Retinoic Acid Receptor Responder Research Articles

Abstract A18: Expression of the tumor suppressor gene RARRES1 in the differentiation hierarchy of breast cancer is regulated by DNA methylation

Abstract The tumor-suppressive function of the retinoic acid (RA)-inducible gene, retinoic acid receptor responder 1 (RARRES1), has been reported in many cancer types; however, in inflammatory breast cancer, RARRES1 is pro-tumorigenic. Furthermore, high transcript levels of RARRES1 are associated with more aggressive triple-negative breast cancers (TNBCs) and poorer patient outcomes. This suggests that RARRES1 may also be oncogenic and requires investigation to determine its role in breast cancer. Expression analyses of 20 breast cancer cell lines (including 18 TNBC and two estrogen-receptor-positive cell lines) revealed that RARRES1 is predominantly expressed in basal-like breast cancer cells, but is often methylated and silenced in claudin-low breast cancer cells. We have identified possible sites of regulation by methylation in RARRES1 using Illumina 450K methylation arrays and 5-methylcytosine ChIP. Basal-like cells express higher levels of the cancer stem cell (CSC) marker ALDH1A3. Expression of RARRES1 is dependent on, and strongly correlates with ALDH1A3 expression in fixed breast cancer patient samples. Immunohistochemistry of the same patient tumor samples revealed RARRES1 expression is localized to the endoplasmic reticulum. Finally, knockdown of RARRES1 in claudin-low MDA-MB-231 and basal-like MDA-MB-468 and HCC1937 significantly increased cell proliferation and tumor growth, suggesting RARRES1 has a tumor suppressive function regardless of position on the differentiation hierarchy. We conclude that RARRES1 is a tumor suppressor in TNBC with methylation and expression profiles distinct to the differentiation hierarchy observed in breast cancer. Citation Format: Krysta Mila Coyle, Dejan Vidovic, Cheryl A. Dean, Margaret Lois Thomas, Mohammad Sultan, Derek Clements, Ahmad Vaghar-Kashani, Melissa Wallace, Ian Weaver, Carman A. Giacomantonio, Lucy Helyer, Paola Marcato. Expression of the tumor suppressor gene RARRES1 in the differentiation hierarchy of breast cancer is regulated by DNA methylation. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A18.

Abstract B66: The immunoproteasome: A point of convergence between immune response chemokine signaling and metastasis in breast cancer

Abstract The aim of this study is to identify gene modules that mediate organ-specific metastasis in breast cancer. Epidemiological studies indicate that breast cancer subtype, based on estrogen receptor (ER) and epidermal growth factor receptor 2 (HER2/ERBB2) levels, is associated with distinct organ-specific patterns of metastasis. At a molecular level, homeostatic chemokines and their receptors have been described as one of the most important mechanisms operating in organotropic metastasis. How these seemingly independent factors might act in concert is yet to be fully understood. To address this question we adopted a novel network integration approach. We generated a metastatic driver gene set representing factors known to contribute to organotropic metastasis. Using gene expression data derived from breast cancer cells and tumor tissue, we computed the Pearson's correlation between the metastatic driver genes and all others on each microarray to develop networks, in which nodes represent genes and the edges represent the Pearson's correlation between connected genes. Given that some chemokines implicated in breast cancer metastasis are also known to play key roles in bone marrow-derived mesenchymal stem cell (BM-MSC) function during immune response, we also created a BM-MSC network. This allows us to identify parallels between BM-MSC gene expression profiles and those from breast cancer cells and tissue. This approach reveals gene modules that are conserved across, or unique to, one or more context-specific networks: thus providing insight into the influence of breast cancer subtype on immune-related chemokine signaling. Of particular interest is a gene module that links the chemokine ligands CCL2 and CCL5 with genes related to the immunoproteasome: transporter for antigen processing genes TAP1 and TAP2, and the catalytic ‘immunosubunits' PSMB8, PSMB9 and PSMB10 (the latter are implicated in immune escape). This i-proteasome gene module is conserved between the BM-MSC network and networks derived from independent datasets representing tumors that metastasized to the lung and HER2+ tumors from patients with one or more positive lymph nodes. We further explored expression patterns linking the i-proteasome gene module to known metastatic drivers. For example, the role of chemokine ligand-receptor pair CCL17-CCR4 has been previously described: CCR4 enhances the chemotactic response of breast cancer cells to the CCL17 ligand; promotes breast tumor growth and lung metastasis in mice; and is positively correlated with HER2 expression, tumor recurrence and lymph node, lung and bone metastasis. In the BM-MSC network, CCR4 is correlated, and CCL17 anti-correlated, with genes in the i-proteasome gene module. A reverse pattern is observed in tumors that metastasize to lung while both the ligand and receptor show positive correlation with the i-proteasome gene module in HER2+ tumors. In the BM-MSC network, i-proteasome genes correlate with the estrogen receptor alpha (ESR1) and are anti-correlated with the ERBB2 interacting protein gene (ERBB2IP), indicating that the i-proteasome function is likely sensitive to breast cancer subtype-related receptor levels. The Retinoic Acid Receptor Responder (Tazarotene Induced) 3 gene (RARRES3) is downregulated in breast cancer and has been shown to suppress metastasis to lung. Here, RARRES3 shows correlated expression with PSMB8 and PSMB9 in eight and seven networks respectively suggesting that the i-proteasome function is also sensitive to retinoic-related signaling. This study demonstrates a knowledge-based and data-driven approach to discovery of gene modules underlying organotropic metastasis in breast cancer. This network resource will serve as a foundation for future research into both the underlying mechanisms driving organotropic metastasis and the development of subtype-specific therapies. Citation Format: Alison M. Anderson. The immunoproteasome: A point of convergence between immune response chemokine signaling and metastasis in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B66.

