Abiraterone shows alternate activity in models of endocrine resistant and sensitive disease

BACKGROUND: Resistance to endocrine therapy remains a major clinical problem in the treatment of oestrogen-receptor positive (ER+) breast cancer. Studies show androgen-receptor (AR) remains present in 80-90% of metastatic breast cancers providing support for blockade of AR-signalling. However, clinical studies with abiraterone, which blocks cytochrome P450 17A1 (CYP17A1) showed limited benefit. METHODS: In order to address this, we assessed the impact of abiraterone on cell-viability, cell-death, ER-mediated transactivation and recruitment to target promoters. together with ligand-binding assays in a panel of ER+ breast cancer cell lines that were either oestrogen-dependent, modelling endocrine-sensitive disease, or oestrogen-independent modelling relapse on an aromatase inhibitor. The latter, harboured wild-type (wt) or naturally occurring ESR1 mutations. RESULTS: Similar to oestrogen, abiraterone showed paradoxical impact on proliferation by stimulating cell growth or death, depending on whether the cells are hormone-dependent or have undergone prolonged oestrogen-deprivation, respectively. Abiraterone increased ER-turnover, induced ER-mediated transactivation and ER-degradation via the proteasome. CONCLUSIONS: Our study confirms the oestrogenic activity of abiraterone and highlights its differential impact on cells dependent on oestrogen for their proliferation vs. those that are ligand-independent and harbour wt or mutant ESR1. These properties could impact the clinical efficacy of abiraterone in breast cancer

PD01-07: AR Overexpression and Aromatase Inhibitor Resistance in Breast Cancer.

Abstract Background: Aromatase inhibitors (AIs) have emerged as the therapy of choice for the treatment of estrogen receptor alpha (ERα)-positive breast cancer. However, many patients develop resistance to AI treatment. Although the involvement of the ERα in AI resistance is well established, the role of the androgen receptor (AR) is not known. It has been estimated that about 60%-70% of ERα-positive breast cancer co-express the AR, and that AR agonists can either inhibit or stimulate breast cancer cell proliferation. Thus it is important to determine if there are biomarkers predicting AR's effects in breast tumors. We have previously shown a role for AR-overexpression in tamoxifen resistance in ERα-positive MCF-7 breast cancer cells; here we hypothesized that AR overexpression might similarly be involved in resistance to the AI anastrazole (Anas). Materials and Methods: Stable transfection of MCF-7 cells was performed to generate cell lines that express the aromatase gene (MCF-7 BK Arom) and then co-transfected with an AR expression vector (MCF-7 AR Arom). Aromatase and AR expression levels were evaluated by western blot analysis, and the enzyme activity was evaluated using aromatase activity assays. Proliferation was tested using anchorage independent soft agar assays and MTT in the presence of the androgen substrate androstenedione (AD), or AD plus Anas. ERα and AR transcriptional activities were tested with ERE-luciferase reporter assays. Localization of ERα and AR within the cells was visualized using immunofluorescence microscopy. Results: ERα-positive MCF-7 cells were stably transfected with either aromatase, or aromatase plus AR. MCF-7 aromatase clones overexpressing AR were resistant to the growth inhibitory effects of Anas when stimulated with the androgen AD. Resistance was not mediated through changes in aromatase expression or activity. The growth of several of the AR Arom-overexpressing cells was stimulated with treatment of Anas alone, suggesting that Anas was acting as an agonist. As expected, AD treatment stimulated ERα transcriptional activity, but Anas was unable to block AD-stimulated activity in AR Arom-overexpressing cells using ERE-Luciferase reporter assay. Anas was able to enhance AR and ERα colocalization in AR-overxpressing cells. Resistance was not associated with activation of known mechanisms of resistance, such as HER2, IGF-1R, or MAPK. However AR-overexpressing cells had higher constitutive phosphorylation of Akt. Accordingly, resistance to Anas was blocked using an Akt1/2 inhibitor. Conclusion: Using a model of ERα-positive breast cancer cells expressing exogenous aromatase and AR, we have demonstrated that AR overexpression confers resistance to the AI Anas. These results suggest that in patients recurring on hormonal therapy whose tumors express elevated levels of AR, targeted therapy to Akt might restore hormone sensitivity. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr PD01-07.</jats:p

Abstract P4-08-09: Targeting Thyroid Receptor b in Estrogen Receptor Negative Breast Cancer

