Liu et al. Clinical Epigenetics (2019) 11:16 https://doi.org/10.1186/s13148-019-0620-6 SHORT REPORT Open Access Epigenetic biomarker screening by FLIM- FRET for combination therapy in ER+ breast cancer Wenjie Liu1,2,3†, Yi Cui1,2,4†, Wen Ren1,2 and Joseph Irudayaraj1,2,3* Abstract Hormone-dependent gene expression involves dynamic and orchestrated regulation of epigenome leading to a cancerous state. Estrogen receptor (ER)-positive breast cancer rely on chromatin remodeling and association with epigenetic factors in inducing ER-dependent oncogenesis and thus cell over-proliferation. The mechanistic differences between epigenetic regulation and hormone signaling provide an avenue for combination therapy of ER-positive breast cancer. We hypothesized that epigenetic biomarkers within single nucleosome proximity of ER- dependent genes could serve as potential therapeutic targets. We described here a Fluorescence lifetime imaging- based Förster resonance energy transfer (FLIM-FRET) methodology for biomarker screening that could facilitate combination therapy based on our study. We screened 11 epigenetic-related markers which include oxidative forms of DNA methylation, histone modifications, and methyl-binding domain proteins. Among them, we identified H4K12acetylation (H4K12ac) and H3K27 acetylation (H3K27ac) as potential epigenetic therapeutic targets. When histone acetyltransferase inhibitor targeting H4K12ac and H3K27ac was combined with tamoxifen, an enhanced therapeutic outcome was observed against ER-positive breast cancer both in vitro and in vivo. Together, we demonstrate a single molecule approach as an effective screening tool for devising targeted epigenetic therapy. Keywords: Fluorescence lifetime, Förster resonance energy transfer, Epigenetic markers, Combination therapy, Breast cancer, Histone acetylation, Anacardic acid Introduction therapy provides an alternative to further improve the Breast cancer poses a tremendous burden on health care outcomes of conventional treatments such as surgery due to its high prevalence and mortality [1, 2]. Even and chemotherapy [8]. To screen for molecular targets though substantial progress has been made to detect the in cancer therapy, traditional methods rely on estab- disease early and prolong patient survival, currently lished libraries comprising of tens of thousands of com- available treatments are far from satisfactory [3, 4]. Of pounds to assess their effects on a model cell line, which the breast cancer population, around 80% of patients are too empirical, low in efficiency, and do not account bear ER-positive tumors, and this makes hormone ther- for molecular interaction [9, 10]. Direct interactions be- apy that block the estrogen pathways a standard option tween ER and various epigenetic modifications could of treatment [5]. However, long-term hormonal therapy provide insights into its role in breast cancer develop- would result in side effects and even tumor recurrence ment as a potential therapeutic target. Such interactions which are resistant to subsequent treatments. Such could be readily and precisely evaluated by fluorescence inadequacies require more in-depth evaluation and lifetime imaging-based Förster resonance energy transfer optimization of therapies [6, 7]. Targeted molecular (FLIM-FRET) at single molecule resolution. FLIM-FRET allows for the observation of interaction * Correspondence: [email protected] and other proximity-based evaluation directly unlike †Wenjie Liu and Yi Cui contributed equally to this work. 1Department of Agricultural and Biological Engineering, Purdue University, intensity-based FRET which is susceptible to reversible West Lafayette, IN 47907, USA photobleaching or photoconversion of donor molecules 2Bindley Bioscience Center, Purdue University, West Lafayette 47907, IN, USA Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Liu et al. Clinical Epigenetics (2019) 11:16 Page 2 of 9 [11]. In drug discovery, FRET screening has been used to ER-dependent breast cancer. Moreover, the combin- identify molecules or drugs that dissociate DNA-protein ation of tamoxifen and anacardic acid could potentially or protein-protein interactions in search of possible in- yield a promising therapeutic strategy for ER-positive hibitors [12, 13]. Other applications of FRET include breast cancers. evaluation of apoptosis or protein aggregation for drug screening with known anticancer molecules [14, 15]. Results and discussion These