Epidermal Growth Factor Receptor Promoter Research Articles

SOX9 Induces Orbital Fibroblast Activation in Thyroid Eye Disease Via MAPK/ERK1/2 Pathway.

To evaluate the expression of sry-box transcription factor 9 (SOX9) in orbital fibroblasts (OFs) of thyroid eye disease (TED) and to find its potential role and underlying mechanism in orbital fibrosis. OFs were cultured from orbital connective tissues obtained from patients with TED (n = 10) and healthy controls (n = 6). SOX9 was depleted by small interfering RNA or overexpressed through lentivirus transduction in OFs. Fibroblast contractile activity was measured by collagen gel contraction assay and proliferation was examined by EdU assay. Transcriptomic changes were assessed by RNA sequencing. The mRNA and protein levels of SOX9 were significantly higher in OFs cultured from patients with TED than those from healthy controls. Extracellular matrix-related genes were down-regulated by SOX9 knockdown and up-regulated by SOX9 overexpression in TED-OFs. SOX9 knockdown significantly decrease the contraction and the antiapoptotic ability of OFs, whereas the overexpression of SOX9 increased the ability of transformation, migration, and proliferation of OFs. SOX9 knockdown suppressed the expression of phosphorylated ERK1/2, whereas its overexpression showed the opposite effect. Epidermal growth factor receptor (EGFR) is one of the notably down-regulated genes screened out by RNA sequencing. Chromatin immunoprecipitation-qPCR demonstrated SOX9 binding to the EGFR promoter. A high expression of SOX9 was found in TED-OFs. SOX9 can activate OFs via MAPK/ERK1/2 signaling pathway, which in turn promotes proliferation and differentiation of OFs. EGFR was a downstream target gene of SOX9. SOX9/EGFR can be considered as therapeutic targets for the treatment of orbital fibrosis in TED.

Serine-arginine protein kinase 1 (SRPK1) promotes EGFR-TKI resistance by enhancing GSK3β Ser9 autophosphorylation independent of its kinase activity in non-small-cell lung cancer

Resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) is a major challenge for clinicians and patients with non-small cell lung cancer (NSCLC). Serine-arginine protein kinase 1 (SRPK1) is a key oncoprotein in the EGFR/AKT pathway that participates in tumorigenesis. We found that high SRPK1 expression was significantly associated with poor progression-free survival (PFS) in patients with advanced NSCLC undergoing gefitinib treatment. Both in vitro and in vivo assays suggested that SRPK1 reduced the ability of gefitinib to induce apoptosis in sensitive NSCLC cells independently of its kinase activity. Moreover, SRPK1 facilitated binding between LEF1, β-catenin and the EGFR promoter region to increase EGFR expression and promote the accumulation and phosphorylation of membrane EGFR. Furthermore, we verified that the SRPK1 spacer domain bound to GSK3β and enhanced its autophosphorylation at Ser9 to activate the Wnt pathway, thereby promoting the expression of Wnt target genes such as Bcl-X. The correlation between SRPK1 and EGFR expression was confirmed in patients. In brief, our research suggested that the SRPK1/GSK3β axis promotes gefitinib resistance by activating the Wnt pathway and may serve as a potential therapeutic target for overcoming gefitinib resistance in NSCLC.

CRNDE acts as an epigenetic modulator of the p300/YY1 complex to promote HCC progression and therapeutic resistance

BackgroundHepatocellular carcinoma (HCC) is one of the most common primary liver malignancies worldwide. The long-term prognosis for HCC remains extremely poor, with drug resistance being the major underlying cause of recurrence and mortality. The lncRNA colorectal neoplasia differentially expressed (CRNDE) is an epigenetic mediator and plays an important role to drive proliferation and drug resistance in HCC. However, CRNDE as an epigenetic regulator with influences sorafenib resistance in HCC is unclear. Thus, we explore the potential of targeting the CRNDE/p300/YY1 axis as a novel therapeutic strategy to overcome sorafenib resistance of HCC.MethodDetection of the expression level of CRNDE and EGFR in clinical specimens of HCC. CRNDE, EGFR, p300, and YY1expression were altered in HCC cells through transfection with different plasmids, and cell proliferation, migration, invasion, and sorafenib resistance were subsequently observed. Immunoprecipitation, chromatin immunoprecipitation, re-chromatin immunoprecipitation, site-directed mutagenesis, RNA Immunoprecipitation, immune fluorescence, qRT-PCR, and western blotting were performed to uncover the mechanisms of CRNDE regulation. The xenograft nude mice model was used to investigate the tumor growth and sorafenib resistance.ResultsIn this study, we showed that CRNDE expression is significantly positively correlated with that of epidermal growth factor receptor (EGFR) in clinical specimens of HCC and induces proliferation and sorafenib resistance of HCC via EGFR-mediated signaling. Mechanistically, CRNDE stabilized the p300/YY1 complex at the EGFR promoter and simultaneously enhanced histone H3K9 and H3K27 acetylation, which serve as markers of relaxed chromatin. EGFR was positively upregulated by the epigenetic complex, p300/YY1, in a manner dependent on CRNDE expression, leading to enhanced tumor cell proliferation and sorafenib resistance. Furthermore, C646, a p300 inhibitor, suppressed EGFR transcriptional activity by decreasing chromatin relaxation and YY1 binding, which effectively reduced proliferation/sorafenib resistance and prolonged overall survival.ConclusionOur collective findings support the potential of targeting the CRNDE/p300/YY1 axis as a novel therapeutic strategy to overcome sorafenib resistance of HCC.

