Epidermal Growth Factor Receptor mRNA Expression Research Articles

Effect of the molecular targeted drug, erlotinib, against endometrial cancer expressing high levels of epidermal growth factor receptor.

BackgroundThe epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, erlotinib, has been clinically applied for the treatment of a variety of tumors with EGFR overexpression. A phase II clinical study of erlotinib (NCIC IND-148) for recurrent or metastatic endometrial carcinoma (EC) resulted in an unfavorable result. However, in that study, the expression levels of EGFR were not accurately analyzed. Thus, the aim of this study was to re-examine the efficacy of erlotinib in EC cells by utilizing in vitro and in vivo models.MethodsTissue samples obtained from patients histologically diagnosed with EC of the uterine corpus were subjected to immunohistochemistry and RT-PCR to determine the protein and mRNA expression levels of EGFR. Western blot and WST-1 assays of EGFR siRNA-transfected HEC-1A, KLE, and Ishikawa cells were used to evaluate the efficacy of erlotinib in tumor cell lines expressing different EGFR levels. Furthermore, HEC-1A and Ishikawa cells were implanted into athymic mice treated with either erlotinib or trastuzumab.ResultsAt our institution, 20.9 % of endometrial cancer patients with low grade endometrioid histology have been diagnosed as stage III and IV. Immunohistochemical analysis and RT-PCR revealed the presence of significant EGFR and EGFR mRNA expression in low-grade endometrioid carcinoma in comparison with high-grade endometrioid carcinoma. In vitro study, WST-1 assay and Western blot analysis revealed that EGFR expression levels were correlated with tumor cell viability. Erlotinib reduced the proliferation of HEC-1A expressing high levels of EGFR, while trastuzumab showed similar effect in Ishikawa cells dominantly expressing human epidermal growth factor receptor type2 (HER2). In vivo erlotinib decreased tumor growth in mice xenografted with HEC-1A cells, whereas this tumor-growth inhibition was not observed in trastuzumab-treated mice xenografted with Ishikawa cell.ConclusionsEGF contributed to tumor proliferation in EC cell lines along with EGFR expression in vitro. Erlotinib also demonstrated anti-tumor effects in xenograft mice models. Our results suggest that erlotinib continues to have clinical usefulness in specific cases, after taking into consideration the EGFR expression levels.

Cell Free EGFR mRNA Expression and Implications for Survival and Metastasis in Non-Small Cell Lung Cancer Cases.

NSCLC is a disease involving uncontrolled cell growth, which could result in metastases into nearby tissues beyond the lungs. The aim of the present study was to analyze the influence of epidermal growth factor receptor (EGFR) gene expression on metastasis and survival in NSCLC patients. The present case-control study included 100 cases of NSCLC patients and 100 age and sex matched controls. EGFR gene expression was analyzed by quantitative real time PCR using serum RNA. Association with NSCLC patient survival was analyzed by the Kaplan-Meier method. We analyzed EGFR gene expression and observed mean increased gene expression of 13.5 fold in NSCLC patients. Values reflected overall survival of patients with a median of 15.8 months in the cases of <13 fold increased gene expression vs 6.7 months with >13 fold increased EGFR gene expression (p=0.005). Distant metastatic patients with <13 fold increased EGFR gene expression had 7.9 months of median survival time while>13 fold increased EGFR gene expression had only 5 months of median survival time (p=0.03). Non metastatic patients with <13 fold increased EGFR gene expression had 18 months of median survival time as compared to only 7.1 months with >13 fold increased expression. Higher cell free EGFR mRNA expression may play an important role in causing distant metastases and reducing overall survival of NSCLC patients in the Indian population.

Phosphatidic Acid Increases Epidermal Growth Factor Receptor Expression by Stabilizing mRNA Decay and by Inhibiting Lysosomal and Proteasomal Degradation of the Internalized Receptor.

