The HDAC inhibitor,MPT0E028, enhances erlotinib-induced cell death in EGFR-TKI-resistant NSCLC cells

Epidermal growth factor receptor (EGFR), which promotes cell survival and division, is found at abnormally high levels on the surface of many cancer cell types, including many cases of non-small cell lung cancer. Erlotinib (Tarceva), an oral small-molecule tyrosine kinase inhibitor, is a so-called targeted drug that inhibits the tyrosine kinase domain of EGFR, and thus targets cancer cells with some specificity while doing less damage to normal cells. However, erlotinib resistance can occur, reducing the efficacy of this treatment. To develop more effective therapeutic interventions by overcoming this resistance problem, we combined the histone deacetylase inhibitor, MPT0E028, with erlotinib in an effort to increase their antitumor effects in erlotinib-resistant lung adenocarcinoma cells. This combined treatment yielded significant growth inhibition, induced the expression of apoptotic proteins (PARP, γH2AX, and caspase-3), increased the levels of acetylated histone H3, and showed synergistic effects in vitro and in vivo. These effects were independent of the mutation status of the genes encoding EGFR or K-Ras. MPT0E028 synergistically blocked key regulators of the EGFR/HER2 signaling pathways, attenuating multiple compensatory pathways (e.g., AKT, extracellular signal-regulated kinase, and c-MET). Our results indicate that this combination therapy might be a promising strategy for facilitating the effects of erlotinib monotherapy by activating various networks. Taken together, our data provide compelling evidence that MPT0E028 has the potential to improve the treatment of heterogeneous and drug-resistant tumors that cannot be controlled with single-target agents.     

Drug localization in different lung cancer phenotypes by MALDI mass spectrometry imaging

Lung cancer is a common cause of cancer mortality in the world, largely due to the risk factor of tobacco smoking. The drug therapy at the molecular level includes targeting the epidermal growth factor receptor (EGFR) tyrosine kinase activity by using inhibitors, such as erlotinib (Tarceva) and gefitinib (Iressa). The heterogeneity of disease phenotypes and the somatic mutations presented in patient populations have a great impact on the efficacy of treatments using targeted personalized medicine. In this study, we report on basic physical and chemical properties of erlotinib and gefitinib in three different lung cancer tumor phenotypes, using MALDI instrumentation in imaging mode, providing spatial localization of drugs without chemical labeling. Erlotinib and gefitinib were analyzed in i) planocellular lung carcinoma, ii) adenocarcinoma and iii) large cell lung carcinoma following their deposition on the tissue surfaces by piezo-dispensing, using a controlled procedure. The importance of high-resolution sampling was crucial in order to accurately localize the EGFR tyrosine kinase inhibitors deposited in heterogeneous cancer tissue compartments. This is the first report on personalized drug characterization with localizations at a lateral resolution of 30μm, which allowed us to map these compounds at attomolar concentrations within the lung tumor tissue microenvironments.     

Epidermal Growth Factor Receptor Inhibition Modulates the Microenvironment by Vascular Normalization to Improve Chemotherapy and Radiotherapy Efficacy

Background

Epidermal growth factor receptor (EGFR) inhibitors have shown only modest clinical activity when used as single agents to treat cancers. They decrease tumor cell expression of hypoxia-inducible factor 1-α (HIF-1α) and vascular endothelial growth factor (VEGF). Hypothesizing that this might normalize tumor vasculature, we examined the effects of the EGFR inhibitor erlotinib on tumor vascular function, tumor microenvironment (TME) and chemotherapy and radiotherapy sensitivity.

