Involvement of HDAC2-mediated kcnq2/kcnq3 genes transcription repression activated by EREG/EGFR-ERK-Runx1 signaling in bone cancer pain.

Bone cancer pain (BCP) represents a prevalent symptom among cancer patients with bone metastases, yet its underlying mechanisms remain elusive. This study investigated the transcriptional regulation mechanism of Kv7(KCNQ)/M potassium channels in DRG neurons and its involvement in the development of BCP in rats. We show that HDAC2-mediated transcriptional repression of kcnq2/kcnq3 genes, which encode Kv7(KCNQ)/M potassium channels in dorsal root ganglion (DRG), contributes to the sensitization of DRG neurons and the pathogenesis of BCP in rats. Also, HDAC2 requires the formation of a corepressor complex with MeCP2 and Sin3A to execute transcriptional regulation of kcnq2/kcnq3 genes. Moreover, EREG is identified as an upstream signal molecule for HDAC2-mediated kcnq2/kcnq3 genes transcription repression. Activation of EREG/EGFR-ERK-Runx1 signaling, followed by the induction of HDAC2-mediated transcriptional repression of kcnq2/kcnq3 genes in DRG neurons, leads to neuronal hyperexcitability and pain hypersensitivity in tumor-bearing rats. Consequently, the activation of EREG/EGFR-ERK-Runx1 signaling, along with the subsequent transcriptional repression of kcnq2/kcnq3 genes by HDAC2 in DRG neurons, underlies the sensitization of DRG neurons and the pathogenesis of BCP in rats. These findings uncover a potentially targetable mechanism contributing to bone metastasis-associated pain in cancer patients.

The growth factors amphiregulin and epiregulin are novel stimulators of feline ovarian granulosa cell functions.

This study aimed to investigate the impact of the epidermal growth factor receptor ligands amphiregulin (AREG) and epiregulin (EREG) on the fundamental functions of feline ovarian granulosa cells. Granulosa cells isolated from feline ovaries were incubated with AREG and EREG (0, 0.1, 1 or 10 ng/mL). The effects of these growth factors on cell viability, proliferation (assessed through BrdU incorporation), nuclear apoptosis (evaluated through nuclear DNA fragmentation) and the release of progesterone and estradiol were determined using Cell Counting Kit-8 assays, BrdU analysis, TUNEL assays and ELISAs, respectively. Both AREG and EREG increased cell viability, proliferation and steroid hormone release and reduced apoptosis. The present findings suggest that these epidermal growth factor receptor ligands may serve as physiological stimulators of feline ovarian cell functions.

Role of Epiregulin on Lipopolysaccharide-Induced Hepatocarcinogenesis as a Mediator via EGFR Signaling in the Cancer Microenvironment.

Lipopolysaccharides (LPSs) have been reported to be important factors in promoting the progression of hepatocellular carcinoma (HCC), but the corresponding molecular mechanisms remain to be elucidated. We hypothesize that epiregulin (EREG), an epidermal growth factor (EGF) family member derived from hepatic stellate cells (HSCs) and activated by LPS stimulation, is a crucial mediator of HCC progression with epidermal growth factor receptor (EGFR) expression in the tumor microenvironment. We used a mouse xenograft model of Huh7 cells mixed with half the number of LX-2 cells, with/without intraperitoneal LPS injection, to elucidate the role of EREG in LPS-induced HCC. In the mouse model, LPS administration significantly enlarged the size of xenografted tumors and elevated the expression of EREG in tumor tissues compared with those in negative controls. Moreover, CD34 immunostaining and the gene expressions of angiogenic markers by a reverse transcription polymerase chain reaction revealed higher vascularization, with increased interleukin-8 (IL-8) expression in the tumors of the mice group treated with LPS compared to those without LPS. Our data collectively suggested that EREG plays an important role in the cancer microenvironment under the influence of LPS to increase not only the tumor cell growth and migration/invasion of EGFR-positive HCC cells but also tumor neovascularization via IL-8 signaling.

Induction of apically mistrafficked epiregulin disrupts epithelial polarity via aberrant EGFR signaling.

