Aromatase Inhibitor Resistance Research Articles

Mammary adipose stromal cells derived from obese women reduce sensitivity to the aromatase inhibitor anastrazole in an organotypic breast model.

Aromatase inhibitors are the preferred treatment for certain women with estrogen receptor (ER)-positive breast cancer, but evidence suggests that women with obesity experience aromatase inhibitor resistance at higher rates. To compare how stromal cells derived from women who are lean or obese influence response to the aromatase inhibitor (anastrazole), we incorporated patient-derived stroma in a previously characterized MCF7-derived in vitro duct model. Coculture with adipose stromal cells enabled the metabolism of testosterone (T) to E2, which induced estrogen response element activity, epithelial proliferation, and hyperplasia in MCF7 cells. The effects of T were inhibited by the ER antagonist tamoxifen and aromatase inhibitor anastrazole and were increased by the aromatase inducer dexamethasone. Primary mammary adipose stromal cells derived from women with obesity displayed increased aromatase mRNA compared with lean controls. MCF7-derived ducts cocultured with obese stromal cells exhibited higher maximal aromatization-induced ER transactivation and reduced anastrazole sensitivity, a difference not seen in 2-dimensional coculture. Finally, tamoxifen was more effective than anastrazole at reducing aromatization-induced ER transactivation and proliferation. These findings suggest that patient-specific responses to hormone therapies can be modeled and studied organotypically in vitro and add to evidence advocating obesity as a parameter to consider when identifying treatments for patients with ER-positive breast cancer.-Morgan, M. M., Arendt, L. M., Alarid, E. T., Beebe, D. J., Johnson, B. P. Mammary adipose stromal cells derived from obese women reduce sensitivity to the aromatase inhibitor anastrazole in an organotypic breast model.

The lncRNA MIR2052HG regulates ER\u03b1 levels and aromatase inhibitor resistance through LMTK3 by recruiting EGR1

BackgroundOur previous genome-wide association study using the MA.27 aromatase inhibitors adjuvant trial identified SNPs in the long noncoding RNA MIR2052HG associated with breast cancer-free interval. MIR2052HG maintained ERα both by promoting AKT/FOXO3-mediated ESR1 transcription and by limiting ubiquitin-mediated ERα degradation. Our goal was to further elucidate MIR2052HG’s mechanism of action.MethodsRNA-binding protein immunoprecipitation assays were performed to demonstrate that the transcription factor, early growth response protein 1 (EGR1), worked together with MIR2052HG to regulate that lemur tyrosine kinase-3 (LMTK3) transcription in MCF7/AC1 and CAMA-1 cells. The location of EGR1 on the LMTK3 gene locus was mapped using chromatin immunoprecipitation assays. The co-localization of MIR2052HG RNA and the LMTK3 gene locus was determined using RNA-DNA dual fluorescent in situ hybridization. Single-nucleotide polymorphisms (SNP) effects were evaluated using a panel of human lymphoblastoid cell lines.ResultsMIR2052HG depletion in breast cancer cells results in a decrease in LMTK3 expression and cell growth. Mechanistically, MIR2052HG interacts with EGR1 and facilitates its recruitment to the LMTK3 promoter. LMTK3 sustains ERα levels by reducing protein kinase C (PKC) activity, resulting in increased ESR1 transcription mediated through AKT/FOXO3 and reduced ERα degradation mediated by the PKC/MEK/ERK/RSK1 pathway. MIR2052HG regulated LMTK3 in a SNP- and aromatase inhibitor-dependent fashion: the variant SNP increased EGR1 binding to LMTK3 promoter in response to androstenedione, relative to wild-type genotype, a pattern that can be reversed by aromatase inhibitor treatment. Finally, LMTK3 overexpression abolished the effect of MIR2052HG on PKC activity and ERα levels.ConclusionsOur findings support a model in which the MIR2052HG regulates LMTK3 via EGR1, and LMTK3 regulates ERα stability via the PKC/MEK/ERK/RSK1 axis. These results reveal a direct role of MIR2052HG in LMTK3 regulation and raise the possibilities of targeting MIR2052HG or LMTK3 in ERα-positive breast cancer.

Estrogen-Induced Apoptosis in Breast Cancers Is Phenocopied by Blocking Dephosphorylation of Eukaryotic Initiation Factor 2 Alpha (eIF2α) Protein.

Approximately 30% of aromatase-inhibitor-resistant, estrogen receptor-positive patients with breast cancer benefit from treatment with estrogen. This enigmatic estrogen action is not well understood and how it occurs remains elusive. Studies indicate that the unfolded protein response and apoptosis pathways play important roles in mediating estrogen-triggered apoptosis. Using MCF7:5C cells, which mimic aromatase inhibitor resistance, and are hypersensitive to estrogen as evident by induction of apoptosis, we define increased global protein translational load as the trigger for estrogen-induced apoptosis. The protein kinase RNA-like endoplasmic reticulum kinase pathway was activated followed by increased phosphorylation of eukaryotic initiation factor-2 alpha (eIF2α). These actions block global protein translation but preferentially allow high expression of specific transcription factors, such as activating transcription factor 4 and C/EBP homologous protein that facilitate apoptosis. Notably, we recapitulated this phenotype of MCF7:5C in two other endocrine therapy-resistant cell lines (MCF7/LCC9 and T47D:A18/4-OHT) by increasing the levels of phospho-eIF2α using salubrinal to pharmacologically inhibit the enzymes responsible for dephosphorylation of eIF2α, GADD34, and CReP. RNAi-mediated ablation of these genes induced apoptosis that used the same signaling as salubrinal treatment. Moreover, combining 4-hydroxy tamoxifen with salubrinal enhanced apoptotic potency. IMPLICATIONS: These results not only elucidate the mechanism of estrogen-induced apoptosis but also identify a drugable target for potential therapeutic intervention that can mimic the beneficial effect of estrogen in some breast cancers.