Methodology/Principal Findings

Erlotinib treatment of human tumor cells in vitro and mice bearing xenografts in vivo led to decreased HIF-1α and VEGF expression. Treatment altered xenograft vessel morphology assessed by confocal microscopy (following tomato lectin injection) and decreased vessel permeability (measured by Evan''s blue extravasation), suggesting vascular normalization. Erlotinib increased tumor blood flow measured by Power Doppler ultrasound and decreased hypoxia measured by EF5 immunohistochemistry and tumor O₂ saturation measured by optical spectroscopy. Predicting that these changes would improve drug delivery and increase response to chemotherapy and radiation, we performed tumor regrowth studies in nude mice with xenografts treated with erlotinib and either radiotherapy or the chemotherapeutic agent cisplatin. Erlotinib therapy followed by cisplatin led to synergistic inhibition of tumor growth compared with either treatment by itself (p<0.001). Treatment with erlotinib before cisplatin led to greater tumor growth inhibition than did treatment with cisplatin before erlotinib (p = 0.006). Erlotinib followed by radiation inhibited tumor regrowth to a greater degree than did radiation alone, although the interaction between erlotinib and radiation was not synergistic.

Conclusions/Significance

EGFR inhibitors have shown clinical benefit when used in combination with conventional cytotoxic therapy. Our studies show that targeting tumor cells with EGFR inhibitors may modulate the TME via vascular normalization to increase response to chemotherapy and radiotherapy. These studies suggest ways to assess the response of tumors to EGFR inhibition using non-invasive imaging of the TME.     

Effect of simvastatin on the resistance to EGFR tyrosine kinase inhibitors in a non-small cell lung cancer with the T790M mutation of EGFR

Although non-small cell lung cancer (NSCLC) tumors with activating mutations in the epidermal growth factor receptor (EGFR) are highly responsive to EGFR tyrosine kinase inhibitors (TKIs) including gefitinib and erlotinib, development of acquired resistance is almost inevitable. Statins show antitumor activity, but it is unknown whether they can reverse EGFR-TKIs resistance in NSCLC with the T790M mutation of EGFR. This study investigated overcoming resistance to EGFR-TKI using simvastatin. We demonstrated that addition of simvastatin to gefitinib enhanced caspase-dependent apoptosis in T790M mutant NSCLC cells. Simvastatin also strongly inhibited AKT activation, leading to suppression of β-catenin activity and the expression of its targets, survivin and cyclin D1. Both insulin treatment and AKT overexpression markedly increased p-β-catenin and survivin levels, even in the presence of gefitinib and simvastatin. However, inhibition of AKT by siRNA or LY294002 treatment decreased p-β-catenin and survivin levels. To determine the role of survivin in simvastatin-induced apoptosis of gefitinib-resistant NSCLC, we showed that the proportion of apoptotic cells following treatment with survivin siRNA and the gefitinib–simvastatin combination was greater than the theoretical additive effects, whereas survivin up-regulation could confer protection against gefitinib and simvastatin-induced apoptosis. Similar results were obtained in erlotinib and simvastatin-treated HCC827/ER cells. These findings suggest that survivin is a key molecule that renders T790M mutant NSCLC cells resistant to apoptosis induced by EGFR-TKIs and simvastatin. Overall, these data indicate that simvastatin may overcome EGFR-TKI resistance in T790M mutant NSCLCs via an AKT/β-catenin signaling-dependent down-regulation of survivin and apoptosis induction.     

Magnetic resonance imaging of the response of a mouse model of non-small cell lung cancer to tyrosine kinase inhibitor treatment

Mutational activation of the gene for epidermal growth factor receptor (EGFR) is 1 of the main ways by which this receptor induces non-small cell lung cancers (NSCLC). Variant III EGFR (EGFRvIII) is a potential therapeutic target in NSCLC treatment because of the high frequency of deletion mutations in this protein. This study used noninvasive magnetic resonance imaging (MRI) to investigate the role of an EGFRvIII mutant in lung tumorigenesis and tumor maintenance as well as its response to the EGFR small molecule inhibitor erlotinib (Tarceva) on bitransgenic mice. Both spin-echo and gradient-echo sequences with and without cardiac and respiratory gating were performed to image the invasive mouse lung tumor driven by EGFRvIII mutation. Tumor volumes were measured based on 2-dimensional axial MRI; 3-dimensional rendering of the images were obtained to demonstrate the spatial location and distribution of the tumor in the lung. The MRI results indicated that the tumor driven by the EGFRvIII mutation was generated and maintained in the bitransgenic mice with the use of doxycycline. Tumor monitoring via MRI showed that Erlotinib can significantly inhibit the growth of tumor in vivo. MRI has the ability to image mouse lung tumor with different sequences focusing on tissue contrasts between tumor and surroundings. The MRI approaches in this work can be applied on other antitumor drug treatment evaluation in vivo when appropriate sequences are chosen.     