In polarized MDCK cells, disruption of the tyrosine-based YXXΦ basolateral trafficking motif (Y156A) in the epidermal growth factor receptor (EGFR) ligand epiregulin (EREG), results in its apical mistrafficking and transformation in vivo. However, the mechanisms underlying these dramatic effects are unknown. Using a doxycycline-inducible system in 3D Matrigel cultures, we now show that induction of Y156A EREG in fully formed MDCK cysts results in direct and complete delivery of mutant EREG to the apical cell surface. Within 3 days of induction, ectopic lumens were detected in mutant, but not wild-type, EREG-expressing cysts. Of note, these structures resembled histological features found in subcutaneous xenografts of mutant EREG-expressing MDCK cells. These ectopic lumens formed de novo rather than budding from the central lumen and depended on metalloprotease-mediated cleavage of EREG and subsequent EGFR activity. Moreover, the most frequent EREG mutation in human cancer (R147stop) resulted in its apical mistrafficking in engineered MDCK cells. Thus, induction of EREG apical mistrafficking is sufficient to disrupt selective aspects of polarity of a preformed polarized epithelium. This article has an associated First Person interview with the first author of the paper.

Human Chorionic Gonadotropin-Stimulated Interleukin-4-Induced-1 (IL4I1) Promotes Human Decidualization via Aryl Hydrocarbon Receptor.

Decidualization is necessary for the successful establishment of early pregnancy in rodents and humans. Disturbed decidualization results in recurrent implantation failure, recurrent spontaneous abortion, and preeclampsia. Tryptophan (Trp), one of the essential amino acids in humans, has a positive effect on mammalian pregnancy. Interleukin 4-induced gene 1 (IL4I1) is a recently identified enzyme that can metabolize L-Trp to activate aryl hydrocarbon receptor (AHR). Although IDO1-catalyzed kynurenine (Kyn) from Trp has been shown to enhance human in vitro decidualization via activating AHR, whether IL4I1-catalyzed metabolites of Trp are involved in human decidualization is still unknown. In our study, human chorionic gonadotropin stimulates IL4I1 expression and secretion from human endometrial epithelial cells through ornithine decarboxylase-induced putrescine production. Either IL4I1-catalyzed indole-3-pyruvic acid (I3P) or its metabolite indole-3-aldehyde (I3A) from Trp is able to induce human in vitro decidualization by activating AHR. As a target gene of AHR, Epiregulin induced by I3P and I3A promotes human in vitro decidualization. Our study indicates that IL4I1-catalyzed metabolites from Trp can enhance human in vitro decidualization through AHR-Epiregulin pathway.

Syringic acid mitigates isoproterenol-induced cardiac hypertrophy and fibrosis by downregulating Ereg.

Gallic acid has been reported to mitigate cardiac hypertrophy, fibrosis and arterial hypertension. The effects of syringic acid, a derivative of gallic acid, on cardiac hypertrophy and fibrosis have not been previously investigated. This study aimed to examine the effects of syringic acid on isoproterenol-treated mice and cells. Syringic acid mitigated the isoproterenol-induced upregulation of heart weight to bodyweight ratio, pathological cardiac remodelling and fibrosis in mice. Picrosirius red staining, quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting analyses revealed that syringic acid markedly downregulated collagen accumulation and fibrosis-related factors, including Fn1. The results of RNA sequencing analysis of Ereg expression were verified using qRT-PCR. Syringic acid or transfection with si-Ereg mitigated the isoproterenol-induced upregulation of Ereg, Myc and Ngfr. Ereg knockdown mitigated the isoproterenol-induced upregulation of Nppb and Fn1 and enhancement of cell size. Mechanistically, syringic acid alleviated cardiac hypertrophy and fibrosis by downregulating Ereg. These results suggest that syringic acid is a potential therapeutic agent for cardiac hypertrophy and fibrosis.

Antibody Screening System Using a Herpes Simplex Virus (HSV)-Based Probe To Identify a Novel Target for Receptor-Retargeted Oncolytic HSVs.