Abstract Background: The treatment of estrogen receptor (ER)-negative breast cancer (BC) is a major clinical problem due to the lack of useful therapeutic targets. Nuclear receptors (NRs) are potential targets in these patients because they regulate global transcriptional events and many already have agonists/antagonists available. Material and Methods: We used microarray analysis of 227 ER-negative tumors to identify NR targets, and performed hierarchical clustering using 41 NRs. Expressed receptors were scored using prediction analysis of microarrays (PAM) across clustered groups. Cell lines were matched to subtypes using previously described data (Neve et al. 2006). Candidate gene expression levels were confirmed by qRT-PCR using TaqMan probes. pGIPZ lentiviral vectors encoding shRNA were used to knockdownselected candidates. MTT and soft agar assays were used to measure chemosensitivity and growth following treatment with Docetaxel (Doc), Doxorubicin (Dox), or Cisplatin (Cis). Statistical analysis was performed using Red-R. Results: The 41 NRs clustered tumors into 5 groups. For each group we selected genes representing the highest ranked discriminators, and examined their effects in cell lines matching each groups' gene signature. Thyroid hormone receptor b (THRβ) was selected from group V. The expression levels of this receptor were confirmed by qRT-PCR and Western blot analysis. Knockdown of THRβ in ER-negative HCC2185 cells rendered cells more resistant to all chemotherapeutics by using MTT assay. Similar results were confirmed in ER-negative MDA-MB-453 and HCC202 cells. Knockdown of THRβ enhanced colony forming potential in anchorage-independent soft agar assays in MDA-MB-453 and HCC202 cells. Statistical analysis using clinical data from Sabatier et al. (BCRT 2011) showed that patients with low THRb have a worse clinical outcome. In order to translate these findings into the clinic, we treated cells with a specific THRβ agonists, GC-1 and KB-141. GC-1 inhibited cell growth in growth assays, and synergistic effects were observed when cells were treated with GC-1 and Docetaxel in combination. Re-expression of ERα protein was observed in ER-nagative cells lines after treatment with GC-1 and KB141, suggesting that modulation of THRβ may also extend hormonal therapy to this hormonally insensitive group of patients. Conclusion: Clinical targeting of NRs in ER-negative BCs is a novel strategy since receptors can be specifically targeted with ligands. Our data suggest that chemotherapy response in ER-negative patients overexpressing THRβ could be enhanced with a THRβ agonist. Similarly, functional re-activation of ERα by activating THRβ might extend hormonal therapies to these patients as well. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-08-09.</jats:p

Abstract S4-02: The Y537S ESR1 mutation is a dominant driver of distant ER-positive breast cancer metastasis

Abstract Background: Estrogen receptor (ESR1) mutations occur at a high frequency in metastatic breast tumors in patients treated with hormonal therapy in the metastatic setting. We do not know if these mutations are involved in metastasis. Experimental design and methods: We generated ESR1 Y537S homozygous mutations using CRISPR Casp-9 technology. Treatment synergy was evaluated using Compusyn. Athymic mice were used in tumor xenograft studies. ChIP-Seq and transcriptome analyses were performed. Results: We generated CRISPR ESR1 Y537S mutation homozygous knock-in clones and lentiviral stable pools in MCF-7 cells. Transcriptome profiling revealed elevated expression of Hallmark pathways, including EMT and estrogen-regulated gene expression. The EMT in mutant cells was associated with a switch from E-cadherin to vimentin, and increased expression of SNAIL and TWIST. Mutant cell growth was resistant to tamoxifen, but responsive to fulvestrant treatment. Synergistic treatment effects were observed with fulvestrant and everolimus or palbociclib. CRISPR Y537S mutant knock-in cells grown in the mammary fat-pad of athymic mice spontaneously metastasized to distant organs including the lung, intestine, and kidneys. In the presence of estrogen, there was no difference in the frequency of distant macrometastases between parental wild-type ER and CRISPR Y537S mutant ER mice. However, in the absence of estrogen, 80% of CRISPR Y537S mutant ER mice displayed overt distant macrometastases, but none were observed in parental wild-type ER mice (p=0.04). Interestingly, although CRISPR Y537S mutant ER tumors grown in the mammary fat-pad were unresponsive to tamoxifen treatment, tamoxifen significantly inhibited the growth of mutant tumors at the distant microenvironment (8-fold). Distant tumors retained ER expression and hormone sensitivity. Comparison of residual tamoxifen-treated metastatic tumors with tumors grown at the primary mammary fat-pad site using immunoblot analysis demonstrated significant reduction in estrogen-regulated gene expression, but no effect on the expression of biomarkers associated with EMT, suggesting a disconnect between EMT and distant metastasis in mutant cells. EMT genes were also identified as direct binding site targets in Y537S mutant cells compared with wide-type ER using ChIPSeq. We discovered that expression of the Y537S mutant was dominant, driving the growth of distant metastatic tumors when co-expressed with wild-type ER cells. A Y537S ER mutant-specific gene expression signature predicted poor disease-free survival of ER-positive patients using the METABRIC database, and lung-specific metastasis-free survival in a Memorial Sloan Kettering dataset. Conclusion: The Y537S ER mutation is a driver of distant metastasis in ER-positive breast cancer cells. Although tamoxifen treatment was ineffective at reducing the growth of mutant cells grown at the primary site, it was effective at reducing distant metastasis. A Y537S ER mutant-specific gene expression signature predicted poor disease-free, and distant lung metastasis in ER-positive patients. Mutation status is a potential new predictive factor for hormone therapy of metastatic breast cancer patients on maintenance hormonal therapy. Citation Format: Fuqua SAW, Gu G, Rechoum Y, Gelsomino L, Dustin DJ, Corona-Rodriguez A, Beyer AR, Pejerrey SM, Gao M, Tsimelzon A, Tian L, Zhang X, Nagi C, Ando' S. The Y537S ESR1 mutation is a dominant driver of distant ER-positive breast cancer metastasis [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 S4-02.</jats:p