Association of Polymorphisms in STRA6 and RARRES2 Genes with Type 2 Diabetes in Southern Han Chinese.

Stimulated by retinoic acid gene homolog 6 (STRA6) and retinoic acid receptor responder 2 (RARRES2) are candidate genes involved in the pathogenesis of type 2 diabetes mellitus (T2DM). Three tag-SNPs in STRA6 and one in RARRES2 gene were selected and genotyped with TaqMan or PCR-RFLP method in 603 populations (571 patients with T2D versus 632 control subjects) in Southern Han Chinese. We estimated the interactions between T2DM risk and genetic variants in the STRA6 and RARRES2 genes using polymerase chain reaction. Rs736118 in STRA6 gene were significantly associated with T2DM occurrence in the recessive genetic model. The genotype of rs974456 was significantly associated with T2DM in the dominant genetic model correlated to sex, MBI, and triglyceride. However, the association of other SNPs with T2DM was not found. Furthermore, smoking history and other factors may be independent risk factors for the incidence of T2DM. This study suggested that a role of STRA6 polymorphism could also be of value in predicting the risk of T2DM while RARRES2 polymorphism could not predict the risk of T2DM.

Identification of CCL2, RARRES2 and EFNB2 as host cell factors that influence the multistep replication of respiratory syncytial virus

Human respiratory syncytial virus (RSV) is a major causative agent of respiratory tract infections in children worldwide. Preterm children or those with underlying cardiopulmonary disorders are at particularly high risk of developing severe and lethal RSV respiratory tract infections; however, there are currently no effective vaccines or anti-viral drugs. To identify targets for the development of drugs to treat RSV infections, we investigated host cell factors involved in the replication of RSV. To this end, MDCK cells with low susceptibility to RSV were transfected with cDNA libraries derived from RSV-susceptible human lung or HeLa cells. A microarray analysis was subsequently performed on parental MDCK cells and MDCK cells that were converted to an RSV-susceptible form. Among the genes identified, chemokine (C–C motif) ligand 2 (CCL2), retinoic acid receptor responder protein 2 (RARRES2) and ephrin-B2 (EFNB2) had a positive effect on RSV replication. Expression of these genes in MDCK cells resulted in a 10- to 100-fold increase in RSV replication. CCL2 expression also disrupted the distribution of claudin-1, a tight junction protein, suggesting that CCL2 plays a role in claudin-based tight junction formation during RSV replication. The knockdown of EFNB2 and RARRES2 by siRNA in RSV-susceptible cell lines (HEp-2 and A549) resulted in reduced RSV replication, suggesting that EFNB2 and RARRES2 participate in RSV replication. Together, our findings suggest that CCL2, RARRES2 and EFNB2 are host cell factors involved in RSV replication.

Molecular evolution and expression profile of the chemerine encoding gene RARRES2 in baboon and chimpanzee.

