RXRalpha Research Articles

Genomic case report of a low grade bladder tumor metastasis to lung

BackgroundWe present a rare case where distant metastasis of a low grade bladder tumor was observed. We carried out detailed genomic analysis and cell based experiments on patient tumor samples to study tumor evolution, possible cause of disease and provide personalized treatment strategies.Case presentationA man with a smoking history was diagnosed with a low-grade urothelial carcinoma of the bladder and a concurrent high-grade upper urinary tract tumor. Seven years later he had a lung metastasis. We carried out exome sequencing on all the patient’s tumors and peripheral blood (germline) to identify somatic variants. We constructed a phylogenetic tree to capture how the tumors are related and to identify somatic changes important for metastasis. Although distant metastasis of low-grade bladder tumor is rare, the somatic variants in the tumors and the phylogenetic tree showed that the metastasized tumor had a mutational profile most similar to the low grade urothelial carcinoma. The primary and the metastatic tumors shared several important mutations, including in the KMT2D and the RXRA genes. The metastatic tumor also had an activating MTOR mutation, which may be important for tumor metastasis. We developed a mutational signature to understand the biologic processes responsible for tumor development. The mutational signature suggests that the tumor mutations are associated with tobacco carcinogen exposure, which is concordant with the patient’s smoking history. We cultured cells from the lung metastasis to examine proliferation and signaling mechanisms in response to treatment. The mTOR inhibitor Everolimus inhibited downstream mTOR signaling and induced cytotoxicity in the metastatic tumor cells.ConclusionWe used genomic analysis to examine a rare case of low grade bladder tumor metastasis to distant organ (lung). Our analysis also revealed exposure to carcinogens found is tobacco as a possible cause in tumor development. We further validated that the patient might benefit from mTOR inhibition as a potential salvage therapy in an adjuvant or recurrent disease setting.

Regulatory effects of lycopene on the key signaling receptors in human cutaneous squamous cell carcinoma cell line COLO16

Objective To evaluate the regulatory effects of lycopene on the key signaling receptors in human cutaneous squamous cell carcinoma cell line COLO16. Methods Cultured COLO16 cells were divided into 6 groups to be treated with lycopene at different concentrations of 0, 5, 10, 15, 20, and 25 μmol/L, respectively, for 24 hours (control group and 5, 10, 15, 20, 25 μmol/L lycopene groups) , followed by estimation of the cell viability by lactate dehydrogenase (LDH) assay. Lycopene at a safe concentration was selected based on the LDH assay, and used for the determination of expression of signaling receptors, and Western blot analysis was performed to measure the expression of key signaling receptor proteins, including epidermal growth factor receptor (EGFR) , glucocorticoid receptor (GR) , retinoic acid receptor-alpha (RAR-α) , retinoid X receptor-alpha (RXR-α) , androgen receptor (AR) and progesterone receptor (PR) . Statistical analysis was carried out by one-way analysis of variance (ANOVA), Tukey multiple comparison teat and Brown-Forsythe test with the SPSS software. Results After 24-hour treatment with lycopene at different concentrations, there were significant differences in the rate of cell death among these groups (F= 13.116, P 0.05) compared with those without lycopene treatment. Conclusion Lycopene can decrease the viability of COLO16 cells, inhibit the activation of EGFR protein, and up-regulate the expression of GR, and these effects may be specific for tumor cells. Key words: Carcinoma, squamous cell; Cell line, tumor; Receptor, epidermal growth factor; Receptors, glucocorticoid; COLO16 cells; Lycopene

Smoking and Parkinson disease: Evidence for gene-by-smoking interactions.

