The Ah receptor regulates growth factor expression in head and neck squamous cell carcinoma cell lines.
ABSTRACT: Previous studies in head and neck squamous cell carcinoma (HNSCC) cell lines have revealed that the Ah receptor (AHR) plays a significant role in mediating the "aggressive" phenotype of these cells, which includes enhanced inflammatory signaling (e.g., IL6) and migratory potential. Here we sought to identify putative novel targets of the AHR associated with enhanced tumor invasiveness. Global gene expression analysis identified a number of genes that are repressed upon treatment of OSC-19 or HN30 cells with an AHR antagonist. Three growth factors were targets of AHR activity; amphiregulin (AREG), epiregulin (EREG), and platelet-derived growth factor A (PDGFA) were repressed by an AHR antagonist and further examined. Quantitative PCR analysis, ELISA, and siRNA-mediated knock down of AHR revealed an attenuation of basal and/or induced levels of expression of these growth factors in two HNSCC lines, following AHR antagonism. In silico analysis revealed that these growth factors possess dioxin-like response elements. Two other AHR ligands, 6-formylindolo[3,2-b]carbazole and benzo(a)pyrene (BP) also elicited similar responses. In conclusion, this study identified AREG, EREG, and PDGFA as growth factor targets of AHR activity associated with metastatic phenotype of HNSCC cells, suggesting that attenuation of AHR activity may be a therapeutic strategy.
Project description:Background: The EGFR (epithelial growth factor receptor) ligands amphiregulin (AREG) and epiregulin (EREG) have been considered as predictors for EGFR-antibody efficacy. The effect of AREG and EREG expression levels in primary tumor samples on the outcome of bevacizumab-treated patients is unknown. Patients and Methods: Formalin-fixed paraffin-embedded (FFPE) tumor samples from surgically removed primaries of the AIO KRK-0207 trial have been tested for AREG and EREG expression. The AIO KRK-0207 trial was a randomized phase-3 study to investigate the best maintenance strategy after oxaliplatin/fluoropyrimidine plus bevacizumab induction treatment in patients with mCRC. Association of AREG and EREG levels with outcome parameters were investigated, taking into account RAS and BRAF mutations. Results: A total of 331 tumor samples had measurable AREG and EREG tissue levels. In the total cohort using continuous expression levels, higher logAREG and logEREG levels were associated with a significant longer overall survival (OS) (HR 0.80; p = 0.003 and HR 0.78; p = 0.001, respectively). The subgroup of BRAF mutant tumors displayed significantly lower AREG and EREG levels compared to wild-type tumors. The prognostic effect of AREG and EREG expression was limited to the double wild-type subpopulation, whereas in the RAS mutant and BRAF mutant subgroups no prognostic effect was detected. Conclusion: Low logAREG and logEREG levels are associated with a shorter OS in oxaliplatin/fluoropyrimidine plus bevacizumab treated patients. As low AREG and EREG level are associated with BRAF mutations, the prognostic value of EREG and AREG levels is limited to the RAS and BRAF wild-type subpopulation.
Project description:Epidermal growth factor receptor (EGFR) and its ligands amphiregulin (AREG) and epiregulin (EREG) play a central role in the development of colorectal cancer, but the prognostic values of AREG and EREG are controversial. We conducted a meta-analysis of studies that investigated AREG and/or EREG mRNA levels in primary tumors to determine their prognostic value in metastatic colorectal cancer (mCRC). In addition, RAS status was assessed. Relevant articles were identified by searching the EMBASE, PubMed, and Cochrane Library databases. Hazard ratios (HR) with 95% confidence intervals (CIs) were calculated using a random-effects model. Nine studies involving 2167 patients were included in this meta-analysis. High AREG expression was associated with longer overall survival (OS) and progression-free survival (PFS). High EREG expression was also associated with prolonged OS and PFS. In RAS wild-type (WT) patients who received anti-EGFR therapy, high AREG and EREG expression was associated with longer OS. Our results indicate that high AREG and EREG mRNA expression are independent favorable prognostic biomarkers in mCRC. The expression of these ligands should be considered when evaluating prognoses in RAS-WT patients receiving anti-EGFR therapy.
