Abstract A37: Cholesterol biosynthesis pathway as a novel mechanism of resistance identified in ER+ long-term estrogen deprived cells
Authors: Simigdala Nikiana , Gao Qiong , Pancholi Sunil , Zvelebil Marketa , Ribas Ricardo , Dowsett Mitch , Martin Lesley-Ann 
Published: May 2016

Abstract
Over 80% of breast cancers (BC) at primary diagnosis express the estrogen receptor (ER+). Therapies targeting the estrogenic stimulation of tumor growth reduce mortality from ER+ BC but resistance remains a major clinical problem. Data from large studies such as TCGA indicate that other than a small number of high frequency mutations such as TP53, PIK3CA and GATA3 that have little association with endocrine resistance, primary ER+ BC shows very low frequency of individual mutations making targeting difficult. In contrast, expression profiling of primary ER+ BC samples have identified several promising signatures/networks for targeting. In order to identify common adaptive mechanisms associated with resistance to aromatase inhibitors, 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, MDA-MB-361 and ZR75.1) or 3D on collagen to model the stromal compartment (MCF7). At the point of resistance MCF7-LTED, HCC1428-LTED and SUM44-LTED retained ER expression whilst T47D-LTED, ZR75.1-LTED and MDA-MB-361-LTED reduced or lost ER. In order to identify common adaptive mechanisms, ingenuity pathway was used. Genes submitted for analysis were selected on the basis of a p-value of 0.001, FDR 5% and fold change 1.5 relative to the parental cell lines. Strikingly, the cholesterol biosynthesis pathway was the common up-regulated pathway in the ER+ LTED cell lines but not in the ER- LTED cell lines, suggesting a potential mechanism dependent on continued ER expression. Further interrogation showed increased expression of MSMO1 (x2.27), EBP (x1.57), SQLE (x2.37) and IDI1 (x2.74) in the MCF7-LTED. As mRNA does not always correlate with protein, we applied quantitative proteomics in wt-MCF7 and MCF7-LTED cells using dimethyl labelling followed by LC-MS/MS. Several enzymes within the cholesterol biosynthesis pathway were higher in abundance in the MCF7-LTED such as EBP (x1.54), SQLE (x3.24) and LBR (x1.86).
Evidence suggests that patients with cancer accumulate cholesteryl esters as a result of loss of PTEN or PI3K/AKT/mTOR activation, stimulating SREBP, ACAT1, HMGSCR and LDLR. As 40% of ER+ BC harbor PTEN loss or PIK3CA mutations, we assessed levels of SREBP, HMGCR, LDLR or ACAT1 in our data sets. Neither SREBP nor HMGCR mRNA or protein levels changed in the ER+ LTED or ER- LTED. Furthermore, ACAT1 was downregulated in the ER+ LTED. Moreover, levels of free and esterified cholesterol in wt-MCF7 and MCF7-LTED cell lysates were unchanged.
Previous studies have suggested oxysterols can act as selective ER-modulators (SERM). We subsequently investigated the function of these cholesterol metabolites in our LTED models. Treatment with 25-hydroxycholesterol (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. Targeting the individual genes of the cholesterol biosynthesis pathway with siRNAs caused a c. 30-60% drop in proliferation and concomitant drop in TFF1 expression in the ER+ LTED cells together with a reduction in recruitment of the ER and CREB binding protein (CBP) to the TFF1 promoter.
Gene expression data from 704 primary ER+ BC from patients treated with adjuvant tamoxifen showed that MSMO1 (p=0.047), EBP (p=0.043), SQLE (p=0.0001), DHCR7 (p=0.002) and IDI1 (p=0.0005), enzymes required for cholesterol synthesis and up-regulated in our in-vitro models, were associated with poor relapse free survival. Of note, DHCR7 forms part of the EndoPredict gene signature. Taken together, these data provide support for the role of the cholesterol biosynthesis pathway and its metabolites as a novel mechanism of resistance to endocrine therapy in ER+ BC and its potential as a therapeutic target.
Citation Format: Nikiana Simigdala, Qiong Gao, Sunil Pancholi, Marketa Zvelebil, Ricardo Ribas, Mitch Dowsett, Lesley-Ann Martin. Cholesterol biosynthesis pathway as a novel mechanism of resistance identified in ER+ long-term estrogen deprived cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A37.