To investigate whether cigarette smoking interacts with genes involved in individual susceptibility to xenobiotics for the risk of Parkinson disease (PD). Two French population-based case-control studies (513 patients, 1,147 controls) were included as a discovery sample to examine gene-smoking interactions based on 3,179 single nucleotide polymorphisms (SNPs) in 289 genes involved in individual susceptibility to xenobiotics. SNP-by-cigarette smoking interactions were tested in the discovery sample through an empirical Bayes (EB) approach. Nine SNPs were selected for replication in a population-based case-control study from California (410 patients, 845 controls) with standard logistic regression and the EB approach. For SNPs that replicated, we performed pooled analyses including the discovery and replication datasets and computed pooled odds ratios and confidence intervals (CIs) using random-effects meta-analysis. Nine SNPs interacted with smoking in the discovery dataset and were selected for replication. Interactions of smoking with rs4240705 in the RXRA gene and rs1900586 in the SLC17A6 gene were replicated. In pooled analyses (logistic regression), the interactions between smoking and rs4240705-G and rs1900586-G were 1.66 (95% CI 1.28-2.14, p = 1.1 × 10-4, p for heterogeneity = 0.366) and 1.61 (95% CI 1.17-2.21, p = 0.003, p for heterogeneity = 0.616), respectively. For both SNPs, while smoking was significantly less frequent in patients than controls in AA homozygotes, this inverse association disappeared in G allele carriers. We identified and replicated suggestive gene-by-smoking interactions in PD. The inverse association of smoking with PD was less pronounced in carriers of minor alleles of both RXRA-rs4240705 and SLC17A6-rs1900586. These findings may help identify biological pathways involved in the inverse association between smoking and PD.

Genomic Activation of PPARG Reveals a Candidate Therapeutic Axis in Bladder Cancer.

The PPARG gene encoding the nuclear receptor PPARγ is activated in bladder cancer, either directly by gene amplification or mutation, or indirectly by mutation of the RXRA gene, which encodes the heterodimeric partner of PPARγ. Here, we show that activating alterations of PPARG or RXRA lead to a specific gene expression signature in bladder cancers. Reducing PPARG activity, whether by pharmacologic inhibition or genetic ablation, inhibited proliferation of PPARG-activated bladder cancer cells. Our results offer a preclinical proof of concept for PPARG as a candidate therapeutic target in bladder cancer. Cancer Res; 77(24); 6987-98. ©2017 AACR.

Trichoplax adhaerens reveals a network of nuclear receptors sensitive to 9-cis-retinoic acid at the base of metazoan evolution.

Trichoplax adhaerens, the only known species of Placozoa is likely to be closely related to an early metazoan that preceded branching of Cnidaria and Bilateria. This animal species is surprisingly well adapted to free life in the World Ocean inhabiting tidal costal zones of oceans and seas with warm to moderate temperatures and shallow waters. The genome of T. adhaerens (sp. Grell) includes four nuclear receptors, namely orthologue of RXR (NR2B), HNF4 (NR2A), COUP-TF (NR2F) and ERR (NR3B) that show a high degree of similarity with human orthologues. In the case of RXR, the sequence identity to human RXR alpha reaches 81% in the DNA binding domain and 70% in the ligand binding domain. We show that T. adhaerens RXR (TaRXR) binds 9-cis retinoic acid (9-cis-RA) with high affinity, as well as high specificity and that exposure of T. adhaerens to 9-cis-RA regulates the expression of the putative T. adhaerens orthologue of vertebrate L-malate-NADP+ oxidoreductase (EC 1.1.1.40) which in vertebrates is regulated by a heterodimer of RXR and thyroid hormone receptor. Treatment by 9-cis-RA alters the relative expression profile of T. adhaerens nuclear receptors, suggesting the existence of natural ligands. Keeping with this, algal food composition has a profound effect on T. adhaerens growth and appearance. We show that nanomolar concentrations of 9-cis-RA interfere with T. adhaerens growth response to specific algal food and causes growth arrest. Our results uncover an endocrine-like network of nuclear receptors sensitive to 9-cis-RA in T. adhaerens and support the existence of a ligand-sensitive network of nuclear receptors at the base of metazoan evolution.

Association of a 3' untranslated region polymorphism in proprotein convertase subtilisin/kexin type 9 with HIV viral load and CD4+ levels in HIV/hepatitis C virus coinfected women.

