Tamoxifen is the standard-of-care treatment for estrogen receptor-positive premenopausal breast cancer. We examined tamoxifen metabolism via blood metabolite concentrations and germline variations of CYP3A5, CYP2C9, CYP2C19 and CYP2D6 in 587 premenopausal patients (Asians, Middle Eastern Arabs, Caucasian-UK; median age 39 years) and clinical outcome in 306 patients. N-desmethyltamoxifen (DM-Tam)/(Z)-endoxifen and CYP2D6 phenotype significantly correlated across ethnicities (R2: 53%, P<10−77). CYP2C19 and CYP2C9 correlated with norendoxifen and (Z)-4-hydroxytamoxifen concentrations, respectively (P<0.001). DM-Tam was influenced by body mass index (P<0.001). Improved distant relapse-free survival (DRFS) was associated with decreasing DM-Tam/(Z)-endoxifen (P=0.036) and increasing CYP2D6 activity score (hazard ratio (HR)=0.62; 95% confidence interval (CI), 0.43–0.91; P=0.013). Low (<14 nM) compared with high (>35 nM) endoxifen concentrations were associated with shorter DRFS (univariate P=0.03; multivariate HR=1.94; 95% CI, 1.04–4.14; P=0.064). Our data indicate that endoxifen formation in premenopausal women depends on CYP2D6 irrespective of ethnicity. Low endoxifen concentration/formation and decreased CYP2D6 activity predict shorter DRFS.

g-Glutamyl transpeptidases (GGTs) are essential for hydrolysis of the tripeptide glutathione (g-glutamate-cysteine-glycine) and glutathione S-conjugates since they are the only enzymes known to cleave the amide bond linking the g-carboxylate of glutamate to cysteine. In Arabidopsis thaliana, four GGT genes have been identified based on homology with animal GGTs. They are designated GGT1 (At4g39640), GGT2 (At4g39650), GGT3 (At1g69820), and GGT4 (At4g29210). By analyzing the expression of each GGT in plants containing GGT:b-glucuronidase fusions, the temporal and spatial pattern of degradation of glutathione and its metabolites was established, revealing appreciable overlap among GGTs. GGT2 exhibited narrow temporal and spatial expression primarily in immature trichomes, developing seeds, and pollen. GGT1 and GGT3 were coexpressed in most organs/ tissues. Their expression was highest at sites of rapid growth including the rosette apex, floral stem apex, and seeds and might pinpoint locations where glutathione is delivered to sink tissues to supplement high demand for cysteine. In mature tissues, they were expressed only in vascular tissue. Knockout mutants of GGT2 and GGT4 showed no phenotype. The rosettes of GGT1 knockouts showed premature senescence after flowering. Knockouts of GGT3 showed reduced number of siliques and reduced seed yield. Knockouts were used to localize and assign catalytic activity to each GGT. In the standard GGT assay with g-glutamyl p-nitroanilide as substrate, GGT1 accounted for 80% to 99% of the activity in all tissues except seeds where GGT2 was 50% of the activity. Protoplasting experiments indicated that both GGT1 and GGT2 are localized extracellularly but have different physical or chemical associations.

During minus-strand DNA synthesis, RNase H degrades viral RNA sequences, generating potential plus-strand DNA primers. However, selection of the 3′ polypurine tract (PPT) as the exclusive primer is required for formation of viral DNA with the correct 5′-end and for subsequent integration. Here we show a new function for the nucleic acid chaperone activity of HIV-1 nucleocapsid protein (NC) in reverse transcription: blocking mispriming by non-PPT RNAs. Three representative 20-nt RNAs from the PPT region were tested for primer extension. Each primer had activity in the absence of NC, but less than the PPT. NC reduced priming by these RNAs to essentially base-line level, whereas PPT priming was unaffected. RNase H cleavage and zinc coordination by NC were required for maximal inhibition of mispriming. Biophysical properties, including thermal stability, helical structure and reverse transcriptase (RT) binding affinity, showed significant differences between PPT and non-PPT duplexes and the trends were generally correlated with the biochemical data. Binding studies in reactions with both NC and RT ruled out a competition binding model to explain NC's observed effects on mispriming efficiency. Taken together, these results demonstrate that NC chaperone activity has a major role in ensuring the fidelity of plus-strand priming.

