Epidermal Growth Factor Receptor Antagonists Research Articles

The Agonistic Activity of the Human Epidermal Growth Factor is Reduced by the D46G Substitution.

Resistance to anti-tumor agents targeting the epidermal growth factor receptor (EGFR) reduces treatment response and requires the development of novel EGFR antagonists. Mutant epidermal growth factor (EGF) forms with reduced agonistic activity could be promising agents in cancer treatment. EGF D46G affinity to EGFR domain III was assessed with affinity chromatography. EGF D46G acute toxicity in Af albino mice at 320 and 3200 μg/kg subcutaneous doses was evaluated. EGF D46G activity in human epidermoid carcinoma cells at 10 ng/mL concentration in serum-free medium and in subcutaneous Ehrlich ascites carcinoma mice model at 320 μg/kg dose was studied. The D46G substitution decreases the thermal stability of EGF complexes with EGFR domain III by decreasing the ability of the C-terminus to be released from the intermolecular β- sheet. However, with remaining binding sites for EGFR domain I, EGF D46G effectively competes with other EGF-like growth factors for binding to EGFR and does not demonstrate toxic effects in mice. EGF D46G inhibits the proliferation of human epidermoid carcinoma cells compared to native EGF. A single subcutaneous administration of EGF D46G along with Ehrlich carcinoma cells injection inhibits the proliferation of these cells and delays tumor formation for up to seven days. EGF D46G can be defined as a partial EGFR agonist as this mutant form demonstrates reduced agonistic activity compared to native EGF. The study emphasizes the role of the EGF C-terminus in establishing interactions with EGFR domain III, which are necessary for EGFR activation and subsequent proliferation of cells.

The role of epidermal growth factor and cholinergic receptor agonists and antagonists in MAPK signal transduction in K562 cells

Objective: Muscarinic receptors (M1-M5) are members of the G protein-coupled receptor superfamily and are effective in physiological
 functions through G proteins. Recent studies suggested that cholinergic receptors mediate cellular activities in hematopoietic cells.
 The aim of this study was to investigate the potential role of mitogen-activated protein kinases (MAPK) signaling extracellular signalregulated
 kinases 1 and 2 (ERK1/2)/phosphorylated ERK1/ (pERK1/2) pathways in chronic myeloid leukemia (K562) cells.
 Materials and Methods: Chronic myeloid leukemia cells were cultured. Cells were incubated in the presence of muscarinic receptor
 agonist, antagonist and epidermal growth factor (EGF). To detect MAPK activation, ERK/pERK protein expression levels were
 determined by western blot method techniques.
 Results: Our study results showed that cholinergic agents and EGFs affect the MAPK pathway in the human K562 cell line.
 Conclusion: Cholinergic and EGF receptors may affect the MAPK pathway in K562 cells.
 Conclusion: Cholinergic and EGF receptors may affect the MAPK pathway in K562 cells.

Current and Perspective Approaches to the Treatment of Prolactinomas.

Along with the presence of the 2011 Endocrine Society Clinical Practice Guidelines and numerous large-scale studies on the treatment of hyperprolactinemia of different origin, there are some unresolved questions, ambiguous and sometimes contradictory points of view regarding the management of patients with prolactinomas. This overview is devoted to the analysis of the results of modern clinical studies and the approaches towards the management of hyperprolactinemia caused by prolactinoma. A systematic research of the literature for the appropriate keywords published mainly for the last 10 years was done; also, a reference list of each selected article was analysed. We included to our review the articles reporting controversial issues or new data on the treatment of hyperprolactinemia. The review describes various problems arising during the treatment of prolactinoma. The presence of primary and secondary dopamine agonist resistance in each case requires an individual approach, and sometimes may include the use of the antineoplastic agent t emozolomide. The side effects of dopamine agonists are discussed, with quite rare ones, including valvulopathy, pathological psychological conditions and cerebrospinal rhinorrhea. The controversial issue of the duration and doses of the drug used to achieve a lasting effect in the treatment of prolactinomas is considered. There are some points connected with the frequency of relapses. Thus, recurrence is correlated to the duration of treatment with dopamine agonists, prolactin levels at diagnosis, and the initial tumor size. Metformin, somatostatin analogues, selective estrogen receptor modulators, tyrosine kinase inhibitors, inhibitors of the mammalian target of rapamycin, epidermal growth factor receptor antagonists are investigated nowadays as potential alternative methods of drug treatment of prolactinomas. Drug therapy with dopamine agonists makes it possible to achieve the desired results in the vast majority of patients. However, despite the proven safety of this group of medicines, the risk of side effects should still be taken into account. The therapy regimen should be determined by the clinical course of prolactinoma and the patient's response to treatment. Other options of treatment should be considered in patients intolerant to medical therapy, with contraindication or resistance to dopamine agonists, in the case of a malignant tumor. The presence of refractory to any of the applied methods of treatment and aggressive prolactinomas leads to the search for new drugs.