To assess variation in genes that regulate cholesterol metabolism in relation to the natural history of HIV infection. Cross-sectional and longitudinal analysis of the Women's Interagency HIV Study. We examined 2050 single nucleotide polymorphisms (SNPs) in 19 genes known to regulate cholesterol metabolism in relation to HIV viral load and CD4 T-cell levels in a multiracial cohort of 1066 antiretroviral therapy-naive women. Six SNPs were associated with both HIV viral load and CD4 T-cell levels at a false discovery rate of 0.01. Bioinformatics tools did not predict functional activity for five SNPs, located in introns of nuclear receptor corepressor 2, retinoid X receptor alpha (RXRA), and tetratricopeptide repeat domain 39B. Rs17111557 located in the 3' untranslated region of proprotein convertase subtilisin/kexin type 9 (PCSK9) putatively affects binding of hsa-miR-548t-5p and hsa-miR-4796-3p, which could regulate PCSK9 expression levels. Interrogation of rs17111557 revealed stronger associations in the subset of women with HIV/hepatitis C virus (HCV) coinfection (n = 408, 38% of women). Rs17111557 was also associated with low-density lipoprotein cholesterol levels in HIV/HCV coinfected (β: -10.4; 95% confidence interval: -17.9, -2.9; P = 0.007), but not in HIV monoinfected (β:1.2; 95% confidence interval: -6.3, 8.6; P = 0.76) women in adjusted analysis. PCSK9 polymorphism may affect HIV pathogenesis, particularly in HIV/HCV coinfected women. A likely mechanism for this effect is PCSK9-mediated regulation of cholesterol metabolism. Replication in independent cohorts is needed to clarify the generalizability of the observed associations.

SNP rs11185644 of RXRA gene is identified for dose-response variability to vitamin D3 supplementation: a randomized clinical trial

The level of serum 25-Hydroxyvitamin D [25(OH)D] has high heritability, suggesting that genes may contribute to variations in serum 25(OH)D level and vitamin D dose-response. As vitamin D deficiency has been linked to numerous diseases, understanding how genetic variation contributes to vitamin D dose-response is important for personalized vitamin D treatment and cost-effective disease prevention. To identify genetic variants responsible for vitamin D status and dose-response, we performed two vitamin D3 and calcium clinical supplementation trials in 2,207 postmenopausal Caucasian women. We examined the association of 291 SNPs with baseline serum 25(OH)D levels and 25(OH)D dose-response. Five SNPs, rs10500804 (P = 4.93 × 10−7), rs2060793 (P = 6.63 × 10−7), rs10741657 (P = 1.49 × 10−6), rs10766197 (P = 1.05 × 10−5) and rs11023380 (P = 7.67 × 10−5) in the CYP2R1 gene, as well as 6 SNPs, rs4588 (P = 7.86 × 10−7), rs2298850 (P = 1.94 × 10−6), rs1155563 (P = 6.39 × 10−6), rs705119 (P = 2.80 × 10−5), rs705120 (P = 1.08 × 10−4) and rs222040 (P = 1.59 × 10−4) in the GC gene were associated with baseline serum 25(OH)D levels. SNP rs11185644 near the RXRA was significantly associated with 25(OH)D dose-response (P = 1.01 × 10−4). Our data suggest that polymorphisms in the CYP2R1 and GC gene may contribute to variation in baseline serum 25(OH)D concentration, and that polymorphism rs11185644 may contribute to variation in 25(OH)D dose-response in healthy postmenopausal Caucasian women.

The Integration of Epistasis Network and Functional Interactions in a GWAS Implicates RXR Pathway Genes in the Immune Response to Smallpox Vaccine

Although many diseases and traits show large heritability, few genetic variants have been found to strongly separate phenotype groups by genotype. Complex regulatory networks of variants and expression of multiple genes lead to small individual-variant effects and difficulty replicating the effect of any single variant in an affected pathway. Interaction network modeling of GWAS identifies effects ignored by univariate models, but population differences may still cause specific genes to not replicate. Integrative network models may help detect indirect effects of variants in the underlying biological pathway. In this study, we used gene-level functional interaction information from the Integrative Multi-species Prediction (IMP) tool to reveal important genes associated with a complex phenotype through evidence from epistasis networks and pathway enrichment. We test this method for augmenting variant-based network analyses with functional interactions by applying it to a smallpox vaccine immune response GWAS. The integrative analysis spotlights the role of genes related to retinoid X receptor alpha (RXRA), which has been implicated in a previous epistasis network analysis of smallpox vaccine.