Overexpression of epidermal growth factor receptor (EGFR) is one of the frequent mechanisms implicated in cancer progression, and so is the overexpression of the enzyme phospholipase D (PLD) and its reaction product, phosphatidic acid (PA). However, an understanding of how these signaling molecules interact at the level of gene expression is lacking. Catalytically active PLD enhanced expression of EGFR in human breast cancer cells. Overexpression of the PLD2 isoform increased EGFR mRNA and protein expression. It also negated an EGFR downregulation mediated by small interfering RNA targeting EGFR (siEGFR). Several mechanisms contributed to the alteration in EGFR expression. First was the stabilization of EGFR transcripts as PLD2 delayed mRNA decay, which prolonged their half-lives. Second, RNase enzymatic activity was inhibited by PA. Third, protein stabilization also occurred, as indicated by PLD resistance to cycloheximide-induced EGFR protein degradation. Fourth, PA inhibited lysosomal and proteasomal degradation of internalized EGFR. PLD2 and EGFR colocalized at the cell membrane, and JAK3 phosphorylation at Tyr980/Tyr981 followed receptor endocytosis. Further, the presence of PLD2 increased stabilization of intracellular EGFR in large recycling vesicles at ∼15 min of EGF stimulation. Thus, PLD2-mediated production of PA contributed to the control of EGFR exposure to ligand through a multipronged transcriptional and posttranscriptional program during the out-of-control accumulation of EGFR signaling in cancer cells.

RNA interference for epidermal growth factor receptor enhances the radiosensitivity of esophageal squamous cell carcinoma cell line Eca109.

The present study investigated the effects of small interfering RNAs (siRNAs) specific to the epidermal growth factor receptor (EGFR) gene, on the radiosensitivity of esophageal squamous cell carcinoma cells. EGFR gene siRNAs (EGFR-siRNA) were introduced into esophageal cancer Eca109 cells using Lipofectamine® 2000. The EGFR messenger (m)RNA expression levels, EGFR protein expression and cell growth were assessed using reverse transcription-polymerase chain reaction analysis, western blot analysis and a Cell Counting Kit-8 (CCK-8), respectively. In addition, colony assays were used to determine the inhibitory effects of X-ray radiation on EGFR-silenced cells. EGFR mRNA and protein levels were reduced in the Eca109 cells transfected with EGFR-siRNA. The relative EGFR mRNA expression levels were reduced to 26.74, 9.52 and 4.61% in Eca109 cells transfected with EGFR-siRNA1, 2 and 3, respectively. These mRNA levels were significantly reduced compared with the those of the control group (42.44%; P<0.0001). Transfection with siRNA3 resulted in the greatest reduction in EGFR mRNA expression, with an inhibition rate of 85%. The relative EGFR protein expression levels were reduced to 24.05, 34.91 and 34.14% in Eca109 cells transfected with EGFR-siRNA1, 2 and 3, respectively. These protein levels were significantly reduced compared with those of the control group (78.57%; P<0.0001). Transfection with siRNA1 resulted in the greatest reduction in EGFR protein expression, with an inhibition rate of 72.84%. This reduction in EGFR expression inhibited the proliferation of Eca109 cells, which was identified using the CCK-8 assay. The proliferation inhibition ratio was 28.2%. The cells treated with irradiation in addition to EGFR-siRNA, demonstrated reduced radiobiological parameters (D0, Dq and SF2) compared with those of cells treated with irradiation only, with a sensitization enhancing ratio of 1.5. In conclusion, suppression of EGFR expression may enhance the radiosensitivity of esophageal cancer Eca109 cells and therefore may represent a promising approach for future clinical practice.

FGF-2 induces the proliferation of human periodontal ligament cells and modulates their osteoblastic phenotype by affecting Runx2 expression in the presence and absence of osteogenic inducers.

The exact phenotype of human periodontal ligament cells (hPDLCs) remains a controversial area. Basic fibroblast growth factor (FGF-2) exhibits various functions and its effect on hPDLCs is also controversial. Therefore, the present study examined the effect of FGF-2 on the growth and osteoblastic phenotype of hPDLCs with or without osteogenic inducers (dexamethasone and β-glycerophosphate). FGF-2 was added to defined growth culture medium and osteogenic inductive culture medium. Cell proliferation, osteogenic differentiation and mineralization were measured. The selected differentiation markers, Runx2, collagen type I, α1 (Col1a1), osteocalcin (OCN) and epidermal growth factor receptor (EGFR), were investigated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Runx2 and OCN protein expression was measured by western blotting. FGF-2 significantly increased the proliferation of hPDLCs, but did not affect alkaline phosphatase activity. RT-qPCR analysis revealed enhanced mRNA expression of Runx2, OCN and EGFR, but suppressed Col1a1 gene expression in the absence of osteogenic inducers, whereas all these gene levels had no clear trend in their presence. The Runx2 protein expression was clearly increased, but the OCN protein level showed no evident trend. The mineralization assay demonstrated that FGF-2 inhibited mineralized matrix deposition with osteogenic inducers. These results suggested that FGF-2 induces the growth of immature hPDLCs, which is a competitive inhibitor of epithelial downgrowth, and suppresses their differentiation into mineralized tissue by affecting Runx2 expression. Therefore, this may lead to the acceleration of periodontal regeneration.

