EGF Ligand Research Articles

A preliminary study on the prognostic significance of cysteine-rich EGF ligand domain 2 protein (CRELD2) in patients with triple negative breast cancer.

The cysteine-rich epidermal growth factor ligand domain 2 protein (CRELD2) is associated with pathways that regulate epithelial-to-mesenchymal transition, a critical process driving cancer metastasis. This study aimed to determine the prognostic value of CRELD2 status on survival outcomes in triple-negative breast cancer (TNBC). Seventy patients were included in the study. Thirty-four patients were metastatic, and 36 patients were non-metastatic. CRELD2 protein expression in tumor tissue was determined by immunohistochemical staining (IHC). The patients were divided into two groups: CRELD2 positive and negative groups. Clinicopathological features and survival outcomes were compared between the groups. In the survival analysis of the non-metastatic patient group, five-year overall survival (OS) rate was 91.7% in the CRELD2-positive patient group and 91% in the negative group (P = 0.91). Median progression free survival (PFS) was 9.4 (95% confidence interval [CI]: 6.4-12.4) months in the CRELD2-positive group and 11.9 (95% CI: 8.2-18.6) months in the CRELD2-negative group (P = 0.04). The median OS was 17.2 (95% CI: 13.7-22.3) months in the CRELD2-positive group and 24.7 (95% CI: 21.8-29.6) months in the CRELD2-negative group (P = 0.02). In multivariate analysis, CRELD2 status (negative vs positive) (hazard ratio [HR]: 0.50, 95% CI: 0.38-0.96, P = 0.02) was determined to be a risk factor for OS and CRELD2 status (negative vs positive) (HR: 0.82, 95% CI: 0.33-0.96, P = 0.01) was defined as a risk factor for PFS in patients with metastatic TNBC. This is the first clinical study to determine the effect of CRELD2 on survival and as a prognostic marker in patients with triple metastatic breast cancer. These results need to be validated prospectively with a large sample size.

P0036 NRG1, a gene significantly detected from intestinal mucosa eccDNA, may predict therapeutic response in Inflammatory Bowel Disease

Abstract Background Many patients with Inflammatory Bowel Diseases do not respond to biological therapies. Extrachromosomal circular DNA(eccDNA) has been showed to play a role in the pathophysiology of IBD, as there is a higher presence of eccDNA in the intestinal mucosa of these patients. By characterizing the gene content of eccDNA, up-regulation of certain genes is observed; one of these is NRG1 (Neuregulin 1), an EGF ligand that guides tissue repair after injury and is associated with tissue plasticity. This gene is located on chromosome 8p12 and it is expressed in macrophages, Paneth cells, and stromal mesenchymal cells of the stem cell niche. In the context of IBD, where chronic inflammation is a hallmark, NRG1 could potentially modulate the immune response to reduce inflammation and promote healing. It is important to understand whether its expression within eccDNA may correlate with a particular course of the disease. Methods eccDNA was extracted from intestinal mucosa samples derived from patients diagnosed with IBD and amplified using the Circle-Seq analysis. Following sequencing, bioinformatic interpretation of the data was performed, through which a list of gene fragments present on the circles of individual patients and their localization was obtained. Finally, the presence of the gene found up-regulated by the bioinformatic analysis was confirmed using Real Time PCR. Results Starting from 99 intestinal mucosa samples from IBD patients, whose eccDNA was sequenced, circles containing fragments of NRG1 were found in 22 of them; its presence was compared to samples of diseased mucosa biopsies versus healthy mucosa from the same IBD patient. The number of circles from NRG1 is greater in diseased mucosa samples compared to healthy ones, in both UC and CD patients. Furthermore stratifying patients by response to therapy, about 70% of responders to IBD-therapy were NRG1- , with Odds Ratio <1. Conclusion Preliminary results show that NRG1, a gene significantly detected from intestinal mucosa eccDNA, may predict therapeutic response in Inflammatory Bowel Disease. Further analysis are needed to confirm these data.

