Inhibition Of Estrogen Synthesis Research Articles

Abstract P3-04-22: RAD1901 demonstrates anti-tumor activity in multiple models of ER-positive breast cancer treatment resistance

Abstract Estrogen receptor positive (ER+) breast cancer makes up approximately 70% of all newly diagnosed breast cancers and ER remains the single most useful predictive and prognostic marker for these patients. In fact, dependence on ER signaling is retained in recurrent disease and even in disease treated with multiple lines of anti-endocrine agents. Indeed, inhibiting estrogen synthesis (e.g. with aromatase inhibitors) and modulating ER pathway activity (e.g. with tamoxifen) continue to be mainstays in the standard of care for ER+ breast cancer patients both in adjuvant and metastatic setting. While patients initially respond well to these agents, a majority of patients with metastatic disease will relapse. Therefore, it is critical to improve our understanding and model the specific mechanisms that can lead to treatment resistance. Mutations in the ligand binding domain of ESR1 have been described as a mechanism that contributes to clinical resistance to aromatase inhibitors. In addition, some ESR1 mutations can lead to a conformational change of the receptor, leading to decreased binding of tamoxifen, thereby reducing its activity. Recently, selective estrogen receptor degraders, or SERDs, have gained widespread attention as a novel treatment strategy for ER+ disease. Indeed, fulvestrant has been shown to degrade ER and cause tumor growth inhibition in many ER+ breast cancer models. However, in the clinic, fulvestrant appears to be limited by PK exposure properties and this, combined with its intramuscular route of administration underscore the need for an oral SERD with improved PK properties. Recently, we have described RAD1901, a novel, orally available, non-steroidal small molecule SERD that is able to degrade ER and effectively cause tumor growth inhibition in multiple patient-derived xenograft models (PDX) models, including two that harbor a Y537S mutation in ESR1. To further validate that RAD1901 has the potential to be used in the advanced disease setting, we tested its anti-tumor activity in preclinical models representing the most common ESR1 mutations (D538G, E380Q, S463P, Y537N/C). In addition, we have tested RAD1901 in multiple cell line models of anti-endocrine resistance. These include long-term estrogen deprived cells that model progression of disease on aromatase inhibitors, as well as tamoxifen-resistant models. Here, we present anti-tumor activity of RAD1901 both as a single agent and as part of rational clinical combinations (eg. CDK4/6, PI3K/Akt/mTOR inhibitors) in multiple models of anti-endocrine resistance. These results combined with previous studies demonstrating RAD1901 efficacy in treatment naïve models provide strong preclinical evidence for clinical testing of RAD1901 in both adjuvant and metastatic setting. RAD1901 is currently under clinical investigation for the treatment of ER+ breast cancers in post-menopausal women (NCT02650817, NCT02338349). Citation Format: Bihani T, Arlt H, Brechbuhl H, Patel H, Tao N, Purandare DM, Hattersley G, Kabos P, Garner F. RAD1901 demonstrates anti-tumor activity in multiple models of ER-positive breast cancer treatment resistance [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-04-22.

The nutritional herb Epimedium grandiflorum inhibits the growth in a model for the Luminal A molecular subtype of breast cancer.

The Luminal A subtype of breast cancer expresses the estrogen receptor (ER)-α and progesterone receptor (PR), but not the human epidermal growth factor receptor (HER)-2 oncogene. This subtype of breast cancer responds to endocrine therapy involving the use of selective estrogen receptor modulators and/or inhibitors of estrogen biosynthesis. However, these therapeutic agents are frequently associated with long-term systemic toxicity and acquired tumor resistance, emphasizing the need to identify non-toxic alternative treatments for chemo-endocrine therapy responsive breast cancer. The present study utilized the human mammary carcinoma-derived, ER+/PR+/HER-2- MCF-7 cell line as a model of the Luminal A subtype of breast cancer to examine the growth inhibitory effect of the Chinese nutritional herb Epimedium grandiflorum (EG) and determine the mechanisms underlying this effect. MCF-7 cells maintained in a serum-depleted culture medium retained their ability to grow in response to 17β-estradiol (E2). Treatment of the MCF-7 cells with EG resulted in dose-dependent inhibition of E2-promoted growth. Mechanistically, EG inhibited E2-promoted cell cycle progression through G1 stage arrest and modulated the cellular metabolism of E2, increasing the formation of the anti-proliferative metabolites 2-hydroxyestrone and estriol. Long-term treatment of MCF-7 cells with EG inhibited E2-promoted anchorage independent growth, a surrogate in vitro biomarker of tumorigenesis. In conclusion, the results of the present study demonstrate the growth inhibitory effects of EG on MCF-7 cells and identified clinically relevant mechanistic leads for its anti-tumorigenic efficacy.

