Epidermal Growth Factor Receptor Downstream Signaling Research Articles

Development and evaluation of a single domain antibody against human epidermal growth factor receptor (EGFR)

Epidermal growth factor receptor (EGFR) plays an important role in cell growth, multiplication and differentiation. Over expression of EGFR is associated with carcinogenesis and seen in variety of cancers. Anti-EGFR monoclonal antibodies can block EGFR downstream signaling pathway resulting in inhibition of uncontrolled cell proliferation. Antibody fragments have a variety of advantages. In comparison to full length antibodies they have smaller size and therefor exhibit better tumor penetration ability. The aim of this study was to prepare a single domain antibody to target extracellular domain of EGFR. mRNA was extracted from C225 hybridoma cells producing anti-EGFR antibody and subjected to reverse transcription reaction (RT-PCR) to obtain cDNA molecules encoding VH domain of mAb C225. The cDNA encoded VH domain was in frame introduced into the pET-22b(+) vector and expressed in BL21 (DE3) bacterial cells. The resultant antibody was purified via Ni- NTA column and its reactivity was assessed by ELISA and western blot techniques using A431 cell lysate. Analysis by ELISA revealed that this single domain antibody was able to bind EGFR on A431cells. This result was further confirmed by western blotting. In conclusion, the results of this study indicated that single domain antibody can identify and bind to EGFR of A431 carcinoma cells. This recombinant fragment antibody would potentially be used for targeting of cancer cells with high EGFR expression.

EGFR is not a major driver for osteosarcoma cell growth in vitro but contributes to starvation and chemotherapy resistance.

BackgroundEnhanced signalling via the epidermal growth factor receptor (EGFR) is a hallmark of multiple human carcinomas. However, in recent years data have accumulated that EGFR might also be hyperactivated in human sarcomas. Aim of this study was to investigate the influence of EGFR inhibition on cell viability and its interaction with chemotherapy response in osteosarcoma cell lines.MethodsWe have investigated a panel of human osteosarcoma cell lines regarding EGFR expression and downstream signalling. To test its potential applicability as therapeutic target, inhibition of EGFR by gefitinib was combined with osteosarcoma chemotherapeutics and cell viability, migration, and cell death assays were performed.ResultsOsteosarcoma cells expressed distinctly differing levels of functional EGFR reaching in some cases high amounts. Functionality of EGFR in osteosarcoma cells was proven by EGF-mediated activation of both MAPK and PI3K/AKT pathway (determined by phosphorylation of ERK1/2, AKT, S6, and GSK3β). The EGFR-specific inhibitor gefitinib blocked EGF-mediated downstream signal activation. At standard in vitro culture conditions, clinically achievable gefitinib doses demonstrated only limited cytotoxic activity, however, significantly reduced long-term colony formation and cell migration. In contrast, under serum-starvation conditions active gefitinib doses were distinctly reduced while EGF promoted starvation survival. Importantly, gefitinib significantly supported the anti-osteosarcoma activities of doxorubicin and methotrexate regarding cell survival and migratory potential.ConclusionOur data suggest that EGFR is not a major driver for osteosarcoma cell growth but contributes to starvation- and chemotherapy-induced stress survival. Consequently, combination approaches including EGFR inhibitors should be evaluated for treatment of high-grade osteosarcoma patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-015-0251-5) contains supplementary material, which is available to authorized users.

244-MPT overcomes gefitinib resistance in non-small cell lung cancer cells.

The epidermal growth factor receptor (EGFR) is known to play a critical role in non-small cell lung cancer(NSCLC). Several EGFR tyrosine kinase inhibitors(TKIs), such as gefitinib, have been used as effective clinical therapies for patients with NSCLC. Unfortunately, acquired resistance to gefitinib commonly occurs after 6-12 months of treatment. The resistance is associated with the appearance of the L858R/T790M double mutation of the EGFR. In our present study, we discovered a compound,referred to as 244-MPT, which could suppress either gefitinib-sensitive or -resistant lung cancer cell growth and colony formation, and also suppressed the kinase activity of both wildtype and double mutant (L858R/T790M) EGFR. The underlying mechanism reveals that 244-MPT could interact with either the wildtype or double-mutant EGFR in an ATP-competitive manner and inhibit activity. Treatment with 244-MPT could substantially reduce the phosphorylation of EGFR and its downstream signaling pathways, including Akt and ERK1/2 in gefitinib-sensitive and -resistant cell lines. It was equally effective in suppressing EGFR phosphorylation and downstream signaling in NL20 cells transfected with wildtype, single-mutant (L858R) or mutant (L858R/T790M) EGFR. 244-MPT could also induce apoptosis in a gefitinib-resistant cell line and strongly suppress gefitinib-resistant NSCLC tumor growth in a xenograft mouse model. In addition, 244-MPT could effectively reduce the size of tumors in a gefitinib-resistant NSCLC patient-derived xenograft (PDX) SCID mouse model. Overall, 244-MPT could overcome gefitinib-resistance by directly targeting the EGFR.

