EGFR Pathway Research Articles

IGFBP2 from a novel copper metabolism-associated biomarker promoted glioma progression and response to immunotherapy.

Copper metabolism encompasses all cellular metabolic processes involving copper ions and plays a significant role in the pathogenesis of diseases, including cancer. Furthermore, copper is intricately involved in various processes related to nucleotide metabolism. However, a comprehensive analysis of copper metabolism in gliomas remains lacking despite its importance. To address this gap, glioma patients were stratified based on the expression levels of copper metabolism-related genes. By utilizing machine learning techniques, a novel copper metabolism-associated biomarker was developed. The potential of this biomarker in prognosis, mutation analysis, and predicting immunotherapy response efficiency in gliomas was systematically investigated. Notably, IGFBP2, identified as a glioma tumor promoter, was found to promote disease progression and influence immunotherapy response. Additionally, glioma-derived IGFBP2 was observed to enhance microglial migration. High IGFBP2 expression in GBM cells facilitated macrophage interactions through the EGFR, CD63, ITGB1, and CD44 signaling pathways. Discussion: Overall, the copper metabolism-associated biomarker shows promising potential to enhance the clinical management of gliomas, offering valuable insights into disease prognosis and treatment strategies.

The Role of Aplnr Signaling in the Developmental Regulation of Mesenchymal Stem Cell Differentiation from Human Pluripotent Stem Cells.

Stem cells are invaluable resources for personalized medicine. Mesenchymal stem cells (MSCs) have received great attention as therapeutic tools due to being a safe, ethical, and accessible option with immunomodulatory and controlled differentiation properties. Apelin receptor (Aplnr) signaling is reported to be involved in biological events, including gastrulation, mesoderm migration, proliferation of MSCs. However, the knowledge about the exact role and mechanism of Aplnr signaling during mesoderm and MSCs differentiation is still primitive. The current study aims to unveil the role of Aplnr signaling during mesoderm and MSC differentiation from pluripotent stem cells (PSCs) through peptide/small molecule activation, overexpression, knock down or CRISPR/Cas9 mediated knock out of the pathway components. Morphological changes, gene and protein expression analysis, including antibody array, LC/MS, mRNA/miRNA sequencing, reveal that Aplnr signaling promotes mesoderm commitment possibly via EGFR and TGF-beta signaling pathways and enhances migration of cells during mesoderm differentiation. Moreover, Aplnr signaling positively regulates MSCs differentiation from hPSCs and increases MSC characteristics and differentiation capacity by regulating pathways, such as EGFR, TGFβ, Wnt, PDGF, and FGF. Osteogenic, chondrogenic, adipogenic, and myogenic differentiations are significantly enhanced with Aplnr signaling activity. This study generates an important foundation to generate high potential MSCs from PSCs to be used in personalized cell therapy.

SEC61G knockdown enhances the sensitivity of gastric cancer cells to etoposide through EGFR and glycolytic-mediated pathways

SEC61G knockdown enhances the sensitivity of gastric cancer cells to etoposide through EGFR and glycolytic-mediated pathways

3-Acetyl-11-keto-β-boswellic Acid-Based Hybrids Alleviate Acetaminophen-Induced Hepatotoxicity in HepG2 by the Regulation of Inflammatory and Oxidative Stress Pathways: An Integrated Approach.

In an effort to develop new compounds for managing drug-induced liver injury, we prepared 23 novel hybrids based on 3-acetyl-11-keto-β-boswellic acid (AKBA) using various biocompatible linkers. A bioguided approach was employed to identify the most promising hybrid. Eight compounds exhibited superior anti-inflammatory activity compared to the parent compound. Two of these hybrids (5b and 18) were able to reduce gene expression of TNF-α in LPS-induced inflammation in RAW 264.7 cells, similar to dexamethasone. Subsequently, the hepatoprotective potential of these hybrids was evaluated against acetaminophen (APAP) toxicity in HepG2 cells at doses of 1 and 10 μM. Both hybrids effectively restored cytokine levels, which had been elevated by APAP, to normal levels. Furthermore, they normalized depleted superoxide dismutase and reduced glutathione levels while significantly reducing malondialdehyde (MDA) levels. Network pharmacology analysis suggested that AKBA-based hybrids exert their action by regulating PI3K and EGFR pathways, activating anti-inflammatory mechanisms, and initiating tissue repair and regeneration. Molecular docking studies provided insights into the interaction of the hybrids with PI3K. Additionally, the hybrids demonstrated good stability at different pH levels, following first-order kinetics, with relatively long half-lives, suggesting potential for absorption into circulation without significant degradation.