Abstract OT1-03-02: CICLADES: Monitoring of ESR1, PIK3CA and AKT1 ctDNA mutations during real-life follow-up of patients with advanced breast cancer treated with endocrine therapy

Abstract Activating ESR1 mutations have recently been reported as a key mechanism leading to Aromatase Inhibitor (AI) resistance. ESR1 mutations occur rarely in primary breast cancers. However, in large retrospective studies, ESR1 mutations occurred in up to 39% of Estrogen-Receptor(ER)-positive metastatic breast cancer resistant to AI. Numerous hotspot mutations have been identified, most of them affecting the ligand-binding domain (LBD) and leading to ligand-independent activation of the ER and to resistance to AI. Phosphatidylinositol 3-kinase (PI3K)/AKT pathway is involved in key Cellular Mechanisms and mutations in PIK3CA and AKT1 are frequently reported in breast cancer. In this study, we propose to use a capture-based Next Generation Sequencing (NGS) assay and to use the barcoding and polishing features in our analysis pipeline. This assay will be able to detect all mutations on AKT1, PIK3CA, ESR1 and other genes on circulating tumor DNA (ctDNA) extracted from blood samples of patients with breast cancer. We consider that this exon-screening strategy is relevant according to the recent knowledge. We plan to prospectively include women with advanced breast cancer about to begin standard-of-care first line endocrine therapy (ET). Patients will be required to have histologically confirmed ER-positive, HER2-negative breast cancer and documented loco-regionally advanced or metastatic disease, not amenable to surgery or radiation with curative intent. Patients with endocrine sensitive disease (no prior ET or relapse more than 12 months after completing adjuvant ET) as well as patients with endocrine resistant disease (relapse while on adjuvant ET or within 12 months of completing adjuvant ET) will be enrolled. ET can be prescribed alone or in combination with a targeted therapy. Nevertheless, we will recruit at least 25% of patients with exclusive ET in the endocrine sensitive group. Peripheral-blood samples, for analysis of ctDNA, will be obtained from participating patients at pre-specified time points: at start of ET to determine the baseline mutational status of ESR1, PIK3CA, AKT1 and other genes included in a panel of genes of interest in solid tumors, and then, at evaluation of response to therapy until disease progression or end of study. Patients will be followed for 36 months or until disease progression. Determination of progression will be done per local investigator. The primary objective is to describe the prevalence of activating ESR1 mutations affecting the LBD, using NGS, from the start of ET to progression or end of study. Secondary objectives include to describe the prevalence of ESR1 mutations affecting other domains, the prevalence of ESR1 mutations in patients with and without endocrine resistance at enrolment and the prevalence of PIK3CA and AKT1 mutations, to demonstrate that ESR1, PIK3CA and AKT1 mutations whatever their times of onset are predictors of progression free survival. As of June 2018, 8 sites were opened to recruitment and 18 pts were included; the target enrollment is 146. The trial is supported by AstraZeneca. Citation Format: Massard V, Uwer L, Salleron J, Deblock M, Kieffer A, Rios M, Gilson P, Lesur A, Harle A, Merlin JL. CICLADES: Monitoring of ESR1, PIK3CA and AKT1 ctDNA mutations during real-life follow-up of patients with advanced breast cancer treated with endocrine therapy [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr OT1-03-02.

Retraction: Aromatase Inhibitor-mediated Downregulation of INrf2 (Keap1) Leads to Increased Nrf2 and Resistance in Breast Cancer.

Aromatase inhibitors (AIs) are effective drugs that reduce or eliminate hormone sensitive breast cancer. However, despite their efficacy, resistance to these drugs can occur in some patients. The INrf2 (Keap1):Nrf2 complex serves as a sensor of drug/radiation-induced oxidative/electrophilic stress. INrf2 constitutively suppresses Nrf2 by functioning as an adapter protein for the Cul3/Rbx1-mediated ubiquitination/degradation of Nrf2. Upon stress, Nrf2 dissociates from INrf2, is stabilized, translocates to the nucleus, and coordinately induces a battery of cytoprotective gene expression. Current studies investigated the role of Nrf2 in AI resistance. RT-PCR and immunoblot assays showed that AI-resistant breast cancer LTLTCa and AnaR cells express lower INrf2 and higher Nrf2 protein levels, as compared to drug sensitive MCF-7Ca and AC1 cells, respectively. The increase in Nrf2 was due to lower ubiquitination/degradation of Nrf2 in AI-resistant cells. Higher Nrf2-mediated levels of biotransformation enzymes, drug-transporters and anti-apoptotic proteins contributed to reduced efficacy of drugs and aversion to apoptosis that led to drug resistance. shRNA inhibition of Nrf2 in LTLTCa (LTLTCa-Nrf2KD) cells reduced resistance and sensitized cells to AI exemestane. Interestingly, LTLTCa-Nrf2KD cells also showed reduced levels of aldehyde dehydrogenase, a marker of Tumor-Initiating Cells and significantly decreased mammosphere formation, as compared to LTLTCa-Vector control cells. The results together suggest that persistent AI treatment down-regulated INrf2 leading to higher expression of Nrf2 and Nrf2 regulated cytoprotective proteins that resulted in increased AI drug resistance. These findings provide a rationale for the development of Nrf2 inhibitors to overcome resistance and increase efficacy of AI.