BackgroundChemerin, encoded by the retinoic acid receptor responder 2 (RARRES2) gene is an adipocytesecreted protein with autocrine/paracrine functions in adipose tissue, metabolism and inflammation with a recently described function in vascular tone regulation, liver, steatosis, etc. This molecule is believed to represent a critical endocrine signal linking obesity to diabetes. There are no data available regarding evolution of RARRES2 in non-human primates and great apes. Expression profile and orthology in RARRES2 genes are unknown aspects in the biology of this multigene family in primates. Thus; we attempt to describe expression profile and phylogenetic relationship as complementary knowledge in the function of this gene in primates. To do that, we performed A RT-PCR from different tissues obtained during necropsies. Also we tested the hypotheses of positive evolution, purifying selection, and neutrality. And finally a phylogenetic analysis was made between primates RARRES2 protein.ResultsRARRES2 transcripts were present in liver, lung, adipose tissue, ovary, pancreas, heart, hypothalamus and pituitary tissues. Expression in kidney and leukocytes were not detectable in either species. It was determined that the studied genes are orthologous.ConclusionsRARRES2 evolution fits the hypothesis of purifying selection. Expression profiles of the RARRES2 gene are similar in baboons and chimpanzees and are also phylogenetically related.

Genome wide meta-analysis highlights the role of genetic variation in RARRES2 in the regulation of circulating serum chemerin.

Chemerin is an adipokine proposed to link obesity and chronic inflammation of adipose tissue. Genetic factors determining chemerin release from adipose tissue are yet unknown. We conducted a meta-analysis of genome-wide association studies (GWAS) for serum chemerin in three independent cohorts from Europe: Sorbs and KORA from Germany and PPP-Botnia from Finland (total N = 2,791). In addition, we measured mRNA expression of genes within the associated loci in peripheral mononuclear cells by micro-arrays, and within adipose tissue by quantitative RT-PCR and performed mRNA expression quantitative trait and expression-chemerin association studies to functionally substantiate our loci. Heritability estimate of circulating chemerin levels was 16.2% in the Sorbs cohort. Thirty single nucleotide polymorphisms (SNPs) at chromosome 7 within the retinoic acid receptor responder 2 (RARRES2)/Leucine Rich Repeat Containing (LRRC61) locus reached genome-wide significance (p<5.0×10−8) in the meta-analysis (the strongest evidence for association at rs7806429 with p = 7.8×10−14, beta = −0.067, explained variance 2.0%). All other SNPs within the cluster were in linkage disequilibrium with rs7806429 (minimum r2 = 0.43 in the Sorbs cohort). The results of the subgroup analyses of males and females were consistent with the results found in the total cohort. No significant SNP-sex interaction was observed. rs7806429 was associated with mRNA expression of RARRES2 in visceral adipose tissue in women (p<0.05 after adjusting for age and body mass index). In conclusion, the present meta-GWAS combined with mRNA expression studies highlights the role of genetic variation in the RARRES2 locus in the regulation of circulating chemerin concentrations.

ATRA Upregulates Cell Surface CD1D on Myeloma Cells and Sensitizes Them to iNKT Cell-Mediated Lysis

