Epidermal Growth Factor Receptor Phosphorylation Research Articles

Osimertinib exacerbates immune checkpoint inhibitor-related severe adverse events by activating the IL-6/JAK/STAT3 pathway in macrophages.

The combination of epithelial growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) and immune checkpoint inhibitors (ICIs) leads to an increased incidence of severe immune-related adverse events (irAEs). However, the mechanisms underlying macrophages in irAEs have not been elucidated. An osimertinib and ICI-induced irAE mouse model was constructed. Lung micro-CT scans were used to assess the degree of inflammatory infiltration. Hematoxylin-eosin staining was used to analyze the histopathologic inflammatory infiltration in mouse liver and lung tissues. Flow cytometry was used to detect the percentages of T cells, NK cells, and macrophages and the expression of EGFR. Enzyme-linked immunosorbent assay (ELISA) was used to detect the serum interleukin (IL)-6, alanine transaminase (ALT), ferritin, and tumor necrosis factor (TNF)-α levels. Total RNA extracted from mouse liver macrophages was analyzed by RNA-seq. Simple Western blot analysis was used to detect the IL-6/JAK/STAT3 pathway activation state. Osimertinib combined with ICIs upregulated EGFR expression on macrophages with increased serum IL-6, ALT, and ferritin levels. RNA-seq and simple Western blot analysis of mouse liver macrophages confirmed that that the IL-6/JAK/STAT3 pathway was activated in the combination treatment group. Ruxolitinib blocked the IL-6/JAK/STAT3 pathway and significantly decreased the serum IL-6, ALT, and ferritin levels in the combination treatment group. An osimertinib and ICI-induced irAE mouse model was constructed that showed osimertinib combined with ICIs inhibited EGFR phosphorylation and activated the IL-6/JAK/STAT3 signaling pathway in mouse liver macrophages, which led to the release of relevant cytokines.

Targeting EGFR Activation to Overcome Gemcitabine Resistance in Cholangiocarcinoma.

Chemotherapy resistance is an important problem in the treatment of patients with cholangiocarcinoma (CCA) who are not eligible for surgery. This study aimed to overcome gemcitabine (Gem) resistance in CCA by investigating and targeting Gem resistance-associated molecules. Three stable Gem-resistant CCA cell lines (CCA-GemR) were established by gradually exposing CCA cell lines to Gem. The cells were characterized in terms of growth, cross-resistance to chemotherapeutic drugs, cell cycle distribution, and colony formation. The molecular mechanisms related to Gem resistance were assessed using a phosphorylation array assay and protein expression was confirmed using western blotting analysis. The targeted molecules were subsequently analyzed using PanDrugs to identify potential targeted therapies. The drug was used to enhance Gem sensitivity. The results demonstrated that CCA-GemR cells grow more slowly compared to their parental cell lines. Cell cycle analysis revealed an increase in KKU-213A-GemR and KKU-213B-GemR cell accumulation in the G1 phase. Moreover, cross-resistance to 5-FU and cisplatin was observed in all CCA-GemR cells. The Proteome Profiler Human Phospho-Kinase Array showed increased phosphorylation of EGFR in CCA-GemR cells. Erlotinib, a specific inhibitor of EGFR, significantly enhanced the anti-tumor activity of Gem with a synergistic effect (Combination index <1). Western blot analysis confirmed that phosphorylation of EGFR increased in cells treated with Gem, whereas the expression was significantly decreased in cells treated with either erlotinib alone or in combination with Gem. EGFR is a potential target molecule for reducing Gem resistance and enhancing its anti-tumor effects in patients with CCA.

Transcription factor PRRX1-activated ANXA6 facilitates EGFR-PKCα complex formation and enhances cisplatin sensitivity in bladder cancer