The relationship between the expression of VEGF, EGFR, and HER-2 mRNA in esophageal squamous cell carcinoma (ESCC) and clinicopathological features of different ethnic groups in Xinjiang.

The purpose of this study was to explore the relationship between the expression of vascular endothelial growth factor (VEGF), human epidermal growth factor receptor 2 (HER-2), and epidermal growth factor receptor (EGFR) mRNA in esophageal squamous cell carcinoma (ESCC) and the clinicopathological characteristics of the Han, Uyghur, and Kazakh in Xinjiang. Real-time quantitative polymerase chain reaction technology (RT-PCR) was used to detect the expression of VEGF, HER-2, and EGFR mRNA in esophageal squamous cancer tissue of 60 cases and 30 cases of VEGF, HER-2, and EGFR mRNA in esophageal cancer adjacent tissues, and analyze its relationship with clinicopathological features. The results were as follows: (1) There was no statistically significant difference in esophageal tissue VEGF, HER-2, and EGFR mRNA gene expression levels of the Han, Uyghur, and Kazakh patients (P > 0.05). (2) The expression levels of VEGF, HER-2, and EGFR mRNA in ESCC group were higher than those in adjacent esophageal tissue group (P < 0.05). (3) The expression levels of VEGF and HER-2 mRNA in ESCC of the Han patients were higher than those of the Uyghur and Kazakh patients (P < 0.05). (4) The expression levels of VEGF, HER-2, and EGFR mRNA in lymph node metastases were higher than those without lymph node metastasis (P < 0.05). (5) The expression level of HER-2 mRNA was related with the degrees of pathological differentiation, and the higher pathologic degree, the lower expression level in HER-2 mRNA (P < 0.05). Therefore, the following conclusions were drawn: (1) There were ethnic differences in the VEGF and HER-2 gene mRNA expression levels of the Uyghur, Han, and Kazakh patients in Xinjiang. (2) The expressions of VEGF, HER-2, and EGFR mRNA were related to the lymph node metastasis in ESCC and pathologic differentiation degree.

Single nucleotide polymorphisms of MAGE-A3 gene and its clinical implications in Chinese patients with non-small cell lung cancer (NSCLC).

Available study revealed advanced tumors have a higher expression rate of MAGE-A3 gene which has a lot of single nucleotide polymorphism (SNP) loci with polymorphisms. This study aimed to analyze the allele frequency of SNP loci in MAGE-A3 gene and investigate the relationship between MAGE-A3 gene polymorphisms and clinical factors. Tumor samples of a cohort of 191 NSCLC patients were collected. EGFR mRNA expression were detected by qRT-PCR. SNPs in whole length of MAGE-A3 gene were detected by direct sequencing. Frequencies of the SNPs were correlated to gene expression, mutation status of EGFR and clinical factors. Sequencing analysis confirmed that allele frequencies of genotypes on SNP loci rs5970360, rs5925210, rs5970361, rs5925211 and rs35123853 were CC (0.681)/CT (0.319), CC (0.660)/CG (0.340), CC (0.681)/CA (0.319), AA (0.984)/AT (0.016) and GG (1.000)/GA (0.000), respectively, which were different from the frequencies and genotypes of MAGE-A3 in SNP database. Chi-square tests showed the EGFR mRNA expression level had significant correlation with the genotypes of SNP loci rs5970360 and rs5925210. But all frequencies of each MAGE-A3 SNPs were not found significantly different between EGFR mutant and wild type patients. MAGE-A3 gene polymorphisms had no significant effects on survival of NSCLC patients. Chinese patients with NSCLC had different SNP patterns of MAGE-A3 in comparison with those in international SNP database. These MAGE-A3 SNP loci might have not prognostic significance. MAGE-A3 SNP loci rs5970360 and rs5925210 might be predictive for EGFR mRNA expression levels and helpful to the selection of patients for epidermal growth factor receptor (EGFR) targeted immunotherapy.

NEU3 activity enhances EGFR activation without affecting EGFR expression and acts on its sialylation levels.