208 Proposed Revision to Meningioma Classification System to Utilize Methylation Status of EGFR

INTRODUCTION: Meningiomas are among the most common brain tumors, making up about 13-26% of all intracranial tumors. They arise from the arachnoid cap and most commonly appear along the lining of the skull base and the venous cavities. The current classification system for meningiomas was established by the World Health Organization (WHO) and designates tumors as WHO grades I-III depending on histological characteristics. While WHO grade does strongly correlate with tumor recurrence, it has serious shortcomings. In particular, some WHO grade I tumors, which are supposedly benign, end up becoming highly aggressive and recurrent. A more accurate method of classifying tumors to provide better insight into prognosis is needed. Epidermal growth factor receptor (EGFR) is a highly expressed protein in meningiomas that has been linked to angiogenesis and other tumor-promoting properties. METHODS: A retrospective chart analysis was first performed to gather clinical and demographic information for 291 patients diagnosed with meningioma and treated with surgical intervention. Primary tissue samples were available for 25 patients and an analysis of EGFR promoter methylation status was performed. RESULTS: A significant association was found between EGFR promoter methylation and tumor recurrence (p = 0.02176*). Of note, there was a 100% positivity rate for EGFR hypermethylation in patients with tumor progression within 10 months post-surgery (n =9). Every tumor sample classified as WHO grade II-III had EGFR hypermethylation (n = 10), as well as 7 out of the 15 WHO grade I tumors. In total, over half of the meningioma patients with EGFR hypermethylation had tumor progression within 10 months of surgery. CONCLUSION: These findings suggest that assessing methylation status of EGFR could be a valuable tool for identifying highly aggressive meningiomas and determining patient prognosis early in the game. This would allow for improved postoperative treatment regimens to be developed, reducing the chances of tumor progression.

EGFR DNA Methylation Correlates With EGFR Expression, Immune Cell Infiltration, and Overall Survival in Lung Adenocarcinoma.

BackgroundThe epidermal growth factor receptor (EGFR) is a primary target of molecular targeted therapy for lung adenocarcinoma (LUAD). The mechanisms that lead to epigenetic abnormalities of EGFR in LUAD are still unclear. The purpose of our study was to evaluate the abnormal methylation of EGFR CpG sites as potential biomarkers for LUAD.MethodsTo assess the differentially methylation CpG sites of EGFR in LUAD, we used an integrative study of Illumina HumanMethylation450K and RNA-seq data from The Cancer Genome Atlas (TCGA). We evaluated and compared EGFR multiple-omics data to explore the role of CpG sites located in EGFR promoter regions and gene body regions and the association with transcripts, protein expression levels, mutations, and somatic copy number variation. We calculated the correlation coefficients between CpG sites of EGFR and immune infiltration fraction (by MCPcounter and ESTIMATE) and immune-related pathways in LUAD. Finally, we validated the differential methylation of clinically and prognostically relevant CpG sites using quantitative methylation-specific PCR (qMSP).ResultsWe found that the methylation level of many EGFR CpGs in the promoter region was negatively correlated with the transcription level, protein expression, and SCNV, while the methylation at the gene body region was positively correlated with these features. The methylation level of EGFR CpGs in the promoter region was positively correlated with the level of immune infiltration and IFN-γ signature, while the opposite was found for methylation of the gene body region. The qMSP results showed that cg02316066 had a high methylation level, while cg02166842 had a low methylation level in LUAD. There was a high degree of co-methylation between cg02316066 and cg03046247.ConclusionOur data indicate that EGFR is an epigenetic regulator in LUAD acting through DNA methylation. Our research provides a theoretical basis for the further detection of EGFR DNA methylation as a predictive biomarker for LUAD survival and immunotherapy.

