PD-L1 induces autophagy and primary resistance to EGFR-TKIs in EGFR-mutant lung adenocarcinoma via the MAPK signaling pathway.

Resistance to epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) is a significant cause of treatment failure and cancer recurrence in non-small cell lung cancer (NSCLC). Approximately 30% of patients with EGFR-activating mutations exhibit primary resistance to EGFR-TKIs. However, the potential mechanisms of primary resistance to EGFR-TKIs remain poorly understood. Recent studies have shown that increased expression of programmed death ligand-1 (PD-L1) is associated with EGFR-TKIs resistance. Therefore, the present study aimed to investigate the mechanism of PD-L1 in primary resistance to EGFR-TKIs in EGFR-mutant lung adenocarcinoma (LUAD) cells. We found that PD-L1 was associated with poor prognosis in patients with EGFR-mutant LUAD, while the combination of EGFR-TKIs with chemotherapy could improve its therapeutic efficacy. In vitro and in vivo experiments revealed that PD-L1 promoted the proliferation and autophagy and inhibited the apoptosis of LUAD cells. Mechanistic studies demonstrated that upregulation of PD-L1 was critical in inducing autophagy through the mitogen-activated protein kinase (MAPK) signaling pathway, which was beneficial for tumor progression and the development of gefitinib resistance. Furthermore, we found that gefitinib combined with pemetrexed could synergistically enhance antitumor efficacy in PD-L1-overexpression LUAD cells. Overall, our study demonstrated that PD-L1 contributed to primary resistance to EGFR-TKIs in EGFR-mutant LUAD cells, which may be mediated by inducing autophagy via the MAPK signaling pathway. These findings not only help improve the prognosis of patients with EGFR-mutant LUAD but also provide a reference for the research of other cancer types.

METTL1/FOXM1 promotes lung adenocarcinoma progression and gefitinib resistance by inhibiting PTPN13 expression.

INTRODUCTION: Lung adenocarcinoma (LUAD) is the most common malignant tumor in respiratory system. Methyltransferase-like 1 (METTL1) is a driver of m7G modification in mRNA. This study aimed to demonstrate the role of METTL1 in the proliferation, invasion and Gefitinib-resistance of LUAD. METHODS: Public datasets were downloaded from the Gene Expression Profiling Interactive Analysis (GEPIA) and GSE31210 datasets. Malignant tumor phenotypes were tested in vitro and in vivo through biological function assays and nude mouse with xenograft tumors. RNA immunoprecipitation assays were conducted to determine the interaction between METTL1 protein and FOXM1 mRNA. Public transcriptional database, Chromatin immunoprecipitation and luciferase report assays were conducted to detect the downstream target of a transcriptional factor FOXM1. Half maximal inhibitory concentration (IC50) was calculated to evaluate the sensitivity to Gefitinib in LUAD cells. RESULTS: The results showed that METTL1 was upregulated in LUAD, and the high expression of METTL1 was associated with unfavorable prognosis. Through the m7G-dependent manner, METTL1 improved the RNA stability of FOXM1, leading to the up-regulation of FOXM1. FOXM1 transcriptionally suppressed PTPN13 expression. The METTL1/FOXM1/PTPN13 axis reduced the sensitivity of LUAD cells to Gefitinib. Taken together, our data suggested that METTL1 plays oncogenic role in LUAD through inducing the m7G modification of FOXM1, therefore METTL1 probably is a new potential therapeutic target to counteract Gefitinib resistance in LUAD.

Impact of Interleukin-6 Activation and Arthritis on Epidermal Growth Factor Receptor (EGFR) Activation in Sensory Neurons and the Spinal Cord.

In tumor cells, interleukin-6 (IL-6) signaling can lead to activation of the epidermal growth factor receptor (EGFR), which prolongs Stat3 activation. In the present experiments, we tested the hypothesis that IL-6 signaling activates EGFR signaling in peripheral and spinal nociception and examined whether EGFR localization and activation coincide with pain-related behaviors in arthritis. In vivo in anesthetized rats, spinal application of the EGFR receptor blocker gefitinib reduced the responses of spinal cord neurons to noxious joint stimulation, but only after spinal pretreatment with IL-6 and soluble IL-6 receptor. Using Western blots, we found that IL-6-induced Stat3 activation was reduced by gefitinib in microglial cells of the BV2 cell line, but not in cultured DRG neurons. Immunohistochemistry showed EGFR localization in most DRG neurons from normal rats, but significant downregulation in the acute and most painful arthritis phase. In the spinal cord of mice, EGFR was highly activated mainly in the chronic phase of inflammation, with localization in neurons. These data suggest that spinal IL-6 signaling may activate spinal EGFR signaling. Downregulation of EGFR in DRG neurons in acute arthritis may limit nociception, but pronounced delayed activation of EGFR in the spinal cord may be involved in chronic inflammatory pain.

Influence of Tyrosine Kinase Inhibitors and Everolimus on TGFß1 and TGF-ß Receptor 2 in Squamous Cell Carcinoma Cells.