applications focus on FRET screening of known FLIM-FRET screening identifies epigenetic biomarker compounds and have not been utilized to discover novel candidates for therapy therapeutic targets. We first assessed the local epigenetic changes associated Herein, we evaluate the interaction of epigenetic targets with ERα in MCF7 cells and ER-positive tumor tissue with ERα by FLIM-FRET to reveal secondary therapeutic samples from patients. The co-localization of ERα and targets. Based on our screening results, we find that epigenetic biomarkers evaluated by FRET suggests a pos- H3K27 acetylation (H3K27ac) and H4K12 acetylation sible interaction and plays a role in ER binding to ERE (H4K12ac) usually co-localize with ERα at the nanoscale genes to induce cancer cell proliferation (Fig. 1a). Utiliz- resolution and could serve as potential therapeutic targets. ing FLIM-FRET, we evaluated the molecular interactions Therefore, we assessed the effects of histone acetyltrans- of 11 different epigenetic relevant targets for possible as- ferase inhibitor (HATi) on these potential epigenetic tar- sociation with ERα in MCF7 cells. We set the FRET effi- gets. Our results show that a combination of tamoxifen ciency threshold to above 5% for target identification, and anacardic acid (AA) resulted in significant tumor sup- which indicates that the targets (ERα and epigenetic pression on MCF7 cell growth both in vitro and in mice marks) are co-localized within 10 nm from each other. A xenograft than treatment with either drug alone. Anacar- typical FRET-induced fluorescence lifetime change in dic acid not only reduces the amount of these epigenetic the ERα–ALEXA488 complex is shown in Fig. 1b. FRET modifications in vitro but also inhibits H4K12ac-estrogen analysis of the 11 epigenetic markers with ERα based on receptor element (ERE) interactions, revealing a possible immunostaining was performed to determine possible mechanism of action of AA in breast cancer treatment. As molecular interactions as epigenetic drug targets (Fig. 2a, proof of principle, we demonstrated that a FLIM-FRET b, Additional file 1: Figure S1A). Results from quantita- platform could serve as a robust and reliable method for tive screening suggest that 5-formylcytosine (5-fC), discovering novel epigenetic therapeutic targets in methyl-CpG-binding domain protein 2 (MBD2), Fig. 1 FLIM-FRET screening identifies potential epigenetic treatment targets. a Schematic illustration of possible ERα-epigenetic biomarker association in a nucleosome scale that facilitates ER-regulated gene transcription. b Typical FLIM images show control (left) and FRET (right) results as lifetime reduces with distance between donor and acceptor fluorophores. c Schematic and prototype of biopsy tissue arrays subjected to FLIM analysis Liu et al. Clinical Epigenetics (2019) 11:16 Page 3 of 9 Fig. 2 FLIM-FRET screening results from MCF7 cell and patient tissue. a Summarized lifetime information of the FRET donor (mean ± s.d.). b Calculated average FRET efficiency based on lifetime values. FRET efficiency of 5% is set as the threshold. c FLIM-FRET screening results from MCF7 cell immunostaining indicates H4K12ac, H3K27ac, MBD2, and 5fC are in close proximity with ERα. Data from 3 independent immunostains with 15 cells analyzed in each experiment. d FLIM-FRET screening from ER+ patient tissue array shows H4K12ac, H3K27ac, and MBD2 as potential treatment targets, n = 30. Data shown as boxplot with values of minimum, 5% percentile, 25% percentile, median, 75% percentile, maximum, and mean (center square marks) H3K27ac, and H4K12ac are potential candidates for epi- studies showed that H4K12ac had increasing occupancy genetic therapy (Fig. 2c). near the transcription start sites of estrogen-induced genes To further facilitate the screening outcome, we evalu- and had a stronger correlation with the ER-positive phe- ated patient tissue arrays for H4K12ac, H3K27ac, and notypes [21]. E2 stimulated H3K27ac was abundant at the MBD2 (Fig. 1c, Additional file 1: Figure S1B). We ob- distal ERE of TFF1 gene, suggesting a potential recruiting served a consistent outcome for all of these targets to be role of H3K27ac at ERE [22–24]. These findings support within 10 nm from ERα (Fig. 2d). The tissue-screening our FLIM-FRET screening results which implicate array showed excellent potential for high throughput H4K12ac and H3K27ac as potential targets in ER+ breast screening and automation with further