Invariant natural killer T (iNKT) cells recognize lipid antigens in the context of CD1D and have been shown to effectively kill CD1D-expressing multiple myeloma (MM) cells. It has been reported that iNKT cells in MM patients are deficient and dysfunctional and that CD1D is variably expressed among MM patients and is completely lost in MM cell lines. Recent clinical study suggests that targeting iNKT cells may prevent progression of asymptomatic MM (AMM) to active disease (Richter at al. Blood 2013). All-Trans Retinoic Acid (ATRA) has been used for the treatment of MM but its modes of action have not been completely elucidated. The aims of this study were to shed light on the mechanism of action of ATRA and to identify ATRA target genes in MM. ATRA at clinically relevant concentrations (1-2 μM, 4 days) had minimal growth inhibitory effect on the majority of MM cell lines (10±13% and 14±16%, respectively; n=10). Gene expression profile (GEP) of MM cell lines and primary cases (n=13) treated with ATRA (1 μM, 2 days) revealed upregulation of CD1D (381±204 vs. 5692±1675 mean±SEM GEP signal in CONT and ATRA groups, respectively; p<0.006). Flow cytometry analysis for cell surface CD1D in 21 MM cases treated in vitro with ATRA revealed significant CD1D induction in 11 cases, intermediate induction in 2 cases and no induction in 8 cases. Clinically, MM cells from patients who received ATRA as part of their salvage therapy also had induced CD1D gene expression compared to levels prior to ATRA therapy. Analysis of abnormal plasma cells based on a single 8-color markers flow cytometry showed higher CD1D coefficient of variation (CV) in AMM cases molecularly classified as high-risk (n=5) than low-risk cases (n=9, p<0.003), while mean florescence intensity (MFI) was insignificantly lower in high-risk AMM cases. In vitro ATRA treatment of BM cells from high-risk AMM patient resulted in increased CD1D MFI by 2 folds and reduced CV in abnormal plasma cell restricted population. To test the functional consequences of CD1D upregulation in MM cells, iNKT cells were purified from healthy donors’ peripheral blood mononuclear cells using anti-Vα24Jα18 immunomagnetic beads and expanded in vitro via stimulation with irradiated autologous α-GalCer-loaded mature dendritic cells (DCs) in the presence of IL-2. ATRA treated (1 μM, 2 days) compared to vehicle treated MM cells (n=4) were more sensitive to iNKT cell-mediated lysis in 5 hour 51chromium-release assays or bioluminescence viability assays (e.g. 21% vs. 75% lysis of H929 cells, p<0.0001, effector: target ratio 2.5:1). ATRA-induced MM cell lysis by iNKT cells was markedly blocked by CD1D neutralizing antibody (1-40 μg/ml) indicating that the enhanced cell lysis was mediated through CD1D. Further GEP analysis identified RARRES3 (retinoic acid receptor responder protein 3) as an additional top ATRA target gene in MM cells. RARRES3 gene expression was commonly but variably induced by ATRA whereas MM cells expressing high baseline RARRES3 were resistant to ATRA-induced CD1D gene and protein expression. Expression levels of CD1D and RARRES3 were inversely correlated in MM cells from newly diagnosed patients (n=449, r=-0.4, p<0.0001). Among molecularly classified groups, the MF subtype, known to be associated with poor survival, had the lowest CD1D/highest RARRES3 expression, whereas the favorable CD2 subtype had a reversed pattern. We conclude that CD1D and RARRES3 are ATRA target genes in MM cells and that stimulation of cell surface CD1D expression on MM cells enhances sensitivity to iNKT cell-mediated lysis. Disclosuresvan Rhee:Senesco: PI Other.

Abstract 196: Targeting breast cancer stem cells with a panel of potential ALDH1A3 inhibitors

Abstract Aldehyde dehydrogenase (ALDH) isoform ALDH1A3 is selectively expressed in breast cancer stem cells (CSCs) and is a key mediator in breast cancer progression via initiation of retinoic acid (RA) signaling and expression of RA-inducible genes. Expression of ALDH1A3 and its induced RA-inducible genes are associated with less favorable patient outcomes. This suggests that ALDH1A3 represents a novel anti-breast cancer therapeutic target. To target the specific ALDH1A3 isoform, a panel of general ALDH inhibitors was attained and the compounds’ effectiveness in targeting ALDH1A3 activity was quantified by their ability to decrease expression of the downstream genes retinoic acid receptor beta (RARβ) and retinoic acid receptor responder protein 1 (RARRES1). Eleven compounds were tested in total, and the essential oil citral was shown to significantly decrease ALDH1A3-mediated expression of both RA-inducible genes in a dose dependent manner. Citral is already used in cosmetics and food additives and recent work shows that it can induce apoptosis of breast cancer cells making it a promising therapeutic agent and a potential subject of a future xenograft study in mice to determine its effects on CSCs in vivo. Citation Format: Margaret L. Thomas, Melissa Wallace, Carman Giacomantonio, Paola Marcato. Targeting breast cancer stem cells with a panel of potential ALDH1A3 inhibitors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 196. doi:10.1158/1538-7445.AM2014-196

Abstract 578: RARRES1 is a tumor suppressor in triple-negative breast cancer cell lines