BACKGROUND/AIM: Transforming growth factor-ß (TGF-ß) plays a significant role in the formation of different cancer subtypes. There is evidence that TGF-ß pathways promote cancerogenic cell characteristics but also have tumor-suppressor capabilities. The tyrosine kinase inhibitors nilotinib, dasatinib, erlotinib, gefitinib, and everolimus are approved as targeted therapies for several tumor entities, including head and neck squamous cell carcinoma (HNSCC). This study aimed to investigate the effects of these substances on the expression levels of TGFß1 and TGF-ß receptor type 2 (TGFßR2) in HPV-negative and HPV-positive SCC cell cultures. MATERIALS AND METHODS: Expression patterns of TGFß1 and TGFßR2 were determined using enzyme-linked immunosorbent assay (ELISA) in three HNSCC cell lines (i.e., HNSCC-11A, HNSCC-14C, and CERV196). These cells were incubated with nilotinib, dasatinib, erlotinib, gefitinib, and everolimus (20 µmol/l) and compared to a chemonaive control. An assessment of concentration levels was conducted after 24, 48, 72, and 96 h of treatment. RESULTS: Statistically significant changes in the expression levels of TGFß1 and TGFßR2 were found in all tested cell cultures (p<0.05) compared to the negative control. An increase in TGFß-R2 expression was detected after treatment with most of the tested tyrosine kinase inhibitors, whereas a reduction in TGFß1 was observed. The addition of everolimus had the opposite effect on both TGFßR2 and TGF-B1- expression. CONCLUSION: Expression of TGFß1 and TGFßR2 was detected in all cultured HNSCC cell lines. Nilotinib, dasatinib, erlotinib, gefitinib, and everolimus had an impact on the expression levels of TGFß1 and TGFßR2 in vitro.

Silencing of lncRNA LOC105376794 promotes migration, invasion, and Gefitinib resistance of lung adenocarcinoma cells with EGFR 19Del mutation by ATF4/CHOP axis and ERK phosphorylation.

Epidermal growth factor receptor (EGFR) gene exon 19 in-frame deletion (19del) and exon 21 L858R point mutation (21L858R mutation) are prevalent mutations in lung adenocarcinoma. Lung adenocarcinoma patients with 19del presented with a better prognosis than the 21L858R mutation under the same epidermal growth factor receptor tyrosine kinase inhibitor treatment. Our study aimed to uncover the expression of long non-coding RNA LOC105376794 between 19del and 21L858R mutation, and explore the mechanism that regulates cells' biological behavior and gefitinib sensitivity in lung adenocarcinoma cells with 19del. Transcriptome sequencing was conducted to identify differentially expressed lncRNAs between EGFR 19del and 21L858R mutation in serum through the DNBSEQ Platform. Protein-protein interaction network and Kyoto Encyclopedia of Genes and Genomes pathway were conducted to analyze the relationship between lncRNAs and mRNAs through STRING and Dr. TOM. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to measure the expression of lncRNA LOC105376794 in serum and cells. Loss-of-function experiments were used to validate the biological function and gefitinib sensitivity of LOC105376794 in lung adenocarcinoma cells. Protein levels were detected by western blotting. Through transcriptome resequencing and RT-qPCR, we found the expression levels of LOC105376794 in serum were increased in the 19del group compared with the 21L858R mutation group. Inhibition of LOC105376794 promoted proliferation, migration and invasion, and reduced apoptosis of HCC827 and PC-9 cells. The low expression of LOC105376794 reduced gefitinib sensitivity in PC-9 cells. Mechanistically, we found that the knockdown of LOC105376794 suppressed activating transcription factor 4 (ATF4)/C/EBP homologous protein (CHOP) signaling pathway and facilitated the expression of extracellular signal-regulated kinase 1/2 (ERK) phosphorylation. LOC105376794 altered cell biological behavior and gefitinib sensitivity of lung adenocarcinoma cells with 19del through the ATF4/CHOP signaling pathway and the expression of ERK phosphorylation. The results further illustrated the fact that lung adenocarcinoma patients with 19del presented with a more favorable clinical outcome and provided a theoretical basis for treatment strategy for lung adenocarcinoma patients with 19del.

Interactive Analysis of UTX-114 Family With EGFR-tyk: Molecular Features of Acetyl Glycosylated Gefitinib.

