Retinoic Acid Receptor Responder Research Articles

Abstract 2846: Chemerin, as a serum biomarker, promotes tumorigenesis and metastasis in oral squamous cell carcinoma

Abstract Chemerin encoded by retinoic acid receptor responder 2 (RARRES2) has been found to be related with malignant tumors, but its causal role in the development of oral squamous cell carcinoma (OSCC) is largely unexplored. In the present study, clinical data showed that higher serum levels of chemerin were observed in OSCC patients compared to the healthy and significantly decreased after tumor resection, and were positively associated with advanced tumor stage and lymph node metastasis. The expression level of chemerin was positive correlated with the migration and invasion of OSCC cell lines. Recombinant chemerin dose-dependently enhanced the migration, invasion and proliferation of OSCC cells in vitro, and shRNA targeting RARRES2 inhibited chemerin expression, OSCC cells metastasis and proliferation in vitro and vivo. Additionally, Chemerin activated the Manganese superoxide dismutase (SOD2) activity and increased intracellular H2O2, led to a significant decrease of E-cadherin expression and a dramatically increasing expression of p-ERK1/2, Slug, Vimentin and N-cadherin. This study suggested that chemerin was a novel biomarker for OSCC patients. Chemerin promoted the tumorigenesis and metastasis of OSCC and would be a new therapeutic target for OSCC. Keywords: Chemerin; Oral squamous cell carcinoma; Tumorgenesis; Metastasis; SOD2 Citation Format: Anxun Wang. Chemerin, as a serum biomarker, promotes tumorigenesis and metastasis in oral squamous cell carcinoma [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 2846.

The serum biomarker chemerin promotes tumorigenesis and metastasis in oral squamous cell carcinoma.

Chemerin, which is encoded by retinoic acid receptor responder 2 (RARRES2), has been found to be related to malignant tumours, but its role in the development of oral squamous cell carcinoma (OSCC) is largely unexplored. In the present study, a higher serum level of chemerin was evident in patients with OSCC than in healthy individuals, and this high level of chemerin significantly decreased after tumour resection. In addition, high chemerin levels were positively associated with advanced tumour stage and lymph node metastasis. The expression levels of chemerin and Chemerin Receptor 23 (ChemR23) were positively correlated with the migration and invasion of OSCC cell lines. Recombinant chemerin (R-chemerin) enhanced the in vitro migration, invasion and proliferation of OSCC cells in a concentration-dependent manner, and short hairpin RNAs (shRNAs) targeting RARRES2 decreased chemerin expression and inhibited OSCC cell metastasis and proliferation both in vitro and in vivo Additionally, R-chemerin activated manganese superoxide dismutase (SOD2) and increased the amount of intracellular hydrogen peroxide (H2O2), leading to a significant decrease in E-cadherin expression and dramatic increase in the expression of phosphorylated ERK1/2 (p-ERK1/2), Slug, Vimentin and N-cadherin, but shRNAs targeting RARRES2 reversed these effects. Moreover, knockdown of ChemR23 with small interfering RNAs (siRNA) significantly inhibited chemerin-induced OSCC cell migration/invasion and SOD2 activity. Our results revealed that chemerin is a novel biomarker for OSCC. Chemerin/ChemR23 promotes tumorigenesis and metastasis in OSCC and may be a new therapeutic target for OSCC.

Possible involvement of the RARRES2/CMKLR1-system in metabolic and reproductive parameters in Holstein dairy cows

