Therapeutic Efficacy Of Tamoxifen Research Articles

Cross-Ancestry Genome-Wide Association Study Defines the Extended CYP2D6 Locus as the Principal Genetic Determinant of Endoxifen Plasma Concentrations.

The therapeutic efficacy of tamoxifen is predominantly mediated by its active metabolites 4-hydroxy-tamoxifen and endoxifen, whose formation is catalyzed by the polymorphic cytochrome P450 2D6 (CYP2D6). Yet, known CYP2D6 polymorphisms only partially determine metabolite concentrations in vivo. We performed the first cross-ancestry genome-wide association study with well-characterized patients of European, Middle-Eastern, and Asian descent (n=497) to identify genetic factors impacting active and parent metabolite formation. Genome-wide significant variants were functionally evaluated in an independent liver cohort (n=149) and in silico. Metabolite prediction models were validated in two independent European breast cancer cohorts (n=287, n=189). Within a single 1-megabase (Mb) region of chromosome 22q13 encompassing the CYP2D6 gene, 589 variants were significantly associated with tamoxifen metabolite concentrations, particularly endoxifen and metabolic ratio (MR) endoxifen/N-desmethyltamoxifen (minimal P=5.4E-35 and 2.5E-65, respectively). Previously suggested other loci were not confirmed. Functional analyses revealed 66% of associated, mostly intergenic variants to be significantly correlated with hepatic CYP2D6 activity or expression (ρ=0.35 to -0.52), and six hotspot regions in the extended 22q13 locus impacting gene regulatory function. Machine learning models based on hotspot variants (n=12) plus CYP2D6 activity score (AS) increased the explained variability (~ 9%) compared with AS alone, explaining up to 49% (median R2 ) and 72% of the variability in endoxifen and MR endoxifen/N-desmethyltamoxifen, respectively. Our findings suggest that the extended CYP2D6 locus at 22q13 is the principal genetic determinant of endoxifen plasma concentration. Long-distance haplotypes connecting CYP2D6 with adjacent regulatory sites and nongenetic factors may account for the unexplained portion of variability.

Pharmacogenomics of breast cancer: highlighting CYP2D6 and tamoxifen.

To review recent pharmacogenomics studies on breast cancer patients undergoing tamoxifen therapy, highlighting how our knowledge on cytochrome P450 2D6 (CYP2D6) can help to guide the development of adjuvant therapies for these patients. A comprehensive literature search was conducted. Articles reporting findings pertaining to the effect of CYP2D6 on the therapeutic efficacy of tamoxifen, those reporting how targeting CYP2D6 could inform tamoxifen-based therapy development, and those on the tamoxifen effects on cell lines and animal models were included in the review. With CYP2D6 being the primary enzyme for tamoxifen metabolism, single-nucleotide polymorphisms (SNPs) in this gene were one of the determinants in the rate of tamoxifen metabolism, thereby potentially having an effect on the efficacy of tamoxifen-based therapies. Our review indicates the potential effectiveness of targeting these SNPs, including those for the CYP2D6*10 allele (c. 100C > T), in modifying the level of tamoxifen metabolism. These findings suggest the importance of pharmacogenomics research in our understanding of the efficacy of adjuvant therapies. However, the involvement of multiple enzymes in tamoxifen metabolism, dietary factors, ethnic differences in gene frequencies, and patients' compliance to tamoxifen therapies in studies do present challenges in pharmacogenomics research. Pharmacogenomics could play important roles in mediating the advancement of tamoxifen-based adjuvant therapies. Research efforts should be directed towards the exploration of further SNPs of CYP2D6 that affect tamoxifen metabolism, as well as epigenetic changes in CYP2D6, enabling the design of precision medicine and confirming clinical validity in the use of pharmacogenomics for tamoxifen.

Topical tamoxifen in the therapy of cutaneous leishmaniasis.

