Matthew J. Sikora

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.

Cytochrome P450 2D6 Activity Predicts Discontinuation of Tamoxifen Therapy in Breast Cancer Patients

The selective estrogen receptor modulator tamoxifen is routinely used for treatment and prevention of estrogen-receptor-positive breast cancer. Studies of tamoxifen adherence suggest that over half of patients discontinue treatment before the recommended 5 years. We hypothesized that polymorphisms in CYP2D6, the enzyme responsible for tamoxifen activation, predict for tamoxifen discontinuation. Tamoxifen-treated women (n=297) were genotyped for CYP2D6 variants and assigned a ‘score’ based on predicted allele activities from 0 (no activity) to 2 (high activity). Correlation between CYP2D6 score and discontinuation rates at 4 months was tested. We observed a strong nonlinear correlation between higher CYP2D6 score and increased rates of discontinuation (r2=0.935, P=0.018). These data suggest that presence of active CYP2D6 alleles may predict for higher likelihood of tamoxifen discontinuation. Therefore, patients who may be most likely to benefit from tamoxifen may paradoxically be most likely to discontinue treatment prematurely.

The Androgen Metabolite 5α-androstane-3β,17β-diol (3βAdiol) Induces Breast Cancer Growth via Estrogen Receptor: Implications for Aromatase Inhibitor Resistance

The aromatase inhibitors (AIs) are used to treat estrogen receptor-positive (ER+) breast tumors in post-menopausal women, and function by blocking the conversion of adrenal androgens to estrogens by the enzyme CYP19 aromatase. Breast cancer patients receiving AI therapy have circulating estrogen levels below the level of detection; however, androgen concentrations remain unchanged. We were interested in studying the effects of androgens on breast cancer cell proliferation under profound estrogen-deprived conditions. Using in vitro models of estrogen-dependent breast cancer cell growth we show that the androgens testosterone and 5α-dihydrotestosterone induce the growth of MCF-7, T47D and BT-474 cells in the absence of estrogen. Furthermore, we demonstrate that under profound estrogen-deprived conditions these breast cancer cells up-regulate steroidogenic enzymes that can metabolize androgens to estrogens. Lastly, we found that the downstream metabolite of 5α-dihydrotestosterone, 5α-androstane-3β,17β-diol (3βAdiol), is estrogenic in breast cancer cells, and induces growth and ER-signaling via activation of ERα. In conclusion, our results show that breast cancer cells deprived of estrogen up-regulate steroidogenic enzymes and metabolize androgens to estrogen-like steroids. The generation of estrogen-like steroids represents a potential mechanism of resistance to aromatase inhibitors.

Invasive Lobular Carcinoma Cell Lines Are Characterized by Unique Estrogen-Mediated Gene Expression Patterns and Altered Tamoxifen Response

Invasive lobular carcinoma (ILC) is a histologic subtype of breast cancer that is frequently associated with favorable outcomes, as approximately 90% of ILC express the estrogen receptor (ER). However, recent retrospective analyses suggest that patients with ILC receiving adjuvant endocrine therapy may not benefit as much as patients with invasive ductal carcinoma. On the basis of these observations, we characterized ER function and endocrine response in ILC models. The ER-positive ILC cell lines MDA MB 134VI (MM134) and SUM44PE were used to examine the ER-regulated transcriptome via gene expression microarray analyses and ER ChIP-Seq, and to examine response to endocrine therapy. In parallel, estrogen response was assessed in vivo in the patient-derived ILC xenograft HCI-013. We identified 915 genes that were uniquely E2 regulated in ILC cell lines versus other breast cancer cell lines, and a subset of these genes were also E2 regulated in vivo in HCI-013. MM134 cells were de novo tamoxifen resistant and were induced to grow by 4-hydroxytamoxifen, as well as other antiestrogens, as partial agonists. Growth was accompanied by agonist activity of tamoxifen on ER-mediated gene expression. Though tamoxifen induced cell growth, MM134 cells required fibroblast growth factor receptor (FGFR)-1 signaling to maintain viability and were sensitive to combined endocrine therapy and FGFR1 inhibition. Our observation that ER drives a unique program of gene expression in ILC cells correlates with the ability of tamoxifen to induce growth in these cells. Targeting growth factors using FGFR1 inhibitors may block survival pathways required by ILC and reverse tamoxifen resistance.

Invasive lobular carcinoma of the breast: patient response to systemic endocrine therapy and hormone response in model systems.