Tissue kallikrein-related peptidase8 accentuates cardiac fibrosis after myocardial ischemia-reperfusion injury via regulation of cardiac fibroblasts

AimsTissue kallikrein-related peptidase8 (KLK8) has been found to mitigate acute myocardial ischemia-reperfusion (IR) injury. However, the effect of KLK8 on cardiac remodeling in response to IR injury has not been determined. Materials and methodsKLK8 overexpressing transgenic rat (KLK8-TG) was used as the animal model. IR injury was induced by ligating the left anterior descending coronary artery for 1 h and subsequent reperfusion. The functional and morphological changes of the heart were examined 14 days after the injury. Neonatal rat cardiac fibroblasts (CFs) were used to investigate the molecular mechanisms in vitro. Key findingsKLK8 overexpression enhanced cardiac diastolic dysfunction, fibrosis, and hypertrophy after IR injury, indicating that KLK8 accentuated cardiac remodeling in response to IR injury. Moreover, KLK8 overexpression increased epidermal growth factor (EGF) release and promoted the phosphorylation of EGF receptor (EGFR) and ERK1/2 in the heart after IR injury. It was interesting to find that both EGFR antagonist (AG 1478) and MEK inhibitor (PD98059) attenuated the KLK8-induced proliferation and activation of CFs in vitro, indicating that EGFR signaling might mediate the pro-fibrotic action of KLK8. SignificanceKLK8 plays a crucial role in cardiac remodeling after myocardial infarction. KLK8 accentuates cardiac fibrosis after IR injury, possibly mediated by EGFR signaling in CFs.

O-141 Combination of gefitinib and methotrexate to treat tubal ectopic pregnancy (GEM3): a multicentre, randomised, double-blind, placebo-controlled trial

Abstract Study question Is a combination of parenteral methotrexate and oral gefitinib more effective than methotrexate alone in the treatment of tubal ectopic pregnancy? Summary answer In women with a tubal ectopic pregnancy, adding oral gefitinib to parental methotrexate does not offer clinical benefit over methotrexate and increases minor adverse reactions. What is known already Current treatment of tubal ectopic pregnancies is with methotrexate or surgery. Methotrexate treatment fails in ∼30% of women and they require rescue surgery. At the time of the design of the trial, it had been shown in preclinical studies that tubal implantation sites express high levels of epidermal growth factor receptor (EGFR) and that gefitinib (an EGFR antagonist) augments methotrexate-induced regression of pregnancy-like tissue. There was also evidence from uncontrolled phase I and II trials that raised the possibility that combination methotrexate and gefitinib could be a more effective medical treatment than methotrexate alone to treat stable ectopic pregnancies. Study design, size, duration Between 2nd November 2016 and 6th October 2021, we performed a multicentre, randomised, double-blind, placebo-controlled trial across 50 UK hospitals. Eligible participants were women with a tubal ectopic pregnancy deemed suitable for medical management with methotrexate. Inclusion criteria were: aged 18-50 years; pre-treatment serum hCG level of 1000–5000 IU/L; and either a definite diagnosis of tubal ectopic pregnancy or a clinical judgement of ‘probable’ tubal ectopic pregnancy. Participants/materials, setting, methods Participants were administered a single dose of intramuscular methotrexate (50 mg/m2) and randomised (1:1 ratio) to seven days of additional oral gefitinib (250mg daily) or placebo. The primary outcome, analysed by intention to treat, was surgical intervention to resolve the ectopic pregnancy. Secondary outcomes included time to resolution of ectopic pregnancy and serious adverse events. Main results and the role of chance 328 participants were allocated to methotrexate and gefitinib (n = 165) or methotrexate and placebo (n = 163). Three participants in the placebo group withdrew. Surgical intervention occurred in 30% (50/165) of the gefitinib group and in 29% (47/160) of the placebo group (adjusted risk ratio 1.15, 95% confidence interval [CI] 0.85-1.58; adjusted risk difference -0.01, 95% CI -0.10-0.09; p = 0.37). Without surgical intervention, median time to resolution was 28.0 days in the gefitinib group and 28.0 days in the placebo group (subdistribution hazard ratio 1.03, 95% CI 0.75-1.40). Serious adverse events occurred in 3% (5/165) of the gefitinib group and in 4% (6/162) of the placebo group. Diarrhoea and rash were more common in the gefitinib group. Limitations, reasons for caution Limitations of the trial include the fact that we only tested one dose regimen. It is possible gefitinib may be effective if a different protocol were used, such as a longer period of administration. Also, we did not carry out pharmacodynamic studies to determine optimal drug bioavailability. Wider implications of the findings Our results show that the addition of gefitinib to standard medical management with methotrexate to treat tubal ectopic pregnancy is not clinically effective as it does not reduce subsequent surgical intervention and is associated with higher rates of reported symptoms than placebo. Trial registration number ISRCTN67795930

