating heart throughout life. How the primitive endocardium-layered myocardial projections called trabeculae form and mature into the adult ventricles is of great interest for biology and regenerative medicine. Trabeculation is dependent on the signaling protein Nrg1 (neuregulin-1). However, the mechanism of action of Nrg1 and its role in ventricular wall maturation are poorly understood. METHODS: We investigated the functions and downstream mechanisms of Nrg1 signaling during ventricular chamber development using confocal imaging, transcriptomics, and biochemical approaches in mice with cardiac-specific inactivation or overexpression of Nrg1. RESULTS: Analysis of cardiac-specific Nrg1 mutant mice showed that the transcriptional program underlying cardiomyocyte-oriented cell division and trabeculae formation depends on endocardial Nrg1 to myocardial ErbB2 (erb-b2 receptor tyrosine kinase 2) signaling and phospho-Erk (phosphorylated extracellular signal-regulated kinase; pErk) activation. Early endothelial loss of Nrg1 and reduced pErk activation diminished cardiomyocyte Pard3 and Crumbs2 (Crumbs Cell Polarity Complex Component 2) protein and altered cytoskeletal gene expression and organization. These alterations are associated with abnormal gene expression related to mitotic spindle organization and a shift in cardiomyocyte division orientation. Nrg1 is crucial for trabecular growth and ventricular wall thickening by regulating an epithelial-to-mesenchymal transition-like process in cardiomyocytes involving migration, adhesion, cytoskeletal actin turnover, and timely progression through the cell cycle G2/M phase. Ectopic cardiac Nrg1 overexpression and high pErk signaling caused S-phase arrest, sustained high epithelial-to-mesenchymal transition-like gene expression, and prolonged trabeculation, blocking compact myocardium maturation. Myocardial trabecular patterning alterations resulting from above- or below-normal Nrg1-dependent pErk activation were concomitant with sarcomere actin cytoskeleton disorganization. The Nrg1 loss- and gain-of-function transcriptomes were enriched for Yap1 (yes-associated protein-1) gene signatures, identifying Yap1 as a potential downstream effector. Furthermore, biochemical and imaging data reveal that Nrg1 influences pErk activation and Yap1 nuclear-cytoplasmic distribution during trabeculation. CONCLUSIONS: These data establish the Nrg1-ErbB2/ErbB4-Erk axis as a crucial regulator of cardiomyocyte cell cycle progression and migration during ventricular development.

Stromal-derived NRG1 enables oncogenic KRAS bypass in pancreas cancer.

Activating KRAS mutations (KRAS*) in pancreatic ductal adenocarcinoma (PDAC) drive anabolic metabolism and support tumor maintenance. KRAS* inhibitors show initial antitumor activity followed by recurrence due to cancer cell-intrinsic and immune-mediated paracrine mechanisms. Here, we explored the potential role of cancer-associated fibroblasts (CAFs) in enabling KRAS* bypass and identified CAF-derived NRG1 activation of cancer cell ERBB2 and ERBB3 receptor tyrosine kinases as a mechanism by which KRAS*-independent growth is supported. Genetic extinction or pharmacological inhibition of KRAS* resulted in up-regulation of ERBB2 and ERBB3 expression in human and murine models, which prompted cancer cell utilization of CAF-derived NRG1 as a survival factor. Genetic depletion or pharmacological inhibition of ERBB2/3 or NRG1 abolished KRAS* bypass and synergized with KRASG12D inhibitors in combination treatments in mouse and human PDAC models. Thus, we found that CAFs can contribute to KRAS* inhibitor therapy resistance via paracrine mechanisms, providing an actionable therapeutic strategy to improve the effectiveness of KRAS* inhibitors in PDAC patients.

Compensatory role of neuregulin-1 in diabetic cardiomyopathy.

Diabetes mellitus is a prevalent risk factor for congestive heart failure. Diabetic cardiomyopathy patients present with left ventricular (LV) diastolic dysfunction at an early stage, then systolic dysfunction as the disease progresses. The mechanism underlying the development of diabetic cardiomyopathy has not yet been fully understood. This study aimed to elucidate the mechanisms by which diastolic dysfunction precedes systolic dysfunction at the early stage of diabetic cardiomyopathy. We hypothesized that the downregulation of cardioprotective factors is involved in the pathogenesis of diabetic cardiomyopathy. LV diastolic dysfunction, but not systolic dysfunction, was observed in type-1 diabetes mellitus model mice 4 weeks after STZ administration (STZ-4W), mimicking the early stage of diabetic cardiomyopathy. Counter to expectations, neuregulin-1 (NRG1) was markedly upregulated in the vascular endothelial cell in the ventricles of STZ-4W mice. To clarify the functional significance of the upregulated NRG1, we blocked its receptor ErbB2 with trastuzumab (TRZ). In STZ-4W mice, TRZ significantly reduced the systolic function without affecting diastolic function and caused a more prominent reduction in Akt phosphorylation levels. These results indicate that the compensatory upregulated NRG1 contributes to maintaining the LV systolic function, which explains why diastolic dysfunction precedes systolic dysfunction at the early stage of diabetic cardiomyopathy.