BackgroundIn dairy cows, the energy cost of milk yield results in a negative energy balance (EB) and body fat mobilization that impairs reproductive efficiency. Emerging evidence suggests that the novel adipokines, Retinoic acid receptor responder protein 2 (RARRES2), and its main receptor, Chemokine-like receptor 1 (CMKLR1) are involved in the regulation of metabolic and ovarian functions. So, we investigated in a first experiment the plasma RARRES2, and RARRES2 and CMKLR1 mRNA expression levels in subcutaneous adipose tissue (SAT) and granulosa cells (GC) at different times of body fat mobilization in dairy cows (4, 8, 20 and 44 weeks postpartum, wk. pp. for SAT and 8, 20 and 44 wk. pp. for GC). Then, in a second experiment we examined the effect of high (HE) and low energy (LE) diets on the RARRES2 system and its links with metabolic and reproductive parameters.MethodsThe first experiment included 9 animals fed with HE diet from 4 to 44 wk. pp. and the second one included animals fed either a HE diet (n = 8) or a LE diet (n = 8) from − 4 to 16 wk. peripartum. In both experiments, various metabolic and reproductive parameters were determined and associated with plasma RARRES2 as measured by bovine ELISA. RARRES2 and CMKLR1 mRNA expression levels were analyzed by RT-qPCR in SAT after biopsy and GC after aspiration of follicles.ResultsPlasma RARRES2 levels were higher at 4 wk. pp. as compared to 20 and 44 wk. pp. and they were positively correlated with body fat mobilization and milk yield. RARRES2 and CMKLR1 mRNA expression levels increased from 4 to 8 wk. pp. (fat mobilization, EB < 0) and remained unchanged at 20 and 44 wk. pp. (fat reconstitution, EB > 0) as compared to 4 wk. pp. in SAT. RARRES2 and CMKLR1 mRNA levels decreased from 8 to 44 wk. pp. in GC from small follicles. In the second experiment, plasma RARRES2 increased from − 4 to 8 wk. peripartum similarly in both LE and HE cows. In addition, the area under of plasma RARRES2 curve was highly negatively associated with the number of small follicles obtained in HE animals during the cycle before the first artificial insemination. In SAT of HE cows, RARRES2 mRNA expression decreased at 1 wk. pp. compared to − 4 and 16 wk. peripartum whereas opposite expression patterns were obtained for CMKLR1. Similar results were observed for CMKLR1 mRNA expression in LE cows while there was no variation in RARRES2 mRNA expression. Moreover, RARRES2 mRNA was higher expressed in LE than in HE cows at 1 wk. pp.ConclusionsThe lactation-induced fat and energy mobilization influenced plasma RARRES2 profile and mRNA expression pattern of RARRES2 and CMKLR1 similarly in both SAT and GC. In addition, the energy content of the diet did not affect plasma RARRES2 but it altered RARRES2 mRNA expression in SAT and the area under the curve of plasma RARRES2 that was negatively associated to the number of small follicles in HE animals. Thus, RARRES2 could be a metabolic or ovarian signal involved in the interactions between metabolic and reproductive functions in dairy cows.

Tumor suppressor RARRES1- A novel regulator of fatty acid metabolism in epithelial cells.

Retinoic acid receptor responder 1 (RARRES1) is silenced in many cancers and is differentially expressed in metabolism associated diseases, such as hepatic steatosis, hyperinsulinemia and obesity. Here we report a novel function of RARRES1 in metabolic reprogramming of epithelial cells. Using non-targeted LC-MS, we discovered that RARRES1 depletion in epithelial cells caused a global increase in lipid synthesis. RARRES1-depleted cells rewire glucose metabolism by switching from aerobic glycolysis to glucose-dependent de novo lipogenesis (DNL). Treatment with fatty acid synthase (FASN) inhibitor, C75, reversed the effects of RARRES1 depletion. The increased DNL in RARRES1-depleted normal breast and prostate epithelial cells proved advantageous to the cells during starvation, as the increase in fatty acid availability lead to more oxidized fatty acids (FAO), which were used for mitochondrial respiration. Expression of RARRES1 in several common solid tumors is also contextually correlated with expression of fatty acid metabolism genes and fatty acid-regulated transcription factors. Pathway enrichment analysis led us to determine that RARRES1 is regulated by peroxisome proliferating activated receptor (PPAR) signaling. These findings open up a new avenue for metabolic reprogramming and identify RARRES1 as a potential target for cancers and other diseases with impaired fatty acid metabolism.

Mechanisms and Functions of Chemerin in Cancer: Potential Roles in Therapeutic Intervention.

Chemerin [RARRES2 [retinoic acid receptor responder 2], TIG2 [tazarotene induced gene 2 (TIG2)]] is a multifunctional cytokine initially described in skin cultures upon exposure to the synthetic retinoid tazarotene. Its secreted pro-form, prochemerin, is widely expressed, found systemically, and is readily converted into active chemerin by various proteases. Subsequent studies elucidated major roles of chemerin as both a leukocyte chemoattractant as well as an adipokine. Chemerin's main chemotactic receptor, the G-protein coupled receptor CMKLR1, is expressed on macrophages, dendritic, and NK cells. With respect to its role in immunology, chemerin mediates trafficking of these cells to sites of inflammation along its concentration gradient, and likely helps coordinate early responses, as it has been shown to have antimicrobial and angiogenic properties, as well. Recently, there has been mounting evidence that chemerin is an important factor in various cancers. As with its role in immune responses—where it can act as both a pro- and anti-inflammatory mediator—the potential functions or correlations chemerin has in or with cancer appears to be context dependent. Most studies, however, suggest a downregulation or loss of chemerin/RARRES2 in malignancies compared to the normal tissue counterparts. Here, we perform a comprehensive review of the literature to date and summarize relevant findings in order to better define the roles of chemerin in the setting of the tumor microenvironment and tumor immune responses, with an ultimate focus on the potential for therapeutic intervention.

