Epidermal Growth Factor Receptor Protein Research Articles

Targeting epidermal growth factor receptor and its downstream signaling pathways by natural products: A mechanistic insight.

The epidermal growth factor receptor (EGFR) is a transmembrane receptor tyrosine kinase (RTK) that maintains normal tissues and cell signaling pathways. EGFR is overactivated and overexpressed in many malignancies, including breast, lung, pancreatic, and kidney. Further, the EGFR gene mutations and protein overexpression activate downstream signaling pathways in cancerous cells, stimulating the growth, survival, resistance to apoptosis, and progression of tumors. Anti-EGFR therapy is the potential approach for treating malignancies and has demonstrated clinical success in treating specific cancers. The recent report suggests most of the clinically used EGFR tyrosine kinase inhibitors developed resistance to the cancer cells. This perspective provides a brief overview of EGFR and its implications in cancer. We have summarized natural products-derived anticancer compounds with the mechanistic basis of tumor inhibition via the EGFR pathway. We propose that developing natural lead molecules into new anticancer agents has a bright future after clinical investigation.

Cetuximab–Toxin Conjugate and NPe6 with Light Enhanced Cytotoxic Effects in Head and Neck Squamous Cell Carcinoma In Vitro

Background: Photodynamic therapy (PDT) is a cancer-targeted treatment that uses a photosensitizer (PS) and irradiation of a specific wavelength to exert cytotoxic effects. To enhance the antitumor effect against head and neck squamous cell carcinoma (HNSCC), we developed a new phototherapy, intelligent targeted antibody phototherapy (iTAP). This treatment uses a combination of immunotoxin (IT) and a PS for PDT and light irradiation. In our prior study, we demonstrated that an immunotoxin (IT) consisting of an anti-ROBO1 antibody conjugated to saporin, when used in combination with the photosensitizer (PS) disulfonated aluminum phthalocyanine (AlPcS2a) and irradiated with light at the appropriate wavelength, resulted in increased cytotoxicity against head and neck squamous cell carcinoma (HNSCC) cells. ROBO1 is a receptor known to be involved in the progression of cancer. In this study, we newly investigate the iTAP targeting epidermal growth factor receptor (EGFR) which is widely used as a therapeutic target for HNSCC. Methods: We checked the expression of EGFR in HNSCC cell lines, SAS, HO-1-u-1, Sa3, and HSQ-89. We analyzed the cytotoxicity of saporin-conjugated anti-EGFR antibody (cetuximab) (IT-Cmab), mono-L-aspartyl chlorin e6 (NPe6, talaporfin sodium), and light (664 nm) irradiation (i.e., iTAP) in SAS, HO-1-u-1, Sa3, and HSQ-89 cells. Results: EGFR was expressed highly in Sa3, moderately in HO-1-u-1, SAS, and nearly not in HSQ-89. Cmab alone or IT-Cmab alone did not show cytotoxic effects in Sa3, HO-1-u-1, and HSQ-89 cells, which have moderate or low expression levels of EGFR protein. However, the iTAP method enhanced the cytotoxicity of IT-Cmab by the photodynamic effect in Sa3 and HO-1-u-1 cells, which have moderate levels of EGFR expression. Conclusion: Our study is the first to report on the iTAP method using IT-Cmab and NPe6 for HNSCC. The cytotoxic effects are enhanced in cell lines with moderate levels of EGFR protein expression, but not in nonexpressing cell lines, which is expected to expand the range of therapeutic windows and potentially reduce complications.

EGFR-dependent endocytosis of Wnt9a and Fzd9b promotes β-catenin signaling during hematopoietic stem cell development in zebrafish.

Cell-to-cell communication through secreted Wnt ligands that bind to members of the Frizzled (Fzd) family of transmembrane receptors is critical for development and homeostasis. Wnt9a signals through Fzd9b, the co-receptor LRP5 or LRP6 (LRP5/6), and the epidermal growth factor receptor (EGFR) to promote early proliferation of zebrafish and human hematopoietic stem cells during development. Here, we developed fluorescently labeled, biologically active Wnt9a and Fzd9b fusion proteins to demonstrate that EGFR-dependent endocytosis of the ligand-receptor complex was required for signaling. In human cells, the Wnt9a-Fzd9b complex was rapidly endocytosed and trafficked through early and late endosomes, lysosomes, and the endoplasmic reticulum. Using small-molecule inhibitors and genetic and knockdown approaches, we found that Wnt9a-Fzd9b endocytosis required EGFR-mediated phosphorylation of the Fzd9b tail, caveolin, and the scaffolding protein EGFR protein substrate 15 (EPS15). LRP5/6 and the downstream signaling component AXIN were required for Wnt9a-Fzd9b signaling but not for endocytosis. Knockdown or loss of EPS15 impaired hematopoietic stem cell development in zebrafish. Other Wnt ligands do not require endocytosis for signaling activity, implying that specific modes of endocytosis and trafficking may represent a method by which Wnt-Fzd specificity is established.

