Changes In Epidermal Growth Factor Receptor Research Articles

Coevolution-based computational approach to detect resistance mechanism of epidermal growth factor receptor

Tyrosine kinase epidermal growth factor receptor (EGFR) correlates the neoplastic cell metastasis, angiogenesis, neoplastic incursion, and apoptosis. Due to the involvement of EGFR in these biological processes, it becomes a most potent target for treating non-small cell lung cancer (NSCLC). The tyrosine kinase inhibitors (TKI) have endorsed high efficacy and anticipation to patients but unfortunately, within a year of treatment, drug targets develop resistance due to mutations. The present study detected the compensatory mutations in EGFR to know the evolutionary mechanism of drug resistance. The results of this study demonstrate that compensatory mutations enlarge the drug-binding pocket which may lead to the altered orientation of the ligand (gefitinib and erlotinib) causing drug resistance. This indicates that coevolutionary forces play a significant role in fine-tuning the structure of EGFR protein against the drugs. The analysis provides insight into the evolution-induced structural aspects of drug resistance changes in EGFR which in turn be useful in designing drugs with better efficacy.

Comparative study of cigarette smoke, Klebsiella pneumoniae, and their combination on airway epithelial barrier function in mice.

The airway epithelium acts as a physical barrier to protect pulmonary airways against pathogenic microorganisms and toxic substances, such as cigarette smoke (CS), bacteria, and viruses. The disruption of the structural integrity and dysfunction of the airway epithelium is related to the occurrence and progression of chronic obstructive pulmonary disease. The aim of this study is to compare the effects of CS, Klebsiella pneumoniae (KP), and their combination on airway epithelial barrier function. The mice were exposed to CS, KP, and their combination from 1 to 8 weeks. After the cessation of CS and KP at Week 8, we observed the recovery of epithelial barrier function in mice for an additional 16 weeks. To compare the epithelial barrier function among different groups over time, the mice were sacrificed at Weeks 4, 8, 16, and 24 and then the lungs were harvested to detect the pulmonary pathology, inflammatory cytokines, and tight junction proteins. To determine the underlying mechanisms, the BEAS-2B cells were treated with an epidermal growth factor receptor (EGFR) inhibitor (AG1478). The results of this study suggested that the decreased lung function, increased bronchial wall thickness (BWT), elevated inflammatory factors, and reduced tight junction protein levels were observed at Week 8 in CS-induced mice and these changes persisted until Week 16. In the KP group, increased BWT and elevated inflammatory factors were observed only at Week 8, whereas in the CS + KP group, decreased lung function, lung tissue injury, inflammatory cell infiltration, and epithelial barrier impairment were observed at Week 4 and persisted until Week 24. To further determine the mechanisms of CS, bacteria, and their combination on epithelial barrier injury, we investigated the changes of EGFR and its downstream protein in the lung tissues of mice and BEAS-2B cells. Our research indicated that CS, KP, or their combination could activate EGFR, which can phosphorylate and activate ERK1/2, and this effect was more pronounced in the CS + KP group. Furthermore, the EGFR inhibitor AG1478 suppressed the phosphorylation of ERK1/2 and subsequently upregulated the expression of ZO-1 and occludin. In general, these results indicated that the combination of CS and KP caused more severe and enduring damage to epithelial barrier function than CS or KP alone, which might be associated with EGFR/ERK1/2 signaling. Epithelial barrier injury occurred earlier, was more severe, and had a longer duration when induced by the combination of CS and KP compared with the exposure to CS or KP alone, which might be associated with EGFR/ERK signaling.

A Multiscale Molecular Dynamic Analysis Reveals the Effect of Sialylation on EGFR Clustering in a CRISPR/Cas9-Derived Model.

