Epidermal Growth Factor Receptor Binding Research Articles

PDZK1 suppresses TNBC development and sensitizes TNBC cells to erlotinib via the EGFR pathway

Epidermal growth factor receptor (EGFR)-targeted drugs (erlotinib, etc.) are used to treat multiple types of tumours. EGFR is highly expressed in most triple-negative breast cancer (TNBC) patients. However, only a small proportion of TNBC patients benefit from EGFR-targeted drugs in clinical trials, and the resistance mechanism is unclear. Here, we found that PDZ domain containing 1 (PDZK1) is downregulated in erlotinib-resistant TNBC cells, suggesting that PDZK1 downregulation is related to erlotinib resistance in TNBC. PDZK1 binds to EGFR. Through this interaction, PDZK1 promotes EGFR degradation by enhancing the binding of EGFR to c-Cbl and inhibits EGFR phosphorylation by hindering EGFR dimerisation. We also found that PDZK1 is specifically downregulated in TNBC tissues and correlated with a poor prognosis in TNBC patients. In vitro and in vivo functional assays showed that PDZK1 suppressed TNBC development. Restoration of EGFR expression or kinase inhibitor treatment reversed the degree of cell malignancy induced by PDZK1 overexpression or knockdown, respectively. PDZK1 overexpression sensitised TNBC cells to erlotinib both in vitro and in vivo. In conclusion, PDZK1 is a significant prognostic factor for TNBC and a potential molecular therapeutic target for reversing erlotinib resistance in TNBC cells.

Synthesis and biological evaluation of EGFR binding peptides for near-infrared photoimmunotherapy

Near-infrared photoimmunotherapy (NIR-PIT) is a new cancer treatment that involves photoimmunotherapy drug injection and NIR light exposure. In NIR-PIT, antibodies are commonly used as target-directed molecules carrying IRDye700DX (IR700). However, antibodies have disadvantages, such as high cost, complex development strategies, and poor tumor penetration. In contrast, peptides have lower production costs, can be easy to chemically synthesize and modify, and can also be used for tumor-targeting like antibodies. In this study, we developed a novel PIT drug using a peptide as the target-directed molecule. Epidermal growth factor receptor (EGFR) was selected as the target, and monovalent and bivalent EGFR-binding peptides were synthesized. The bivalent peptide showed sufficient binding to EGFR-positive cells, and a bivalent peptide-IR700 conjugate with a long linker induced morphological changes in EGFR-positive cells. Additionally, the drug significantly reduced cell viability in vitro in an NIR light-dose- and drug-concentration-dependent manner. These results indicate the feasibility of NIR-PIT in treating cancer using peptide-based drugs.

Association between skin toxicity and efficacy of necitumumab in squamous non-small-cell lung cancer: a pooled analysis of two randomized clinical trials-SQUIRE and JFCM

Efficacy of necitumumab [recombinant human monoclonal antibody that blocks the ligand binding epidermal growth factor receptor (EGFR)] in patients with squamous (SQ) non-small-cell lung cancer (NSCLC) has been confirmed in two randomized clinical trials (SQUIRE and JFCM). This study evaluated the association between efficacy and initial skin toxicity with necitumumab treatment by analyzing pooled data from two clinical trials (SQUIRE and JFCM). Data of 635 patients with SQ-NSCLC (intent-to-treat population) treated with necitumumab plus gemcitabine and cisplatin (N+ GC) were pooled from two clinical trials (SQUIRE and JFCM). The relationship between skin toxicities developed by the end of the second cycle and efficacy was evaluated. Efficacy endpoints included overall survival (OS), progression-free survival (PFS), and objective response rate (ORR). Univariate and multivariate analyses were carried out for these endpoints. OS and ORR were associated with skin toxicity, whereas PFS was not. Patients with grade ≥2 or grade 1 skin toxicity had significantly longer OS compared to patients without skin toxicity (grade 0) in the N+ GC group [median= 15.0 (grade ≥2); 12.7 (grade 1); 9.4 (grade 0) months; hazard ratio (HR)= 0.51 (grade ≥2 to grade 0); 95% confidence interval (CI) 0.40-0.64, P < 0.001 and HR= 0.64 (grade 1 to grade 0); 95% CI 0.52-0.80, P < 0.001]. In multivariate analysis, OS was significantly associated with skin toxicity. A significant association was found between necitumumab-induced skin toxicity and efficacy. These results are consistent with the previously reported association between other EGFR inhibitors-induced skin toxicity and efficacy.