The Relative Expression of Mig6 and EGFR Is Associated with Resistance to EGFR Kinase Inhibitors

The sensitivity of only a few tumors to anti-epidermal growth factor receptor EGFR tyrosine kinase inhibitors (TKIs) can be explained by the presence of EGFR tyrosine kinase (TK) domain mutations. In addition, such mutations were rarely found in tumor types other than lung, such as pancreatic and head and neck cancer. In this study we sought to elucidate mechanisms of resistance to EGFR-targeted therapies in tumors that do not harbor TK sensitizing mutations in order to identify markers capable of guiding the decision to incorporate these drugs into chemotherapeutic regimens. Here we show that EGFR activity was markedly decreased during the evolution of resistance to the EGFR tyrosine kinase inhibitor (TKI) erlotinib, with a concomitant increase of mitogen-inducible gene 6 (Mig6), a negative regulator of EGFR through the upregulation of the PI3K-AKT pathway. EGFR activity, which was more accurately predicted by the ratio of Mig6/EGFR, highly correlated with erlotinib sensitivity in panels of cancer cell lines of different tissue origins. Blinded testing and analysis in a prospectively followed cohort of lung cancer patients treated with gefitinib alone demonstrated higher response rates and a marked increased in progression free survival for patients with a low Mig6/EGFR ratio (approximately 100 days, P = 0.01).     

Antitumor activity of sorafenib in human cancer cell lines with acquired resistance to EGFR and VEGFR tyrosine kinase inhibitors

Treatment of non small cell lung cancer (NSCLC) and colorectal cancer (CRC) have substantially changed in the last years with the introduction of epidermal growth factor receptor (EGFR) inhibitors in the clinical practice. The understanding of mechanisms which regulate cells sensitivity to these drugs is necessary for their optimal use.An in vitro model of acquired resistance to two tyrosine kinase inhibitors (TKI) targeting the EGFR, erlotinib and gefitinib, and to a TKI targeting EGFR and VEGFR, vandetanib, was developed by continuously treating the human NSCLC cell line CALU-3 and the human CRC cell line HCT116 with escalating doses of each drug. MTT, western blot analysis, migration, invasion and anchorage-independent colony forming assays were conducted in vitro and experiments with established xenografts in athymic nude mice were performed in vivo in sensitive, wild type (WT) and TKI-resistant CALU-3 and HCT116 cell lines.As compared to WT CALU-3 and HCT116 human cancer cells, TKI-resistant cell lines showed a significant increase in the levels of activated, phosphorylated AKT, MAPK, and of survivin. Considering the role of RAS and RAF as downstream signals of both the EGFR and VEGFR pathways, we treated resistant cells with sorafenib, an inhibitor of C-RAF, B-RAF, c-KIT, FLT-3, RET, VEGFR-2, VEGFR-3, and PDGFR-β. Sorafenib reduced the activation of MEK and MAPK and caused an inhibition of cell proliferation, invasion, migration, anchorage-independent growth in vitro and of tumor growth in vivo of all TKI-resistant CALU-3 and HCT116 cell lines.These data suggest that resistance to EGFR inhibitors is predominantly driven by the RAS/RAF/MAPK pathway and can be overcame by treatment with sorafenib.     