Abstract Increasing evidence suggests that retinoic acid (RA) signaling is of great importance in the progression and metastasis of different cancer types. We have previously reported that MDA-MB-231 and MDA-MB-468 triple-negative breast cancer cell lines display different responses to increased retinoic acid: retinoic acid increases MDA-MB-231 xenograft growth and decreases growth MDA-MB-468 xenografts. To investigate what may be responsible for these opposite responses to RA, we have investigated the role of retinoic acid receptor responder protein 1 (RARRES1). RARRES1 is an RA-inducible gene which has not yet been fully characterized. Despite its unknown function, many independent studies have reported the tumor suppressive function of RARRES1 in multiple cancer types. Interestingly, our previous data indicates that high levels of RARRES1 in clinical breast cancer samples is correlated with poorer outcomes for patients. In particular, high expression of RARRES1 is associated with triple-negative breast cancers as well as with more aggressive cancers (by Ki67 positivity, as well as nuclear, mitotic, and overall grade). Thus, to determine the role of RARRES1 in breast cancers, we have performed in vitro cell proliferation and in vivo xenograft assays. We report that knockdown of RARRES1 in MDA-MB-231 and MDA-MB-468 cells leads to increased cell proliferation in vitro and in in vivo xenografts. RARRES1 is proposed to regulate a number of genes, including the hypothesized tumor suppressors discs large homolog 2 and protein phosphatase 2A, the proto-oncogene valosin-containing protein (p97), and ankyrin repeat domain-containing protein 26 which is of unknown function. We are investigating the regulation of these genes by RARRES1 in MDA-MB-231 and MDA-MB-468 triple-negative cell lines. We expect that the RARRES1 tumor suppressor is one of many genes induced by RA which form a complex signaling pathway regulating the response of cells to RA. We conclude that RARRES1 is a tumor suppressor in MDA-MB-231 and MDA-MB-468 triple-negative breast cancer cell lines; however, we continue to investigate the discrepancy between this finding and our previous clinical data. Citation Format: Krysta M. Coyle, Ahmad Vaghar-Kashani, Florence Wong, Cheryl Dean, Carman Giacomantonio, Paola Marcato. RARRES1 is a tumor suppressor in triple-negative breast cancer cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 578. doi:10.1158/1538-7445.AM2014-578

Abstract 564: Reduction of Blood Pressure with Antisense Oligodeoxynucleotides Against Chemerin

Chemerin promotes dendritic cells and macrophage migration to sites of inflammation. The liver and adipose tissues are sources of chemerin. There is a direct relationship of BMI, plasma chemerin concentration and blood pressure. Chemerin causes arterial contraction that is amplified in tissues with endothelial dysfunction. We hypothesized that if chemerin supported hypertension through such mechanisms, removal of chemerin should decrease blood pressure. Ten antisense oligodeoxynucleotides (ASOs) were developed against the retinoic acid receptor responder protein 2 (RARRES2, chemerin gene). ASO642647 was chosen as a lead based on excellent tolerance and minimal changes in a standard clinical panel. The model chosen was the deoxycorticosterone acetate (DOCA) salt hypertensive rat as this model shows profound endothelial dysfunction. Rats were uninephrectomized, given DOCA (s.c., 150 mg/kg) and placed on 1.0% NaCl + 0.2 % KCl. One week later, radiotelemeters were implanted for measurement of systolic, diastolic, mean arterial blood pressure and heart rate. ASOs (control nonsense or chemerin) were injected ip weekly over 3.5 weeks (25 mg/kg week 1 and 2; 12.5 mg/kg week 3 and 4) when DOCA-salt hypertension was stable (week 3.5). A group of DOCA-salt rats also received injections of PBS (ASO vehicle). Two days after the 4th injection, tissues were dissected for measurement of chemerin mRNA and blood taken for measures of catecholamines. ASO Chemerin reduced mean arterial blood pressure vs ASO control and PBS group within the first week, and remained reduced by ~17+/- 2 mm Hg at the time of sacrifice. Target organ mass (liver, heart and kidneys) was not different between ASO control and ASO chemerin group. HPLC revealed that circulating norepinephrine (NE) and dopamine (DA) were reduced 50% in the ASO chemerin vs control or PBS group. Real time RT-PCR validated knockdown of chemerin in ASO chemerin treated animals, with reductions as follows (vs ASO control, +/-3%): Liver (94%), epididymal fat (90%), retroperitoneal fat (90% ) and mesenteric perivascular adipose tissue (85%). These experiments provide powerful support for chemerin playing a role in blood pressure elevation.

Wide-gene expression analysis of lipid-relevant genes in nutritionally challenged gilthead sea bream (Sparus aurata).