Persistent NRG1 Type III Overexpression in Spinal Motor Neurons Has No Therapeutic Effect on ALS-Related Pathology in SOD1G93A Mice.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease affecting upper and lower motor neurons (MNs). Neuregulin-1 (NRG1) is a pleiotropic growth factor that has been shown to be potentially valuable for ALS when supplemented by means of viral-mediated gene therapy. However, these results are inconsistent with other reports. An alternative approach for investigating the therapeutic impact of NRG1 on ALS is the use of transgenic mouse lines with genetically defined NRG1 overexpression. Here, we took advantage of a mouse line with NRG1 type III overexpression in spinal cord α motor neurons (MN) to determine the impact of steadily enhanced NRG1 signalling on mutant superoxide dismutase 1 (SOD1)-induced disease. The phenotype of SOD1G93A-NRG1 double transgenic mice was analysed in detail, including neuropathology and extensive behavioural testing. At least 3 animals per condition and sex were histopathologically assessed, and a minimum of 10 mice per condition and sex were clinically evaluated. The accumulation of misfolded SOD1 (mfSOD1), MN degeneration, and a glia-mediated neuroinflammatory response are pathological hallmarks of ALS progression in SOD1G93A mice. None of these aspects was significantly improved when examined in double transgenic NRG1-SOD1G93A mice. In addition, behavioural testing revealed that NRG1 type III overexpression did not affect the survival of SOD1G93A mice but accelerated disease onset and worsened the motor phenotype.

Durable response to afatinib in advanced lung adenocarcinoma harboring a novel NPTN-NRG1 fusion: a case report.

BACKGROUND: NRG1 fusions are rare oncogenic drivers in solid tumors, and the incidence of NRG1 fusions in non-small cell lung cancer (NSCLC) was 0.26%. It is essential to explore potential therapeutic strategies and efficacy predictors for NRG1 fusion-positive cancers. CASE PRESENTATION: We report an advanced lung adenocarcinoma patient harboring a novel NPTN-NRG1 fusion identified by RNA-based next-generation sequencing (NGS), which was not detected by DNA-based NGS at initial diagnosis. Transcriptomics data of the tissue biopsy showed NRG1α isoform accounted for 30% of total NRG1 reads, and NRG1ß isoform was undetectable. The patient received afatinib as fourth-line treatment and received a progression-free survival (PFS) of 14 months. CONCLUSIONS: This report supports afatinib can provide potential benefit for NRG1 fusion patients, and RNA-based NGS is an accurate and cost-effective strategy for fusion detection and isoform identification.

Clinicopathological Characteristics of NRG1 Fusion-Positive Solid Tumors in Korean Patients.

PURPOSE: Neuregulin 1 (NRG1) gene fusion is a potentially actionable oncogenic driver. The oncoprotein binds to ERBB3-ERBB2 heterodimers and activates downstream signaling, supporting a therapeutic approach for inhibiting ERBB3/ERBB2. However, the frequency and clinicopathological features of solid tumors harboring NRG1 fusions in Korean patients remain largely unknown. MATERIALS AND METHODS: We reviewed archival data from next-generation sequencing panel tests conducted at a single institution, specifically selecting patients with in-frame fusions that preserved the functional domain. The clinicopathological characteristics of patients harboring NRG1 fusions were retrospectively reviewed. RESULTS: Out of 8,148 patients, NRG1 fusions were identified in 22 patients (0.27%). The average age of the patients was 59 years (range, 32 to 78 years), and the male-to-female ratio was 1:1.2. The lung was the most frequently observed primary site (n=13), followed by the pancreaticobiliary tract (n=3), gastrointestinal tract (n=2, stomach and rectum each), ovary (n=2), breast (n=1), and soft tissue (n=1). Histologically, all tumors demonstrated adenocarcinoma histology, with the exception of one case of sarcoma. CD74 (n=8) and SLC3A2 (n=4) were the most frequently identified fusion partners. Dominant features included the presence of fewer than three co-occurring genetic alterations, a low tumor mutation burden, and low programmed death-ligand 1 expression. Various clinical responses were observed in patients with NRG1 fusions. CONCLUSION: Despite the rarity of NRG1 fusions in Korean patients with solid tumors, identification through next-generation sequencing enables the possibility of new targeted therapies.

Androgen receptor transcriptional activity is required for heregulin-1ß-mediated nuclear localization of the HER3/ErbB3 receptor tyrosine kinase.