Characterization and response of newly developed high-grade glioma cultures to the tyrosine kinase inhibitors, erlotinib, gefitinib and imatinib

High-grade gliomas (HGG), are the most common aggressive brain tumours in adults. Inhibitors targeting growth factor signalling pathways in glioma have shown a low clinical response rate. To accurately evaluate response to targeted therapies further in vitro studies are necessary. Growth factor pathway expression using epidermal growth factor receptor (EGFR), mutant EGFR (EGFRvIII), platelet derived growth factor receptor (PDGFR), C-Kit and C-Abl together with phosphatase and tensin homolog (PTEN) expression and downstream activation of AKT and phosphorylated ribosomal protein S6 (P70S6K) was analysed in 26 primary glioma cultures treated with the tyrosine kinase inhibitors (TKIs) erlotinib, gefitinib and imatinib. Response to TKIs was assessed using 50% inhibitory concentrations (IC(50)). Response for each culture was compared with the EGFR/PDGFR immunocytochemical pathway profile using hierarchical cluster analysis (HCA) and principal component analysis (PCA). Erlotinib response was not strongly associated with high expression of the growth factor pathway components. PTEN expression did not correlate with response to any of the three TKIs. Increased EGFR expression was associated with gefitinib response; increased PDGFR-α expression was associated with imatinib response. The results of this in vitro study suggest gefitinib and imatinib may have therapeutic potential in HGG tumours with a corresponding growth factor receptor expression profile.     

Activated epidermal growth factor receptor as a novel target in pancreatic cancer therapy

Pancreatic cancer is one of the most fatal among all solid malignancies. Targeted therapeutic approaches have the potential to transform cancer therapy as exemplified by the success of several tyrosine kinase inhibitors. Prompted by this, comprehensive profiling of tyrosine kinases and their substrates was carried out using a panel of low passage pancreatic cancer cell lines. One of the pancreatic cancer cell lines, P196, which showed dramatic upregulation of tyrosine kinase activity as compared to non-neoplastic cells, was systematically studied using a quantitative proteomic approach called stable isotope labeling with amino acids in cell culture (SILAC). A careful analysis of activated tyrosine kinase pathways revealed aberrant activation of epidermal growth factor receptor pathway in this cell line. Mouse xenograft based studies using EGFR inhibitor erlotinib confirmed EGFR pathway to be responsible for proliferation in these tumors. By a systematic study across low passage pancreatic cancer cell lines and mice carrying pancreatic cancer xenografts, we have demonstrated activated epidermal growth factor receptor as an attractive candidate for targeted therapy in a subset of pancreatic cancers. Further, we propose immunohistochemical labeling of activated EGFR (pEGFR (1068)) as an efficient screening tool to select patients who are more likely to respond to EGFR inhibitors.     

Exogenous Restoration of TUSC2 Expression Induces Responsiveness to Erlotinib in Wildtype Epidermal Growth Factor Receptor (EGFR) Lung Cancer Cells through Context Specific Pathways Resulting in Enhanced Therapeutic Efficacy

Expression of the tumor suppressor gene TUSC2 is reduced or absent in most lung cancers and is associated with worse overall survival. In this study, we restored TUSC2 gene expression in several wild type EGFR non-small cell lung cancer (NSCLC) cell lines resistant to the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor erlotinib and analyzed their sensitivity to erlotinib in vitro and in vivo. A significant inhibition of cell growth and colony formation was observed with TUSC2 transient and stable expression. TUSC2-erlotinib cooperativity in vitro could be reproduced in vivo in subcutaneous tumor growth and lung metastasis formation lung cancer xenograft mouse models. Combination treatment with intravenous TUSC2 nanovesicles and erlotinib synergistically inhibited tumor growth and metastasis, and increased apoptotic activity. High-throughput qRT-PCR array analysis enabling multi-parallel expression profile analysis of eighty six receptor and non-receptor tyrosine kinase genes revealed a significant decrease of FGFR2 expression level, suggesting a potential role of FGFR2 in TUSC2-enhanced sensitivity to erlotinib. Western blots showed inhibition of FGFR2 by TUSC2 transient transfection, and marked increase of PARP, an apoptotic marker, cleavage level after TUSC2-erlotinb combined treatment. Suppression of FGFR2 by AZD4547 or gene knockdown enhanced sensitivity to erlotinib in some but not all tested cell lines. TUSC2 inhibits mTOR activation and the latter cell lines were responsive to the mTOR inhibitor rapamycin combined with erlotinib. These results suggest that TUSC2 restoration in wild type EGFR NSCLC may overcome erlotinib resistance, and identify FGFR2 and mTOR as critical regulators of this activity in varying cellular contexts. The therapeutic activity of TUSC2 could extend the use of erlotinib to lung cancer patients with wildtype EGFR.     