Bacterial and viral pathogens can modulate the glycosylation of key host proteins to facilitate pathogenesis by using various glycosidases, particularly sialidases. Epidermal growth factor receptor (EGFR) signaling is activated by ligand-induced receptor dimerization and oligomerization. Ligand binding induces conformational changes in EGFR, leading to clusters and aggregation. However, information on the relevance of EGFR clustering in the pattern of glycosylation during bacterial and viral invasion remains unclear. In this study, (1) we established CRISPR/Cas9-mediated GFP knock-in (EGFP-KI) HeLa cells expressing fluorescently tagged EGFR at close to endogenous levels to study EGF-induced EGFR clustering and molecular dynamics; (2) We studied the effect of sialylation on EGF-induced EGFR clustering and localization in live cells using a high content analysis platform and raster image correlation spectroscopy (RICS) coupled with a number and brightness (N&B) analysis; (3) Our data reveal that the removal of cell surface sialic acids by sialidase treatment significantly decreases EGF receptor clustering with reduced fluorescence intensity, number, and area of EGFR-GFP clusters per cell upon EGF stimulation. Sialylation appears to mediate EGF-induced EGFR clustering as demonstrated by the change of EGFR-GFP clusters in the diffusion coefficient and molecular brightness, providing new insights into the role of sialylation in EGF-induced EGFR activation; and (4) We envision that the combination of CRISPR/Cas9-mediated fluorescent tagging of endogenous proteins and fluorescence imaging techniques can be the method of choice for studying the molecular dynamics and interactions of proteins in live cells.

Disease monitoring of epidermal growth factor receptor (EGFR)-mutated non-small-cell lung cancer patients treated with tyrosine kinase inhibitors via EGFR status in circulating tumor DNA.

ObjectiveCirculating tumor DNA (ctDNA) monitoring proves to be a promising approach to assess response and predict survival in epidermal growth factor receptor (EGFR)‐mutated non‐small‐cell lung cancer (NSCLC) patients treated with tyrosine kinase inhibitors (TKIs). However, whether the dynamic changes in ctDNA EGFR mutation status have the same predictive value as ctDNA remains unknown. This study aims to explore the predictive value of dynamic changes in both ctDNA and ctDNA EGFR status.MethodsA retrospective analysis was performed using 91 ctDNA samples from a cohort of 28 patients who were diagnosed with EGFR‐mutated NSCLC and treated with EGFR‐TKIs, including 14 patients treated with first‐/second‐generation TKIs and 14 treated with osimertinib. Blood samples at baseline (BL), within 4 weeks after TKI initiation (Week4), within 12 weeks before progression (pre‐PD), and at progression were collected. The relationship alternatives in ctDNA status, ctDNA EGFR status and response to EGFR‐TKIs as well as progression‐free survival (PFS) were analyzed.ResultsWe categorized 20 BL‐ctDNA positive patients with available Week4‐ctDNA into two groups: ctDNA‐clearance (N = 7, 35%) and ctDNA‐non‐clearance (N = 13, 65%). The ctDNA‐clearance group had better PFS than the ctDNA‐non‐clearance group (ctDNA‐clearance vs. ctDNA‐non‐clearance, p = 0.091, hazard ratio [HR] = 0.42, 95% confidence interval [CI] = 0.15–1.19). According to Week4‐EGFR status, we observed that PFS was significantly longer in EGFR‐clearance patients than EGFR‐non‐clearance groups, (p = 0.011, HR = 0.23, 95% CI = 0.08–0.72). We then categorized patients into three subgroups according to Week4‐ctDNA and Week4‐EGFR status: non‐clearance (N = 9), only‐EGFR‐clearance (concomitant alterations non‐clearance) (N = 4), and all‐clearance (N = 7). The nonclearance group had a significantly worse PFS than the all‐clearance group (median PFS = 5.07 vs. 11.40 months, p = 0.029, HR = 3.45, 95% CI = 1.05–11.49). The only‐EGFR‐clearance group had a similar PFS to the all‐clearance group (p = 0.607), which was longer than that of the non‐clearance group (median PFS = 9.20 vs. 5.07 months, p = 0.060, HR = 0.25, 95% CI = 0.05–1.18). We found that the all‐clearance group had a similar objective response rate (ORR) to the only‐EGFR‐clearance group (p = 1.000) and a higher ORR than the non‐clearance group (p = 0.012).ConclusionMonitoring of EGFR clearance in ctDNA is promising and cost‐effective in assessing response and predicting survival in EGFR‐mutated NSCLC patients treated with EGFR‐TKIs, with similar predictive value to ctDNA surveillance.

The Functional Interaction of EGFR with AT1R or TP in Primary Vascular Smooth Muscle Cells Triggers a Synergistic Regulation of Gene Expression.