Prostate cancer is initially regulated by the androgen receptor (AR), a ligand-activated, transcription factor, and is in a hormone-dependent state (hormone-sensitive prostate cancer (HSPC)), but eventually becomes androgen-refractory (castration-resistant prostate cancer (CRPC)) because of mechanisms that bypass the AR, including by activation of ErbB3, a member of the epidermal growth factor receptor family. ErbB3 is synthesized in the cytoplasm and transported to the plasma membrane for ligand binding and dimerization, where it regulates downstream signaling, but nuclear forms are reported. Here, we demonstrate in prostatectomy samples that ErbB3 nuclear localization is observed in malignant, but not benign prostate, and that cytoplasmic (but not nuclear) ErbB3 correlated positively with AR expression but negatively with AR transcriptional activity. In support of the latter, androgen depletion upregulated cytoplasmic, but not nuclear ErbB3, while in vivo studies showed that castration suppressed ErbB3 nuclear localization in HSPC, but not CRPC tumors. In vitro treatment with the ErbB3 ligand heregulin-1ß (HRG) induced ErbB3 nuclear localization, which was androgen-regulated in HSPC but not in CRPC. In turn, HRG upregulated AR transcriptional activity in CRPC but not in HSPC cells. Positive correlation between ErbB3 and AR expression was demonstrated in AR-null PC-3 cells where stable transfection of AR restored HRG-induced ErbB3 nuclear transport, while AR knockdown in LNCaP reduced cytoplasmic ErbB3. Mutations of ErbB3's kinase domain did not affect its localization but was responsible for cell viability in CRPC cells. Taken together, we conclude that AR expression regulated ErbB3 expression, its transcriptional activity suppressed ErbB3 nuclear translocation, and HRG binding to ErbB3 promoted it.

Exploring the Binding Mechanism of NRG1-ERBB3 Complex and Discovery of Potent Natural Products to Reduce Diabetes-Assisted Breast Cancer Progression.

Diabetes mellitus significantly contributes to breast cancer progression, where hyperglycemia upregulates specific genes, leading to more aggressive tumor growth. In patients with BC that develop diabetes, neuregulin 1 (NRG1) and epidermal growth factor receptor 3 (ERBB3) overexpression exacerbate tumor growth and progression. Since the interaction between NRG1 and ERBB3 is critical for tumor growth, understanding the molecular mechanisms underlying NRG1-ERBB3 complex formation is essential for elucidating diabetes-assisted breast cancer progression. However, the key residues forming the NRG1-ERBB3 complex remain unknown. Here, we substituted specific residues in NRG1 with alanine and studied its interactions with ERBB3 using computational structural biology tools. We further screened the South African natural compounds database to target the complex's interface residues to discover potential inhibitors. The conformational stability and dynamic features of NRG1-WT, -H2A, -L3A, and -K35A complexed with ERBB3 were subjected to 400 ns molecular dynamics simulations. The free binding energies of all NRG1-ERBB3 complexes were calculated using the molecular mechanics-generalized Born surface area (MM/GBSA). The H2 and L3 alanine substitutions caused a loss of interaction with ERBB3 residue D73, weakening the interaction with ERBB3. Screening 1300 natural compounds identified four (SANC00643, SANC00824, SANC00975, and SANC00335) with the best potential to inhibit ERRB3-NRG1 coupling. The binding free energies for each complex were - 48.55 kcal/mol for SANC00643, - 47.68 kcal/mol for SANC00824, - 46.04 kcal/mol for SANC00975, and - 45.29 kcal/mol for SANC00335, showing their overall stronger binding with ERBB3 than NRG1 and their potential to act as ERBB3-NRG1 complex inhibitors. In conclusion, this complex may represent a residue-specific drug target to inhibit BC progression.

Pathogenic HER3 dimerization domain mutations create a structural bias towards un-conventional EGFR-HER3 signalling axis in breast cancer.

Among the EGFR family of receptors, HER3 is considered as a pseudo-kinase which primarily interacts with HER2 in presence of heregulin-1ß. We identified two hotspot mutations i.e. G284R and D297Y and one double mutant HER2-S310F/HER3-G284R in breast cancer patients. Long term MDS (7.5 µs) revealed that HER3-D297Y and HER2-S310F:HER3-G284R do not allow the interaction with HER2 as these mutations cause dramatic conformational changes in its flanking regions. This results in formation of an unstable HER2-WT:HER3-D297Y heterodimer, thereby abrogating the downstream signalling by AKT. We found that His228 and Ser300 of HER3-D297Y form stable interactions with Glu245 and Tyr270 of EGFR-WT, in the presence of either EGF or heregulin-1ß. Applying TRIM-ing mediated direct knockdown of endogenous EGFR protein, specificity of the unconventional EGFR:HER3-D297Y interaction was validated. Due to this unusual ligand mediated interaction, cancer cells were found susceptible to EGFR targeted therapeutics i.e. Gefitinib and Erlotinib. Further, in TCGA analysis, BC patients harbouring HER3-D297Y mutation showed increased p-EGFR levels as compared to the patients harbouring HER3-WT and HER3-G284R mutations. For the first time, this comprehensive study showed the importance of specific hotspot mutations in HER3 dimerization domain can defy the Trastuzumab therapy, rather cells become susceptible to the EGFR inhibitors.