Herpes simplex virus (HSV) is a promising tool for developing oncolytic virotherapy. We recently reported a platform for receptor-retargeted oncolytic HSVs that incorporates single-chain antibodies (scFvs) into envelope glycoprotein D (gD) to mediate virus entry via tumor-associated antigens. Therefore, it would be useful to develop an efficient system that can screen antibodies that might mediate HSV entry when they are incorporated as scFvs into gD. We created an HSV-based screening probe by the genetic fusion of a gD mutant with ablated binding capability to the authentic HSV entry receptors and the antibody-binding C domain of streptococcal protein G. This engineered virus failed to enter cells through authentic receptors. In contrast, when this virus was conjugated with an antibody specific to an antigen on the cell membrane, it specifically entered cells expressing the cognate antigen. This virus was used as a probe to identify antibodies that mediate virus entry via recognition of certain molecules on the cell membrane other than authentic receptors. Using this method, we identified an antibody specific to epiregulin (EREG), which has been investigated mainly as a secreted growth factor and not necessarily for its precursor that is expressed in a transmembrane form. We constructed an scFv from the anti-EREG antibody for insertion into the retargeted HSV platform and found that the recombinant virus entered cells specifically through EREG expressed by the cells. This novel antibody-screening system may contribute to the discovery of unique and unexpected molecules that might be used for the entry of receptor-retargeted oncolytic HSVs.IMPORTANCE The tropism of the cellular entry of HSV is dependent on the binding of the envelope gD to one of its authentic receptors. This can be fully retargeted to other receptors by inserting scFvs into gD with appropriate modifications. In theory, upon binding to the engineered gD, receptors other than authentic receptors should induce a conformational change in the gD, which activates downstream mechanisms required for viral entry. However, prerequisite factors for receptors to be used as targets of a retargeted virus remain poorly understood, and it is difficult to predict which molecules might be suitable for our retargeted HSV construct. Our HSV-based probe will allow unbiased screening of antibody-antigen pairs that mediate virus entry and might be a useful tool to identify suitable pairs for our construct and to enhance our understanding of virus-cell interactions during infection by HSV and possibly other viruses.

3-Nitrobenzanthrone promotes malignant transformation in human lung epithelial cells through the epiregulin-signaling pathway.

Exposure to environmental and occupational contaminants leads to lung cancer. 3-Nitrobenzanthrone (3-nitro-7H-benz[de]anthracen-7-one, 3-NBA) is a potential carcinogen in ambient air or diesel particulate matter. Studies have revealed that short-term exposure to 3-NBA induces cell death, reactive oxygen species activation, and DNA adduct formation and damage. However, details of the mechanism by which chronic exposure to 3-NBA influences lung carcinogenesis remain largely unknown. In this study, human lung epithelial BEAS-2B cells were continuously exposed to 0-10-µM 3-NBA for 6 months. NanoString analysis was conducted to evaluate gene expression in the cells, revealing that 3-NBA-mediated transformation results in a distinct gene expression signature including carbon cancer metabolism, metastasis, and angiogenesis. Alterations in tumor-promoting genes such as EREG (epiregulin), SOX9, E-cadherin, TWIST, and IL-6 were involved in epithelial cell aggressiveness. Kaplan-Meier plotter analyses indicated that increased EREG and IL-6 expressions in early-stage lung cancer cells are correlated with poor survival. In vivo xenografts on 3-NBA-transformed cells exhibited prominent tumor formation and metastasis. EREG knockout cells exposed to 3-NBA for a short period exhibited high apoptosis and low colony formation. By contrast, overexpression of EREG in 3-NBA-transformed cells markedly activated the PI3K/AKT and MEK/ERK signaling pathways, resulting in tumorigenicity. Furthermore, elevated IL-6 and EREG expressions synergistically led to STAT3 signaling activation, resulting in clonogenic cell survival and migration. Taken together, chronic exposure of human lung epithelial cells to 3-NBA leads to malignant transformation, in which the EREG signaling pathway plays a pivotal mediating role. • Short-term exposure of lung epithelial cells to 3-NBA can lead to ROS production and cell apoptosis. • Long-term chronic exposure to 3-NBA upregulates the levels of tumor-promoting genes such as EREG and IL-6. • Increased EREG expression in 3-NBA-transformed cells markedly contributes to tumorigenesis through PI3K/AKT and MEK/ERK activation and synergistically enhances the IL-6/STAT3 signaling pathway, which promotes tumorigenicity.

Epiregulin, epigen and betacellulin antibodies and axial elongation in young guinea pigs with lens-induced myopization.