In vivo, cells are simultaneously exposed to multiple stimuli whose effects are difficult to distinguish. Therefore, they are often investigated in experimental cell culture conditions where stimuli are applied separately. However, it cannot be presumed that their individual effects simply add up. As a proof-of-principle to address the relevance of transcriptional signaling synergy, we investigated the interplay of the Epidermal Growth Factor Receptor (EGFR) with the Angiotensin-II (AT1R) or the Thromboxane-A2 (TP) receptors in murine primary aortic vascular smooth muscle cells. Transcriptome analysis revealed that EGFR-AT1R or EGFR-TP simultaneous activations led to different patterns of regulated genes compared to individual receptor activations (qualitative synergy). Combined EGFR-TP activation also caused a variation of amplitude regulation for a defined set of genes (quantitative synergy), including vascular injury-relevant ones (Klf15 and Spp1). Moreover, Gene Ontology enrichment suggested that EGFR and TP-induced gene expression changes altered processes critical for vascular integrity, such as cell cycle and senescence. These bioinformatics predictions regarding the functional relevance of signaling synergy were experimentally confirmed. Therefore, by showing that the activation of more than one receptor can trigger a synergistic regulation of gene expression, our results epitomize the necessity to perform comprehensive network investigations, as the study of individual receptors may not be sufficient to understand their physiological or pathological impact.

Advances in Treatment of Non-small Cell Lung Cancer Harboring EGFR Exon 20 Insertion Mutations

表皮生长因子受体（epidermal growth factor receptor, EGFR）外显子20插入突变是EGFR突变的第三大常见类别，约占所有EGFR突变阳性非小细胞肺癌（non-small cell lung cancer, NSCLC）的5%-12%。携带EGFR外显子20插入突变的NSCLC患者临床特征与“经典”EGFR激活突变人群相似，但预后很差。EGFR外显子20插入突变异质性高，可导致EGFR不同构象变化。大多数（几乎90%的病例）发生在α-C-螺旋后的Loop环结构区，最常报告的插入突变亚型为D770_N771 > ASVDN（A767_V769dupASV），突变频率为21%-28%。目前已知EGFR外显子20插入突变的NSCLC对既往已批准的EGFR酪氨酸激酶抑制剂原发性耐药，而且对传统化疗和免疫治疗也不敏感。近期Amivantamab与Mobocertinib在美国的获批改变了EGFR外显子20插入突变的NSCLC患者的治疗模式。此外，针对NSCLC EGFR外显子20插入突变的其他新型靶向药物正在研发中，期待为该类患者带来更多生存获益。本文就近年来伴有EGFR外显子20插入突变的NSCLC的临床研究进展进行归纳总结，旨在为该类患者的临床治疗提供参考。

XHL11, a novel selective EGFR inhibitor, overcomes EGFRT790M-mediated resistance in non-small cell lung cancer

The first-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), gefitinib and erlotinib significantly improved the therapeutic effect in non–small cell lung cancer (NSCLC) patients with EGFR mutation. However, the EGFRT790M mutation occurs and results in acquired resistance. Consequently, mutant selective third-generation EGFR TKIs represented by AZD9291 (Osimertinib) have been developed to offer more effective therapeutic treatment, but the clinical application is limited by the acquired resistance and the high costs. A series of 5-chloropyrimidine-2,4-diamine derivatives were synthesized and screened for in vitro antitumor activity on H1975 and A431 cells. XHL11 showed the strongest antineoplastic activity. Compared to AZD9291, XHL11 suppressed cellular proliferation and colony formation and induced apoptosis in H1975 cells with EGFRL858R/T790M mutation. In addition, XHL11 caused expression changes in EGFR and apoptosis-related pathways. Moreover, oral administration of XHL11 suppressed tumor progression in vivo in a H1975 subcutaneous xenograft model. These data demonstrated that XHL11 might be developed as a promising EGFR TKI for the therapeutic use of NSCLC patients.

Changes of EGFR and SMC4 expressions in triple-negative breast cancer and their early diagnostic value.