Role of Anti Angiogenic Therapy in Prevention of Recurrence in Hormonal Positive Breast Cancer: A Secondary Prevention Strategy and Method of Therapy

Existing literature supports the role of signaling protein vascular endothelial growth factor (VEGF) in tumor growth and metastasis and furthers its involvement in recurrence. In both experimental and clinical studies, VEGF has been shown to be a significant factor involved for aberrant blood vessel growth, and in fact is the target of several classes of antineoplastic drugs [1] [2] [3] [4]. That said, the current standard of care for estrogen receptor positive breast cancer (although improved over the last decade), has not provided a “meaningful preventive shift” since the discovery of angiogenesis and its role in induction of recurrence. In this article, we discuss an anti angiogenic therapy implementing natural compounds to inhibit the production of VEGF. We applied our preclinical data to justify the predicted effect on VEGF. We used liquid biopsy to monitor patients response to therapy as a surrogate for recurrence. We hypothesize that by inhibition of angiogenesis through this protocol, we are able to positively impact tumor recurrence. It is our experience that patients in our sample even with high recurrence scores (based on Oncotype Dx testing) had a major reduction in recurrence when estrogen blockers were combined with this protocol. We also propose longitudinal studies to compare outcomes with combinational therapies with estrogen blockers in highly expected to recur disease.

Extended AI Therapy in Postmenopausal Women With Early Breast Cancer

Extended AI Therapy in Postmenopausal Women With Early Breast Cancer

A novel approach to breast cancer prevention: reducing excessive ovarian androgen production in elderly women.

Minimizing endogenous estrogen production and activity in women at high risk for breast cancer is a prominent approach to prevention of the disease. A number of clinical trials have shown that the administration of selective-estrogen receptor modulators or aromatase inhibitors significantly reduces the incidence of breast cancer in healthy women. Unfortunately, these drugs often produce adverse effects on the quality of life and are, therefore, poorly accepted by many women, even those who are at high risk for breast cancer. We propose a novel alternative approach to decreasing estrogen production: suppression of ovarian synthesis of the androgen precursors of estrogens by administration of long-acting gonadotropin-releasing hormone analogs to women with ovarian stromal hyperplasia. The specific target population would be elderly postmenopausal women, at increased risk of breast cancer, and with high blood levels of testosterone, marker of ovarian hyperandrogenemia, and recognized factor of risk for breast cancer. Testosterone levels are measured at baseline to identify women at risk and during the follow-up to evaluate the effectiveness of therapy. The postmenopausal ovary is an important source of excessive androgen production which originates from the ovarian interstitial cell hyperplasia frequently present in breast cancer patients. We propose to counter the source of androgen excess in women with ovarian stromal hyperplasia, thus reducing the substrate for estrogen formation without completely inhibiting estrogen synthesis. Available evidence indicates that gonadotropin-releasing hormone analogs can be safely used for breast cancer prevention in postmenopausal women.

Inhibition of TrkB limits development of the zebra finch song system

Large sexual dimorphisms exist in the zebra finch song system. Masculinization may be mediated by both estradiol and expression of one or more Z-genes (males: ZZ; females: ZW). Roles of the Z-gene tyrosine kinase B (TrkB) in HVC in masculinizing both HVC and one of its targets the robust nucleus of the arcopallium (RA), were tested using siRNA administration in juvenile males at two ages (post-hatching days 15–17 or 25–27). Birds were euthanized 10 days later. Potential interactions or additive effects with estradiol were evaluated by treating males with the estrogen synthesis inhibitor fadrozole. Females treated with estradiol were also exposed to the siRNA at the later age. Local inhibition of TrkB in males of both ages reduced the volume of HVC, an effect due to a change in cell number and not cell size. In the older males, in which the treatment spanned the period when the projection from HVC to RA grows, TrkB inhibition reduced the volume of RA and the relative number of cells within it. TrkB siRNA in HVC decreased the volume of and soma size in the RA of females, and the projection from HVC to RA in both sexes. Estradiol in females masculinized various aspects of the song system, and its effect in masculinizing the volume of RA was decreased by TrkB inhibition. However, effects of fadrozole in males were limited. The data indicate that TrkB is involved in masculinizing the song system, but for most measures it probably does not work in concert with E2.