EXPRESSION OF NUCLEOTIDE EXCISION REPAIR PROTEIN ERCC1 IN TUMOR TISSUE AS A PROGNOSTIC FACTOR IN COLORECTAL CANCER

At present, biomarkers of tumor resistance to platinum drugs in colorectal cancer are of much concern. Relapses and metastases occur in 35 % of patients with colorectal cancer (stages I–III) after adjuvant polychemotherapy with platinum-based anticancer drugs within the first 5 years. Resistance to platinum-based drugs is determined by a number of factors, including increased DNA repair capacity.
 The aim of the study is to evaluate the prognostic role of ERCC1 expression in tumor tissue in colorectal cancer.
 Materials and Methods. The authors studied the expression of the nucleotide excision repair protein ERCC1 in tumor tissue as a prognostic marker for adjuvant chemotherapy FOLFOX/XELOX in patients with colorectal cancer (stages II–IV). The correlation between ERCC1 expression in tumor tissue and chemotherapy effectiveness, clinical stage, age, survival, differentiation, and EGFR pathway mutations (NRAS, KRAS, BRAF) was assessed.
 Results. ERCC1 expression was detected in 46.9 % of tumor tissue samples and in 88.9 % of resection line tissue samples. In patients with ERCC1 overexpression, the median survival was significantly lower than in patients with low ERCC1 expression. In tumors with NRAS and BRAF mutations, no ERCC1 expression was observed. No differences were determined in patient according to the disease stage, tumor differentiation, and mutations.
 Cancer tumors is an independent predictor of FOLFOX/XELOX chemotherapy resistance.

β2-AR inhibition enhances EGFR antibody efficacy hampering the oxidative stress response machinery

The β2-Adrenergic receptor (β2-ARs) is a cell membrane-spanning G protein-coupled receptors (GPCRs) physiologically involved in stress-related response. In many cancers, the β2-ARs signaling drives the tumor development and transformation, also promoting the resistance to the treatments. In HNSCC cell lines, the β2-AR selective inhibition synergistically amplifies the cytotoxic effect of the MEK 1/2 by affecting the p38/NF-kB oncogenic pathway and contemporary reducing the NRF-2 mediated antioxidant cell response. In this study, we aimed to validate the anti-tumor effect of β2-AR blockade and the synergism with MEK/ERK and EGFR pathway inhibition in a pre-clinical orthotopic mouse model of HNSCC. Interestingly, we found a strong β2-ARs expression in the tumors that were significantly reduced after prolonged treatment with β2-Ars inhibitor (ICI) and EGFR mAb Cetuximab (CTX) in combination. The β2-ARs down-regulation correlated in mice with a significant tumor growth delay, together with the MAPK signaling switch-off caused by the blockade of the MEK/ERK phosphorylation. We also demonstrated that the administration of ICI and CTX in combination unbalanced the cell ROS homeostasis by blocking the NRF-2 nuclear translocation with the relative down-regulation of the antioxidant enzyme expression. Our findings highlighted for the first time, in a pre-clinical in vivo model, the efficacy of the β2-ARs inhibition in the treatment of the HNSCC, remarkably in combination with CTX, which is the standard of care for unresectable HNSCC.

Design, Synthesis, and Biological Evaluation of Novel 3-Cyanopyridone/Pyrazoline Hybrids as Potential Apoptotic Antiproliferative Agents Targeting EGFR/BRAFV600E Inhibitory Pathways.

A series of novel 3-cyanopyridone/pyrazoline hybrids (21-30) exhibiting dual inhibition against EGFR and BRAFV600E has been developed. The synthesized target compounds were tested in vitro against four cancer cell lines. Compounds 28 and 30 demonstrated remarkable antiproliferative activity, boasting GI50 values of 27 nM and 25 nM, respectively. These hybrids exhibited dual inhibitory effects on both EGFR and BRAFV600E pathways. Compounds 28 and 30, akin to Erlotinib, displayed promising anticancer potential. Compound 30 emerged as the most potent inhibitor against cancer cell proliferation and BRAFV600E. Notably, both compounds 28 and 30 induced apoptosis by elevating levels of caspase-3 and -8 and Bax, while downregulating the antiapoptotic Bcl2 protein. Molecular docking studies confirmed the potential of compounds 28 and 30 to act as dual EGFR/BRAFV600E inhibitors. Furthermore, in silico ADMET prediction indicated that most synthesized 3-cyanopyridone/pyrazoline hybrids exhibit low toxicity and minimal adverse effects.