To explore the diagnostic value of epidermal growth factor receptor (EGFR) and structural maintenance of chromosome protein 4 (SMC4) for triple-negative breast cancer. A total of 213 breast cancer patients were selected and divided into triple-negative breast cancer (100 cases) and non-triple-negative breast cancer (113 cases) according to the expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Patient information including age, body mass index (BMI), smoking history, drinking history, menopause, tumor classification, lymph node metastasis, distant metastasis, clinical stage, and EGFR and SMC4 expression were collected for all subjects. Logistic regression analysis was then used to evaluate the risk factors for triple-negative breast cancer. The ROC curve was also used to evaluate the clinical value of EGFR and SMC4 in the diagnosis of triple-negative breast cancer. Logistic regression analysis showed that high expression of SMC4 and high expression of EGFR were both risk factors for triple-negative breast cancer, with an odds ratio (OR) of 1.72 and 1.56, respectively (both P<0.05). ROC curve analysis results showed that the areas under the curve with high SMC4 expression and high EGFR expression for the diagnosis of triple-negative breast cancer were 0.84 and 0.78, respectively. High expression of SMC4 and EGFR is significantly correlated with triple-negative breast cancer, and can be used as an auxiliary diagnostic indicator for triple-negative breast cancer.

A rotamer relay information system in the epidermal growth factor receptor-drug complexes reveals clues to new paradigm in protein conformational change.

Cancer cells can escape the effects of chemotherapy through mutations and upregulation of a tyrosine kinase protein called the epidermal growth factor receptor (EGFR). In the past two decades, four generations of tyrosine kinase inhibitors targeting EGFR have been developed. Using comparative structure analysis of 116 EGFR-drug complex crystal structures, cluster analysis produces two clans of 73 and 43 structures, respectively. The first clan of 73 structures is larger and is comprised mostly of the C-helix-IN conformation while the second clan of 43 structures correlates with the C-helix-OUT conformation. A deep rotamer analysis identifies 43 residues (18%) of the total of 237 residues spanning the kinase structures under investigation with significant rotamer variations between the C-helix-IN and C-helix-OUT clans. The locations of these rotamer variations take on the appearance of side chain conformational relays extending out from points of EGFR mutation to different regions of the EGFR kinase. Accordingly, we propose that key EGFR mutations act singly or together to induce drug resistant conformational changes in EGFR that are communicated via these side chain conformational relays. Accordingly, these side chain conformational relays appear to play a significant role in the development of tumour resistance. This phenomenon also suggests a new paradigm in protein conformational change that is mediated by supportive relays of rotamers on the protein surface, rather than through conventional backbone movements.

Acute Statin Treatment Improves Antibody Accumulation in EGFR- and PSMA-Expressing Tumors.

Statins are cholesterol-depleting drugs used to treat patients with hypercholesterolemia. Preclinically, statins disrupt trafficking of receptors present at the cell membrane. Membrane receptors, defined as tumor biomarkers and therapeutic targets, are often internalized by an endocytic pathway. Indeed, receptor endocytosis and recycling are dynamic mechanisms that often affect receptor density at the cell surface. In therapies using monoclonal antibodies (mAb), a downregulation in receptor density at the cell surface decreases antibody binding to the extracellular domain of the membrane receptor. Here, we determined the potential of lovastatin, simvastatin, and rosuvastatin in preclinically modulating epidermal growth factor receptor (EGFR) and prostate-specific membrane antigen (PSMA) receptor density at the tumor cell surface. Small-animal PET was used to study the binding of 89Zr-labeled antibodies in ectopic xenografts. Ex vivo analyses were performed to determine changes in endocytic proteins, EGFR, and PSMA surface levels. Acute statin treatment using lovastatin, simvastatin, or rosuvastatin enhanced tumors' avidity for the mAbs panitumumab, cetuximab, and huJ591. Statins temporarily modulated caveolin-1, cavin-1, endophilin, clathrin, and dynamin proteins in EGFR- and PSMA-overexpressing xenografts. These data show the potential of statins as pharmacologic modulators of endocytic proteins for improved tumors' accumulation of mAbs. The translational significance of these findings lies in the potential of statins to temporarily modulate the heterogeneous presence of receptors at the cell membrane, a characteristic often associated with poor response in tumors to therapeutic antibodies.