Abstract 4888: Disrupting MUC1/IFITM1 crosstalk reverses the aggressive phenotype of aromatase inhibitor-resistant breast cancer

Abstract Breast cancer is the most commonly diagnosed and second most lethal cancer among women, affecting over 252,000 women in 2017. The ER+/PR+ subtype of breast cancer is the most frequently diagnosed, representing approximately 70% of all cases. Estrogen depletion through aromatase inhibitors (AIs) is the standard of care to cause tumor regression in the ER+ subtype. Our research focuses on targeting the AI-resistant tumors that develop in 30% of women. The human oncoprotein, mucin 1 (MUC1), drives tumorigenesis in breast carcinomas promoting the epithelial-mesenchymal transition, epigenetic reprogramming, and evasion of the immune response. MUC1 interacts with the STAT proteins, via the JAK/STAT signaling pathway, and stimulates transcription of interferon-stimulated genes (ISGs) including IFITM1. Our laboratory has shown that IFITM1 is overexpressed in AI-resistant breast cancer cells and AI-resistant tumors and promotes an aggressive phenotype. In this study, we demonstrate that MUC1 is differentially regulated in endocrine-sensitive (MCF-7 and T-47D) compared to AI-resistant (MCF-7:5C) cells and that it plays a critical role in enhancing IFITM1 expression in the resistant cells. Analysis of a tumor microarray of 94 ER+ human breast tumors indicated that co-expression of both MUC1 and IFITM1 correlated with poor recurrence-free survival, poor overall survival, and AI resistance. To investigate how disruption of the MUC1/IFITM1 crosstalk effects cell survival and proliferation, we used an AI-resistant cell line (MCF-7:5C) that overexpresses MUC1, P-STAT1, and IFITM1. We genetically manipulated breast cancer cells by knocking down the levels of MUC1 with siRNA and observed lowered expression of IFITM1 at the mRNA and protein level and reduced IFITM1 promoter activation. Additionally, knockdown of MUC1 and IFITM1 levels with siRNA induced cell death in AI-resistant cells (MCF-7:5C). We verified this effect with the pharmacologic inhibitors GO-201 (a MUC1 inhibitor) and ruxolitinib (a JAK/STAT inhibitor) to interfere with the MUC1/IFITM1 crosstalk in vitro. Both agents significantly reduced cell proliferation in resistant cells (MCF-7:5C) but not in estrogen-sensitive cell lines (MCF-7 and T-47D). Co-IP confirmed the physical interaction between MUC1 and STAT1/2 and ruxolitinib treatment blocked this interaction by inhibiting STAT phosphorylation in both AI-resistant and -sensitive cells. In vivo studies using ruxolitinib significantly reduced tumor size in NSG mice and decreased expression of MUC1, P-STAT1, and IFITM1. The research discussed here highlights a novel mechanism by which breast cancer cells develop AI resistance and suggests that disrupting MUC1/IFITM1 crosstalk might be therapeutically beneficial to patients with AI-resistant disease. Citation Format: Taylor E. Escher, Eric Geanes, Asona Lui, Joan Lewis-Wambi. Disrupting MUC1/IFITM1 crosstalk reverses the aggressive phenotype of aromatase inhibitor-resistant breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4888.

Abstract SY01-01: Endocrine resistance in metastatic breast cancer: Mechanisms and new therapeutic strategies

Abstract SY01-01: Endocrine resistance in metastatic breast cancer: Mechanisms and new therapeutic strategies

Abstract 3432: Evolving genomes of breast cancer aromatase inhibitor resistance models