Activity of pure flavonoid-based green silver nano particles against breast cancer: A combined experimental and computational investigation

Because of the wide range of applications in various industries, the development of novel nanomaterials is receiving more and more attention nowadays. A green route to synthesize one such versatile nanomaterial, silver nanoparticles (AgNPs) with a pure flavonoid, quercetin has been explored in this study. The reducing and capping mechanism of quercetin was examined using density functional theory (DFT). Dynamic light scattering (DLS), UV–vis spectra (UV–Vis), and Transmission Electron Microscopy (TEM) studies confirmed that the synthesized AgNPs are stable, monodispersed, spherical shaped with an average size of 16 nm. A molecular docking study has identified the epidermal growth factor receptor (EGFR) protein as a potential target for quercetin-capped AgNPs to fight against breast cancer. Molecular dynamics (MD) simulation has quantified the efficiency of quercetin-capped AgNPs over quercetin only in targeting breast cancer. These findings will be helpful in using quercetin-based AgNPs for drug discovery against breast cancer.

Active substance and mechanisms of Actinidia chinensis Planch for the treatment of breast cancer was explored based on network pharmacology and in silico study.

In this paper, our objective was to investigate the potential mechanisms of Actinidia chinensis Planch (ACP) for breast cancer treatment with the application of network pharmacology, molecular docking, and molecular dynamics. "Mihoutaogen" was used as a key word to query the Traditional Chinese Medicine Systems Pharmacology database for putative ingredients of ACP and its related targets. DrugBank, GeneCards, Online Mendelian Inheritance in Man, and therapeutic target databases were used to search for genes associated with "breast cancer." Using Cytoscape 3.9.0 we then constructed the protein-protein interaction and drug-ingredient-target-disease networks. An enrichment analysis of Kyoto encyclopedia of genes and genomes pathway and gene ontology were performed to exploration of the signaling pathways associated with ACP for breast cancer treatment. Discovery Studio software was applied to molecular docking. Finally, the ligand-receptor complex was subjected to a 50-ns molecular dynamics simulation using the Desmond_2020.4 tools. Six main active ingredients and 176 targets of ACP and 2243 targets of breast cancer were screened. There were 118 intersections of targets for both active ingredients and diseases. Tumor protein P53 (TP53), AKT serine/threonine kinase 1 (AKT1), estrogen receptor 1 (ESR1), Erb-B2 receptor tyrosine kinase 2 (ERBB2), epidermal growth factor receptor (EGFR), Jun Proto-Oncogene (JUN), and Heat Shock Protein 90 Alpha Family Class A Member 1 (HSP90AA1) selected as the most important genes were used for verification by molecular docking and molecular dynamics simulation. The primary active compounds of ACP against breast cancer were predicted preliminarily, and its mechanism was studied, thereby providing a theoretical basis for future clinical studies.

DRD1 suppresses cell proliferation and reduces EGFR activation and PD-L1 expression in NSCLC.