Abstract 3274: Neuroblastoma express a novel EGFR extracellular mutation, EGFRΔ768, which possesses distinct biological and biochemical properties

Abstract Background: Epidermal Growth Factor Receptor (EGFR) is a biologic target for cancer therapy. Clinical response to EGFR inhibitors is variable. Recently, EGFR mutations were found to predict response to anti-EGFR therapy. Since a few children with Neuroblastoma (NB) benefited from EGFR inhibitors in clinical trials, we screened for EGFR mutations in primary NB. Interestingly, we identified the expression of both EGFRvIII and a novel mutation, EGFRΔ768, in these tumors. In this study, we characterized the EGFRΔ768 mutation by comparing its biological and biochemical effect with that of wild type (WT)-EGFR and EGFRvIII. Hypothesis: EGFRΔ768 expression confers an aggressive cancer phenotype in NB cells Materials and Methods: We obtained 62 Snap Frozen primary neuroblastic tumors from the Columbus Bipathology Repository Center and 80 NB RNA from the Children Oncology Group (COG). Primary NB were prepared for RT-PCR and sequencing analyses using EGFR exon 1 & 8 primers. Five NB cell lines were obtained and confirmed by STR profiling. The EGFRΔ768 and EGFRvIII deletions were subcloned into a WT-EGFR expression vector with a C-terminal GFP tag. Next, stable 3T3 and NB clones expressing WT and mutant EGFR were generated. The clones were visualized under fluorescence microscopy. Invasion and XTT assays were performed with the stable clones. Responses to Erlotinib from 0.01 - 1 ng/ul and Etoposide from 0.5 - 2 uM were compared among the clones by XTT and western analyses. EGFR phosphorylation and downstream signaling pathways were examined by Western Blot and phosphokinase array. Results: We previously reported that 32% (20/62) of the Columbus neuroblastic tumors expressed EGFR extracellular deletion mutants, EGFRΔ768 (an in-frame deletion from nucleotides 102 of exon 2 to 869 of exon 7) and EGFRvIII (Δ801). In the 80 COG NB RNA, 7 expressed EGFRΔ768, 7 expressed EGFRvIII and 1 expressed both. EGFRΔ768 expression was also detected in the NB cell line, BE2M17. This novel mutant localized to the cell surface and enhanced the invasion and proliferation of NB cells in vitro when compared to control GFP+ cells. Its expression also confers sensitivity to Erlotinib, but the biochemical inhibition of Erlotinib on EGFRΔ768 is significantly less effective than that on activated WT-EGFR from 0.01 - 0.1 ng/ul. EGFRΔ768+ cells were also significantly more resistant to Etoposide than the GFP and WT-EGFR+ cells. Although both EGFRΔ768 and EGFRvIII autophosphorylated without ligand, the cellular proliferation rate and baseline autophosphorylation of EGFRΔ768+ cells were significantly higher than those of EGFRvIII+ cells. Finally, we found that EGFRΔ768 did not activate downstream signaling pathways of WT-EGFR such as MAPK or STAT. It has a different tyrosine phosphorylation profile than WT-EGFR. Conclusion: EGFRΔ768 confers aggressive cancer cell behaviors and has distinct biological as well as biochemical properties. Citation Format: James Keller, Kristen A. VanHeyst, Anjaruwee S. Nimnual, Mathew Varghese, Michael J. Hayman, Edward L. Chan. Neuroblastoma express a novel EGFR extracellular mutation, EGFRΔ768, which possesses distinct biological and biochemical properties. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3274. doi:10.1158/1538-7445.AM2015-3274

Abstract 4998: Novel N-glycosilation inhibitor blocks proliferation of EGFR-dependent NSCLC