BACKGROUND/AIM: Acetyl glucose adducts (UTX-114, -115, and -116) were prepared from gefitinib, and their characteristics (e.g., anticancer activity, structural property) were analyzed. MATERIALS AND METHODS: Cytotoxicity and radiosensitizing properties of the UTX-114 family were examined using A431 cells. Supramolecular associations between the UTX-114 family compounds and the tyrosine kinase domain of epidermal growth factor receptor (EGFR-tyk) were also examined. The interactive analyses of the UTX-114 family compounds with EGFR-tyk were performed using docking simulation technique. RESULTS: The UTX-114 family showed a similar cytotoxicity as gefitinib, yielding IC50 values of 31.2 µM (gefitinib), 34.3 µM (UTX-114), 36.8 µM (UTX-115), and 39.4 µM (UTX-116). The EGFR-tyk inhibition ratios (IR) of UTX-114, -115, and -116 to gefitinib were 1.515, 0.983, and 0.551, respectively. The EGFR-tyk inhibitory activity of UTX-114 was higher than that of gefitinib. UTX-114 also showed the highest radiosensitizing activity among the tested compounds. UTX-114 expressed 1841 conformers (-8.989~15.718 kcal/mol) with the solvation free energy (dGW) of UTX-114 decreasing with increasing conformational energy, ranging between -354.955~ -260.815 kJ/mol. Interactive energies of gefitinib, UTX-114, -115, and -116 with EGFR-tyk were -123.640, -144.053, -120.830, and -124.658 kcal/mol, respectively. CONCLUSION: UTX-114 yielded the lowest interaction energy with EGFR-tyk among tested compounds. Given the association behavior between UTX-114 and EGFR-tyk, along with its other observed properties, UTX-114 appears to be a viable therapeutic possibility.

HB-EGF activates EGFR to induce reactive neural stem cells in the mouse hippocampus after seizures.

Hippocampal seizures mimicking mesial temporal lobe epilepsy cause a profound disruption of the adult neurogenic niche in mice. Seizures provoke neural stem cells to switch to a reactive phenotype (reactive neural stem cells, React-NSCs) characterized by multibranched hypertrophic morphology, massive activation to enter mitosis, symmetric division, and final differentiation into reactive astrocytes. As a result, neurogenesis is chronically impaired. Here, using a mouse model of mesial temporal lobe epilepsy, we show that the epidermal growth factor receptor (EGFR) signaling pathway is key for the induction of React-NSCs and that its inhibition exerts a beneficial effect on the neurogenic niche. We show that during the initial days after the induction of seizures by a single intrahippocampal injection of kainic acid, a strong release of zinc and heparin-binding epidermal growth factor, both activators of the EGFR signaling pathway in neural stem cells, is produced. Administration of the EGFR inhibitor gefitinib, a chemotherapeutic in clinical phase IV, prevents the induction of React-NSCs and preserves neurogenesis.

IGFBP2/ITGA5 promotes gefitinib resistance via activating STAT3/CXCL1 axis in non-small cell lung cancer.

There is a paucity of comprehensive knowledge pertaining to the underlying mechanisms leading to gefitinib resistance in individuals diagnosed NSCLC harboring EGFR-sensitive mutations who inevitably develop resistance to gefitinib treatment within six months to one year. In our preceding investigations, we have noted a marked upregulation of IGFBP2 in the neoplastic tissues of NSCLC, predominantly in the periphery of the tissue, implying its plausible significance in NSCLC. Consequently, in the current research, we delved into the matter and ascertained the molecular mechanisms that underlie the participation of IGFBP2 in the emergence of gefitinib resistance in NSCLC cells. Firstly, the expression of IGFBP2 in the bronchoalveolar lavage fluid and lung cancer tissues of 20 NSCLC patients with gefitinib tolerance was found to be significantly higher than that of non-tolerant patients. Furthermore, in vitro and in vivo experiments demonstrated that IGFBP2 plays a significant role in the acquisition of gefitinib resistance. Mechanistically, IGFBP2 can activate STAT3 to enhance the transcriptional activity of CXCL1, thereby increasing the intracellular expression level of CXCL1, which contributes to the survival of lung cancer cells in the gefitinib environment. Additionally, we identified ITGA5 as a key player in IGFBP2-mediated gefitinib resistance, but it does not function as a membrane receptor in the process of linking IGFBP2 to intracellular signaling transduction. In conclusion, this study demonstrates the promoting role and mechanism of IGFBP2 in acquired gefitinib resistance caused by non-EGFR secondary mutations, suggesting the potential of IGFBP2 as a biomarker for gefitinib resistance and a potential intervention target.

PCK2 induces gefitinib resistance by suppresses ferroptosis in non-small cell lung cancer.