Dopamine (DA) acts in various key neurological and physiological processes as both a neurotransmitter and circulating hormone. Over the past several decades, the DA signaling network has been shown to regulate the progression of several types of solid tumors, and considerable evidence has shown it is a druggable pathway in the cancer cell context. However, the specific activity and effect of these pathway components appears to be tissue-type and cell-context-dependent. In the present study, expression and methylation of dopamine receptor D1 (DRD1) were measured using RNA sequencing (RNAseq) and reverse transcription polymerase chain reaction (RT-PCR) in non-small cell lung cancer (NSCLC) samples, and validated using publicly available datasets, including The Cancer Genome Atlas (TCGA). In vitro and in vivo functional experiments were performed for cell proliferation and tumor growth, respectively. Mechanistic analyses of the transcriptome and kinome in DRD1-modulated cells informed further experiments, which characterized the effects on the epidermal growth factor receptor (EGFR) pathway and programmed cell death 1 ligand 1 (PD-L1) proteins. Through these experiments, we identified the DRD1 gene as a negative regulator of disease progression in NSCLC. We show that DRD1, as well as other DA pathway components, are expressed in normal human lung tissue, and that loss of DRD1 expression through promoter hypermethylation is a common feature in NSCLC patients and is associated with worse survival. At the cellular level, DRD1 affects proliferation by inhibiting the activation of EGFR and mitogen-activated protein kinase 1/2 (ERK1/2). Interestingly, we also found that DRD1 regulates the expression of PD-L1 in lung cancer cells. Taken together, these results suggest that DRD1 methylation may constitute a biomarker of poor prognosis in NSCLC patients while other components of this pathway could be targeted to improve response to EGFR- and PD-L1-targeted therapies.

Hedgehog ligand and receptor cooperatively regulate EGFR stability and activity in non-small cell lung cancer.

The hyperactivation of epidermal growth factor receptor (EGFR) plays a crucial role in non-small cell lung cancer (NSCLC). Hedgehog (Hh) signaling has been implicated in the tumorigenesis and progression of various cancers, however, its function in NSCLC cells remains controversial. Herein, we present a novel finding that challenges the current understanding of Hh signaling in tumor growth. Expression of Hh ligands and receptor were assessed using TCGA datasets, immunoblotting and immunohistochemical. Biological function of Hh ligands and receptor in NSCLC were tested using colony formation, cell count kit-8 (CCK-8) and xenograft assays. Biochemical effect of Hh ligands and receptor on regulating EGFR stability and activity were checked via immunoblotting. Expression of Hh ligands and receptor was suppressed in NSCLC tissues, and the lower expression levels of these genes were associated with poor prognosis. Ptch1 binds to EGFR and facilitates its poly-ubiquitylation and degradation independent of downstream transcriptional signaling. Moreover, Hh ligands cooperate with Ptch1 to regulate the protein stability and activity of EGFR. This unique mechanism leads to a suppressive effect on NSCLC tumor growth. Non-canonical Hh signaling pathway, involving cooperation between Hh ligands and their receptor Ptch1, facilitates the degradation of EGFR and attenuates its activity in NSCLC. These findings provide novel insights into the regulation of EGFR protein stability and activity, offer new diagnostic indicators for molecular typing of NSCLC and identify potential targets for targeted therapy of this challenging disease.

New Theobromine Apoptotic Analogue with Anticancer Potential Targeting the EGFR Protein: Computational and In Vitro Studies.

The aim of this study was to design and examine a novel epidermal growth factor receptor (EGFR) inhibitor with apoptotic properties by utilizing the essential structural characteristics of existing EGFR inhibitors as a foundation. The study began with the natural alkaloid theobromine and developed a new semisynthetic derivative (T-1-PMPA). Computational ADMET assessments were conducted first to evaluate its anticipated safety and general drug-likeness. Deep density functional theory (DFT) computations were initially performed to validate the three-dimensional (3D) structure and reactivity of T-1-PMPA. Molecular docking against the EGFR proteins was conducted to investigate T-1-PMPA's binding affinity and inhibitory potential. Additional molecular dynamics (MD) simulations over 200 ns along with MM-GPSA, PLIP, and principal component analysis of trajectories (PCAT) experiments were employed to verify the binding and inhibitory properties of T-1-PMPA. Afterward, T-1-PMPA was semisynthesized to validate the proposed design and in silico findings through several in vitro examinations. DFT studies indicated T-1-PMPA's reactivity using electrostatic potential, global reactive indices, and total density of states. Molecular docking, MD simulations, MM-GPSA, PLIP, and ED suggested the binding and inhibitory properties of T-1-PMPA against the EGFR protein. The in silico ADMET predicted T-1-PMPA's safety and general drug-likeness. In vitro experiments demonstrated that T-1-PMPA effectively inhibited EGFRWT and EGFR790m, with IC50 values of 86 and 561 nM, respectively, compared to Erlotinib (31 and 456 nM). T-1-PMPA also showed significant suppression of the proliferation of HepG2 and MCF7 malignant cell lines, with IC50 values of 3.51 and 4.13 μM, respectively. The selectivity indices against the two cancer cell lines indicated the overall safety of T-1-PMPA. Flow cytometry confirmed the apoptotic effects of T-1-PMPA by increasing the total percentage of apoptosis to 42% compared to 31, and 3% in Erlotinib-treated and control cells, respectively. The qRT-PCR analysis further supported the apoptotic effects by revealing significant increases in the levels of Casp3 and Casp9. Additionally, T-1-PMPA controlled the levels of TNFα and IL2 by 74 and 50%, comparing Erlotinib's values (84 and 74%), respectively. In conclusion, our study's findings suggest the potential of T-1-PMPA as a promising apoptotic anticancer lead compound targeting the EGFR.