Regulation of Chemerin and CMKLR1 Expression by Nutritional Status, Postnatal Development, and Gender.

Chemerin (also known as tazarotene-induced gene 2 and retinoic acid receptor responder 2) has been identified as an adipokine that exerts effects on many biological processes, including adipogenesis, angiogenesis, inflammation, immune responses, and food intake. This variety of effects has led to its implication in obesity and co-morbidities including diabetes and a risk of cardiovascular disease. The biological effects are mostly mediated by a so-called G protein-coupled receptor, chemokine-like receptor 1 (CMKLR1). Given the association of chemerin with obesity and related diseases, we decided to study in detail the regulation of chemerin and CMKLR1 expression in white adipose tissue (WAT). Specifically, we focused on their expression levels in physiological and pathophysiological settings involved in energy balance: e.g., fasting, postnatal development, and gender. We used Sprague Dawley rats with different nutritional statuses, levels of hormonal deficiency, and states of development as well as ob/ob (leptin-deficient) mice. We analysed the protein expression of both the ligand and receptor (chemerin and CMKLR1) in gonadal WAT by western blotting. We found that chemerin and CMKLR1 protein levels were regulated in WAT by different conditions associated with metabolic changes such as nutritional status, sex steroids, pregnancy, and food composition. Our data indicate that regulation of the expression of this new adipokine and its receptor by nutritional status and gonadal hormones may be a part of the adaptive mechanisms related to altered fat mass and its metabolic complications.

Seminal GSTM3, CRISPLD2, and RARRES1 diagnose sperm functional alterations

Sperm functional alterations are considered the main cellular mechanisms of male infertility, observed in 25% of infertile men. We have previously shown, using a shotgun proteomics approach, that Glutathione S-transferase Mu3 (GSTM3), Cysteine-rich secretory protein LCCL domain-containing 2 (CRISPLD2) and Retinoic acid receptor responder protein 1 (RARRES1) seminal proteins are associated with sperm abnormalities. Current limitations in the quantitative aspect of a shotgun proteomics approach demand these results be validated in a larger cohort. In this study, thus, we sought to evaluate, using a confirmatory method, GSTM3, CRISPLD2 and RARRES1 levels in seminal plasma in association with sperm mitochondrial activity and DNA fragmentation. Prospective study. 197 normozoospermic men were recruited for this study. Semen was collected by masturbation following 2 to 5 days of ejaculatory abstinence. An aliquot was used for semen analysis, another for sperm functional evaluation, and the remaining semen volume was centrifuged for separation of seminal plasma. Sperm functional data were used to separate the experimental groups: High (control, n=27) and Low (study, n=27) sperm mitochondrial activity, and Low (control, n=29) and High (study, n=29) sperm DNA fragmentation. Seminal plasma of all samples was utilized to evaluate the levels of GSTM3, CRISPLD2 and RARRES1 by Western blotting. Data were normalized using total protein (Ponceau S staining) and multiplied by the ejaculate volume, to obtain the total protein levels in the ejaculate. Groups were compared using an unpaired Student’s t test (α=5%). GSTM3 was observed as two different bands (27 kDa, its expected molecular mass, and 38 kDa). Total levels of both bands were twice as high in samples with sperm mitochondrial alterations. CRISPLD2 was observed as three different bands (17, 28 and 38 kDa, expected molecular mass = 51 kDa), suggesting its digestion by seminal plasma proteases during semen liquefaction. Total seminal levels of the 17 and 28 kDa bands were decreased in samples with high sperm DNA fragmentation (1.6-fold and 2.6-fold decrease, respectively). Likewise, RARRES1 was identified as two bands (50 and 62 kDa, expected molecular mass = 33 kDa), suggesting post-translational modifications and/or dimer formation. The 50 kDa band was 1.9-fold decreased in the seminal plasma of men with sperm DNA fragmentation. Seminal plasma proteins directly reflect sperm functional alterations and, thus, male infertility. Seminal plasma proteins may be used as markers of functional alterations. In this study, we confirmed that this is the case for GSTM3 (sperm mitochondrial activity), and CRISPLD2 and RARRES1 (sperm DNA fragmentation).