BackgroundTumor resistance to cisplatin represents a major clinical challenge, particularly in bladder cancer (BC). ANXA6 is a member of annexin family, and its role in cisplatin resistance remains unclear. This study explores ANXA6's role in promoting cisplatin sensitivity. MethodsBioinformatics analyses and clinical specimen verifications assessed the correlation between ANXA6 and cisplatin treatment. A series of assays, including CCK-8, clone formation assay, flow cytometry assays for reactive oxygen species (ROS) and apoptosis, and comet assays, were used to confirm ANXA6's role in enhancing cisplatin sensitivity and re-sensitizing resistant BC cells. Mass spectrometry, immunofluorescence, and co-immunoprecipitation experiments elucidated ANXA6's role in enhancing PKCα/EGFR complex formation and inhibiting the EGFR pathway. ChIP-PCR and dual-luciferase assays determined PRRX1's regulatory role on ANXA6 transcription. Finally, the impact of ANXA6 in vivo was evaluated using xenograft models. ResultsBioinformatics analyses showed a significant correlation between ANXA6 expression and cisplatin sensitivity. In vitro and in vivo experiments confirmed that ANXA6 was a new target for cisplatin treatment. ANXA6 overexpression not only enhanced cell viability inhibition, DNA damage and apoptosis caused by cisplatin, but also re-sensitized cisplatin-resistant cells. Mechanistically, ANXA6 promotes PKCα/EGFR complex formation, inhibiting EGFR phosphorylation and downstream AKT and ERK1/2. Moreover, PRRX1 was identified as a transcription factor promoting ANXA6 expression, thereby augmenting the cytotoxic effects of cisplatin. ConclusionOur study reveals the mechanism by which ANXA6 enhances cisplatin sensitivity and re-sensitizes resistant cells. The roles of PRRX1 and ANXA6 in cisplatin resistance offer new therapeutic targets to overcome cisplatin resistance in clinical practice.

Loss of ERα involved-HER2 induction mediated by the FOXO3a signaling pathway in fulvestrant-resistant breast cancer

Patients with estrogen receptor alpha (ERα)-positive breast cancer are commonly treated with anti-estrogen drugs as an initial treatment strategy. Fulvestrant, an estrogen receptor antagonist, effectively blocks ERα signaling; however, long-term fulvestrant treatment induces drug resistance in the absence of ERα. In this study, we investigated the molecular mechanism underlying the loss of ERα, FOXO3a, and induction of HER2 in fulvestrant-resistant breast cancer. Short-term fulvestrant treatment degraded ERα proteins via the ubiquitin-proteasome degradation pathway in MCF7 cells. MCF7 cells turn into highly proliferative cells (fulvestrant-resistant cells: Ful-R) after long-term fulvestrant treatment. These cells exhibit markedly suppressed estrogen and progesterone receptor levels. The phosphorylation of EGFR, HER2, and ERK was induced in Ful-R, and these phosphorylation inhibitors suppressed cell proliferation in Ful-R. FOXO3a, a transcriptional regulator of ERα was decreased in Ful-R, and a ubiquitin-proteasome inhibitor restored the expression of FOXO3a. These results suggest that the suppression of FOXO3a and ERα led to the increased expression of TGF-α, EGFR, and HER2 and subsequent cell proliferation in Ful-R. This study highlights the potential development of therapeutic drugs targeting FOXO3a for the treatment of HER2-positive, estrogen, and progesterone receptor-negative her2-type proliferative breast cancers.

Investigating the anti-cancer potential of sulfatase 1 and its underlying mechanism in non-small cell lung cancer

Objective: Patients with non-small cell lung cancer (NSCLC) have poor prognoses. Sulfatase 1 (SULF1) is an extracellular neutral sulfatase and is involved in multiple physiological processes. Hence, this study investigated the function and possible mechanisms of SULF1 in NSCLC. Material and Methods: Difference in SULF1 expression level between tumors and normal lung tissues was analyzed through bioinformatics and clinical sampling, and the effects of SULF1 expression on prognosis were investigated through Kaplan–Meier analysis. SULF1 level in NSCLC cells was modulated through small interfering ribonucleic acid interference. NSC228155, which is an epidermal growth factor receptor (EGFR)/mitogen-activated protein kinase (MAPK) signaling pathway agonist, was for handling NSCLC cells. SULF1 expression level was tested through quantitative reverse transcriptase real-time polymerase chain reaction. Cell proliferation, migration, and invasion were evaluated with cell counting kit-8, 5-ethynyl-2-deoxyuridine, and transwell assays, and the levels of epithelial-to-mesenchymal transition (EMT)- and EGFR/MAPK pathway-related proteins were detected through Western blot. Results: Bioinformatics and clinical samples showed that NSCLC tumor tissues had elevated SULF1 expression levels relative to those of normal tissues (P &lt; 0.05). Patients with NSCLC and high SULF1 expression levels experienced poorer prognosis than those of low SULF1 expression levels (P &lt; 0.05). SULF1 knockdown repressed the malignant biological behavior, including proliferation, migration, and invasion, of the NSCLC cells (P &lt; 0.05). Mechanistically, SULF1 knockdown augmented E-cadherin level and abated N-cadherin and vimentin protein levels (P &lt; 0.05). These results confirmed that EMT was inhibited. In addition, the knockdown of SULF1 reduced the phosphorylation of EGFR, extracellular signal-regulated kinase, p38 MAPK and c-Jun N-terminal kinase, and NSC228155 partially reversed these changes, which were affected by SULF1 knockdown. Meanwhile, NSC228155 partially reversed the inhibition of EMT, migration, and invasion affected by SULF1 knockdown. Conclusion: SULF1 knockdown inhibits the proliferation, migration, invasion, and EMT of NSCLC cells by inactivating EGFR/MAPK pathway.

