James M. Rae

Activating ESR1 mutations in hormone-resistant metastatic breast cancer

Breast cancer is the most prevalent cancer in women, and over two-thirds of cases express estrogen receptor-α (ER-α, encoded by ESR1). Through a prospective clinical sequencing program for advanced cancers, we enrolled 11 patients with ER-positive metastatic breast cancer. Whole-exome and transcriptome analysis showed that six cases harbored mutations of ESR1 affecting its ligand-binding domain (LBD), all of whom had been treated with anti-estrogens and estrogen deprivation therapies. A survey of The Cancer Genome Atlas (TCGA) identified four endometrial cancers with similar mutations of ESR1. The five new LBD-localized ESR1 mutations identified here (encoding p.Leu536Gln, p.Tyr537Ser, p.Tyr537Cys, p.Tyr537Asn and p.Asp538Gly) were shown to result in constitutive activity and continued responsiveness to anti-estrogen therapies in vitro. Taken together, these studies suggest that activating mutations in ESR1 are a key mechanism in acquired endocrine resistance in breast cancer therapy.

Pharmacological characterization of 4-hydroxy-N-desmethyl tamoxifen, a novel active metabolite of tamoxifen.

The antiestrogen tamoxifen is extensively metabolized in patients to form a series of compounds with altered affinity for estrogen receptors (ERs), the primary target of this drug. Furthermore, these metabolites exhibit a range of partial agonist and antagonist activities for ER mediated effects that do not depend directly on their absolute affinity for ERs. Thus, clinical response to tamoxifen therapy is likely to depend on the aggregate effect of these different metabolites resulting from their abundance in the patient, their affinity for the receptors, and their agonist/antagonist profile. A recent study has shown that plasma concentrations of the tamoxifen metabolite 4-hydroxy-N-desmethyl tamoxifen (endoxifen), in patents undergoing tamoxifen therapy, are dependent on the cytochrome P450 (CYP) 206 genotype of the patient and that medications commonly prescribed to patients on tamoxifen therapy can also inhibit endoxifen production. In this study we characterized the properties of this metabolite with respect to binding to ERs, ability to inhibit estrogen stimulated breast cancer cell proliferation and the regulation of estrogen responsive genes. We demonstrate that endoxifen has essentially equivalent activity to the potent metabolite 4-hydroxy tamoxifen (4-OH-tam) often described as the active metabolite of this drug. Since plasma levels of endoxifen in patients with functional CYP2D6 frequently exceed the levels of 4-OH-tam, it seems likely that endoxifen is at least as important as 4-OH-tam to the overall activity of this drug and suggests that CYP2D6 status and concomitant administration of drugs that inhibit CYP2D6 activity have the potential to affect response to tamoxifen therapy.

Association of genetic variation in tamoxifen-metabolizing enzymes with overall survival and recurrence of disease in breast cancer patients

SummaryTamoxifen has been a mainstay of adjuvant therapy for breast cancer for many years. We sought to determine if genetic variability in the tamoxifen metabolic pathway influenced overall survival in breast cancer patients treated with tamoxifen. We examined functional polymorphisms in CYP2D6, the P450 catalyzing the formation of active tamoxifen metabolites, and UGT2B15, a Phase II enzyme facilitating the elimination of active metabolite in a retrospective study of breast cancer patients. We also examined whether the combination of variant alleles in SULT1A1 and UGT2B15 had more of an impact on overall survival in tamoxifen-treated patients than when the genes were examined separately.We conducted a retrospective study using archived paraffin blocks for DNA extraction and data from pathology reports and hospital tumor registry data for information on clinical characteristics, treatment, and outcomes (162 patients receiving tamoxifen and 175 who did not). Genotypes for CYP2D6 and UGT2B15 were obtained and Cox proportional hazards modeling was performed.After adjusting for age, race, stage of disease at diagnosis, and hormone receptor status, we found no significant association between CYP2D6 genotype and overall survival in either group of breast cancer patients. Tamoxifen-treated patients with UGT2B15 high activity genotypes had increased risk of recurrence and poorer survival. When UGT2B15 and SULT1A1 ‘at-risk’ alleles were combined, women with two variant alleles had significantly greater risk of recurrence and poorer survival than those with common alleles. These studies indicate that genetic variation in Phase II conjugating enzymes can influence the efficacy of tamoxifen therapy for breast cancer.