Presence of EGF ligand restricts the binding ability of EgB4 nanobody to EGFR extracellular domain

EgB4 is a nanobody that could facilitate the development of drug–nanobody conjugates or drug delivery in cancer treatment due to its specific binding ability to the EGFR transmembrane protein. More significantly, EgB4 does not hamper the EGFR function and associates with EGFR in both the presence and absence of an EGF ligand. However, the difference in EgB4–EGFR interaction with and without EGF ligand is not clear. To explore this aspect, we carried out computations including classical molecular dynamics simulations, steered molecular dynamics simulations and the umbrella sampling method. The three main objectives of the study are: (i) how EgB4 interacts with EGFR, (ii) the role of an EGF molecule, and (iii) how to mutate EgB4 to efficiently construct a stronger nanobody. Our computed results showed that, in addition to the residues asp98 and asp110 that were previously reported, two other residues, arg105 and asp108, are also highly interactive with the EGFR extracellular domain. Notably, the absolute binding free energy of the EgB4–EGFR complex decreases from − 17.1 to -13.3 kcal/mol in going from an absence to a presence of EGF. With the EGF presence, the interacting space between both EgB4 and EGFR entities is also reduced. When EGF stabilizes the dimerization of EGFR, there is less opportunity for EgB4 to bind. This crucial aspect has not been reported before.

H Antigen expression modulates epidermal Keratinocyte Integrity and differentiation

BackgroundABO blood group antigens (ABH antigens) are carbohydrate chains glycosylated on epithelial and red blood cells. Recent findings suggest reduced ABH expression in psoriasis and atopic dermatitis, a chronic inflammatory skin disease with retained scale. H antigen, a precursor for A and B antigens, is synthesized by fucosyltransferase 1 (FUT1). Desmosomes, critical for skin integrity, are known to require N-glycosylation for stability. We investigate the impact of H antigens, a specific type of glycosylation, on desmosomes in keratinocytes.MethodPrimary human keratinocytes were transfected with FUT1 siRNA or recombinant adenovirus for FUT1 overexpression. Cell adhesion and desmosome characteristics and their underlying mechanisms were analyzed.ResultThe knockdown of FUT1, responsible for H2 antigen expression in the skin, increased cell-cell adhesive strength and desmosome size in primary cultured keratinocytes without altering the overall desmosome structure. Desmosomal proteins, including desmogleins or plakophilin, were upregulated, suggesting enhanced desmosome assembly. Reduced H2 antigen expression via FUT1 knockdown led to increased keratinocyte differentiation, evidenced by elevated expression of differentiation markers. Epidermal growth factor receptor (EGFR) has been described to be associated with FUT1 and promotes cell migration and differentiation. The effects of FUT1 knockdown were recapitulated by an EGFR inhibitor concerning desmosomal proteins and cellular differentiation. Further investigation demonstrated that the FUT1 knockdown reduced EGFR signaling by lowering the levels of EGF ligands rather than directly regulating EGFR activity. Moreover, FUT1 overexpression reversed the effects observed in FUT1 knockdown, resulting in the downregulation of desmosomal proteins and differentiation markers while increasing both mRNA and protein levels of EGFR ligands.ConclusionThe expression level of FUT1 in the epidermis appears to influence cell-cell adhesion and keratinocyte differentiation status, at least partly through regulation of H2 antigen and EGFR ligand expression. These observations imply that the fucosylation of the H2 antigen by FUT1 could play a significant role in maintaining the molecular composition and regulation of desmosomes and suggest a possible involvement of the altered H2 antigen expression in skin diseases, such as psoriasis and atopic dermatitis.