Abstract Acquired endocrine resistance is a severe barrier in the anti-hormonal treatment of breast cancer and is responsible for its failure in about half of the treated patients. In vitro models of long-term estrogen deprived breast cancer cells serve as laboratory tools to explore new therapeutic strategies. The models MCF-7:5C and MCF-7:2A mimic clinical AI resistance but regress with E2 due to the reconfiguration of survival signaling known as E2-inducible apoptosis (5C rapid, 2A delayed reaction). Alterations of stress related pathways including the accumulation of endoplasmic reticulum stress, oxidative stress, and inflammatory stress that occur prior to E2-induced apoptosis (Ariazi et al. 2011; Fan et al. 2013, 2015; Sweeney et al. 2014) and specific miRNA expression profiles (Hoppe et al Oncotarget 2016) have been linked with these resistance and E2-inducible apoptosis phenotypes. Little is known on their underlying genetic variability. Here we compare the mutational profiles of 5C and 2A clones with those of the estradiol stimulated growth sensitive MCF-7:WS8 and standard MCF-7 (ATCC) cells. Whole genome libraries were sequenced on the Illumina HiSeq platform (2x150bp reads). Alignments were manually inspected (bcftools mpileup) to avoid false negative variant calling in poorly (<20x) covered regions. Loci with at least 10x coverage and absence of any variant reads were classified as wildtype. The coding genome positions defined as mutant or wildtype in all four cell types served as a basis to select candidate mutations for the verification of MCF-7 variants via comparison to the COSMIC database (http://cancer.sanger.ac.uk/cosmic) and identification of novel mutations by variant calling (GATK HaplotypeCaller). Putative protein effects of variants were predicted by the SnpEff tool. We verified 421 point mutations in MCF-7 (COSMIC) of which 203 mutations were lost in our models, with additional specific losses of 4, 4, and 18 mutations in WS8, 2A and 5C, respectively. In 5C cells new mutations affected 261 genes with 31 high impact mutations based on splice site alterations in 7 genes (i.e. CERS2, SEMA3A, TDRD7, ATP5H) and gain of stop codons in 24 genes (i.e. CACNA1S, NOTCH4, SUN1, JPH3, SERPINB2). Similarly, in 2A cells there were 12 high impact mutations among 122 affected genes (e.g. ARHGEF5, ARHGAP35, ARAP2). We will report on the integrative genomic analysis including larger structural variants (short indels and CNVs) towards the evolution of these models for a comprehensive understanding of the biology of E2-inducible apoptosis. Citation Format: Reiner Hoppe, Siarhei Kandabarau, Ping Fan, V Craig Jordan, Hiltrud B. Brauch. Evolving genomes of breast cancer aromatase inhibitor resistance models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3432.

Targeting tumour re-wiring by triple blockade of mTORC1, epidermal growth factor, and oestrogen receptor signalling pathways in endocrine-resistant breast cancer

BackgroundEndocrine therapies are the mainstay of treatment for oestrogen receptor (ER)-positive (ER+) breast cancer (BC). However, resistance remains problematic largely due to enhanced cross-talk between ER and growth factor pathways, circumventing the need for steroid hormones. Previously, we reported the anti-proliferative effect of everolimus (RAD001-mTORC1 inhibitor) with endocrine therapy in resistance models; however, potential routes of escape from treatment via ERBB2/3 signalling were observed. We hypothesised that combined targeting of three cellular nodes (ER, ERBB, and mTORC1) may provide enhanced long-term clinical utility.MethodsA panel of ER+ BC cell lines adapted to long-term oestrogen deprivation (LTED) and expressing ESR1wt or ESR1Y537S, modelling acquired resistance to an aromatase-inhibitor (AI), were treated in vitro with a combination of RAD001 and neratinib (pan-ERBB inhibitor) in the presence or absence of oestradiol (E2), tamoxifen (4-OHT), or fulvestrant (ICI182780). End points included proliferation, cell signalling, cell cycle, and effect on ER-mediated transactivation. An in-vivo model of AI resistance was treated with monotherapies and combinations to assess the efficacy in delaying tumour progression. RNA-seq analysis was performed to identify changes in global gene expression as a result of the indicated therapies.ResultsHere, we show RAD001 and neratinib (pan-ERBB inhibitor) caused a concentration-dependent decrease in proliferation, irrespective of the ESR1 mutation status. The combination of either agent with endocrine therapy further reduced proliferation but the maximum effect was observed with a triple combination of RAD001, neratinib, and endocrine therapy. In the absence of oestrogen, RAD001 caused a reduction in ER-mediated transcription in the majority of the cell lines, which associated with a decrease in recruitment of ER to an oestrogen-response element on the TFF1 promoter. Contrastingly, neratinib increased both ER-mediated transactivation and ER recruitment, an effect reduced by the addition of RAD001. In-vivo analysis of an LTED model showed the triple combination of RAD001, neratinib, and fulvestrant was most effective at reducing tumour volume. Gene set enrichment analysis revealed that the addition of neratinib negated the epidermal growth factor (EGF)/EGF receptor feedback loops associated with RAD001.ConclusionsOur data support the combination of therapies targeting ERBB2/3 and mTORC1 signalling, together with fulvestrant, in patients who relapse on endocrine therapy and retain a functional ER.

PO-178 Androstenedione (4AD) activates an androgen receptor (AR) mediated transcriptome and AR interactions associated with cell-cell adhesion in aromatase inhibitor (AI) resistant breast cancer cells

Introduction AI’s are the front line treatment for endocrine postmenopausal breast cancer. However, response rates vary from 35%–70% and recurrence occurs in almost all advanced disease. In order to identify a target for overcoming AI treatment resistance, research has turned to the most widely expressed sex steroid receptor in breast cancer the AR. AI therapies, by their mechanism of action, create an unopposed androgenic steroid environment. 4AD is the major circulating steroid in postmenopausal women and AI therapy prevents it’s conversion to estrogens. We hypothesise that cancer cells adapt to utilise 4AD to promote resistance and survival in AI resistance. Material and methods Molecular changes in a breast cancer cell model which have transitioned from AI sensitive (MCF7 cells) to AI resistant (LetR cells) were assessed using RNA sequencing. Alterations in the steroid microenvironment occur as a result of AI therapy and responses to these steroids was confirmed using MTS, mammosphere and invasion assays. Mass spectrometry (LC–MS/MS) analysis identified androgen-mediated, AR protein interactions.The clinical relevance of AR in AI resistance was also evaluated in a cohort of primary breast cancer patients (n=375). Results and discussions LetR cell growth is AR dependent as the anti-AR drug Enzalutamide inhibits cell growth. Furthermore, Enzalutamide abrogates mammosphere formation and self-renewal of LetR cells. We have found LetR cells to be responsive to 4AD, and treatment with this steroid drives a more aggressive phenotype in vitro. RNA sequencing data highlighted a non-canonical cytoplasmic AR signalling mechanism. Our research suggests that 4AD initiates rapid second messenger signalling in the AI resistant setting. LC–MS/MS analysis identified androgen-mediated, AR protein interactors unique to our resistant model particularly associated with cell-cell adhesion and invasion. Evaluation of AR protein in a cohort of primary breast cancer patients found AR expression in 90% of patients, with 25% expressing high intensity AR staining. Analysis is ongoing into this subset and its association with clinical pathological parameters. Conclusion AR and its ligand 4AD may be a significant driving factor in AI resistance and may play a role in facilitating a more invasive phenotype. Further investigations into AR interactors identified will help elucidate mechanisms of resistance to AI therapy, and these novel AR protein partners may aid the identification of patients who would benefit from anti-AR therapy.

