This chapter outlines the materials, methods, and procedures for the in vitro biological evaluation of retinoid-X-receptor (RXR) agonists including 6-(ethyl(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)amino)nicotinic acid (NEt-TMN), as well as several NEt-TMN analog compounds recently reported by our group. These methods have general applicability beyond this NEt-TMN case study, and can be employed to characterize and biologically evaluate other putative RXR agonists (rexinoids), and benchmarked against perhaps the most common rexinoid known as bexarotene (Bex), a drug awarded FDA approval for the treatment of cutaneous T-cell lymphoma in 1999 but that is also prescribed for non-small cell lung cancer and continues to be explored in multiple human cancer types. The side-effect profile of Bex treatment includes hypothyroidism and hypertriglyceridemia arising from the inhibition or activation of additional nuclear receptors that partner with RXR. Because rexinoids often exhibit selectivity for RXR activation, versus activating the retinoic-acid-receptor (RAR), rexinoid treatment avoids the cutaneous toxicity commonly associated as a side effect with retinoids. There are many examples of other potent rexinoids, where biological evaluation has contributed useful insight into qSAR studies on these compounds, often also benchmarked to Bex, as potential treatments for cancer. Because of differential pleiotropy in other pathways, even closely related rexinoids display unique side-effect and activity profiles. Notable examples of potent rexinoids in addition to Bex and NEt-TMN include CD3254, LGD100268, and 9-cis-UAB30. Indeed, the methods described herein to evaluate NEt-TMN and analogous rexinoids are generally applicable to a wider variety of potent, moderate, and even weak RXR ligands.In terms of in vitro biological evaluation, methods for a rapid and preliminary assessment of rexinoid activity are described by employing a biologically relevant, RXR-responsive element (RXRE)-mediated transcription assay in mammalian cells. In addition, a second, more sensitive assay is also detailed that utilizes activation of RXR-RXR homodimers in the context of a mammalian two-hybrid (M2H) luciferase assay. Methods for applying the M2H assay at different rexinoid concentrations are further described for the determination of EC50 values for rexinoids from dose-response curves.

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.

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.

OBJECTIVE: Polymorphisms in peroxisome proliferator activated receptor-γ (PPAR-γ) and retinoid X receptor-α (RXR-α) gene may alter metabolic syndrome (MetS) risks by increasing or decreasing the human adiponectin promoter activity in cells. To test this statement, three potentially functional SNPs of PPAR-γ and four SNPs of RXR-α with minor allele frequency (MAF) ≥0.05 in the Chinese Han population were identified from NCBI dbSNPs database to evaluate their associations with MetS.
METHODS: TaqMan assay was performed to test the genotypes in MetS patients (n = 901) and normal controls (n = 1009). Serum adiponectin concentration was measured by ELISA kit.
RESULTS: The variant genotypes rs2920502CG and CG/CC, rs4240711GG and AG/GG, rs4842194CC and CT/CC, rs3132291CT, CC and CT/CC were associated with MetS. Furthermore, in the haplotype of PPAR-γ gene, compared with the most common haplotype GC, haplotype CC was associated with an increased risk of MetS (crude p = 0.017). In the haplotype of RXR-α gene, haplotype GCGC was associated with a significant protective effect for MetS [adjusted p = 0.002, OR (95% CI) = 0.718 (0.585-0.882)] compared with the most common haplotype GTAT. After taking smoking, alcohol consumption and physical activity as environmental adjustment factors into the analysis, the result showed A1 A2 A4 A5 A6 A7 B1 (rs3856806, rs2920502, rs180128, rs1045570, rs3132291, rs4240711, rs4842194) was the best model (cross-validation consistency 10/10, p = 0.0107).
CONCLUSIONS: The present study suggested that the variant genotypes in PPAR-γ gene could increase the risk of MetS; however, genotypes in RXR-α gene could decrease the risk of MetS in a Chinese Han population.

The study reported by Lee and colleagues in this issue of the journal (beginning on page 185) incorporated global genetic variation within a new assessment of the outcome of a previously reported phase-III trial of low-dose 13-cis-retinoic acid (13-cRA) for preventing second primary tumors (SPT) or the recurrence of head-and-neck cancer. This analysis identified genotypes of common single-nucleotide polymorphisms (SNP) and cumulative effect and potential gene-gene interactions that were highly associated with increased placebo-arm risk (prognostic) and/or with reduced treatment-arm risk and longer event-free survival (predictive). For example, the wild-type rs3118570 SNP of the retinoid X receptor alpha gene (carried by 71% of the 13-cRA trial population) marked a 3.33-fold increased SPT/recurrence risk in the placebo arm and a 38% reduced risk in the treatment arm. Adding two other informative genotypes strengthened the treatment-arm risk reduction to 76%, although the genotype trio reflected only 13% of the trial population. This report extends the concept of personalized therapy to cancer prevention.

BACKGROUND: The serum levels of adiponectin are paradoxically decreased in obesity and may play important roles in the development of type 2 diabetes mellitus (T2DM). Potentially functional polymorphisms in the peroxisome proliferator-activated receptor-γ (PPAR-γ) and retinoid X receptor-α (RXR-α) genes may alter T2DM risks by increasing the human adiponectin promoter activity in cells. Therefore, we hypothesized that single nucleotide polymorphisms (SNPs) in PPAR-γ and RXR-α were associated with risk of T2DM. To test this hypothesis, three potentially functional SNPs of PPAR-γ and four of RXR-α with a minor allele frequency of ≥ 0.05 in the Chinese Han population were identified from the National Center for Biotechnology Information dbSNPs database to evaluate their association with T2DM.
METHODS: Polymerase chain reaction-restriction fragment length polymorphism was performed to test the genotypes in T2DM patients (n = 540) and normal controls (n = 604).
RESULTS: The variant genotypes rs2920502CC, rs3856806CT, rs3856806CT/TT, and rs4240711AG/GG were associated with T2DM. Furthermore, the prevalences of haplotype GTC and CTG in PPAR-γ and GTAC in RXR-α were less frequent in cases (17.1%, 2.6%, and 2.4%, respectively) than in controls (22.3%, 3.8%, and 6.6%, respectively), whereas GTGT in RXR-α was more frequent in cases (6.9%) than in controls (4.4%) (P < 0.05 for both two-sided χ(2) test and thousand times permutation tests). Patients with genotype CT/TT of rs3856806 and genotype AG/GG of rs4240711 had higher levels of serum adiponectin than those with the genotype CC and genotype AA (P = 0.026 and 0.021, respectively). Model X2 X5 X6 X7 (rs3856806, rs3132291, rs4240711, and rs4842194) was the best model with the highest test balanced accuracy (0.5764) (cross-validation consistency = 10/10) in the multifactor dimensionality reduction method.
CONCLUSIONS: The PPAR-γ and RXR-α gene variants associated with the development of T2DM in this study must be investigated in a larger population to reveal any potential effects on metabolism.