Abstract 549: PDZK1 sensitizes TNBC cells to erlotinib via promoting c-Cbl-mediated EGFR degradation and inhibiting EGFR phosphorylation

Abstract Background: Triple-negative breast cancer (TNBC) is lack of effective targeted therapies. Epidermal growth factor receptor (EGFR) is a potential therapeutic target for TNBC. However, TNBC is resistant to EGFR-targeted drugs in clinical trials, and the tolerance mechanism remains unclear. This study aimed to elucidate the possible resistance mechanism for TNBC to EGFR-targeted therapy and how sensitize TNBC cells to erlotinib. Methods: PDZ domain containing 1 (PDZK1) correlated with TNBC development was screened out by bioinformatics analysis and its correlation with resistance to erlotinib was identified by western blotting (WB). Its interaction with EGFR and structural basis were determined by GST-Pull down and coimmunoprecipitation. Its regulation on EGFR signaling activation and expression level were detected by WB in PDZK1 overexpression and knockdown TNBC cells, xenograft tissues and correlation analyses. Correlation was performed by gene set enrichment analysis (GSEA) and immunohistochemistry of tissue microarray followed by Pearson analysis. TNBC-suppressing effects of PDZK1 and its sensitizing effects on erlotinib were investigated using cell viability assay, colony-forming assay, wound healing assay, Matrigel invasion assay and xenograft model in combination with PDZK1 wild type/mutant transfection and rescue experiment. Results: Here we found that PDZK1 was correlated with TNBC development and its level was downregulated in erlotinib-resistant TNBC cells, suggesting that PDZK1 downregulation was related to erlotinib resistance in TNBC. PDZK1 bound with EGFR. Through the interaction, PDZK1 promoted EGFR degradation by enhancing the binding of EGFR with c-Cbl and inhibited EGFR phosphorylation by hindering EGFR dimerization. PDZK1 was specifically downregulated in TNBC tissues and was correlated with poor prognosis of TNBC. Functional assays in vitro and in vivo showed that PDZK1 suppressed TNBC development. Restoring EGFR expression and kinase inhibitor treatment reversed the malignancy caused by PDZK1 overexpression and knockdown, respectively. PDZK1 wild type, but not mutant overexpression enhanced the inhibitory effect of erlotinib on EGFR signaling and TNBC malignancy in vitro and in vivo. Conclusion: PDZK1 is a significant prognostic factor for TNBC and a potential molecular therapeutic target for TNBC to reverse the tolerance of TNBC cells to erlotinib. Citation Format: Junfang Zheng, Yuanzhen Ma, Zhiyu Fang, Yijun Qi. PDZK1 sensitizes TNBC cells to erlotinib via promoting c-Cbl-mediated EGFR degradation and inhibiting EGFR phosphorylation [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 549.

Chemical mixture that targets the epidermal growth factor pathway impairs human trophoblast cell functions

Pregnant women are exposed to complex chemical mixtures, many of which reach the placenta. Some of these chemicals interfere with epidermal growth factor receptor (EGFR) activation, a receptor tyrosine kinase that modulates several placenta cell functions. We hypothesized that a mixture of chemicals (Chem-Mix) known to reduce EGFR activation (polychlorinated biphenyl (PCB)-126, PCB-153, atrazine, trans-nonachlor, niclosamide, and bisphenol S) would interfere with EGFR-mediated trophoblast cell functions. To test this, we determined the chemicals' EGFR binding ability, EGFR and downstream effectors activation, and trophoblast functions (proliferation, invasion, and endovascular differentiation) known to be regulated by EGFR in extravillous trophoblasts (EVTs). The Chem-Mix competed with EGF for EGFR binding, however only PCB-153, niclosamide, trans-nonachlor, and BPS competed for binding as single chemicals. The effects of the Chem-Mix on EGFR phosphorylation were tested by exposing the placental EVT cell line, HTR-8/SVneo to control (0.1% DMSO), Chem-Mix (1, 10, or 100 ng/ml), EGF (30 ng/ml), or Chem-Mix + EGF. The Chem-Mix - but not the individual chemicals - reduced EGF-mediated EGFR phosphorylation in a dose dependent manner, while no effect was observed in its downstream effectors (AKT and STAT3). None of the individual chemicals affected EVT cell invasion, but the Chem-Mix reduced EVT cell invasion independent of EGF. In support of previous studies that have explored chemicals targeting a specific pathway (estrogen/androgen receptor), current findings indicate that exposure to a chemical mixture that targets the EGFR pathway can result in a greater impact compared to individual chemicals in the context of placental cell functions.