Development and recovery of histopathological alterations in the gonads of zebrafish (Danio rerio) after single and combined exposure to endocrine disruptors (17α-ethinylestradiol and fadrozole)

Exposure of wildlife to endocrine disrupting chemicals (EDCs) is not necessarily continuous. Due to seasonal changes and variable industrial and agricultural activities it often occurs intermittently. Thus, it is possible that aquatic organisms may be more affected by periodic peak exposure than by chronic exposure. Therefore, an experimental scenario including an exposure from 2h to 90 days post-fertilization (dpf) and a subsequent recovery period until 150 dpf was chosen to assess the potential reversibility of the effects of sex steroids on sexual and gonad development of zebrafish (Danio rerio). The aim of this study was to investigate the persistence of the endocrine effects of an estrogen (EE2-17α-ethinylestradiol, 4ng/L), an inhibitor of estrogen synthesis (Fad-fadrozole, 50μg/L) or their binary mixture (Mix-EE2+ Fad, 4ng/L+50μg/L). Afterwards, a semi-quantitative histological assessment was used to investigate histopathological changes on gonad differentiation and development. The data showed that fadrozole, alone or in combination with EE2, permanently disrupts the sexual development, inducing masculinization and causing severe pathological alterations in testis, such as intersex associated to the enlargement of sperm ducts, interstitial changes, asynchronous development and detachment of basal membrane. After exposures to both EDCs and their mixture, the gonad histopathology revealed interstitial proteinaceous fluid deposits and, in ovaries, there were atretic oocytes, and presumably degenerative mineralization. On the other hand, the gonadal changes induced by EE2 alone seem to be partially reversible when the exposure regime changed to a recovery period. In addition, EE2 enhanced zebrafish growth in both genders, with male fish presenting signs of early obesity such as the presence of adipocytes in testis. Moreover, sex ratio was slightly skewed toward females, at 90 and 105 dpf, in zebrafish exposed to EE2. The data further indicate that long-term studies on impacts of single EDCs and their mixtures with recovery periods are crucial to reveal the possibility of sex reversal and pathological changes of gonads that can adversely affect breeding.

Abstract P5-12-04: Directed exercise intervention in breast cancer patients with arthralgias receiving aromatase inhibitors: A randomized pilot study

Abstract Purpose: Early discontinuation or non-adherence of AI therapy occurs in 25-50 % of patients due to treatment associated toxicities, including musculoskeletal symptoms such as arthralgias and myalgias. The goal of this pilot study is to evaluate the effect of a directed physical therapy regimen including joint mobility and stretching exercises on musculoskeletal symptoms resulting from AI therapy, in comparison to normal physical activity alone. Patients and methods: Eligibility criteria included post-menopausal women with histological evidence of hormone receptor positive breast cancer who were currently receiving adjuvant AI therapy and experiencing significant joint discomfort/stiffness when attempting activities of daily living which began or significantly increased after initiation of AI therapy. Patients were excluded if they had preexisting rheumatoid arthritis or fibromyalgia, systemic metastasis or ECOG performance status of greater than 2. Patients were randomly assigned to an 8 week directed exercise program under the supervision of a physical therapist (Group A, n=15) or were told to participate in moderate physical activity (walking) (Group B, n=12). Before beginning the study and at the end of the 8 week intervention, patients in both groups underwent a physical therapy evaluation including performance based measures such as functional lower extremity strength and grip strength and a Patient Specific Functional Scale (PSFS) to assess functional ability to complete specific activities. Additionally, all patients answered a series of questionnaires including a pain disability index (PDI), pain scale (PS) and the PHQ4 depression scale. Effects of the directed exercise regimen were evaluated using non-parametric analysis to determine differences between the two groups. Results: Significant improvement was observed in both left and right grip strength and right pinch in the intervention group (Group A) as compared to the control group (Group B). Additionally, significant improvement in lower body measures including 2-minute step tests and chair raises were observed in patients who underwent the directed exercise program compared to the control group. No significant differences were observed in PS, PDI or PHQ4 between the groups. However, Group A showed a slight improvement on the PS and PDI. Conclusions: Directed exercise regimens involving joint mobility and stretching exercises may have benefits over moderate physical activity alone for joint pain and arthralgia associated with AI therapy. Citation Format: Varadarajan R, Helm E, Arnold C, Huelsenbeck-Dill L, Ingraham-Lopresto B, Sonaad S, Swanson P, Sims-Mourtada J, Dickson-Whitmer D. Directed exercise intervention in breast cancer patients with arthralgias receiving aromatase inhibitors: A randomized pilot study. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-12-04.