Disturbances of lipid metabolism are a major problem in livestock fish and the present study analysed the different tissue expression patterns and regulations of 40 lipid-relevant genes in gilthead sea bream. Nineteen sequences, including fatty acid elongases (4), phospholipases (7), acylglycerol lipases (8) and lipase-maturating enzymes (1), were new for gilthead sea bream (GenBank, JX975700-JX975718JX975700JX975701JX975702JX975703JX975704JX975705JX975706JX975707JX975708JX975709JX975710JX975711JX975712JX975713JX975714JX975715JX975716JX975717JX975718). Up to six different lipase-related enzymes were highly expressed in adipose tissue and liver, which also showed a high expression level of Δ6 and Δ9 desaturases. In the brain, the greatest gene expression level was achieved by the very long chain fatty acid elongation 1, along with relatively high levels of Δ9 desaturases and the phospholipase retinoic acid receptor responder. These two enzymes were also expressed at a high level in white skeletal muscle, which also shared a high expression of lipid oxidative enzymes. An overall down-regulation trend was observed in liver and adipose tissue in response to fasting following the depletion of lipid stores. The white skeletal muscle of fasted fish showed a strong down-regulation of Δ9 desaturases in conjunction with a consistent up-regulation of the "lipolytic machinery" including key enzymes of tissue fatty acid uptake and mitochondrial fatty acid transport and oxidation. In contrast, the gene expression profile of the brain remained almost unaltered in fasted fish, which highlights the different tissue plasticity of lipid-related genes. Taken together, these findings provide new fish genomic resources and contribute to define the most informative set of lipid-relevant genes for a given tissue and physiological condition in gilthead sea bream.

Rax regulates hypothalamic tanycyte differentiation and barrier function in mice

The wall of the ventral third ventricle is composed of two distinct cell populations: tanycytes and ependymal cells. Tanycytes regulate many aspects of hypothalamic physiology, but little is known about the transcriptional network that regulates their development and function. We observed that the retina and anterior neural fold homeobox transcription factor (Rax) is selectively expressed in hypothalamic tanycytes, and showed a complementary pattern of expression to markers of hypothalamic ependymal cells, such as Rarres2 (retinoic acid receptor responder [tazarotene induced] 2). To determine whether Rax controls tanycyte differentiation and function, we generated Rax haploinsufficient mice and examined their cellular and molecular phenotype in adulthood. These mice appeared grossly normal, but careful examination revealed a thinning of the third ventricular wall and reduction of both tanycyte and ependymal markers. These experiments show that Rax is required for hypothalamic tanycyte and ependymal cell differentiation. Rax haploinsufficiency also resulted in the ectopic presence of ependymal cells in the α2 tanycytic zone, where few ependymal cells are normally found, suggesting that Rax is selectively required for α2 tanycyte differentiation. These changes in the ventricular wall were associated with reduced diffusion of Evans Blue tracer from the ventricle to the hypothalamic parenchyma, with no apparent repercussion on the gross anatomical or behavioral phenotype of these mice. In conclusion, we have provided evidence that Rax is required for the normal differentiation and patterning of hypothalamic tanycytes and ependymal cells, as well as for maintenance of the cerebrospinal fluid-hypothalamus barrier.

Retinoic acid receptor responder (tazarotene induced) 1 (RARRES1; TIG1); AXL receptor tyrosine kinase (AXL; UFO)

Retinoic acid receptor responder (tazarotene induced) 1 (RARRES1; TIG1); AXL receptor tyrosine kinase (AXL; UFO)

Retinoic acid represses invasion and stem cell phenotype by induction of the metastasis suppressors RARRES1 and LXN

The mouse haematopoietic stem cell (SC) regulator Latexin (LXN) is the only known homologue of the retinoic acid receptor responder 1 (RARRES1) gene. Both genes lie adjacent on chromosome 3 and differ mostly by the presence of a transmembrane domain in RARRES1. Despite their homology, it is not known whether they possess similar regulatory mechanisms, cellular localization and function. Here, we identified RARRES1 and LXN as highly significantly downregulated genes in human prostate SCs, whose expression was induced by the pro-differentiation agent all-trans retinoic acid (atRA). AtRA induced expression in the most differentiated cells compared with the SC fraction, suggesting that this subpopulation was less responsive to atRA. Small interfering RNA suppression of RARRES1 and LXN enhanced the SC properties of primary prostate cultures, as shown by a significant increase in their colony-forming ability. Expression of both RARRES1 and LXN was co-ordinately repressed by DNA methylation in prostate cancer cell lines and inhibition of RARRES1 and LXN increased the invasive capacity of primary prostate cultures, which also fully rescued an inhibitory effect induced by atRA. Moreover, we showed that RARRES1 and LXN reside within different sub-cellular compartments, providing evidence that RARRES1 is not a plasma membrane protein as previously supposed but is located primarily in the endoplasmic reticulum; whereas LXN was detected in the nucleus of prostate epithelial cells. Thus, LXN and RARRES1 are potential tumour suppressor genes, which are co-ordinately regulated, SC-silenced genes functioning to suppress invasion and colony-forming ability of prostate cancer cells; yet the proteins reside within different sub-cellular compartments.