Abstract Purpose: Receptor Tyrosine Kinase (RTK) are transmembrane glycoproteins that are known to drive cellular proliferation in non-small cell lung cancer (NSCLC). Asparagine (N) linked glycosylation (NLG) is an endoplasmic reticulum (ER) post-translational modification common to RTKs that is crucial for receptor stability, localization, and activation. We hypothesized that blockade of NLG would be a novel strategy for reducing RTK signaling in NSCLC and therefore initiated a small molecule screening program to identify novel inhibitors of this biosynthetic process that have the potential for clinical translation. Experimental Procedures: The ER-LucT NLG reporter was used to perform a cell-based high throughput screen of 358,301 compounds and identified a novel small molecule with NLG inhibitory activity (NGI-1). Effects on protein localization were determined using confocal microscopy and by surface biotin labeling. Cell proliferation was assessed with MTT assays over 5 days of drug treatment and cell cycle analysis was performed by flow cytometry. Senescence was determined by autofluorescence and p21 and p53 protein levels measured by western blot. Results: NGI-1 is a first in class inhibitor of the oligosaccharyltransferase that is cell permeable and has a submicromolar IC50. This small molecule partially inhibits NLG and blocks epidermal growth factor receptor (EGFR) trafficking to the cell surface. Partially glycosylated receptors are retained in the secretory pathway as demonstrated by colocalization with the ER marker calreticulin. The loss of EGFR cell surface expression correlated with a significant reduction of EGFR phosphorylation and downstream signaling through MAPK and AKT. After 48hrs, NGI-1 treatment reduced proliferation of EGFR kinase domain mutant cell lines (PC9, HCC827, H3255) by >90% (p<0.001) but did not significantly block proliferation of KRAS mutant A549 cells or normal fibroblasts, suggesting a preferential effect on RTK addicted NSCLC. Cell cycle analysis also showed a differential effect on G1 arrest for EGFR addicted cell lines relative to controls. This proliferative block was shown to correlate with increased levels of p53 phosphorylation and increased expression of p21, as well as accumulation of lipofuscin and cell autofluorescence. Together these data suggest that NGI-1 induces a senescence-like phenotype in EGFR addicted NSCLC. Similar results were obtained in PC9 cells with T790M mutation, demonstrating the potential advantage of this approach for overcoming TKI acquired resistance in NSCLC. Conclusion: This study suggests that partial inhibition of NLG with NGI-1 is a novel approach for blocking proliferation of RTK dependent NSCLC. Citation Format: Cecilia Lopez Sambrooks, Joseph N. Contessa. Novel N-glycosilation inhibitor blocks proliferation of EGFR-dependent NSCLC. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4998. doi:10.1158/1538-7445.AM2015-4998

Lewisy promotes migration of oral cancer cells by glycosylation of epidermal growth factor receptor.

Aberrant glycosylation changes normal cellular functions and represents a specific hallmark of cancer. Lewisy (Ley) carbohydrate upregulation has been reported in a variety of cancers, including oral squamous cell carcinoma (OSCC). A high level of Ley expression is related to poor prognosis of patients with oral cancer. However, it is unclear how Ley mediates oral cancer progression. In this study, the role of Ley in OSCC was explored. Our data showed that Ley was upregulated in HSC-3 and OC-2 OSCC cell lines. Particularly, glycosylation of epidermal growth factor receptor (EGFR) with Ley was found in OC-2 cells, and this modification was absent upon inhibition of Ley synthesis. The absence of Ley glycosylation of EGFR weakened phosphorylation of AKT and ERK in response to epidermal growth factor (EGF). Additionally, EGF-triggered cell migration was reduced, but cell proliferation was not affected. Ley modification stabilized EGFR upon ligand activation. Conversely, absence of Ley glycosylation accelerated EGFR degradation. In summary, these results indicate that increased expression of Ley in OSCC cells is able to promote cell migration by modifying EGFR which in turn stabilizes EGFR expression and downstream signaling. Targeting Ley on EGFR could have a potential therapeutic effect on oral cancer.

Functional cyclic AMP response element in the breast cancer resistance protein (BCRP/ABCG2) promoter modulates epidermal growth factor receptor pathway- or androgen withdrawal-mediated BCRP/ABCG2 transcription in human cancer cells

Phosphorylated cyclic-AMP (cAMP) response element binding protein (p-CREB) is a downstream effector of a variety of important signaling pathways. We investigated whether the human BCRP promoter contains a functional cAMP response element (CRE). 8Br-cAMP, a cAMP analogue, increased the activity of a BCRP promoter reporter construct and BCRP mRNA in human carcinoma cells. Epidermal growth factor receptor (EGFR) pathway activation also led to an increase in p-CREB and in BCRP promoter reporter activity via two major downstream EGFR signaling pathways: the phosphotidylinositol-3-kinase (PI3K)/AKT pathway and the mitogen-activated protein kinase (MAPK) pathway. EGF treatment increased the phosphorylation of EGFR, AKT, ERK and CREB, while simultaneously enhancing BCRP mRNA and functional protein expression. EGF-stimulated CREB phosphorylation and BCRP induction were diminished by inhibition of EGFR, PI3K/AKT or RAS/MAPK signaling. CREB silencing using RNA interference reduced basal levels of BCRP mRNA and diminished the induction of BCRP by EGF. Chromatin immunoprecipitation assays confirmed that a putative CRE site on the BCRP promoter bound p-CREB by a point mutation of the CRE site abolished EGF-induced stimulation of BCRP promoter reporter activity. Furthermore, the CREB co-activator, cAMP-regulated transcriptional co-activator (CRTC2), is involved in CREB-mediated BCRP transcription: androgen depletion of LNCaP human prostate cancer cells increased both CREB phosphorylation and CRTC2 nuclear translocation, and enhanced BCRP expression. Silencing CREB or CRTC2 reduced basal BCRP expression and BCRP induction under androgen-depletion conditions. This novel CRE site plays a central role in mediating BCRP gene expression in several human cancer cell lines following activation of multiple cancer-relevant signaling pathways.