OBJECTIVES: This study aimed to explore the involvement of phosphoenolpyruvate carboxykinase 2 (PCK2) in gefitinib-resistant non-small cell lung cancer (NSCLC) cells and assess its feasibility as a therapeutic target against gefitinib resistance. METHODS: Gefitinib-resistant cell lines, PC9GR and HCC827GR, were generated through progressive exposure of parental cells to escalating concentrations of gefitinib. Transcriptomic analysis encompassed the treatment of PC9 and PC9GR cells with gefitinib or vehicle, followed by RNA extraction, sequencing, and subsequent bioinformatic analysis. Cell viability was determined via CCK-8 assay, while clonogenic assays assessed colony formation. Apoptosis was detected utilizing the Annexin V-FITC/7AAD kit. Iron ion concentrations were quantified using FerroOrange. mRNA analysis was conducted through quantitative RT-PCR. Western blotting was employed for protein analysis. H&E and immunohistochemical staining were performed on tumor tissue sections. RESULTS: The results revealed that depletion or inhibition of PCK2 significantly enhanced gefitinib's efficacy in inducing cell growth arrest, apoptosis, and ferroptosis in resistant NSCLC. Moreover, PCK2 knockdown led to the downregulation of key ferroptosis-related proteins, GPX4 and SLC7A11, while upregulating ASCL4. Conversely, overexpression of PCK2 in gefitinib-sensitive cells rendered resistance to gefitinib. In vivo experiments using a gefitinib-resistant xenograft model demonstrated that PCK2 silencing not only reduced tumor growth but also considerably increased the anti-tumor effect of gefitinib. CONCLUSIONS: In conclusion, our study presents compelling evidence indicating that PCK2 plays a pivotal role in gefitinib resistance in NSCLC. The modulation of ferroptosis-related proteins and the involvement of Akt activation further elucidate the mechanisms underlying this resistance. Consequently, PCK2 emerges as a promising therapeutic target for overcoming gefitinib resistance in NSCLC, offering a new avenue for the development of more effective treatment strategies.

Nanoenabled intracellular zinc bursting for efficacious reversal of gefitinib resistance in lung cancer.

Following the identification of specific epidermal growth factor receptor (EGFR)-activating mutations, gefitinib, one of the first-generation tyrosine kinase inhibitors (TKIs), has proven efficacious in targeting NSCLC that is driven by specific EGFR-activating mutations. However, most patients who initially respond to gefitinib, develop acquired resistance. In the current study, we devised a novel strategy to enhance the efficacy of gefitinib. We developed a simple and effective, nano-interrupter termed zeolitic imidazolate framework-8@Gefitinib@hyaluraonic nanoparticle (ZIF-8@G@HA NP). This nanoparticle was prepared by loading gefitinib onto a ZIF-8 nanoplatform followed by coating with hyaluronic acid (HA). The burst of Zn2+ release triggered by pH-sensitive degradation of ZIF-8@G@HA NPs was shown to enhance the efficacy of gefitinib in parental lung carcinoma HCC827 cells and overcame acquired gefitinib resistance in gefitinib drug resistant (GDR) HCC827 cells. We found that when treated with ZIF-8@G@HA NPs, Zn2+ acts synergistically with gefitinib via increased apoptosis in both parental and GDR HCC827 cells. Consistently, this in vitro activity was correlated with in vivo tumor growth inhibition. Interestingly, GDR cells were more sensitive to Zn2+ when compared with parental cells. We further found that ZIF-8 NPs overcame gefitinib resistance by triggering reactive oxygen species (ROS) generation and consequent cell cycle arrest at the G2/M phase, resulting in cancer cell apoptosis. Zn2+ was also found to block P-gp activity, facilitating the accumulation of gefitinib in GDR cells, thus enhancing the anti-tumor efficacy of gefitinib resulting in reversal of gefitinib resistance. Thus, this study offers a novel and promising strategy to surmount acquired gefitinib resistance via cell cycle arrest at the G2/M phase by facilitating gefitinib accumulation in GDR cells.

Gefitinib metabolism-related lncRNAs for the prediction of prognosis, tumor microenvironment and drug sensitivity in lung adenocarcinoma.

The complete compound of gefitinib is effective in the treatment of lung adenocarcinoma. However, the effect on lung adenocarcinoma (LUAD) during its catabolism has not yet been elucidated. We carried out this study to examine the predictive value of gefitinib metabolism-related long noncoding RNAs (GMLncs) in LUAD patients. To filter GMLncs and create a prognostic model, we employed Pearson correlation, Lasso, univariate Cox, and multivariate Cox analysis. We combined risk scores and clinical features to create nomograms for better application in clinical settings. According to the constructed prognostic model, we performed GO/KEGG and GSEA enrichment analysis, tumor immune microenvironment analysis, immune evasion and immunotherapy analysis, somatic cell mutation analysis, drug sensitivity analysis, IMvigor210 immunotherapy validation, stem cell index analysis and real-time quantitative PCR (RT-qPCR) analysis. We built a predictive model with 9 GMLncs, which showed good predictive performance in validation and training sets. The calibration curve demonstrated excellent agreement between the expected and observed survival rates, for which the predictive performance was better than that of the nomogram without a risk score. The metabolism of gefitinib is related to the cytochrome P450 pathway and lipid metabolism pathway, and may be one of the causes of gefitinib resistance, according to analyses from the Gene Set Enrichment Analysis (GSEA), Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Immunological evasion and immunotherapy analysis revealed that the likelihood of immune evasion increased with risk score. Tumor microenvironment analysis found most immune cells at higher concentrations in the low-risk group. Drug sensitivity analysis found 23 sensitive drugs. Twenty-one of these drugs exhibited heightened sensitivity in the high-risk group. RT-qPCR analysis validated the characteristics of 9 GMlncs. The predictive model and nomogram that we constructed have good application value in evaluating the prognosis of patients and guiding clinical treatment.