Abstract A040: Characterization of a humanized monoclonal antibody targeting cancer-expressed EGFR

Abstract The epidermal growth factor receptor (EGFR), an established oncology target, is a transmembrane protein highly expressed on the surface of malignant tumors such as glioblastoma, lung, liver, bladder and breast cancers. In addition to overexpression, EGFR mutants are frequently associated with malignancy. For example, EGFR variant III (EGFRvIII), deletion of exons 2-7, is only expressed on cancer cells and is associated with poor prognosis and chemotherapy resistance. To target cancer-expressed EGFR, including EGFRvIII, we generated the 40H3 monoclonal antibody which is reactive with the 287-302 loop that is fully exposed on the extracellular domain of EGFRvIII. The 40H3 antibody also showed strong reactivity for cells expressing gene-amplified EGFR but little or no reactivity for cells expressing normal levels of wild type EGFR. When conjugated with toxic payloads (either tesirine or deruxtecan), the resulting antibody-drug conjugates (ADCs) exhibited antitumor activity in several xenograft models. To improve its utility as a clinical candidate, we humanized the variable portions of the heavy and light chains of 40H3, without changing the original CDRs. The variable regions of 40H3, heavy and light chains, were compared to the closest human immunoglobulin families and then framework residues were altered to produce candidate humanized antibodies. The humanized variable chains were then fused with a human IgG1 heavy chain and a kappa light chain to generate full-sized humanized antibodies. A total of 9 humanized antibodies were generated by pairing 3 variable humanized heavy chains (VH1, VH2, VH3) with 3 variable humanized light chains (VL1, VL2, VL3). To evaluate the binding activity of humanized antibodies, we used either immobilized EGFRvIII (by ELISA or Octet) or various cancer cells expressing either EGFR or EGFRvIII (by flow cytometry). All humanized antibodies retained binding activity to immobilized EGFRvIII but bound with different apparent affinities to cells overexpressing EGFR or cells transfected with EGFRvIII. The ‘A10’ antibody was the best candidate. Mutations introduced in A10 preserved the binding affinity of the 40H3 antibody but exhibited an increased affinity compared to the corresponding chimeric antibody constructed with the original mouse VH and VL. The other eight antibodies retained EGFRvIII binding activity, albeit with a lower cell binding affinity. Specifically, A10 showed improved binding to cells with EGFR gene amplification including two TNBC lines, MDA-MB-468 and BT-20, epithelial cancer cells, A431, head and neck squamous carcinoma, UMSCC17B, as well to cells expressing EGFRvIII, F98npEGFRvIII. No binding of A10 was detected to cell lines expressing wild type EGFR, WI-38 or U87MG. In summary, humanized candidates of the 40H3 monoclonal antibody retained EGFR binding. Of 9 antibodies, A10 exhibited the best cell binding activity elevating it to our top clinical candidate for the production of ADCs or similar antibody-based therapeutics. Citation Format: Tamara G Fernandes Costa, Robert Sarnovsky, Maria Teresa Bilotta, Antonella Antignani, David FitzGerald. Characterization of a humanized monoclonal antibody targeting cancer-expressed EGFR [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A040.

Identification of angiogenesis-related genes in diabetic foot ulcer using machine learning algorithms