Forskolin increases the effect of everolimus on aromatase inhibitor-resistant breast cancer cells.

Aromatase inhibitor (AI) resistance is a major obstacle in the treatment of estrogen receptor-positive breast cancer. Everolimus (EVE) ameliorates AI-resistant breast cancer and is therefore used in cancer treatment. However, some patients show resistance to EVE. Here, we used 30 clones of long-term estrogen-deprived (LTED) MCF-7 cells as a model of AI-resistant breast cancer. We examined changes in protein phosphatase type 2A (PP2A) and cancerous inhibitor of PP2A (CIP2A), a negative regulator of PP2A, in LTED cells treated with EVE. In LTED cells with high sensitivity to EVE, CIP2A expression decreased at low EVE concentrations; however, in LTED cells poorly sensitive to EVE, CIP2A and PP2A did not change upon exposure to EVE. Therefore, we hypothesized that there is a relation between expression of CIP2A and sensitivity to EVE. Knockdown of CIP2A increased the sensitivity to EVE in three clones poorly sensitive to EVE. Additionally, we found that treatment with FSK, which activates PP2A, increased the sensitivity of the cells to EVE. Our data point to CIP2A and PP2A as novel therapeutic targets for AI-resistant breast cancer.

Acquired resistance to everolimus in aromatase inhibitor-resistant breast cancer.

We previously reported the establishment of several types of long-term estrogen-depleted-resistant (EDR) cell lines from MCF-7 breast cancer cells. Type 1 EDR cells exhibited the best-studied mechanism of aromatase inhibitor (AI) resistance, in which estrogen receptor (ER) expression remained positive and PI3K signaling was upregulated. Type 2 EDR cells showed reduced ER activity and upregulated JNK-related signaling. The mTOR inhibitor everolimus reduced growth in cells similar to Type 1 EDR cells. The present study generated everolimus-resistant (EvR) cells from Types 1 and 2 EDR cells following long-term exposure to everolimus in vitro. These EvR cells modeled resistance to AI and everolimus combination therapies following first-line AI treatment failure. In Type 1 EvR cells, everolimus resistance was dependent on MAPK signaling; single agents were not effective, but hormonal therapy combined with a kinase inhibitor effectively reduced cell growth. In Type 2 EvR cells, ER expression remained negative and a JNK inhibitor was ineffective, but a Src inhibitor reduced cell growth. The mechanism of acquired everolimus resistance appears to vary depending on the mechanism of AI resistance. Strategies targeting resistant tumors should be tailored based on the resistance mechanisms, as these mechanisms impact therapeutic efficacy.

Tracking evolution of aromatase inhibitor resistance with circulating tumour DNA analysis in metastatic breast cancer

Selection of resistance mutations may play a major role in the development of endocrine resistance. ESR1 mutations are rare in primary breast cancer but have high prevalence in patients treated with aromatase inhibitors (AI) for advanced breast cancer. We investigated the evolution of genetic resistance to the first-line AI therapy using sequential ctDNA sampling in patients with advanced breast cancer. Eighty-three patients on the first-line AI therapy for metastatic breast cancer were enrolled in a prospective study. Plasma samples were collected every 3 months to disease progression and ctDNA analysed by digital droplet PCR and enhanced tagged-amplicon sequencing (eTAm-Seq). Mutations identified in progression samples by sequencing were tracked back through samples before progression to study the evolution of mutations on therapy. The frequency of novel mutations was validated in an independent cohort of available baseline plasma samples in the Study of Faslodex versus Exemestane with or without Arimidex (SoFEA) trial, which enrolled patients with prior sensitivity to AI. Of the 39 patients who progressed on the first-line AI, 56.4% (22/39) had ESR1 mutations detectable at progression, which were polyclonal in 40.9% (9/22) patients. In serial tracking, ESR1 mutations were detectable median 6.7 months (95% confidence interval 3.7-NA) before clinical progression. Utilising eTAm-Seq ctDNA sequencing of progression plasma, ESR1 mutations were demonstrated to be sub-clonal in 72.2% (13/18) patients. Mutations in RAS genes were identified in 15.4% (6/39) of progressing patients (4 KRAS, 1 HRAS, 1 NRAS). In SoFEA, KRAS mutations were detected in 21.2% (24/113) patients although there was no evidence that KRAS mutation status was prognostic for progression free or overall survival. Cancers progressing on the first-line AI show high levels of genetic heterogeneity, with frequent sub-clonal mutations. Sub-clonal KRAS mutations are found at high frequency. The genetic diversity of AI resistant cancers may limit subsequent targeted therapy approaches.