Abstract 3003: Exon skipping for the tumor suppressor Annexin A7 promotes EGFR signaling in stem-like brain tumor initiating cells through receptor recycling

Abstract Background: Glioblastoma (GBM) is one of the most lethal forms of cancer. The majority of GBMs express aberrant receptor tyrosine kinase (RTK) activity, which upon activation, are canonically endocytosed and degraded. This process is disrupted in GBM to promote receptor recycling resulting in hyperactive RTK signaling. We demonstrated that the alternative splicing of the tumor suppressor Annexin A7 (ANXA7) supports the impaired endosomal trafficking of RTKs to favor recycling in GBM. Exon six of ANXA7 can be alternatively spliced to yield two distinct protein isoforms through its inclusion (I1) or exclusion (I2). Methods: We investigated ANXA7 isoform differences for epidermal growth factor receptor (EGFR) trafficking fates first in U251 malignant glioma followed by stem-like brain tumor initiating cells (BTICs) originating from patient-derived xenografts (PDXs). U251s and primary BTICs (JX14) were transduced with lentivirus to overexpress ANXA7 and GFP for enrichment by flow cytometry. JX14 empty vector (EV; I2 only) and I1 (I2 and I1 expressing) cells were plated onto Geltrex and ligand starved overnight in serum-free media to maintain BTICs. We assessed EGFR protein levels following EGF stimulation in a time dependent manner. Immunofluorescence experiments were conducted to measure EGFR-ANXA7 colocalization (Pearson) with markers of the endocytic pathway, which included the early endosomes (EEA1), recycling endosomes (Rab4/Rab11), and lysosomes (LAMP1). EV and I1 cells were also incubated with siRNA targeting both ANXA7 isoforms to determine the impact of ANXA7 loss on endosomal EGFR trafficking. Results: Interestingly, we found drastic differences in endosomal EGFR trafficking in I1 expressing cells. EGFR colocalized with EEA1 in both EV and I1 cells (R2=0.01336, P>0.6275). In EV cells, EGFR was recycled via fast/slow recycling endosomes demonstrated by Rab4/Rab11 colocalization while I1 did not (Rab11: R2=0.9267, P<0.0001). Immunoblotting revealed a significant reduction in EGFR protein in I1 cells only. Furthermore, I1 cells and not EV preferentially degraded EGFR as seen by LAMP1 colocalization (R2=0.9398, P<0.0001). ANXA7 siRNA in EV cells did not alter EGFR trafficking, which implicates I2 as nonfunctional. On the other hand, ANXA7 KD in I1 cells impaired EGFR sorting via a 40% increase (P<0.0010) in total EGFR protein and a 40% reduction (P<0.0001) in colocalization with EEA1. Loss of I1 also shifted the degradation of EGFR back towards recycling. Conclusion: GBMs express I2, which supports EGFR hyperactivity by favoring receptor recycling. I1 overexpression significantly reduced EGFR signaling by promoting degradation. I1-induced reduction in EGFR signaling suggests a new therapeutic vulnerability for BTICs by potentially reducing tumorigenicity or improving the efficacy of EGFR inhibition. Citation Format: Aran Merati, Sindhu Nair, Rajani Rajbhandari, Nicholas Johnson, Markus Bredel. Exon skipping for the tumor suppressor Annexin A7 promotes EGFR signaling in stem-like brain tumor initiating cells through receptor recycling [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 3003.

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.