A phase 3 open-label, randomized, multicenter study of Imprime PGG in combination with cetuximab in patients with KRAS wild type metastatic colorectal cancer.

TPS787 Background: Imprime PGG (Imprime) is a novel immune modulator (complex carbohydrate biologic), which harnesses innate immune cells to enhance killing of antibody-targeted tumor cells. In a phase 2 single-arm clinical trial in mCRC, the combination of Imprime with cetuximab resulted in 24% ORR, 62% disease control rate (DCR), and median time to progression (TTP) of 12 wks (Tamayo ME, Ann Onc 2010), representing approximate 100% increases vs historical control (Cunningham, NEJM 2004). ORR was 45%, DCR, 82% and TTP, 24 wks in pts with KRAS WT tumors (post hoc analysis). Single-agent cetuximab has been shown to improve objective response rate (ORR), progression-free survival (PFS) and overall survival (OS) in patients (pts) with epidermal growth factor receptor (EGFR) expressing, KRAS wild-type (WT) metastatic colorectal cancer (mCRC) who failed oxaliplatin- and irinotecan-based therapy or are intolerant to irinotecan. The mechanism of action of cetuximab is thought to rely on competitive blockade of endogenous ligand binding and downstream signaling, internalization and down regulation of EGFR, as well as antibody-dependent cellular cytotoxicity (ADCC) (Erbitux SmPC). The current trial, sponsored by Biothera (registered with ClinicalTrials.gov NCT01309126) is to confirm these findings in phase 3. Methods: Eligible pts will have had prior oxaliplatin- and irinotecan-based therapy or be intolerant to irinotecan, and will meet key inclusion criteria including measurable disease and ECOG performance status of 0 or 1. In a 2:1 randomization, stratified by geographic region, prior chemotherapy and site, approximately 795 pts will receive weekly open-label Imprime plus cetuximab or cetuximab alone. The primary endpoint of the study is OS and primary analysis will occur when ~709 deaths have occurred. Secondary endpoints include PFS, ORR (based on RECIST 1.1), quality of life, safety and pharmacokinetics. Exploratory endpoints include biomarker analyses. Pt screening and enrollment is underway in the United States and Europe. Clinical trial information: NCT01309126.

Erlotinib enhances the CIK cell-killing sensitivity of lung adenocarcinoma A549 cells.

We examined the effects and molecular mechanism of the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor erlotinib on NKG2D ligand expression in human lung adenocarcinoma A549 cells and the cytotoxicity of cytokine-induced killer cells. Flow cytometry was used to detect NKG2D ligand expression in A549 cells under effects of erlotinib and EGFR downstream molecules, including LY294002 (phosphoinositide 3-kinase inhibitor), SB203580 (mitogen-activated protein kinase inhibitor), and STAT21 (signal transduction and transcription 3 inhibitor) after 24 h. A lactate dehydrogenase release assay was used to detect, at different effector-to-target ratios, the A549 cell killing activity of cytokine-induced killer cells before and after treatment with 10 mM erlotinib. Erlotinib suppressed MICA expression in A549 cells and upregulated MICB and UL16 binding protein 1 expression. EGFR downstream molecules mitogen-activated protein kinase and signal transduction and transcription 3 inhibitor did not affect the expression of NKG2D ligands in A549 cells. The phosphoinositide 3-kinase inhibitor reduced MICA expression in A549 cells, while erlotinib enhanced the killing sensitivity of cytokine-induced killer cells in A549 cells. The anti-lung carcinoma effects of EGFR tyrosine kinase inhibitor were associated with the sensitivity of lung cancer cells to enhanced immune cell killing.

Concurrent Targeting of KRAS and AKT by MiR-4689 Is a Novel Treatment Against Mutant KRAS Colorectal Cancer.