Estradiol suppresses phosphorylation of ERα serine 167 through upregulation of PP2A in breast cancer cells.

Aromatase inhibitors (AIs) are effective endocrine therapeutics for postmenopausal women with estrogen receptor (ER)α-positive breast cancer. However, the efficacy of the treatment is often limited by the onset of AI resistance, owing to the phosphorylation of ERα serine 167 (Ser167). Previous studies have indicated that hyperactivation of the phosphoinositide-3 kinase/RAC serine/threonine-protein kinase signaling pathway occurs in AI-resistant breast cancer models, which coincides with elevated levels of ERα phosphorylation at Ser167. The tumor suppressor serine/threonine-protein phosphatase 2A (PP2A) regulates the phosphatidylinositol 3-kinase/RAC serine/threonine-protein kinase signaling pathway. A previous study indicated that PP2A inhibition decreased ERα Ser167 phosphorylation and estradiol (E2)-independent cell growth. The present study investigated the potential relevance of PP2A in E2 deprivation-resistant MCF-7 cells. E2 depletion reduced the susceptibility of MCF-7 cells to inhibitors of mechanistic target of rapamycin (mTOR) and significantly increased ERα Ser167 phosphorylation and decreased expression of PP2A. Conversely, long-term E2-deprived (LTED) MCF-7 cells, a model of AI-resistant breast cancer, exhibited decreased ERα Ser167 phosphorylation and further upregulation of PP2A in E2-containing medium. The PP2A activator forskolin (FSK) significantly inhibited LTED cell proliferation by increasing the effect of everolimus (Eve), an mTOR inhibitor. In summary, the present study provides further evidence that PP2A represents a therapeutic target for AI-resistant breast cancer.

ISY15-4 - Mechanisms of resistance to hormonal therapy in breast cancer

Luminal-type breast cancers are preferentially treated with hormonal therapy, while not a few patients relapse. The mechanisms of the hormone refractoriness have been investigated by numerous studies so far. However, it has not been fully clarified yet. We have been investigating the mechanisms using cancer specimens and cell lines by monitoring transcription activity of estrogen receptor (ER). We so far established six different types of cell lines which mimic aromatase inhibitor (AI) resistance from ERE-GFP introduced ER-positive breast cancer cell lines. They revealed that multiple and alternative ER activating pathways were involved in the resistance. The mechanisms are currently proposed to be three categories, such as (1) an aromatase-independent estrogen-producing pathway, (2) estrogen-independent ER function, and (3) ER-independent growth signaling. The established cell lines of category (1) showed increased expression of steroid metabolizing enzymes to produce agonist for ER. The category (2) was ligand-independent activation of ER, mainly via intracellular phosphorylation signaling pathway, which has been well investigated so far in many laboratory. The category (3) showed decreased ER expression or loss of ER function. Some of them were depended no androgen signaling pathway. As a study for therapeutic approach for AI-resistance, the effect of SERD, mTOR inhibitor, pan- or specific-PI3K inhibitors, and CDK4/6 inhibitor on these resistant cell lines were explored. Furthermore, fulvestrant-resistant cell lines, evelorimus-resistant cell lines, PI3K inhibitor-resistant cell lines, and CDK4/6 inhibitor-resistant cell lines were established and examined the efficacy of the other drugs and their cross-resistances. The results of these investigation will provide useful information for overcoming the hormonal therapy resistance.

Jagged1 promotes aromatase inhibitor resistance by modulating tumor-associated macrophage differentiation in breast cancer patients.

Endocrine resistance limits the efficacy of anti-estrogen therapies. Notch signaling is involved in modulating tumor-associated macrophage (TAM) differentiation and is upregulated in endocrine-resistant breast cancer cells. Here, we analyzed the role of Jagged1 in the regulation of TAM polarization to investigate whether the Jagged1-Notch pathway promotes the acquisition of aromatase inhibitor (AI) resistance by upregulating TAM infiltration. The Jagged1 expression levels and M2 TAM infiltration density, in 203 tumor samples from ER-positive postmenopausal patients, who received AI treatment, were evaluated by immunohistochemical staining and the results were compared with clincopathological parameters and survival. The Jagged1 protein and mRNA levels were analyzed in MCF-7 and long-term endocrine-depleted (LTED) cell lines. The phenotypes of macrophage after macrophages were co-cultured with either MCF-7 or LTED cells, were evaluated using flow cytometry. Cell migration assay was performed to evaluate the mobility of cancer cells. Notch gama secretase inhibitor (GSI) RO4929097 was employed to investigate whether modulation of Notch signaling affects M2 polarization. In the tumor samples, Jagged1 expression was found to be associated with a large tumor size, high histological grade, lymphatic invasion, and high Ki67 expression. Jagged1 expression was also correlated with reduced disease-free and overall survival and was positively associated with the stromal M2 TAM infiltration density in primary tumor tissues. In AI-resistance patients, M2 TAM infiltration was denser in metastatic lesions than in primary tumors. Higher Jagged1 protein and mRNA levels were also found in LTED cells, which model AI-resistant conditions in patients, compared with MCF-7 cells. Macrophages co-cultured with LTED cells expressed higher levels of M2 marker and IL-10. M2 TAM proportion was reduced when macrophages were pre-treated with GSI before co-culture. The Jagged1-Notch pathway showed elevated expression in AI-resistant breast cancer cells, resulting in macrophage differentiation towards M2 TAMs and there contributing to the acquisition of AI resistance.