The mineralocorticoid receptor leads to increased expression of EGFR and T-type calcium channels that support HL-1 cell hypertrophy

The EGF receptor (EGFR) has been extensively studied in tumor biology and recently a role in cardiovascular pathophysiology was suggested. The mineralocorticoid receptor (MR) is an important effector of the renin–angiotensin–aldosterone-system and elicits pathophysiological effects in the cardiovascular system; however, the underlying molecular mechanisms are unclear. Our aim was to investigate the importance of EGFR for MR-mediated cardiovascular pathophysiology because MR is known to induce EGFR expression. We identified a SNP within the EGFR promoter that modulates MR-induced EGFR expression. In RNA-sequencing and qPCR experiments in heart tissue of EGFR KO and WT mice, changes in EGFR abundance led to differential expression of cardiac ion channels, especially of the T-type calcium channel CACNA1H. Accordingly, CACNA1H expression was increased in WT mice after in vivo MR activation by aldosterone but not in respective EGFR KO mice. Aldosterone- and EGF-responsiveness of CACNA1H expression was confirmed in HL-1 cells by Western blot and by measuring peak current density of T-type calcium channels. Aldosterone-induced CACNA1H protein expression could be abrogated by the EGFR inhibitor AG1478. Furthermore, inhibition of T-type calcium channels with mibefradil or ML218 reduced diameter, volume and BNP levels in HL-1 cells. In conclusion the MR regulates EGFR and CACNA1H expression, which has an effect on HL-1 cell diameter, and the extent of this regulation seems to depend on the SNP-216 (G/T) genotype. This suggests that the EGFR may be an intermediate for MR-mediated cardiovascular changes and that SNP analysis can help identify subgroups of patients that will benefit most from MR antagonists.

Somatic Focal Copy Number Gains of Noncoding Regions of Receptor Tyrosine Kinase Genes in Treatment-Resistant Epilepsy.

Epilepsy is a heterogenous group of disorders defined by recurrent seizure activity due to abnormal synchronized activity of neurons. A growing number of epilepsy cases are believed to be caused by genetic factors and copy number variants (CNV) contribute to up to 5% of epilepsy cases. However, CNVs in epilepsy are usually large deletions or duplications involving multiple neurodevelopmental genes. In patients who underwent seizure focus resection for treatment-resistant epilepsy, whole genome DNA methylation profiling identified 3 main clusters of which one showed strong association with receptor tyrosine kinase (RTK) genes. We identified focal copy number gains involving epidermal growth factor receptor (EGFR) and PDGFRA loci. The dysplastic neurons of cases with amplifications showed marked overexpression of EGFR and PDGFRA, while glial and endothelial cells were negative. Targeted sequencing of regulatory regions and DNA methylation analysis revealed that only enhancer regions of EGFR and gene promoter of PDGFRA were amplified, while coding regions did not show copy number abnormalities or somatic mutations. Somatic focal copy number gains of noncoding regulatory represent a previously unrecognized genetic driver in epilepsy and a mechanism of abnormal activation of RTK genes. Upregulated RTKs provide a potential avenue for therapy in seizure disorders.

KLF4 defines the efficacy of the epidermal growth factor receptor inhibitor, erlotinib, in triple-negative breast cancer cells by repressing the EGFR gene