Genetic characterisation of PPARG, CEBPA and RXRA, and their influence on meat quality traits in cattle

BackgroundPeroxisome proliferator-activated receptor gamma (PPARG), CCAAT/enhancer binding protein alpha (CEBPA) and retinoid X receptor alpha (RXRA) are nuclear transcription factors that play important roles in regulation of adipogenesis and fat deposition. The objectives of this study were to characterise the variability of these three candidate genes in a mixed sample panel composed of several cattle breeds with different meat quality, validate single nucleotide polymorphisms (SNPs) in a local crossbred population (Angus - Hereford - Limousin) and evaluate their effects on meat quality traits (backfat thickness, intramuscular fat content and fatty acid composition), supporting the association tests with bioinformatic predictive studies.ResultsGlobally, nine SNPs were detected in the PPARG and CEBPA genes within our mixed panel, including a novel SNP in the latter. Three of these nine, along with seven other SNPs selected from the Single Nucleotide Polymorphism database (SNPdb), including SNPs in the RXRA gene, were validated in the crossbred population (N = 260). After validation, five of these SNPs were evaluated for genotype effects on fatty acid content and composition. Significant effects were observed on backfat thickness and different fatty acid contents (P < 0.05). Some of these SNPs caused slight differences in mRNA structure stability and/or putative binding sites for proteins.ConclusionsPPARG and CEBPA showed low to moderate variability in our sample panel. Variations in these genes, along with RXRA, may explain part of the genetic variation in fat content and composition. Our results may contribute to knowledge about genetic variation in meat quality traits in cattle and should be evaluated in larger independent populations.Electronic supplementary materialThe online version of this article (doi:10.1186/s40781-016-0095-3) contains supplementary material, which is available to authorized users.

Cryptochrome 1 functions as nuclear co-receptor independence of circadian clock

Cryptochrome 1 (CRY1) is well known as a circadian clock repressor. Recent studies have shown that it could intimately associate with nuclear receptors and either activate or inhibit its targets at the transcriptional level. In the present study, we applied publicly available transcriptional factors Chromatin Immunoprecipitation coupled high throughput sequencing data to prove that CRY1 associates with various nuclear receptors at the genome-wide level, which is independent of its canonical role as a circadian clock repressor. Furthermore, we have also shown that CRY1 is a confident co-receptor of retinoid X receptor, alpha (RXRa). We have also further shown that CRY1 could directly interact with RXRa. The interaction was regulated by retinoic acid treatment in a dose-dependent manner. In addition, CRY activator could antagonized RA-medicated PCK1 and G6PC expression, which in turn antagonize glucose production. Our findings indicate that CRYs could be potential targets in relieving side effects of RA-oriented treatment for diseases such as leukemia, acne, and psoriasis.

Genetic Polymorphisms in Vitamin D Metabolism and Signaling Genes and Risk of Breast Cancer: A Nested Case-Control Study.

Genetic polymorphisms in vitamin D metabolism and signaling genes have been inconsistently associated with risk of breast cancer, though few studies have examined SNPs in vitamin D-related genes other than the vitamin D receptor (VDR) gene and particularly have not examined the association with the retinoid X receptor alpha (RXRA) gene which may be a key vitamin D pathway gene. We conducted a nested case-control study of 734 cases and 1435 individually matched controls from a population-based prospective cohort study, the Northern Sweden Mammary Screening Cohort. Tag and functional SNPs were genotyped for the VDR, cytochrome p450 24A1 (CYP24A1), and RXRA genes. We also genotyped specific SNPs in four other genes related to vitamin D metabolism and signaling (GC/VDBP, CYP2R1, DHCR7, and CYP27B1). SNPs in the CYP2R1, DHCR7, and VDBP gene regions that were associated with circulating 25(OH)D concentration in GWAS were also associated with plasma 25(OH)D in our study (p-trend <0.005). After taking into account the false discovery rate, these SNPs were not significantly associated with breast cancer risk, nor were any of the other SNPs or haplotypes in VDR, RXRA, and CYP24A1. We observed no statistically significant associations between polymorphisms or haplotypes in key vitamin D-related genes and risk of breast cancer. These results, combined with the observation in this cohort and most other prospective studies of no association of circulating 25(OH)D with breast cancer risk, do not support an association between vitamin D and breast cancer risk.