C1GALT1 high expression enhances the progression of glioblastoma through the EGFR-AKT/ERK cascade

Core1 β1,3-galactosyltransferase (C1GALT1) is an essential glycotransferase controlling the elongation of GalNAc-type O-glycosylation and its altered expression contributes tumor progression in various cancers. However, the mechanism how C1GALT1 influences gliomas remains unclear. Here，our results from The Cancer Genome Atlas (TCGA) database, The Chinese Glioma Genome Atlas (CGGA) database and the Clinical Proteomic Tumor Analysis Consortium (CPTAC) database showed that the expression of C1GALT1 was increased in higher grade gliomas namely glioblastoma compared with low grade gliomas or non-tumor tissues and significantly associated with poor survival. Downregulation of C1GALT1 suppressed cell proliferation, invasion, and migration in glioma cell lines. Consistent with the result in vitro, C1GALT1 knockdown distinctly inhibited the weight and tumor growth in nude mice. Mechanistically, C1GALT1 knockdown decreased the level of terminal galactose O-glycosylation and phosphorylation on epidermal growth factor receptor (EGFR). Moreover, The AKT/ERK phosphorylation was attenuated in C1GALT1 knockdown cells. And C1GALT1 knockdown decreased the expression of cyclinD1, matrix metalloproteinase 9 (MMP9) through the AKT/ERK signaling pathway Furthermore, transcription factor SP1 which the expression was found to be associated the C1GALT1 expression could bind to the promoter of C1GALT1 gene and regulated its expression. In conclusion, our data show that C1GALT1 enhances the progression of glioma by regulated the O-glycosylation and phosphorylation of EGFR and the subsequent downstream AKT/ERK signaling pathway. Therefore, C1GALT1 represents a potential target for the diagnosis and treatment of glioma.

Inhibitory effect of 1,4,5,6-tetrahydroxy-7,8-diprenylxanthone against NSCLC with L858R/T790M/C797S mutant EGFR

Epidermal growth factor receptor (EGFR)-activating mutations are critical factors in the development of EGFR-driven non-small-cell lung cancer (NSCLC), and molecular targeted therapies have focused on inhibiting these mutations. EGFR tyrosine kinase inhibitors (TKIs) have remarkable inhibitory effects on NSCLC with EGFR mutations. However, acquired resistance limits the clinical application of EGFR-TKIs, highlighting the need for discovery of novel therapeutic strategies. 1,4,5,6-tetrahydroxy-7,8-diprenylxanthone is a natural product derived from Garcinia xanthochymus, has shown potential anti-tumor activity, but the underlying mechanism needs further elucidation. In this study, we developed Ba/F3 and NIH/3T3 cells harboring EGFR L858R/T790M/C797S mutation, and then found that 1,4,5,6-tetrahydroxy-7,8-diprenylxanthone exerted inhibitory effects against cells with EGFR L858R/T790M/C797S mutation. Additionally, 1,4,5,6-tetrahydroxy-7,8-diprenylxanthone exhibited potent anti-tumor activity against cells harboring the triple-mutant EGFR by promoting apoptosis and inducing changes in cell cycle distribution. Furthermore, 1,4,5,6-tetrahydroxy-7,8-diprenylxanthone was found to significantly reduce tumor growth via suppressing the phosphorylation of EGFR in tumor tissues. These effects are associated with binding of 1,4,5,6-tetrahydroxy-7,8-diprenylxanthone to EGFR resulting in the suppression of extracellular signal-regulated kinase (Erk) phosphorylation. In conclusion, our results suggest that 1,4,5,6-tetrahydroxy-7,8-diprenylxanthone may be a potential novel candidate for further investigation and treatment of NSCLC with the triple-mutant EGFR.

Inhibition of EGFR Pathway Suppresses M1 Macrophage Polarization and Osteoclastogenesis, Mitigating Titanium Particle-Induced Bone Resorption.