Several studies performed over the last decade have focused on the role of sialylation in the progression of cancer and, in particular, on the association between deregulation of sialidases and tumorigenic transformation. The plasma membrane-associated sialidase NEU3 is often deregulated in colorectal cancer (CRC), and it was shown that this enzyme co-immunoprecipitates in HeLa cells with epidermal growth factor receptor (EGFR), the molecular target of most recent monoclonal antibody-based therapies against CRC. To investigate the role of NEU3 sialidase on EGFR deregulation in CRC, we first collected data on NEU3 gene expression levels from a library of commercial colon cell lines, demonstrating that NEU3 transcription is upregulated in these cell lines. We also found EGFR to be hyperphosphorylated in all cell lines, with the exception of SW620 cells and the CCD841 normal intestinal cell line. By comparing the effects induced by overexpression of either the wild-type or the inactive mutant form of NEU3 on EGFR, we demonstrated that the active form of NEU3 enhanced receptor activation without affecting EGFR mRNA or protein expression. Moreover, through western blots and mass spectrometry analysis, we found that EGFR immunoprecipitated from cells overexpressing active NEU3, unlike the receptor from mock cells and cells overexpressing inactive NEU3, is desialylated. On the whole, our data demonstrate that, besides the already reported indirect EGFR activation through GM3, sialidase NEU3 could also play a role on EGFR activation through its desialylation.

Relationship between EGFR Promoter Region Methylation and Secondary Resistance Which may be Induced by Gefitinib

背景与目的在肺腺癌靶向治疗中的吉非替尼继发性耐药是临床遇到的重要问题。本研究旨在探讨吉非替尼是否可诱导肺腺癌PC9细胞发生继发性耐药，以及表皮生长因子受体（epidermal growth factor receptor, EGFR）启动子甲基化在耐药过程中的作用，拟为肺腺癌耐药提供新的治疗靶点。方法体外培养肺腺癌吉非替尼敏感细胞株PC9，应用不同浓度吉非替尼进行干预。MTT法检测PC9细胞株对吉非替尼的敏感性。亚硫酸氢盐处理后测序法（bisulfite sequencing polymerase chain reaction, BSP）检测肺腺癌PC9细胞株EGFR启动子甲基化水平。使用5-氮杂-2’-脱氧胞苷（5-Aza-dc）对吉非替尼耐药细胞株PC9/GR细胞株进行干预。MTT法检测耐药株PC9/GR对吉非替尼敏感性的变化。结果经过从0.01 μmol/L逐渐提升吉非替尼诱导浓度至3 μmol/L之后，MTT显示耐药细胞株PC9/GR细胞株半抑制浓度（half maximal inhibitory concentration, IC50）[（3.95±0.23）μmol/L]较之前敏感株PC9[(0.01±0.002) μmol/L]明显升高（P < 0.05），BSP显示发生异常甲基化的位点结果比较：PC9/GR甲基化水平明显升高（74% vs 59%, P < 0.05）。RT-PCR显示PC9/GR细胞株较PC9细胞株EGFR mRNA表达量升高（P < 0.05）。使用5-Aza-dc处理PC9/GR细胞后，PC9/GR细胞株IC50较对照组降低[（2.55±0.14）μmol/L vs（3.87±0.034）μmol/L, P < 0.05）]。结论吉非替尼体外浓度递增诱导法可诱导PC9细胞株产生吉非替尼继发性耐药，成功构建PC9/GR耐药细胞株。PC9细胞EGFR启动子甲基化异常可能参与了吉非替尼继发性耐药机制。

Elevated expression of Notch-1 and EGFR induced apoptosis in glioblastoma multiforme patients

ObjectiveThe Notch signaling pathway has been well recognized as important adjuster in glioma tumorigenesis and could regulate the glioma cell proliferation through downstream factors such as epidermal growth factor receptor (EGFR). Our current study was aim to investigate the clinical association between Notch-1 gene and EGFR gene as well as cell survival rate in human glioblastoma multiforme (GBM) samples. Patients and methodsSamples from 90 patients with GBMs and 20 normal brain tissues were analyzed in our study. Western blot and immunohistochemistry was used to detect Notch and EGFR protein expression. RT-PCR was used to detect Notch and EGFR mRNA expression. Apoptosis was detected with flow cytometry. ResultsResults demonstrated that the Notch and EGFR gene mRNA and protein levels were dramatically higher in GBM tissues compared to normal brain. Further analysis found these increased mRNA levels were only associated with patient survival period, but not related to patient age, gender and tumor size. A positive correlation was observed between Notch and EGFR protein expression. The positive correlations were also exhibited between Notch-1, EGFR gene expression and apoptosis percentage. ConclusionOur study verified both Notch-1 and EGFR involved in GBM tumorigenesis and may provide important information for GBM clinical treatment and prognosis.