The aims of the present work were to test the effect of tamoxifen administered topically and the therapeutic efficacy of tamoxifen and pentavalent antimonial combinations in an experimental model of cutaneous leishmaniasis. BALB/c mice infected with a luciferase expressing line of Leishmania amazonensis were treated with topical tamoxifen in two different formulations (ethanol or oil-free cream) as monotherapy or in co-administration with pentavalent antimonial. Treatment efficacy was evaluated by lesion size and parasite burden, quantified through luminescence, at the end of treatment and 4 weeks later. Topical tamoxifen, formulated in ethanol or as a cream, was shown to be effective. The interaction between tamoxifen and pentavalent antimonial was additive in vitro. Treatment with combined schemes containing tamoxifen and pentavalent antimonial was effective in reducing lesion size and parasite burden. Co-administration of tamoxifen and pentavalent antimonial was superior to monotherapy with antimonial.

Abstract 896: Correlation between modulation of RNA expression and the preventive and therapeutic efficacy of Tamoxifen in an ER+ model of breast cancer

Abstract The SERMs, primarily Tamoxifen, have been employed in both the therapy and prevention of ER+ breast cancer for many decades. In the present study we examined the effects of the SERM tamoxifen as a preventive and therapeutic agent in an ER+ Methylnitrosourea (MNU) -induced model of mammary cancer in rats. Fifty day old female Sprague Dawley rats were administered MNU i.v. and tamoxifen was administered continually in diet beginning either 5 days post MNU (prevention) or when an animal developed its first palpable tumor (150mm2) [therapy]. The approximate ED50 (dose required to reduce tumor multiplicity 50%) and ED90 in the preventive assays were 0.66 and 3.3 ppm (mg of tamoxifen per g of diet) for Tamoxifen. Interestingly the 3.3 ppm dose was ineffective as a therapy at these doses although it slowed growth of palpable lesions. Rather one had to administer a dose of roughly 100 ppm achieve a strong therapeutic response. We also examined the short term effects (5 days of treatment) of 3.3 and 100 ppm on gene expression in palpable lesions. We compared each treatment group with the control to find the up or down regulated genes. The number of differentially expressed (DE) genes selected at FDR = 0.2 increased from 584 to 2267 when the dose increased from 3.3ppm to 100ppm. The 3.3ppm treatment yielded fewer DE genes compared to the 100ppm treatment, but the majority (60%) of the genes identified in the 3.3 ppm group were identified in the high dose group. The higher dose profoundly decreased a variety of specific genes in the proliferative pathways, particularly in APC (Anaphase Prometaphase Condensation Complex), which includes Ccnb1 and Top2A. In contrast, the low dose failed to significantly change expression of these specific genes. The effects of the higher dose of tamoxifen were confirmed by the use of RT-PCR. Using the human Tamoxifen treatment dataset GSE6532 in GEO, we found 633 DE genes by comparing the treated (n = 277) and untreated (n = 237) patients. We found 68 genes shared between human and rat derived from 100ppm treatment and 11 genes from 3.3ppm treatment. 9 of the 11 genes regulated by the low dose were also regulated by the high dose. These genes included RAB5B, a member of the RAS oncogene family, and GDF9, the growth differentiation factor 9. By gene enrichment analysis (GSEA), we found several pathways, including RAS, TGF beta signaling, DNA damage signaling, and cell cycle pathways, which were affected by the tamoxifen treatment. Our study suggests that the rat gene expression was regulated by tamoxifen even at low dose, and the DE genes at the low dose are a subset of these DE genes altered at the high dose. These data provide a rationale for testing low dose tamoxifen as a chemo preventive agent for women at high risk of developing breast cancer and offer potential genomic biomarkers associated with efficacy at these lower doses. Citation Format: Howard H. Yang, Huaitian Liu, Ronald Lubet, Clinton J. Grubbs, Holly Nicastro, Maxwell P. Lee. Correlation between modulation of RNA expression and the preventive and therapeutic efficacy of Tamoxifen in an ER+ model of breast cancer. [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 896. doi:10.1158/1538-7445.AM2015-896

Abstract PD6-7: In-silico discovery of novel estrogen receptor-α inhibitors as potential therapeutics for tamoxifen resistant breast cancer