Erlotinib-induced dry eye.

Erlotinib is a newer, second-line oral antineoplastic drug mainly used in the treatment of non-small cell lung carcinoma, in addition to carcinomas of the pancreas, esophagus, and ovaries.[1] It targets protein tyrosine kinase and inhibits phosphorylation of epidermal growth factor receptor (EGFR), thereby blocking tumor cell proliferation. Other previously reported ocular side effects of erlotinib include trichomegaly, periorbital inflammation, ectropion, and epiphora.[2–4] Case Report A 58-year-old female presented with complaints of persistent irritation and redness in both eyes since a month. She was diagnosed with adenocarcinoma of the lung and had been under treatment with oral EGFR antagonist erlotinib for 6 months. Ocular examination revealed sparse eyelashes, blepharitis, and diffuse conjunctival congestion in both eyes [Fig. 1a]. Fluorescein staining showed punctate epithelial erosions and a tear film breakup time of 9 s [Fig. 1b]. She was diagnosed with erlotinib-induced blepharitis and dry eye; she was started on treatment with topical azithromycin ointment and preservative-free lubricants and was advised lid hygiene and hot fomentation as supportive therapy. Discontinuation of erlotinib was not advised as the tumor was responsive to the therapy and the side effects were tolerable and improved with treatment.Figure 1: (a) Diffuse slit-lamp image showing sparse eyelashes and blepharitis with decreased tear film height. (b) Diffuse slit-lamp image with cobalt blue filter showing punctate epithelial erosions on corneaEGFR receptors are found in the sebaceous glands, hair follicles, capillary system, and basal epidermal cells, and the abundance of these in the periorbital region has been hypothesized to be the reason for the ocular side effects of erlotinib.[2,4] A murine study with topical erlotinib showed it to cause a reduction in goblet cell numbers, ultrastructural changes to the corneal epithelium, and a significant reduction in the tear film breakup time.[5] With foreseeable increase in the use of erlotinib in cancer therapy, it is essential to understand its ocular side effects for appropriate patient education and treatment, which may warrant a concerted management from the ophthalmologist and the oncologist. Declaration of patient consent The authors certify that they have obtained all appropriate patient consent forms. In the form the patient (s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.

Insights from the molecular docking analysis of EGFR antagonists.

Overexpression of the epidermal growth factor receptor (EGFR) has been shown to be a critical factor in tumor development and cancer progression. Although established EGFR inhibitors have been effective in the treatment of cancer, they are associated with several side effects. As a result, there is an urgent need to develop novel EGFR inhibitors that can effectively target the receptor while causing no adverse side effects. Here, the bioactive compounds of Glycyrrhiza glabra and established EGFR inhibitors have been screened against the EGFR catalytic site. The compounds LTS0058805, LTS0114552, LTS0128805, LTS0174203, LTS0007447, and LTS0164690 exhibited binding energies to the EGFR that were comparable to those of established EGFR inhibitors. Further, these hit compounds were observed to interact with critical residues of the EGFR, suggesting their potential as inhibitors of the receptor. In addition, these hits possess good drug-like properties and merit further exploration for their potential application in cancer management.