2261 Tumor suppressor RARRES1 regulates cell survival by modulating mitochondrial energetics

OBJECTIVES/SPECIFIC AIMS: One of the driving mechanisms of cancer progression is the reprogramming of metabolic pathways in intermediary metabolism. Cancers increase their energy expenditure by increasing ATP production for utilization in anabolic pathways to increase production of proteins, nucleic acids and lipids. The Warburg effect, where cancer cells predominantly use aerobic glycolysis rather than oxidative phosphorylation to produce ATP, was long thought to be the main initiating pathway in increasing tumor burden. However, compelling new evidence shows that there exists metabolic heterogeneity among and within tumors. Mitochondrial respiration often plays a major role in tumor progression, as many different cancers contain a subpopulation of slow-cycling tumor-initiating cells that are multidrug-resistant and dependent on oxidative phosphorylation. These cells represent a target for cancer therapy. In this study, we identification a novel endogenous regulator of mitochondrial respiration, retinoic acid receptor responder 1 (RARRES1). METHODS/STUDY POPULATION: We assessed the metabolic phenotype of RARRES1-depleted normal epithelial cells through metabolomics, a flux analyzer and blotting for phosphorylation of AMP kinase, a major regulator of energy homeostasis. We further examined mitochondrial energetics by staining the mitochondria with TMRM and Mito-Tracker. We then analyzed the apoptotic phenotype of epithelial cells with depletion of RARRES1 with fluorescence-activated cell sorting analysis of annexin V-staining. RESULTS/ANTICIPATED RESULTS: Remarkably, fluorescence-activated cell sorting analysis of annexin V-stained epithelial cells with depletion of RARRES1 were resistant to all studied modes of cell death, implying an effect on a fundamental cell process. By using proteomics, metabolomics, cellular and molecular analyses, our data show that RARRES1 regulates mitochondrial membrane potential and subsequently alters 1-carbon metabolism by modulating the function of the mitochondrial voltage-dependent anion channel. We believe this is the first example of a tumor suppressor protein that functions to directly regulate mitochondrial energetics. Using an extracellular flux analyzer, our data also show that depletion of RARRES1 causes an increase in mitochondrial respiration and ATP production, thus enhancing biosynthetic pathways that drive the pathogenicity and survival of cancer. The metabolic and anti-apoptotic phenotype of RARRES1-depleted cells was reversed by treatment of metformin, a mitochondrial inhibitor. DISCUSSION/SIGNIFICANCE OF IMPACT: These data lay the foundation for metabo-therapy of the many tumor types that exhibit RARRES1 depletion and may have the added benefit of targeting drug-resistant tumor-initiating cells.

Pleiotropic Associations of RARRES2 Gene Variants and Circulating Chemerin Levels: Potential Roles of Chemerin Involved in the Metabolic and Inflammation-Related Diseases.

Chemerin, an adipokine and inflammatory mediator, is associated with metabolic, inflammation- and immune-mediated diseases. The genetic, clinical, and biomarker correlates of circulating chemerin levels have not been completely elucidated. We analyzed the determinants and correlates of retinoic acid receptor responder 2 (RARRES2; encoding chemerin) gene variants and chemerin levels in the Taiwanese population. In total, 612 individuals were recruited. Clinical and metabolic phenotypes, 13 inflammatory markers, 5 adipokines, and 6 single-nucleotide polymorphisms (SNPs) covering the RARRES2 region were analyzed. High chemerin levels and chemerin level tertiles were positively associated with multiple metabolic phenotypes and circulating inflammatory marker and adipokine levels and negatively associated with high-density lipoprotein cholesterol and adiponectin levels and estimated glomerular filtration rates (eGFRs). Genotype and haplotype analyses showed that RARRES2 SNPs were significantly associated with chemerin, fibrinogen, interleukin 6, and lipocalin 2 levels. Stepwise logistic regression analysis showed that C-reactive protein level, leptin level, triglyceride level, eGFR, rs3735167 genotypes, sex, and soluble P-selectin level were independently associated with chemerin levels. In conclusion, pleiotropic associations were noted between RARRES2 variants, circulating chemerin levels and multiple metabolic phenotypes and inflammatory marker levels. This study provides further evidence for the potential roles of chemerin in metabolic and inflammation-related diseases.