Distinct gene expression profiles of proximal and distal colorectal cancer: implications for cytotoxic and targeted therapy

Colorectal cancer (CRC) is a heterogeneous disease with genetic profiles and clinical outcomes dependent on the anatomic location of the primary tumor. How location impacts the molecular makeup of a tumor and how prognostic and predictive biomarkers differ between proximal versus distal colon cancers is not well established. We investigated the associations between tumor location, KRAS and BRAF mutation status, and the mRNA expression of proteins involved in major signaling pathways, including tumor growth (EGFR), angiogenesis (VEGFR2), DNA repair (ERCC1) and fluoropyrimidine metabolism (TS). FFPE tumor specimens from 431 advanced CRC patients were analyzed. The presence of 7 different KRAS base substitutions and the BRAF V600E mutation was determined. ERCC1, TS, EGFR and VEGFR2 mRNA expression levels were detected by RT-PCR. BRAF mutations were significantly more common in the proximal colon (p<0.001), whereas KRAS mutations occurred at similar frequencies throughout the colorectum. Rectal cancers had significantly higher ERCC1 and VEGFR2 mRNA levels compared to distal and proximal colon tumors (p=0.001), and increased TS levels compared to distal colon cancers (p=0.02). Mutant KRAS status was associated with lower ERCC1, TS, EGFR, and VEGFR2 gene expression in multivariate analysis. In a subgroup analysis, this association remained significant for all genes in the proximal colon and for VEGFR2 expression in rectal cancers. The mRNA expression patterns of predictive and prognostic biomarkers as well as associations with KRAS and BRAF mutation status depend on primary tumor location. Prospective studies are warranted to confirm these findings and determine the underlying mechanisms.

Lentiviral vector-mediated RBM5 overexpression downregulates EGFR expression in human non-small cell lung cancer cells.

BackgroundRNA binding motif 5 (RBM5) is a tumor suppressor gene that modulates apoptosis through the regulation of alternative splicing of apoptosis-related genes. Our previous studies suggested that RBM5 expression was negatively correlated with the expression of epidermal growth factor receptor (EGFR) in non-small cell lung cancer (NSCLC) tissues. This study was aimed at determining whether RBM5 is able to regulate EGFR expression.MethodsBoth in vitro and in vivo studies were carried out to determine the effect of RBM5 on the expression of EGFR. Lentiviral vector-mediated RBM5 overexpression was employed in lung adenocarcinoma cell line A549. A549 xenograft mice were treated with recombinant RBM5 plasmid carried by attenuated Salmonella typhi Ty21a. Real-time quantitative polymerase chain reaction and Western blot were carried out to detect RBM5 and EGFR expression.ResultsBoth in vivo and in vitro studies indicated that the expression of EGFR mRNA and protein was decreased significantly in the RBM5 overexpression group compared to control groups as shown by real-time PCR and Western blot analysis. We identified that RBM5 overexpression inhibited EGFR expression both in A549 cells and in A549 xenograft mice model.ConclusionsOur study demonstrated that EGFR expression is regulated by RBM5 in lung adenocarcinomas cells either in a direct or indirect way, which might be meaningful with regards to target therapy in lung cancer.

Biomarker Analyses from a Placebo-Controlled Phase II Study Evaluating Erlotinib ± Onartuzumab in Advanced Non–Small Cell Lung Cancer: MET Expression Levels Are Predictive of Patient Benefit