Epidermal growth factor receptor mediates the basolateral uptake of phosphorothioate-modified antisense oligonucleotides in the kidney

Antisense oligonucleotides (ASOs) are highly biologically stable and specific therapeutic molecules that interfere with mRNA transcription and as a result effectively reduce the expression of a protein of interest. ASOs have low drug clearance and are retained in tissues. This is particularly evident in the kidneys where they accumulate in the renal proximal tubules. Receptor-mediated endocytosis (RME) plays a role in ASO uptake, however the membrane receptors facilitating the process and the full mechanism behind these transport and subsequent renal retention is still poorly understood. In the present study we employ a proximal tubule-on-a-chip (PTOC) that recapitulates the highly polarized nature of the renal epithelium and can discriminate between basal and apical uptake processes. The PTOC was used to determine the impact of cellular polarization in ASO uptake in the kidney and elucidate which receptors predominantly facilitated uptake. In the PTOC the uptake occurred predominantly from the basolateral side and was extensively inhibited using the selective epidermal growth factor receptor (EGFR) antagonist cetuximab. These results demonstrate that ASO uptake in a physiologically relevant model is predominately mediated by the EGFR and takes place from the basolateral side of the proximal tubules. In comparison to the 2D culture, PTOC could differentiate between distinct ASO uptake routes mediated by either Megalin or EGFR, which proper membrane expression is highly dependent on cellular polarity. Our results highlight the limitations of renal 2D models and demonstrate how an organ on a chip model can fill the physiological gap and is a powerful tool to study ASO, which have intricate transport mechanisms that ultimately impact both their efficacy and safety. • Continuously perfused proximal tubule-on-a-chip model recreates a highly polarized renal epithelium. • Uptake of antisense-oligonucleotides occurs from the basolateral side of the renal proximal tubules. • EGFR drives antisense-oligonucleotide uptake in our proximal tubule-on-a-chip model, an effect not observed in 2D culture.

Pharmacophore-based virtual screening approaches to identify novel molecular candidates against EGFR through comprehensive computational approaches and in-vitro studies.

Alterations to the EGFR (epidermal growth factor receptor) gene, which primarily occur in the axon 18-21 position, have been linked to a variety of cancers, including ovarian, breast, colon, and lung cancer. The use of TK inhibitors (gefitinib, erlotinib, lapatinib, and afatinib) and monoclonal antibodies (cetuximab, panitumumab, and matuzumab) in the treatment of advanced-stage cancer is very common. These drugs are becoming less effective in EGFR targeted cancer treatment and developing resistance to cancer cell eradication, which sometimes necessitates stopping treatment due to the side effects. One in silico study has been conducted to identify EGFR antagonists using other compounds, databases without providing the toxicity profile, comparative analyses, or morphological cell death pattern. The goal of our study was to identify potential lead compounds, and we identified seven compounds based on the docking score and four compounds that were chosen for our study, utilizing toxicity analysis. Molecular docking, virtual screening, dynamic simulation, and in-vitro screening indicated that these compounds' effects were superior to those of already marketed medication (gefitinib). The four compounds obtained, ZINC96937394, ZINC14611940, ZINC103239230, and ZINC96933670, demonstrated improved binding affinity (-9.9kcal/mol, -9.6kcal/mol, -9.5kcal/mol, and -9.2kcal/mol, respectively), interaction stability, and a lower toxicity profile. In silico toxicity analysis showed that our compounds have a lower toxicity profile and a higher LD50 value. At the same time, a selected compound, i.e., ZINC103239230, was revealed to attach to a particular active site and bind more tightly to the protein, as well as show better in-vitro results when compared to our selected gefitinib medication. MTT assay, gene expression analysis (BAX, BCL-2, and β-catenin), apoptosis analysis, TEM, cell cycle assay, ELISA, and cell migration assays were conducted to perform the cell death analysis of lung cancer and breast cancer, compared to the marketed product. The MTT assay exhibited 80% cell death for 75µM and 100µM; however, flow cytometry analysis with the IC50 value demonstrated that the selected compound induced higher apoptosis in MCF-7 (30.8%) than in A549.