BackgroundTriple-negative breast cancer (TNBC) is characterized by high rates of recurrence and poor overall survival. This is due, in part, to a deficiency of targeted therapies, making it essential to identify therapeutically targetable driver pathways of this disease. While epidermal growth factor receptor (EGFR) is expressed in 60% of TNBCs and drives disease progression, attempts to inhibit EGFR in unselected TNBC patients have had a marginal impact on outcomes. Hence, we sought to identify the mechanisms that dictate EGFR expression and inhibitor response to provide a path for improving the utility of these drugs. In this regard, the majority of TNBCs express low levels of the transcription factor, Krüppel-like factor 4 (KLF4), while a small subset is associated with high expression. KLF4 and EGFR have also been reported to have opposing actions in TNBC. Thus, we tested whether KLF4 controls the expression of EGFR and cellular response to its pharmacological inhibition.MethodsKLF4 was transiently overexpressed in MDA-MB-231 and MDA-MB-468 cells or silenced in MCF10A cells. Migration and invasion were assessed using modified Boyden chamber assays, and proliferation was measured by EdU incorporation. Candidate downstream targets of KLF4, including EGFR, were identified using reverse phase protein arrays of MDA-MB-231 cells following enforced KLF4 expression. The ability of KLF4 to suppress EGFR gene and protein expression and downstream signaling was assessed by RT-PCR and western blot, respectively. ChIP-PCR confirmed KLF4 binding to the EGFR promoter. Response to erlotinib in the context of KLF4 overexpression or silencing was assessed using cell number and dose-response curves.ResultsWe report that KLF4 is a major determinant of EGFR expression and activity in TNBC cells. KLF4 represses transcription of the EGFR gene, leading to reduced levels of total EGFR, its activated/phosphorylated form (pEGFR), and its downstream signaling intermediates. Moreover, KLF4 suppression of EGFR is a necessary intermediary step for KLF4 to inhibit aggressive TNBC phenotypes. Most importantly, KLF4 dictates the sensitivity of TNBC cells to erlotinib, an FDA-approved inhibitor of EGFR.ConclusionsKLF4 is a major regulator of the efficacy of EGFR inhibitors in TNBC cells that may underlie the variable effectiveness of such drugs in patients.

MiRNA-9 Inhibits Proliferation and Migration of Lung Squamous Cell Carcinoma Cells by Regulating NRSF/EGFR.

Background:To investigate the mechanism of microRNA9 in inhibiting proliferation and migration of lung squamous cell carcinoma cells via neuron-restricted silencing factor/epidermal growth factor receptor.Material and Methods:Detection of microRNA9, neuron-restricted silencing factor, and epidermal growth factor receptor expression levels in lung cancer patients’ tissues and lung cancer cells by Western blotting and quantitative polymerase chain reaction. Detection of cell proliferation by colony formation assay and cell counting kit-8 assay. Detection of cell migration by wound-healing assay and Transwell assay. And detection of the regulatory effect between neuron-restricted silencing factor and epidermal growth factor receptor by Luciferase reporter gene system. Subcutaneous implantation mouse models of NCI-H520 cells were constructed to detect cell proliferation in vivo, and Kaplan–Meier method calculated patient survival.Results:The expression of microRNA9 and epidermal growth factor receptor was higher in lung cancer tissues than in normal lung tissues, while the expression of neuron-restricted silencing factor was lower in lung cancer tissues than in normal lung tissues. MicroRNA9 higher expression was strongly related to tumor size, and TNM stage and predicted showed reduced overall survival in patients with lung cancer. Further loss of function and enhancement experiments revealed that inhibition of microRNA9 could significantly inhibit lung squamous carcinoma cell proliferation and migration. Luciferase reporter assay demonstrated that microRNA9 could bind to NRSF messenger RNA and inhibit its expression, neuron-restricted silencing factor overexpression also exerted inhibitory effects on cell proliferation and migration. Moreover, Luciferase reporter assay showed that neuron-restricted silencing factor downregulate epidermal growth factor receptor expression levels by binding to epidermal growth factor receptor promoter regions, and Pearson’s correlation analysis indicated that the levels of microRNA9 in lung cancer tissues were correlated with neuron-restricted silencing factor and epidermal growth factor receptor. Combined microRNA9 with neuron-restricted silencing factor or epidermal growth factor receptor to predict the prognosis of patients with lung cancer may be more accurate.Conclusion:MicroRNA9 inhibits proliferation and migration of lung squamous cell carcinoma cells by inhibiting neuron-restricted silencing factor/epidermal growth factor receptor axis. MicroRNA9 can be a new prognostic marker and therapeutic target for lung squamous cell carcinoma.

Intron 1–Mediated Regulation of EGFR Expression in EGFR-Dependent Malignancies Is Mediated by AP-1 and BET Proteins

The epidermal growth factor receptor (EGFR) is overexpressed in numerous solid tumors and is the subject of extensive therapeutic efforts. Much of the research on EGFR is focused on protein dynamics and downstream signaling; however, few studies have explored its transcriptional regulation. Here, we identified two enhancers (CE1 and CE2) present within the first intron of the EGFR gene in models of glioblastoma (GBM) and head and neck squamous cell carcinoma (HNSCC). CE1 and CE2 contain open chromatin and H3K27Ac histone marks, enhance transcription in reporter assays, and interact with the EGFR promoter. Enhancer genetic deletion by CRISPR/Cas9 significantly reduces EGFR transcript levels, with double deletion exercising an additive effect. Targeted repression of CE1 and CE2 by dCas9-KRAB demonstrates repression of transcription similar to that of genomic deletion. We identify AP-1 transcription factor family members in concert with BET bromodomain proteins as modulators of CE1 and CE2 activity in HNSCC and GBM through de novo motif identification and validate their presence. Genetic inhibition of AP-1 or pharmacologic disruption of BET/AP-1 binding results in downregulated EGFR protein and transcript levels, confirming a role for these factors in CE1 and CE2. Our results identify and characterize these novel enhancers, shedding light on the role that epigenetic mechanisms play in regulating EGFR transcription in EGFR-dependent cancers. IMPLICATIONS: We identify critical constituent enhancers present in the first intron of the EGFR gene, and provide a rationale for therapeutic targeting of EGFR intron 1 enhancers through perturbation of AP-1 and BET in EGFR-positive malignancies.