The polarization of macrophages towards the pro-inflammatory M1 phenotype and osteoclast overactivation play a significant role in the pathogenesis of aseptic loosening of orthopedic implants. This study sought to examine the expression and activation of macrophages and osteoclasts in implant biopsies with respect to epidermal growth factor receptor (EGFR) signaling and to assess the potential of EGFR inhibition in mitigating titanium particle-induced bone resorption in a cranial resorption murine model. Bone marrow-derived macrophages (BMDMs) were stimulated with Tumor Necrosis Factor-alpha (TNF-α) and Interferon-gamma (IFN-γ) initially. Subsequently, Osteoclast differentiation was initiated after Gefitinib was added to the treatment groups. Male C57BL/6 mice were treated with Gefitinib or 0.5% Carboxymethyl Cellulose-Sodium (CMC-Na) by oral gavage daily for two weeks. A sham group received no further intervention, while the other groups had titanium particles implanted. Tissues were collected and analyzed by measurements such as micro-computed tomography (micro-CT) analyses, histology, immunofluorescence stainings, cell viability assays, assays for resorption pit formation, Reverse Transcription-Polymerase Chain Reactions (RT-PCRs), and Western blots were conducted. The study demonstrated a significant upregulation of EGFR in response to titanium particle exposure. Inhibition of EGFR phosphorylation with gefitinib effectively reduced bone degradation at osteolytic sites in a murine model. Gefitinib treatment led to a notable reduction in M1 macrophage polarization, as indicated by immunofluorescence staining and Western blot analysis of macrophage markers. Mechanistically, selective EGFR inhibitors mitigate osteoclastogenesis and osteoclast resorption by inhibiting the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) and nuclear factor-κB (NF-κB) signaling pathways. Our findings provide compelling evidence of the essential role of EGFR-related pathways in M1 polarization, osteoclast activation, and ensuing periprosthetic osteolysis. Overall, EGFR presents a novel target for addressing bone resorption-associated conditions triggered by particles or modulated by macrophages and osteoclasts.

Fully human monoclonal antibody targeting the cysteine-rich substrate-interacting region of ADAM17 on cancer cells

ADAM17 sheds EGFR/erbB ligands and triggers oncogenic pathways that lead to the progression of solid tumors. We targeted the ADAM17 disintegrin and cysteine rich domain region (D+C) to generate a panel of single-chain antibody fragments (scFvs) that selectively bind to the D or C domains of ADAM17, but not of ADAM10 or ADAM19. From the panel, we selected one scFv, referred to as C12, based on its high binding affinity towards the target, and re-formatted it to a full IgG for further studies. High-resolution cryo-electron microscopy studies documented that the mAb binds to the ADAM17 C-domain that in ADAM proteases, notably ADAM10 and ADAM17, is known to impart substrate-specificity. The C12 mAb significantly inhibited EGFR phosphorylation in cancer cell lines by hindering the cleavage of EGFR ligands tethered to the cell surface. This inhibition provides a mechanism for potential anti-tumor effects, and indeed C12 diminished the viability of a variety of EGFR-expressing cancer cell lines. Cell-based ELISA studies revealed that C12 preferentially bound to activated ADAM17 present on tumor cells, as compared to the autoinhibited ADAM17 that is the predominant form on HEK293 and other non-tumor cells. C12 also exhibited tumor growth inhibition in an ovarian cancer xenograft mouse model. Consistent with its selective tumor cell binding in vitro, radioimmuno PET (positron emission tomography) imaging with 89Zr-DFO-C12 in mouse xenograft models confirmed tumoral accumulation of the C12 mAb.

Emodin Inhibited MUC5AC Mucin Gene Expression via Affecting EGFR-MAPK-Sp1 Signaling Pathway in Human Airway Epithelial Cells.

The aim of this study was to evaluate emodin, a natural trihydroxyanthraquinone compound found in the roots and barks of several plants including rhubarb and buckthorn, might attenuate epidermal growth factor (EGF)-induced airway MUC5AC mucin gene expression. The human pulmonary mucoepidermoid NCI-H292 cells were pretreated with for 30 min and then stimulated with EGF for the following 24 h. The effect of emodin on EGF-induced mitogen-activated protein kinase (MAPK) signaling pathway was examined. As a result, emodin blocked the expression of MUC5AC mucin mRNA and production of mucous glycoprotein via suppressing the phosphorylation of EGF receptor (EGFR), phosphorylation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) 1 and 2 (MEK1/2), phosphorylation of p38 MAPK, phosphorylation of ERK 1/2 (p44/42), and the nuclear expression of specificity protein-1 (Sp1). These findings imply that emodin has a potential to mitigate EGF-stimulated mucin gene expression by inhibiting the EGFR-MAPK-Sp1 signaling pathway, in NCI-H292 cells.