The efficacy of TKIs in treatment of human primary small cell lung cancer xenograft model in vivo

Objective: To study the treatmaient of non-small cell lung cancer, we established the HU-Prim allograft transplantation tumor model. Methods: The fresh tumor samples were transplanted in the right scapular subcutaneous layer of the severe combined immunodeficient Non-obese diabetic/severe combined immunodeficient(NOD/SCID) mice. The pathological features of the tumors were observed. Nonnecrotic tissue was inoculated subcutaneously into the right axillary. When the tumor in burdened rat grew approximately 100 mm3, according to the tumor size all the animals were divided into the following four groups, eight rats in each group: solvent control group, gefitinib group(100 mg/kg), erlotinib group(50 mg/kg), afatinib group(20 mg/kg). Aniamals were treated with drugs by intragastric(i.g.) administrated, once daily, for consecutively 14 days. Measure the tumor size 2-3 times every week. Results: Hu Prime1-NSCLC mutant sensitive xenograft model research data showed that reversible tyrosine kinase inhibitors gefitinib, erlotinib and irreversible tyrosine kinase inhibitor afatinib could effectively inhibit tumor growth in EGFR positive NSCLC allografts model. The pharmacodynamic activity of irreversible inhibitor was better than that of the reversible inhibitor. Specimens from clinical anthropogenic tumor retain characteristics of the human primary malignancy, histopathology, biological characteristics, and tumor markers, etc., which can more accurately reflect the characteristics of the tumor and the impact of interventions. Conclusion: The model is not only a good antitumor drug experimental platform, but also a new evaluation tool of individualized medication.     

Combination of mTOR and EGFR Kinase Inhibitors Blocks mTORC1 and mTORC2 Kinase Activity and Suppresses the Progression of Colorectal Carcinoma

Mammalian target of rapamycin complex 1 and 2 (mTORC1/2) are overactive in colorectal carcinomas; however, the first generation of mTOR inhibitors such as rapamycin have failed to show clinical benefits in treating colorectal carcinoma in part due to their effects only on mTORC1. The second generation of mTOR inhibitors such as PP242 targets mTOR kinase; thus, they are capable of inhibiting both mTORC1 and mTORC2. To examine the therapeutic potential of the mTOR kinase inhibitors, we treated a panel of colorectal carcinoma cell lines with PP242. Western blotting showed that the PP242 inhibition of mTORC2-mediated AKT phosphorylation at Ser 473 (AKT^S473) was transient only in the first few hours of the PP242 treatment. Receptor tyrosine kinase arrays further revealed that PP242 treatment increased the phosphorylated epidermal growth factor receptor (EGFR) at Tyr 1068 (EGFR^T1068). The parallel increase of AKT^S473 and EGFR^T1068 in the cells following PP242 treatment raised the possibility that EGFR phosphorylation might contribute to the PP242 incomplete inhibition of mTORC2. To test this notion, we showed that the combination of PP242 with erlotinib, an EGFR small molecule inhibitor, blocked both mTORC1 and mTORC2 kinase activity. In addition, we showed that the combination treatment inhibited colony formation, blocked cell growth and induced apoptotic cell death. A systemic administration of PP242 and erlotinib resulted in the progression suppression of colorectal carcinoma xenografts in mice. This study suggests that the combination of mTOR kinase and EGFR inhibitors may provide an effective treatment of colorectal carcinoma.     