Abstract 474: Estrogen-responsive miRNAs as modulators of E2-induced apoptosis in AI-resistant breast cancer

Abstract Long-term estrogen deprivation with tamoxifen or aromatase inhibitors (AI) is the basic principle of endocrine treatment of ER-positive breast cancer. Acquired resistance is however a major obstacle in treatment success. Based on clinical observations and in vitro as well as animal in vivo experiments it has been suggested that tumor cell clones evolve over time and become vulnerable to E2-induced apoptosis, thereby potentially providing a promising second line treatment option. In vitro, this vulnerability is mimicked in the AI-resistant breast cancer models MCF-7:5C and MCF-7:2A, which during long-term E2-deprivation reconfigure their survival signaling including endoplasmic reticulum, oxidative and inflammatory stress related pathways. E2-stimulation of these cells initiates an unfolded protein response which in turn triggers apoptosis through the intrinsic and subsequently extrinsic pathway (Jordan VC 2015). Recently, we identified miRNA profiles matching the biology of AI resistance and vulnerability to E2-induced apoptosis (Hoppe R et al. 2016). Here we investigate the modulatory role of miRNAs in E2-induced apoptosis through the identification of their global expression changes in E2-stimulated 5C and 2A models compared to MCF-7:WS8 reference. Each cell line was treated with 10-9 M E2 or vehicle over a 72h time course (6, 12, 24, 72 h). miRNA profiles were generated using Affymetrix GeneChip miRNA2.0 arrays. At each time point relative miRNA expression changes (E2/control) were evaluated resulting in a total of 72 (5C), 104 (2A) and 94 (WS8) differentially expressed miRNAs (FC > 1.5 or < 1/1.5, Pt-test < 0.05). Differential expression analyses between consecutive time points revealed 16 (5C), 43 (2A) and 27 (WS8) miRNAs (maximum absolute FC difference > 1.5, PF-test < 0.05). Differential area under the curve (dAUC) analyses at 6-72, 6-24, and 24-72 h mined the overall, early, and late-responding miRNAs of the different phenotypes (Ppermutation test < 0.05). Representative early down-regulated miRNA candidates potentially modulating E2-induced apoptosis in 5C cells are miR-543 and miR-432 of the DLK1-DIO3 locus on Chr. 14q32.31. Late responding up-regulated miRNA candidates are miR-150* and miR-149* the low expression of which in tumor tissues registered in The Cancer Genome Atlas (miRNA-Seq v. 3.1.17.0) was associated with worse outcome in all PAM50 breast cancer subtypes and Lum B, respectively (HR = 2.1, 95% CI: 1.4-3.2; P = 0.00017; HR = 6.8, 95% CI: 1.5-3.0; P = 0.0036). We are currently in the process to subject respective miRNA sets to functional enrichment analyses (KEGG, GO) and to correlate E2-responsive miRNA candidates with respective transcriptome signatures. In summary, we will present miRNAs matching to the biological processes inherent to E2-induced apoptosis. These may serve as potential targets for the amplification of the apoptotic trigger upon E2-treatment. Citation Format: Reiner Hoppe, Ping Fan, Stefan Winter, Florian Büttner, V. Craig Jordan, Hiltrud B. Brauch. Estrogen-responsive miRNAs as modulators of E2-induced apoptosis in AI-resistant breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 474. doi:10.1158/1538-7445.AM2017-474

Abstract 4157: Co-blockade of mTORC1, ERBB and estrogen receptor signalling pathways in endocrine resistance breast cancer: Combating tumor plasticity