Ecotropic viral integration site 1 regulates EGFR transcription in glioblastoma cells

PurposeEcotropic viral integration site-1 (EVI1) is a transcription factor that contributes to the unfavorable prognosis of leukemia, some epithelial cancers, and glial tumors. However, the biological function of EVI1 in glioblastoma multiforme (GBM) remains unclear. Based on microarray experiments, EVI1 has been reported to regulate epidermal growth factor receptor (EGFR) transcription. Signal transduction via EGFR plays an essential role in glioblastoma. Therefore, we performed this study to clarify the importance of EVI1 in GBM by focusing on the regulatory mechanism between EVI1 and EGFR transcription.MethodsWe performed immunohistochemical staining and analyzed the EVI1-expression in glioma tissue. To determine the relationship between EVI1 and EGFR, we induced siRNA-mediated knockdown of EVI1 in GBM cell lines. To investigate the region that was essential for the EVI1 regulation of EGFR expression, we conducted promoter reporter assays. We performed WST-8 assay to investigate whether EVI1 affected on the proliferation of GBM cells or not.ResultsIt was observed that 22% of GBM tissues had over 33% of tumor cells expressing EVI1, whereas no lower-grade glioma tissue had over 33% by immunohistochemistry. In A172 and YKG1 cells, the expression levels of EGFR and EVI1 correlated. Analysis of the EGFR promoter region revealed that the EGFR promoter (from − 377 to − 266 bp) was essential for the EVI regulation of EGFR expression. We showed that EVI1 influenced the proliferation of A172 and YKG1 cells.ConclusionThis is the first study reporting the regulation of EGFR transcription by EVI1 in GBM cells.

Abstract 3329: Lung cancer screening initiative and identification of novel blood biomarkers for early detection of lung cancer

Abstract In lung cancer, 81% of patients detected are late-stage, since early-stage patients are generally asymptomatic. Winnebago County has a 14% higher mortality rate for lung cancer when compared to the national rate; hence, to improve early detection of lung cancer in the Winnebago County, we implemented low-dose computed tomography (LDCT) screening. Currently, LDCT is used to detect lung cancer at early-stages, and the CDC has proposed new guidelines for early screening of lung cancer in individuals between 55-77 years of age with a 30 pack/year smoking history. Using circulating DNA from lung cancer patients and examining changes in methylation patterns in genes that are expressed in lung cancer patients can be used for early detection. Epidermal growth factor receptor (EGFR) is shown to be expressed in lung cancer patients’ serum in all stages; hence, it can be used as a potential biomarker for early detection. To improve early detection of lung cancer in the Winnebago County, we educated physicians/smokers about LDCT screenings to promote early detection of lung cancer. We evaluated the number of LDCTs performed and recorded the number of lung cancer cases detected in the Winnebago County between June 2015 to October 2017. To explore EGFR as a promising biomarker, plasma was separated from whole blood and collected. Circulating DNA was extracted with the MagMax cell-free circulating kit and bisulfite converted. Identification of percent methylation patterns in the EGFR promoter region in CpG islands was obtained after next generation sequencing (NGS) followed by bioinformatics analysis. In our study, 1,116 LDCT screenings occurred in the Winnebago County. Lung cancer was diagnosed in 19 patients, out of which 11 patients (57.8%) were early-stage patients. We detected 45.4% of patients screened that were present with nodules compared to 24.2% of patients in the National Lung Cancer Screening Trial. We observed that 1.70% of patients referred for LDCT screening were diagnosed with lung cancer, compared to 0.87% of patients in the National Lung Cancer Screening Trial. This may be due to the higher incidence of smoking in Winnebago County (18%) compared to the nation (15.5%). Our screening study had 96.3% false positives, which is comparable (96.4%) to the results obtained in the National Lung Cancer Screening Trial. Using NGS, methylation in the promoter region of EGFR was studied using degenerate EGFR primers. In the promoter region of EGFR, our analysis showed that there are regions of hypermethylation (68-86%) in three CpG islands in sequences at the beginning of the promoter (8-241 bases). However, there was hypomethylation (6-21%) at four CpG island further downstream (375-689 bases from the beginning of the promoter). Currently we are studying the methylation patterns in normal subjects to determine whether these regions which could be used as predictive biomarkers for early detection. Citation Format: Adijan Kuckovic, Joseph Berei, Shylendra Sreenivasappa, Joseph Ross, Sandra Martell, Connie Vitali, William Schulz, Neelu Puri. Lung cancer screening initiative and identification of novel blood biomarkers for early detection of lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3329.