Hypoxia-Inducible Expression of Annexin A6 Enhances the Resistance of Triple-Negative Breast Cancer Cells to EGFR and AR Antagonists.

Physiological changes such as hypoxia in the tumor microenvironment (TME) endow cancer cells with malignant properties, leading to tumor recurrence and rapid progression. Here, we assessed the effect of hypoxia (1% Oxygen) on the tumor suppressor Annexin A6 (AnxA6) and the response of triple-negative breast cancer (TNBC) cells to epidermal growth factor receptor (EGFR) and androgen receptor (AR) targeted therapies. We demonstrate that brief exposure of TNBC cells to hypoxia (within 24 h) is associated with down regulation of AnxA6 while > 24 h exposure cell type dependently stimulated the expression of AnxA6. Hypoxia depicted by the expression and stability of HIF-1/2α led to up regulation of the HIF target genes SLC2A1, PGK1 as well as AR and the AR target genes FABP-4 and PPAR-γ, but the cellular levels of AnxA6 protein decreased under prolonged hypoxia. Down regulation of AnxA6 in TNBC cells inhibited, while AnxA6 over expression enhanced the expression and cellular levels of HIF-1/2α, SLC2A1 and PGK1. RNAi mediated inhibition of hypoxia induced AnxA6 expression also strongly inhibited glucose uptake and ROS production in AnxA6 expressing TNBC cells. Using a luciferase reporter assay, we confirm that short-term exposure of cells to hypoxia inhibits while prolonged exposure of cells to hypoxia enhances AnxA6 promoter activity in HEK293T cells. Compared to cells cultured under normoxia, TNBC cells were more resistant to lapatinib under hypoxic conditions, and the downregulation of AnxA6 sensitized the cells to EGFR as well as AR antagonists. These data suggest that AnxA6 is a hypoxia inducible gene and that targeting AnxA6 upregulation may be beneficial in overcoming TNBC resistance to EGFR and/or AR targeted therapies.

Identification and functional analysis of epidermal growth factor receptor (EGFR) from Scylla paramamosain: The first evidence of two EGFR genes in animal and their involvement in immune defense against pathogen infection

The epidermal growth factor receptor (EGFR) is a pleiotropic glycoprotein which plays a role in regulating cell proliferation, migration and differentiation. However, the genetic diversity of EGFR in crustaceans as well as its function, such as whether it is involved in immune regulation, remains obscure. In this study, two EGFR genes, including EGFR1 and EGFR2, and three transcripts were identified and characterized in Scylla Paramamosain for the first time. To our knowledge, this is the first time that more than one EGFR gene was identified in a single species. The complete open reading frames (ORFs) of SpEGFR1, SpEGFR2a and SpEGFR2b were 4377 bp, 4404 bp and 4341 bp encoding deduced proteins of 1458 amino acids (aa), 1467 aa and 1446 aa, respectively. All EGFR had a signal peptide region and two Recep_L_domain region, followed by a transmembrane region and a conserved tyrosine kinase domain (TyrKc), and phylogenetic analysis demonstrated three SpEGFRs clustered together with invertebrate EGFR branch. Tissue specific expression analysis depicted that all SpEGFRs presented similar transcription patterns. The expression levels of SpEGFR1 and SpEGFR2s in hepatopancreas and gills were significantly altered after the stimulation of bacterial and viral pathogens including Staphylococcus aureus, Vibrio alginolyticus, White spot syndromre virus and Polycytidylinic acid. The in vivo RNA interference assays demonstrated that expression levels of SpIKK, two members of NF-κB (SpRelish and SpDorsal) and six antimicrobial peptide (AMP) genes (SpCrustin and SpALF1–5) were significantly reduced when SpEGFR1 or SpEGFR2 was silenced, respectively. The transcription patterns of SpIKK, SpRelish, SpDorsal and AMPs exhibited similar down- or up-regulation trend when the primary cultured hemocytes were treated with EGFR antagonist or agonist for 24 h. These results suggested that SpEGFR might play an important role in innate immune responses to bacterial and viral infections by regulating the NF-κB pathway. It also provided a better understanding of the origin or evolution of EGFR in crustaceans and even invertebrates.