KRAS mutations are a major cause of drug resistance to molecular-targeted therapies. Aberrant epidermal growth factor receptor (EGFR) signaling may cause dysregulation of microRNA (miRNA) and gene regulatory networks, which leads to cancer initiation and progression. To address the functional relevance of miRNAs in mutant KRAS cancers, we transfected exogenous KRASG12V into human embryonic kidney 293 and MRC5 cells with wild-type KRAS and BRAF genes, and we comprehensively profiled the dysregulated miRNAs. The result showed that mature miRNA oligonucleotide (miR)-4689, one of the significantly down-regulated miRNAs in KRASG12V overexpressed cells, was found to exhibit a potent growth-inhibitory and proapoptotic effect both in vitro and in vivo. miR-4689 expression was significantly down-regulated in cancer tissues compared to normal mucosa, and it was particularly decreased in mutant KRAS CRC tissues. miR-4689 directly targets v-ki-ras2 kirsten rat sarcoma viral oncogene homolog (KRAS) and v-akt murine thymoma viral oncogene homolog 1(AKT1), key components of two major branches in EGFR pathway, suggesting KRAS overdrives this signaling pathway through inhibition of miR-4689. Overall, this study provided additional evidence that mutant KRAS functions as a broad regulator of the EGFR signaling cascade by inhibiting miR-4689, which negatively regulates both RAS/mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/AKT pathways. These activities indicated that miR-4689 may be a promising therapeutic agent in mutant KRAS CRC.

Abstract 4401: A novel drug suppresses proliferation of lung cancer cells via increasing the CBL activity and down-regulating epidermal growth factor receptor

Abstract Aberrant epidermal growth factor receptor (EGFR) signaling is one of the most critical oncogenic pathways in NSCLC that may trigger the tumor progression. Thus, how to suppress the EGFR downstream signaling cascade or to reduce the EGFR expression level has long been considered an important therapeutic approach for cancer interventions. Here, we found a regulatory mechanism by which the degradation of EGFR is enhanced by a novel promising drug, compound 22. First, the exposure to compound 22 induced cell death via apoptosis in lung cancer cell lines, including EGFR wild type A549 as well as EGFR mutant PC9 and H1975 cells. Compound 22 decreased the EGFR protein level in a dose-dependent manner and facilitated dephosphorylation of its downstream targets, including AKT and ERK, both of which play a critical role for cancer cell survival. Second, we observed that compound 22 induced EGFR degradation through proteasome degradation in the cycloheximide chase plus MG132 assay. Additionally, compound 22 increased the CBL (the ubiquitin E3 ligase)-mediated ubiquitination of EGFR, which was accompanied by the enhancement of Y1045 phosphorylation in the EGFR kinase domain. Overall, these pieces of evidence suggest that compound 22 is a novel class of anticancer drug which is able to inhibit the TKI-resistant NSCLC cells by inducing the degradation of EGFR. Citation Format: Kuo-Yen Huang, Szu-Hua Pan, Wen-Lung Wang, Ching-Shih Chen, Tse-Ming Hong, Pan-Chyr Yang. A novel drug suppresses proliferation of lung cancer cells via increasing the CBL activity and down-regulating epidermal growth factor receptor. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4401. doi:10.1158/1538-7445.AM2014-4401

A Phase 3 Open-Label, Randomized, Multicenter Study of Imprime Pgg in Combination with Cetuximab in Patients with Kras Wild Type Metastatic Colorectal Cancer

ABSTRACT Background: For patients (pts) with epidermal growth factor receptor (EGFR) expressing, KRAS wild-type (WT) metastatic colorectal cancer (mCRC) who failed oxaliplatin- and irinotecan-based therapy or who are intolerant to irinotecan, single-agent cetuximab has been shown to improve objective response rate (ORR), progression-free survival (PFS) and overall survival (OS). The mechanism of action of cetuximab is thought to rely on competitive blockade of endogenous ligand binding and downstream signaling, internalization and downregulation of EGFR, as well as antibody-dependent cellular cytotoxicity (ADCC) (Erbitux SmPC). Imprime PGG (Imprime) is a novel immune modulator (complex carbohydrate biologic), which harnesses innate immune cells to enhance killing of antibody-targeted tumor cells. In a phase 2 single-arm clinical trial in mCRC, the combination of Imprime with cetuximab resulted in 24% ORR, 62% disease control rate (DCR), and median time to progression (TTP) of 12 wks (Tamayo ME, Ann Onc 2010), representing approximate 100% increases vs historical control (Cunningham, NEJM 2004). ORR was 45%, DCR 82% and TTP 24 wks in pts with KRAS WT tumors (post hoc analysis). The current trial, sponsored by Biothera and registered with ClinicalTrials.gov NCT01309126, EudraCT 2010-023562-51, is to confirm these findings in phase 3. Trial design: Approximately 795 pts with previously treated, KRAS WT mCRC will be randomized 2:1 to weekly open-label Imprime plus cetuximab or cetuximab alone, until disease progression. Randomization will be stratified by geographic region, prior chemotherapy and site. The primary endpoint of the study is OS. Secondary endpoints are PFS, ORR (based on RECIST 1.1), quality of life, safety and pharmacokinetics. Exploratory endpoints include biomarker analyses. The primary analysis will occur when ∼709 deaths have occurred. Eligible pts have measurable disease, an ECOG performance status of 0 or 1 and have received prior oxaliplatin- and irinotecan-based therapy or are intolerant to irinotecan. Pts with known hypersensitivity to cetuximab or yeast are excluded. Pt screening and enrollment is underway in Europe and the United States. Disclosure: M.L. Patchen, M. Gargano, M. Grady, J. Lowe, R.D. Huhn and A. Braun: Author is a Biothera employee and has stock options and/or grants.