Single molecule tracking based drug screening

The single-molecule tracking of transmembrane receptors in living cells has provided significant insights into signaling mechanisms, such as mobility and clustering upon their activation/inactivation, making it a potential screening method for drug discovery. Here we show that single-molecule tracking-based screening can be used to explore compounds both detectable and undetectable by conventional methods for disease-related receptors. Using an automated system for a fast large-scale single-molecule analysis, we screen for epidermal growth factor receptor (EGFR) from 1134 of FDA approved drugs. The 18 hit compounds include all EGFR-targeted tyrosine kinase inhibitors (TKIs) in the library that suppress any phosphorylation-dependent mobility shift of EGFR, proving the concept of this approach. The remaining hit compounds are not reported as EGFR-targeted drugs and do not inhibit EGF-induced EGFR phosphorylation. These non-TKI compounds affect the mobility and/or clustering of EGFR without EGF and induce EGFR internalization, to impede EGFR-dependent cell growth. Thus, single-molecule tracking provides an alternative modality for discovering therapeutics on various receptor functions with previously untargeted mechanisms.

Assembly‐Glued Ligands and Warheads for Hypoxia‐Activatable Supramolecular Covalent Inhibitors

AbstractTargeted covalent inhibitors are promising for drug discovery and challenged by the strict structural features for target proteins to sustain proximity‐induced conjugations. Herein, an assembly‐glued strategy is reported to create surface‐displayed supramolecular covalent inhibitors that leverage the display‐induced proximity between noncovalent ligands and warheads. The ligand and warhead are obtained via attaching an epidermal growth factor receptor (EGFR)‐binding segment or nitroreductase (NTR)‐activated moiety to bola‐amphiphilic peptides, respectively. Co‐assembling the ligand and warhead with a filler peptide glues them and forms the surface‐displayed covalent inhibitor. While the ligand associates with EGFR, the warhead undergoes NTR‐induced cysteine conjugation facilitated by the ligand‐EGFR association. In vitro studies show the reliable hypoxia‐activated inhibition of EGFR phosphorylation and enhanced cytotoxicity of the covalent inhibitor under hypoxic condition. In addition, this strategy also allows for creating covalent inhibitors targeting proteins without small molecular covalent drugs, due to the display‐induced proximity between ligands and warheads. In vivo results illustrate the prolonged retention of the covalent inhibitor and its efficacy in inhibiting tumor growth. These findings demonstrate that simultaneous surface display of ligands and warheads via assembly adhesives allows for implementation of covalent inhibition functions, thus providing a new strategy for developing covalent inhibitors in the future.

Jiedu recipe, a compound Chinese herbal medicine, suppresses hepatocellular carcinoma metastasis by inhibiting the release of tumor-derived exosomes in a hypoxic microenvironment

ObjectiveTumor-derived exosomes (TDEs) play crucial roles in intercellular communication. Hypoxia in the tumor microenvironment enhances secretion of TDEs and accelerates tumor metastasis. Jiedu recipe (JR), a traditional Chinese medicinal formula, has demonstrated efficacy in preventing the metastasis of hepatocellular carcinoma (HCC). However, the underlying mechanism remains largely unknown. MethodsAnimal experiments were performed to investigate the metastasis-preventing effects of JR. Bioinformatics analysis and in vitro assays were conducted to explore the potential targets and active components of JR. TDEs were assessed using nanoparticle tracking analysis (NTA) and Western blotting (WB). Exosomes derived from normoxic or hypoxic HCC cells (H-TDEs) were collected to establish premetastatic mouse models. JR was intragastrically administered to evaluate its metastasis-preventive effects. WB and lysosomal staining were performed to investigate the effects of JR on lysosomal function and autophagy. Bioinformatics analysis, WB, NTA, and immunofluorescence staining were used to identify the active components and potential targets of JR. ResultsJR effectively inhibited subcutaneous-tumor-promoted lung premetastatic niche development and tumor metastasis. It inhibited the release of exosomes from tumor cells under hypoxic condition. JR treatment promoted both lysosomal acidification and suppressed secretory autophagy, which were dysregulated in hypoxic tumor cells. Quercetin was identified as the active component in JR, and the epidermal growth factor receptor (EGFR) was identified as a potential target. Quercetin inhibited EGFR phosphorylation and promoted the nuclear translocation of transcription factor EB (TFEB). Hypoxia-impaired lysosomal function was restored, and secretory autophagy was alleviated by quercetin treatment. ConclusionJR suppressed HCC metastasis by inhibiting hypoxia-stimulated exosome release, restoring lysosomal function, and suppressing secretory autophagy. Quercetin acted as a key component of JR and regulated TDE release through EGFR-TFEB signaling. Our study provides a potential strategy for retarding tumor metastasis by targeting H-TDE secretion.Please cite this article as: Jia WT, Xiang S, Zhang JB, Yuan JY, Wang YQ, Liang SF, Lin WF, Zhai XF, Shang Y, Ling CQ, Cheng BB. Jiedu recipe, a compound Chinese herbal medicine, suppresses hepatocellular carcinoma metastasis by inhibiting the release of tumor-derived exosomes in a hypoxic microenvironment through the EGFR-TFEB signaling pathway. J Integr Med. 2024; Epub ahead of print.