EGF receptor tyrosine kinase inhibitors diminish transforming growth factor-alpha-induced pulmonary fibrosis

Transforming growth factor-alpha (TGF-alpha) is a ligand for the EGF receptor (EGFR). EGFR activation is associated with fibroproliferative processes in human lung disease and animal models of pulmonary fibrosis. We determined the effects of EGFR tyrosine kinase inhibitors gefitinib (Iressa) and erlotinib (Tarceva) on the development and progression of TGF-alpha-induced pulmonary fibrosis. Using a doxycycline-regulatable transgenic mouse model of lung-specific TGF-alpha expression, we determined effects of treatment with gefitinib and erlotinib on changes in lung histology, total lung collagen, pulmonary mechanics, pulmonary hypertension, and expression of genes associated with synthesis of ECM and vascular remodeling. Induction in the lung of TGF-alpha caused progressive pulmonary fibrosis over an 8-wk period. Daily administration of gefitinib or erlotinib prevented development of fibrosis, reduced accumulation of total lung collagen, prevented weight loss, and prevented changes in pulmonary mechanics. Treatment of mice with gefitinib 4 wk after the induction of TGF-alpha prevented further increases in and partially reversed total collagen levels and changes in pulmonary mechanics and pulmonary hypertension. Increases in expression of genes associated with synthesis of ECM as well as decreases of genes associated with vascular remodeling were also prevented or partially reversed. Administration of gefitinib or erlotinib did not cause interstitial fibrosis or increases in lavage cell counts. Administration of small molecule EGFR tyrosine kinase inhibitors prevented further increases in and partially reversed pulmonary fibrosis induced directly by EGFR activation without inducing inflammatory cell influx or additional lung injury.     

Mass spectrometry mapping of epidermal growth factor receptor phosphorylation related to oncogenic mutations and tyrosine kinase inhibitor sensitivity

The epidermal growth factor receptor (EGFR) plays an important role in cancer by activating downstream signals important in growth and survival. Inhibitors of EGFR are frequently selected as treatment for cancer including lung cancer. We performed an unbiased and comprehensive search for EGFR phosphorylation events related to somatic activating mutations and EGFR inhibitor (erlotinib) sensitivity. EGFR immunoprecipitation combined with high resolution liquid chromatography-mass spectrometry and label free quantitation characterized EGFR phosphorylation. Thirty (30) phosphorylation sites were identified including 12 tyrosine (pY), 12 serine (pS), and 6 threonine (pT). Site-specific phosphorylation was monitored by comparing ion signals from the corresponding unmodified peptide. Phosphorylation sites related to activating mutations in EGFR as well as sensitivity to erlotinib were identified using 31 lung cancer cell lines. We identified three sites (pY1092, pY1110, pY1172) correlated with activating mutations and three sites (pY1110, pY1172, pY1197) correlated with erlotinib sensitivity. Five sites (pT693, pY1092, pY1110, pY1172, and pY1197) were inhibited by erlotinib in concentration-dependent manner. Erlotinib sensitivity was confirmed using liquid chromatography coupled to multiple reaction monitoring (LC-MRM) and quantitative Western blotting. This LC-MS/MS strategy can quantitatively assess site-specific EGFR phosphorylation and can identify relationships between somatic mutations or drug sensitivity and protein phosphorylation.     