The Impact of Inadequate Exposure to Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitors on the Development of Resistance in Non-Small-Cell Lung Cancer Cells.

Epidermal growth factor receptor (EGFR)-mutant non-small-cell lung cancer (NSCLC) patients treated with EGFR-tyrosine kinase inhibitors (TKIs) inevitably develop resistance through several biological mechanisms. However, little is known on the molecular mechanisms underlying acquired resistance to suboptimal EGFR-TKI doses, due to pharmacodynamics leading to inadequate drug exposure. To evaluate the effects of suboptimal EGFR-TKI exposure on resistance in NSCLC, we obtained HCC827 and PC9 cell lines resistant to suboptimal fixed and intermittent doses of gefitinib and compared them to cells exposed to higher doses of the drug. We analyzed the differences in terms of EGFR signaling activation and the expression of epithelial-mesenchymal transition (EMT) markers, whole transcriptomes byRNA sequencing, and cell motility. We observed that the exposure to low doses of gefitinib more frequently induced a partial EMT associated with an induced migratory ability, and an enhanced transcription of cancer stem cell markers, particularly in the HCC827 gefitinib-resistant cells. Finally, the HCC827 gefitinib-resistant cells showed increased secretion of the EMT inducer transforming growth factor (TGF)-ß1, whose inhibition was able to partially restore gefitinib sensitivity. These data provide evidence that different levels of exposure to EGFR-TKIs in tumor masses might promote different mechanisms of acquired resistance.

Polyphyllin I ameliorates gefitinib resistance and inhibits the VEGF/VEGFR2/p38 pathway by targeting HIF-1a in lung adenocarcinoma.

BACKGROUND: Lung adenocarcinoma (LUAD) is the most common pathological type of lung cancer. Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have been administered as the first-line therapy for patients with EGFR mutations in LUAD, but it is almost inevitable that resistance to EGFR-TKIs therapy eventually arises. Polyphyllin I (PPI), derived from Paris polyphylla rhizomes, has been shown to have potent anti-cancer properties in a range of human cancer types including LUAD. However, the role of PPI in gefitinib resistance and the underlying mechanism remain elusive. PURPOSE: To evaluate the antitumor impacts of PPI on gefitinib resistance cells and investigate its molecular mechanism. METHODS: CCK-8, wound healing, transwell assay, and xenograft model were performed to determine the anti-cancer effects of PPI as well as its ability to overcome gefitinib resistance. Immunoblotting, co-immunoprecipitation, phospho-RTK antibody array, qRT-PCR, and immunofluorescence were utilized to explore the mechanism by which PPI overrides gefitinib resistance. RESULTS: PPI inhibited cell survival, growth, and migration/invasion in both gefitinib-sensitive (PC9) and -resistant (PC9/GR) LUAD cells (IC50 at 2.0 µM). Significantly, treatment with PPI at 1.0 µM resensitized the resistant cells to gefitinib. Moreover, cell-derived xenograft experiments revealed that the combination of PPI and gefitinib overcame gefitinib resistance. The phospho-RTK array and immunoblotting analyses showed PPI significant inhibition of the VEGFR2/p38 pathway. In addition, molecular docking suggested the interaction between PPI and HIF-1α. Mechanistically, PPI reduced the protein expression of HIF-1α in both normoxia and hypoxia conditions by triggering HIF-1α degradation. Moreover, HIF-1α protein but not mRNA level was elevated in gefitinib-resistant LUAD. We further demonstrated that PPI considerably facilitated the binding of HIF-1α to VHL. CONCLUSIONS: We present a novel discovery demonstrating that PPI effectively counteracts gefitinib resistance in LUAD by modulating the VEGF/VEGFR2/p38 pathway. Mechanistic investigations unveil that PPI facilitates the formation of the HIF-1α /VHL complex, leading to the degradation of HIF-1α and subsequent inhibition of angiogenesis. These findings uncover a previously unidentified mechanism governing HIF-1α expression in reaction to PPI, providing a promising method for therapeutic interventions targeting EGFR-TKI resistance in LUAD.

Effects of EGFR-TKI on epidermal melanin unit integrity: Therapeutic implications for hypopigmented skin disorders.

Epidermal melanin unit integrity is crucial for skin homeostasis and pigmentation. Epidermal growth factor (EGF) receptor (EGFR) is a pivotal player in cell growth, wound healing, and maintaining skin homeostasis. However, its influence on skin pigmentation is relatively unexplored. This study investigates the impact and underlying mechanisms of EGFR inhibitors on skin pigmentation. We evaluated EGF and EGFR expression in various skin cells using quantitative real-time PCR, Western blot, and immunofluorescence. EGF and EGFR were predominantly expressed in epidermal keratinocytes, and treatment with the EGFR tyrosine kinase inhibitors (EGFR-TKIs) gefitinib and PD153035 significantly increased stem cell factor (SCF) and endothelin-1 (ET-1) expression in cultured keratinocytes. Enhanced melanocyte migration and proliferation were observed in co-culture, as evidenced by time-lapse live imaging and single-cell tracking assays. Furthermore, topical application of gefitinib to guinea pig dorsal skin induced increased pigmentation and demonstrated efficacy in mitigating rhododendrol-induced leukoderma. Suppression of EGF signaling indirectly enhanced skin pigmentation by upregulating SCF and ET-1 in epidermal keratinocytes. This novel mechanism highlights the pivotal role of EGF signaling in regulating skin pigmentation, and topical EGFR-TKI therapy at an appropriate dose may be a promising approach for depigmentation disorder management.