Vitexin Inhibits TNBC Progression and Metastasis by Modulating Macrophage Polarization Through EGFR Signaling.

Triple-negative breast cancer (TNBC) lacks sensitivity to endocrine and targeted therapies, exhibiting high recurrence and poor prognosis postchemotherapy. Tumor-associated macrophages (TAMs) play a crucial role in cancer progression. Vitexin, a compound with diverse pharmacological effects including anti-cancer activity, remains unexplored in its impact on TAMs during TNBC development. This study aimed to investigate vitexin's effect on TNBC, its regulation of macrophage polarization (M1 vs. M2), and the underlying EGFR/PI3K/AKT/mTOR pathway. Our results demonstrated that vitexin suppressed the proliferation and invasion of TNBC cells (MDA-MB-231 and BT549) while inducing macrophage mediators that further inhibited cancer cell migration. Vitexin also promoted M1 polarization and suppressed M2 polarization, affecting EGFR phosphorylation and downstream signaling. In vivo, vitexin inhibited tumor growth, favoring M1 polarization and suppressing M2 polarization, with synergistic effects when combined with doxorubicin (Dox). These findings offer novel insights into vitexin's potential in TNBC treatment.

A novel protein SPECC1-415aa encoded by N6-methyladenosine modified circSPECC1 regulates the sensitivity of glioblastoma to TMZ

BackgroundCircular RNAs (circRNAs) can influence a variety of biological functions and act as a significant role in the progression and recurrence of glioblastoma (GBM). However, few coding circRNAs have been discovered in cancer, and their role in GBM is still unknown. The aim of this study was to identify coding circRNAs and explore their potential roles in the progression and recurrence of GBM.MethodsCircSPECC1 was screened via circRNAs microarray of primary and recurrent GBM samples. To ascertain the characteristics and coding ability of circSPECC1, we conducted a number of experiments. Afterward, through in vivo and in vitro experiments, we investigated the biological functions of circSPECC1 and its encoded novel protein (SPECC1-415aa) in GBM, as well as their effects on TMZ sensitivity.ResultsBy analyzing primary and recurrent GBM samples via circRNAs microarray, circSPECC1 was found to be a downregulated circRNA with coding potential in recurrent GBM compared with primary GBM. CircSPECC1 suppressed the proliferation, migration, invasion, and colony formation abilities of GBM cells by encoding a new protein known as SPECC1-415aa. CircSPECC1 restored TMZ sensitivity in TMZ-resistant GBM cells by encoding the new protein SPECC1-415aa. The m6A reader protein IGF2BP1 can bind to circSPECC1 to promote its expression and stability. Mechanistically, SPECC1-415aa can bind to ANXA2 and competitively inhibit the binding of ANXA2 to EGFR, thus resulting in the inhibition of the phosphorylation of EGFR (Tyr845) and its downstream pathway protein AKT (Ser473). In vivo experiments showed that the overexpression of circSPECC1 could combine with TMZ to treat TMZ-resistant GBM, thereby restoring the sensitivity of TMZ-resistant GBM to TMZ.ConclusionsCircSPECC1 was downregulated in recurrent GBM compared with primary GBM. The m6A reader protein IGF2BP1 could promote the expression and stability of circSPECC1. The sequence of SPECC1-415aa, which is encoded by circSPECC1, can inhibit the binding of ANXA2 to EGFR by competitively binding to ANXA2 and inhibiting the phosphorylation of EGFR and AKT, thereby restoring the sensitivity of TMZ-resistant GBM cells to TMZ.