Project description:A local autocrine/paracrine role for progesterone is an absolute requirement for corpus luteum formation in primates. Despite this, the mechanism(s) remain obscure, although existing data suggest an anti-apoptotic action to be central. There are a limited number of progestin-regulated gene targets identified in the luteinizing primate follicle, suggesting that a small number of important genes may mediate progesterone action. Possible gene targets could be the epidermal growth factor (EGF) family members amphiregulin (AREG) and epiregulin (EREG). Using macaques undergoing controlled ovarian stimulation cycles, we show that the phosphorylation of EGF receptor (EGFR), ERK 1/2, and AKT increases 6 h after an ovulatory human chorionic gonadotropin (hCG) stimulus and remains activate through 24 h. Immunoreactive EREG and AREG ligands in the follicular fluid both increased in a time frame commensurate with EGFR phosphorylation. The mRNA expression of AREG and EREG in nonluteinized granulosa cells (NLGC) was induced in culture with hCG, an effect blocked by progesterone receptor (PGR) antagonists. Overexpression of PGR B in NLGC and treatment with a nonmetabolizable progestin did not increase either gene, indicating both progesterone and luteinizing hormone/CG are necessary. Addition of EGF and EGF-like ligands did not promote steroidogenesis in vitro by granulosa cells in the presence of gonadotropin, but were able to partially reverse RU486-induced cell death. These data suggest that progesterone promotes the expression of AREG and EREG, which in turn maintain viability of luteinizing granulosa cells, representing one possible mechanism whereby progesterone promotes corpus luteum formation in the primate.
Project description:Rationale: The oncogenesis of head and neck squamous cell carcinoma (HNSCC) is believed to result from oncogene activation and tumor suppressor inactivation. Here, we identified a new oncogenic role for the EREG gene in HNSCC. Methods: The TCGA database and immunohistochemistry assay were used to analyze expression of EREG in HNSCC tissues. Immunoblotting was performed to identify the EGFR-mediated pathways altered by EREG. The role of EREG in oncogenesis was investigated in vivo and in vitro. Results: Upregulated EREG expression predicted a poor prognosis and triggered HNSCC oncogenic transformation by activating the epidermal growth factor receptor (EGFR) signaling pathway. We also demonstrated the direct association of EREG with EGFR and that this binding required EGFR domains I and III and the N57 residue of EREG. Moreover, EREG overexpression was shown to promote HNSCC oncogenesis by inducing C-Myc expression, and the pharmacological inhibition of C-Myc rescued EREG-promoted HNSCC oncogenesis. Unlike other EGFR ligands, EREG could mimic EGFR mutations by sustaining the activation of the EGFR-Erk pathway, and high EREG expression was positively associated with the response to treatment with the EGFR inhibitor erlotinib. Furthermore, knockdown of EREG decreased sensitivity to erlotinib treatment in vitro and in vivo. Conclusions: These results identify the EREG-EGFR-C-Myc pathway as a crucial axis that drives HNSCC oncogenesis and show that EREG expression could be a predictive functional marker of sensitivity to erlotinib therapy in HNSCC.
Project description:Toll-like receptors (TLRs) trigger intestinal inflammation when the epithelial barrier is breached by physical trauma or pathogenic microbes. Although it has been shown that TLR-mediated signals are ultimately protective in models of acute intestinal inflammation [such as dextran sulfate sodium (DSS)-induced colitis], it is less clear which cells mediate protection. Here we demonstrate that TLR signaling in the nonhematopoietic compartment confers protection in acute DSS-induced colitis. Epithelial cells of MyD88/Trif-deficient mice express diminished levels of the epidermal growth factor receptor (EGFR) ligands amphiregulin (AREG) and epiregulin (EREG), and systemic lipopolysaccharide administration induces their expression in the colon. N-ethyl-N-nitrosourea (ENU)-induced mutations in Adam17 (which is required for AREG and EREG processing) and in Egfr both produce a strong DSS colitis phenotype, and the Adam17 mutation exerts its deleterious effect in the nonhematopoietic compartment. The effect of abrogation of TLR signaling is mitigated by systemic administration of AREG. A TLR?MyD88?AREG/EREG?EGFR signaling pathway is represented in nonhematopoietic cells of the intestinal tract, responds to microbial stimuli once barriers are breached, and mediates protection against DSS-induced colitis.