EGFR tyrosine kinase inhibitors activate autophagy as a cytoprotective response in human lung cancer cells

Epidermal growth factor receptor tyrosine kinase inhibitors gefitinib and erlotinib have been widely used in patients with non-small-cell lung cancer. Unfortunately, the efficacy of EGFR-TKIs is limited because of natural and acquired resistance. As a novel cytoprotective mechanism for tumor cell to survive under unfavorable conditions, autophagy has been proposed to play a role in drug resistance of tumor cells. Whether autophagy can be activated by gefitinib or erlotinib and thereby impair the sensitivity of targeted therapy to lung cancer cells remains unknown. Here, we first report that gefitinib or erlotinib can induce a high level of autophagy, which was accompanied by the inhibition of the PI3K/Akt/mTOR signaling pathway. Moreover, cytotoxicity induced by gefitinib or erlotinib was greatly enhanced after autophagy inhibition by the pharmacological inhibitor chloroquine (CQ) and siRNAs targeting ATG5 and ATG7, the most important components for the formation of autophagosome. Interestingly, EGFR-TKIs can still induce cell autophagy even after EGFR expression was reduced by EGFR specific siRNAs. In conclusion, we found that autophagy can be activated by EGFR-TKIs in lung cancer cells and inhibition of autophagy augmented the growth inhibitory effect of EGFR-TKIs. Autophagy inhibition thus represents a promising approach to improve the efficacy of EGFR-TKIs in the treatment of patients with advanced non-small-cell lung cancer.     

Retracted: Nimotuzuma restrains proliferation and induces apoptosis in human osteosarcoma cells by regulation of EGFR/PI3K/AKT signal pathway

Osteosarcoma (OS) is a conversant malignant bone tumor, commonly occurs in children and adolescents. Nimotuzuma is an epidermal growth factor receptor (EGRF) monoclonal antibody agent, which has been exploited in varied solid tumors. Nevertheless, the functions of Nimotuzuma in OS remain blurry. We attempted to disclose the impacts of Nimotuzuma on OS cells proliferation and apoptosis. OS MG-63 and U2OS cells were stimulated with the disparate doses of Nimotuzuma. Then, cell viability, cell cycle, and apoptosis were appraised through executing CCK-8 and flow cytometry assays. Moreover, the change of mitochondrial membrane potential (ΔΨm) was estimated via JC-1 fluorescent probe to further probe the impacts of Nimotuzuma on cell apoptosis. The proteins of cell apoptosis, cell cycle, and EGFR/PI3K/AKT were appraised via western blot. Eventually, Nimotuzuma together EGRF or PI3K inhibitor (LY294002) were utilized to dispose MG-63 to further uncover the latent mechanism. We found that Nimotuzuma remarkably repressed cell viability at a time- and dose-dependent manners in MG-63 and U2OS cells. The percentage of the S phase cells was evidently reduced by Nimotuzuma through regulating P21, Cyclin E1, and Cyclin D1. In addition, Nimotuzuma obviously evoked cell apoptosis, meanwhile elevated Bid, Bax, and cleaved-caspase-3. Further exploration showed that Nimotuzuma decreased ΔΨm in a dose-dependent manner in MG-63 and U2OS cells. Besides, we discovered the repressive functions of Nimotuzuma in OS cells proliferation and apoptosis via hindering the EGFR/PI3K/AKT pathway. These investigations testified that Nimotuzuma repressed cell growth by restraining the EGFR/PI3K/AKT pathway in OS cells, hinting the antitumor activity of Nimotuzuma in OS.     

The epidermal growth factor receptor modulates DNA double-strand break repair by regulating non-homologous end-joining