Human Blood Serum Can Diminish EGFR-Targeted Inhibition of Squamous Carcinoma Cell Growth through Reactivation of MAPK and EGFR Pathways.

Regardless of the presence or absence of specific diagnostic mutations, many cancer patients fail to respond to EGFR-targeted therapeutics, and a personalized approach is needed to identify putative (non)responders. We found previously that human peripheral blood and EGF can modulate the activities of EGFR-specific drugs on inhibiting clonogenity in model EGFR-positive A431 squamous carcinoma cells. Here, we report that human serum can dramatically abolish the cell growth rate inhibition by EGFR-specific drugs cetuximab and erlotinib. We show that this phenomenon is linked with derepression of drug-induced G1S cell cycle transition arrest. Furthermore, A431 cell growth inhibition by cetuximab, erlotinib, and EGF correlates with a decreased activity of ERK1/2 proteins. In turn, the EGF- and human serum-mediated rescue of drug-treated A431 cells restores ERK1/2 activity in functional tests. RNA sequencing revealed 1271 and 1566 differentially expressed genes (DEGs) in the presence of cetuximab and erlotinib, respectively. Erlotinib- and cetuximab-specific DEGs significantly overlapped. Interestingly, the expression of 100% and 75% of these DEGs restores to the no-drug level when EGF or a mixed human serum sample, respectively, is added along with cetuximab. In the case of erlotinib, EGF and human serum restore the expression of 39% and 83% of DEGs, respectively. We further assessed differential molecular pathway activation levels and propose that EGF/human serum-mediated A431 resistance to EGFR drugs can be largely explained by reactivation of the MAPK signaling cascade.

Antimicrobial, Cytotoxic, and α-Glucosidase Inhibitory Activities of Ethanol Extract and Chemical Constituents Isolated from Homotrigona apicalis Propolis-In Vitro and Molecular Docking Studies.

The chemical investigation of Homotrigona apicalis propolis collected in Binh Dinh province, Vietnam, led to the isolation of nine compounds, including four sesquiterpenes: spathulenol (1), 1αH,5βH-aromandendrane-4β,10α-diol (2), 1β,6α-dihydroxy-4(15)-eudesmene (3), and 1βH,5βH-aromandendrane-4α,10β-diol (4); three triterpenes: acetyl oleanolic acid (5), 3α-hydroxytirucalla-8,24-dien-21-oic acid (6), and ursolic acid (7); and two xanthones: cochinchinone A (8) and α-mangostin (9). Sesquiterpens 1-4 and triterpene 6 were isolated for the first time from stingless bee propolis. Plants in the Cratoxylum and Aglaia genus were suggested as resin sources of the propolis sample. In the antibacterial activity evaluation, the EtOH extract only showed moderate activity on S. aureus, while the isolated compounds 7-9 showed good antibacterial activity, with IC50 values of 0.56 to 17.33 µg/mL. The EtOH extract displayed selective cytotoxicity against the A-549 cancer cell line, with IC50 values of 22.82 ± 0.86 µg/mL, and the xanthones 8 and 9 exhibited good activity against the KB, HepG-2, and A-549 cancer cell lines, with IC50 values ranging from 7.55 ± 0.25 µg/mL to 29.27 ± 2.07 µg/mL. The cytotoxic effects of xanthones 8 and 9 were determined by the inhibition of the EGFR and HER2 pathways using a molecular docking study. Compounds 8 and 9 displayed strong binding affinity with EFGR and HER2, with values of -9.3 to -9.9 kcal/mol. Compounds 5, 8, and 9 showed potential α-glucosidase inhibitory activities, which were further confirmed by computational studies. The binding energies of compounds 5, 8, and 9 were lower than that of arcabose.