Investigating NanoLuc-EGFR engineered cell lines for real-time monitoring of EGFR protein dynamics in live cells

Evaluating the steady-state protein level of the EGFR in live cells presents significant challenges compared to measuring its kinase activity. Traditional testing methods, such as immunoblotting, ELISA, and immunofluorescence assays, are generally restricted to fixed cells or cell lysates. Despite their utility, these methods are cumbersome and provide only intermittent snapshots of EGFR levels at specific time points. With emerging trends in drug development shifting toward engineering novel agents that promote protein degradation, rather than simply inhibiting kinase activity, a tool that enables real-time, quantitative detection of drug effects in live cells could catalyze advances in the field. Such an innovation would expedite the drug development process, enhancing the translation of research findings into effective, patient-centered therapies. The NanoLuc-EGFR cell line, created through CRISPR genome editing, allows for the continuous tracking and analysis of EGFR protein levels and their degradation within live cells. This approach provides quantitative monitoring of protein dynamics in real time, offering insights that go beyond absolute protein levels to include aspects such as protein stability and degradation rate. Using this cell line model, we observed that AT13387 and H84T BanLec induce EGFR degradation in A549-HiBiT cells, with the results confirmed by immunoblotting. In contrast, Erlotinib, Osimertinib, and Cetuximab inhibit EGFR phosphorylation without altering total EGFR levels, as validated by the HiBiT luciferase assay. The NanoLuc-EGFR cell line marks a significant advancement in understanding protein regulation and serves as an instrumental platform for investigating targeted therapies that modulate protein kinases, especially those that induce protein degradation.

Synthesis and Biological Evaluation of Novel Cationic Rhenium and Technetium-99m Complexes Bearing Quinazoline Derivative for Epidermal Growth Factor Receptor Targeting.

Background/Objectives: Epidermal growth factor receptor (EGFR) plays a vital role in cell proliferation and survival, with its overexpression linked to various malignancies, including non-small cell lung cancer (NSCLC). Although EGFR tyrosine kinase inhibitors (TKIs) are a key therapeutic strategy, acquired resistance and relapse remain challenges. This study aimed to synthesize and evaluate novel rhenium-based complexes incorporating EGFR TKIs to enhance anticancer efficacy, particularly in radiosensitization. Methods: We synthesized a rhenium tricarbonyl complex (Complex 2) and its 99mTc analog (Complex 2') by incorporating triphenylphosphine instead of bromine as the monodentate ligand and PF6- as the counter-ion, resulting in a positively charged compound that forms cationic structures. Cytotoxicity and EGFR inhibition were evaluated in A431 cells overexpressing EGFR using MTT assays, Western blotting, and flow cytometry. Radiosensitization was tested through MTT and clonogenic assays. The 99mTc complex's radiochemical yield, stability, and lipophilicity were also assessed. Results: Complex 2 exhibited significant cytotoxicity with an IC50 of 2.6 μM and EGFR phosphorylation inhibition with an IC50 of 130.6 nM. Both complex 1 and 2 induced G0/G1 cell cycle arrest, with Complex 2 causing apoptosis. Radiosensitization was observed at doses above 2 Gy. Complex 2' demonstrated high stability and favorable lipophilicity (LogD7.4 3.2), showing 12% cellular uptake after 30 min. Conclusions: Complexes 2 and 2' show promise as dual-function anticancer agents, offering EGFR inhibition, apoptosis induction, and radiosensitization. Their potential as radiopharmaceuticals warrants further in-depth investigation in preclinical models.

Phase II Clinical Chemoprevention Trial of Weekly Erlotinib before Bladder Cancer Surgery.

We performed a clinical trial in non-muscle invasive urothelial cancer (NMIUC) patients randomized (2:1) to the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor erlotinib or placebo (either orally once weekly x 3 doses prior to scheduled surgery) to assess for a difference in EGFR phosphorylation in tumor adjacent normal urothelium <24 hours post-study dose and tolerance of weekly erlotinib. Thirty-seven volunteers (6 female/31 male, mean age 70, 35 white/2 non-white) with confirmed or suspected NMIUC were enrolled into either erlotinib (n=24; 900 mg-13, 600 mg-11) or placebo (n=13). Immunohistochemical assessment of phosphorylated and total EGFR in adjacent normal urothelium (20 erlotinib; 9 placebo) or tumor (21 erlotinib and 11 placebo subjects) at study end observed no significant difference between those receiving erlotinib or placebo. This was also true for other assessed tissue biomarkers (phosphorylated ERK, ERK, e-cadherin, p53 and Ki67). Adverse events were more common, in a dose-related fashion, in participants receiving erlotinib, e.g. 38% experienced Grade 1 with rare grade 2 diarrhea and skin toxicity vs 8% in placebo. Clinically insignificant, but statistically significant (p=0.001) elevations in serum total bilirubin and creatinine were observed in erlotinib participants. Serum erlotinib and metabolite concentrations (OSI-420) confirmed compliance in all erlotinib subjects and did not significantly differ between the 600 and 900 mg doses. Despite compelling pre-clinical and clinical data for targeted EGFR inhibition in bladder cancer prevention, these data do not support the use of weekly erlotinib to prevent progression of NMI bladder cancer.