Genetic variant in the osteoprotegerin gene is associated with aromatase inhibitor-related musculoskeletal toxicity in breast cancer patients

BackgroundAromatase inhibitor (AI) therapy is associated with musculoskeletal (MS) toxicity, which adversely affects quality of life and therapy adherence. Our objective was to evaluate whether genetic variants may predict endocrine therapy-related MS pain and hot flashes in a prospective observational cohort study. Patients & methods254 early breast cancer patients starting AI (n = 159) or tamoxifen therapy (n = 95) were included in this genetic biomarker study. MS and vasomotor symptoms were assessed at baseline and after 3, 6 and 12 months of therapy. AI-induced MS pain was defined as an increase in arthralgia or myalgia relative to baseline. Single nucleotide polymorphisms (SNP) in candidate genes involved in oestrogen signalling or previously associated with AI-related MS pain or oestrogen levels were selected. ResultsOverall, 13 SNPs in CYP19, CYP17, osteoprotegerin (OPG) and oestrogen receptor 1 exhibited an allele frequency >0.05 and were included in the analysis. Patients carrying the G allele of rs2073618 in OPG experienced significantly more AI-induced MS toxicity compared to the wildtype allele, after correction for multiple testing (P = 0.046). Furthermore, this SNP was associated with severity of pain (P = 0.018). No association was found with regard to the other SNPs, both in AI and tamoxifen-treated patients. Neither could an association with vasomotor symptoms be demonstrated. ConclusionThe SNP rs2073618 in OPG is associated with an increased risk of MS symptoms and pain with AI therapy, which has not been reported previously. Validation of this finding in larger cohorts and further functional studies are required.

Inhibitory Aromatase Effects of Flavonoids from Ginkgo Biloba Extracts on Estrogen Biosynthesis.

Ginkgo biloba extract (GBE) is a popular phytomedicine and has been used for disorders of the central nervous system, cardiovascular, renal, respiratory, and circulatory diseases. Although GBE is a complex mixture of over 300 compounds, its major components are 24% flavonoids and 6% terpene lactones. In this study, we tested the inhibitory effects of the three major flavonoids (kaempferol, quercetin, and isorhamnetin) from GBE, independently and as mixtures, on aromatase activity using JEG-3 cells (human placental cells) and recombinant proteins (human placental microsome). In both systems, kaempferol showed the strongest inhibitory effects among the three flavonoids; the flavanoid mixtures exerted increased inhibitory effects. The results of exon I.1-driven luciferase reporter gene assays supported the increased inhibitory effects of flavonoid mixtures, accompanied by suppression of estrogen biosynthesis. In the RT-PCR analysis, decreased patterns of aromatase promoter I.1 mRNA expressions were observed, which were similar to the aromatase inhibition patterns of flavonoids and their mixtures. The present study demonstrated that three flavonoids synergistically inhibit estrogen biosynthesis through aromatase inhibition, decrease CYP19 mRNA, and induce transcriptional suppression. Our results support the usefulness of flavonoids in adjuvant therapy for breast cancer by reducing estrogen levels with reduced adverse effects due to estrogen depletion.

Single-Molecule Sequencing Reveals Estrogen-Regulated Clinically Relevant lncRNAs in Breast Cancer.

Estrogen receptor (ER)α-positive tumors are commonly treated with ERα antagonists or inhibitors of estrogen synthesis, but most tumors develop resistance, and we need to better understand the pathways that underlie the proliferative and tumorigenic role of this estrogen-activated transcription factor. We here present the first single-molecule sequencing of the estradiol-induced ERα transcriptome in the luminal A-type human breast cancer cell lines MCF7 and T47D. Sequencing libraries were prepared from the polyadenylated RNA fraction after 8 hours of estrogen or vehicle treatment. Single-molecule sequencing was carried out in biological and technical replicates and differentially expressed genes were defined and analyzed for enriched processes. Correlation analysis with clinical expression and survival were performed, and follow-up experiments carried out using time series, chromatin immunoprecipitation and quantitative real-time PCR. We uncovered that ERα in addition to regulating approximately 2000 protein-coding genes, also regulated up to 1000 long noncoding RNAs (lncRNAs). Most of these were up-regulated, and 178 lncRNAs were regulated in both cell lines. We demonstrate that Long Intergenic Non-protein Coding RNA 1016 (LINC01016) and LINC00160 are direct transcriptional targets of ERα, correlate with ERα expression in clinical samples, and show prognostic significance in relation to breast cancer survival. We show that silencing of LINC00160 results in reduced proliferation, demonstrating that lncRNA expression have functional consequences. Our findings suggest that ERα regulation of lncRNAs is clinically relevant and that their functions and potential use as biomarkers for endocrine response are important to explore.