In mammalian cells repair of radiation-induced DNA damage appears to be also controlled by the epidermal growth factor receptor (EGFR) with a special impact on DNA double-strand break (DSB) repair. Aim of this study was to demonstrate this interaction between EGFR signalling and DNA DSB repair and to identify the underlying downstream pathways. We especially wanted to know in how far non-homologous end-joining (NHEJ) as the most important DSB repair pathway is involved in this interaction. Overall DSB repair was determined by counting γH2AX foci remaining 24 after irradiation, while NHEJ activity was monitored by using a specially designed repair construct stably integrated into the genome. The overall DSB repair capacity was clearly enhanced when EGFR was activated by its natural ligand EGF and, vice versa, was reduced when EGFR was blocked either by the specific antibody Cetuximab or the tyrosine kinase inhibitor erlotinib, whereby reduction was clearly stronger for erlotinib. There was also a difference in the pathways affected. While erlotinib lead to a block of both, MAPK as well as AKT signalling, Cetuximab only affected MAPK. As demonstrated by specific inhibitors (PD98059, AKTIII) EGFR interacts with DSB repair mostly via MAPK pathway. Also for NHEJ activity, there was a substantial increase, when EGFR was activated by EGF as determined for two different reporter cell lines (A549.EJ and H1299.EJ) and, vice versa, a reduction was seen when EGFR signalling was blocked by Cetuximab or erlotinib. There was, however, no difference for the two inhibitors used. This regulation of NHEJ by EGFR was only blocked when ERK was affected by siRNA but not when AKT was knocked down. These data indicate that EGFR modulates DSB repair by regulating NHEJ via MAPK signalling.     

Osimertinib for lung cancer cells harboring low-frequency EGFR T790M mutation

Osimertinib, a third-generation EGFR tyrosine kinase inhibitor, shows significant benefit among patients with EGFR T790M mutation at disease progression. We analyzed the whole exome sequence of 48 samples obtained from 16 lung cancer patients with a longitudinal follow-up: treatment-naïve-baseline primary tumors positive for EGFR activating-mutations, paired re-biopsies upon disease progression but negative for EGFR T790M mutation based on qPCR, and their matched normal blood samples. Our Next generation sequencing (NGS) analysis identified an additional set of 25% re-biopsy samples to harbor EGFR T790M mutation occurring at a low-allele frequency of 5% or less, undetectable by conventional qPCR-based assays. Notably, the clinical utility of osimertinib among patients harboring low-allele frequency of EGFR T790M in tissue biopsy upon disease progression remains less explored. We established erlotinib-resistant PC-9R cells and twenty single-cell sub-clones from erlotinib-sensitive lung cancer PC-9 cells using in vitro drug-escalation protocol. NGS and allele-specific PCR confirmed the low-allele frequency of EGFR T790M present at 5% with a 100-fold higher resistance to erlotinib in the PC-9R cells and its sub-clones. Additionally, luciferase tagged PC-9, and PC-9R cells were orthotopically injected through the intercostal muscle into NOD-SCID mice. The orthotopic lung tumors formed were observed by non-invasive bioluminescence imaging. Consistent with in vitro data, osimertinib, but not erlotinib, caused tumor regression in mice injected with PC-9R cells, while both osimertinib and erlotinib inhibited tumors in mice injected with PC-9 cells. Taken together, our findings could extend the benefit of osimertinib treatment to patients with low EGFR T790M mutation allele frequency on disease progression.     

Structural and mechanistic underpinnings of the differential drug sensitivity of EGFR mutations in non-small cell lung cancer

EGFR and other ErbB-family tyrosine kinases are overexpressed in many human tumors, and their aberrant expression and mutational activation is associated with the development, progression and aggressiveness of a number of malignancies. Thus the EGFR kinase has long been recognized as a potential drug target in oncology, and small-molecule inhibitors have been under development for more than two decades. As a result of their effectiveness in treating non-small cell lung cancers (NSCLCs) driven by somatic mutations in the EGFR kinase, gefitinib and erlotinib were the first EGFR tyrosine kinase inhibitors (TKIs) approved for clinical use. Ironically, these drugs found their target against mutant forms of the EGFR kinase, which have altered enzyme active sites, and not against the wild type (WT) kinase against which their potency and selectivity was carefully honed. Here we review recent structural and enzymological studies that explore the exquisite sensitivity of a subset of these lung cancer mutants to gefitinib and erlotinib. We discuss available structural evidence for the mechanisms of activation of the EGFR kinase by these mutants, and compare it to physiologic activation of the kinase by ligand-induced dimerization. Finally, we consider the mechanisms by which the secondary T790M “gatekeeper” mutation confers resistance to gefitinib and erlotinib.