BACKGROUND: To examine an effect of intravitreally applied antibodies against epidermal growth factor family members, namely epiregulin, epigen and betacellulin, on ocular axial elongation. METHODS: The experimental study included 30 guinea pigs (age:3-4 weeks) which underwent bilateral lens-induced myopization and received three intraocular injections of 20 µg of epiregulin antibody, epigen antibody and betacellulin antibody in weekly intervals into their right eyes, and of phosphate-buffered saline into their left eyes. Seven days after the last injection, the animals were sacrificed. Axial length was measured by sonographic biometry. RESULTS: At baseline, right eyes and left eyes did not differ (all P > 0.10) in axial length in neither group, nor did the interocular difference in axial length vary between the groups (P = 0.19). During the study period, right and left eyes elongated (P < 0.001) from 8.08 ± 0.07 mm to 8.59 ± 0.06 mm and from 8.08 ± 0.07 mm to 8.66 ± 0.07 mm, respectively. The interocular difference (left eye minus right eye) in axial elongation increased significantly in all three groups (epiregulin-antibody:from 0.03 ± 0.06 mm at one week after baseline to 0.16 ± 0.08 mm at three weeks after baseline;P = 0.001); epigen-antibody group:from -0.01 ± 0.06 mm to 0.06 ± 0.08 mm;P = 0.02; betacellulin antibody group:from -0.05 ± 0.05 mm to 0.02 ± 0.04 mm;P = 0.004). Correspondingly, interocular difference in axial length increased from -0.02 ± 0.04 mm to 0.13 ± 0.06 mm in the epiregulin-antibody group (P < 0.001), and from 0.01 ± 0.05 mm to 0.07 ± 0.05 mm in the epigen-antibody group (P = 0.045). In the betacellulin-antibody group the increase (0.01 ± 0.04 mm to 0.03 ± 0.03 mm) was not significant (P = 0.24). CONCLUSIONS: The EGF family members epiregulin, epigen and betacellulin may be associated with axial elongation in young guinea pigs, with the effect decreasing from epiregulin to epigen and to betacellulin.

The Role of EREG/EGFR Pathway in Tumor Progression.

Aberrant activation of the epidermal growth factor receptor (EGFR/ERBB1) by erythroblastic leukemia viral oncogene homolog (ERBB) ligands contributes to various tumor malignancies, including lung cancer and colorectal cancer (CRC). Epiregulin (EREG) is one of the EGFR ligands and is low expressed in most normal tissues. Elevated EREG in various cancers mainly activates EGFR signaling pathways and promotes cancer progression. Notably, a higher EREG expression level in CRC with wild-type Kirsten rat sarcoma viral oncogene homolog (KRAS) is related to better efficacy of therapeutic treatment. By contrast, the resistance of anti-EGFR therapy in CRC was driven by low EREG expression, aberrant genetic mutation and signal pathway alterations. Additionally, EREG overexpression in non-small cell lung cancer (NSCLC) is anticipated to be a therapeutic target for EGFR-tyrosine kinase inhibitor (EGFR-TKI). However, recent findings indicate that EREG derived from macrophages promotes NSCLC cell resistance to EGFR-TKI treatment. The emerging events of EREG-mediated tumor promotion signals are generated by autocrine and paracrine loops that arise from tumor epithelial cells, fibroblasts, and macrophages in the tumor microenvironment (TME). The TME is a crucial element for the development of various cancer types and drug resistance. The regulation of EREG/EGFR pathways depends on distinct oncogenic driver mutations and cell contexts that allows specific pharmacological targeting alone or combinational treatment for tailored therapy. Novel strategies targeting EREG/EGFR, tumor-associated macrophages, and alternative activation oncoproteins are under development or undergoing clinical trials. In this review, we summarize the clinical outcomes of EREG expression and the interaction of this ligand in the TME. The EREG/EGFR pathway may be a potential target and may be combined with other driver mutation targets to combat specific cancers.

Depletion of EREG enhances the osteo/dentinogenic differentiation ability of dental pulp stem cells via the p38 MAPK and Erk pathways in an inflammatory microenvironment.