Distinct biological responses of metastatic castration resistant prostate cancer cells upon exposure to G-quadruplex interacting naphthalenediimide derivatives

Small molecules able to bind non-canonical G-quadruplex DNA structures (G4) have been recently tested as novel potential agents for the treatment of prostate cancer thanks to their repression of aberrant androgen receptor gene. However, metastatic castration-resistant prostate cancer (mCRPC), a letal form of prostate cancer, is still incurable. Here we tested two naphthalenediimide derivatives, previously reported as multitarget agents, on a couple of relevant mCRPC cell models (DU145 and PC-3). We showed that these compounds interfere with the RAS/MEK/ERK and PI3K/AKT pathways. Interestingly, both these two biological processes depend upon Epidermal Growth Factor Receptor (EGFR) activation. By means of biological and analytical tools we showed that our compounds are efficient inducers of the structural transition of the EGFR promoter towards a G-quadruplex conformation, ultimately leading to a reduction of the receptor production. The overall result is an interesting cytotoxic profile for these two derivatives. Thanks to their activity at different steps, these compounds can open the way to novel therapeutic approaches for mCRPC that could contribute to escape resistance to selective treatments.

LXRα-mediated downregulation of EGFR suppress colorectal cancer cell proliferation.

Liver X receptors (LXRs) are members of the nuclear receptor family, including the LXRα (NR1H3) and LXRβ (NR1H2) subtypes, which are related to the metabolism of glucose and cholesterol and possess anti-inflammatory functions. Mounting evidence has linked LXRs to the inhibition of cell proliferation in a variety of cancers. We revealed a differential distribution for NR1H3, but not for NR1H2, in colorectal cancer and adjacent normal tissues. We found that NR1H3 enhanced the inhibitory action of GW3965, an agonist of LXRs, on the proliferation of colorectal cancer cells. Upregulation of NR1H3 enhanced the inhibition of cell proliferation by GW3965 while silencing of NR1H3 attenuated the inhibitory effect of GW3965 on cell proliferation. Bioinformatic prediction and luciferase assays showed that NR1H3 was able to inhibit the activity of the epidermal growth factor receptor (EGFR) promoter. Moreover, we demonstrated that activation of NR1H3 inhibited the growth of transplanted tumors in an animal experiment, with the inhibition accompanied by downregulation of EGFR. Our findings suggest that NR1H3 controls cell proliferation by affecting EGFR promoter activity. The high expression of EGFR was due to the downregulation of NR1H3 which is a novel molecular mechanism in the development of colorectal cancer.

Activin A regulates the epidermal growth factor receptor promoter by activating the PI3K/SP1 pathway in oral squamous cell carcinoma cells

Epidermal growth factor receptor (EGFR) and activin A are both overexpressed in oral cavity squamous cell carcinoma (OSCC). We evaluated their clinical correlation and activin A-mediated EGFR regulation in this study. Overexpression of both transcripts/proteins indicated a poorer prognosis in OSCC patients. Knockdown of endogenous INHBA repressed the expression of EGFR and inhibited activin A-mediated canonical Smads, noncanonical phosphorylation of AKT (ser473) (p-AKT ser473) and SP1. Inhibition of PI3K signaling via its inhibitor attenuated p-AKT ser473 and in turn reduced SP1 and EGFR expression in the presence of recombinant activin A (rActivin A) in OSCC cells, as revealed via a luciferase assay and western blotting. However, canonical Smad signaling repressed the EGFR promoter, as revealed by a luciferase assay. The transcription factor SP1, its coactivator CBP/p300, and Smad proteins were recruited to the EGFR proximal promoter following rActivin A treatment, as revealed by chromatin immunoprecipitation (ChIP). Smad2/3/4 dramatically outcompeted SP1 binding to the EGFR proximal promoter following mithramycin A treatment. Activin A activates the PI3K and Smad pathways to compete for binding to overlapping SP1 consensus sequences on the EGFR proximal promoter. Nevertheless, canonical p-Smad2 was largely repressed in OSCC tumor tissues, suggesting that the activin A-mediated noncanonical pathway is essential for the carcinogenesis of OSCC.