Molecular and clonal evolution in vivo reveal a common pathway of distant relapse gliomas

The evolutionary trajectories of genomic alterations underlying distant recurrence in glioma remain largely unknown. To elucidate glioma evolution, we analyzed the evolutionary trajectories of matched pairs of primary tumors and relapse tumors or tumor in situ fluid (TISF) based on deep whole-genome sequencing data (ctDNA). We found that MMR gene mutations occurred in the late stage in IDH-mutant glioma during gene evolution, which activates multiple signaling pathways and significantly increases distant recurrence potential. The proneural subtype characterized by PDGFRA amplification was likely prone to hypermutation and distant recurrence following treatment. The classical and mesenchymal subtypes tended to progress locally through subclonal reconstruction, trunk genes transformation, and convergence evolution. EGFR and NOTCH signaling pathways and CDNK2A mutation play an important role in promoting tumor local progression. Glioma subtypes displayed distinct preferred evolutionary patterns. ClinicalTrials.gov, NCT05512325.

Poliovirus receptor (PVR) mediates carboplatin-induced PD-L1 expression in non-small-cell lung cancer cells

Programmed death-ligand-1 (PD-L1) is an immune suppressor that inhibits T cell based immunity. Anti-PD-L1/PD-1 immunotherapy benefits those patients receiving platinum-based combinational chemotherapy. However, the underlying mechanism is still largely unknown. In this study, we found that carboplatin could induce PD-L1 expression in NSCLC H292, A549 and H1299 cells in a dose-dependent manner. mRNA sequencing and the subsequent validation assays found that carboplatin significantly induced PVR expression, which is considered as an immuno-adhesion molecule. Mechanistically, PVR knockdown significantly abrogated carboplatin-induced PD-L1 expression. Functionally, knockdown of PVR significantly reversed the CD3+ T cells proliferation inhibition caused by carboplatin increased PD-L1. Moreover, the carboplatin-induced PVR and subsequent up-regulation of PD-L1 might be mediated via the EGFR, PI3K/AKT, and ERK signaling pathways. Immunohistochemical staining results showed that the PD-L1 expression was positively associated with PVR expression in clinical NSCLC samples. Our study reveals a novel regulatory mechanism of PD-L1 expression, provides evidence that carboplatin inhibits tumor immune response by up-regulating PD-L1 expression and explains the rationale for combining platinum-based chemotherapy with PD-L1/PD-1 inhibitors.

Retraction Note: Hsa_circ_0005379 regulates malignant behavior of oral squamous cell carcinoma through the EGFR pathway

This article has been retracted. Please see the Retraction Notice for more detail: https://doi.org/10.1186/s12885-019-5593-5.

TRAIL and EGFR Pathways Targeting MicroRNAs are Predominantly Regulated in Human Diabetic Nephropathy.

Unbiased microRNA profiling of renal tissue and urinary extracellular vesicles (uEVs) from diabetic nephropathy (DN) subjects may unravel novel targets with diagnostic and therapeutic potential. Here we used the miRNA profile of uEVs and renal biopsies from DN subjects available on the GEO database. The miR expression profiles of kidney tissue (GSE51674) and urinary exosomes (GSE48318) from DN and control subjects were obtained by GEO2R tools from Gene Expression Omnibus (GEO) databases. Differentially expressed miRNAs in DN samples, relative to controls, were identified using a bioinformatic pipeline. Targets of miRs commonly regulated in both sample types were predicted by miRWalk, followed by functional gene enrichment analysis. Gene targets were identified by MiRTarBase, TargetScan and MiRDB. Eight miRs, including let-7c, miR-10a, miR-10b and miR-181c, were significantly regulated in kidney tissue and uEVs in DN subjects versus controls. The top 10 significant pathways targeted by these miRs included TRAIL, EGFR, Proteoglycan syndecan, VEGF and Integrin Pathway. Gene target analysis by miRwalk upon validation using ShinyGO 70 targets with significant miRNA-mRNA interaction. In silico analysis showed that miRs targeting TRAIL and EGFR signaling are predominately regulated in uEVs and renal tissue of DN subjects. After wet-lab validation, the identified miRs- target pairs may be explored for their diagnostic and/or therapeutic potential in diabetic nephropathy.