BACKGROUND: Epiregulin (EREG) is an important component of EGF and was demonstrated to promote the osteo/dentinogenic differentiation of stem cells from dental apical papilla (SCAPs). Whether EREG can stimulate the osteo/dentinogenic differentiation of dental pulp stem cells (DPSCs) in inflammatory environment is not clear. The purpose of the present study is to investigate the role of EREG on the osteo/dentinogenic differentiation ability of DPSCs in inflammatory environment. METHODS: DPSCs were isolated from human third molars. Short hairpin RNAs (shRNAs) were used to knock down EREG expression in DPSCs. Recombinant human EREG (rhEREG) protein was used in the rescue experiment. TNF-α was employed to mimic the inflammatory environment in vitro. Alkaline phosphatase (ALP) staining, Alizarin red staining, quantitative calcium analysis, and real-time RT-PCR were performed to detect osteo/dentinogenic differentiation markers and related signalling pathways under normal and inflammatory conditions. RESULTS: EREG depletion promoted the ALP activity and mineralization ability of DPSCs. The expression of BSP, DMP-1, and DSPP was also enhanced. Moreover, 50 ng/mL rhEREG treatment decreased the osteo/dentinogenic differentiation potential of DPSCs, while treatment with 10 ng/mL TNF-α for 4 h increased the expression of EREG in DPSCs. Conversely, EREG knockdown rescued the impaired osteo/dentinogenic differentiation ability caused by TNF-α treatment. Further mechanistic studies showed that EREG depletion activated the p38 MAPK and Erk signalling pathways in DPSCs under normal and inflammatory conditions. CONCLUSIONS: Our results demonstrated that EREG could inhibit the osteo/dentinogenic differentiation potential of DPSCs via the p38 MAPK and Erk signalling pathways. Under inflammatory environment, EREG depletion enhanced osteo/dentinogenic differentiation potential of DPSCs by improving the expression of p-p38 MAPK and p-Erk.

Clinical and molecular characteristics and treatment outcomes of advanced right-colon, left-colon and rectal cancers: data from 1180 patients in a phase III trial of panitumumab with an extended biomarker panel.

BACKGROUND: Primary tumour location (PTL) is being adopted by clinicians to guide treatment decisions in metastatic colorectal cancer (mCRC). Here we test PTL as a predictive marker for panitumumab efficacy, and examine its relationship with an extended biomarker profile. We also examine rectal tumours as a separate location. PATIENTS AND METHODS: mCRC patients from the second-line PICCOLO trial of irinotecan versus irinotecan/panitumumab (IrPan). PTL was classified as right-PTL, left-PTL or rectal-PTL. PTL was assessed as a predictive biomarker for IrPan effect in RAS-wild-type (RAS-wt) patients (compared with irinotecan alone), then tested for independence alongside an extended biomarker profile (BRAF, epiregulin/amphiregulin (EREG/AREG) and HER3 mRNA expression). RESULTS: PTL data were available for 1180 patients (98.5%), of whom 558 were RAS-wt. High HER3 expression was independently predictive of panitumumab overall survival improvement, but PTL and EREG/AREG were not. IrPan progression-free survival (PFS) improvement compared with irinotecan was seen in left-PTL [hazard ratio (HR) = 0.61, P = 0.002) but not right-PTL (HR = 0.98, P = 0.90) (interaction P = 0.05; RAS/BRAF-wt interaction P = 0.10), or in rectal-PTL (HR = 0.82, P = 0.20) (interaction P = 0.14 compared with left-PTL; RAS/BRAF-wt interaction P = 0.04). Patients with right-PTL and high EREG/AREG or HER3 expression, had IrPan PFS improvement (high EREG/AREG HR = 0.20, P = 0.04; high HER3 HR = 0.33, P = 0.10) compared with irinotecan. Similar effect was seen for rectal-PTL patients (high EREG/AREG HR = 0.44, P = 0.03; high HER3 HR = 0.34, P = 0.05). CONCLUSIONS: RAS-wt patients with left-PTL are more likely to have panitumumab PFS advantage than those with right-PTL or rectal-PTL. However, an extended biomarker panel demonstrated significant heterogeneity in panitumumab PFS effect within a tumour location. AREG/EREG and HER3 mRNA expression identifies patients with right-PTL or rectal-PTL who achieve similar PFS effect with panitumumab as left-colon patients. Testing could provide a more reliable basis for clinical decision making. Further validation and development of these biomarkers is required to optimise routine patient care. CLINICAL TRIAL REGISTRATION: ISRCTN identifier: ISRCTN93248876.

High EREG Expression Is Predictive of Better Outcomes in Rectal Cancer Patients Receiving Neoadjuvant Concurrent Chemoradiotherapy.