T helper cell‑related cytokine gene polymorphisms and vitamin D pathway gene polymorphisms as predictors of survival probability in patients on renal replacement therapy.

Negative outcomes in patients on renal replacement therapy (RRT) may have a source in T helper (Th)-cell imbalance or vitamin D deficiency. We examined the association of genes encoding cytokines related to Th1 and Th2 cells and vitamin D pathway genes with survival probability of patients on RRT. The study included 1253 patients on hemodialysis. IL13, IL4R, IL18, IL12A, IL12B, IL28B, MCP1, GC, VDR, and RXRA gene polymorphisms were tested. The Kaplan-Meier method with the log‑rank test was used to estimate significance of survival probabilities. Patients carrying the IL13 minor T allele had an increased risk of death compared with CC subjects (log‑rank test, P = 0.005; hazard ratio [HR], 1.40; 95% confidence interval [CI], 1.11-1.76; P = 0.005). IL28B rs8099 917 GG patients had higher mortality rates compared with IL28B GT+TT carriers (log‑rank test, P = 0.04; HR, 1.82; 95% CI, 1.10-3.01; P =0.02). IL28B rs12979860 TT carriers had an increased risk of death compared with CC+CT carriers (log‑rank test, P = 0.02; HR, 1.62; 95% CI, 1.09-2.42; P = 0.02) only when they were negative for hepatitis B virus (HBV) or hepatitis C virus (HCV) infection. The prevalence of coronary artery disease differed significantly among patients with the IL28B rs12979860 TT genotype compared with CC+CT carriers (odds ratio, 1.87; 95% CI, 1.14-3.09; P = 0.01). There was no association between the GC, VDR, and RXRA nucleotide variants and survival. The IL13 rs20541 T allele and IL28B rs8099917 GG genotype are negative predictors of survival in patients on RRT, while the IL28B rs12979860 TT genotype increases the risk of death only in patients negative for HBV or HCV infection.

Disruption of the TSLP-TSLPR-LAP signaling between epithelial and dendritic cells through hyperlipidemia contributes to regulatory T-Cell defects in atherosclerotic mice

Regulatory T-Cells (Tregs) play a protective role against the development of atherosclerosis. Moreover, thymic stromal lymphopoietin (TSLP)/thymic stromal lymphopoietin receptor (TSLPR) signaling in myeloid dendritic cells (DCs) promote Treg differentiation. Here, we examined the potential role of TSLP/TSLPR on Treg homeostasis in atherosclerosis. The frequencies of both latency-associated peptide (LAP)+ and Foxp3+ Tregs were reduced in the thymus and spleen of ApoE−/− mice compared with C57BL/6 mice, and this effect was associated with decreased thymic output. The tolerogenic function of DCs obtained from ApoE−/− mice was compromised compared with those from C57BL/6 mice. The expression of TSLP and TSLPR was also inhibited in ApoE−/− mice. In addition, we found that ox-LDL attenuated TSLP expression in cultured thymic epithelial cells (TECs) through the activation of retinoid X receptor alpha (RXRA) and IL-1β and decreased LAP and PD-L1 expression in oxLDL-activated DCs while both were up-regulated in TSLP-activated DCs. We also observed that the TSLP-DCs mediated differentiation of Tregs was abrogated through LAP neutralization. Furthermore, TSLP injection rescued Treg defects in ApoE−/− mice. These findings suggest that Treg defects in ApoE−/− mice might partially be attributed to the disruption of TSLP-TSLPR-LAP signaling in epithelial cells (ECs) and DCs.

Polymorphisms in Genes Involved in Fatty Acid β-Oxidation Interact with Dietary Fat Intakes to Modulate the Plasma TG Response to a Fish Oil Supplementation