In vitro effects of glutathione on Transforming Growth Factor beta and Epidermal Growth Factor Receptor genes expression in the protoscoleces and strobilated worms of Echinococcus granulosus

Cystic Echinococcosis (CE) caused by the small taeniid cestode Echinococcus granulosus, is a globally distributed zoonosis. Administration of some chemicals or natural compounds could lead to significant effects on the expression of some developmentally important genes including Transforming Growth Factor beta (TGF-β) and Epidermal Growth Factor Receptor (EGFR) in other parasitic organisms. The main purpose of this study was to describe the effect of glutathione (GSH) on the expression of TGF-β and EGFR genes in different developmental stages of E. granulosus.Protoscoleces of hydatid cysts collected from naturally infected sheep liver were cultured in diphasic CMRL1066 medium. Glutathione Mono-ethyl Ester (GME) at 250 μg/ml concentration was applied on the invaginated protoscoleces (PSCi), evaginated protoscoleces (PSCe) and strobilated worms (SW3) in vitro. TGF-β and EGFR genes expression were evaluated by using Real Time qPCR analysis compared to the controls.In response to GME treatment TGF-β expression was affected, however no significant effect was observed in EGFR expression. The results indicate a significant difference of TGF-β expression in the intact protoscoleces and the strobilated worms comparing to the controls. In intact invaginated protoscoleces TGF-β expression was significantly increased (p < 0.01) while in the strobilated worms a significant decrease was observed comparing to no-treatment controls (p < 0.001). None of the three developmental stages of E. granulosus demonstrated significant changes in EGFR expression.The results indicated that administration of GSH modified TGF-β expression in the protoscoleces and strobilar stages of E. granulosus. To improve our understanding of the physiology and biochemistry of the parasite more in depth in vitro and in vivo studies on the morphological and molecular effects of glutathione on the parasite is recommended. Further investigation on the gene profiles in other stages including microcysts and germinal layer cells is also suggested. This paves the way for the effective treatment and control of cystic echinococcosis.

Anti-EGFR therapy in metastatic colorectal cancer: mechanisms and potential regimens of drug resistance.

Cetuximab and panitumumab, as the highly effective antibodies targeting epidermal growth factor receptor (EGFR), have clinical activity in the patients with metastatic colorectal cancer (mCRC). These agents have good curative efficacy, but drug resistance also exists at the same time. The effects of KRAS, NRAS, and BRAF mutations and HER2 amplification on the treatment of refractory mCRC have been elucidated and the corresponding countermeasures have been put forward. However, the changes in EGFR and its ligands, the mutations or amplifications of PIK3CA, PTEN, TP53, MET, HER3, IRS2, FGFR1, and MAP2K1, the overexpression of insulin growth factor-1, the low expression of Bcl-2-interacting mediator of cell death, mismatch repair-deficient, and epigenetic instability may also lead to drug resistance in mCRC. Although the emergence of drug resistance has genetic or epigenetic heterogeneity, most of these molecular changes relating to it are focused on the key signaling pathways, such as the RAS/RAF/mitogen-activated protein kinase or phosphatidylinositol 3-kinase/Akt/mammalian target of the rapamycin pathway. Accordingly, numerous efforts to target these signaling pathways and develop the novel therapeutic regimens have been carried out. Herein, we have reviewed the underlying mechanisms of the resistance to anti-EGFR therapy and the possible implications in clinical practice.

Conformational Insight on WT- and Mutated-EGFR Receptor Activation and Inhibition by Epigallocatechin-3-Gallate: Over a Rational Basis for the Design of Selective Non-Small-Cell Lung Anticancer Agents.