Epidermal growth factor receptor antagonists

Epidermal growth factor receptor antagonists

Galvanotactic Migration of Glioblastoma and Brain Metastases Cells.

Galvanotaxis, the migration along direct current electrical fields, may contribute to the invasion of brain cancer cells in the tumor-surrounding tissue. We hypothesized that pharmacological perturbation of the epidermal growth factor (EGF) receptor and downstream phosphatidylinositol 3-kinase (PI3K)/AKT pathway prevent galvanotactic migration. In our study, patient-derived glioblastoma and brain metastases cells were exposed to direct current electrical field conditions. Velocity and direction of migration were estimated. To determine the effects of EGF receptor antagonist afatinib and AKT inhibitor capivasertib, assays of cell proliferation, apoptosis and immunoblot analyses were performed. Both inhibitors attenuated cell proliferation in a dose-dependent manner and induced apoptosis. We found that most of the glioblastoma cells migrated preferentially in an anodal direction, while brain metastases cells were unaffected by direct current stimulations. Afatinib presented only a mild attenuation of galvanotaxis. In contrast, capivasertib abolished the migration of glioblastoma cells without genetic alterations in the PI3K/AKT pathway, but not in cells harboring PTEN mutation. In these cells, an increase in the activation of ERK1/2 may in part substitute the inhibition of the AKT pathway. Overall, our data demonstrate that glioblastoma cells migrate in the electrical field and the PI3K/AKT pathway was found to be highly involved in galvanotaxis.

Cross-talk between EGFR and BMP signals regulates chondrocyte maturation during endochondral ossification.

Progressive maturation of growth plate chondrocytes drives long bone growth during endochondral ossification. Signals from the epidermal growth factor receptor (EGFR), and from bone morphogenetic protein-2 (BMP2), are required for normal chondrocyte maturation. Here, we investigated cross-talk between EGFR and BMP2 signals in developing and adult growth plates. Using in vivo mouse models of conditional cartilage-targeted EGFR or BMP2 loss, we show that canonical BMP signal activation is increased in the hypertrophic chondrocytes of EGFR-deficient growth plates; whereas EGFR signal activation is increased in the reserve, prehypertrophic and hypertrophic chondrocytes of BMP2-deficient growth plates. EGFR-deficient chondrocytes displayed increased BMP signal activation in vitro, accompanied by increased expression of IHH, COL10A1, and RUNX2. Hypertrophic differentiation and BMP signal activation were suppressed in normal chondrocyte cultures treated with the EGFR ligand betacellulin, effects that were partially blocked by simultaneous treatment with BMP2 or a chemical EGFR antagonist. Cross-talk between EGFR and BMP2 signals occurs during chondrocyte maturation. In the reserve and prehypertrophic zones, BMP2 signals unilaterally suppress EGFR activity; in the hypertrophic zone, EGFR and BMP2 signals repress each other. This cross-talk may play a role in regulating chondrocyte maturation in developing and adult growth plates.

Changes in Eustachian Tube Mucosa in Mice After Short-Term Tobacco and E-cigarette Smoke Exposure.

To evaluate histologic changes in middle ear and eustachian tube (ET) mucosa of mice after exposure to tobacco or electronic cigarette (e-cigarette) smoke. To determine whether there were any mitigating effects of middle ear application of anti-IL-13 or the epidermal growth factor receptor antagonist AG1478 on noted changes within ET mucosa. Controlled animal study. Fifty BALB/cJ mice were randomly assigned to one of five groups: A control group with no smoke exposure, two groups exposed to tobacco smoke, and two groups exposed to e-cigarette vapor. Within the exposed groups after 4 weeks of exposure, one ear was infiltrated with a saline hydrogel and the other ear with hydrogel of either Anti-IL-13 or AG1478. After four more weeks of exposure, the animals were euthanized and the ETs were evaluated for mucosal changes. Compared to control animals with no smoke exposure, there were significant decreases in the numbers of goblet cells within the ET mucosa of mice exposed to tobacco smoke and e-cigarette vapor. No significant differences in cilia, mucin, or squamous metaplasia were noted. Neither anti-IL-13 nor AG178 significantly altered goblet cell count in the ET mucosa of mice exposed to tobacco smoke; however, both agents significantly increased goblet cells within the ET mucosa of mice exposed to e-cigarette vapor. Short-term tobacco smoke and e-cigarette vapor significantly decrease goblet cell count in mouse ET mucosa. Middle ear application of both anti-IL-13 and AG1478 resulted in an increase in goblet cell count among mice exposed to e-cigarette vapor, but not to tobacco smoke. NA Laryngoscope, 132:648-654, 2022.