Abstract 4552: EGFR inhibitors increase LGR5 expression and enhance potency of LGR5 antibody-drug conjugates targeting colorectal cancer stem cells

Abstract Colorectal cancer (CRC) is the second leading cause of cancer-associated deaths in the United States. One of the primary challenges in treating CRC is therapy resistance thought to be mediated by cancer stem cells (CSCs), a subpopulation of cancer cells with infinite replicative potential that can differentiate to drive tumorigenesis and disease relapse. Antibody-drug conjugates (ADCs) are amongst the fastest growing classes of anticancer drugs and utilize the specificity of monoclonal antibodies (mAbs) to hone cytotoxic payloads to cancer cells. We generated ADCs directed against leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), a well-defined biomarker of CSCs that is highly overexpressed in CRCs. LGR5 ADCs demonstrated high specificity and efficacy in CRC cells and xenograft models, though tumor relapse after treatment withdrawal was a major obstacle. Interestingly, FDA-approved therapy targeting epidermal growth factor receptor (EGFR) has been shown to increase LGR5 mRNA expression levels in patient-derived models of CRC. This study aims to determine the mechanism of EGFR-mediated regulation of LGR5 expression and evaluate the therapeutic efficacy of combination treatments targeting EGFR and LGR5 for the improved treatment of CRC. To first analyze the effect of EGFR inhibition on LGR5 expression, a panel of CRC cell lines of different KRAS and PI3CKA mutational statuses were treated with FDA-approved EGFR-targeted mAbs or small molecule inhibitors. LGR5 protein expression was shown to be upregulated by EGFR-targeted therapies in a dose- and time-dependent manner, independent of mutation status. Notably, treatment with EGF, MEK1/2 inhibitor trametinib, and EGFR-directed siRNA all resulted in concomitant reduction in EGFR and LGR5 protein levels, suggesting EGFR and LGR5 are co-degraded. Co-immunoprecipitation and immunocytochemistry experiments identified a novel EGFR-LGR5 interaction and co-internalization mechanism in CRC cells, respectively. Furthermore, we showed EGFR-LGR5 interaction is enhanced by treatment with the EGFR-targeting mAb, cetuximab (CTX). To evaluate efficacy of combination therapies targeting EGFR and LGR5, CRC cells were treated with LGR5 ADCs with or without CTX. Importantly, we showed CTX significantly enhanced the potency of LGR5 ADCs incorporating different classes of cytotoxic payloads. These results suggest combining LGR5 ADCs with EGFR inhibitors may be a more effective approach for the treatment of CRC and eliminating CSCs to overcome resistance and relapse. Citation Format: Peyton C. High, Zhengdong Liang, Kendra S. Carmon. EGFR inhibitors increase LGR5 expression and enhance potency of LGR5 antibody-drug conjugates targeting colorectal cancer stem cells [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 4552.

Abstract 5774: Are 19del and L858R really not the same disease entities

Abstract IntroductionThe similarities and differences between the two subtypes of common epidermal growth factor receptor (EGFR) mutations have been recognized by clinicians, but actual treatment strategies remain unchanged. The L858R mutation can be explained by the pharmacological conformational plasticity of the receptor protein and other co-occurring mutations, which may be subtypes of EGFR mutations or non-EGFR mutations, and differences in downstream signaling pathways. As long as we know that molecular differences lead to biological differences, it is a challenge for all of us that our treatment strategies must changeWe investigated the RWD, conformational structure and RNA expression profile about two types of common mutations in the mEGFR+ NSCLC patients.MethodWe collected the medical information for stage I-IIIA mEGFR+ NSCLC patients and explored the conformational structure by crystalized radiologic technique. And we performed RNA expression assay using multiplex IF and High-through-output Profiling. ResultOverall, the static structures of wild type (WT), L858R, and 19del (ΔL747-E749) EGFR did not show any significant differences. However, the difference in the static structure between WT-19del and L858R-19del was subtly greater than that of WT-L858R. In addition, further analyses demonstrated no significant structural differences in the phosphorylation sites. However, it should be noted that proteins do not exist as static structures in vivo, and they have a dynamic, flexible structure that changes over time, i.e., plasticity of proteins. Therefore, we investigated the X-ray B-factor value, which is an indicator of flexibility, dynamics, and plasticity of proteins. As expected, the loop or disordered regions mainly shows higher B-factor values, which indicates an active structure, highly changeable and dynamic. Together, the three types of the EGFR protein manifest wholly similar B-factor values, while several local regions present appreciably distinct B-factors. Physical properties such as the dynamic structure may affect intermolecular recognition of EGFR proteins and their function Recent biochemical and biophysical evidence suggests that oncogenic mutations have an impact on the conformational dynamics of enzymesConclusionIn conclusion, our results suggest that Patients with 19del and L858R mutations demonstrated a different clinical course. It is necessary to consider the dynamic structural characteristics and functional differences of EGFR membrane proteins. Citation Format: Yunseok Heo, Joo Ri Kim, Yeoun Eun Sung, Seoree Kim, Chan Kwon Jung, Jeong-Oh Kim, Jeong-yeon Shin, Guk Jin Lee, Sang hoon Chun, Seong Hyeon Bu, Young Ho Lee, Jin Hyoung Kang. Are 19del and L858R really not the same disease entities [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 5774.