BackgroundDiabetic foot ulcers (DFUs) are among the most prevalent and dangerous complications of diabetes. Angiogenesis is pivotal for wound healing; however, its role in the chronic wound healing process in DFU requires further investigation. We aimed to investigate the pathogenic processes of angiogenesis in DFU from a molecular biology standpoint and to offer insightful information about DFU prevention and therapy. MethodsDifferential gene and weighted gene co-expression network analyses (WGCNA) were employed to screen for genes related to DFU using the downloaded and collated GSES147890 datasets. With the goal of identifying hub genes, an interaction among proteins (PPI) network was constructed, and enrichment analysis was carried out. Utilizing a variety of machine learning techniques, including Boruta, Support Vector Machine Recursive Feature Elimination (SVM-RFE), and Least Absolute Shrinkage and Selection Operator (LASSO), we were able to determine which hub genes most strongly correspond to DFU. This allowed us to create an ideally suited DFU forecasting model that was validated via an external dataset. Finally, by merging 36 angiogenesis-related genes (ARGs) and machine learning models, we identified the genes involved in DFU-related angiogenesis. ResultsBy merging 260 genes located in the green module and 59 differentially expressed genes (DEGs), 35 candidate genes highly associated with DFU were found for more investigation. 35 candidate genes were enriched in epidermal growth factor receptor binding, nuclear division regulation, fluid shear stress, atherosclerosis, and negative regulation of chromosomal structure for the enrichment study. Fifteen hub genes were found with the aid of the CytoHubba plug. The LASSO method scored better in terms of prediction performance (GSE134341) (LASSO:0.89, SVM:0.65, Boruta:0.66) based on the validation of the external datasets. We identified thrombomodulin (THBD) as a key target gene that potentially regulates angiogenesis during DFU development. Based on the external validation dataset (GSE80178 and GSE29221), receiver operating characteristic (ROC) curves with higher efficiency were generated to confirm the potential of THBD as a biomarker of angiogenesis in DFU. Furthermore supporting this finding were the results of Western blot and real-time quantitative polymerase chain reaction (RT-qPCR), which showed decreased THBD expression in human umbilical vein endothelial cells (HUVECs) cultivated under high glucose. ConclusionsThe findings implicate that THBD may influence DFU progression as a potential target for regulating angiogenesis, providing a valuable direction for future studies.

Experimental study on the therapeutic effect and mechanism of erlotinib on non-proliferative diabetic retinopathy

Experimental study on the therapeutic effect and mechanism of erlotinib on non-proliferative diabetic retinopathy

Cbl and Cbl-b independently regulate EGFR through distinct receptor interaction modes.

Highly homologous E3 ubiquitin ligases, Cbl and Cbl-b, mediate ubiquitination of EGF receptor (EGFR), leading to its endocytosis and lysosomal degradation. Cbl and Cbl-b, are thought to function in a redundant manner by binding directly to phosphorylated Y1045 (pY1045) of EGFR and indirectly via the Grb2 adaptor. Unexpectedly, we found that inducible expression of Cbl or Cbl-b mutants lacking the E3 ligase activity but fully capable of EGFR binding does not significantly affect EGFR ubiquitination and endocytosis in human oral squamous cell carcinoma (HSC3) cells which endogenously express Cbl-b at a relatively high level. Each endogenous Cbl species remained associated with ligand-activated EGFR in the presence of an overexpressed counterpart species or its mutant, although Cbl-b overexpression partially decreased Cbl association with EGFR. Binding to pY1045 was the preferential mode for Cbl-b:EGFR interaction, whereas Cbl relied mainly on the Grb2-dependent mechanism. Overexpression of the E3-dead mutant of Cbl-b slowed down EGF-induced degradation of active EGFR, while this mutant and a similar mutant of Cbl did not significantly affect MAPK/ERK1/2 activity. EGF-guided chemotaxis migration of HSC3 cells was diminished by overexpression of the E3-dead Cbl-b mutant but was not significantly affected by the E3-dead Cbl mutant. By contrast, the inhibitory effect of the same Cbl mutant on the migration of OSC-19 cells expressing low Cbl-b levels was substantially stronger than that of the Cbl-b mutant. Altogether, our data demonstrate that Cbl and Cbl-b may operate independently through different modes of EGFR binding to jointly control receptor ubiquitination, endocytic trafficking, and signaling.

Dual targeting pH responsive chitosan nanoparticles for enhanced active cellular internalization of gemcitabine in non-small cell lung cancer

Lung cancer (LC), related with the enhanced expression of epidermal growth factor receptor (EGFR) and sialic acid binding receptors (glycan) brought about the development of EGFR and glycan receptor specific anticancer therapeutics. The current study assessed the formulation, physiochemical characterization, in vitro and in vivo effects of sialic acid (SA) and cetuximab (Cxmab) decorated chitosan nanoparticles (CSN-NPs) loaded with gemcitabine (GMC) targeted to glycan and EGFR over-expressing non-small-cell lung-cancer (NSCLC) A-549 cells. Chitosan (CSN) was conjugated with sialic acid via EDC/NHS chemistry followed by gemcitabine loaded sialic acid conjugated chitosan nanoparticles (GMC-CSN-SA-NPs) were prepared by ionic gelation method decorated with Cxmab by electrostatic interaction. In vitro cytotoxicity of NPs quantified using cell based MTT, DAPI and Annexing-V/PI apoptosis assays showed superior antiproliferative activity of targeted nanoformulations (GMC-CSN-SA-Cxmab-NPs ≫ GMC-CSN-SA-NPs, GMC-CSN-Cxmab-NPs) over non-targeted nanoformulation (GMC-CSN-NPs) against A-549 cells. In vivopharmacokinetic study showed superior bioavailability and in vivo therapeutic efficacy investigation exhibited strongest anticancer activity of glycan and EGFR targeted NPs (GMC-CSN-SA-Cxmab-NPs). GMC-CSN-SA-Cxmab-NPs demonstrated enhanced cellular internalization and better therapeutic potential, by specifically targeting glycan and EGFR on NSCLC A-549 cells and B[a]P induced lung cancer mice model, hence it might be a good substitute for non-targeted, conventional chemotherapy.

