Pia Wegman

Genotype of metabolic enzymes and the benefit of tamoxifen in postmenopausal breast cancer patients

BackgroundTamoxifen is widely used as endocrine therapy for oestrogen-receptor-positive breast cancer. However, many of these patients experience recurrence despite tamoxifen therapy by incompletely understood mechanisms. In the present report we propose that tamoxifen resistance may be due to differences in activity of metabolic enzymes as a result of genetic polymorphism. Cytochrome P450 2D6 (CYP2D6) and sulfotransferase 1A1 (SULT1A1) are polymorphic and are involved in the metabolism of tamoxifen. The CYP2D6*4 and SULT1A1*2 genotypes result in decreased enzyme activity. We therefore investigated the genotypes of CYP2D6 and SULT1A1 in 226 breast cancer patients participating in a trial of adjuvant tamoxifen treatment in order to validate the benefit from the therapy.MethodsThe patients were genotyped using PCR followed by cleavage with restriction enzymes.ResultsCarriers of the CYP2D6*4 allele demonstrated a decreased risk of recurrence when treated with tamoxifen (relative risk = 0.28, 95% confidence interval = 0.11–0.74, P = 0.0089). A similar pattern was seen among the SULT1A1*1 homozygotes (relative risk = 0.48, 95% confidence interval = 0.21–1.12, P = 0.074). The combination of CYP2D6*4 and/or SULT1A1*1/*1 genotypes comprised 60% of the patients and showed a 62% decreased risk of distant recurrence with tamoxifen (relative risk = 0.38, 95% confidence interval = 0.19–0.74, P = 0.0041).ConclusionThe present study suggests that genotype of metabolic enzymes might be useful as a guide for adjuvant endocrine treatment of postmenopausal breast cancer patients. However, results are in contradiction to prior hypotheses and the present sample size is relatively small. Findings therefore need to be confirmed in a larger cohort.

A Mechanistic Hypothesis for the Cytochrome P450-Catalyzed Cis–Trans Isomerization of 4-Hydroxytamoxifen: An Unusual Redox Reaction

We provide a detailed description of the cis-trans isomerization of 4-hydroxytamoxifen/endoxifen catalyzed by several isoforms from the cytochrome P450 (CYP) superfamily, including CYP1B1, CYP2B6, and CYP2C19. We show that the reactions mainly involve redox processes catalyzed by CYP. DFT calculation results strongly suggest that the isomerization occurs via a cationic intermediate. The cationic cis-isomer is more than 3 kcal/mol more stable than the trans form, resulting in an easier conversion from trans-to-cis than cis-to-trans. The cis-trans isomerization is a rarely reported CYP reaction and is ascribed to the lack of a second abstractable proton on the ethenyl group of the triarylvinyl class of substrates. The cationic intermediates thus formed instead of the stable dehydrogenation products allow for isomerization to occur. As a comparison, the reactions for the tamoxifen derivatives are compared to those of other substrates, 4-hydroxyacetanilide and raloxifene, for which the stable dehydrogenation products are formed.

Conformational Enantiomerization and Estrogen Receptor α Binding of Anti-Cancer Drug Tamoxifen and Its Derivatives

The anticancer drug tamoxifen (TAM) displays two chiral vinyl propeller structures, which interconvert so rapidly that the process is undetectable on the NMR time scale. In the present work, the enantiomerization processes were investigated with molecular modeling techniques. The threshold mechanisms probed at the different rings were shown to be identical, i.e., involving a synchronous three-ring flip, with a correlated rotation of the rings. In order to reveal the pharmacological profiles of the two chiral forms, we performed structural studies on the ligand binding domain of estrogen receptor α (ERα LBD) and associated ligands. The enantiomers, with opposite torsional twist, were found to be discriminated by ERα. For TAM and its main metabolites, the effects of the stereoselectivity of ERα are overcome by the low energy cost for helical inversion between the two torsional enantiomers, estimated to be ∼3 kcal/mol.

HER4 tumor expression in breast cancer patients randomized to treatment with or without tamoxifen

The human epidermal growth factor receptor (HER) 4 is a relative of HER2 and has been associated to endocrine breast cancer and prediction of tamoxifen response. In addition to PI3K/Akt and MAPK pathway activation, ligand binding to HER4 triggers proteolytic cleavage and release of an intracellular receptor domain (4ICD) with signaling properties. The aim of the present study was to analyze HER4 protein expression and intracellular localization in breast cancer tissue from patients randomized to treatment with or without adjuvant tamoxifen. To investigate HER4 expression and localization in response to estradiol (E2) and 4-hydroxytamoxifen (4-OHT) exposure, we also performed in vitro studies. Cytoplasmic, nuclear and membrane expression of HER4 protein was evaluated by immunohistochemical staining in tumor tissue from 912 breast cancer patients. Three different breast epithelia cancer cell lines were exposed to E2 and 4-OHT and mRNA expression was analyzed using qPCR. Further, nuclear and cytoplasmic proteins were separated and analyzed with western blotting. We found an association between nuclear HER4 protein expression and ER-positivity (P=0.004). Furthermore, significant association was found between cytoplasmic HER4 and ER-negativity (P<0.0005), PgR-negativity (P<0.0005), tumor size >20 mm (P=0.001) and HER2-negativity (P=0.008). However, no overall significance of HER4 on recurrence-free survival was found. After E2 exposure, HER4 mRNA and protein expression had decreased in two cell lines in vitro yet no changes in nuclear or cytoplasmic protein fractions were seen. In conclusion, nuclear HER4 seem to be co-located with ER, however, we did not find support for overall HER4 expression in independently predicting response of tamoxifen treatment. The possible influence of separate isoforms was not tested and future studies may further evaluate HER4 significance.

Biological significance of allele specific loss of the p53 gene in breast carcinomas.

CTRC-AACR San Antonio Breast Cancer Symposium: 2008 Abstracts Abstract #4064 Background: The p53 tumour suppressor gene has a central role in the defence against cancer, including breast cancer, and contains a polymorphic variant (Arg/Pro) at codon 72 that has been shown to have different biological properties regarding apoptosis and cell cycle arrest. Earlier studies have shown allele specific loss of heterozygosity (LOH) at this particular site and we aimed to investigate its biological relevance in codon 72 heterozygous breast cancer patients (i.e. survival and age of disease onset). Method: 199 postmenopausal cases were analyzed for LOH using MegaBACE1000 and statistics was performed using Statistical Package for Social Sciences. As reference for loss/retention of heterozygosity at the Arg or Pro allele DNA from 30 heterozygous blood donors included in a population based DNA bank was used. Results: LOH was found in totally 124 (62.3%) patients and the Pro allele (n = 103) was significantly more often deleted compared to the Arg allele (n = 21) ( p = 0.001). Patients with LOH of the Arg allele were diagnosed at an earlier age (mean age 62.5 years) than those with loss of the Pro allele (mean age 69.2 years) ( p = 0.011). LOH of the Arg allele was also associated with worse survival ( p = 0.05). Tumour size or number of positive lymph nodes was not associated with LOH. Conclusion: We found a non-random loss of the codon 72 Pro allele, implying the existence of a selective pressure during carcinogenesis that confers a growth advantage to cells lacking the Pro allele. We also found that earlier age of onset was associated with loss of the Arg allele. These results were achieved from primary tumors without any influence of adjuvant treatment, possibly showing a picture of the natural history of carcinogenesis. The worsen prognosis associated with loss of the Arg allele is somewhat more complex to interpret since recurrence free survival could be influenced by genotype, mutational status and adjuvant treatment. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4064.