Molecular Docking Analysis of Hydroxy Chalcones and Flavones from Anisaldehyde and Veratraldehyde as EGFR Inhibitors: Predicting Anticancer Potential

This study aimed to investigate the potential of hydroxy chalcone and flavone derivatives as inhibitors of the epidermal growth factor receptor (EGFR) with anticancer properties. Molecular docking simulations were conducted using Autodock Tools 1.5.6 and Discovery Studio visualizer. The EGFR protein structure with the PDB code 1M17 was utilized as the receptor, explicitly targeting the binding pocket. Redocking of the reference ligand erlotinib yielded a binding energy of -7.51 kcal mol-1 with an RMSD of 0.54 Å, confirming the accuracy of the docking protocol. The hydroxy chalcone and flavone derivatives exhibited binding energies ranging from -6.50 to -7.67 kcal mol-1 when interacting with the EGFR protein. Among the studied compounds, compound 2',5'-dihydroxy-3,4-dimethoxychalcone (1g) displayed the lowest binding energy. Interactions involving amino acids such as Met769, Ala719, Thr766, Lys721, and Glu738 were identified as crucial hydrogen bonding interactions between the ligands and the EGFR protein. These findings suggest that 2',5'-dihydroxy-3,4-dimethoxychalcone holds strong potential as a tyrosine kinase inhibitor, positioning it as a promising candidate for further development as an anticancer agent. The outcomes of the computational analysis conducted through the pkCSM online platform indicated that the chemical 2',5'-dihydroxy-3,4-dimethoxychalcone had favorable pharmacokinetic characteristics and showed low toxicity levels.
 
 Keywords: molecular docking, hydroxy chalcone, flavone, egfr, ADMET

Epidermal growth factor receptor (EGFR) is a target of the tumor-suppressor E3 ligase FBXW7

FBXW7 is an E3 ubiquitin ligase that targets proteins for proteasome-mediated degradation and is mutated in various cancer types. Here, we use CRISPR base editors to introduce different FBXW7 hotspot mutations in human colon organoids. Functionally, FBXW7 mutation reduces EGF dependency of organoid growth by ~10,000-fold. Combined transcriptomic and proteomic analyses revealed increased EGFR protein stability in FBXW7 mutants. Two distinct phosphodegron motifs reside in the cytoplasmic tail of EGFR. Mutations in these phosphodegron motifs occur in human cancer. CRISPR-mediated disruption of the phosphodegron motif at T693 reduced EGFR degradation and EGF growth factor dependency. FBXW7 mutant organoids showed reduced sensitivity to EGFR-MAPK inhibitors. These observations were further strengthened in CRC-derived organoid lines and validated in a cohort of patients treated with panitumumab. Our data imply that FBXW7 mutations reduce EGF dependency by disabling EGFR turnover.

Intraventricular CARv3-TEAM-E T Cells in Recurrent Glioblastoma

SummaryIn this first-in-human, investigator-initiated, open-label study, three participants with recurrent glioblastoma were treated with CARv3-TEAM-E T cells, which are chimeric antigen receptor (CAR) T cells engineered to target the epidermal growth factor receptor (EGFR) variant III tumor-specific antigen, as well as the wild-type EGFR protein, through secretion of a T-cell–engaging antibody molecule (TEAM). Treatment with CARv3-TEAM-E T cells did not result in adverse events greater than grade 3 or dose-limiting toxic effects. Radiographic tumor regression was dramatic and rapid, occurring within days after receipt of a single intraventricular infusion, but the responses were transient in two of the three participants. (Funded by Gateway for Cancer Research and others; INCIPIENT ClinicalTrials.gov number, NCT05660369.)

Potential of targeting signal-transducing adaptor protein-2 in cancer therapeutic applications.