A large inter-individual variability in the plasma triglyceride (TG) response to an omega-3 polyunsaturated fatty acid (n-3 PUFA) supplementation has been observed. The objective was to examine gene-diet interaction effects on the plasma TG response after a fish oil supplementation, between single-nucleotide polymorphisms (SNPs) within genes involved in fatty acid β-oxidation and dietary fat intakes. Two hundred and eight (208) participants were recruited in the greater Quebec City area. The participants completed a six-week fish oil supplementation (5 g fish oil/day: 1.9–2.2 g EPA and 1.1 g DHA). Dietary fat intakes were measured using three-day food records. SNPs within RXRA, CPT1A, ACADVL, ACAA2, ABCD2, ACOX1 and ACAA1 genes were genotyped using TAQMAN methodology. Gene-diet interaction effects on the plasma TG response were observed for SNPs within RXRA (rs11185660, rs10881576 and rs12339187) and ACOX1 (rs17583163) genes. For rs11185660, fold changes in RXRA gene expression levels were different depending on SFA intakes for homozygotes T/T. Gene-diet interaction effects of SNPs within genes involved in fatty acid β-oxidation and dietary fat intakes may be important in understanding the inter-individual variability in plasma TG levels and in the plasma TG response to a fish oil supplementation.

Influence of PPARA, RXRA, NR1I2 and NR1I3 gene polymorphisms on the lipid-lowering efficacy and safety of statin therapy

The aim of the present study was investigate the association between six genetic variants in the nuclear receptor genes PPARA, RXRA, NR1I2 and NR1I3 and the lipid-lowering efficacy and safety of statin therapy. The study was carried out on 240 Brazilian hypercholesterolemic patients on simvastatin and atorvastatin therapy. The polymorphisms were analyzed by PCR-based methods. The NR1I3 rs2307424 genotype distribution was different between subjects with and without adverse drug reactions. Among subjects in the ADR group, no T/T homozygotes were observed for this polymorphism, while in the non-ADR group the frequency of this genotype was 19.4% (P = 0.007, after multiple testing corrections P = 0.042). The polymorphisms investigated in PPARA (rs1800206), RXRA (rs11381416), and NR1I2 (rs1523130) did not influence the lipid-lowering efficacy and safety of statin. Our results show the possible influence of NR1I3 genetic variant on the safety of statin.

Childhood bone mineral content is associated with methylation status of the RXRA promoter at birth.

Maternal vitamin D deficiency has been associated with reduced offspring bone mineral accrual. Retinoid-X receptor-alpha (RXRA) is an essential cofactor in the action of 1,25-dihydroxyvitamin D (1,25[OH]2 -vitamin D), and RXRA methylation in umbilical cord DNA has been associated with later offspring adiposity. We tested the hypothesis that RXRA methylation in umbilical cord DNA collected at birth is associated with offspring skeletal development, assessed by dual-energy X-ray absorptiometry, in a population-based mother-offspring cohort (Southampton Women's Survey). Relationships between maternal plasma 25-hydroxyvitamin D (25[OH]-vitamin D) concentrations and cord RXRA methylation were also investigated. In 230 children aged 4 years, a higher percent methylation at four of six RXRA CpG sites measured was correlated with lower offspring bone mineral content (BMC) corrected for body size (β = -2.1 to -3.4 g/SD, p = 0.002 to 0.047). In a second independent cohort (n = 64), similar negative associations at two of these CpG sites, but positive associations at the two remaining sites, were observed; however, none of the relationships in this replication cohort achieved statistical significance. The maternal free 25(OH)-vitamin D index was negatively associated with methylation at one of these RXRA CpG sites (β = -3.3 SD/unit, p = 0.03). Thus, perinatal epigenetic marking at the RXRA promoter region in umbilical cord was inversely associated with offspring size-corrected BMC in childhood. The potential mechanistic and functional significance of this finding remains a subject for further investigation.

Genetic Variants in Vitamin D Pathway Genes and Risk of Pancreas Cancer; Results from a Population-Based Case-Control Study in Ontario, Canada