Project description:Amphiregulin (AREG) and epiregulin (EREG) are important ligands to the epithelial growth factor receptor, which is involved in the regulation of progression and stemness in gastric cancer (GC). This study investigated whether frequent single nucleotide polymorphisms (SNPs) in genes of AREG and EREG are associated with recurrence-free survival and overall survival in patients with locally advanced GC.SNPs with a minor allele frequency of at least 10% were analyzed using direct DNA sequencing in two independent study populations.The minor allele of AREG rs1615111 was associated with a significantly higher 3-year recurrence rate and lower 3-year survival rate [hazard ratio (HR)=2.21 and 2.35, respectively] compared with patients homozygous for the dominant allele G. The value for overall survival could be validated with a HR of 2.54 (P=0.018) in an independent cohort. Patients homozygous for the minor allele A of EREG rs12641042 had a significantly higher 3-year survival rate than patients with allele C (HR 0.48; P=0.034), but significance was lost in multivariable analysis (P=0.066). The value of rs12641042 could not be validated (P=0.98). Exploratory multivariable subgroup analysis showed the strongest prognostic value for rs1615111 in tumors with a diffuse histology (Pfor interaction=0.004).AREG rs1615111, located in the AREG genomic region, can significantly define different prognostic cohorts in locally advanced GC. This value is most evident in GC patients with diffuse histology, which might be relevant as none of the trials testing epithelial growth factor receptor inhibitors has been enriched for diffuse histology or a molecularly defined population.
Project description:BACKGROUND: More than half of patients with KRAS-wild type advanced colorectal cancer (CRC) fail anti-EGFR monoclonal antibodies. We studied EGFR-axis messenger RNA (mRNA) expression and RAS, RAF, PIK3CA mutations in order to identify additional biomarkers of cetuximab efficacy. METHODS: Previously genotyped (KRAS, NRAS, BRAF, PIK3CA mutations) formalin-fixed paraffin-embedded tumour biopsies of 226 cetuximab-treated CRC patients (1st to 3rd line therapy) were assessed for mRNA expression of epidermal growth factor receptor (EGFR) and its ligands EGF, Transofrming Growth Factor-a (TGFA), Amphiregulin (AREG) and Epiregulin (EREG) with real time quantitative PCR. Mutations were detected in 72 (31.9%) tumours for KRAS, in 6 (2.65%) for BRAF, in 7 (3.1%) for NRAS and in 37 (16.4%) for PIK3CA. RESULTS: Only PIK3CA mutations occasionally coexisted with other gene mutations. In univariate analysis, prognostic significance for survival ( from metastases until death) was seen for BRAF mutations (Hazard Ratio HR 8.1, 95% CI 3.4-19), codon 12-only KRAS mutations (HR 1.62, 95% CI 1.1-2.4), high AREG mRNA expression only in KRAS wild type CRC (HR 0.47, 95% CI 0.3-0.7) and high EREG mRNA expression irrespective of KRAS mutation status (HR 0.45, 95% CI 0.28-0.7). EREG tumoural mRNA expression was significantly associated with a 2.26-fold increased likelihood of objective response to cetuximab therapy (RECIST 1.1). In multivariate analysis, favourable predictive factors were high AREG mRNA in KRAS wild type tumours, high EREG mRNA, low Ephrin A2 receptor mRNA. Cetuximab-treated patients with AREG-low KRAS wild type CRC fared very poorly, their survival being similar to KRAS mutant CRC. Patients with KRAS codon 13 or other non-codon 12 mutations had a median survival (30 months, 95% CI 20-35) similar to that of patients with KRAS wild-type (median survival 29 months, 95% CI 25-35), in contrast to patients with KRAS codon 12 mutations who fared worse (median survival 19 months, 95% CI 15-26). CONCLUSIONS: BRAF and codon 12 KRAS mutations predict for adverse outcome of CRC patients receiving cetuximab. AREG mRNA reflects EGFR signalling in KRAS wild type tumours, predicting for cetuximab efficacy when high and failure when low. EREG may have a prognostic role independent of KRAS mutation.