Testosterone Mediates Seasonal Growth of the Song Control Nuclei in a Tropical Bird

In mid- to high-latitude songbirds, seasonal reproduction is stimulated by increasing day length accompanied by elevated plasma sex steroid levels, increased singing, and growth of the song control nuclei (SCN). Plasticity of the SCN and song behavior are primarily mediated by testosterone (T) and its metabolites in most species studied thus far. However, the majority of bird species are tropical and have less pronounced seasonal reproductive cycles. We have previously documented that equatorial rufous-collared sparrows (Zonotrichia capensis) exhibit seasonal neuroplasticity in the SCN. Manipulating T in these birds, however, did not alter singing behavior. In the current study, we investigated whether T mediates plasticity of the SCN in a similar manner to temperate songbirds. In the first experiment, we treated captive male birds with T or blank implants during the nonbreeding season. In a second experiment, we treated captive male birds with either blank implants, T-filled implants, T with flutamide (FLU; an androgen receptor antagonist) or T with FLU and 1,4,6-androstatriene-3,17-dione (ATD; an estrogen synthesis inhibitor) during the breeding season. In both experiments, the volumes of the brain areas high vocal center (HVC), Area X, and robust nucleus of the arcopallium (RA) were measured along with singing behavior. In summary, T stimulated growth of HVC and RA, and the combined effect of FLU and ATD reversed this effect in HVC. Area X was not affected by T treatment in either experiment. Neither T-treated birds nor controls sang in captivity during either experiment. Together, these data indicate that T mediates seasonal changes in the HVC and RA of both tropical and higher- latitude bird species even if the environmental signals differ. However, unlike most higher-latitude songbirds, we found no evidence that motivation to sing or growth of Area X are stimulated by T under captive conditions.

Adjuvant endocrine therapy for postmenopausal women: Type and duration

Postmenopausal women with ER positive breast cancers benefit from adjuvant endocrine therapy. Most such patients should consider aromatase inhibitor therapy at some point in their treatment course as there are modest additional risk reductions over tamoxifen therapy, alone. Longer durations of therapy – beyond five years of tamoxifen – with either ongoing tamoxifen or an AI also improve cancer outcomes. Markers of risk for early recurrence are the same as those for late recurrence. Decisions regarding the optimal type and duration of therapy reflect patient preferences in light of well described side effects of tamoxifen and AI therapy, tolerability of treatment, and established clinical, pathological and genomic markers of risk.

Abstract 3993: MicroRNA analysis of aromatase inhibitor-resistant breast cancer cells reveals upregulation of a unique miRNA cluster on chromosome 14

Abstract Estrogen signaling has been shown to play a large role in the development and progression of breast tumors that express the estrogen receptor (ERα). Thus, endocrine therapies that either inhibit ERα action (tamoxifen or fulvestrant) or inhibit estrogen synthesis (aromatase inhibitors) have been increasingly valuable for the management of ER+ breast cancer. A frequent obstacle, however, is acquired resistance to endocrine therapies, often characterized by aberrant ERα expression and heightened growth factor signaling. Another aspect of acquired endocrine resistance that is now being examined is the role of microRNA (miRNA). miRNAs are 18-22 base pair RNA molecules whose primary role is to inhibit expression of mRNA targets by inhibition of translation or by degradation of the mRNA. Here, we have conducted next generation sequencing of the miRNA from three breast cancer cell lines - one ER+ line that is estrogen-dependent and AI-sensitive (MCF-7), and two ER+ long-term estrogen deprived (LTED) models of AI resistance (MCF-7:5C and MCF-7:2A). We found that in LTED/AI-resistant MCF-7:5C and MCF-7:2A cells there were over 330 and 240 miRNAs differentially expressed, respectively, compared to AI-sensitive MCF-7 breast cancer cells. Initially, we used qPCR to validate the expression of miRNAs that that were differentially expressed in both LTED cell lines vs. MCF-7. Notably, LTED cell lines had upregulated miR-181a (> 4 to 8-fold) and downregulated miR-221/222 expression compared to MCF-7. Elevated miR-181a has been shown to be associated with more aggressive breast cancers whereas miR-221/222 play a role in tamoxifen resistance through targeting ERα. Further bioinformatic analysis also revealed that the LTED cells had very unique expression profiles compared to one another, indicating that they have adapted to an estrogen-depleted environment through potentially different mechanisms. In MCF-7:5C cells, which have lost expression of the progesterone receptor (PR), a miRNA cluster on chromosome 14 is upregulated. This upregulated cluster, which encodes miR-127, miR-654, miR-889 and others, has been shown to be associated with Luminal B (ER+/PR-) breast cancer, consistent with the hormone receptor status of the MCF-7:5C cells. The oncogenic, estrogen-responsive miR-17/92 cluster on chromosome 13 is upregulated in the MCF-7:2A cells and these miRNAs target proteins including PTEN, p21, and Bim. Overall, these findings reveal altered expression of miRNAs in AI-resistant breast cancer cells and they suggest that targeting these miRNAs might be an effective strategy to reverse AI resistance in breast cancer patients. Citation Format: Erin L. Hayes, Jennifer Knapp, Joan Lewis-Wambi. MicroRNA analysis of aromatase inhibitor-resistant breast cancer cells reveals upregulation of a unique miRNA cluster on chromosome 14. [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 3993. doi:10.1158/1538-7445.AM2015-3993