Adaptor proteins play essential roles in various intracellular signaling pathways. Signal-transducing adaptor protein-2 (STAP-2) is an adaptor protein that possesses pleckstrin homology (PH) and Src homology 2 (SH2) domains, as well as a YXXQ signal transducer and activator of transcription 3 (STAT3)-binding motif in its C-terminal region. STAP-2 is also a substrate of breast tumor kinase (BRK). STAP-2/BRK expression is deregulated in breast cancers and enhances STAT3-dependent cell proliferation. In prostate cancer cells, STAP-2 interacts with and stabilizes epidermal growth factor receptor (EGFR) after stimulation, resulting in the upregulation of EGFR signaling, which contributes to cancer-cell proliferation and tumor progression. Therefore, inhibition of the interaction between STAP-2 and BRK/EGFR may be a possible therapeutic strategy for these cancers. For this purpose, peptides that interfere with STAP-2/BRK/EGFR binding may have great potential. Indeed, the identified peptide inhibitor successfully suppressed the STAP-2/EGFR protein interaction, EGFR stabilization, and cancer-cell growth. Furthermore, the peptide inhibitor suppressed tumor formation in human prostate- and lung-cancer cell lines in a murine xenograft model. This review focuses on the inhibitory peptide as a promising candidate for the treatment of prostate and lung cancers.

Enhancing targeted cancer therapy through multiple drug delivery by silk peptide nanoparticles

AbstractTargeted therapy drugs exert an essential role in precision therapy in cancer treatments. However, their therapeutic efficiency is greatly limited by the poor water solubility and low bioavailability. Herein, by using an epidermal growth factor receptor (EGFR) targeted drug, erlotinib (ERL), as a representative of targeted therapy drugs, we demonstrate silk peptide (SP) extracted from silkworm cocoons can be used as a drug carrier to co‐load curcumin (CUR) and ERL to achieve dramatically improved antitumor therapeutic efficiency. The dual‐drug loaded nanoparticles (CUR/ERL@SP) with a mean size within 150 nm show significantly enhanced cell internalization, and thus induce more effective inhibition of cancer cell growth. As compared with free drugs, CUR/ERL@SP can more efficiently downregulate the expression of EGFR and other proteins promoting tumor invasion, immunosuppression and cell fusion in EGFR mutant PC‐9 lung cancer cells. The evaluation by using the circulating malignant cells (CMCs) from a lung cancer patient further confirms that CUR/ERL@SP can more effectively inhibit EGFR expression. This study provides a convenient strategy to enhance the therapeutic efficacy of targeted therapy drugs and a facile ex vivo method for evaluating treatment outcomes in personalized therapy.

Development of 1,5-diarylpyrazoles as EGFR/JNK-2 dual inhibitors: design, synthesis, moleecular docking, and bioactivity evaluation

The eradication of multifactorial diseases, such as cancer, requires the design of drug candidates that attack multiple targets that contribute to the progression and proliferation of such diseases. Here, 1,5-diarylpyrazole derivatives bearing vanillin or sulfanilamide are developed as potential dual inhibitors of epidermal growth factor receptor (EGFR)/c-Jun N-terminal kinase 2 (JNK-2) for possible anticancer activity. These derivatives inhibited the growths of DLD-1, HeLa, K-562, SUIT-2 and HepG2 cancer cell lines, with minimum concentration required to inhibit half of the cellular growth (IC50) values of 2.7–63 μM. The tests confirmed that 5b and 5d were potent JNK-2 inhibitors, with IC50 of 2.0 and 0.9 μM, respectively, whereas 6 h selectively inhibited EGFR protein kinase (EGFR-PK) (IC50 = 1.7 μM). Notably, 6c inhibited both kinases, with IC50 values of 2.7 and 3.0 μM against EGFR-PK and JNK-2, respectively, offering a reference for designing mutual inhibitors of EGFR/JNK-2. The docking studies revealed the ability of the pyrazole ring to bind to the hinge region of the ATP binding site, thereby supporting the experimental inhibitory results. Furthermore, the developed compounds could induce apoptosis and induce cell cycle arrest at different cell phases.