Decline in arylsulfatase B expression increases EGFR expression by inhibiting the protein-tyrosine phosphatase SHP2 and activating JNK in prostate cells

Epidermal growth factor receptor (EGFR) has a crucial role in cell differentiation and proliferation and cancer, and its expression appears to be up-regulated when arylsulfatase B (ARSB or GalNAc-4-sulfatase) is reduced. ARSB removes 4-sulfate groups from the nonreducing end of dermatan sulfate and chondroitin 4-sulfate (C4S), and its decreased expression has previously been reported to inhibit the activity of the ubiquitous protein-tyrosine phosphatase, nonreceptor type 11 (SHP2 or PTPN11). However, the mechanism by which decline in ARSB leads to decline in SHP2 activity is unclear. Here, we show that SHP2 binds preferentially C4S, rather than chondroitin 6-sulfate, and confirm that SHP2 activity declines when ARSB is silenced. The reduction in ARSB activity, and the resultant increase in C4S, increased the expression of EGFR (Her1/ErbB1) in human prostate stem and epithelial cells. The increased expression of EGFR occurred after 1) the decline in SHP2 activity, 2) enhanced c-Jun N-terminal kinase (JNK) activity, 3) increased nuclear DNA binding by c-Jun and c-Fos, and 4) EGFR promoter activation. In response to exogenous EGF, there was increased bromodeoxyuridine incorporation, consistent with enhanced cell proliferation. These findings indicated that ARSB and chondroitin 4-sulfation affect the activation of an important dual phosphorylation threonine-tyrosine kinase and the mRNA expression of a critical tyrosine kinase receptor in prostate cells. Restoration of ARSB activity with the associated reduction in C4S may provide a new therapeutic approach for managing malignancies in which EGFR-mediated tyrosine kinase signaling pathways are active.

Identification and analysis of key genes in osteosarcoma using bioinformatics

Osteosarcoma (OS) is an invasive malignant neoplasm of the bones. The present study identified and analyzed key genes associated with OS. Expression profiling of the dataset GSE49003, which included 6 metastatic and 6 non-metastatic OS cell lines and was obtained from the Gene Expression Omnibus, was performed. Following data preprocessing, the differentially expressed genes (DEGs) were selected using the limma package in R. Subsequently, bidirectional hierarchical clustering using the pheatmap package in R and an unpaired Students' t-test was performed for the DEGs. Based on the Search Tool for the Retrieval of Interacting Genes database and Cytoscape software, a protein-protein interaction (PPI) network for the DEGs was constructed. Using Database for Annotation, Visualization and Integrated Discovery software and the Kyoto Encyclopedia of Genes and Genomes Orthology Based Annotation System server, functional and pathway enrichment analyses were performed for the DEGs corresponding to the proteins of the network. In addition, the transcription factors (TFs) and CpG islands of the gene promoter were searched for using the TRANSFAC database and CpG Island Searcher software, respectively. A total of 323 DEGs were identified between the metastatic and non-metastatic samples. In the PPI network, upregulated epidermal growth factor receptor (EGFR) exhibits a high degree and was therefore highly interconnected with other proteins. Enrichment analysis revealed that EGFR was enriched in cytoskeleton organization, organic substance response and the signaling pathway of focal adhesion. The TFs early growth response 1, nuclear factor-κB complex subunits, peroxisome proliferator activated receptor α, signal transducer and activator of transcription 3 and MYC proto-oncogene were identified in the EGFR promoter region. Furthermore, multiple CpG islands, starting from the 400 bp of the EGFR promoter sequence, were predicted. Methylated modification of the CpG islands in the EGFR promoter may help to regulate EGFR expression. The TFs identified in the EGFR promoter may function in the progression of OS.