Hypermethylation and loss of retinoic acid receptor responder 1 expression in human choriocarcinoma

BackgroundHuman placental development resembles tumorigenesis, due to the invasive and fusogenic potential of trophoblasts. However, these features are tightly controlled in trophoblasts. Disturbance of this spatial and temporal regulation is thought to contribute to the rare formation of choriocarcinomas. Promoter hypermethylation and loss of the tumor suppressor Retinoic acid receptor responder 1 (RARRES1) were shown to contribute to cancer progression. Our study investigated the epigenetic and transcriptional regulation of RARRES1 in healthy human placenta in comparison to choriocarcinoma cell lines and cases.MethodsThree choriocarcinoma cell lines (Jeg-3, JAR and BeWo) were treated with three different retinoic acid derivates (Am580, Tazarotene and all-trans retinoic acid) and 5-aza-2′-deoxycytidine. We analyzed RARRES1 promoter methylation by pyrosequencing and performed realtime-PCR quantification to determine RARRES1 expression in placental tissue and trophoblastic cell lines. Additionally, RARRES1 was stained in healthy placentas and in biopsies of choriocarcinoma cases (n = 10) as well as the first trimester trophoblast cell line Swan71 by immunofluorescence and immunohistochemistry.ResultsIn the choriocarcinoma cell lines, RARRES1 expression could not be induced by sole retinoic acid treatment. Stimulation with 5-aza-2′-deoxycytidine significantly induced RARRES1 expression, which then could be further increased with Am580, Tazarotene and all-trans retinoic acid. In comparison to healthy placenta, choriocarcinoma cell lines showed a hypermethylation of the RARRES1 promoter, which correlated with a reduced RARRES1 expression. In concordance, RARRES1 protein expression was lost in choriocarcinoma tissue. Additionally, in the trophoblastic cell line Swan71, we found a significant induction of RARRES1 expression with increased cell density, during mitosis and in syncytial knots.ConclusionsOur findings showed that RARRES1 expression is absent in choriocarcinoma due to promoter methylation. Based on our analysis, we hypothesize that RARRES1 might exert tumor suppressive functions in multiple cellular processes (e.g. cell cycle regulation, adhesion, invasion and apoptosis).

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OBJECTIVES/SPECIFIC AIMS: The goal of this study is to examine bioenergetic phenotype of retinoic acid receptor responder 1 (RARRES1)-depleted epithelial cells and to facilitate the discovery of personalized metabo-therapeutics in the context of cancers characterized with loss of or low expression of RARRES1. METHODS/STUDY POPULATION: Anoikis assay and annexinV labeling were used to assess drug resistance and apoptotic phenotype in RARRES1-depleted epithelial cells. Metabolomics, AMP kinase activity, mito-tracker, and extracellular flux assays were used to examine the bioenergetic profile of RARRES1-depleted epithelial cells. Extracellular flux assays were used to assess the phenotype of RARRES1-depleted epithelial cells treated with or without metformin. RESULTS/ANTICIPATED RESULTS: RARRES1 is a major regulator of mitochondrial function. Its depletion in tumors induces an oxidative phosphorylation dependent phenotype and subsequently increases ATP abundance in the cell, enhances anabolic pathways and increases survival. Treatment with FDA approved mitochondrial respiration inhibitor, metformin, reversed the metabolic phenotype of RARRES1 depleted-epithelial cells. Metformin could be the ideal therapeutics to reduce tumor burden in cancers with loss of or low expression of RARRES1. DISCUSSION/SIGNIFICANCE OF IMPACT: Bioenergetic dynamics are emerging as a basis for understanding the pathology of cancer. The malignancy progresses as its metabolic pattern and mitochondrial respiration become more dysfunctional. The regulatory pathways of bioenergetic dynamics are currently poorly understood, and the characterization of proteins implicated in those processes must be assessed. One understudied protein and tumor suppressor is RARRES1. RARRES1 is induced by retinoic acid (a major metabolic regulator) and functions as a putative carboxypeptidase inhibitor. Understanding the connection between this carboxypeptidase inhibitor and intermediary metabolism will enlighten our understanding of the bioenergetic profile of cells and facilitate the discovery of personalized metabo-therapeutics in the context of cancer.

Dietary vitamin B6 modulates the gene expression of myokines, Nrf2-related factors, myogenin and HSP60 in the skeletal muscle of rats.