Correlation of MRI quantitative perfusion parameters with EGFR, VEGF and EGFR gene mutations in non-small cell cancer

To explore the relationship between quantitative perfusion histogram parameters of dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) with the expression of tumor tissue epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF) and EGFR gene mutations in non-small cell lung cancer (NSCLC). A total of 44 consecutive patients with known NSCLC were recruited from March 2018 to August 2021. Histogram parameters (mean, uniformity, skewness, energy, kurtosis, entropy, percentile) of each (Ktrans, Kep, Ve, Vp, Fp) were obtained by Omni Kinetics software. Immunohistochemistry staining was used in the detection of the expression of VEGF and EGFR protein, and the mutation of EGFR gene was detected by PCR. Corresponding statistical test was performed to compare the parameters and protein expression between squamous cell carcinoma (SCC) and adenocarcinoma (AC), as well as EGFR mutations and wild-type. Correlation analysis was used to evaluate the correlation between parameters with the expression of VEGF and EGFR protein. Fp (skewness, kurtosis, energy) were statistically significant between SCC and AC, and the area under the ROC curve were 0.733, 0.700 and 0.675, respectively. The expression of VEGF in AC was higher than in SCC. Fp (skewness, kurtosis, energy) were negatively correlated with VEGF (r = − 0.527, − 0.428, − 0.342); Ktrans (Q50) was positively correlated with VEGF (r = 0.32); Kep (energy), Ktrans (skewness, kurtosis) were positively correlated with EGFR (r = 0.622, r = 0.375, 0.358), some histogram parameters of Kep, Ktrans (uniformity, entropy) and Ve (kurtosis) were negatively correlated with EGFR (r = − 0.312 to − 0.644). Some perfusion histogram parameters were statistically significant between EGFR mutations and wild-type, they were higher in wild-type than mutated (P < 0.05). Quantitative perfusion histogram parameters of DCE-MRI have a certain value in the differential diagnosis of NSCLC, which have the potential to non-invasively evaluate the expression of cell signaling pathway-related protein.

Exploring Bioactive Phytochemicals in Gymnema sylvestre: Biomedical Uses and Computational Investigations

The main objective of this research was to perform Gymnema sylvestre (Asclepiadaceae) extract’s phytochemical screening and identify its therapeutic potential. Using a Soxhlet apparatus, the powdered plant material was extracted using ethyl acetate. The preliminary phytochemical analysis confirmed the presence of alkaloids, flavonoids, phenols, glycosides, and steroids. Gas chromatography–mass spectroscopy analysis of the extract was performed and confirmed the presence of 11 compounds. As per the quantitative analysis, the extract exhibited a phenolic content of 948 µg gallic acid equivalent/g dry weight, a total flavonoid content of 398 µg quercetin equivalent/g dry weight, and an alkaloid content of 487 µg atropine equivalent/g dry weight. As per the in vitro cytotoxicity test using A549 cells, the IC50 (half-maximal inhibitory concentration) value for the extract was found to be 76.06 ± 1.26 µg/mL, indicating its cytotoxic effect on the cells. The ethyl acetate extract showed significant antibacterial efficacy, as evidenced by a zone of clearance measuring 3 mm against Escherichia coli and 6 mm against Bacillus subtilis. For anthelmintic activity, the earthworm paralysis time induced by G. sylvestre extract (10 mg/mL) was 28.13 ± 0.8 min, and the time of death was 68.21 ± 1.72 min. In comparison, the reference drug, piperazine citrate (10 mg/mL), caused paralysis in 22.18 ± 1.02 min and resulted in death at 66.22 ± 2.35 min. Similarly, the coagulation time was notably prolonged, with blood clot formation observed at 1 min and 40 s, at a concentration of 1 mg/mL, which underscores the potential anticoagulant or hemostatic modulation properties of G. sylvestre extract. The test extract showed good inhibition of alpha-amylase activity and exhibited an IC50 value of 15.59 µg/mL. The IC50 value for DPPH (2,2-diphenyl-1-picrylhydrazyl)-scavenging activity for the extract was 19.19 µg/mL. Based on the GCMS results, the compound 2,7-dimethyl-undecane was selected for its anticancer potential. Docking studies were conducted with the epidermal growth factor receptor (EGFR) protein, specifically the 5WB7 variant associated with lung cancer. The docking score was −4.5, indicating a potential interaction. Key interaction residues such as ASN328, VAL350, and THR358 were identified. Overall, this research provides valuable insights into the phytochemical composition and diverse biological activities of G. sylvestre extract, offering a foundation for further exploration of its medicinal and pharmacological potential.