Non-small cell lung cancer (NSCLC) represents a difficult condition to treat, due to epidermal growth factor receptor (EGFR) kinase domain mutations, which lead to ligand-independent phosphorylation. Deletion of five amino acids (ELREA) in exon 19 and mutational change from leucine to arginine at position 858 (L858R) are responsible for tyrosine kinase domain aberrant activation. These two common types of EGFR-mutated forms are clinically associated with high response with Tyrosine Kinase Inhibitors (TKI); however, the secondary T790M mutation within the Tyrosine Kinase Domain (TKD) determines a resistance to these EGFR-TKIs. Using molecular dynamic simulation (MD), the present study investigated the architectural changes of wild-type and mutants EGFR’s kinase domains in order to detect any conformational differences that could be associated with a constitutively activated state and thus to evaluate the differences between the wild-type and its mutated forms. In addition, in order to evaluate to which extent the EGFR mutations affect its inhibition, Epigallocatechin 3-Gallate (EGCG) and Erlotinib (Erl), known EGFR-TKI, were included in our study. Their binding modes with the EGFR-TK domain were elucidated and the binding differences between EGFR wild-type and the mutated forms were evidenced. The aminoacids mutations directly influence the binding affinity of these two inhibitors, resulting in a different efficacy of Erl and EGCG inhibition. In particular, for the T790M/L858R EGFR, the binding modes of studied inhibitors were compromised by aminoacidic substitution confirming the experimental findings. These results may be useful for novel drug design strategies targeting the dimerization domain of the EGFR mutated forms, thus preventing receptor activation.

Ang-(1-7)/ MAS1 receptor axis inhibits allergic airway inflammation via blockade of Src-mediated EGFR transactivation in a murine model of asthma.

The angiotensin-(1–7) [Ang-(1–7)]/MAS1 receptor signaling axis is a key endogenous anti-inflammatory signaling pathway. However, the mechanisms by which its mediates the anti-inflammatory effects are not completely understood. Using an allergic murine model of asthma, we investigated whether Ang-1(1–7)/MAS1 receptor axis a): inhibits allergic inflammation via modulation of Src-dependent transactivation of the epidermal growth factor receptor (EGFR) and downstream signaling effectors such as ERK1/2, and b): directly inhibits neutrophil and/or eosinophil chemotaxis ex vivo. Ovalbumin (OVA)-induced allergic inflammation resulted in increased phosphorylation of Src kinase, EGFR, and ERK1/2. In addition, OVA challenge increased airway cellular influx, perivascular and peribronchial inflammation, fibrosis, goblet cell hyper/metaplasia and airway hyperresponsiveness (AHR). Treatment with Ang-(1–7) inhibited phosphorylation of Src kinase, EGFR, ERK1/2, the cellular and histopathological changes and AHR. Ang-(1–7) treatment also inhibited neutrophil and eosinophil chemotaxis ex vivo. These changes were reversed following pre-treatment with A779. These data show that the anti-inflammatory actions of Ang-(1–7)/ MAS1 receptor axis are mediated, at least in part, via inhibition of Src-dependent transactivation of EGFR and downstream signaling molecules such as ERK1/2. This study therefore shows that inhibition of the Src/EGRF/ERK1/2 dependent signaling pathway is one of the mechanisms by which the Ang-(1–7)/ MAS1 receptor axis mediates it anti-inflammatory effects in diseases such as asthma.

Label-Free Surface Protein Profiling of Extracellular Vesicles by an Electrokinetic Sensor

Small extracellular vesicles (sEVs) generated from the endolysosomal system, often referred to as exosomes, have attracted interest as a suitable biomarker for cancer diagnostics, as they carry valuable biological information and reflect their cells of origin. Herein, we propose a simple and inexpensive electrical method for label-free detection and profiling of sEVs in the size range of exosomes. The detection method is based on the electrokinetic principle, where the change in the streaming current is monitored as the surface markers of the sEVs interact with the affinity reagents immobilized on the inner surface of a silica microcapillary. As a proof-of-concept, we detected sEVs derived from the non-small-cell lung cancer (NSCLC) cell line H1975 for a set of representative surface markers, such as epidermal growth factor receptor (EGFR), CD9, and CD63. The detection sensitivity was estimated to be ∼175000 sEVs, which represents a sensor surface coverage of only 0.04%. We further validated the ability of the sensor to measure the expression level of a membrane protein by using sEVs displaying artificially altered expressions of EGFR and CD63, which were derived from NSCLC and human embryonic kidney (HEK) 293T cells, respectively. The analysis revealed that the changes in EGFR and CD63 expressions in sEVs can be detected with a sensitivity in the order of 10% and 3%, respectively, of their parental cell expressions. The method can be easily parallelized and combined with existing microfluidic-based EV isolation technologies, allowing for rapid detection and monitoring of sEVs for cancer diagnosis.

Effect of double mutations T790M/L858R on conformation and drug-resistant mechanism of epidermal growth factor receptor explored by molecular dynamics simulations.