Targeting Cardiomyocytes with Adeno Associated Viruses to Protect Hearts from Trastuzumab-induced Cardiotoxicity

Amplification of HER2 occurs in 20% of breast cancers. HER2 (known as ErbB2 in mouse) belongs to the Epidermal Growth Factor Receptor (EGFR) family that is responsible for cell growth and survival. While HER2 does not bind ligands, it forms heterodimers with HER1 (also known as EGFR) in the presence of its ligand EGF, and HER4 following stimulation with Neuregulin. In HER2-positive breast cancer where HER2 expression is high, HER2 homodimerizes leading to excessive activation of downstream signalling and cell proliferation. Current HER2-positive breast cancer treatment involves chemotherapy with drugs called anthracyclines, such as Doxorubicin, and an antibody targeting HER2 called Trastuzumab. This combination is effective in killing HER2-positive breast tumours, however, Trastuzumab alone or in combination with anthracyclines leads to severe cardiotoxicity and heart failure in 2.6-11% of cases, making it a dose-limiting side effect. We hypothesise that cardiac toxicity can be mitigated by targeting cardiomyocytes exclusively to express a mutated (but functional) version of HER2 that is not recognised by trastuzumab. We designed 3 HER2 variants wherein the trastuzumab binding domain was mutated to disrupt binding. These HER2 mutants were transfected into HEK293T cells, and we confirmed that all 3 successfully localised to the cell membrane, and did not bind trastuzumab. We have used bioluminescence resonance energy transfer to examine the capacity of all 3 mutants to activate recruitment of Grb2 to the HER2 as part of HER1/HER2 and HER4/HER2 heterodimers; Western blotting also confirming mutants were capable of stimulating ERK and Akt phosphorylation. Future studies will evaluate the effcacy of using AAVs to instruct cardiomyocytes to express these HER2 mutants in a murine model of breast cancer. We predict that this new therapy would not only mitigate cardiac toxicity, but would also permit higher doses of trastuzumab to be used to treat breast cancer. This work is supported by Australia's National Health and Medical Research Council and the Australian Research Council. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Hypoxia delays steroid-induced developmental maturation in Drosophila by suppressing EGF signaling.

Animals often grow and develop in unpredictable environments where factors like food availability, temperature, and oxygen levels can fluctuate dramatically. To ensure proper sexual maturation into adulthood, juvenile animals need to adapt their growth and developmental rates to these fluctuating environmental conditions. Failure to do so can result in impaired maturation and incorrect body size. Here we describe a mechanism by which Drosophila larvae adapt their development in low oxygen (hypoxia). During normal development, larvae grow and increase in mass until they reach critical weight (CW), after which point a neuroendocrine circuit triggers the production of the steroid hormone ecdysone from the prothoracic gland (PG), which promotes maturation to the pupal stage. However, when raised in hypoxia (5% oxygen), larvae slow their growth and delay their maturation to the pupal stage. We find that, although hypoxia delays the attainment of CW, the maturation delay occurs mainly because of hypoxia acting late in development to suppress ecdysone production. This suppression operates through a distinct mechanism from nutrient deprivation, occurs independently of HIF-1 alpha and does not involve dilp8 or modulation of Ptth, the main neuropeptide that initiates ecdysone production in the PG. Instead, we find that hypoxia lowers the expression of the EGF ligand, spitz, and that the delay in maturation occurs due to reduced EGFR/ERK signaling in the PG. Our study sheds light on how animals can adjust their development rate in response to changing oxygen levels in their environment. Given that hypoxia is a feature of both normal physiology and many diseases, our findings have important implications for understanding how low oxygen levels may impact animal development in both normal and pathological situations.

Abstract 4365: Mechanistic effects of N361 glycosylation in epidermal growth factor receptor protein