Induced skin aging by blue-light irradiation in human skin fibroblasts via TGF-β, JNK and EGFR pathways

BackgroundStudies indicate that blue light (BL) irradiation can damage human skins, but the impact of BL irradiation on skin aging is unknown. ObjectivesThis study aimed to give an insight to phenotypic characteristics and molecular mechanism of blue light-induced skin aging, and thus provide a theoretical basis for the precise protection of photodermatosis. MethodsThe effect of BL on skin photoaging in mice was evaluated by non-invasive measurement equipment and histopathology analysis. The effect of BL irradiation on the proliferation of HFF-1 cells was detected by the Real-Time Cell Analyzer. The expression and protein levels of genes associated with skin aging were examined. ResultsOur studies indicated photoaging caused by BL irradiation, including collagen disorder and increased MMP1. BL irradiation also inhibited cell proliferation and collagen expression in human skin fibroblasts by inhibiting TGF-β signaling pathway, based on in vitro experiments. Importantly, BL irradiation promoted the degradation of collagen by increasing MMP1 activated by the JNK/c-Jun and EGFR pathways. Moreover, ROS levels were significantly increased after BL irradiation in human skin fibroblasts. Yet, the transcriptional change in human skin fibroblasts caused by BL irradiation was unable to be completely restored by ROS scavenger. ConclusionBL irradiation down-regulated expression of type I collagen genes and up-regulated MMP1 expression to inhibit the proliferation of human skin fibroblasts. Multiple key pathways including TGF-β, JNK, and EGFR signaling were involved in BL-induced skin aging. Our results provide theoretical bases for the protection of photoaging caused by BL irradiation.

Combinations of PRI-724 Wnt/β-Catenin Pathway Inhibitor with Vismodegib, Erlotinib, or HS-173 Synergistically Inhibit Head and Neck Squamous Cancer Cells.

The Wnt/β-catenin, EGFR, and PI3K pathways frequently undergo upregulation in head and neck squamous carcinoma (HNSCC) cells. Moreover, the Wnt/β-catenin pathway together with Hedgehog (Hh) signaling regulate the activity of cancer stem cells (CSCs). The aim of this study was to investigate the effects of the combinatorial use of the Wnt/β-catenin and Hh pathway inhibitors on viability, cell cycle progression, apoptosis induction, cell migration, and expression of CSC markers in tongue (CAL 27) and hypopharynx (FaDu) cancer cells. Co-inhibition of Wnt signaling with EGFR or PI3K pathways was additionally tested. The cells were treated with selective inhibitors of signaling pathways: Wnt/β-catenin (PRI-724), Hh (vismodegib), EGFR (erlotinib), and PI3K (HS-173). Cell viability was evaluated by the resazurin assay. Cell cycle progression and apoptosis induction were tested by flow cytometric analysis after staining with propidium iodide and Annexin V, respectively. Cell migration was detected by the scratch assay and CSC marker expression by the R-T PCR method. Mixtures of PRI-724 and vismodegib affected cell cycle distribution, greatly reduced cell migration, and downregulated the transcript level of CSC markers, especially POU5F1 encoding OCT4. Combinations of PRI-724 with erlotinib or HS-173 were more potent in inducing apoptosis.

Injury prevents Ras mutant cell expansion in mosaic skin

Healthy skin is a mosaic of wild-type and mutant clones1,2. Although injury can cooperate with mutated Ras family proteins to promote tumorigenesis3–12, the consequences in genetically mosaic skin are unknown. Here we show that after injury, wild-type cells suppress aberrant growth induced by oncogenic Ras. HrasG12V/+ and KrasG12D/+ cells outcompete wild-type cells in uninjured, mosaic tissue but their expansion is prevented after injury owing to an increase in the fraction of proliferating wild-type cells. Mechanistically, we show that, unlike HrasG12V/+ cells, wild-type cells respond to autocrine and paracrine secretion of EGFR ligands, and this differential activation of the EGFR pathway explains the competitive switch during injury repair. Inhibition of EGFR signalling via drug or genetic approaches diminishes the proportion of dividing wild-type cells after injury, leading to the expansion of HrasG12V/+ cells. Increased proliferation of wild-type cells via constitutive loss of the cell cycle inhibitor p21 counteracts the expansion of HrasG12V/+ cells even in the absence of injury. Thus, injury has a role in switching the competitive balance between oncogenic and wild-type cells in genetically mosaic skin.