EGFR feedback-inhibition by Ran-binding protein 6 is disrupted in cancer

Transport of macromolecules through the nuclear pore by importins and exportins plays a critical role in the spatial regulation of protein activity. How cancer cells co-opt this process to promote tumorigenesis remains unclear. The epidermal growth factor receptor (EGFR) plays a critical role in normal development and in human cancer. Here we describe a mechanism of EGFR regulation through the importin β family member RAN-binding protein 6 (RanBP6), a protein of hitherto unknown functions. We show that RanBP6 silencing impairs nuclear translocation of signal transducer and activator of transcription 3 (STAT3), reduces STAT3 binding to the EGFR promoter, results in transcriptional derepression of EGFR, and increased EGFR pathway output. Focal deletions of the RanBP6 locus on chromosome 9p were found in a subset of glioblastoma (GBM) and silencing of RanBP6 promoted glioma growth in vivo. Our results provide an example of EGFR deregulation in cancer through silencing of components of the nuclear import pathway.

Abstract 1032: Identification of Ran binding protein 6 as a novel negative regulator of EGFR and candidate tumor suppressor in glioblastoma

Abstract Amplification and overexpression of the epidermal growth factor receptor (EGFR) are common in glioblastoma (GBM) and frequently associated with silencing of the phosphatase and tensin homologue (PTEN) tumor suppressor. PTEN silencing has been associated with clinical resistance to EGFR tyrosine kinase inhibitors, in part by raising EGFR levels. Here, we investigated the effect of PTEN on the EGFR signaling complex by EGFR affinity immunopurification and mass spectrometry with and without PTEN knockdown. We identified Ran binding protein 6 (RanBP6), a 125-kDa protein of previously unknown functions, as EGFR interacting protein in PTEN expressing, but not PTEN knockdown cells. Further studies of the effect of RanBP6 on EGFR revealed that RanBP6 depletion by shRNA or CRISPR/Cas9-mediated gene silencing resulted in increased EGFR mRNA levels and upregulation of EGFR promoter activity. Consistent with a model of a negative EGFR regulation by RanBP6, we observed an inverse correlation between RanBP6 and EGFR mRNA levels in PTEN wildtype but not PTEN altered cancer cells in a large panel of human cancer cell lines (Cancer Cell Line Encyclopedia). To further understand the mechanism of how RanBP6 negatively regulates EGFR mRNA level, we found that RanBP6 interacted with nuclear Ran-GTPase and repressed EGFR transcription by promoting nuclear import of Signal transducer and activator of transcription 3 (STAT3). Lastly, RanBP6 appeared to be frequently deleted on chromosome 9p in GBM. We showed that RanBP6 silencing raised EGFR levels and signal output and accelerated in-vivo glioma growth. Our results establish a novel function of RanBP6 as a link between EGFR signaling and the Ran-mediated nuclear import pathway, and identify RanBP6 as candidate tumor suppressor on chromosome 9p. Citation Format: Wan-Ying Hsieh, Barbara Oldrini, Hediye Erdjument-Bromage, Paolo Codega, Maria S. Carro, Igor Vivanco, Dan Rohle, Carl Campos, Craig Bielski, Barry Taylor, Paul Tempst, Massimo Squatrito, Ingo K. Mellinghoff. Identification of Ran binding protein 6 as a novel negative regulator of EGFR and candidate tumor suppressor in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1032. doi:10.1158/1538-7445.AM2017-1032

Expression of HIF-1α ODD domain fused canine caspase 3 by EGFR promoter-driven adenovirus vector induces cytotoxicity in canine breast tumor cells under hypoxia.

Adenovirus (Ad) vectors are widely used in cancer gene therapies. However, compared to human patients, relatively limited information is available on gene transduction efficiency or cell-specific cytotoxicity in canine tumor cells transduced with Ad vectors. Since epidermal growth factor receptor (EGFR) is highly expressed on canine breast tumor cells, we sought to develop an Ad vector based on the RGD fiber-mutant adenovirus vector (AdRGD) that expresses canine caspase 3 under the control of EGFR promoter. The aims of this study were to achieve high transduction efficiency with transgene expression restricted to canine breast tumor cells. Using EGFR promoter-driven AdRGD, we were able to restrict transgene expression to canine breast tumor cells with no evidence of expression in normal cells. Canine breast tumor cells transduced with EGFR promoter-driven AdRGD carrying canine caspase 3 gene showed cytotoxic activity. We constructed a second AdRGD vector that expressed oxygen-dependent degradation (ODD)-caspase 3 under the control of the EGFR promoter; the fusion protein contains a core part of the ODD domain of hypoxia inducible factor-1 alpha (HIF-1α) fused to caspase 3. Transduction of canine breast tumor cells with EGFR promoter-driven AdRGD expressing ODD-caspase 3 induced a higher rate of cell death under hypoxic conditions compared with under normoxia. The results indicate that the EGFR promoter-driven AdRGD vectors will be of value for tumor-specific transgene expression and safe cancer gene therapy in dogs.