Abstract Glycosylation is a tightly regulated post-translational modification where structured sugar groups are added to protein amino acid backbones, most commonly on asparagine for N-glycosylation. The glycosylation-regulating metabolic enzyme uridine diphosphate-glucose pyrophosphorylase 2 (UGP2) is required in oncogene-transformed cancer cells, but not in normal cells. UGP2 heavily regulates glycosylation of epidermal growth factor receptor (EGFR) at asparagine 361 (N361), a key site near the ligand binding domain. EGFR is a transmembrane tyrosine kinase receptor that promotes cell proliferation via growth cascades. Mutations and amplifications in EGFR account for a significant fraction of non-small cell lung carcinomas and breast adenocarcinoma, with L858R being the most common oncogenic hotspot. Other glycosylation sites on EGFR have been implicated in ligand binding, dimerization, and phosphorylation of downstream targets. Molecular dynamic simulations suggested that N361 may be important for dimerization or ligand binding. However, the functional relevance of how glycosylation of EGFR at N361 impacts EGFR protein and cellular behaviors remains unclear. To determine the functional relevance of glycosylation at N361, we created glycosylation-defective EGFR N361A mutant, with or without an additional oncogenic EGFR L858R mutant. We expressed these constructs in MCF10A and 293T cells, which do not have pre-existing activation of this pathway. Immunofluorescence and flow cytometry showed that the mutants were each well expressed at the cell membrane. Proximity ligation assays measuring dimerization of EGFR in cells showed that EGFR N361A greatly increased dimerization relative to wildtype controls. Loss of glycosylation of EGFR N361 impacted cell viability when grown in dose courses of its high affinity ligand EGF or lower affinity ligand amphiregulin. Furthermore, EGFR N361A desensitized cells expressing the oncogenic EGFR L858R to inhibitors targeting the extracellular domain, possibly because the mutation alters the antibody binding interface. N361A sensitized EGFR L858R to inhibitors targeting the cytoplasmic domain of EGFR. These findings help us understand the intricate relationship between EGFR and N-glycosylation, reinforcing the critical functional relevance of post-translational modifications on oncogenes. Citation Format: Dennis Lam, Ariel N. Liberchuk, Brandon Arroyo, Andrew L. Wolfe. Mechanistic effects of N361 glycosylation in epidermal growth factor receptor protein [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4365.

EGFR signaling and pharmacology in oncology revealed with innovative BRET-based biosensors

Mutations of receptor tyrosine kinases (RTKs) are associated with the development of many cancers by modifying receptor signaling and contributing to drug resistance in clinical settings. We present enhanced bystander bioluminescence resonance energy transfer-based biosensors providing new insights into RTK biology and pharmacology critical for the development of more effective RTK-targeting drugs. Distinct SH2-specific effector biosensors allow for real-time and spatiotemporal monitoring of signal transduction pathways engaged upon RTK activation. Using EGFR as a model, we demonstrate the capacity of these biosensors to differentiate unique signaling signatures, with EGF and Epiregulin ligands displaying differences in efficacy, potency, and responses within different cellular compartments. We further demonstrate that EGFR single point mutations found in Glioblastoma or non-small cell lung cancer, impact the constitutive activity of EGFR and response to tyrosine kinase inhibitor. The BRET-based biosensors are compatible with microscopy, and more importantly characterize the next generation of therapeutics directed against RTKs.

Proximity extracellular protein-protein interaction analysis of EGFR using AirID-conjugated fragment of antigen binding

Receptor proteins, such as epidermal growth factor receptor (EGFR), interact with other proteins in the extracellular region of the cell membrane to drive intracellular signalling. Therefore, analysis of extracellular protein-protein interactions (exPPIs) is important for understanding the biological function of receptor proteins. Here, we present an approach using a proximity biotinylation enzyme (AirID) fusion fragment of antigen binding (FabID) to analyse the proximity exPPIs of EGFR. AirID was C-terminally fused to the Fab fragment against EGFR (EGFR-FabID), which could then biotinylate the extracellular region of EGFR in several cell lines. Liquid Chromatography-Mass Spectrometry (LC-MS/MS) analysis indicated that many known EGFR interactors were identified as proximity exPPIs, along with many unknown candidate interactors, using EGFR-FabID. Interestingly, these proximity exPPIs were influenced by treatment with EGF ligand and its specific kinase inhibitor, gefitinib. These results indicate that FabID provides accurate proximity exPPI analysis of target receptor proteins on cell membranes with ligand and drug responses.

Piezo1 activates noncanonical EGFR endocytosis and signaling.