In a recent phase II study of onartuzumab (MetMAb), patients whose non-small cell lung cancer (NSCLC) tissue scored as positive for MET protein by immunohistochemistry (IHC) experienced a significant benefit with onartuzumab plus erlotinib (O+E) versus erlotinib. We describe development and validation of a standardized MET IHC assay and, retrospectively, evaluate multiple biomarkers as predictors of patient benefit. Biomarkers related to MET and/or EGF receptor (EGFR) signaling were measured by IHC, FISH, quantitative reverse transcription PCR, mutation detection techniques, and ELISA. A positive correlation between IHC, Western blotting, and MET mRNA expression was observed in NSCLC cell lines/tissues. An IHC scoring system of MET expression taking proportional and intensity-based thresholds into consideration was applied in an analysis of the phase II study and resulted in the best differentiation of outcomes. Further analyses revealed a nonsignificant overall survival (OS) improvement with O+E in patients with high MET copy number (mean≥5 copies/cell by FISH); however, benefit was maintained in "MET IHC-positive"/MET FISH-negative patients (HR, 0.37; P=0.01). MET, EGFR, amphiregulin, epiregulin, or HGF mRNA expression did not predict a significant benefit with onartuzumab; a nonsignificant OS improvement was observed in patients with high tumor MET mRNA levels (HR, 0.59; P=0.23). Patients with low baseline plasma hepatocyte growth factor (HGF) exhibited an HR for OS of 0.519 (P=0.09) in favor of onartuzumab treatment. MET IHC remains the most robust predictor of OS and progression-free survival benefit from O+E relative to all examined exploratory markers.

Chronopharmacology and mechanism of antitumor effect of erlotinib in Lewis tumor-bearing mice.

The epidermal growth factor receptor (EGFR), a ubiquitously expressed receptor tyrosine kinase, is recognized as a key mediator of tumorigenesis in many human epithelial tumors. Erlotinib is tyrosine kinase inhibitor approved by FDA for use in oncology. It inhibits the intracellular phosphorylation of tyrosine kinase associated with the EGFR to restrain the development of the tumor. To investigate the antitumor effect of erlotinib at different dosing times and the underlying molecular mechanism via the PI3K/AKT pathway, we established a mouse model of Lewis lung cancer xenografts. The tumor-bearing mice were housed four or five per cage under standardized light-dark cycle conditions (light on at 7:00 AM, 500 Lux, off at 7:00 PM, 0 Lux) with food and water provided ad libitum. The mice were observed for quality of life, their body weight and tumor volume measured, and the tumor growth curves drawn. After being bled, the mice were sacrificed by cervical dislocation. The tumor masses were removed at different time points and weighed. The mRNA expression of EGFR, AKT, Cyclin D1 and CDK-4 were assayed by quantitative real-time PCR (qRT-PCR). Protein expression levels of AKT, P-AKT and Cyclin D1 were determined by Western blot analysis. The results suggest that erlotinib has a significant antitumor effect on xenografts of non-small cell lung cancer in mice, and its efficacy and toxicity is dependent on the time of day of administration. Its molecular mechanism of action might be related to the EGFR-AKT-Cyclin D1-CDK-4 pathway which plays a crucial role in the development of pathology. Therefore, our findings suggest that the time of day of administration of Erlotinib may be a clinically important variable.

Choline deficiency decreases proliferation of embryonic neural progenitor cells through inhibiting epidermal growth factor pathway (827.2)

Choline deficiency can alter the development of fetal hippocampus. Epidermal growth factor (EGF) is pivotal for neural progenitor cell (NPC) proliferation in the embryonic ventricular and subventricular zones. To address the possibility that EGF signaling pathway plays a functional role in the effect of choline deficiency, we cultured NPCs derived from E14 mouse hippocampus and treated them with low choline (5 µM) or medium choline (70 µM). Low choline significantly suppressed the phosphorylation of EGF receptor (EGFR) and activation of downstream effectors ERK1/2 and Akt after stimulation of NPCs with EGF. Moreover, low choline decreased the EGFR protein expression and half‐life in NPCs. There were no changes observed on the EGFR mRNA expression and degradation of EGFR protein. However, low choline remarkably inhibited the EGFR nascent protein synthesis. Our findings reveal that choline deficiency influences NPC proliferation by suppressing EGFR signaling pathway, which might have long‐term effects in the brain.Grant Funding Source: North Carolina Biotechnology Center (NCBC)

Case-control study of epidermal growth factor receptor (EGFR) gene in head and neck cancer (HNC)