Previous studies have suggested that vitamin B6 is an ergogenic factor. However, the role of dietary vitamin B6 in skeletal muscle has not been widely researched. The aim of the present study was to investigate the effects of dietary vitamin B6 on the gene expression of 19 myokines, 14 nuclear factor erythroid 2-related factor 2 (Nrf2)-regulated factors, 8 myogenesis-related factors and 4 heat shock proteins (HSPs), which may serve important roles in skeletal muscles. Rats were fed a diet containing 1 (marginal vitamin B6 deficiency), 7 (recommended dietary level) or 35 mg/kg of pyridoxine (PN) HCl/ for 6 weeks. Gene expressions were subsequently analysed using reverse transcription-quantitative polymerase chain reaction. Food intake and growth were unaffected by this dietary treatment. The rats in the 7 and 35 mg/kg PN HCl groups exhibited a significant increase in the concentration of pyridoxal 5'-phosphate in the gastrocnemius muscle compared with the 1 mg/kg PN HCl diet (P<0.01). The expressions of myokines, such as IL-7, IL-8, secreted protein acidic and rich in cysteine, IL-6, growth differentiation factor 11, myonectin, leukaemia inhibitory factor, apelin and retinoic acid receptor responder (tazarotene induced) 1, the expression of Nrf2 and its regulated factors, such as heme oxygenase 1, superoxide dismutase 2, glutathione peroxidase 1 and glutathione S-transferase, and the expression of myogenin and HSP60 were significantly elevated in the 7 mg/kg PN HCl group compared with the 1 mg/kg PN HCl diet (P<0.05). No significant differences in levels of these genes were observed between the 35 and 1 mg/kg PN HCl, with the exception of GDF11 and myonectin, whose expressions were significantly increased in the 35 mg/kg PN HCl (P<0.05). Notably, the majority of gene expressions that were affected responded to dietary supplemental vitamin B6 in a similar manner. The results suggest that compared with the marginal vitamin B6 deficiency, the recommended dietary intake of vitamin B6 upregulates the gene expression of a number of factors that promote the growth and repair of skeletal muscle.

Multiple roles of RARRES1 in prostate cancer: Autophagy induction and angiogenesis inhibition.

BackgroundProstate cancer (PCa) poses a major health concern in men worldwide. Retinoic Acid Receptor Responder (RARRES1)/ Tazarotene-induced gene-1 (TIG-1) is a putative tumor suppressor gene that exerts its tumor suppressor function via unknown mechanisms. Epigenetic silencing of RARRES1 leads to its loss in several types of cancer, including PCa. Determining the molecular mechanisms that mediate the tumor suppressor role of RARRES1 in PCa is the focus of our study.FindingsOur data indicates that RARRES1 over expression in PCa cell lines represses mitogen-activated protein kinase (MAPK) activation. RARRES1 expression induces the levels of autophagy-related genes, beclin, ATG3 and increases LC3B-II conversion. A significant induction of SIRT1 along with mTOR inhibition is noted on RARRES1 expression. Furthermore, RARRES1 over expression elevates the levels of the antioxidant enzyme, catalase. Our results also indicate that RARRES1 expression inhibits angiogenesis in endothelial cells.ConclusionsIn summary, the data presented here indicate that forced expression of RARRES1 in PCa cells (a) induces ER stress and autophagic response; (b) increases SIRT1 levels; and (c) higher levels of anti-oxidant enzymes. Our study also implicates the role of RARRES1 as a novel anti-angiogenic molecule. Overall this study reports the molecular players that RARRES1 modulates to serve as a tumor suppressor molecule. Future studies will help determine the in vivo mechanisms by which RARRES1 may serve as a target for therapeutic intervention both in cancer and in angiogenesis-related disorders.

RARRES2 functions as a tumor suppressor by promoting β-catenin phosphorylation/degradation and inhibiting p38 phosphorylation in adrenocortical carcinoma.

Tumor suppressor genes and the immune system are critical players in inhibiting cancer initiation and/or progression. However, little is known about whether a tumor suppressor gene can function through both immune-dependent and -independent mechanisms. Retinoic acid receptor responder 2 (RARRES2) is transcriptionally downregulated in multiple cancer types. Previous studies suggested that it can serve as an immune-dependent tumor suppressor by acting as a chemoattractant to recruit anti-cancer immune cells expressing its receptor, the chemerin chemokine receptor 1 (CMKLR1), to sites of tumor. In this study, we investigated the role of RARRES2 in adrenocortical carcinoma (ACC), a rare lethal malignancy in which aberrant Wnt/β-catenin signaling is frequently detected. We show that RARRES2 expression is downregulated in ACC as compared to normal and benign adrenocortical tissues, which is a result of CpG hypermethylation. Despite minimal CMKLR1 expression and lack of phenotypic tumor-suppressive effect with exogenous RARRES2 treatment, RARRES2 overexpression in ACC cell lines not only reduced cell proliferation, cell invasion and tumorigenicity in vitro, but also inhibited tumor growth in vivo in two immunodeficient mouse xenograft models. Mechanistically, RARRES2 overexpression in ACC cells inhibited Wnt/β-catenin pathway activity by promoting β-catenin phosphorylation and degradation, it also inhibited the phosphorylation of p38 mitogen-activated protein kinase. Thus our study identifies RARRES2 as a novel tumor suppressor for ACC, which can function through an immune-independent mechanism.