Cytotoxicity of the bisphenolic honokiol from Magnolia officinalis against multiple drug-resistant tumor cells as determined by pharmacogenomics and molecular docking

A main problem in oncology is the development of drug-resistance. Some plant-derived lignans are established in cancer therapy, e.g. the semisynthetic epipodophyllotoxins etoposide and teniposide. Their activity is, unfortunately, hampered by the ATP-binding cassette (ABC) efflux transporter, P-glycoprotein. Here, we investigated the bisphenolic honokiol derived from Magnolia officinalis. P-glycoprotein-overexpressing CEM/ADR5000 cells were not cross-resistant to honokiol, but MDA-MB-231 BRCP cells transfected with another ABC-transporter, BCRP, revealed 3-fold resistance. Further drug resistance mechanisms analyzed study was the tumor suppressor TP53 and the epidermal growth factor receptor (EGFR). HCT116 p53−/− did not reveal resistance to honokiol, and EGFR-transfected U87.MG EGFR cells were collateral sensitive compared to wild-type cells (degree of resistance: 0.34). To gain insight into possible modes of collateral sensitivity, we performed in silico molecular docking studies of honokiol to EGFR and EGFR-related downstream signal proteins. Honokiol bound with comparable binding energies to EGFR (−7.30±0.01kcal/mol) as the control drugs erlotinib (−7.50±0.30kcal/mol) and gefitinib (−8.30±0.10kcal/mol). Similar binding affinities of AKT, MEK1, MEK2, STAT3 and mTOR were calculated for honokiol (range from −9.0±0.01 to 7.40±0.01kcal/mol) compared to corresponding control inhibitor compounds for these signal transducers. This indicates that collateral sensitivity of EGFR-transfectant cells towards honokiol may be due to binding to EGFR and downstream signal transducers. COMPARE and hierarchical cluster analyses of microarray-based transcriptomic mRNA expression data of 59 tumor cell lines revealed a specific gene expression profile predicting sensitivity or resistance towards honokiol.

Regulation of EGFR nanocluster formation by ionic protein-lipid interaction.

The abnormal activation of epidermal growth factor receptor (EGFR) is strongly associated with a variety of human cancers but the underlying molecular mechanism is not fully understood. By using direct stochastic optical reconstruction microscopy (dSTORM), we find that EGFR proteins form nanoclusters in the cell membrane of both normal lung epithelial cells and lung cancer cells, but the number and size of clusters significantly increase in lung cancer cells. The formation of EGFR clusters is mediated by the ionic interaction between the anionic lipid phosphatidylinositol-4,5-bisphosphate (PIP2) in the plasma membrane and the juxtamembrane (JM) region of EGFR. Disruption of EGFR clustering by PIP2 depletion or JM region mutation impairs EGFR activation and downstream signaling. Furthermore, JM region mutation in constitutively active EGFR mutant attenuates its capability of cell transformation. Collectively, our findings highlight the key roles of anionic phospholipids in EGFR signaling and function, and reveal a novel mechanism to explain the aberrant activation of EGFR in cancers.

Expression and activity of EGFR in human cutaneous melanoma cell lines and influence of vemurafenib on the EGFR pathway.

Data regarding the expression of epidermal growth factor receptor (EGFR) in melanoma and its role in the tumor biology are conflicting. In BRAF V600-mutant melanomas, the expression of EGFR has been associated with acquired resistance to BRAF inhibitors. In this study, we assessed EGFR expression and downstream signaling activity in a panel of melanoma cell lines and we investigated the effects of the BRAF inhibitor vemurafenib on expression of EGFR and its downstream effectors in a subgroup of BRAF-mutant melanoma cells. Three out of 10 melanoma cell lines expressed EGFR. Downstream signaling via ERK and AKT was responsive to either stimulation by EGF or inhibition by erlotinib. Constitutive activation of ERK occurred in all the cell lines investigated whereas constitutive activation of AKT only in three cell lines. Constitutive activation of ERK and AKT was independent from EGFR expression. Vemurafenib did not affect EGFR expression in general, but it increased EGFR phosphorylation in the cell line SkMel5. Induced EGFR phosphorylation was sensitive to treatment with erlotinib. Vemurafenib efficiently blocked ERK activation in all the BRAF-mutant cell lines tested, whereas its effects on AKT activation were dissimilar in the different cell lines. Our data suggest that EGFR is functional but usually inactive in EGFR high-expressing cell lines. Basal EGFR expression unlikely represents a biomarker for predicting the sensitivity to vemurafenib in melanoma, but EGFR activation might represent a mechanism of vemurafenib resistance in a subset of melanoma cells.