Abstract Estrogen receptor-α (ER) positive breast cancer (BCa) represents 75% of all invasive BCas. Conventional ER-directed drug Tamoxifen targets the estrogen binding pocket (EBP) of the receptor. However, over prolonged periods of treatment, the therapeutic efficacy of Tamoxifen declines due to development of resistance. Numerous factors are causative for this phenomenon, including recently reported mutations in the receptor (T537S). Therefore, there is an urgency to develop novel anti-ER therapeutics that exhibit entirely different mode of ER inhibition. A promising alternative strategy is to prevent receptor-coactivator interaction and block further crucial steps in ER activity. ER-coactivator interface should be less prone to adaptive mutations as any mutations at this site would also likely block the coactivator recruitment, we therefore targeted the Activation Function-2 (AF2) site, a coactivator binding pocket on ER, called to overcome the limitations of Tamoxifen. Although AF2 is a shallow surface pocket, the pharmacophore-rich features of this site make it a druggable target. To identify potential ER AF2 inhibitors, virtual screening was performed. Initial hits were subjected to lead optimization and more potent analogues were rationally designed by exploiting critical features of this site. Potential compounds were tested for their ability to inhibit ER transcriptional activity using the T47D-KBluc cell line stably transfected with an ER-specific luciferase reporter. Consequently, the lead compound VPC-16339 (IC50=8.24µM) was identified. The direct binding between VPC-16339 and the receptor was confirmed by Biolayer Interferometry assay. More importantly, VPC-16339 prevents coactivator recruitment at the AF2 pocket in a dose dependent manner as measured by TR-FRET coactivator recruitment assay. Increasing concentrations of estradiol did not affect the IC50 of the lead compound, thereby ruling out the possibility of VPC-16339 binding to EBP. VPC-16339 demonstrated a strong anti-proliferative effect on MCF7 and Tamoxifen resistant cells, with no effect on ER- HeLa cells, suggesting its selective ER-mediated action, as further validated by ER luciferase assay in Tamoxifen resistant cells. VPC-16339 effectively inhibits mRNA and protein expression levels of the estrogen dependent genes such as pS2,CathD and CDC2. Due to AF2-guided mechanism, VPC-16339 successfully overcomes Tamoxifen resistance and inhibits the constitutively active Tamoxifen resistant form of ER (T537S). In summary, we report VPC-16339 as an ER AF2 specific inhibitor with promising anti-proliferative effect in BCa cell lines including Tamoxifen resistant cell lines. VPC-16339 effectively inhibits the mutant form of the receptor (T537S) which is responsible for acquired endocrine resistance. It can be anticipated that ER AF2 inhibitors will provide an alternative therapeutic strategy that can be applied concurrently or simultaneously with current anti-ER treatments for BCa patients with advanced disease. Citation Format: Kriti Singh, Ravi SN Munuganti, Miriam Butler, Artem Cherkasov, Paul S Rennie. In-silico discovery of novel estrogen receptor-α inhibitors as potential therapeutics for tamoxifen resistant breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr PD6-7.

Effects of Ginkgo biloba on chemically-induced mammary tumors in rats receiving tamoxifen

BackgroundGinkgo biloba extract (GbE) is used extensively by breast cancer patients undergoing treatment with Tamoxifen (TAM). Thus, the present study investigated the effects of GbE in female Sprague–Dawley (SD) rats bearing chemically-induced mammary tumors and receiving TAM.MethodsAnimals bearing mammary tumors (≥1 cm in diameter) were divided into four groups: TAM [10 mg/kg, intragastrically (i.g.)], TAM plus GbE [50 and 100 mg/kg, intraperitoneally (i.p.)] or an untreated control group. After 4 weeks, the therapeutic efficacy of the different treatments was evaluated by measuring the tumor volume (cm3) and the proportions of each tumor that were alive, necrotic or degenerative (mm2). In addition, labeling indexes (LI%) were calculated for cell proliferation (PCNA LI%) and apoptosis (cleaved caspase-3 LI%), expression of estrogen receptor-alpha (ER-α) and p63 biomarkers.ResultsOverall, the tumor volume and the PCNA LI% within live tumor areas were reduced by 83% and 99%, respectively, in all TAM-treated groups when compared to the untreated control group. GbE treatment (100 mg/kg) reduced the proportions of live (24.8%) and necrotic areas (2.9%) (p = 0.046 and p = 0.038, respectively) and significantly increased the proportion of degenerative areas (72.9%) (p = 0.004) in mammary tumors when compared to the group treated only with TAM. The expression of ER-α, p63 and cleaved caspase-3 in live tumor tissues was not modified by GbE treatment.ConclusionsCo-treatment with 100 mg/kg GbE presented a slightly beneficial effect on the therapeutic efficacy of TAM in female SD rats bearing mammary tumors.