Estrogen-receptor positive breast cancer accounts for 75% of diagnosed breast cancers worldwide. There are currently two major options for adjuvant treatment: tamoxifen and aromatase inhibitors. Variability in metabolizing enzymes determines their pharmacokinetic profile, possibly affecting treatment response. Therefore, prediction of therapy outcome based on genotypes would enable a more personalized medicine approach, providing optimal therapy for each patient. In this review, the authors will discuss the current evidence on the most important metabolizing enzymes in endocrine therapy, with a special focus on CYP2D6 and its role in tamoxifen metabolism.

The presence or absence of estrogen and progesterone steroid hormone receptor expression (ER, PR) is an essential feature of invasive breast cancer and determines prognosis and endocrine treatment decisions. Among the four ER/PR receptor phenotypes, the ER-/PR+ is infrequent, and its clinical relevance has been controversially discussed. Thus, we investigated its clinical significance and gene expression pattern in large datasets. In a retrospective clinical study of 15,747 breast cancer patients, we determined the ER/PR subtype survival probabilities using Kaplan-Meier and Cox regression analyses. From The Cancer Genome Atlas (TCGA) breast cancer dataset, PAM50 expression signature and pathway analyses were performed to test for distinct molecular features. In our cohort, the ER-/PR+ phenotype has been observed at a frequency of 4.1 % and was associated with an improved 10-year survival for stage I cancers compared to the ER+/PR+ reference subtype (median; 95 % CI 88.1 %; 83-93 vs. 84.3 %; 82-86 %, P = 0.024) as was confirmed by multivariate analysis over the entire follow-up (HR 0.59, 95 % CI 0.38-0.92, P = 0.021). This association lacked significance when including all stages. ER-/PR+ patients treated with antihormonal agents (34.5 %) had shorter survival compared to their non-treated counterparts (Log-rank P = 0.0001). PAM50 signatures suggest a distinct configuration for the ER-/PR+ phenotype. This specific phenotype has been further separated by a set of 59 uniquely expressed genes. Our study supports the notion of the existence of an ER-/PR+ phenotype with clinical and molecular features distinct from the large group of ER+/PR+ patients.

IntroductionNot all breast cancer patients respond to tamoxifen treatment, possibly due to genetic predisposition. As tamoxifen-induced reductions in percent mammographic density (PMD) have been linked to the risk and prognosis of breast cancer, we conducted a candidate gene study to investigate the association between germline CYP2D6 polymorphisms and PMD change.MethodsBaseline and follow-up mammograms were retrieved for 278 tamoxifen-treated subjects with CYP2D6 metabolizer status (extensive (EM), heterozygous extensive/intermediate (hetEM/IM) or poor metabolizer (PM)). Logistic regression analyses were conducted comparing subjects who experienced >10% reduction in PMD to those who experienced ≤10% reduction or increase.ResultsAfter multivariate adjustment, PMD change was found to be significantly associated with the degree of CYP2D6 enzyme functionality (Ptrend = 0.021). Compared with EM, hetEM/IM and PM were 72% (95% confidence interval (CI): 0.10 to 0.79) and 71% (0.03 to 2.62) less likely to experience a >10% reduction, respectively.ConclusionsTamoxifen-induced change in PMD appears to have a genetic component.