Chemerin in Non-Alcoholic Fatty Liver Disease ? Up or Down?

Chemerin (previously known as tazarotene-induced gene-2 or retinoic acid receptor responder-2) is an adipokine whose systemic levels are increased in human and rodent obesity [1-5]. Chemerin is an attractant for immune cells and may play a role in the recruitment of tissue macrophages [6]. This adipokine also regulates adipogenesis and glucose metabolism [2,7,8]. Chemerin is released as a proprotein with low biological activity, and extracellular C-terminal processing by distinct proteases generates bioactive polypeptides which exert proor antiinflammatory effects [6,7]. Hence, local and systemic levels of bioactive chemerin depend on proteolytic processing and are not simply related to chemerin protein concentrations determined by commercially available ELISAs or immunoblot techniques.

Secretome Survey of Human Plexiform Neurofibroma Derived Schwann Cells Reveals a Secreted form of the RARRES1 Protein

To bring insights into neurofibroma biochemistry, a comprehensive secretome analysis was performed on cultured human primary Schwann cells isolated from surgically resected plexiform neurofibroma and from normal nerve tissue. Using a combination of SDS-PAGE and high precision LC-MS/MS, 907 proteins were confidently identified in the conditioned media of Schwann cell cultures combined. Label free proteome profiling revealed consistent release of high levels of 22 proteins by the four biological replicates of NF1 Schwann cell cultures relative to the two normal Schwann cell cultures. Inversely, 9 proteins displayed decreased levels in the conditioned media of NF1 relative to normal Schwann cells. The proteins with increased levels included proteins involved in cell growth, angiogenesis and complement pathway while proteins with decreased levels included those involved in cell adhesion, plasminogen pathway and extracellular matrix remodeling. Retinoic acid receptor responder protein-1 (RARRES1), previously described as an integral membrane tumor suppressor, was found exclusively secreted by NF1 Schwann cells but not by normal Schwann cells. All-trans retinoic acid modulated secretion of RARRES1 in a dose dependent manner. This study shows altered secretion of key proteins in NF1 derived Schwann cells. The potential implication of these proteins in neurofibroma biology is discussed.

Epigenetic Repression of RARRES1 Is Mediated by Methylation of a Proximal Promoter and a Loss of CTCF Binding

BackgroundThe cis-acting promoter element responsible for epigenetic silencing of retinoic acid receptor responder 1 (RARRES1) by methylation is unclear. Likewise, how aberrant methylation interplays effectors and thus affects breast neoplastic features remains largely unknown.Methodology/Principal FindingsWe first compared methylation occurring at the sequences (−664∼+420) flanking the RARRES1 promoter in primary breast carcinomas to that in adjacent benign tissues. Surprisingly, tumor cores displayed significantly elevated methylation occurring solely at the upstream region (−664∼−86), while the downstream element (−85∼+420) proximal to the transcriptional start site (+1) remained largely unchanged. Yet, hypermethylation at the former did not result in appreciable silencing effect. In contrast, the proximal sequence displayed full promoter activity and methylation of which remarkably silenced RARRES1 transcription. This phenomenon was recapitulated in breast cancer cell lines, in which methylation at the proximal region strikingly coincided with downregulation. We also discovered that CTCF occupancy was enriched at the unmethylayed promoter bound with transcription-active histone markings. Furthermore, knocking-down CTCF expression hampered RARRES1 expression, suggesting CTCF positively regulated RARRES1 transcription presumably by binding to unmethylated promoter poised at transcription-ready state. Moreover, RARRES1 restoration not only impeded cell invasion but also promoted death induced by chemotherapeutic agents, denoting its tumor suppressive effect. Its role of attenuating invasion agreed with data generated from clinical specimens revealing that RARRES1 was generally downregulated in metastatic lymph nodes compared to the tumor cores.Conclusion/SignificanceThis report delineated silencing of RARRES1 by hypermethylation is occurring at a proximal promoter element and is associated with a loss of binding to CTCF, an activator for RARRES1 expression. We also revealed the tumor suppressive roles exerted by RARRES1 in part by promoting breast epithelial cell death and by impeding cell invasion that is an important property for metastatic spread.