Zebrafish sex differentiation and gonad development after exposure to 17α-ethinylestradiol, fadrozole and their binary mixture: A stereological study

Current knowledge on zebrafish (Danio rerio) sex determination suggests that this trait has a polygenic genetic basis, although environmental factors, such as endocrine disrupting chemicals (EDC), may also be involved in modeling or disturbing the species sex differentiation and development.This study aimed to assess how sex steroids imbalance triggers impact on sex differentiation and gonad development in zebrafish. Fish where exposed to an estrogen (EE2, i.e. 17α-ethinylestradiol, 4ng/L), to an inhibitor of estrogen synthesis (Fad, i.e. fadrozole, 50μg/L) or to their binary mixture (Mix-EE2+ Fad, 4ng/L+50μg/L), from 2h to 60 days post-fertilization (dpf). Afterwards, a quantitative (stereological) analysis using light microscopy, based on systematic sampling, was made at 35 and 60dpf, to identify alterations on gonad differentiation and development. During the sex differentiation period, our histological data showed that not all zebrafish males develop a “juvenile ovary”, contrarily to what is currently taken for granted. Furthermore, the stereological analysis suggests that EE2 alone enhanced both zebrafish growth and gonad development. On the other hand, exposure to Fad affected the sexual development in zebrafish, inducing masculinization of the specimens, with some degree of intersex observed in males. In addition, the binary mixture allowed identifying sex-dependent roles of steroid hormones in the general growth and gonad development of zebrafish, with estrogens acting as growth promoters in females and being essential for ovary development. Data further support that sex-specific and single EDC impact studies are important, but clearly not sufficient to understand what may occur in the environment.

Abstract P3-05-24: Adaptation to AI therapy in breast cancer can induce dynamic alterations in ER activity resulting in estrogen independent metastatic tumours

Abstract Acquired resistance to aromatase inhibitor therapy is a major clinical problem in the treatment of breast cancer. The detailed mechanisms of how tumour cells develop this resistance remain unclear. Here, discovery of pro-survival adaptations to the estrogen receptor (ER) signalling pathway in response to AI treatment is reported. Global ChIPseq analysis shows that the ER retains binding activity in AI resistant cells under steroid-depleted conditions. In AI treated patients, evidence of steroid independent adaptive ER signalling was demonstrated by the ER target gene Early Growth Response 3 (EGR3). Expression of EGR3 decreased initially upon AI treatment but subsequently recovered following as little as 12 weeks of AI treatment. In vitro data indicates that this increased expression of EGR3 may enhance cell growth and motility of tumour cells. Finally, evidence from established metastatic tumours suggests that the ER signalling network may undergo further adaptations with disease progression as EGR3 expression is routinely lost in the established metastatic tumour. Overall, these data provide evidence of a dynamic ER response to AI treatment which may provide vital clues for overcoming the clinical issue of AI resistance. Citation Format: Damir Vareslija, Jean McBryan, Ailis Fagan, Aisling Redmond, Yuan Hao, Arnold DK Hill, Leonie S Young. Adaptation to AI therapy in breast cancer can induce dynamic alterations in ER activity resulting in estrogen independent metastatic tumours [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 P3-05-24.

Economic evaluation of hormonal therapies for postmenopausal women with estrogen receptor-positive early breast cancer in Canada.

Aromatase inhibitor (ai) therapy has been subjected to numerous cost-effectiveness analyses. However, with most ais having reached the end of patent protection and with maturation of the clinical trials data, a re-analysis of ai cost-effectiveness and a consideration of ai use as part of sequential therapy is desirable. Our objective was to assess the cost-effectiveness of the 5-year upfront and sequential tamoxifen (tam) and ai hormonal strategies currently used for treating patients with estrogen receptor (er)-positive early breast cancer. The cost-effectiveness analysis used a Markov model that took a Canadian health system perspective with a lifetime time horizon. The base case involved 65-year-old women with er-positive early breast cancer. Probabilistic sensitivity analyses were used to incorporate parameter uncertainties. An expected-value-of-perfect-information test was performed to identify future research directions. Outcomes were quality-adjusted life-years (qalys) and costs. The sequential tam-ai strategy was less costly than the other strategies, but less effective than upfront ai and more effective than upfront tam. Upfront ai was more effective and less costly than upfront tam because of less breast cancer recurrence and differences in adverse events. In an exploratory analysis that included a sequential ai-tam strategy, ai-tam dominated based on small numerical differences unlikely to be clinically significant; that strategy was thus not used in the base-case analysis. In postmenopausal women with er-positive early breast cancer, strategies using ais appear to provide more benefit than strategies using tam alone. Among the ai-containing strategies, sequential strategies using tam and an ai appear to provide benefits similar to those provided by upfront ai, but at a lower cost.