Fibulin7 Mediated Pathological Cardiac Remodeling through EGFR Binding and EGFR-Dependent FAK/AKT Signaling Activation.

Adverse remodeling after myocardial infarction (MI) result in heart failure and sudden cardiac death. Fibulin7 (FBLN7) is an adhesion protein excreted into the extracellular matrix that functions in multiple biological processes. However, whether and how FBLN7 affects post-MI cardiac remodeling remains unclear. Here, the authors identify FBLN7 as a critical profibrotic regulator of adverse cardiac remodeling. They observe significantly upregulated serum FBLN7 levels in MI patients with left ventricular remodeling compared to those without MI. Microarray dataset analysis reveal FBLN7 is upregulated in human heart samples from patients with dilated and hypertrophic cardiomyopathy compared with non-failing hearts. The authors demonstrate that FBLN7 deletion attenuated post-MI cardiac remodeling, leading to better cardiac function and reduced myocardial fibrosis, whereas overexpression of FBLN7 results in the opposite effects. Mechanistically, FBLN7 binds to the epidermal growth factor receptor (EGFR) through its EGF-like domain, together with the EGF-like calcium-binding domain, and induces EGFR autophosphorylation at tyrosine (Y) 1068 and Y1173, which activates downstream focal adhesion kinase/AKT signaling, thereby leading to fibroblast-to-myofibroblast transdifferentiation. In addition, FBLN7-EGFR mediates this signal transduction, and the fibrotic response is effectively suppressed by the inhibition of EGFR activity. Taken together, FBLN7 plays an important role in cardiac remodeling and fibrosis after MI.

Tauroursodeoxycholic Acid Enhances Osteogenic Differentiation through EGFR/p-Akt/CREB1 Pathway in Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are pluripotent stromal cells that are among the most appealing candidates for regenerative medicine and may aid in the repair and regeneration of skeletal disorders through multiple mechanisms, including angiogenesis, differentiation, and response to inflammatory conditions. Tauroursodeoxycholic acid (TUDCA) has recently been used in various cell types as one of these drugs. The mechanism of osteogenic differentiation by TUDCA in hMSCs remains unknown. Cell proliferation was performed by the WST-1 method, and alkaline phosphatase activity and alizarin red-sulfate staining were used to confirm the osteogenic differentiation indicator. Expression of genes related to bone differentiation and specific genes related to signaling pathways was confirmed by quantitative real-time polymerase chain reaction. We found that cell proliferation was higher as the concentration increased, and showed that the induction of osteogenic differentiation was significantly enhanced. We also show that osteogenic differentiation genes were upregulated, with the expression of the epidermal growth factor receptor (EGFR) and cAMP responsive element binding protein 1 (CREB1) being specifically high. To confirm the participation of the EGFR signaling pathway, the osteogenic differentiation index and expression of osteogenic differentiation genes were determined after using an EGFR inhibitor. As a result, EGFR expression was remarkably low, and that of CREB1, cyclin D1, and cyclin E1 was also significantly low. Therefore, we suggest that TUDCA-induced osteogenic differentiation of human MSCs is enhanced through the EGFR/p-Akt/CREB1 pathway.