RARRES3 (retinoic acid receptor responder (tazarotene induced) 3)

Review on RARRES3 (retinoic acid receptor responder (tazarotene induced) 3), with data on DNA, on the protein encoded, and where the gene is implicated.

Abstract 4044: Epigenetic repression of RARRES1 is mediated by methylation of a critical proximal promoter and a loss of CTCF binding

Abstract Background: The critical promoter element responsible for epigenetic silencing of retinoic acid receptor responder 1 (RARRES1) by methylation is unclear. Likewise, how aberrant methylation interplays effectors and thus affects breast neoplastic features remains largely unknown. Methodology/Principal Findings: We first compared methylation occurring at the sequences flanking the RARRES1 promoter in primary breast carcinomas to that in adjacent benign tissues. Surprisingly, tumor cores displayed significantly elevated methylation occurring solely at the upstream region, while the downstream element proximal to the transcription start site (+1) remained largely unchanged. Yet, hypermethylation at the former did not result in appreciable silencing effect. In contrast, the proximal sequence displayed full promoter activity and methylation of which remarkably silenced RARRES1 transcription. This phenomenon was recapitulated in breast cancer cell lines, in which methylation at the proximal region strikingly coincided with downregulation. We also discovered that CTCF occupancy was enriched at the unmethylayed promoter enriched with transcription-active histone markings. Furthermore, knocking-down CTCF expression hampered RARRES1 expression, suggesting CTCF positively regulated RARRES1 transcription presumably by binding to unmethylated promoter poised at transcription-ready state. Moreover, RARRES1 restoration not only impeded cell invasion but also promoted death induced by chemotherapeutic agents, denoting its tumor suppressive effects. Its role of attenuating invasion agreed with data generated from clinical specimens revealing that RARRES1 was generally downregulated in metastatic lymph nodes compared to the tumor cores. Conclusion/Significance: For the first time, this report delineated that silencing of RARRES1 by hypermethylation is occurring at a crucial proximal promoter element and is associated with a loss of binding to CTCF, an activator for RARRES1 expression. We also revealed the tumor suppressive roles exerted by RARRES1 in part by promoting breast epithelial cell death and by impeding cell invasion that is an important property for metastatic spread. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4044. doi:1538-7445.AM2012-4044

Abstract 501: The carboxypeptidase inhibitor TIG1 interacts with cathepsin L2 and inhibits cathepsin L2-stimulated cellular invasion

Abstract The tazarotene-induced gene 1 (TIG1), also known as retinoic acid receptor responder 1 (RARRES1), is a putative tumor suppressor gene. The gene encodes a carboxypeptidase inhibitor for ATP/GTP binding protein-like 2 (AGBL2). This study identified TIG1 binding proteins and characterized the activities of TIG1 on cellular invasion of HCT116 colon cancer cells stimulated by the cathepsin L2 (CTSL2). The cytoplasmic portion of the TIG1A isoform was used for identification of TIG1 binding proteins using the yeast two-hybrid screening, and six genes, CTSL2, DNAJC8, SPATA22, SPINK2, VAC14, and C6orf225, were selected. CTSL2, a cysteine protease with an increased expression shown in breast and colon carcinomas, was selected for analysis. Immunoprecipitation analysis using transiently transfected cytosols confirmed the binding between both TIG1 isoforms and CTSL2. Both TIG1A and CTSL2 were secreted proteins; however, the interaction of both proteins in culture supernatants was not detected. Further immunoprecipitation analysis using purified recombinant proteins demonstrated direct binding between both TIG1 isoforms and CTSL2. Cellular invasion of HCT116 cells was increased by 52 to 71% in CTSL2 transiently transfected cells. Both TIG1 isoforms had no effects on cellular invasion of HCT116 cells. However, TIG1A and TIG1B significantly inhibited CTSL2-stimulated cellular invasion by 65.1 and 62.9%, respectively. In conclusion, both TIG1 isoforms interact with CTSL2 and inhibited CTSL2-stimulated cellular invasion of HCT116 cells. The results suggest that TIG1 may suppress CTSL2-mediated cellular invasion through inhibiting the activation of CTSL2 protease, and merit for further investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 501. doi:1538-7445.AM2012-501