BACKGROUND/AIM: A great proportion of patients with rectal cancer initially present with locally advanced disease and can potentially benefit from neoadjuvant concurrent chemoradiotherapy (CCRT) for downstaging before surgery. However, risk and clinical outcome stratification remain a great challenge. We aimed to find the potential biomarker to predict the effect of neoadjuvant CCRT on rectal cancer. METHODS: We identified epiregulin (EREG) as the most significant predictive marker for neoadjuvant CCRT response from the published rectal cancer transcriptome data set GSE35452. We collected 172 biopsy specimens from rectal cancer patients who received neoadjuvant CCRT followed by radical proctectomy, performed EREG immunohistochemistry, and analyzed the H-scores. We further examined the correlations between the expression level of EREG and clinicopathological features, tumor regression grade, and survival, including disease-specific survival (DSS), locoregional recurrence-free survival (LRFS), and metastasis-free survival (MeFS). RESULTS: High EREG expression was significantly related to early pretreatment (pre-Tx) and posttreatment (post-Tx) tumor status (T1, T2, p = 0.047 and p < 0.001), pre-Tx and post-Tx negative nodal status (N0, p < 0.001 and p = 0.004), less vascular and perineurial invasion (p = 0.015 and p = 0.023), and higher tumor regression grade (p < 0.001). In the survival analysis, high EREG expression was significantly associated with better DSS (p < 0.0001), LRFS (p = 0.0004), and MeFS (p < 0.0001). In the multivariate analysis, high EREG expression remained prognostically significant for better DSS (p = 0.003; hazard ratio: 5.599). CONCLUSION: These data suggest that EREG is a potential predictive marker and therapeutic target in rectal cancer patients receiving neoadjuvant CCRT.

Epiregulin promotes osteogenic differentiation and inhibits neurogenic trans-differentiation of adipose-derived mesenchymal stem cells via MAPKs pathway.

Mesenchymal stem cells (MSCs) exists low efficiency to trans-differentiate into other germinal layer cell types. One key issue is to discover the effect of important factor on MSCs differentiation abiltiy. In this study, we investigated the role and mechanism of epiregulin (EREG) on the osteogenic differentiation and neurogenic trans-differentiation in adipose-derived stem cells (ADSCs). We discovered that the depletion of EREG inhibited the osteogenic differentiation in vitro. And 25 ng/mL recombinant human epiregulin protein (rhEREG) effectively improved the osteogenic differentiation of EREG-depleted-ADSCs. Depletion of EREG promoted the formation of neural spheres, and increased the expressions of nestin, ßIII-tubulin, NeuroD, NCAM, TH, and NEF in ADSCs. Then, 25 ng/mL rhEREG significantly inhibited these neurogenic differentiation indicators. Inhibition of p38 MAPK, JNK, or Erk1/2 signaling pathway separately, blocked the rhEREG-enhanced osteogenic differentiation ability and the rhEREG-inhibited neurogenic trans-differentiation ability of ADSCs. In conclusions, EREG promoted the osteogenic differentiation and inhibited the neurogenic trans-differentiation potentials of ADSCs via MAPK signaling pathways.

Human papillomavirus-positivity is associated with EREG down-regulation and promoter hypermethylation in head and neck squamous cell carcinoma.

BACKGROUND: Human papillomavirus (HPV) etiology has become evident in head and neck cancers (HNCs) and HPV positivity showed a strong association with its malignant progression. Since aberrant DNA methylation is known to drive carcinogenesis and progression in HNCs, we investigated to determine target gene(s) associated with this modification. METHODS: We characterized epigenetic changes in tumor-related genes (TRGs) that are known to be associated with HNC development and its progression. RESULTS: The expression levels of 42 candidate HNC-associated genes were analyzed. Of these, 7 TGRs (CHFR, RARß, GRB7, EREG, RUNX2, RUNX3, and SMG-1) showed decreased expressions in HPV-positive (+) HNC cells compared with HPV-negative (-) HNC cells. When gene expression levels were compared corresponding to the DNA methylation conditions, GRB7 and EREG showed significant differential expression between HPV+ and HPV- cells, which suggested these genes as primary targets of epigenetic regulation in HPV-induced carcinogenesis. Furthermore, treatment with a demethylation agent, 5-aza-2'-deoxycytidine (5-aza-dc), caused restoration of EREG expression and was associated with hypomethylation of its promoter in HPV+ cells, while no changes was noted in HPV- cells. EREG promoter hypermethylation in HPV+ cells was confirmed using methylation-specific PCR (MS-PCR). CONCLUSION: We conclude that EREG is the target of epigenetic regulation in HPV+ HNCs and its suppressed expression through promoter hypermethylation is associated with the development of HPV-associated HNCs.