Recent studies of 25-hydroxyvitamin D (25(OH)D) levels and pancreas cancer have suggested a potential role of the vitamin D pathway in the etiology of this fatal disease. Variants in vitamin-D related genes are known to affect 25(OH)D levels and function and it is unknown if these variants may influence pancreatic cancer risk. The association between 87 single nucleotide polymorphisms (SNPs) in 11 genes was evaluated within the Ontario Pancreas Cancer Study, a population-based case-control study. Pancreatic cancer cases with pathology confirmed adenocarcinoma were identified from the Ontario Cancer Registry (n = 628) and controls were identified through random digit dialing (n = 1193). Age and sex adjusted odds ratios (OR) and 95% confidence intervals (CI) were estimated by multivariate logistic regression. SNPs in the CYP24A1, CYP2R1, calcium sensing receptor (CASR), vitamin D binding protein (GC), retinoid X receptor-alpha (RXRA) and megalin (LRP2) genes were significantly associated with pancreas cancer risk. For example, pancreas cancer risk was inversely associated with CYP2R1 rs10741657 (AA versus GG, OR = 0.70; 95%CI: 0.51–0.95) and positively with CYP24A1 rs6127119 (TT versus CC. OR = 1.94; 95%CI: 1.28–2.94). None of the associations were statistically significant after adjustment for multiple comparisons. Vitamin D pathway gene variants may be associated with pancreas cancer risk and future studies are needed to understand the possible role of vitamin D in tumorigenesis and may have implications for cancer-prevention strategies.

Abstract 428: Decreased RXRα dampened APC-independent proteosomal targeting of β-catenin to cause increased intestinal tumorigenesis after 56-Fe radiation.

Abstract Purpose: Radiation exposure is a risk factor for colorectal cancer (CRC) and high linear energy transfer radiation (high-LET) radiation such as 56-Fe in outer space is expected to pose greater risk of CRC to space travelers. Earlier, we have reported increased intestinal tumorigenesis in heavy ion radiation (HZE) exposed APCMin/+ mice, a well studied model of CRC, and higher tumor incidence was due to increased stabilization and activation of β-catenin signaling after 56-Fe radiation exposure. However the molecular mechanism behind β-catenin activation is not yet explored. Levels of β-catenin are tightly regulated through its targeting to proteasomal degradation mediated via adenomatous polyposis coli (APC) dependent and independent pathways. The APC independent pathway of β-catenin degradation is facilitated via retinoid-X receptor alpha (RXRα). The present study aimed to investigate alterations in RXRα mediated proteasomal pathways in intestinal tumors induced by 56Fe relative to γ radiation. Materials and Methods: Six to eight weeks old female APCMin/+ mice (n=6 mice/ group) were irradiated with 5 Gy of gamma and isotoxic doses of 56Fe (4 Gy, energy: 1 GeV/nucleon; LET: 148 keV/μ) radiation. Mice were euthanized between 100 and 110 days after radiation and intestinal tumor samples were collected. Tumor samples were analyzed to deliniate the 56-Fe-induced changes in RXRα-proteasomal pathways using activity assays, Western blot and immunohistochemistry (IHC). Results: Higher proteasomal activity was observed in 56Fe-induced tumors accompanied with increased expression of proteasomal subunit 20S-β and increased expression/activation of proteosomal activator-200 (PA200) compared to γ radiation. However, we found decreased phosphorylation and consequent reduction in ubiquitination of β-catenin, leading to its increased cellular level in 56Fe relative to gamma radiation-induced tumors. We also show both by Western blot as well as IHC that exposure to 56-Fe radiation led to decreased RXRα expression, a key element in the APC-independent β-catenin degradation pathway. Conclusions: Although 56Fe radiation led to elevated proteasomal activity via overexpression of proteasomal subunit and its activator, we observed increased rather than decreased active β-catenin. We propose that increased β-catenin activity and consequent higher frequency of intestinal tumors is due to the inability of not only the APC-dependent but also the APC-independent RXRα-mediated pathway to facilitate ubiquitination of β-catenin and its subsequent proteasomal degradation. Taken together our study shows for the first time that 56-Fe radiation exposure modulates β-catenin stability in an APC-independent manner to promote intestinal tumorigenesis. Citation Format: Shubhankar Suman, Kamal Datta, Albert J. Fornace. Decreased RXRα dampened APC-independent proteosomal targeting of β-catenin to cause increased intestinal tumorigenesis after 56-Fe radiation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 428. doi:10.1158/1538-7445.AM2013-428

Abstract 3701: Tomato carotenoids and testosterone modulate mRNA and miRNA profiles during prostate carcinogenesis.