Background The estrogen-related receptors (ERRs) are orphan nuclear receptors (NRs) with high sequence similarity to the estrogen receptor (ER). Peroxisome proliferator-activated receptor gamma coactivator-1 beta (PERC) specifically activates ER and ERRs, thus providing a link with breast cancer growth. Here we functionally characterized a non-synonymous PERC polymorphism, rs7732671, previously associated with increased risk for familial breast cancer, and tested its clinical relevance for breast cancer progression. Methods A mammalian two-hybrid assay was used to identify protein interactions using full-length and truncated NR (ERα, ERRα, ERRγ) and PERC. PERC rs7732671 variant allele (649G&gt;C_A203P) was created by site-directed mutagenesis. Expression of genes involved in cell glycolysis and/or direct ERR targets Eno1, PKM, LDHA and IDH3A, were analyzed in breast cancer cells and in breast tumors (N = 84). Cell supernatants were quantitated for lactate formation using liquid chromatography coupled mass spectrometry. PERC rs7732671 was genotyped in 896 postmenopausal early breast cancer patients treated with adjuvant tamoxifen and clinical outcome was analyzed using Kaplan-Meier and Cox regression analyses. Results Truncated PERC (aa129-426) containing the 649G&gt;C_A203P variant did not interact with ERα, but ERRα and ERRγ ligand-binding domains were activated. The PERC129-426-C allele enhanced ERRα activity by 21% and ERRγ by 67%, compared to the PERC129-426-G major allele (P&lt;0.005). Expression of ERR downstream genes Eno1, LDHA, IDH3A and lactate concentrations were increased in MCF7 and MDA-MB-231 overexpressing PERC-C as compared to PERC-G. In breast tumors, glycolytic or ERR target gene expression was increased in patients carrying the PERC-C allele. Patients carrying the PERC-C showed higher distant recurrence rates compared with patients of the G/G genotype (P = 0.01), which was confirmed in Cox regression adjusted to tumor size, nodal status, and grade (Hazard Ratio 2.06; 95% CI 1.26-3.38, P = 0.005 per C allele). Conclusion PERC rs7732671 variant 649C_203P enhances ERRα and ERRγ signaling independent of ER. Higher lactate concentrations in cells and increased ERR target gene expression in vitro and in patients with the PERC-C allele point to a modulation of aerobic glycolysis. The ERR/PERC axis is possibly linked with disease progression in breast cancer. Citation Format: Pilar H. Saladores, Reiner Hoppe, Wing-Yee Lo, Sibylle Cocciardi, Ute Hofmann, Ute Hamann, Peter Fasching, Marcus Schmidt, Hiltrud Brauch, Werner Schroth. Coactivator PPARGC1B Ala203Pro polymorphism is linked with estrogen-related receptor function and breast cancer outcome. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5485. doi:10.1158/1538-7445.AM2015-5485

Long-term adjuvant tamoxifen therapy for five years is the anti-estrogenic standard of care for ER-positive breast cancer in premenopausal patients. The metabolic activation of tamoxifen into the active metabolite endoxifen is mediated by Cytochrome P450 (CYP) 2D6 and it has been shown that CYP2D6 polymorphims can influence the clinical outcome of postmenopausal breast cancer patients (Schroth et al. JAMA 2009, Mürdter et al.CPT 2011). While this is currently still under debate awaiting the denouement (Brauch et al. JCO 2011), no such relationship has been investigated for premenopausal patients. We used the Prospective Study of Outcomes in Sporadic versus Hereditary Breast Cancer (POSH) collection to explore the pharmacogenetic relationship between CYP2D6 polymorphisms and tamoxifen outcome in premenopausal patients. POSH is an observational cohort study established to explore the effect of genetic factors on breast cancer prognosis (Eccles et al. BMC Cancer 2007). We genotyped genomic DNA of 772 patients with adjuvant tamoxifen (age 18 to 41 years) for CYP2D6 and other CYP enzymes by MALDI TOF-MS. Patients were assigned for CYP2D6 metabolizer status, poor (PM), intermediate (IM), and extensive (EM), and tested for clinical associations using Kaplan-Meier and Cox regression analyses. After a median follow-up of 4 years (0.1 - 8 years) with 24.6% of study participants reporting a relapse, Kaplan Meier survival analysis did not show an association between CYP2D6 metabolizer status and the occurrence of relapse. In contrast, CYP3A5 functional status predicted by the *1 genotype was associated with worse outcome compared to the CYP3A5 deficient status (*3). Since chemotherapy is a suggested confounder of the CYP2D6 tamoxifen pharmacogenetic effect and the majority of POSH patients had received chemotherapy prior to tamoxifen, there is a possibility that variation in the metabolism and cytotoxic effects of these agents may mask CYP2D6 dependent tamoxifen outcome. However, in premenopausal patients due to higher plasma estrogen concentrations, the inhibition of tumor ER signaling may likely depend on factors other than endoxifen formation. While this is currently being explored with respect to the underlying ER pharmacodynamic effects, the polymorphic CYP3A5, one of the major drug metabolizing enzymes, appears to be a candidate marker for the prognosis of premenopausal breast cancer. 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 2671. doi:1538-7445.AM2012-2671