Epidermal growth factor receptor (EGFR) is one of the most promising targets for the treatment of cancers. Double mutations T790M/L858R lead to different degrees of drug resistance toward inhibitors. In this study, molecular dynamics (MD) simulations followed by principal component analysis are performed to study the conformational changes of EGFR induced by T790M/L858R. The results suggest that T790M/L858R cause obvious disturbance of the structural stability of EGFR. Molecular mechanics-Poisson Boltzmann surface area (MM-PBSA) and residue-based free energy decomposition methods are integrated to explore the drug-resistant mechanism of T790M/L858R toward inhibitors. The results show that the decrease in van der Waals interactions of inhibitors with the mutated EFGR relative to the wild-type (WT) one is the main force inducing drug resistance of T790M/L858R toward inhibitors TAK-285, while drug resistance toward W2P and HKI-272 is dominated by the decrease in van der Waals interactions and the increase in polar interactions. We expect that the information obtained from this study can aid rational design of effective drugs to relieve drug resistance of EGFR induced by T790M/L858R.

Cetuximab sensitivity of head and neck squamous cell carcinoma xenografts is associated with treatment-induced reduction in EGFR, pEGFR, and pSrc.

The aims of this study were to validate in vitro drug sensitivity testing of head and neck squamous cell carcinoma (HNSCC) cell lines in an in vivo xenograft model and to identify treatment-induced changes in the epidermal growth factor receptor (EGFR) signaling pathway that could be used as markers for cetuximab treatment response. The in vitro and in vivo cetuximab sensitivity of two HNSCC cell lines, UT-SCC-14 and UT-SCC-45, was assessed using a crystal violet assay and xenografts in nude mice, respectively. The expression of EGFR, phosphorylated EGFR (pEGFR), phosphorylated Src (pSrc), and Ki-67 was investigated by immunohistochemistry. To verify these results, the in vitro expression of EGFR and pEGFR was analyzed with ELISA in a panel of 10 HNSCC cell lines. A close correlation was found between in vitro and in vivo cetuximab sensitivity data in the two investigated HNSCC cell lines. In treatment sensitive UT-SCC-14 xenografts, there was a decrease in EGFR, pEGFR, and pSrc upon cetuximab treatment. Interestingly, in insensitive UT-SCC-45 xenografts, an increased expression of these three proteins was found. The change in EGFR and pEGFR expression in vivo was confirmed in cetuximab-sensitive and cetuximab-insensitive HNSCC cell lines using ELISA. High sensitivity to cetuximab was strongly associated with a treatment-induced reduction in pEGFR both in vivo and in vitro in a panel of HNSCC cell lines, suggesting that EGFR and pEGFR dynamics could be used as a predictive biomarker for cetuximab treatment response.

Elevated H3K18 acetylation in airway epithelial cells of asthmatic subjects.

BackgroundEpigenetic adjustments of the chromatin architecture through histone modifications are reactive to the environment and can establish chromatin states which are permissive or repressive to gene expression. Epigenetic regulation of gene expression is cell specific and therefore, it is important to understand its contribution to individual cellular responses in tissues like the airway epithelium which forms the mucosal barrier to the inhaled environment within the lung. The airway epithelium of asthmatics is abnormal with dysregulation of genes such as epidermal growth factor receptor (EGFR), the ΔN isoform of the transcription factor p63 (ΔNp63), and signal transducer and activator of transcription 6 (STAT6), integral to differentiation, proliferation, and inflammation. It is important to establish in diseases like asthma how histone modifications affect tissue responses such as proliferation and differentiation.ObjectivesTo characterize the global histone acetylation and methylation status in the epithelium of asthmatic compared to healthy subjects and to identify the impact of these variations on genes involved in epithelial functions.MethodsWhole lungs were obtained from healthy and asthmatic subjects (n = 6) from which airway epithelial cells (AECs) were isolated and airway sections were taken for analysis of histone lysine acetylation and methylation by immunohistochemistry. AECs were subjected to chromatin immunoprecipitation (ChIP) using anti-H3K18ac and anti-H3K4me2 antibodies followed by RT-PCR targeting ΔNp63, EGFR, and STAT6. AECs were also treated with TSA and changes in ΔNp63, EGFR, and STAT6 expression were determined.ResultsWe identified an increase in the acetylation of lysine 18 on histone 3 (H3K18ac) and trimethylation of lysine 9 on histone 3 (H3K9me3) in the airway epithelium of asthmatic compared to healthy subjects. We found increased association of H3K18ac around the transcription start site of ΔNp63, EGFR, and STAT6 in AECs of asthmatics. However, we were unable to modify the expression of these genes with the use of the HDAC inhibitor TSA in healthy subjects.DiscussionThe airway epithelium from asthmatic subjects displays increased acetylation of H3K18 and association of this mark around the transcription start site of ΔNp63, EGFR, and STAT6. These findings suggest a complex interaction between histone modifications and gene regulation in asthma.Electronic supplementary materialThe online version of this article (doi:10.1186/s12931-015-0254-y) contains supplementary material, which is available to authorized users.