EGFR-ERK signaling controls cell cycle progression during development, homeostasis, and disease. While EGF ligand and mechanical inputs can activate EGFR-ERK signaling, the molecules linking mechanical force to this axis have remained mysterious. We previously found that stretch promotes mitosis via the stretch-activated ion channel Piezo1 and ERK signaling. Here, we show that Piezo1 provides the missing link between mechanical signals and EGFR-ERK activation. While both EGF- and Piezo1-dependent activation trigger clathrin-mediated EGFR endocytosis and ERK activation, EGF relies on canonical tyrosine autophosphorylation, whereas Piezo1 involves Src-p38 kinase-dependent serine phosphorylation. In addition, unlike EGF, ex vivo lung slices treated with Piezo1 agonist promoted cell cycle re-entry via nuclear ERK, AP-1 (FOS and JUN), and YAP accumulation, typical of regenerative and malignant signaling. Our results suggest that mechanical activation via Piezo1, Src, and p38 may be more relevant to controlling repair, regeneration, and cancer growth than tyrosine kinase signaling via canonical EGF signaling, suggesting an alternative therapeutic approach.

An EGCG Derivative in Combination with Nimotuzumab for the Treatment of Wild-Type EGFR NSCLC.

Inhibiting the tyrosine kinase activity of epidermal growth factor receptor (EGFR) using small-molecule tyrosine kinase inhibitors (TKIs) or monoclonal antibodies is often ineffective in treating cancers harboring wild-type EGFR. Given the fact that EGFR possesses a kinase-independent pro-survival function, more effective inhibition of EGFR-mediated signals is therefore necessary. In this study, we investigated the effects of using a combination of low-dose nimotuzumab and theasinensin A to evaluate whether the inhibitory effect of nimotuzumab on NCI-H441 cancer cells was enhanced. Here, theasinensin A, a novel epigallocatechin-3-gallate (EGCG) derivative, was identified and its potent anticancer activity against wild-type EGFR NSCLC was demonstrated in vitro; the anticancer activity was induced through degradation of EGFR. Mechanistic studies further revealed that theasinensin A bound directly to the EGFR extracellular domain, which decreased interaction with its ligand EGF in combination with nimotuzumab. Theasinensin A significantly promoted EGFR degradation and repressed downstream survival pathways in combination with nimotuzumab. Meanwhile, treatment with theasinensin A and nimotuzumab prevented xenograft growth, whereas the single agents had limited effect. Thus, the combination therapy of theasinensin A with nimotuzumab is a powerful candidate for treatment of wild-type EGFR cancers.

The emerging roles of deep crypt secretory cells in colonic physiology.

Deep crypt secretory (DCS) cells are a population of epithelial cells located at the colonic crypt base that share some similarities to Paneth and goblet cells. They were initially defined as c-Kit expressing cells, though subsequent work showed that they are more specifically marked by Reg4 in the murine colon. The best-understood function of DCS cells at present is supporting the stem cell niche by generating Notch and EGF ligands. However, as these cells also express immunoregulatory (e.g., Ccl6) and host defense (e.g., Retnlb) genes, it is likely they have additional functions in maintaining colonic health outside of maintenance of the stem niche. Recent advances in single-cell transcriptomic profiling hint at additional epithelial and immune roles that may exist for these cells and have aided in elucidating their developmental lineage. This review highlights the emerging evidence supporting a crucial role for DCS cells in intestinal physiology, the current understanding of how these cells are regulated, and their potential role(s) in colonic disease.

Budding uninhibited by benzimidazoles-1 (BUB1) regulates EGFR signaling by reducing EGFR internalization.

