Abstract
BackgroundTherapies targeting estrogenic stimulation in estrogen receptor-positive (ER+) breast cancer (BC) reduce mortality, but resistance remains a major clinical problem. Molecular studies have shown few high-frequency mutations to be associated with endocrine resistance. In contrast, expression profiling of primary ER+ BC samples has identified several promising signatures/networks for targeting.MethodsTo identify common adaptive mechanisms associated with resistance to aromatase inhibitors (AIs), we assessed changes in global gene expression during adaptation to long-term estrogen deprivation (LTED) in a panel of ER+ BC cell lines cultured in 2D on plastic (MCF7, T47D, HCC1428, SUM44 and ZR75.1) or in 3D on collagen (MCF7) to model the stromal compartment. Furthermore, dimethyl labelling followed by LC-MS/MS was used to assess global changes in protein abundance. The role of target genes/proteins on proliferation, ER-mediated transcription and recruitment of ER to target gene promoters was analysed.ResultsThe cholesterol biosynthesis pathway was the common upregulated pathway in the ER+ LTED but not the ER– LTED cell lines, suggesting a potential mechanism dependent on continued ER expression. Targeting the individual genes of the cholesterol biosynthesis pathway with siRNAs caused a 30–50 % drop in proliferation. Further analysis showed increased expression of 25-hydroxycholesterol (HC) in the MCF7 LTED cells. Exogenous 25-HC or 27-HC increased ER-mediated transcription and expression of the endogenous estrogen-regulated gene TFF1 in ER+ LTED cells but not in the ER– LTED cells. Additionally, recruitment of the ER and CREB-binding protein (CBP) to the TFF1 and GREB1 promoters was increased upon treatment with 25-HC and 27-HC. In-silico analysis of two independent studies of primary ER+ BC patients treated with neoadjuvant AIs showed that increased expression of MSMO1, EBP, LBR and SQLE enzymes, required for cholesterol synthesis and increased in our in-vitro models, was significantly associated with poor response to endocrine therapy.ConclusionTaken together, these data provide support for the role of cholesterol biosynthesis enzymes and the cholesterol metabolites, 25-HC and 27-HC, in a novel mechanism of resistance to endocrine therapy in ER+ BC that has potential as a therapeutic target.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-016-0713-5) contains supplementary material, which is available to authorized users.
Highlights
Therapies targeting estrogenic stimulation in estrogen receptor-positive (ER+) breast cancer (BC) reduce mortality, but resistance remains a major clinical problem
Current clinical strategies include the use of endocrine agents, which inhibit estrogen (E) signalling either by blocking the conversion of androgens to E in the case of aromatase inhibitors (AIs), by directly antagonizing estrogen receptor alpha (ER) function with agents such as tamoxifen, which compete with E for the ER and results in the recruitment of nuclear corepressors, or by use of drugs such as fulvestrant (ICI182780), which targets ER for proteasomal degradation [1,2,3,4]
Transcriptomic and proteomic analysis showed upregulation of cholesterol biosynthesis pathway was restricted to ER+ long-term estrogen deprivation (LTED) cells To identify novel mechanisms of resistance to Edeprivation, we first generated five LTED cell lines. wtMCF7, wt-ZR75.1, wt-T47D, wt-HCC1428 and wt-SUM44 cells were cultured in the absence of E2 until their growth rate was shown to be independent of the exogenous E2 (Fig. 1a)
Summary
Therapies targeting estrogenic stimulation in estrogen receptor-positive (ER+) breast cancer (BC) reduce mortality, but resistance remains a major clinical problem. Preclinical and clinical data support cross-talk between ER and growth factor receptor pathways, such as IGF1R and ERBB2/HER2 [7,8,9,10], which can lead to ligand-independent activation of the ER or can alter the phosphorylation state of nuclear co-activators, thereby changing the balance of ER transcription factors and potentiating transcription [11]. Despite this knowledge, few clinical trials have shown benefit from the targeting of endocrine resistance using signal transduction or receptor tyrosine kinase inhibitors. Expression profiling of primary ER+ BC samples has identified several promising signatures/networks for targeting [13]
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