Invasive lobular carcinoma of the breast (ILC) represents 10-15% of all newly diagnosed breast cancers, affecting ∼30,000 women annually in the United States. However, ILC is critically understudied as a breast cancer subtype. Though the vast majority of ILC are estrogen receptor-positive and present with overall favorable biomarkers, ILC patients do not benefit from improved outcomes versus other breast cancer patients. Patient outcomes, in particular in response to endocrine therapies, are not well understood in ILC, due in large part to the lack of prospective identification in large clinical trials. Further, there is a lack of laboratory models to study cell signaling, hormone response, and endocrine resistance in ILC. In this review, we provide an overview of clinicopathological features of ILC tumors, discuss issues with clinical management, and highlight the disconnect between ILC biomarkers and patient outcomes. We review currently available data on ILC patient outcomes, with a focus on response to endocrine therapy. Additionally, we describe currently available laboratory models for understanding hormone response in ILC cells, and review current data on these model systems. The promise for new insight into ILC, based on extensive representation of the disease in recent large scale genomic studies, is also discussed. Increasing understanding of endocrine response in ILC represents a critical area for future research to improve patient outcomes for this understudied breast cancer subtype.

Anti-oxidant treatment enhances anti-tumor cytotoxicity of (-)-gossypol

We showed that tumor cells with wild-type p53 and high levels of Bcl-xL are cisplatin resistant but are induced to undergo apoptosis by (-)-gossypol, making this a promising agent for overcoming cisplatin resistance. However, some cells in a population with this phenotype are not killed and continue to survive. Conversely, tumor cells with low Bcl-xL expression and either wild type or mutant p53 are relatively cisplatin sensitive and do not exhibit such high levels of apoptosis. However, these do undergo progressive loss of viability after (-)-gossypol that may not be tumor specific. We sought to elucidate the basis for these observations using cDNA microarray analysis of (-)-gossypol treated cisplatin sensitive and resistant cells. Genes in the reactive oxygen species (ROS) pathway were highly up-regulated in response to (-)-gossypol. The up-regulation was of much greater magnitude in cisplatin sensitive than resistant cells. Staining with an oxidation reporter dye confirmed differential induction of ROS in tumor cells with low Bcl-xL. As (-)-gossypol is known to undergo oxidative metabolism in vivo, ROS generation may be responsible for both off-target cytotoxicity and inactivation of the drug. In agreement with this hypothesis, oxidation of (-)-gossypol by pre-treatment with hydrogen peroxide eliminated its activity. Combined treatment with the antioxidant N-acetyl-cysteine (NAC) to block ROS increased (-)-gossypol-induced cytotoxicity to tumor but not normal cells. Furthermore, NAC increased the induction of apoptosis as measured by the sub G1 population, in both cisplatin sensitive and resistant cells. We postulate that concurrent treatment with antioxidant to block ROS prevents oxidative inactivation of (-)-gossypol and limits off-target toxicity allowing more potent (-)-gossypol-induced anti tumor activity.

Discovery of naturally occurring ESR1 mutations in breast cancer cell lines modelling endocrine resistance

Resistance to endocrine therapy remains a major clinical problem in breast cancer. Genetic studies highlight the potential role of estrogen receptor-α (ESR1) mutations, which show increased prevalence in the metastatic, endocrine-resistant setting. No naturally occurring ESR1 mutations have been reported in in vitro models of BC either before or after the acquisition of endocrine resistance making functional consequences difficult to study. We report the first discovery of naturally occurring ESR1Y537C and ESR1Y537S mutations in MCF7 and SUM44 ESR1-positive cell lines after acquisition of resistance to long-term-estrogen-deprivation (LTED) and subsequent resistance to fulvestrant (ICIR). Mutations were enriched with time, impacted on ESR1 binding to the genome and altered the ESR1 interactome. The results highlight the importance and functional consequence of these mutations and provide an important resource for studying endocrine resistance.ESR1 mutations occur in endocrine-resistant patients but have not yet been reported in in vitro models of breast cancer. Here, the authors report the discovery of naturally occurring ESR1Y537Cand ESR1Y537S mutations in two breast cancer cell lines after acquisition of resistance to long-term-estrogen-deprivation.