Molecular Docking Analysis on the Designed Benzimidazole Derivatives as EGFR Inhibitors: Comparison between EGFR Wild-Type (EGFRWT) and T790M Mutant

The non-small cell lung (NSCL) and colorectal cancers are frequently linked with the oncogenic activation of the epidermal growth factor receptor (EGFR), a member of the receptor tyrosine kinase (RTK) family. Current tyrosine kinase inhibitors (TKIs) are susceptible to drug resistance mutations and induce cytotoxicity effects on normal EGFRs. The isosteric nature of benzimidazole with purine renders its great potential to imitate the binding mode of the purine-based ATP and prevents its contact with the EGFR active sites. Here, we report the molecular docking of 50 designed benzimidazole derivatives, as well as Gefitinib and ATP, to analyse and compare their binding modes at EGFRwt and T790M active sites. The design of the ligands is based on our previous study, in which we proposed to evaluate keto- and amino-benzimidazoles, attached to a double bond linker and a phenyl group having electron donating and electron withdrawing groups attached at various positions. Docking simulations showed that keto-benzimidazoles dominated the top ten highest binding affinities in both EGFR-ligand complexes. The presence of sulfonyl substituents contributed to more stable complexes compared to others with binding energies of -8.1 (7c) and -7.8 (11c) kcal/mol in EGFRwt, and -8.3 (7d) and -8.4 (1c) kcal/mol for T790M mutant. The substituent effects on the benzimidazole contributed not only to the hydrogen bonding and hydrophobic interaction, but also to the often-disregarded Van der Waals forces that are responsible for shape complementarity of the benzimidazoles with the EGFR binding pocket.

Molecular Docking, Molecular Dynamics Simulation, and Analysis of EGFR-derived Peptides against the EGF

Background: The epidermal growth factor receptor (EGFR) is a member of the tyrosine kinase receptor family known as ErbB. The EGFR signaling pathway is an important regulator of cell proliferation, differentiation, division, and survival, as well as cancer development in humans. Epidermal growth factor, betacellulin, amphiregulin, transforming growth factor and heparin-binding EGF-like growth factor are high-affinity ligands of EGFR. Objective: Tumor progression can be effectively prevented by inhibiting EGF/EGFR interactions. In this study, many anti-EGF peptides targeting EGFR binding regions were designed, modeled, and evaluated. After selecting the peptides with the highest binding energy to the EGF, the interactions between the candidate peptides and all of the key EGFR ligands were investigated. Methods: To identify an EGF-binding peptide capable of blocking EGFR-EGF interactions, large-scale peptide mutation screening was performed. Using the AntiCP server, several possible peptides with anti-cancer properties were identified. The ClusPro analysis was performed in order to analyze the interactions between EGF and all of the library peptides. A total of five peptides with favorable docking scores were identified. The stability of three peptides with the best docking scores in complex with EGF was verified, applying molecular dynamics simulation with the help of the GROMACS software package. Finally, the interaction of candidate peptides with transforming growth factor-alpha, heparin-binding EGF-like growth factor, and betacellulin was investigated using the ClusPro server. Results: After the screening of modeled peptides by the ClusPro server and GROMACS software, two anti-EGF peptides of Pep4 and Pep5 with 31 residues were developed. Then, we demonstrate that both of these peptides can bind to the other high-affinity ligands of EGFR and block TGFA/EGFR, HBEGF/EGFR, and BTC/EGFR interactions. result: After screening of modeled peptides by the ClusPro server and GROMACS software, two anti-EGF peptides of Pep4 and Pep5 with 31 residues were developed. Then, we demonstrate that both of these peptides can bind to the other high-affinity ligands of EGFR and block TGFA/EGFR, HBEGF/EGFR, and BTC/EGFR interactions. Conclusion: The findings suggest novel insights for developing therapies based on peptides for inhibiting the EGF, TGFA, HBEGF, and BTC signaling cascade in cancer cells. Pep4 and Pep5 designed in this work, are recommended as potentially promising anticancer peptides for further experimental evaluation. other: Running title: Anti- EGF Peptides for Cancer Therapeutics

Site-specific encoding of photoactivity and photoreactivity into antibody fragments

Design of biomolecules that perform two or more distinct functions in response to light remains challenging. Here, we have introduced concurrent photoactivity and photoreactivity into an epidermal growth factor receptor (EGFR)-targeting antibody fragment, 7D12. This was achieved by site-specific incorporation of photocaged tyrosine (pcY) for photoactivity and p-benzoyl-ʟ-phenylalanine (Bpa) for photoreactivity into 7D12. We identified a position for installing Bpa in 7D12 that has minimal effect on 7D12–EGFR binding affinity in the absence of light. Upon exposure to 365-nm light, this Bpa-containing 7D12 mutant forms a covalent bond with EGFR in an antigen-specific manner. We then developed a method for site-specific incorporation of pcY and Bpa at two distinct sites in 7D12. Finally, we demonstrated that in the absence of light, this pcY- and Bpa-containing mutant of 7D12 does not bind to EGFR, but irradiation with 365-nm light activates (1) specific binding and (2) covalent bond formation with EGFR.