Synergistic effects of combined platelet-activating factor receptor and epidermal growth factor receptor targeting in ovarian cancer cells

BackgroundGenetic alterations, including the overexpression of epidermal growth factor receptor (EGFR), play a crucial role in ovarian carcinogenesis. To date, EGFR targeting has shown limited antitumor effects in ovarian cancer when administered as monotherapy. We previously identified platelet-activating factor receptor (PAFR) as being overexpressed in ovarian cancer and found that its ligand PAF evoked EGFR phosphorylation. To determine whether PAFR targeting can enhance the antitumor efficacy of EGFR inhibition, we investigated the effects of a PAFR antagonist (WEB2086) in conjunction with an EGFR inhibitor (AG1478).MethodsThe expression of EGFR and PAFR in CAOV-3 and SKOV-3 ovarian cancer cell lines was measured by Western blot and immunocytochemistry. Synergy was determined using isobologram analysis. The effects of combined PAFR and EGFR targeting on both cells were assessed by using CCK-8, transwell, flow cytometry, western blot analysis. In vivo studies were conducted using CAOV-3 cells xenografted in nu/nu mice.ResultsTreatment with combination WEB2086 and AG1478 resulted in significantly greater inhibition of proliferation and invasion compared to either drug alone. When examining equipotent combinations of WEB2086 and AG1478 to determine potential synergy, a combination index (CI) of 0.49 was identified for CAOV-3 cells and a CI of 0.58 for SKOV-3 cells indicating synergy. This co-inhibition induced significantly more apoptosis and arrested the cells at G0/G1 phase in both cell lines. The activation of PAFR and/or EGFR induced phosphorylation of the mTOR, AKT, and MAPK pathways. Combined PAFR and EGFR targeting synergistically diminished the expression of PAFR and EGFR phosphorylation and downstream signaling. In vivo studies further verified the antitumor effects of combined PAFR and EGFR targeting in a CAOV-3 xenograft model.ConclusionsThese results suggest that WEB2086 and AG1478 are synergistic in ovarian cancer cells with high expression of both PAFR and EGFR. The presented approach may have important therapeutic implications in the treatment of ovarian cancer patients.

Weak power frequency magnetic field acting similarly to EGF stimulation, induces acute activations of the EGFR sensitive actin cytoskeleton motility in human amniotic cells.

In this article, we have examined the motility-related effects of weak power frequency magnetic fields (MFs) on the epidermal growth factor receptor (EGFR)-sensitive motility mechanism, including the F-actin cytoskeleton, growth of invasive protrusions and the levels of signal molecules in human amniotic epithelial (FL) cells. Without extracellular EGF stimulation, the field stimulated a large growth of new protrusions, especially filopodia and lamellipodia, an increased population of vinculin-associated focal adhesions. And, an obvious reduction of stress fiber content in cell centers was found, corresponding to larger cell surface areas and decreased efficiency of actin assembly of FL cells in vitro, which was associated with a decrease in overall F-actin content and special distributions. These effects were also associated with changes in protein content or distribution patterns of the EGFR downstream motility-related signaling molecules. All of these effects are similar to those following epidermal growth factor (EGF) stimulation of the cells and are time dependent. These results suggest that power frequency MF exposure acutely affects the migration/motility-related actin cytoskeleton reorganization that is regulated by the EGFR-cytoskeleton signaling pathway. Therefore, upon the MF exposure, cells are likely altered to be ready to transfer into a state of migration in response to the stimuli.