Targeting and Covalently Immobilizing the EGFR through SNAP-Tag Technology for Screening Drug Leads.

Membrane protein immobilization is particularly significant in in vitro drug screening and determining drug-receptor interactions. However, there are still some problems in the immobilization of membrane proteins with controllable direction and high conformational stability, activity, and specificity. Cell membrane chromatography (CMC) retains the complete biological structure of membrane proteins. However, conventional CMC has the limitation of poor stability, which results in its limited life span and low reproducibility. To overcome this limitation, we propose a method for the specific covalent immobilization of membrane proteins in cell membranes. We used the SNAP-tag as an immobilization tag fused to the epidermal growth factor receptor (EGFR), and Cys145 located at the active site of the SNAP-tag reacted with the benzyl group of O6-benzylguanine (BG). The SNAP-tagged EGFR was expressed in HEK293 cells. We captured the SNAP-tagged EGFR from the cell membrane suspension onto a BG-derivative-modified silica gel. Our immobilization strategy improved the life span and specificity of CMC and minimized loss of activity and nonspecific attachment of proteins. Next, a SNAP-tagged EGFR/CMC online HPLC-IT-TOF-MS system was established to screen EGFR antagonists from Epimedii folium. Icariin, magnoflorine, epimedin B, and epimedin C were retained in this model, and pharmacological assays revealed that magnoflorine could inhibit cancer cell growth by targeting the EGFR. This EGFR immobilization method may open up possibilities for the immobilization of other membrane proteins and has the potential to serve as a useful platform for screening receptor-binding leads from natural medicinal herbs.

GABRP sustains the stemness of triple-negative breast cancer cells through EGFR signaling.

Effective treatment regimens for triple-negative breast cancer (TNBC) are relatively scarce due to a lack of specific therapeutic targets. Epidermal growth factor receptor (EGFR) signaling is highly active in TNBC and is associated with poor prognosis. Most EGFR antagonists, which significantly improve outcome in lung and colon cancer, have shown limited clinical effects in breast cancer. However, limiting EGFR expression in TNBC is a potential strategy for improving the clinical efficacy of EGFR antagonists. Here, we found that the gamma-aminobutyric acid type A receptor π subunit (GABRP), as a membrane protein enriched in TNBC stem cells, interacted with EGFR and significantly sustained its expression, resulting in stemness maintenance and chemotherapy resistance. Silencing GABRP induced down-regulation of EGFR signaling, which hindered cell stemness and enhanced sensitivity to chemotherapies, including paclitaxel, doxorubicin, and cisplatin. We also identified that retigabine, an FDA-approved drug for adjunctive treatment of seizures, increased the sensitivity of EGFR to gefitinib in gefitinib-resistant cells. Our findings show that GABRP can sustain the stemness of TNBC via modulating EGFR expression, suggesting that GABRP may be a potential therapeutic target that can address EGFR inhibitor resistance in TNBC.

LL-37-mediated activation of host receptors is critical for defense against group A streptococcal infection

Group A Streptococcus (GAS) causes diverse human diseases, including life-threatening soft-tissue infections. It is accepted that the human antimicrobial peptide LL-37 protects the host by killing GAS. Here, we show that GAS extracellular protease ScpC N-terminally cleaves LL-37 into two fragments of 8 and 29 amino acids, preserving its bactericidal activity. At sub-bactericidal concentrations, the cleavage inhibits LL-37-mediated neutrophil chemotaxis, shortens neutrophil lifespan, and eliminates P2X7 and EGF receptors' activation. Mutations at the LL-37 cleavage site protect the peptide from ScpC-mediated splitting, maintaining all its functions. The mouse LL-37 ortholog CRAMP is neither cleaved by ScpC nor does it activate P2X7 or EGF receptors. Treating wild-type or CRAMP-null mice with sub-bactericidal concentrations of the non-cleavable LL-37 analogs promotes GAS clearance that is abolished by the administration of either P2X7 or EGF receptor antagonists. We demonstrate that LL-37-mediated activation of host receptors is critical for defense against GAS soft-tissue infections.