CYP2D6 Genotype and Tamoxifen Response in Postmenopausal Women with Endocrine-Responsive Breast Cancer: The Breast International Group 1-98 Trial

BACKGROUND Adjuvant tamoxifen therapy is effective for postmenopausal women with endocrine-responsive breast cancer. Cytochrome P450 2D6 (CYP2D6) enzyme metabolizes tamoxifen to clinically active metabolites, and CYP2D6 polymorphisms may adversely affect tamoxifen efficacy. In this study, we investigated the clinical relevance of CYP2D6 polymorphisms. METHODS We obtained tumor tissues and isolated DNA from 4861 of 8010 postmenopausal women with hormone receptor-positive breast cancer who enrolled in the randomized, phase III double-blind Breast International Group (BIG) 1-98 trial between March 1998 and May 2003 and received tamoxifen and/or letrozole treatment. Extracted DNA was used for genotyping nine CYP2D6 single-nucleotide polymorphisms using polymerase chain reaction-based methods. Genotype combinations were used to categorize CYP2D6 metabolism phenotypes as poor, intermediate, and extensive metabolizers (PM, IM, and EM, respectively; n = 4393 patients). Associations of CYP2D6 metabolism phenotypes with breast cancer-free interval (referred to as recurrence) and treatment-induced hot flushes according to randomized endocrine treatment and previous chemotherapy were assessed. Cox proportional hazards models were used to calculate hazard ratios (HRs) and 95% confidence intervals (CIs). All statistical tests were two-sided. RESULTS No association between CYP2D6 metabolism phenotypes and breast cancer-free interval was observed among patients who received tamoxifen monotherapy without previous chemotherapy (P = .35). PM or IM phenotype had a non-statistically significantly reduced risk of breast cancer recurrence compared with EM phenotype (PM or IM vs EM, HR of recurrence = 0.86, 95% CI = 0.60 to 1.24). CYP2D6 metabolism phenotype was associated with tamoxifen-induced hot flushes (P = .020). Both PM and IM phenotypes had an increased risk of tamoxifen-induced hot flushes compared with EM phenotype (PM vs EM, HR of hot flushes = 1.24, 95% CI = 0.96 to 1.59; IM vs EM, HR of hot flushes = 1.23, 95% CI = 1.05 to 1.43). CONCLUSIONS CYP2D6 phenotypes of reduced enzyme activity were not associated with worse disease control but were associated with increased hot flushes, contrary to the hypothesis. The results of this study do not support using the presence or absence of hot flushes or the pharmacogenetic testing of CYP2D6 to determine whether to treat postmenopausal breast cancer patients with tamoxifen.

CYP2D6 and UGT2B7 Genotype and Risk of Recurrence in Tamoxifen-Treated Breast Cancer Patients

BACKGROUND Adjuvant tamoxifen therapy substantially decreases the risk of recurrence and mortality in women with hormone (estrogen and/or progesterone) receptor-positive breast cancer. Previous studies have suggested that metabolic conversion of tamoxifen to endoxifen by cytochrome P450 2D6 (CYP2D6) is required for patient benefit from tamoxifen therapy. METHODS Tumor specimens from a subset of postmenopausal patients with hormone receptor-positive early-stage (stages I, II, and IIIA) breast cancer, who were enrolled in the randomized double-blind Arimidex, Tamoxifen, Alone or in Combination (ATAC) clinical trial, were genotyped for variants in CYP2D6 (N = 1203 patients: anastrozole [trade name: Arimidex] group, n = 615 patients; tamoxifen group, n = 588 patients) and UDP-glucuronosyltransferase-2B7 (UGT2B7), whose gene product inactivates endoxifen (N = 1209 patients; anastrozole group, n = 606 patients; tamoxifen group, n = 603 patients). Genotyping was performed using polymerase chain reaction-based TaqMan assays. Based on the genotypes for CYP2D6, patients were classified as poor metabolizer (PM), intermediate metabolizer (IM), or extensive metabolizer (EM) phenotypes. We evaluated the association of CYP2D6 and UGT2B7 genotype with distant recurrence (primary endpoint) and any recurrence (secondary endpoint) by estimating the hazard ratios (HRs) and corresponding 95% confidence intervals (CIs) using Cox proportional hazards models. All statistical tests were two-sided. RESULTS After a median follow-up of 10 years, no statistically significant associations were observed between CYP2D6 genotype and recurrence in tamoxifen-treated patients (PM vs EM: HR for distant recurrence = 1.25, 95% CI = 0.55 to 3.15, P = .64; HR for any recurrence = 0.99, 95% CI = 0.48 to 2.08, P = .99). A near-null association was observed between UGT2B7 genotype and recurrence in tamoxifen-treated patients. No associations were observed between CYP2D6 and UGT2B7 genotypes and recurrence in anastrozole-treated patients. CONCLUSION The results do not support the hypothesis that CYP2D6 genotype predicts clinical benefit of adjuvant tamoxifen treatment among postmenopausal breast cancer patients.