The first intron of EGFR gene contains a microsatellite sequence of 9 to 23 CA-dinucleotide repeats that is very polymorphic. Transcription of the gene is inversely proportional to number of CA-repeats. Shorter CA-dinucleotide repeats cause over expression of EGFR mRNA and protein products with increased proliferation and survival advantage to the cells. Increased EGFR expression is reported to be associated with HNC susceptibility with poorer prognosis, and polymorphism in the CA-repeats is one of the early genetic alterations in HNC. We extracted DNA from blood and saliva samples from 51 (26 cases, 25 controls) participants of Feasibility Study of Cancer of the Head and Neck, Genetics and Environment in Grampian, and analysed using PCR and ABI 3130×L Genetic Analyser. Subjects' ages ranged from 45-84 years; mean (SD) 63.15 ± 10.46 years. The EGFR risk association with HNC was calculated based on two categories: subjects with short CA-repeats (alleles ≤ 119bp) and those with long CA-repeats (alleles >119bp). After multivariate analysis adjustment for gender, tobacco, alcohol, and diet, there was increased risk of the short CA-dinucleotide repeats for HNC (OR= 1.50, 95% CI 0.34-6.71, <i>p</i>=0.6). The low statistical power is likely due to small sample size. However, the result agrees with what obtains in the literature database.

Phosphatidylinositol 3-Kinase dependent upregulation of the epidermal growth factor receptor upon Flotillin-1 depletion in breast cancer cells.

BackgroundFlotillin-1 and flotillin-2 are two homologous and ubiquitously expressed proteins that are involved in signal transduction and membrane trafficking. Recent studies have reported that flotillins promote breast cancer progression, thus making them interesting targets for breast cancer treatment. In the present study, we have investigated the underlying molecular mechanisms of flotillins in breast cancer.MethodsHuman adenocarcinoma MCF7 breast cancer cells were stably depleted of flotillins by means of lentivirus mediated short hairpin RNAs. Western blotting, immunofluorescence and quantitative real-time PCR were used to analyze the expression of proteins of the epidermal growth factor receptor (EGFR) family. Western blotting was used to investigate the effect of EGFR stimulation or inhibition as well as phosphatidylinositol 3-kinase (PI3K) inhibition on mitogen activated protein kinase (MAPK) signaling. Rescue experiments were performed by stable transfection of RNA intereference resistant flotillin proteins.ResultsWe here show that stable knockdown of flotillin-1 in MCF7 cells resulted in upregulation of EGFR mRNA and protein expression and hyperactivation of MAPK signaling, whereas ErbB2 and ErbB3 expression were not affected. Treatment of the flotillin knockdown cells with an EGFR inhibitor reduced the MAPK signaling, demonstrating that the increased EGFR expression and activity is the cause of the increased signaling. Stable ectopic expression of flotillins in the knockdown cells reduced the increased EGFR expression, demonstrating a direct causal relationship between flotillin-1 expression and EGFR amount. Furthermore, the upregulation of EGFR was dependent on the PI3K signaling pathway which is constitutively active in MCF7 cells, and PI3K inhibition resulted in reduced EGFR expression.ConclusionsThis study demonstrates that flotillins may not be suitable as cancer therapy targets in cells that carry certain other oncogenic mutations such as PI3K activating mutations, as unexpected effects are prone to emerge upon flotillin knockdown which may even facilitate cancer cell growth and proliferation.

MRNA expression of CK19, EGFR and LUNX in patients with lung cancer micrometastasis

To evaluate the clinical significance of mRNA expression of cytokeratin 19 (CK19), epidermal growth factor receptor (EGFR) and lung-specific X protein (LUNX), a total of 42 patients who were diagnosed with non-small cell lung cancer (NSCLC) by pathology were studied retrospectively. The messenger RNA (mRNA) expression levels of CK19, EGFR and LUNX in the peripheral blood were analyzed using reverse transcription-polymerase chain reaction (RT-PCR). The expression of CK19 mRNA did not differ significantly according to the location, size, clinical stage or differentiation of the primary tumor (all P>0.05). However, there was a significant difference in the level of CK19 mRNA expression between squamous carcinoma and adenocarcinoma. The positive rates of EGFR mRNA in the patient and the healthy control groups were 69.0 and 12.5%, respectively, and were significantly different (P<0.05). The positive rates of LUNX mRNA in the two groups were 40.5 and 0%, respectively, and were significantly different (P<0.05). The results indicate that the mRNA expression of CK19, EGFR and LUNX in the peripheral blood is of significant clinical value for the diagnosis of micrometastasis and the prognosis of lung cancer.