RhoG/Rac1 signaling pathway involved in migration and invasion of salivary adenoid cystic carcinoma cells.

OBJECTIVES: This study aimed to explore whether RhoG/Rac1 was involved in migration and invasion of salivary adenoid cystic carcinoma (SACC). MATERIALS AND METHODS: RhoG and Rac1 were evaluated in two SACC cell lines, namely SACC-83 and SACC-LM, with low and high rates of lung metastasis, respectively. Functional changes were evaluated using cell proliferation, transwell, and wound-healing assays, and molecular events were investigated using real-time PCR and Western blot assays. RESULTS: RhoG and Rac1 were highly expressed and more activated in SACC-LM cells than in SACC-83 cells. RhoG overexpression promoted SACC-83 cell migration and invasion through activating Rac1. The knockdown of RhoG or Rac1 partially blocked epiregulin-induced migration and invasion in SACC-83 cells. Epiregulin-induced activation of RhoG/Rac1 in SACC-83 cells was blocked by a Src inhibitor, or an AKT inhibitor or AKT siRNA, or an ERK1/2 inhibitor. Moreover, the epiregulin-induced phosphorylation of AKT and ERK1/2 in SACC-83 cells was blocked by a Src inhibitor, and the epiregulin-induced phosphorylation of ERK1/2 was blocked by an AKT inhibitor or AKT siRNA. Overexpression of activated AKT induced activation of ERK1/2 and RhoG. CONCLUSIONS: RhoG/Rac1 signaling pathway was involved in SACC cell migration and invasion. RhoG/Rac1 at least partially mediated epiregulin/Src/AKT/ERK1/2 signaling to promote SACC cell migration and invasion.

EPA and DHA Fatty Acids Induce a Remodeling of Tumor Vasculature and Potentiate Docetaxel Activity.

n-3 long chain Polyunsaturated Fatty Acids (n-3 LCPUFA) have been shown to improve the efficacy of conventional chemotherapies used for breast cancer treatment. In addition to their reported ability to increase the chemosensitivity of cancer cells, we hypothesized that n-3 LCPUFA could induce a remodeling of the vascular network in mammary tumors. A contrast-enhanced ultrasound method was used to monitor the vascular architecture during docetaxel treatment of mammary tumors in rats fed either a control or an n-3 LCPUFA-enriched diet (docosahexaenoic acid (DHA)/eicosapentaenoic acid (EPA)). The vascular network was remodeled in favor of smaller vessels (microvascularization), which represented 54% of the vasculature in n-3 LCPUFA tumors but only 26% in control tumors after 2 weeks of chemotherapy. Importantly, vascularization changes occurred both before and during docetaxel treatment. The density of smaller vessels quantified before chemotherapy was correlated with improved tumor size reduction by docetaxel treatment. Furthermore, transcript levels of the angiogenesis-specific genes epiregulin and amphiregulin were reduced by ~4.5- and twofold in tumors obtained from rats fed an n-3 LCPUFA-enriched diet compared to those of rats fed a control diet, respectively. Their expression levels were negatively correlated with tumor regression after chemotherapy. Taken together, this preclinical data strengthen the potential usefulness of n-3 LCPUFA as a complementary clinical strategy to improve drug efficiency via remodeling of the tumor vasculature.

Polymorphism in the EREG gene confers susceptibility to tuberculosis.