Endoxifen, a secondary metabolite of tamoxifen, and 4-OH-tamoxifen induce similar changes in global gene expression patterns in MCF-7 breast cancer cells

We recently demonstrated that endoxifen (4-hydroxy-N-desmethyl-tamoxifen), a pharmacogenetically regulated metabolite of tamoxifen, is equipotent to 4-hydroxy-tamoxifen (4-OH-Tam) with respect to estrogen receptor binding and inhibition of 17β-estradiol (E2)-induced cell proliferation. Endoxifen was also found to be more abundant in human plasma than 4-OH-Tam, and its formation has been shown to be primarily catalyzed by cytochrome P450 2D6 (CYP2D6). Here, we report studies evaluating the effects of endoxifen, 4-OH-Tam, and E2 on gene expression in MCF-7 cells using Affymetrix U133A GeneChip Arrays (Santa Clara, CA). We detected 4062 genes that were E2-regulated (1924 induced; 2138 suppressed), and the ratio of E2-induced versus E2-suppressed genes was consistent regardless of the cutoff value. In the presence of E2, 2444 and 2390 genes were affected by 4-OH-Tam and endoxifen, respectively, when no minimal -fold change cutoff was implemented. The majority of genes regulated by the tamoxifen metabolites were also E2-responsive (74.4 and 73.3%, respectively). Endoxifen and 4-OH-Tam had overlapping effects on 1365 E2-sensitive genes, whose -fold effects between these metabolites were highly correlated (R2 = 0.99). A significant correlation was also found between the -fold effects of 249 E2-insensitive genes coregulated by both metabolites (R2 = 0.99). Hierarchical clustering analysis demonstrated similar gene regulation patterns between these metabolites, which were distinct from E2 or vehicle treatment patterns. Using real time-polymerase chain reaction, we validated the gene expression patterns of five genes that were differentially regulated by endoxifen and 4-OH-Tam. We conclude that endoxifen and 4-OH-Tam have similar effects on global gene expression patterns in MCF-7 cells and that the majority of the affected genes are estrogen-regulated genes.

Genes regulated by estrogen in breast tumor cells in vitro are similarly regulated in vivo in tumor xenografts and human breast tumors.

BackgroundEstrogen plays a central role in breast cancer pathogenesis. Although many studies have characterized the estrogen regulation of genes using in vitro cell culture models by global mRNA expression profiling, it is not clear whether these genes are similarly regulated in vivo or how they might be coordinately expressed in primary human tumors.ResultsWe generated DNA microarray-based gene expression profiles from three estrogen receptor α (ERα)-positive breast cancer cell lines stimulated by 17β-estradiol (E2) in vitro over a time course, as well as from MCF-7 cells grown as xenografts in ovariectomized athymic nude mice with E2 supplementation and after its withdrawal. When the patterns of genes regulated by E2 in vitro were compared to those obtained from xenografts, we found a remarkable overlap (over 40%) of genes regulated by E2 in both contexts. These patterns were compared to those obtained from published clinical data sets. We show that, as a group, E2-regulated genes from our preclinical models were co-expressed with ERα in a panel of ERα+ breast tumor mRNA profiles, when corrections were made for patient age, as well as with progesterone receptor. Furthermore, the E2-regulated genes were significantly enriched for transcriptional targets of the myc oncogene and were found to be coordinately expressed with Myc in human tumors.ConclusionOur results provide significant validation of a widely used in vitro model of estrogen signaling as being pathologically relevant to breast cancers in vivo.

