Abstract 2211: Anastrozole metabolism - Effect of UGT1A4 promoter variants on UGT1A4 gene expression and UGT1A4 enzymatic reactions i n vitro

Anastrozole belongs to the non-steroidal triazole-derivative group of aromatase inhibitors. Anastrozole is predominantly metabolized by Phase I oxidation with the potential for further Phase II glucuronidation. It also is subject to direct glucuronidation by UDP-glucuronosyltransferase1A4 (UGT1A4). Genetic variability in anastrozole metabolism may be one putative causal factor associated with inter-patient variability in response to therapy. The main objective of this study is to establish the potential effect of promoter SNPs found in the UGT1A4 promoter region on UGT1A4 expression and UGT1A4 enzymatic function in vitro. We demonstrate that UGT1A4 promoter SNPs -163G>A (p=0.009), -217T>G (p=0.009) and -219 C>T (p=0.014) are associated with the observed interindividual variability in anastrozole glucuronidation. To demonstrate the functional relevance of the -163G>A, -217T>G and -219 C>T variants within the UGT1A4 promoter, a 1500bp sequence fragment representing the most prevalent and the variant genotypes were studied for their ability to drive luciferase expression. Individually, the -163G>A variant and the -219 C>T variant in either or both alleles reduced the basal activity by 60% and 50% respectively. On the other hand, the -217 T>G variant in either or both alleles increased the basal expression by 55%. To explore the effects of multiple variants on promoter activity, a series of mutagenesis permutations was performed, replacing the bases at -163,-217 and -219 with common and variant alleles. Variants in -163 and -219, but not -217, showed a drop in luciferase activity by 60%. Similar reduction in luciferase activity was seen with variants in -163 and -217 but not -219 (47%), and also with variants in -217 and -219 but not -163 (36%) Complete variant haplotype (-163A, -217G and -219T) reduced basal activity by 35% from the reference promoter. In silco analysis revealed these variants are associated with changes in c-Myb, CdxA, S8, SRY, HSF and AP4 transcription factors binding sites. These data show that permutations on the common and variant genotype of -163, -217 and -219 alter transcription by 30-60%,indicating that nucleotide changes at these positions alter the activity of the UGT1A4 promoter and are therefore relevant for UGT1A4 regulation. UGT1A4 -163A,-217G and -219T variations were predicted to be associated with variations in transcriptional activity. However it is clear that transcription factors may differentially affect these SNPs and a