Adiponectin, Retinoic Acid Receptor Responder 2, and Peroxisome Proliferator-Activated Receptor-γ Coativator-1 Genes and the Risk for Obesity.

Obesity is the most common nutritional disorder. This disease is a multifactorial disease influenced by environmental and genetic factors. This study investigated the relationship between common variants of adiponectin (ADIPOQ), retinoic acid receptor responder 2 (RARRES2), and peroxisome proliferator-activated receptor-γ coativator-1 (PPARGC1) and obesity-related traits and susceptibility. A total of 167 individuals with obesity and 165 normal-weight subjects were recruited. Genotype frequencies of rs182052 in ADIPOQ differed significantly between the groups. Genotype AA was observed at a higher frequency in case than in control subjects. Association analysis showed that the A allele was a risk factor for obesity. This polymorphism was associated with body weight, body mass index (BMI), and waist circumference. After stratification by BMI, eutrophic individuals with AA or AG genotypes had higher body weights and waist circumferences than those with GG genotypes. In the case group, no associations were observed, except for stratified subjects with morbid obesity that exhibited a progressive increase of body weight, BMI, and waist circumference when rs182052 A was present. No associations were observed between SNPs in RARRES2 and PPARGC1 and obesity or any other studied variables. The rs182052 polymorphism in ADIPOQ is associated with a higher risk for obesity and obesity-related parameters.

Cilostazol에 의한 뇌혈관내피세포의 세포이동 증진 효과연구

Cilostazol is known to be a selective inhibitor of phosphodiesterase III and is generally used to treat stroke. Our previous findings showed that cilostazol enhanced capillary density through angiogenesis after focal cerebral ischemia. Angiogenesis is an important physiological process for promoting revascularization to overcome tissue ischemia. It is a multistep process consisting of endothelial cell proliferation, migration, and tubular structure formation. Here, we examined the modulatory effect of cilostazol at each step of the angiogenic mechanism by using human brain microvascular endothelial cells (HBMECs). We found that cilostazol increased the migration of HBMECs in a dose-dependent manner. However, it did not enhance HBMEC proliferation and capillary-like tube formation. We used a cDNA microarray to analyze the mechanisms of cilostazol in cell migration. We picked five candidate genes that were potentially related to cell migration, and we confirmed the gene expression levels by real-time PCR. The genes phosphoserine aminotransferase 1 (PSAT1) and CCAAT/enhancer binding protein β (C/EBPβ) were up-regulated. The genes tissue factor pathway inhibitor 2 (TFPI2), retinoic acid receptor responder 1 (RARRES1), and RARRES3 were down-regulated. Our observations suggest that cilostazol can promote angiogenesis by promoting endothelial migration. Understanding the cilostazol-modulated regulatory mechanisms in brain endothelial cells may help stimulate blood vessel formation for the treatment of ischemic diseases.

Impaired gene-expression and epigenetic regulation of Retinoic Acid Receptor Responder 1 in preeclampsia and choriocarcinoma

Impaired gene-expression and epigenetic regulation of Retinoic Acid Receptor Responder 1 in preeclampsia and choriocarcinoma

Abstract 3661: ALDH1A3-inducible RARRES1 is a tumor suppressor in triple-negative breast cancer and is methylated in claudin-low breast cancers