Composite Functional Genetic and Comedication CYP2D6 Activity Score in Predicting Tamoxifen Drug Exposure Among Breast Cancer Patients

Accurate assessment of CYP2D6 phenotypes from genotype is inadequate in patients taking CYP2D6 substrate together with CYP2D6 inhibitors. A novel CYP2D6 scoring system is proposed that incorporates the impact of concomitant medications with the genotype in calculating the CYP2D6 activity score. Training (n = 159) and validation (n = 81) data sets were obtained from a prospective cohort tamoxifen pharmacogenetics registry. Two inhibitor factors were defined: 1 genotype independent and 1 genotype based. Three CYP2D6 gene scoring systems, and their combination with the inhibitor factors, were compared. These 3 scores were based on Zineh, Zanger, and Gaedigks approaches. Endoxifen/NDM‐Tam plasma ratio was used as the phenotype. The overall performance of the 3 gene scoring systems without consideration of CYP2D6‐inhibiting medications in predicting CYP2D6 phenotype was poor in both the training set (r2 = 0.24, 0.22, and 0.18) and the validation set (r2 = 0.30, 0.24, and 0.15). Once the CYP2D6 genotype‐independent inhibitor factor was integrated into the score calculation, the R 2 values in the training and validation data sets were nearly twice as high as the genotype‐only scoring model: (0.44, 0.43, 0.38) and (0.53, 0.50, 0.41), respectively. The integration of the inhibitory effect of concomitant medications with the CYP2D6 genotype into the composite CYP2D6 activity score doubled the ability to predict the CYP2D6 phenotype. However, endoxifen phenotypes still varied substantially, even with incorporation of CYD2D6 genotype and inhibiting factors, suggesting that other, as yet unidentified factors must be involved in tamoxifen activation.

The Endocannabinoid Anandamide Is a Substrate for the Human Polymorphic Cytochrome P450 2D6

Members of the cytochrome P450 (P450) family of drug-metabolizing enzymes are present in the human brain, and they may have important roles in the oxidation of endogenous substrates. The polymorphic CYP2D6 is one of the major brain P450 isoforms and has been implicated in neurodegeneration, psychosis, schizophrenia, and personality traits. The objective of this study was to determine whether the endocannabinoid arachidonoylethanolamide (anandamide) is a substrate for CYP2D6. Anandamide is the endogenous ligand to the cannabinoid receptor CB1, which is also activated by the main psychoactive component in marijuana. Signaling via the CB1 receptor alters sensory and motor function, cognition, and emotion. Recombinant CYP2D6 converted anandamide to 20-hydroxyeicosatetraenoic acid ethanolamide and 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acid ethanolamides (EET-EAs) with low micromolar Km values. CYP2D6 further metabolized the epoxides of anandamide to form novel dioxygenated derivatives. Human brain microsomal and mitochondrial preparations metabolized anandamide to form hydroxylated and epoxygenated products, respectively. An inhibitory antibody against CYP2D6 significantly decreased the mitochondrial formation of the EET-EAs. To our knowledge, anandamide and its epoxides are the first eicosanoid-like molecules to be identified as CYP2D6 substrates. Our study suggests that anandamide may be a physiological substrate for brain mitochondrial CYP2D6, implicating this polymorphic enzyme as a potential component of the endocannabinoid system in the brain. This study also offers support to the hypothesis that neuropsychiatric phenotype differences among individuals with genetic variations in CYP2D6 could be ascribable to interactions of this enzyme with endogenous substrates.

Common origins of MDA-MB-435 cells from various sources with those shown to have melanoma properties

Recently, the tissue origin of MDA-MB-435 cell line has been the subject of considerable debate. In this study, we set out to determine whether MDA-MB-435-DTP cells shown to express melanoma-specific genes were identical to various other MDA-MB-435 cell stocks worldwide. CGH-microarray, genetic polymorphism genotyping, microsatellite fingerprint analysis and/or chromosomal number confirmed that the MDA-MB-435 cells maintained at the Lombardi Comprehensive Cancer Center (MDA-MB-435-LCC) are almost identical to the MDA-MB-435-DTP cells, and showed a very similar profile to those obtained from the same original source (MD Anderson Cancer Center) but maintained independently (MDA-MB-435-PMCC). Gene expression profile analysis confirmed common expression of genes among different MDA-MB-435-LCC cell stocks, and identified some unique gene products in MDA-MB-435-PMCC cells. RT-PCR analysis confirmed the expression of the melanoma marker tyrosinase across multiple MDA-MB-435 cell stocks. Collectively, our results show that the MDA-MB-435 cells used widely have identical origins to those that exhibit a melanoma-like gene expression signature, but exhibit a small degree of genotypic and phenotypic drift.