Arthralgia induced by endocrine treatment for breast cancer: A prospective study of serum levels of insulin like growth factor-I, its binding protein and oestrogens

BackgroundAromatase inhibitors (AIs) frequently induce or enhance musculoskeletal problems (AI-induced musculoskeletal syndrome (AIMSS)) which sometimes are debilitating. Apart from low oestrogen levels, underlying mechanisms are unknown and likely multiple. We previously hypothesised a role for the growth hormone/insulin like growth factor-I (IGF-I) axis. Here, we report the effect of tamoxifen and AI on IGF-I, IGF binding protein-3 (IGFBP-3) and oestrogen levels from a prospective study. Materials and methodsPostmenopausal women with an early breast cancer scheduled to start adjuvant endocrine therapy with an AI or tamoxifen were recruited. A rheumatologic questionnaire was completed and serum was collected for assessment of IGF-I, IGFBP-3 and oestrogen levels. Re-evaluation was done after 3, 6 and 12months of therapy. Results84 patients started on tamoxifen (n=42) or an AI (n=42). 66% of the latter group experienced worsening of pre-existing or de novo complaints in joint and/or muscle, compared to 29% of tamoxifen-treated patients. AI therapy resulted in elevated IGF-I levels with a statistically significant increase at 6months (p=0.0088), whereas tamoxifen users were characterised by a decrease in IGF-I levels at all follow-up times (p<0.0004). No effect on IGFBP-3 was seen in the latter group. AI-users, however, showed decreased IGFBP-3 levels at 12months (p=0.0467). AIMSS was characterised by a decrease in IGFBP-3 levels (p=0.0007) and a trend towards increased IGF-I/IGFBP-3 ratio (p=0.0710). ConclusionThese findings provide preliminary evidence that AI-induced musculoskeletal symptoms are associated with changes in the growth hormone (GH)/IGF-I axis.

Abstract CT319: A randomized, controlled trial of high dose vs. standard dose vitamin D for aromatase inhibitor-induced arthralgia in breast cancer survivors

Abstract Aromatase inhibitors (AI's) are the most effective adjuvant anti-hormonal therapy for estrogen receptor positive (ER+) post-menopausal breast cancer patients, with superior disease free survival over tamoxifen. However, approximately half of the women who take this drug will develop significant joint pains, termed Aromatase Inhibitor-Induced Arthralgia (AIA). Though this medicine should be taken for 5 years, the joint pain can be so troublesome that 13-20% may prematurely discontinue it because of the arthralgia, thus sacrificing their best chance of disease free survival. Furthermore, daily quality of life is significantly affected by this chronic pain while taking AI therapy. Nonetheless, neither the etiology nor the treatment of AIA is well-understood within the medical community. It is known that low estrogen levels, as seen on AI therapy, decrease the amount of available, active vitamin D. Low Vitamin D is implicated in non-specific musculoskeletal pain in non-cancer patients, possibly mediated by higher levels of inflammatory cytokines IL-6 and TNF-alpha in a low vitamin D state. Vitamin D replacement has been shown to be helpful for AIA in some small clinical trials. We therefore propose to test the hypothesis that AIA can be effectively prevented by high dose Vitamin D supplementation, and that effective prophylaxis of the problem will lead to improved compliance with AI therapy. In this clinical trial, we will enroll 184 women who are beginning adjuvant AI therapy and randomize half of them to receive high dose vitamin D (50,000 IU Vitamin D3 per week for 12 weeks, followed by 2000 IU Vitamin D3 daily for 40 weeks), and the other half to receive standard dose vitamin D (800 IU Vitamin D3 daily for 52 weeks). We will assess each woman's baseline joint pains via a questionnaire (Health Assessment Questionnaire-II). We hypothesize that AIA can be effectively prevented with high dose vitamin D, and this will correlate to increased compliance in the high dose vitamin D arm.. The primary endpoint is development of AIA as measured by a defined change in HAQ-II score between baseline and 12 wks. This randomized Phase 2 study is powered to detect an improvement from an expected 33% incidence of AIA in the standard dose group to 18% or less in the high-dose group (alpha=10%, one-tailed). Finally, as an exploratory objective, we will determine whether those in the standard dose vitamin D arm have a correlation between higher levels of inflammatory cytokines and development of AIA. This study targets a very common cause of pain among breast cancer survivors and aims to offer an effective treatment strategy to alleviate pain and improve quality of life as well as medication compliance. Citation Format: Polly A. Niravath, Sue Hilsenbeck, Tao Wang, Mothaffar Rimawi. A randomized, controlled trial of high dose vs. standard dose vitamin D for aromatase inhibitor-induced arthralgia in breast cancer survivors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CT319. doi:10.1158/1538-7445.AM2014-CT319