BACKGROUND: Host genetic factors affect the immune response to Mycobacterium tuberculosis (Mtb) infection as well as the progression of the disease. Epiregulin (EREG) belongs to the epidermal growth factor (EGF) family, which binds to the epidermal growth factor receptor (EGFR) to regulate the immune response of the host during infections. Our study aimed to compare EREG levels in tuberculosis (TB) patients and healthy controls and assess whether polymorphisms in EREG increase the risk of TB. METHODS: We used ELISA to determine the plasma EREG level from 30 healthy controls and 50 tuberculosis patients. By evaluating the EREG gene from 624 TB patients and 600 healthy controls, we determined the allelic and genotypic frequencies for association with susceptibility to TB infections in this group. RESULTS: This paper shows that the pulmonary tuberculosis (PTB) and extrapulmonary tuberculosis (EPTB) groups showed a significantly higher plasma EREG level (1014 ± 733.9 pg/ml, 700.2 ± 676.6 pg/ml, respectively) than the healthy controls (277 ± 105.4 pg/ml). The rs2367707 polymorphism was associated with a higher risk of PTB and EPTB (P = 0.00051, P = 0.0012). Analyses of haplotype frequencies found that people with the haplotype CACAT had a higher risk of PTB and EPTB (P = 0.00031, OR = 1.43; P = 0.000053, OR = 1.65). Moreover, the rs6446993 polymorphism of the EREG gene was found to be associated with EPTB (P = 0.00087, OR = 1.54; 95% CI = 1.23-1.94). CONCLUSIONS: Compared to that of healthy controls, the level of EREG in the plasma of TB patients increased significantly. Based on these data, we demonstrated that EREG polymorphisms are genetic factors for susceptibility to TB and various forms of TB.

Proliferative Pathways of Vascular Smooth Muscle Cells in Response to Intermittent Hypoxia.

Obstructive sleep apnea (OSA) is characterized by intermittent hypoxia (IH) and is a risk factor for cardiovascular diseases (e.g., atherosclerosis) and chronic inflammatory diseases (CID). The excessive proliferation of vascular smooth muscle cells (VSMCs) plays a pivotal role in the progression of atherosclerosis. Hypoxia-inducible factor-1 and nuclear factor-κB are thought to be the main factors involved in responses to IH and in regulating adaptations or inflammation pathways, however, further evidence is needed to demonstrate the underlying mechanisms of this process in VSMCs. Furthermore, few studies of IH have examined smooth muscle cell responses. Our previous studies demonstrated that increased interleukin (IL)-6, epidermal growth factor family ligands, and erbB2 receptor, some of which amplify inflammation and, consequently, induce CID, were induced by IH and were involved in the proliferation of VSMCs. Since IH increased IL-6 and epiregulin expression in VSMCs, the same phenomenon may also occur in other smooth muscle cells, and, consequently, may be related to the incidence or progression of several diseases. In the present review, we describe how IH can induce the excessive proliferation of VSMCs and we develop the suggestion that other CID may be related to the effects of IH on other smooth muscle cells.

Epiregulin (EREG) is upregulated through an IL-1ß autocrine loop in Caco-2 epithelial cells with reduced CFTR function.

CFTR is a cAMP-regulated chloride channel, whose mutations produce cystic fibrosis. The impairment of CFTR activity increases the intracellular Cl- concentration, which in turn produces an increased interleukin-1ß (IL-1ß) secretion. The secreted IL-1ß then induces an autocrine positive feedback loop, further stimulating IL-1ß priming and secretion. Since IL-1ß can transactivate the epidermal growth factor receptor (EGFR), we study here the levels of expression for different EGFR ligands in Caco-2/pRS26 cells (expressing shRNA against CFTR resulting in a reduced CFTR expression and activity). The epiregulin (EREG), amphiregulin (AREG), and heparin binding EGF like growth factor (HBEGF) mRNAs, were found overexpressed in Caco-2/pRS26 cells. The EREG mRNA had the highest differential expression and was further characterized. In agreement with its mRNA levels, Western blots (WB) showed increased EREG levels in CFTR-impaired cells. In addition, EREG mRNA and protein levels were stimulated by incubation with exogenous IL-1ß and inhibited by the Interleukin 1 receptor type I (IL1R1) antagonist IL1RN, suggesting that the overexpression of EREG is a consequence of the autocrine IL-1ß loop previously described for these cells. In addition, the JNK inhibitor SP600125, and the EGFR inhibitors AG1478 and PD168393, also had an inhibitory effect on EREG expression, suggesting that EGFR, activated in Caco-2/pRS26 cells, is involved in the observed EREG upregulation. In conclusion, in Caco-2 CFTR-shRNA cells, the EGFR ligand EREG is overexpressed due to an active IL-1ß autocrine loop that indirectly activates EGFR, constituting new signaling effectors for the CFTR signaling pathway, downstream of CFTR, Cl- , and IL-1ß.