EGFR signaling initiates upon ligand binding which leads to activation and internalization of the receptor-ligand complex. Here, we evaluated if BUB1 impacted EGFR signaling by regulating EGFR receptor internalization and activation. BUB1 was ablated genomically (siRNA) or biochemically (2OH-BNPP1) in cells. EGF ligand was used to initiate EGFR signaling while disuccinimidyl suberate (DSS) was used for cross linking cellular proteins. EGFR signaling was measured by western immunoblotting and receptor internalization was evaluated by fluorescent microscopy (pEGFR (pY1068) colocalization with early endosome marker EEA1). siRNA mediated BUB1 depletion led to an overall increase in total EGFR levels and more phospho-EGFR (Y845, Y1092, and Y1173) dimers while the amount of total EGFR (non-phospho) dimers remained unchanged. BUB1 inhibitor (BUB1i) decreased EGF mediated EGFR signaling including pEGFR Y845, pAKT S473 and pERK1/2 in a time dependent manner. Additionally, BUB1i also reduced EGF mediated pEGFR (Y845) dimers (asymmetric dimers) without affecting total EGFR dimers (symmetric dimers) indicating that dimerization of inactive EGFR is not affected by BUB1. Furthermore, BUB1i blocked EGF mediated EGFR degradation (increase in EGFR half-life) without impacting half-lives of HER2 or c-MET. BUB1i also reduced co-localization of pEGFR with EEA1 positive endosomes suggesting that BUB1 might modulate EGFR endocytosis. Our data provide evidence that BUB1 protein and its kinase activity may regulate EGFR activation, endocytosis, degradation, and downstream signaling without affecting other members of the receptor tyrosine kinase family.

Characterization of a novel recombinant calcium-binding protein from Arca subcrenata and its anti-hepatoma activities in vitro and in vivo

Previous studies demonstrated that ASP-3 was a novel calcium-binding protein from Arca subcrenata that effectively inhibited the proliferation of HepG2 cells. To further study the antitumor activity and mechanism of ASP-3, the cytotoxic effects of recombinant ASP-3 were evaluated in HepG2 cells. The results demonstrated that ASP-3 inhibited the proliferation of HepG2 cells by competitively binding to the EGF binding pocket of EGFR and inhibiting the JAK-STAT, RAS-RAF-MEK-ERK, and PI3K-Akt-mTOR signaling pathways mediated by EGFR. ASP-3 significantly inhibited tumor growth in a HepG2 cell subcutaneous xenograft nude mouse model, and its (25 mg/kg and 75 mg/kg) tumor inhibition rates were 46.92 % and 60.28 %, respectively. Furthermore, the crystal structure of ASP-3 was resolved at 1.4 Å. ASP-3 formed as a stable dimer and folded as an EF-Hand structure. ASP-3 stably bound to domain I and domain III of the EGFR extracellular region by using molecular docking and molecular dynamics simulation analysis. Compared with the endogenous ligand EGF, ASP-3 displayed a stronger interaction with EGFR. These experimental results indicated that recombinant ASP-3 possessed an effective anti-hepatoma effect. So, it might be a potential molecule for liver cancer therapy.

Biased activation of the receptor tyrosine kinase HER2

HER2 belongs to the ErbB sub-family of receptor tyrosine kinases and regulates cellular proliferation and growth. Different from other ErbB receptors, HER2 has no known ligand. Activation occurs through heterodimerization with other ErbB receptors and their cognate ligands. This suggests several possible activation paths of HER2 with ligand-specific, differential response, which has so far remained unexplored. Using single-molecule tracking and the diffusion profile of HER2 as a proxy for activity, we measured the activation strength and temporal profile in live cells. We found that HER2 is strongly activated by EGFR-targeting ligands EGF and TGFα, yet with a distinguishable temporal fingerprint. The HER4-targeting ligands EREG and NRGβ1 showed weaker activation of HER2, a preference for EREG, and a delayed response to NRGβ1. Our results indicate a selective ligand response of HER2 that may serve as a regulatory element. Our experimental approach is easily transferable to other membrane receptors targeted by multiple ligands.Graphical abstract

Abstract 6135: Dynamics of ligand-induced epidermal growth factor receptor internalization in cancer cell lines