High-efficiency genotype analysis from formalin-fixed, paraffin-embedded tumor tissues

Single-nucleotide polymorphisms (SNPs) can be assayed using DNA isolated from archival formalin-fixed, paraffin-embedded (FFPE) samples, making retrospective pharmacogenetic studies possible. In this study, we describe methods that significantly increase the number of SNP determinations possible using FFPE samples. Quantifying the amount of DNA amenable to PCR (amplification-quality DNA, AQ-DNA) allows a significant reduction in the amount of sample required for Taqman-based SNP assays. Optimizing AQ-DNA input increases PCR amplification efficiency and SNP determination accuracy. DNA was extracted from 39 FFPE tumor sections and matched tumor and stromal cores, which were of the type used to generate tissue microarrays. Sections and tumor cores yielded sufficient AQ-DNA for more than 1000 SNP determinations. Seven SNPs were assessed following individual assay optimization for minimal AQ-DNA. Genotypes from tumor cores for single SNPs were 92.3–100% concordant with those obtained from sections. Using these methods, the number of SNP genotypes that can be determined from single FFPE samples is greatly increased expanding the genetic association studies possible from limited archival specimens. The use of tumor cores is of particular importance as the harvesting of tumor cores has minimal impact on the utility of the donor blocks for other purposes.

Active estrogen receptor-alpha signaling in ovarian cancer models and clinical specimens

Purpose: High-grade serous ovarian cancer (HGSOC) is an aggressive disease with few available targeted therapies. Despite high expression of estrogen receptor-alpha (ERα) in approximately 80% of HGSOC and some small but promising clinical trials of endocrine therapy, ERα has been understudied as a target in this disease. We sought to identify hormone-responsive, ERα-dependent HGSOC. Experimental Design: We characterized endocrine response in HGSOC cells across culture conditions [ two-dimensional (2D), three-dimensional (3D), forced suspension] and in patient-derived xenograft (PDX) explants, assessing proliferation and gene expression. Estrogen-regulated transcriptome data were overlapped with public datasets to develop a comprehensive panel of ERα target genes. Expression of this panel and ERα H-score were assessed in HGSOC samples from patients who received endocrine therapy. Time on endocrine therapy was used as a surrogate for clinical response. Results: Proliferation is ERα-regulated in HGSOC cells in vitro and in vivo, and is partly dependent on 3D context. Transcriptomic studies identified genes shared by cell lines and PDX explants as ERα targets. The selective ERα downregulator (SERD) fulvestrant is more effective than tamoxifen in blocking ERα action. ERα H-score is predictive of efficacy of endocrine therapy, and this prediction is further improved by inclusion of target gene expression, particularly IGFBP3. Conclusions: Laboratory models corroborate intertumor heterogeneity of endocrine response in HGSOC but identify features associated with functional ERα and endocrine responsiveness. Assessing ERα function (e.g., IGFBP3 expression) in conjunction with H-score may help select patients who would benefit from endocrine therapy. Preclinical data suggest that SERDs might be more effective than tamoxifen. Clin Cancer Res; 23(14); 3802–12. ©2017 AACR.

The CYP17A1 inhibitor abiraterone exhibits estrogen receptor agonist activity in breast cancer

Abstract Cytochrome P450 17A1 (CYP17A1) is the requisite enzyme for synthesis of sex steroids, including estrogens and androgens. As such, inhibition of CYP17A1 is a target for inhibiting the growth of hormone-dependent cancers including prostate and breast cancer. Abiraterone, is a first in class potent and selective CYP17A1 inhibitor that has been approved for the treatment of castration-resistant prostate cancer. Given that, androgens are the precursors for estrogen production, it has been proposed that abiraterone could be an effective form of treatment for estrogen receptor (ER)-positive breast cancer, though its utility in this context has yet to be established. Abiraterone has a core steroid-like chemical structure, and so we hypothesized that it may bind to nuclear steroid receptors including ER and have estrogenic activity. We tested this hypothesis by investigating abiraterone’s ability to directly modulate ER signaling in breast cancer cell line models. We show that abiraterone directly activates ER, induces ER-target gene expression, and elicits estrogen-response-element reporter activity in the ER-positive cell lines MCF-7 and T47D. Abiraterone also induced cell proliferation by ~2.5-fold over vehicle in both MCF-7 and T47D cells. Importantly, abiraterone-induced cell proliferation and ER-activity was blocked by the selective estrogen receptor downregulator (SERD) fulvestrant, confirming that abiraterone directly acts at the ER. These data suggest that abiraterone should be combined with other ER antagonists when used for the clinical management of ER-positive breast cancer.