Epidermal growth factor receptor and AKT1 gene copy numbers by multi‐gene fluorescence in situ hybridization impact on prognosis in breast cancer

The epidermal growth factor receptor (EGFR)/PI3K/AKT signaling pathway aberrations play significant roles in breast cancer occurrence and development. However, the status of EGFR and AKT1 gene copy numbers remains unclear. In this study, we showed that the rates of EGFR and AKT1 gene copy number alterations were associated with the prognosis of breast cancer. Among 205 patients, high EGFR and AKT1 gene copy numbers were observed in 34.6% and 27.8% of cases by multi-gene fluorescence in situ hybridization, respectively. Co-heightened EGFR/AKT1 gene copy numbers were identified in 11.7% cases. No changes were found in 49.3% of patients. Although changes in EGFR and AKT1 gene copy numbers had no correlation with patients' age, tumor stage, histological grade and the expression status of other molecular makers, high EGFR (P = 0.0002) but not AKT1 (P = 0.1177) gene copy numbers correlated with poor 5-year overall survival. The patients with co-heightened EGFR/AKT1 gene copy numbers displayed a poorer prognosis than those with tumors with only high EGFR gene copy numbers (P = 0.0383). Both Univariate (U) and COX multivariate (C) analyses revealed that high EGFR and AKT1 gene copy numbers (P = 0.000 [U], P = 0.0001 [C]), similar to histological grade (P = 0.001 [U], P = 0.012 [C]) and lymph node metastasis (P = 0.046 [U], P = 0.158 [C]), were independent prognostic indicators of 5-year overall survival. These results indicate that high EGFR and AKT1 gene copy numbers were relatively frequent in breast cancer. Co-heightened EGFR/AKT1 gene copy numbers had a worse outcome than those with only high EGFR gene copy numbers, suggesting that evaluation of these two genes together may be useful for selecting patients for anti-EGFR-targeted therapy or anti-EGFR/AKT1-targeted therapy and for predicting outcomes.

Modulating the structure of EGFR with UV light: new possibilities in cancer therapy.

The epidermal growth factor receptor (EGFR) is a member of the ErbB family of receptor tyrosine kinases. EGFR is activated upon binding to e.g. epidermal growth factor (EGF), leading to cell survival, proliferation and migration. EGFR overactivation is associated with tumor progression. We have previously shown that low dose UVB illumination of cancer cells overexpressing EGFR prior to adding EGF halted the EGFR signaling pathway. We here show that UVB illumination of the extracellular domain of EGFR (sEGFR) induces protein conformational changes, disulphide bridge breakage and formation of tryptophan and tyrosine photoproducts such as dityrosine, N-formylkynurenine and kynurenine. Fluorescence spectroscopy, circular dichroism and thermal studies confirm the occurrence of conformational changes. An immunoassay has confirmed that UVB light induces structural changes in the EGF binding site. A monoclonal antibody which competes with EGF for binding sEGFR was used. We report clear evidence that UVB light induces structural changes in EGFR that impairs the correct binding of an EGFR specific antibody that competes with EGF for binding EGFR, confirming that the 3D structure of the EGFR binding domain suffered conformational changes upon UV illumination. The irradiance used is in the same order of magnitude as the integrated intensity in the solar UVB range. The new photonic technology disables a key receptor and is most likely applicable to the treatment of various types of cancer, alone or in combination with other therapies.