Abstract Antibody-drug conjugates (ADCs), comprised of cancer cell-specific antibodies linked to cytotoxic drugs, represent an innovative and promising class of anticancer agents. Due to its amplified expression in various tumor types, the epidermal growth factor receptor (EGFR) presents an attractive target for ADC therapy, and three anti-EGFR ADCs are currently studied in clinical trials. As ADCs depend on receptor internalization for intracellular release of drugs in the targeted cancer cells, studying the dynamics of receptor internalization can aid in their development and optimization. We studied the internalization of EGFR induced by its ligand EGF, as a mimic of ADCs, in cell lines with different surface receptor densities. BxPC-3 (pancreas) and RT-112 (bladder) cancer cells were seeded in 6-well plates and allowed to adhere for 24 hours. Cells were serum-starved overnight, followed by stimulation with 150 ng/mL EGF for 2, 4, 6, 24 and 48 hours. Internalization was studied by quantification of the number of surface-expressed receptors using flow cytometry with Quantibrite PE beads and EGFR-PE antibody. Moreover, incorporation of the ligand-receptor complex into acidic endo-lysosomes was studied using EGF conjugated to the fluorescent pH sensor pHrodo™ Green. Total EGFR and EGFR phosphorylation levels were assessed by western blot analysis. RNA was isolated and converted into cDNA to perform TaqMan qPCR. Unstimulated BxPC-3 cells showed higher numbers of EGFR per cell compared to unstimulated RT-112 cells. Since high receptor expression is key for some ADCs to induce effective endocytosis, this may indicate BxPC-3 cells as a more attractive model to study EGFR internalization. Stimulation with EGF induced a strong decrease in surface receptor density for both BxPC-3 and RT-112 cells during the first two hours, suggesting receptor internalization. Accordingly, treatment of BxPC-3 cells with pHrodo™ Green EGF demonstrated the incorporation of ligand-receptor complexes into endo-lysosomes. During following hours, the cell surface expression of EGFR remained stable, whereas it partially recovered after 24 to 48 hours. In contrast, the lysosomal incorporation of EGF-EGFR complexes was not decreased at 24 or 48 hours, suggesting that the increase in surface EGFR may be due to de novo protein synthesis rather than recycling. No increase of EGFR mRNA was, however, detected. Finally, increased phosphorylation of EGFR was observed after two to six hours of stimulation, whereas total EGFR levels were highest in unstimulated and 24- or 48-hour-stimulated cells, which is in concordance with the flow cytometry data. In conclusion, both BxPC-3 and RT-112 cells show dynamic changes in EGFR internalization upon EGF stimulation, despite differences in EGFR expression levels. These cell lines can therefore successfully be used for internalization studies, which may contribute to future ADC development. Citation Format: Mandy W. Smeets, Eef F. Smits, Janneke J. Melis, Dimitri Pappaioannou, Guido J. Zaman. Dynamics of ligand-induced epidermal growth factor receptor internalization in cancer cell lines. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6135.

First person – Toni Lemmetyinen

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Disease Models & Mechanisms, helping researchers promote themselves alongside their papers. Toni Lemmetyinen is first author on ‘ Fibroblast-derived EGF ligand neuregulin 1 induces fetal-like reprogramming of the intestinal epithelium without supporting tumorigenic growth’, published in DMM. Toni is a PhD student in the lab of Saara Ollila at the University of Helsinki, investigating how stromal fibroblasts regulate the intestinal epithelium.

Buffered EGFR signaling regulated by spitz-to-argos expression ratio is a critical factor for patterning the Drosophila eye.