Project description:In cultures of normal human epidermal keratinocytes (NHEKs), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces the expression of the epidermal growth factor receptor ligands transforming growth factor-? (TGF-?) and epiregulin (EREG). TCDD also down-regulates EGF receptors (EGFR), suggesting that decreases in signaling contribute to the effects of TCDD. In this study, we treated post-confluent NHEKs with 10 nM TCDD and assessed its effects on EGFR binding, EGFR ligand secretion, basal ERK activity, and proliferation. TCDD caused time-dependent deceases in [(125)I]-EGF binding to levels 78% of basal cell values at 72 h. Amphiregulin (AREG) levels increased with time in culture in basal and TCDD-treated cells, while TGF-? and epiregulin (EREG) secretion were stimulated by TCDD. Inhibiting EGFR ligand release with the metalloproteinase inhibitor batimastat prevented EGFR down-regulation and neutralizing antibodies for AREG and EREG relieved receptor down-regulation. In contrast, neutralizing TGF-? intensified EGFR down-regulation. Treating NHEKs with AREG or TGF-? caused rapid internalization of receptors with TGF-? promoting recycling within 90 min. EREG had limited effects on rapid internalization or recycling. TCDD treatment increased ERK activity, a response reduced by batimastat and the neutralization of all three ligands indicating that the EGFR and its ligands maintain ERK activity. All three EGFR ligands were required for the maintenance of total cell number in basal and TCDD-treated cultures. The EGFR inhibitor PD1530305 blocked basal and TCDD-induced increases in the number of cells labeled by 5-ethynyl-2'-deoxyuridine, identifying an EGFR-dependent pool of proliferating cells that is larger in TCDD-treated cultures. Overall, these data indicate that TCDD-induced EGFR down-regulation in NHEKs is caused by AREG, TGF-?, and EREG, while TGF-? enhances receptor recycling to maintain a pool of EGFR at the cell surface. These receptors are required for ERK activity, maintenance of total cell number, and stimulating the proliferation of a small subset cells.
Project description:MiR-34a is a well-known tumor metastasis inhibitor, but only a few target genes involved in metastasis have been identified. In HNSCC, the role of miR-34a in metastasis has not been fully elaborated, and the target gene of miR-34a is still blind. Here we addressed that, the relative lower expression of miR-34a is associated with HNSCC lymphatic metastasis. HNSCC metastasis was found to be strongly suppressed in vitro and in vivo by over-expressing miR-34a. In order to screen the possible target genes of miR-34a in HNSCC, a microarray-based differential mRNA profiling mediated by miR-34a over-expression was performed, and AREG was identified as a pivotal target. We demonstrated that the mRNA and protein levels of AREG were greatly reduced when forcing miR-34a expression. The correlation between AREG mRNA levels and HNSCC metastatic phenotype was also significant in HNSCC tissues (p < 0.01). Moreover, the results of luciferase assay provided the further evidence that miR-34a degraded AREG mRNA through targeting the 3'-UTR site. Restoration of AREG expression partially rescued miR-34a-mediated cell invasion defects in vivo and in vitro. Additionally, Over-expressing miR-34a greatly reduced EGFR and uPA, which were reversed by re-expression of AREG. Taken together, these findings indicate that miR-34a targets AREG, and is essential in inhibition of HNSCC metastasis.
Project description:Aberrant DNA methylation patterns are a common theme across all cancer types. Specific DNA demethylation of regulatory sequences can result in upregulation of genes that are critical for tumor development and progression. Integrin ?6?4 is highly expressed in pancreatic carcinoma and contributes to cancer progression, in part, through the specific DNA demethylation and upregulation of epidermal growth factor receptor (EGFR) ligands amphiregulin (AREG) and epiregulin (EREG). Whole genome bisulfite sequencing (WGBS) revealed that integrin ?6?4 signaling promotes an overall hypomethylated state and site specific DNA demethylation of enhancer elements within the proximal promoters of AREG and EREG. Additionally, we find that the base excision repair (BER) pathway is required to maintain expression of AREG and EREG, as blocking DNA repair molecules, TET1 GADD45A, TDG, or PARP-1 decreased gene expression. Likewise, we provide the novel finding that integrin ?6?4 confers an enhanced ability on cells to repair DNA lesions and survive insult. Therefore, while many known signaling functions mediated by integrin ?6?4 that promote invasive properties have been established, this study demonstrates that integrin ?6?4 can dramatically impact the epigenome of cancer cells, direct global DNA methylation levels toward a hypomethylated state, and impact DNA repair and subsequent cell survival.