[Resistance to anti-EGFR through the successive and cumulative acquisition of two new oncogenic addictions: BRAF and ALK]. / Résistance aux anti-EGFR via l'acquisition successive et cumulée de deux nouvelles addictions oncogénique BRAF et ALK.

Targeted therapies are the standard first-line treatment for metastatic lung adenocarcinoma with certain molecular abnormalities. These abnormalities are particularly common in Southeast Asia and French Polynesia. A 51-year-old Tahitian female non-smoker was diagnosed in 2018 with stage IV lung adenocarcinoma harboring a p.L858R EGFR mutation. She received gefitinib as first-line treatment. Due to locoregional progression and the presence of a resistance mutation (p.T790M of EFGR), she received osimertinib as second-line treatment, after which chemotherapy was proposed as 3rd-line treatment. An additional biopsy detected not only the previously known EGFR mutation, but also a BRAF p.V600E mutation. Following disease progression during chemotherapy, the patient received targeted therapies combining dabrafenib, trametinib and osimertinib. Due to a dissociated response after four months of treatment, a 5th line of paclitaxel bevacizumab was initiated. Subsequent to additional progression and given the ALK rearrangement shown on the re-biopsy, 6th-line treatment with alectinib was proposed. As the response was once again dissociated, a final line was proposed before stopping active treatments due to their toxicity and overall deterioration in the patient's state of health. This exceptional case is characterized by resistance to anti-EGFR through the successive and cumulative acquisition of two new oncogene addictions. The authors underline the importance of re-biopsy at each progression, leading (if at all feasible) to yet around round of targeted therapy.

[The effects and mechanisms of Gefitinib on airway inflammation and airway remodeling in C57BL/6 mice with asthma].

Objective: To investigate the effects of the epidermal growth factor receptor(EGFR) inhibitor Gefitinib on airway inflammation and airway remodelling in asthmatic C57BL/6 mice, and to analyze its possible mechanisms. Methods: Male C57BL/6 mice, aged 6-8 weeks, were randomly assigned into five groups: Group A (control group), Group B (asthma group), Group C (asthma+20 mg/kg gefitinib group), Group D (asthma+40 mg/kg gefitinib group), and Group E (40 mg/kg gefitinib group), with seven mice per group. Mice were sensitized by intraperitoneal injection of a mixture of 0.2 ml solution containing OVA and Al(OH)3 [20 µg OVA+2 mg Al(OH)3 dissolved in 0.2 ml of physiological saline] at Day 0 and 14. Starting from Day 25 to 31, Group B, C, and D were challenged with nebulization of 1% OVA solution (8 ml) to induce asthma, once a day for approximately 40 minutes, with continuous aerosolization for 7 days. Group C and D were given 0.2 ml of Gefitinib dissolved in 0.5% carboxymethylcellulose sodium (CMCNa) by gavage half an hour before challenging, and Group E was simultaneously given with 0.2 ml of Gefitinib dissolved in 0.5% CMCNa only. Group A and B were given an equivalent volume of 0.5% CMCNa by gavage. After 24 h of final challenge, the bronchoalveolar lavage fluid (BALF) was prepared for the determination of total cell count and eosinophil count. The levels of total immune globulin E (IgE) in serum and interleukin (IL)-4, IL-5 and IL-13 in BALF and lung tissue homogenates were measured by ELISA. The mRNA expression levels of IL-4, IL-5, IL-13 in lung were measured. Immunohistochemistry and Western blot experiments were used to detect the expression levels of EGFR in lung tissues. Results: In Group B, the level of total IgE in serum, total cell count, eosinophil count, the levels of IL-4, IL-5, IL-13 in BALF and the phosphorylation of EGFR and its downstream activation in lung were higher than those in Group A (all P<0.05). The levels of total IgE in serum [(261.32±44.38) ng/ml, (194.09±52.39) ng/ml vs (1 023.70±105.51) ng/ml], total cell count [(23.70±4.08)×105/ml, (14.92±4.06)×105/ml vs (35.36±6.30)×105/ml], eosinophil count [(108.00±13.69)×104/ml, (67.00±17.28)×104/ml vs (147.86±20.06)×104/ml], IL-4 [(36.42±4.48) pg/ml, (30.45±8.12) pg/ml vs (58.72±7.17) pg/ml], IL-5 [(16.20±4.62) pg/ml, (13.38±5.14) pg/ml vs (23.46±5.38) pg/ml], IL-13 [(18.45±7.28) pg/ml, (14.33±7.70) pg/ml vs (104.12±24.66) pg/ml] in BALF of Group C and D were lower than those in Group B (all P<0.05). The levels of IL-4, IL-5, and IL-13 as well as their mRNA levels in the lung tissue of Group C and D were lower than those in Group B (all P<0.05). In Group C and D, the positive expression rate of phosphorylated epidermal growth factor receptor (p-EGFR) in lung tissue [(40.53±6.80)%, (23.60±4.42)% vs (70.78±5.36)%], p-EGFR/EGFR (61.68±7.48, 51.13±5.19 vs 105.90±11.66), phosphorylated extracellular regulated protein kinase (p-Erk)/extracellular regulated protein kinase (Erk) (75.28±7.11, 47.54±4.83 vs 98.76±4.71), and phosphorylated protein kinase B (p-Akt)/protein kinase B (Akt) (96.24±5.40, 68.52±2.73 vs 103.30±4.52) was lower than those of Group B (all P<0.05). There was no statistically significant difference in the relevant indicators between Group A and E (all P>0.05). Conclusion: Gefitinib may alleviate airway inflammation and airway remodeling in asthmatic mice by inhibiting EGFR phosphorylation and affecting the activation of downstream Erk and Akt.

