RNA-Seq Analysis of Anacardic Acid Treated MCF7 and MDA-MB-231 Breast Cancer Cell Lines. Homo sapiens

Anacardic acid (AnAc) is a mixture of 6-alkylbenzoic acid congeners that are produced in a number of plants. Previously, we showed a specific congener AnAc 24:1n5 acts as a nuclear receptor alternate site modulator (NRAM) to inhibit breast cancer cells in an estrogen receptor (ER)-dependent manner by interfering with ER-DNA binding. AnAc 24:1n5 also inhibited the growth of a triple negative breast cancer (TNBC) cell line, through an undefined mechanism. Additional work from our labs indicated AnAc 24:1n5 inhibits prostaglandin synthase and that inhibition is more specific to COX-2. Reports from other investigators indicate AnAc has a number of interesting potential pharmacological targets. We previously used qRT-PCR to investigate expression changes in endogenous estrogen-regulated genes, i.e., TFF1, CCND1, and CTSD in breast cancer cell lines. However, since AnAc has the capacity of effect multiple molecular targets and since we detected an ER-independent inhibition of breast cancer cell proliferation, we suspect additional unknown molecular targets are affected in breast cancer cells. Identification of such targets using RNA-seq would be quite beneficial in targeting TNBC which primarily affects premenopausal women with a predominance in women of African and Latina ancestry. The goal of this portion of the project is to use next-generation RNA-SEQ to identify alterations in molecular target sequence levels in ER-positive and -negative breast cancer cell lines treated with AnAc. Overall design: There are two breast cancer cell lines used in this study, including MCF-7 (invasive breast ductal carcinoma; estrogen receptor positive (ER+); progesterone receptor positive (PR+); human epidermal growth factor 2 negative (HER2-)) and MDA-MB-231 (breast adenocarcinoma; triple negative – ER-; PR-; HER2-). Each of these cell lines was treated with anacardic acid (AnActrt) with three replicates each, resulting in a total of 12 RNA samples. One MDA-MB-231 control sample and one MDA-MB-231 AnActrt treated sample were removed after QA/QC determined they were likely contaminated samples.

漆树酸（Anacardic acid, AnAc）是一类由多种植物合成的6-烷基苯甲酸同系物混合物。既往本团队研究表明，特定同系物AnAc 24:1n5可作为核受体可变位点调节剂（nuclear receptor alternate site modulator, NRAM），通过干扰雌激素受体（estrogen receptor, ER）与DNA的结合，以雌激素受体依赖的方式抑制乳腺癌细胞增殖。此外，AnAc 24:1n5还可通过尚未阐明的分子机制抑制三阴性乳腺癌（triple negative breast cancer, TNBC）细胞系的生长。本实验室后续研究显示，AnAc 24:1n5可抑制前列腺素合酶，且该抑制作用对环氧合酶2（COX-2）具有更高特异性。其他研究者的报道亦指出，AnAc存在多项具有潜力的药理学靶点。本团队此前曾采用定量实时聚合酶链反应（quantitative real-time polymerase chain reaction, qRT-PCR），探究乳腺癌细胞系中内源性雌激素调控基因（如TFF1、CCND1及CTSD）的表达变化。鉴于AnAc可作用于多个分子靶点，且本团队已观测到其以雌激素受体非依赖的方式抑制乳腺癌细胞增殖，我们推测乳腺癌细胞中尚存在未被发现的其他分子靶点受AnAc调控。通过RNA测序（RNA-seq）鉴定此类靶点，将对靶向主要累及绝经前女性（其中非洲裔及拉丁裔女性占比更高）的三阴性乳腺癌具有重要应用价值。本项目此部分的研究目标为：采用下一代RNA测序技术（next-generation RNA-seq），鉴定经AnAc处理的雌激素受体阳性及阴性乳腺癌细胞系中分子靶点的表达水平变化。实验整体设计：本研究选用两种乳腺癌细胞系，分别为MCF-7细胞（侵袭性乳腺导管癌细胞；雌激素受体阳性（ER+）；孕激素受体阳性（PR+）；人表皮生长因子受体2阴性（HER2-））与MDA-MB-231细胞（乳腺腺癌；三阴性乳腺癌——ER-；PR-；HER2-）。每种细胞系均设置AnAc处理组（AnActrt），并设置3次生物学重复，共计获得12份RNA样本。经质量保证/质量控制（quality assurance/quality control, QA/QC）检测后，1份MDA-MB-231对照组样本与1份MDA-MB-231 AnAc处理组样本因疑似污染被移除。