Overcoming EGFR T790M-based Tyrosine Kinase Inhibitor Resistance with an Allele-specific DNAzyme

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are the main therapeutic agents used to treat non–small-cell lung cancer patients harboring EGFR-activating mutations. However, most of these patients will eventually develop resistance, 50% of which are due to a secondary mutation at T790M in the EGFR. In this paper, we describe the development of an allele-specific DNAzyme, DzT, that can specifically silence EGFR T790M mutant messenger RNA while leaving wild-type EGFR intact. Allele-specific silencing of EGFR T790M expression and downstream signaling by DzT triggered apoptosis in non–small-cell lung cancer cells harboring this mutant. Adding a cholesterol-triethylene glycol group on the 3′-end of DzT (cDzT) improved drug efficacy, increasing inhibitory effect on cell viability from 46 to 79% in T790M/L858R-harboring H1975TM/LR non–small-cell lung cancer cells, without loss of allele specificity. Combined treatment with cDzT and BIBW-2992, a second-generation EGFR-tyrosine kinase inhibitor, synergistically inhibited EGFR downstream signaling and suppressed the growth of xenograft tumors derived from H1975TM/LR cells. Collectively, these results indicate that the allele-specific DNAzyme, DzT, may provide an alternative treatment for non–small-cell lung cancer that is capable of overcoming EGFR T790M mutant-based tyrosine kinase inhibitor resistance.

Accelerated and increased joint damage in young mice with global inactivation of mitogen-inducible gene 6 after ligament and meniscus injury

IntroductionLigament and meniscal damage can cause joint disease. Arthritic joints contain increased amounts of epidermal growth factor receptor (EGFR) protein, and polymorphisms in EGFR are associated with arthritis risk. The role of endogenous EGFR regulation during joint disease due to ligament and meniscal trauma is unknown. Mitogen-inducible gene 6 (MIG-6) can reduce EGFR phosphorylation and downstream signaling. We examined the effect of EGFR modulation by MIG-6 on joint disease development after ligament and meniscus injury.MethodsKnee ligament transection and meniscus removal were performed surgically on mice homozygous for a global inactivating mutation in MIG-6 (Mig-6−/−) and in wild-type (WT) animals.ResultsTwo weeks after surgery, Mig-6−/−mice had bone erosion as well as greater fibrous tissue area and serum RANKL concentration than WT mice. Four weeks after surgery, Mig-6−/−mice had less cartilage and increased cell proliferation relative to contralateral control and WT knees. Increased apoptotic cells and growth outside the articulating region occurred in Mig-6−/−mice. Tibia trabecular bone mineral density (BMD) and the number of trabeculae were lower in surgically treated knees relative to the respective control knees for both groups. BMD, as well as trabecular thickness and number, were lower in surgically treated knees from Mig-6−/−mice relative to WT surgically treated knees. Phosphorylated EGFR staining in surgically treated knees decreased for WT mice and increased for Mig-6−/−mice. Fewer inflammatory cells were present in the knees of WT mice.ConclusionMig-6−/−mice have rapid and increased joint damage after ligament and meniscal trauma. Mig-6 modification could lessen degenerative disease development after this type of injury.

Protein Kinase C (PKC) Increases TACE/ADAM17 Enzyme Activity in Porcine Ovarian Somatic Cells, Which Is Essential for Granulosa Cell Luteinization and Oocyte Maturation

During in vitro maturation of porcine cumulus cell-oocyte complexes and in vitro luteinization of porcine granulosa cells, FSH induces the expression of the protease TNFα-converting enzyme/A disintegrin and metalloproteinase domain 17 (TACE/ADAM17) and the epidermal growth factor (EGF)-like factors, which activate the EGF receptor (EGFR)-MAPK3/1 pathway in both cumulus and granulosa cells. FSH is known to activate not only protein kinase A and p38MAPK pathways in both cell types but also activates protein kinase C (PKC). Because PKC-induced association of cellular-Sarcoma (c-Src) and TACE/ADAM17 is required for TACE/ADAM17 enzyme activation in some cancer cells, we hypothesized that PKC and c-Src impact TACE/ADAM17-mediated activation of EGFR signaling pathway in porcine granulosa and cumulus cells. When granulosa cells or cumulus cell-oocyte complexes were cultured with FSH, PKC activity and c-Src phosphorylation increased and were associated with increased TACE/ADAM17 enzyme activity. The PKC inhibitor calphostin C (CalC) and the c-Src inhibitor (4 amino 5 (4 chlorophenyl) 7 (t butyl)pyrazolo[3,4 d]pyrimidine [PP2]) suppressed TACE/ADAM17 enzyme activity, whereas these inhibitors did not affect Tace/Adam17 mRNA expression. Immunoprecipitation analysis showed that FSH mediated the association of c-Src with TACE/ADAM17 via a PKC-dependent mechanism. Either CalC or PP2 suppressed EGFR downstream signaling pathway (MAPK3/1) in these ovarian cell types and reduced cumulus expansion, meiotic maturation of oocytes, and progesterone production. The negative effects were overcome by the addition of amphiregulin. Collectively, these results indicate that activation of TACE/ADAM17 via a PKC-induced c-Src-dependent manner mediates proteolytic activation of the EGF-like factors that are involved in the induction of granulosa cell differentiation, cumulus expansion, and meiotic maturation of porcine oocytes in vitro.