Targeted blocking of EGFR and GLUT1 by compound H reveals a new strategy for treatment of triple-negative breast cancer and nasopharyngeal carcinoma.

BACKGROUND: Cytoplasmic epidermal growth factor receptor (EGFR) is overexpressed in both nasopharyngeal carcinoma (NPC) and triple-negative breast cancer (TNBC), while clinical outcome and prognosis vary greatly among patients treated with gefitinib, and all patients eventually develop resistance to this agent. Therefore, we propose a new concept for synthesizing multitarget compounds and reveal new therapeutic strategies for NPC and TNBC expressing EGFR. METHODS: Compound H was synthesized in our previous study. Molecular docking, and cell thermal shift assays (CETSAs) and drug affinity responsive target stability(DARTS) were used to confirm the binding of compound H to EGFR and GLUT1. Methylthiazolyldiphenyl-tetrazolium bromide(MTT), annexin V-PE assays, mitochondrial membrane potential (MMP) assays, and animal models were used to evaluate the inhibitory effect of compound H on TNBC cell lines. Energy metabolism tests, Western blotting, and immunofluorescence staining were performed to evaluate the synergistic effects on EGFR- and glucose transporter type 1(GLUT1)-mediated energy metabolism. RESULTS: Compound H can simultaneously act on the EGFR tyrosine kinase ATP-binding site and inhibit GLUT1-mediated energy metabolism, resulting in reductions in ATP, MMP, intra-cellular lactic acid, and EGFR nuclear transfer. The anti-tumor activity of compound H is significantly superior to the combination of GLUT1 inhibitor BAY876 and EGFR inhibitor gefitinib. Compound H has remarkable anti-proliferative effects on TNBC MDA-MB231 cells, and importantly, no obvious toxicity effects of compound H were found in vivo. CONCLUSIONS: Synergistic effects of inhibition of EGFR- and GLUT1-mediated energy metabolism by compound H may present a new strategy for the treatment of TNBC and NPC.

Combination of betulinic acid and EGFR-TKIs exerts synergistic anti-tumor effects against wild-type EGFR NSCLC by inducing autophagy-related cell death via EGFR signaling pathway.

BACKGROUND: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of lung cancer patients with mutated EGFR. However, the efficacy of EGFR-TKIs in wild-type EGFR tumors has been shown to be marginal. Methods that can sensitize EGFR-TKIs to EGFR wild-type NSCLC remain rare. Hence, we determined whether combination treatment can maximize the therapeutic efficacy of EGFR-TKIs. METHODS: We established a focused drug screening system to investigate candidates for overcoming the intrinsic resistance of wild-type EGFR NSCLC to EGFR-TKIs. Molecular docking assays and western blotting were used to identify the binding mode and blocking effect of the candidate compounds. Proliferation assays, analyses of drug interactions, colony formation assays, flow cytometry and nude mice xenograft models were used to determine the effects and investigate the molecular mechanism of the combination treatment. RESULTS: Betulinic acid (BA) is effective at targeting EGFR and synergizes with EGFR-TKIs (gefitinib and osimertinib) preferentially against wild-type EGFR. BA showed inhibitory activity due to its interaction with the ATP-binding pocket of EGFR and dramatically enhanced the suppressive effects of EGFR-TKIs by blocking EGFR and modulating the EGFR-ATK-mTOR axis. Mechanistic studies revealed that the combination strategy activated EGFR-induced autophagic cell death and that the EGFR-AKT-mTOR signaling pathway was essential for completing autophagy and cell cycle arrest. Activation of the mTOR pathway or blockade of autophagy by specific chemical agents markedly attenuated the effect of cell cycle arrest. In vivo administration of the combination treatment caused marked tumor regression in the A549 xenografts. CONCLUSIONS: BA is a potential wild-type EGFR inhibitor that plays a critical role in sensitizing EGFR-TKI activity. BA combined with an EGFR-TKI effectively suppressed the proliferation and survival of intrinsically resistant lung cancer cells via the inhibition of EGFR as well as the induction of autophagy-related cell death, indicating that BA combined with an EGFR-TKI may be a potential therapeutic strategy for overcoming the primary resistance of wild-type EGFR-positive lung cancers.