Abstract There is considerable evidence supporting the hypothesis that tomato carotenoids may contribute to prostate cancer prevention however the mechanisms remain uncertain. We propose that bioactive tomato components, such as lycopene, may impact testosterone signaling to inhibit prostate carcinogenesis. Four week-old C57/BL6 WT and TRAMP male mice were fed either control (AIN93G), 10% tomato powder or 0.25% lycopene beadlets. At eight weeks of age, mice were randomized among treatments: intact, castration, or castration + 2.5mg/kg testosterone repletion and animals sacrificed at 10 weeks of age. Prostate gene expression was quantified by Nanostring nCounter was used to quantify prostate gene expression with a 200 gene murine prostate carcinogenesis custom codeset and miRNA with codeset v1.1. Carotenoid analysis is by HPLC and proliferation (Ki67) by immunohistochemical evaluation. Feeding dietary tomato or lycopene increased serum lycopene, for example, in WT mice concentrations reached 335 +/- 33 nmol/L and 337 +/- 34 nmol/L in tomato and lycopene fed animals, respectively. As expected, the TRAMP genotype and testosterone stimulate proliferation with diet as a modulating factor. In the WT intact prostate, the tomato powder and lycopene diet decreased proliferation (p&amp;lt;0.009). The TRAMP genotype and endocrine status impacted the expression of multiple genes and miRNAs in several distinct patterns. For example, Birc5, Ki67, PCNA, BRCA1, ATM, Aurora kinase A / B, Cdc25c, Cyclin B2, cyclin E2, Fox M1,and miRs 15b, 16, 25, 200a, 200b, and 429 are increased in the TRAMP prostate, only when testosterone is present. Alternatively, TMPRSS2, CD31, CD34, Cox 2, nCad, Cyclin D1, Keratin 5, Shh, and p63, and miRs 145, 205, 328, and 423-5p are decreased in the TRAMP prostate in the presence of testosterone. Nkx3.1, miR 148a, and miR 375 are decreased upon castration whereas beta catenin, FGFR1 and TNF alpha are increased. Tomato and / or Lycopene modestly decreased expression of eCad, HIF 1 alpha, Paxillin, RxR alpha, Sca 1 and Vimentin and increased expression of CD133 and miR 22. These highly controlled in vivo studies document the strong interactions between genetic defects in the prostate, the hormonal environment, and diet in modulating early prostate carcinogenesis. Citation Format: Jennifer M. Thomas-Ahner, Lei Wan, Hsueh-Li Tan, Nancy E. Moran, Amy C. Elsen, Dennis K. Pearl, John W. Erdman, Steven K. Clinton. Tomato carotenoids and testosterone modulate mRNA and miRNA profiles during prostate carcinogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3701. doi:10.1158/1538-7445.AM2013-3701

Genetic variants in the vitamin D pathway and breast cancer disease-free survival

Epidemiological studies have investigated the association between vitamin D pathway genes and breast cancer risk; however, little is known about the association between vitamin D pathway genes and breast cancer prognosis. In a retrospective cohort of 1029 patients with early-stage breast cancer, we analyzed the association between 106 tagging single nucleotide polymorphisms (SNPs) in eight vitamin D pathway genes and breast cancer disease-free survival (DFS) using Cox regression analysis adjusted for known prognostic variables. Using a false discovery rate of 10%, six intronic SNPs were significantly associated with poorer DFS: retinoid-X receptor alpha (RXRA) SNPs (rs881658, rs11185659, rs10881583, rs881657 and rs7864987) and plasminogen activator and urokinase receptor (PLAUR) SNP (rs4251864). Treatment received (no systemic therapy, hormone therapy alone or chemotherapy) was an effect modifier of the RXRA SNPs association with DFS (P < 0.05); therefore, we stratified further analysis by treatment group. Among patients who did not receive systemic therapy, RXRA SNP [rs10881583 (P = 0.02)] was associated with poorer DFS, and among patients who received chemotherapy, RXRA SNPs (rs881658, rs11185659, rs10881583, rs881657 and rs7864987) were associated with poorer DFS (P < 0.001 for all SNPs). However, RXRA SNPs: rs10881583 (P < 0.001) and rs881657 (P = 0.02) were associated with improved DFS in patients treated with hormone therapy alone. Our results suggest that SNPs in the RXRA and PLAUR genes in the vitamin D pathway may contribute to breast cancer DFS. In particular, SNPs in RXRA may predict for poorer or improved DFS in patients, according to type of systemic treatment received. If validated, these markers could be used for risk stratification of breast cancer patients.