QSOX1 exerts anti-inflammatory effects in sepsis-induced acute lung injury: Regulation involving EGFR phosphorylation mediated M1 polarization of macrophages

Sepsis is a systemic inflammatory response caused by an infection, which can easily lead to acute lung injury. Quiescin Q6 sulfhydryl oxidase 1 (QSOX1) is a sulfhydryl oxidase involved in oxidative stress and the inflammatory response. However, there are few reports on the role of QSOX1 in sepsis-induced acute lung injury (SALI). In this study, mice model of SALI was constructed by intraperitoneal injection with lipopolysaccharide (LPS). The increased inflammatory response and lactate dehydrogenase activity in bronchoalveolar lavage fluid (BALF) indicated successful modeling. Increased QSOX1 expression was both observed in lung tissues and lung macrophages of sepsis mice accompanied by increased polarization of M1-type macrophages. To explore the role of QSOX1 in the SALI, lentivirus containing QSOX1-specific overexpression or knockdown vectors were used to change QSOX1 expression in LPS-treated RAW264.7 cells. QSOX1 suppressed LPS-induced M1 polarization and further inhibited inflammatory response in RAW264.7 cells. Interestingly, the phosphorylation of epidermal growth factor receptor (EGFR), the promoter of M1 polarization in macrophages, was found to be downregulated upon QSOX1 overexpression in RAW264.7 cells. Mechanically, the binding of QSOX1 to EGFR protein promoted EGFR ubiquitination and degradation, thereby down-regulating EGFR phosphorylation. Moreover, inhibiting EGFR expression or its phosphorylation restored the impact of QSOX1 silencing on M1 polarization and inflammation in the LPS-treated RAW264.7 cells. In summary, QSOX1 may exert anti-inflammatory effects in SALI by inhibiting EGFR phosphorylation-mediated M1 macrophage polarization. This presented a potential target for the treatment and prevention of SALI.

Discovery of flavonoid-containing compound Lupalbigenin as anti-NSCLC cancer agents via suppression of EGFR and ERK1/2 pathway

Epidermal growth factor receptor exon 20 insertions (EGFR Ex20ins) driver mutations in non–small cell lung cancer (NSCLC) is insensitive to EGFR tyrosine kinase inhibitors (TKIs). Therefore, it is necessary to develop more novel strategy to address the limitations of existing therapies targeting EGFR-mutated NSCLC. Lupalbigenin (LB), a flavonoid compound extracted from Derris scandens, has shown preclinical activity in lung cancer. However, the activity of LB in Ex20ins-driven tumors has not yet been elucidated. In this study, a series of stable BaF/3 cell-line that contains a high proportion (>90 %) of EGFR-eGFP Ex20ins were generated using an IL3-deprivation method. Ba/F3 cell models harboring dissimilar Ex20ins were used to characterize the antineoplastic mechanism of LB. Molecular docking confirmed that the LB could effectively bind to key target EGFR. The in vitro anticancer activity of LB was investigated in engineered Ba/F3 cells bearing diverse uncommon EGFR mutations. LB was shown to be more potent in inhibiting the viability of various uncommon EGFR-mutated cell lines. Mechanistic studies disclosed that LB repressed EGFR phosphorylation and downstream survival pathways in Ba/F3 cells expressing EGFR Ex20ins, resulting in caspase activation by activating the intrinsic apoptotic pathway. Further analyses showed that LB significantly induced G0/G1 cell cycle arrest and apoptosis in cells. LB also reduced the protein expression levels of CDK4, CDK6, CDK8, cyclin D1, cyclin A2, and Bcl2 and promoted the expression of cytochrome C, p27, and p53. In summary, we explored the possible potential targets of LB through network pharmacology and verified the target using in vitro experiments. Furthermore, our results demonstrated that LB showed potential anti-Ex20ins cancer activity through suppression of the EGFR and ERK1/2 signaling pathway in Ba/F3 cells bearing two to three amino acid insertion mutations. These findings suggested that LB might be valuable for further investigation as a potential candidate in the treatment of associated diseases.