Mechanistic insights into Yifei Sanjie pill's regulation of EMT to enhance gefitinib treatment effect in NSCLC by in silico analysis and experimental validation.

ETHNOPHARMACOLOGICAL RELEVANCE: The Yi-Fei San-Jie pill (YFSJ) is a well-known Chinese medicine that has been used to treat non-small cell lung cancer in China for decades. AIM OF THE STUDY: Previous studies have shown that YFSJ combined with gefitinib can effectively inhibit the proliferation of gefitinib-resistant non-small cell lung cancer (NSCLC) cell lines by promoting apoptosis and autophagy, but the molecular biological mechanisms involved and whether YFSJ combined with gefitinib can have synergistic effects still need to be further explored. Thus, the present study aimed to establish an in silico and experimental framework to decipher the underlying mechanism by which YFSJ augments the efficacy of gefitinib in treating NSCLC. MATERIALS AND METHODS: Integrated approaches, including microarray analysis, network pharmacology, RNA sequencing, bioinformatics algorithm analysis and in vivo and in vitro experiments, were applied to elucidate the underlying mechanism. RESULTS: Analysis of microarray datasets indicated that gefitinib may play a role in the regulation of the epithelial-mesenchymal transition (EMT) of PC9 cells. EMT-related Gene Ontology (GO) terms and the MAPK pathway were found to be enriched in the differentially expressed genes (DEGs), and a decreasing trend was observed in the EMT score. Network pharmacology analysis revealed that the potential NSCLC-related targets of YFSJ also showed enrichment in EMT-related GO terms and the MAPK pathway. Experimental findings demonstrated that combined YFSJ-treated serum and gefitinib treatment significantly inhibited PC9 cell migration and invasion. In addition, the combined treatment dramatically reduced the tumour volume in an animal model. The effectiveness of the combination treatment surpassed that of gefitinib alone in both cell and animal experiments. RNA sequencing analysis revealed significant enrichment of DEGs in EMT-related GO terms for the gefitinib treatment group, YFSJ treatment group, and combination treatment group compared to the control group. Notably, the negative regulation of EMT showed significant enrichment in the DEGs of the combination treatment group. The MAPK pathway was significantly enriched among the different groups. Moreover, combined treatment with YFSJ and gefitinib may exert synergistic anti-NSCLC effects by inhibiting the p-p38 MAPK/GSK3ß signalling axis, subsequently suppressing downstream EMT processes. CONCLUSION: Combined treatment with YFSJ and gefitinib could enhance the sensitivity of NSCLC cells to gefitinib by suppressing EMT through the EGFR/p-p38 MAPK/GSK3ß signalling axis. YFSJ may serve as an important adjunctive medication for NSCLC patients receiving gefitinib treatment in clinical practice.

Synergistic anticancer effects of ginsenoside CK and gefitinib against gefitinib-resistant NSCLC by regulating the balance of angiogenic factors through HIF-1α/VEGF.

Drug resistance is a serious problem for gefitinib in the treatment of lung cancer. Ginsenoside CK, a metabolite of diol ginsenosides, have many excellent pharmacological activities, but whether ginsenoside CK can overcome gefitinib resistance remains unclear. In our study, the sensitizing activity of ginsenoside CK on gefitinib-resistant non-small cell lung cancer (NSCLC) in vitro and in vivo was investigated. Ginsenoside CK was confirmed to enhance the anti-proliferation, pro-apoptotic and anti-migration effects of gefitinib in primary and acquired resistant NSCLC. Furthermore, the combined administration of CK and gefitinib effectively promoted the sensitivity of lung cancer xenograft to gefitinib in vivo, and the tumor inhibition rate reached 70.97% (vs. gefitinib monotherapy 32.65%). Subsequently, tubule formation experiment and western blot results showed that co-treatment of ginsenoside CK inhibited the angiogenesis ability of HUVEC cells, and inhibited the expression of HIF-1α, VEGF, FGF and MMP2/9. More interestingly, ginsenoside CK co-treatment enhanced the expression of anti-angiogenic factor PF4, increased pericellular envelope, and promoted the normalization of vascular structure. In conclusion, ginsenoside CK improved the resistance of gefitinib by regulating the balance of angiogenic factors through down-regulating the HIF-1α/VEGF signaling pathway, providing a theoretical basis for improving the clinical efficacy of gefitinib and applying combined strategies to overcome drug resistance.