Binding sites in the epidermal growth factor receptor are responsible for bisphenol S effects on trophoblast cell invasion

Bisphenol S (BPS) is an endocrine disrupting chemical and the second most abundant bisphenol detected in humans. We have recently demonstrated that in utero exposure to BPS reduces human placenta cell fusion by interfering with epidermal growth factor (EGF)-dependent EGF receptor (EGFR) activation. Our previous work suggests that this occurs via binding of BPS to the extracellular domain of EGFR. However, whether BPS directly binds to EGFR has not been confirmed. We evaluated the binding ability of BPA, BPF and BPS to EGFR to determine whether EGFR binding is a unique attribute of BPS. To test these hypotheses, we first exposed HTR-8/SVneo cells to BPS, BPA, or BPF, with or without EGF. When co-exposed to EGF, BPS, but not BPA nor BPF, reduced EGFR phosphorylation by ∼60%, demonstrating that only BPS can interfere with EGF-dependent EGFR activation. As this indicates that BPS binding to the extracellular domain is responsible for its effect, we performed a computational search for putative binding sites on the EGFR extracellular domain, and performed ligand docking of BPS, BPA, and BPF at these sites. We identified three sites where polar interactions between positively charged residues and the sulfonyl group of BPS could lead binding selectivity over BPA and BPF. To test whether EGFR mutations at the predicted BPS binding sites (Arg255, Lys454, and Arg297) could prevent BPS's interference on EGFR activation, mutations for each EGFR target amino acids (R255A, R297A, and K454A) were introduced. For variants with R297A or K454A mutations, BPS did not affect EGF-mediated EGFR phosphorylation or EGFR-mediated cell invasion, suggesting that these residues are needed for the BPS antagonism effect on EGFR. In conclusion, BPS, but not BPA or BPF, interferes with EGFR-mediated trophoblast cell functions through binding at Arg297 and Lys454 amino acid residues in the extracellular domain of EGFR.

Expanding the Therapeutic Window of EGFR-Targeted PE24 Immunotoxin for EGFR-Overexpressing Cancers by Tailoring the EGFR Binding Affinity.

Immunotoxins (ITs), which are toxin-fused tumor antigen-specific antibody chimeric proteins, have been developed to selectively kill targeted cancer cells. The epidermal growth factor receptor (EGFR) is an attractive target for the development of anti-EGFR ITs against solid tumors due to its overexpression on the cell surface of various solid tumors. However, the low basal level expression of EGFR in normal tissue cells can cause undesirable on-target/off-tumor toxicity and reduce the therapeutic window of anti-EGFR ITs. Here, based on an anti-EGFR monobody with cross-reactivity to both human and murine EGFR, we developed a strategy to tailor the anti-EGFR affinity of the monobody-based ITs carrying a 24-kDa fragment of Pseudomonas exotoxin A (PE24), termed ER-PE24, to distinguish tumors that overexpress EGFR from normal tissues. Five variants of ER-PE24 were generated with different EGFR affinities (KD ≈ 0.24 nM to 104 nM), showing comparable binding activity for both human and murine EGFR. ER/0.2-PE24 with the highest affinity (KD ≈ 0.24 nM) exhibited a narrow therapeutic window of 19 pM to 93 pM, whereas ER/21-PE24 with an intermediate affinity (KD ≈ 21 nM) showed a much broader therapeutic window of 73 pM to 1.5 nM in in vitro cytotoxic assays using tumor model cell lines. In EGFR-overexpressing tumor xenograft mouse models, the maximum tolerated dose (MTD) of intravenous injection of ER/21-PE24 was found to be 0.4 mg/kg, which was fourfold higher than the MTD (0.1 mg/kg) of ER/0.2-PE24. Our study provides a strategy for the development of IT targeting tumor overexpressed antigens with basal expression in broad normal tissues by tailoring tumor antigen affinities.

A Self-Assembly ICG Nanoparticle Potentiating Targeted Photothermal and Photodynamic Therapy in NSCLC.