Bisphenol S and Epidermal Growth Factor Receptor Signaling in Human Placental Cytotrophoblasts.

Background:Bisphenol S (BPS) is an endocrine-disrupting chemical and the second most abundant bisphenol detected in humans. In vivo BPS exposure leads to reduced binucleate cell number in the ovine placenta. Binucleate cells form by cellular fusion, similar to the human placental syncytiotrophoblast layer. Given that human placental syncytialization can be stimulated through epidermal growth factor (EGF), we hypothesized that BPS would reduce human cytotrophoblast syncytialization through disruption of EGF receptor (EGFR) signaling.Objective:We tested whether BPS interferes EGFR signaling and disrupts human cytotrophoblast syncytialization.Methods:We first tested BPS competition for EGFR using an EGF/EGFR AlphaLISA assay. Using human primary term cytotrophoblast cells (hCTBs) and MDA-MD-231 cells, a breast cancer cell line with high EGFR expression, we evaluated EGFR downstream signaling and tested whether BPS could inhibit the EGF response by blocking EGFR activation. We also evaluated functional end points of EGFR signaling, including EGF endocytosis, cell proliferation, and syncytialization.Results:BPS blocked EGF binding in a dose-dependent manner and reduced EGF-mediated phosphorylated EGFR in both cell types. We further confirmed that BPS acted as an EGFR antagonist as shown by a reduction in EGF internalization in both hCTBs and MDA-MD-231 cells. Finally, we demonstrated that BPS interfered with EGF-mediated cell processes, such as cell proliferation in MDA-MD-231 cells and syncytialization in hCTBs. EGF-mediated, but not spontaneous, hCTB syncytialization was fully blocked by BPS (), a dose within urinary BPS concentrations detected in humans.Conclusions:Given the role of EGFR in trophoblast proliferation and differentiation during placental development, this study suggests that exposures to BPS at environmentally relevant concentrations may result in placenta dysfunction, affecting fetal growth and development. https://doi.org/10.1289/EHP7297

Epidermal growth factor receptor association with β1-adrenergic receptor is mediated via its juxtamembrane domain

β1-adrenergic receptor (β1AR)-mediated transactivation of epidermal growth factor receptor (EGFR) engages downstream signaling events that impact numerous cellular processes including growth and survival. While association of these receptors has been shown to occur basally and be important for relaying transactivation-specific intracellular events, the mechanism by which they do so is unclear and elucidation of which would aid in understanding the consequence of disrupting their interaction. Using fluorescence resonance energy transfer (FRET) and immunoprecipitation (IP) analyses, we evaluated the impact of C-terminal truncations of EGFR on its ability to associate with β1AR. While loss of the last 230 amino acid C-terminal phosphotyrosine-rich domain did not disrupt the ability of EGFR to associate with β1AR, truncation of the entire intracellular domain of EGFR resulted in almost complete loss of its interaction with β1AR, suggesting that either the kinase domain or juxtamembrane domain (JMD) may be required for this association. Treatment with the EGFR antagonist gefitinib did not prevent β1AR-EGFR association, however, treatment with a palmitoylated peptide encoding the first 20 amino acids of the JMD domain (JMD-A) disrupted β1AR-EGFR association over time and prevented β1AR-mediated ERK1/2 phosphorylation, both in general and specifically in association with EGFR. Conversely, neither a mutated JMD-A peptide nor a palmitoylated peptide fragment consisting of the subsequent 18 amino acids of the JMD domain (JMD-B) were capable of doing so. Altogether, the proximal region of the JMD of EGFR is responsible for its association with β1AR, and its disruption prevents β1AR-mediated transactivation, thus providing a new tool to study the functional consequences of disrupting β1AR-EGFR downstream signaling.