P5-05-04: Bipshenol-A Modulates Estrogen Receptor Alpha and Compromises Tamoxifen-Induced Apoptosis.

Abstract BACKGROUND: Antiestrogens are important for treatment and prevention of breast cancer. Such drugs influence multiple pathways involved in regulation of cell survival — including apoptosis. In previous work, exposure of human, benign, high-risk donor breast epithelial cells (HRBECs) to the xenoestrogen (XE) bisphenol-A (BPA) induced gene expression changes predicting disruption of apoptotic signaling (Dairkee et al, Cancer Research 2008). Therefore, we hypothesized that antiestrogenic, pro-apoptotic effects of tamoxifen are likely modulated under the influence of XEs. Because the environmental XE, BPA is routinely detected in a large percentage of random human blood samples, we employed functional tests to determine whether BPA interferes with tamoxifen-induced apoptosis in XE-exposed HRBECs. METHODS: With IRB approval and written informed consent, HRBECs were obtained by random periareolar fine needle aspiration (RPFNA) from women who were high risk on the basis of personal or family history of breast cancer, breast density, or high-risk histology. Primary HRBEC cultures were treated for one week with a low concentrations of BPA corresponding to levels reported in human body fluids. Estrogen Receptor (ER)-α and -β were measured by Western blot (n=6). Altered response to tamoxifen toxicity was measured by analyzing quantitative changes in the Annexin V-positive apoptotic cell population (n=8). RESULTS: After one-week exposure to BPA, ER-α increased in six of six cases, ER-β decreased in 5 of 6 cases, and we observed a 25 percent reduction in tamoxifen-induced apoptosis. Prior exposure to BPA at the high concentration range detected in the human population reduced tamoxifen efficacy by 50 percent. There was a dose response of evasion of apoptosis to increasing concentrations of BPA (P<0.001, two-sided test for slope fixed effect in a linear mixed effects regression). Prior exposure to another XE, methylparaben (MP), caused a similar dose-dependent decline in tamoxifen-induced apoptosis (p<0.001). CONCLUSION: BPA and MP, in a range of concentrations measured in humans, prevent tamoxifen-induced apoptotic cell death. For BPA this involves upregulation of ER-α and downregulation of ER-β. Because these XEs are widespread in the American population, they may play a role in limiting the prophylactic and therapeutic efficacy of tamoxifen and other hormone-based therapies. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-05-04.

Abstract 4603: Role of endoxifen as an antiestrogen: Effects on oncogene expression and polyamine pathway

Abstract The nonsteroid antiestrogen tamoxifen [trans-1-(4-b-dimethylaminoethoxyphenyl)-1,2-diphenylbut-1-ene] is the most commonly used endocrine treatment for estrogen receptor α (ERα)-positive breast cancer in pre- and post-menopausal women, and it has helped to reduce breast cancer death rate by one third. Tamoxifen is extensively metabolized, and several metabolites have been detected in human serum. It is metabolized to 4-hydroxytamoxifen and N-desmethyltamoxifen by the action of CYP2D6 and CYP3A4/5 enzymes, respectively. N-desmethyltamoxifen and 4-hydroxy-tamoxifen are further converted to endoxifen by the action of these enzymes. Steady state levels of serum tamoxifen/metabolite are achieved within 4 weeks of continuous administration of the drug. The therapeutic efficacy of tamoxifen is determined by the distribution of the drug into tissues and the availability of the parent drug and its active metabolites in target tissues. Recent reports indicate that endoxifen might act as an antiestrogen. In order to examine the mechanism of action of endoxifen on an ERα-positive breast cancer cell line, MCF-7, we determined the effects of 100, 250, 500 and 1000 nM concentrations of this agent on cell growth, c-myc oncogene expression and activity of the polyamine biosynthetic enzymes. MCF-7 breast cancer cells were treated for 1, 3 or 5 days with endoxifen. Endoxifen had a growth inhibitory effect on MCF-7 cells by day 3 and 5 of treatment, as measured by cell number. Estradiol increased cell number by >2-fold compared to untreated control by day 5 of treatment, whereas endoxifen inhibited the estrogenic effect. c-myc gene expression was increased by a significant 2- to 3-fold by 4 nM estradiol in 2 and 4 hours of treatment, while 250 and 500 nM endoxifen suppressed the increase in c-myc gene expression, as measured by real-time qPCR experiment. In the absence of estradiol, endoxifen slightly (1.5 fold) increased c-myc gene expression. Endoxifen decreased ornithine decarboxylase (ODC) and S-adenosine methyl decarboxylase (AdoMetDC) activities in a concentration- and time-dependent manner; however, it had little effect on spermidine/spermine N1-acetyl transferase (SSAT) activity. The change in biosynthetic enzyme activities were reflected in polyamine pools, as decreased putrescine and spermidine levels. There were no changes in spermine level. As expected, treatment of the cells with 4 nM estradiol induced ODC and AdoMetDC activities, and slightly decreased SSAT activity. Addition of endoxifen in combination with 4 nM estradiol led to even more dramatic decrease in ODC and AdoMetDC activities and reduction of putrescine and spermidine levels. These results support our hypothesis that endoxifen can act as an antiestrogen and suppress cell growth stimulatory effects of estradiol in ERα-positive breast cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4603.