The Epidermal Growth Factor Receptor (EGFR) signaling pathway plays a critical role in regulating tissue patterning. Drosophila EGFR signaling achieves specificity through multiple ligands and feedback loops to finetune signaling outcomes spatiotemporally. The principal Drosophila EGF ligand, cleaved Spitz, and the negative feedback regulator, Argos are diffusible and can act both in a cell autonomous and non-autonomous manner. The expression dose of Spitz and Argos early in photoreceptor cell fate determination has been shown to be critical in patterning the Drosophila eye, but the exact identity of the cells expressing these genes in the larval eye disc has been elusive. Using single molecule RNA Fluorescence in situ Hybridization (smFISH), we reveal an intriguing differential expression of spitz and argos mRNA in the Drosophila third instar eye imaginal disc indicative of directional non-autonomous EGFR signaling. By genetically tuning EGFR signaling, we show that rather than absolute levels of expression, the ratio of expression of spitz-to-argos to be a critical determinant of the final adult eye phenotype. Proximate effects on EGFR signaling in terms of cell cycle and differentiation markers are affected differently in the different perturbations. Proper ommatidial patterning is robust to thresholds around a tightly maintained wildtype spitz-to-argos ratio, and breaks down beyond. This provides a powerful instance of developmental buffering against gene expression fluctuations.

Egfr signaling promotes juvenile hormone biosynthesis in the German cockroach

BackgroundIn insects, an interplay between the activities of distinct hormones, such as juvenile hormone (JH) and 20-hydroxyecdysone (20E), regulates the progression through numerous life history hallmarks. As a crucial endocrine factor, JH is mainly synthesized in the corpora allata (CA) to regulate multiple physiological and developmental processes, including molting, metamorphosis, and reproduction. During the last century, significant progress has been achieved in elucidating the JH signal transduction pathway, while less progress has been made in dissecting the regulatory mechanism of JH biosynthesis. Previous work has shown that receptor tyrosine kinase (RTK) signaling regulates hormone biosynthesis in both insects and mammals. Here, we performed a systematic RNA interference (RNAi) screening to identify RTKs involved in regulating JH biosynthesis in the CA of adult Blattella germanica females.ResultsWe found that the epidermal growth factor receptor (Egfr) is required for promoting JH biosynthesis in the CA of adult females. The Egf ligands Vein and Spitz activate Egfr, followed by Ras/Raf/ERK signaling, and finally activation of the downstream transcription factor Pointed (Pnt). Importantly, Pnt induces the transcriptional expression of two key enzyme-encoding genes in the JH biosynthesis pathway: juvenile hormone acid methyltransferase (JHAMT) and methyl farnesoate epoxidase (CYP15A1). Dual-luciferase reporter assay shows that Pnt is able to activate a promoter region of Jhamt. In addition, electrophoretic mobility shift assay confirms that Pnt directly binds to the − 941~ − 886 nt region of the Jhamt promoter.ConclusionsThis study reveals the detailed molecular mechanism of Egfr signaling in promoting JH biosynthesis in the German cockroach, shedding light on the intricate regulation of JH biosynthesis during insect development.

Fluorescently tagged nanobodies and NanoBRET to study ligand-binding and agonist-induced conformational changes of full-length EGFR expressed in living cells.

The Epidermal Growth Factor Receptor is a member of the Erb receptor tyrosine kinase family. It binds several ligands including EGF, betacellulin (BTC) and TGF-α, controls cellular proliferation and invasion and is overexpressed in various cancer types. Nanobodies (VHHs) are the antigen binding fragments of heavy chain only camelid antibodies. In this paper we used NanoBRET to compare the binding characteristics of fluorescent EGF or two distinct fluorescently labelled EGFR directed nanobodies (Q44c and Q86c) to full length EGFR. Living HEK293T cells were stably transfected with N terminal NLuc tagged EGFR. NanoBRET saturation, displacement or kinetics experiments were then performed using fluorescently labelled EGF ligands (EGF-AF488 or EGF-AF647) or fluorescently labelled EGFR targeting nanobodies (Q44c-HL488 and Q86c-HL488). These data revealed that the EGFR nanobody Q44c was able to inhibit EGF binding to full length EGFR, while Q86c was able to recognise agonist bound EGFR and act as a conformational sensor. The specific binding of fluorescent Q44c-HL488 and EGF-AF488 was inhibited by a range of EGFR ligands (EGF> BTC>TGF-α). EGFR targeting nanobodies are powerful tools for studying the role of the EGFR in health and disease and allow real time quantification of ligand binding and distinct ligand induced conformational changes.