Abstract Introduction The majority of breast cancers (BC) are estrogen (E) receptor positive (ER+). Endocrine therapies target E stimulation of tumour growth but resistance remains problematic, often a result of enhanced crosstalk between ER and growth factor pathways. Previously we reported the antiproliferative efficacy of combining everolimus (RAD001, mTORC1 inhibitor) with endocrine therapy in resistance models, but potential routes of escape from treatment via ERBB2/3 signalling were observed. We hypothesised that combined targeting of three cellular nodes (ER, ERBB and mTORC1) may target tumour rewiring and provide enhanced long-term clinical utility in endocrine resistant BCs. Methods Several ER+ BC lines adapted to long term E deprivation (LTED), modelling relapse to an aromatase inhibitor (AI), were treated in vitro with a combination of RAD001 and neratinib (pan ERBB inhibitor) in the presence or absence of estradiol (E2), tamoxifen or fulvestrant. Effects on proliferation, cell signalling, cell cycle and transcription were assessed. Additionally, an in vivo model of AI resistance was treated with monotherapies or combinations to evaluate efficacy in delaying tumour progression. Results All cell lines showed dose dependent decreases in proliferation in response to RAD001 (IC50 0.6-50nM without E2; 1-10nM with E2). Neratinib showed a wide range of IC50 values in the presence of E2 (300-1000nM). In the absence of E2, wild type (wt) cell lines showed IC50 values in excess of 1800nM with hormetic response curves, whilst in the LTED IC50 values ranged between 400-900nM. Combination of either agent with endocrine therapy caused a concentration dependent decrease in proliferation in both wt and LTED lines but the maximum effect was observed with a triple combination of RAD001, neratinib and endocrine therapy. Expression of pS6 was suppressed by RAD001 in all cells tested, whilst neratinib caused a cell specific reduction in expression of ERBB family proteins. Upregulation of pAKT was observed in all cell lines upon treatment with RAD001. Combination of RAD001 with neratinib suppressed the upregulation of pAKT and reduced cell cycle progression. In the absence of E2, RAD001 reduced ER mediated transcription and recruitment of ER and CREB binding protein to the TFF1 promoter, contrasting with neratinib, which caused a marked increase. In vivo study using LTED tumour xenografts showed the triple combination of RAD001, neratinib and fulvestrant was the most effective at reducing tumour volume (89%, p<0.001). Antitumor effects persisted after therapy removal with exception of RAD001 alone or in combination with neratinib where a slow increase in tumour growth was observed. Conclusion The combination of RAD001, neratinib and endocrine agents may be effective in patients who have relapsed on endocrine therapy but retain a functional ER by combating ERBB pathway upregulation. Citation Format: Ricardo Ribas, Sunil Pancholi, Stephanie K. Guest, Aradhana Rani, Joanna Nikitorowicz-Buniak, Nikiana Simigdala, Allan Thornhill, Richard E. Cutler Jr, Alshad S. Lalani, Francesca Avogadri-Connors, Mitch Dowsett, Stephen R. Johnston, Lesley-Ann Martin. Co-blockade of mTORC1, ERBB and estrogen receptor signalling pathways in endocrine resistance breast cancer: Combating tumor plasticity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4157. doi:10.1158/1538-7445.AM2017-4157

Corrigendum: Acquired CYP19A1 amplification is an early specific mechanism of aromatase inhibitor resistance in ERα metastatic breast cancer.

Nat. Genet.; 10.1038/ng.3773; corrected online 31 January 2017 In the version of this article initially published online, the names of authors Hermannus Kempe and Pernette J. Verschure were spelled incorrectly. These errors have been corrected in the print, PDF and HTML versions of this article.

Abstract MIP-056: CONSTITUTIVELY ACTIVE ESTROGEN RECEPTOR–ALPHA LIGAND BINDING DOMAIN (ERA–LBD) MUTATIONS IN OVARIAN CARCINOMA

Abstract OBJECTIVES: Mutations in ESR1 that are acquired following treatment with aromatase inhibitors commonly underlie resistance to endocrine therapy in breast cancer. ERa-LBD mutations, especially at ESR1 Y537 and D538, confer constitutive activity and partial resistance to selective estrogen receptor modulators (SERMs), such as tamoxifen, and degraders (SERDs) such as fulvestrant in transcription assays. Some ovarian tumors also respond to anti-hormone therapy, and a subset which initially respond subsequently become resistant. The mechanisms of resistance to hormone therapy in ovarian cancer have not been previously described. Comprehensive genomic profiling (CGP) of ovarian tumors was used to analyze ESR1 and correlate responsiveness to aromatase inhibitors in a subset of these patients. METHODS: DNA was extracted from tumor specimens and sequenced using hybridization-capture (FoundationOne) targeting up to 315 genes, plus select introns frequently rearranged in cancer. Demographic and clinicopathologic data will be presented for a subset of patients whose tumors harbor ERa-LBD mutations associated with activation and resistance.. RESULTS: CGP of 3,641 ovarian and peritoneal carcinomas revealed 31 (0.9%) samples with amplification of ESR1 and 16 (0.4%) samples with acquired or de novo mutations affecting the ERa-LBD: Y537S (10), Y537N (1), D638G (4), and S341L (1). Within the 16 samples, ovarian serous carcinoma (10), ovarian endometrioid adenocarcinoma (3), ovarian epithelial carcinoma NOS (2), and peritoneal serous carcinoma (1) were represented. In cases of acquired mutations following prior hormone therapy, there was evidence of clinical benefit to SERM/SERDs. CaseAgeDiseaseESR1 MutationPrior HRTAcquiredSERM/SERD (*post mut if acquired)Clinical Benefit SERM/SERD159Ov serousY537SYYNN/A258Papillary serousY537NNNYY344Endometrioid ov adenoY537SNNYY445HG ov serousD538GNUYU564Endometrioid ov adenoY537SYUNU646LG ov serousY537SYUYU739Papillary serousY537SYUNN/A827LG ov serousY537SYYYY CONCLUSIONS: Acquired activating mutations in ESR1, previously described in breast cancer, can occur in ovarian serous carcinomas and endometrioid carcinomas initially responsive to hormone therapy and suggest a similar mechanism for acquired aromatase inhibitor resistance. However, ERa mutations can occur in gynecologic malignancies even in the absence of prior endocrine therapy. Tumors harboring these mutations may remain responsive to SERM/SERDs. Citation Format: J. A. Elvin, L. Gay, G. Colon-Otero, M. Jorgensen, L. Havrilesky, D. Zajchowski, L Shawver, F. A. Valea, S Aithal, J. S. Ross, M. Markman, S. Gaillard. CONSTITUTIVELY ACTIVE ESTROGEN RECEPTOR–ALPHA LIGAND BINDING DOMAIN (ERA–LBD) MUTATIONS IN OVARIAN CARCINOMA [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr MIP-056.