RETRACTED: Amphiregulin promotes cardiac fibrosis post myocardial infarction by inducing the endothelial-mesenchymal transition via the EGFR pathway in endothelial cells

RETRACTED: Amphiregulin promotes cardiac fibrosis post myocardial infarction by inducing the endothelial-mesenchymal transition via the EGFR pathway in endothelial cells

An integrated genomic approach identifies follistatin as a target of the p63-epidermal growth factor receptor oncogenic network in head and neck squamous cell carcinoma.

Although numerous putative oncogenes have been associated with the etiology of head and neck squamous cell carcinoma (HNSCC), the mechanisms by which these oncogenes and their downstream targets mediate tumor progression have not been fully elucidated. We performed an integrative analysis to identify a crucial set of targets of the oncogenic transcription factor p63 that are common across multiple transcriptomic datasets obtained from HNSCC patients, and representative cell line models. Notably, our analysis revealed FST which encodes follistatin, a secreted glycoprotein that inhibits the transforming growth factor TGFβ/activin signaling pathways, to be a direct transcriptional target of p63. In addition, we found that FST expression is also driven by epidermal growth factor receptor EGFR signaling, thus mediating a functional link between the TGF-β and EGFR pathways. We show through loss- and gain-of-function studies that FST predominantly imparts a tumor-growth and migratory phenotype in HNSCC cells. Furthermore, analysis of single-cell RNA sequencing data from HNSCC patients unveiled cancer cells as the dominant source of FST within the tumor microenvironment and exposed a correlation between the expression of FST and its regulators with immune infiltrates. We propose FST as a prognostic biomarker for patient survival and a compelling candidate mediating the broad effects of p63 on the tumor and its associated microenvironment.

CircEGFR reduces the sensitivity of pirarubicin and regulates the malignant progression of triple-negative breast cancer via the miR-1299/EGFR axis

Circular RNAs (circRNAs) have been found to be involved in cancer progression and chemotherapy sensitivity. However, the biological function of circRNAs in triple-negative breast cancer (TNBC) and its effect on the sensitivity to pirarubicin (THP) chemotherapy are still unclear. CircEGFR (hsa_circ_0080220) was screened and verified by bioinformatics analysis, proving it was highly expressed in TNBC cell lines, patient tissues, and plasma exosomes, and was associated with poor prognosis of patients. The expression level of circEGFR in patient tissue has potential diagnostic value to distinguish TNBC tissue from normal breast tissue. In vitro studies confirmed that overexpression of circEGFR promoted the proliferation, migration, invasion, and EMT of TNBC cells and decreased the sensitivity of THP treatment while silencing circEGFR showed the opposite effect. The circEGFR/miR-1299/EGFR pathway was cascaded and verified. CircEGFR regulated malignant progression of TNBC by regulating EGFR via sponging miR-1299. THP can inhibit the malignant phenotype of MDA-MB-231 cells by downregulating the expression of circEGFR. In vivo studies confirmed that overexpression of circEGFR can promote tumor growth and EMT and reduce tumor sensitivity to THP treatment. Silencing circEGFR inhibited the malignant progression of the tumor. These results revealed circEGFR is a promising biomarker for TNBC diagnosis, therapeutic and prognosis.

Midazolam impedes lung carcinoma cell proliferation and migration via EGFR/MEK/ERK signaling pathway.

Non-small-cell lung cancer (NSCLC) is a dominating type of lung cancer with high morbidity and mortality. Midazolam has been reported to promote cell apoptosis in NSCLC, but the molecular mechanism of midazolam remains to be further explored. In the current work, cell viability, proliferation, migration, and apoptosis rates of NSCLC cells treated with midazolam were measured using cell counting kit-8 assay, 5-ethynyl-2'-deoxyuridine (EdU) and colony formation assays, transwell, and flow cytometry assay, respectively, to evaluate the malignant behaviors. Western blot was applied to access EGFR/MEK/ERK pathway-related protein levels. The results demonstrated midazolam significantly declined the viability of NSCLC cells. Furthermore, midazolam restrained cell proliferation and migration and contributed to cell apoptosis in NSCLC. Midazolam exerted suppressive function to EGFR pathway during NSCLC development. Moreover, the activation of EGFR/MEK/ERK pathway abrogated the effects of midazolam on NSCLC cell proliferation, apoptosis, and migration. Taken together, midazolam exhibited anti-tumor effects hallmarked by EGFR pathway inhibition, providing a novel insight into the treatment of NSCLC.