Granulosa Cells from Emerged Antral Follicles of the Bovine Ovary Initiate Inflammation in Response to Bacterial Pathogen-Associated Molecular Patterns via Toll-Like Receptor Pathways1

Bacterial infections of the uterus or mammary gland commonly perturb ovarian antral follicle growth and function, causing infertility in cattle. Cells of the innate immune system use Toll-like receptors (TLRs) TLR2, TLR4, and TLR5 to recognize pathogen-associated molecular patterns (PAMPs) of bacteria, leading to production of inflammatory mediators, such as IL-1beta, IL-6, and IL-8. The present study examined whether granulosa cells from emerged antral follicles have functional responses to typical bacterial PAMPs. Granulosa cells from emerged bovine antral follicles expressed mRNA for all 10 TLRs. Cellular expression of mRNA for the cytokines IL1B, IL6, IL10, and TNF, and chemokines IL8 and CCL5, increased after treatment with synthetic bacterial lipoprotein binding TLR2, lipopolysaccharide binding TLR4, or flagellin binding TLR5. Supernatants of granulosa cells accumulated IL-1beta, IL-6, and IL-8 protein in a concentration-dependent manner when treated with lipoprotein or lipopolysaccharide, but not flagellin. Accumulation of IL6 in response to lipoprotein and lipopolysaccharide was attenuated using siRNA targeting TLR2 and TLR4, respectively. Granulosa cells increased phosphorylation of mitogen-activated protein kinase (MAPK) 14 and MAPK3/1 within 30 min of treatment with lipopolysaccharide or lipoprotein, and inhibitors targeting MAPK14 reduced the accumulation of IL-6 in response to the PAMPs. Treatment with hormones follicle-stimulating hormone, luteinizing hormone, estradiol, or progesterone did not significantly affect granulosa cell responses to PAMPs. However, epidermal growth factor enhanced IL-6 accumulation in response to lipoprotein and inhibiting epidermal growth factor receptor (EGFR) abrogated the effect, whereas lipoprotein increased granulosa cell EGFR mRNA expression. In conclusion, bovine granulosa cells from emerged follicles sense bacterial PAMPs and initiate inflammatory responses via TLR2 and TLR4 pathways.

Abstract C37: Reduced annexin A6 expression enhances EGFR degradation and sensitizes invasive breast cancer cells to EGFR-targeted tyrosine kinase inhibitors

Abstract The role of annexin A6 (AnxA6), a calcium-dependent phospholipid binding protein, in breast carcinogenesis remains poorly understood. Some reports suggest that AnxA6 inhibits the activation and down-stream signaling of epidermal growth factor receptor (EGFR). On the contrary, other studies have shown that AnxA6 not only stabilizes activated receptors on the cell surface but also promotes the expression of differentiation markers in chondrocytes. Together with our recent study showing that AnxA6 expression is reduced in breast cancer, we investigated whether AnxA6 expression status influences breast cancer outcome in patients and whether this may be attributed to its modulation of EGFR expression. We show that low AnxA6 expression correlates with reduced distant metastasis-free and overall survival, but is associated with a higher probability for recurrence-free survivalof patients with basal-like breast cancer. To determine whether these effects are mediated via EGFR, a predictive marker of basal-like breast cancer, we examined the effects of altered expression of AnxA6 in AnxA6-high invasive triple negative breast cancer cells. Interestingly, depletion of AnxA6 in invasive BT-549 cells was accompanied by decreased expression of EGFR protein and mRNA. We also show that in invasive breast cancer cells, AnxA6 expression is required for sustained membrane localization of activated EGFR and persistent downstream signaling. Consequently, AnxA6-depleted cells exhibited decreased cell motility, and increased sensitivity to the tyrosine kinase inhibitors (TKIs), lapatinib and PD153035. On the contrary, over-expression of AnxA6 in AnxA6-low HCC1806 cells did not affect their motility but as expected, inhibited their growth in 3D matrigel cultures. These data suggest that AnxA6-modulation of EGFR expression and activity underlies at least in part, the changes in proliferation, metastatic potentials and sensitivity to EGFR-targeted TKIs of invasive breast cancer cells. Our data also suggest that AnxA6 expression status may predict the survival and likelihood to respond to EGFR-targeted therapies of patients with basal-like breast cancer. Citation Format: Rainelli B. Koumangoye, Josiah Ochieng, Amos M. Sakwe. Reduced annexin A6 expression enhances EGFR degradation and sensitizes invasive breast cancer cells to EGFR-targeted tyrosine kinase inhibitors. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C37.