In nonsmall cell lung cancers (NSCLC), near-infrared (NIR) fluorescence imaging using indocyanine green (ICG) has proven to be an efficient approach for locating pulmonary nodules and pulmonary sentinel lymph nodes. However, due to a lack of tumor selectivity, ICG's use as a photosensitizer for photothermal therapy (PTT) and photodynamic therapy (PDT) is restricted. In the current study, we aimed to develop a type of high-performance NIR nanoparticle formulated with ICG to enhance its targeted efficacy and tumor specificity on NSCLC. An ICG-osimertinib nanoparticle (ICG-Osi) was self-assembled through π-π stacking, with a size of 276 nm and a surface charge of -7.4 mV. The NIR visibility and epidermal growth factor receptor (EGFR) targetability of the ICG-Osi was confirmed by its binding efficiency to EGFR-expressing NSCLC cells in vitro and in vivo, regardless of EGFR mutation status. The targeted effect was further confirmed in mouse xenograft models and showed an extended tumor retention time (>96 h). We demonstrated a significantly enhanced hyperthermia effect and a retained reactive oxygen species (ROS) generating ability of ICG-Osi, resulting in a 2-fold higher cell death rate than ICG alone. The ICG-Osi down-regulated GPX4 and p62 expression while up-regulating caspase-3 and beclin1 expression in NSCLC cells, indicating a complex network of cell death-related proteins. Considering the merits of simple assembly, EGFR binding efficacy, improved hyperthermia effect, and efficient cancer cell suppression, the ICG-Osi exhibits great potential for clinical application in EGFR-expressing NSCLC therapy.

MiR-141 exacerbates lung ischemia-reperfusion injury by targeting EGFR/β-catenin axis-mediated autophagy.

Some microRNAs (miRNAs) play important roles in lung ischemia-reperfusion injury (LIRI) injury. Here, this study aimed to examine whether miR-141 was related to lung ischemia-reperfusion injury (IRI) via regulating autophagy and the epidermal growth factor receptor (EGFR), and to explore the underlying signal transduction pathways. To this end, we constructed the LIRI cell model and mouse models, separately. According to RT-qPCR and Western blotting (WB) analysis results, miR-141 up-regulation together with β-catenin and EGFR down-regulation within mouse pulmonary microvascular endothelial cells (PMVECs) or lung tissues was related to lung IRI. Besides, we conducted dual-luciferase reporter assay, which suggested the binding of EGFR to miR-141. In addition, we carried out TUNEL staining, HE staining, and flow cytometric analysis to assess the apoptosis of PMVECs and the injury to mouse lung tissues. Furthermore, we performed light-chain immunofluorescence assay to examine autophagosomes within PMVECs. According to our results, miR-141 suppressed β-catenin level through reducing EGFR level. Besides, the miR-141/EGFR/β-catenin axis enhanced autophagy to aggravate LIRI. To sum up, miR-141 suppresses EGFR expression to inhibit β-catenin level, which subsequently aggravates autophagy and complicates LIRI. The above results offer the candidate therapeutic target for the treatment of lung IRI.

Oxidized tea polyphenol (OTP-3) targets EGFR synergistic nimotuzumab at inhibition of non-small cell lung tumor growth

Aberrant activation of epidermal growth factor receptor (EGFR) plays a pivotal role in cancer initiation and progression and has gained attention as an anticancer drug target. EGFR monoclonal antibodies have been canonically used in non-small cell lung cancer (NSCLC) treatment. However, a basal level of ligand-independent EGFR signaling pro-survival properties limit the clinical efficacy of EGFR monoclonal antibodies. Therefore, targeting EGFR by inducing degraders is a promising approach towards improving therapeutic efficacy and augmenting the effect of nimotuzumab. Here we describe rational discovery of OTP-3, an oxidized (−)-Epigallocatechin gallate (EGCG) derivative that elicits potent anticancer activity in EGFR wild type NSCLC. Mechanistic studies disclosed that OTP-3 directly binds to EGFR extracellular domain decreases EGF and EGFR binding affinities by combination with nimotuzumab. Molecular docking studies revealed that OTP-3-EGFR is a very stable complex. Further analyses showed that nimotuzumab combined with OTP-3 resulted in significantly promoted EGFR degradation and repressed downstream survival pathways. Accordingly, OTP-3 combined with nimotuzumab significantly inhibits tumor growth through degrading EGFR in vivo. Thus, OTP-3 can also serve as an effective therapeutic agent in NSCLC where it can augment the effects of nimotuzumab, a valuable property for combination agents.