Abstract The retinoic acid (RA) signalling pathway plays an important role in breast cancer progression and has either a pro-tumorigenic or tumor-suppressive role depending upon the effector function of RA-inducible genes that are expressed or epigenetically silenced. To study this paradigm in breast cancer, we focused on a controversial RA-inducible gene, the retinoic acid receptor responder 1 (RARRES1) protein that is often hypermethylated in cancer and has been reported to have tumor-suppressive function in prostate and nasopharyngeal carcinomas. However, in a study focused on a rare subtype of breast cancer, inflammatory breast cancer, RARRES1 is pro-tumorigenic. This functional discrepancy requires further investigation to determine its role in breast cancer in general. First, analysis of patient data sets revealed that RARRES1 is predominantly expressed in triple-negative breast cancers (TNBCs). Knockdown of RARRES1 in claudin-low MDA-MB-231 and basal-like MDA-MB-468 and HCC1937 significantly increased tumor growth and cell proliferation, suggesting RARRES1 has a tumor suppressive function in (TNBC), regardless of position on the differentiation hierarchy. Expression analyses of 24 breast cancer cell lines (including 18 TNBC and 2 normal-like cell lines) revealed that RARRES1 is predominantly expressed in basal-like TNBC cells We found that RARRES1 expression is dependent on, and strongly correlates with, the cancer stem cell marker, and RA-producing, ALDH1A3 in fixed breast cancer patient samples. Immunohistochemistry of the same patient tumor samples revealed RARRES1 expression is localized to the endoplasmic reticulum. Importantly, however, the presence of ALDH1A3 or RA is not sufficient to induce RARRES1 expression. RARRES1 is hypermethylated in claudin-low breast cancer cell lines, and release of this silencing is required for full induction of RARRES1 expression. We have identified sites of regulation by methylation in RARRES1 using Illumina 450K methylation arrays and 5-methylcytosine ChIP. We conclude that RARRES1 is an ALDH1A3/RA-inducible tumor suppressor in TNBC with methylation and expression profiles distinct to the differentiation hierarchy observed in breast cancer. Citation Format: Krysta M. Coyle, Patrick Murphy, Dejan Vidovic, Cheryl A. Dean, Margaret L. Thomas, Derek Clements, Mohammad Sultan, Ahmad Vaghar-Kashani, Carman Giacomantonio, Lucy Helyer, Ian Weaver, Shashi Gujar, Patrick WK Lee, Paola Marcato. ALDH1A3-inducible RARRES1 is a tumor suppressor in triple-negative breast cancer and is methylated in claudin-low breast cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3661.

Proteomic analysis of egg white heparin-binding proteins: towards the identification of natural antibacterial molecules.

The chicken egg resists most environmental microbes suggesting that it potentially contains efficient antimicrobial molecules. Considering that some heparin-binding proteins in mammals are antibacterial, we investigated the presence and the antimicrobial activity of heparin-binding proteins from chicken egg white. Mass spectrometry analysis of the proteins recovered after heparin-affinity chromatography, revealed 20 proteins, including known antimicrobial proteins (avidin, lysozyme, TENP, ovalbumin-related protein X and avian bêta-defensin 11). The antibacterial activity of three new egg candidates (vitelline membrane outer layer protein 1, beta-microseminoprotein-like (LOC101750704) and pleiotrophin) was demonstrated against Listeria monocytogenes and/or Salmonella enterica Enteritidis. We showed that all these molecules share the property to inhibit bacterial growth through their heparin-binding domains. However, vitelline membrane outer layer 1 has additional specific structural features that can contribute to its antimicrobial potential. Moreover, we identified potential supplementary effectors of innate immunity including mucin 5B, E-selectin ligand 1, whey acidic protein 3, peptidyl prolyl isomerase B and retinoic acid receptor responder protein 2. These data support the concept of using heparin affinity combined to mass spectrometry to obtain an overview of the various effectors of innate immunity composing biological milieus, and to identify novel antimicrobial candidates of interest in the race for alternatives to antibiotics.

Breast cancer subtype dictates DNA methylation and ALDH1A3-mediated expression of tumor suppressor RARRES1.

Breast cancer subtyping, based on the expression of hormone receptors and other genes, can determine patient prognosis and potential options for targeted therapy. Among breast cancer subtypes, tumors of basal-like and claudin-low subtypes are typically associated with worse patient outcomes, are primarily classified as triple-negative breast cancers (TNBC), and cannot be treated with existing hormone-receptor-targeted therapies. Understanding the molecular basis of these subtypes will lead to the development of more effective treatment options for TNBC. In this study, we focus on retinoic acid receptor responder 1 (RARRES1) as a paradigm to determine if breast cancer subtype dictates protein function and gene expression regulation. Patient tumor dataset analysis and gene expression studies of a 26 cell-line panel, representing the five breast cancer subtypes, demonstrate that RARRES1 expression is greatest in basal-like TNBCs. Cell proliferation and tumor growth assays reveal that RARRES1 is a tumor suppressor in TNBC. Furthermore, gene expression studies, Illumina HumanMethylation450 arrays, and chromatin immunoprecipitation demonstrate that expression of RARRES1 is retained in basal-like breast cancers due to hypomethylation of the promoter. Additionally, expression of the cancer stem cell marker, aldehyde dehydrogenase 1A3, which provides the required ligand (retinoic acid) for RARRES1 transcription, is also specific to the basal-like subtype. We functionally demonstrate that the combination of promoter methylation and retinoic acid signaling dictates expression of tumor suppressor RARRES1 in a subtype-specific manner. These findings provide a precedent for a therapeutically-inducible tumor suppressor and suggest novel avenues of therapeutic intervention for patients with basal-like breast cancer.