Abstract SY34-03: Braking the cycle: Inhibition of the cyclin D-Cdk4/6 pathway in breast cancer

In spite of the dramatically improved outlook for patients with ER+ breast cancer, a subset of patients are intrinsically non-responsive to anti-hormonal therapy alone, and even the responders invariably acquire resistance to this treatment modality. In the setting of established hormone resistance, most of these patients will receive chemotherapy, with limited benefit and considerable impact on quality of life due to toxicity. Given the sheer numbers of patients affected (approximately two-thirds of all breast cancers are ER+), the unmet need for well-tolerated therapies that overcome resistance to anti-hormonal agents remains very high. Strikingly, the vast majority (>90%) of ER+ breast cancers express a functional retinoblastoma protein (Rb), a tumor suppressor that represents a critical regulator of the G1 to S phase transition in mammalian cells. In its hypo-phosphorylated state, Rb suppresses the expression of proteins that are essential for commitment to S-phase entry and progression through the remainder of the eukaryotic cell cycle. The G1 cyclin-dependent kinases 4 and 6 (cdk4 and cdk6) which function in complexes with the D-type cyclins (collectively termed cyclin D) initiate the phosphorylation of Rb and override the repressive effects of Rb on cell-cycle progression. In ER-driven breast cancers, oncogenic signaling through the ER stimulates the cyclin D-cdk4/6 -dependent phosphorylation of Rb, and this proliferative stimulus is frequently augmented by amplification of cyclin D or loss of expression of the cyclin D-cdk4/6 inhibitor, p16 (encoded by the CDKN2A gene), two alterations that lead to elevated and dysregulated cyclin D-cdk4/6 activity. The requirement for cyclin D-cdk4/6 activity to circumvent the cell-cycle braking action of Rb suggested that ER+ breast cancer cells might be vulnerable to drug-induced inhibition of cdk4/6 activity. PD-0332991 is an orally administered, selective inhibitor of cyclin D-cdk4/6 kinase activities. In preclinical models, this drug showed significant anti-proliferative activity in Rb+ breast cancer cell lines, the majority of which also expressed the ER. The most well established substrate for the cyclin D-cdk4/6 is Rb, which is modified at multiple phosphoacceptor sites (including Ser-780, Ser-795, Ser-807, and Ser-811) by these cdks. The phosphorylation status of the Ser-807 and Ser-811 residues in Rb proved to be the most robust pharmacodynamic readouts for PD-0332991 activity in vivo, and serve as appropriate biomarkers for target modulation by this drug under in vitro and in vivo study conditions. The IC50 for reduction of Rb phosphorylation at these sites in two breast cancer cell lines (MCF7 and T47D) was approximately 20 nM. Similarly, we observed that PD-0332991 inhibits S-phase entry (measured by incorporation of thymidine into newly synthesized DNA) and cellular proliferation in panel of Rb+ cancer cell lines, with IC50 values ranging from 40-170 nM. The antiproliferative effects of PD-0332991 were not limited to transformed cells, reinforcing the idea that this drug was capable of suppressing the proliferation of any human cell type that requires cyclin D-cdk4/cdk6 activity to bypass the G1 restriction point imposed by Rb. Conversely, cancer cells that have inactivated Rb through genetic or epigenetic mechanisms are highly resistant (IC50 > 3 uM) to the inhibitory effects of PD-0332991 on cell-cycle progression and cellular proliferation. Xenograft experiments confirmed the favorable in vivo safety profile and antitumor activity of PD-0332991. The maximum tolerated dose (MTD; defined as the dose that induced These preclinical studies provided a launching pad for a clinical trial in post-menopausal women with locally advanced or metastatic ER+ (HER2-) breast cancer. In this Phase 2 study, PD-0332991 was given in combination with letrozole (an inhibitor of estrogen synthesis), with the control arm receiving letrozole only. An interim analysis showed that the PD-0332991 plus letrozole combination yielded a dramatic improvement in treatment outcomes, more than tripling progression-free survival relative to the letrozole-only treatment arm. The drug was generally well-tolerated, due in part to its high degree of selectivity for cdk4/6 relative to other members of the cyclin-dependent kinase family, as well as other members of the extended kinase superfamily. These positive results led to a Breakthrough Therapy designation in 2013 for PD-0332991 in the treatment of patients with ER+ breast cancer. This presentation will provide information on the mechanisms of antitumor action of PD-0332991, together with updates on the current status of clinical development of this new addition to the armentarium of breast cancer treatments. Citation Format: Robert T. Abraham, Todd VanArsdale, David V. Shields, Nathan V. Lee, Maria Koehler, Kim Arndt. Braking the cycle: Inhibition of the cyclin D-Cdk4/6 pathway in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr SY34-03. doi:10.1158/1538-7445.AM2014-SY34-03