Regulation of Aryl Hydrocarbon Receptor Function by Selective Estrogen Receptor Modulators

Selective estrogen receptor modulators (SERMs), such as tamoxifen (TAM), have been used extensively for the treatment and prevention of breast cancer and other pathologies associated with aberrant estrogen receptor (ER) signaling. These compounds exhibit cell-selective agonist/antagonist activities as a consequence of their ability to induce different conformational changes in ER, thereby enabling it to recruit functionally distinct transcriptional coregulators. However, the observation that SERMs can also regulate aspects of calcium signaling and apoptosis in an ER-independent manner in some systems suggests that some of the activity of drugs within this class may also arise as a consequence of their ability to interact with targets other than ER. In this study, we demonstrate that 4-hydroxy-TAM (4OHT), an active metabolite of TAM, directly binds to and modulates the transcriptional activity of the aryl hydrocarbon receptor (AHR). Of specific interest was the observation, that in the absence of ER, 4OHT can induce the expression of AHR target genes involved in estradiol metabolism, cellular proliferation, and metastasis in cellular models of breast cancer. The potential role for AHR in SERM pharmacology was further underscored by the ability of 4OHT to suppress osteoclast differentiation in vitro in part through AHR. Cumulatively, these findings provide evidence that it is necessary to reevaluate the relative roles of ER and AHR in manifesting the pharmacological actions and therapeutic efficacy of TAM and other SERMs.

Estrogen Receptor-dependent and Estrogen Receptor-independent Pathways for Tamoxifen and 4-Hydroxytamoxifen-induced Programmed Cell Death

The therapeutic efficacy of tamoxifen (TAM) in cancer therapy is thought to arise primarily from its ability to compete with estrogens for binding to the estrogen receptor (ER). We show that TAM and its active metabolite, 4-hydroxytamoxifen (OHT), can actively induce programmed cell death through distinct ER-dependent and ER-independent pathways. The ER-independent pathway is activated by 10-20 microm TAM and OHT and by 10-20 microm 17beta-estradiol and raloxifene, and occurs in ER-negative cells. The ER dependence of a second pathway, caused by submicromolar concentrations of TAM and OHT, was demonstrated by the ability of the ER ligands 17beta-estradiol, raloxifene, and ICI 182,780 to effectively block the cell death-inducing effects of TAM and OHT. Because the p38-specific inhibitor SB203580 blocks OHT.ER-induced cell death, stress kinase pathways are likely involved. ER-independent cell death triggers classic caspase-dependent apoptosis. However, although OHT.ER triggers some hallmarks of apoptosis, including Bax translocation and cytochrome c release, the absence of poly(ADP-ribose) polymerase cleavage or DNA laddering indicates that the death pathway involved is caspase-independent. The OHT.ER-dependent cell death pathway appears to diverge from classical apoptosis at the level of caspase 9 activation. The ability to promote ER-dependent programmed cell death represents a novel activity of TAM and OHT.