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Mir-190 Enhances Endocrine Therapy Sensitivity by Regulating SOX9

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Mir-190 Enhances Endocrine Therapy Sensitivity by Regulating SOX9 Yu et al. Journal of Experimental & Clinical Cancer Research (2019) 38:22 https://doi.org/10.1186/s13046-019-1039-9 RESEARCH Open Access miR-190 enhances endocrine therapy sensitivity by regulating SOX9 expression in breast cancer Yue Yu1,2,3,4, Wen Yin1,2,3,4, Zhi-Hao Yu1,2,3,4, Yan-Jun Zhou1,2,3,4, Jiang-Rui Chi1,2,3,4, Jie Ge1,2,3,4 and Xu-Chen Cao1,2,3,4* Abstract Background: Breast cancer is the most common cancer among women worldwide, and approximately 70% of breast cancers are hormone receptor-positive and express estrogen receptor-α (ERα) or/and progesterone receptor. Therapies targeting ERα have been successfully used in patients with ERα+ breast cancer. However, intrinsic or acquired resistance to anti-estrogen therapy presents a major challenge. The Wnt/β-catenin signaling pathway regulates various processes that are important for cancer progression, and emerging evidences have shown a close interaction between Wnt/β-catenin and ERα signaling. miR-190 is also involved in ER signaling and our previous study indicated that miR-190 suppresses breast cancer metastasis. Methods: The effect of miR-190 on breast cancer anti-estrogen sensitivity was investigated both in vitro and in vivo. The protein expression levels and localization were analyzed by western blotting and immunofluorescence, respectively. Chromatin immunoprecipitation and dual-luciferase reporter assays were used to validate the regulation of the zinc-finger E-box binding homeobox 1/ ERα-miR-190-SRY-related high mobility group box 9 (ZEB1/ERα-miR-190-SOX9) axis. Results: miR-190 increased the anti-estrogen sensitivity of breast cancer cells both in vitro and in vivo. miR-190 inhibited Wnt/β-catenin signaling by targeting SOX9, and its expression inversely correlated with that of SOX9 in breast cancer samples. Furthermore, ERα and ZEB1 competitively regulated miR-190 expression. Conclusions: Our data uncover the ZEB1/ERα-miR-190-SOX9 axis and suggest a mechanism by which the Wnt/β-catenin signaling pathway is involved in breast cancer anti-estrogen therapy. Keywords: Breast cancer, Endocrine therapy, Wnt/β-catenin signaling, miR-190, SOX9, ZEB1 Introduction and is a key regulator of breast cancer development and Breast cancer is the most frequently diagnosed malignancy progression. Therapies targeting ERα have been success- in women worldwide [1]. It is the most common malig- fully applied in patients with ERα+ breast cancer [3]. nant tumor, and the third largest cause of cancer-related However, intrinsic or acquired resistance to anti-estrogen deaths in China. Although the incidence of this disease is therapy presents a major challenge. Thus, an improved increasing, the number of deaths caused by it is decreasing understanding of the ERα-related regulation network may [2]. Approximately 70% of breast cancers are hormone reveal new strategies for breast cancer endocrine therapy. receptor-positive and express estrogen receptor-α (ERα) miRNAs are a class of small, endogenous, non-coding or/and progesterone receptor. ERα is a nuclear receptor RNAs that negatively regulate the expression of a wide variety of genes by binding to complementary sequences * Correspondence: [email protected] in the 3′-untranslated regions (UTRs) of target mRNAs 1 The First Department of Breast Cancer, Tianjin Medical University Cancer [4, 5]. A large number of studies have shown that Institute and Hospital, National Clinical Research Center for Cancer, Huan-Hu-Xi Road, Hexi District, Tianjin 300060, China miRNA alteration or dysfunction is involved in cancer 2Key Laboratory of Cancer Prevention and Therapy, Tianjin 300060, China development and progression by regulating cancer cell 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. Yu et al. Journal of Experimental & Clinical Cancer Research (2019) 38:22 Page 2 of 13 proliferation, differentiation, apoptosis, angiogenesis, MDA-MB-435 were obtained from the Cell Bank of the metastasis, and metabolism [6, 7]. Dysregulated miRNAs Chinese Academy of Sciences (Shanghai, China). Culture are involved in breast cancer carcinogenesis and conditions have been described in the Additional file 1: progression and function as oncogenes or tumor sup- Supplementary Materials and Methods. pressors, as well as useful biomarkers in the diagnosis Breast cancer specimens were obtained from Tianjin and prognosis of breast cancer [8, 9]. miR-190 is located Medical University Cancer Institute and Hospital (TMU- in the intron region of the talin2 (TLN2) gene on CIH). Thirty primary breast cancer tissue samples were chromosome 15q22.2. Previous studies have shown that used for this study. All tumor samples were obtained the expression of miR-190 is reduced in aggressive neu- from patients newly diagnosed with breast cancer and roblastomas, and its overexpression leads to repression who had received no therapy before sample collection. of tumor growth and prolonged dormancy periods in This study was approved by the Institutional Review fast-growing tumors [10]. miR-190 suppresses the migra- Board of TMUCIH and written consent was obtained tion, invasion, and angiogenesis abilities of hepatocellu- from all participants. lar carcinoma cells through inhibition of epithelial– mesenchymal transition (EMT) phenotype [11]. In con- Transient and stable transfection of breast cancer cells trast, miR-190 expression is elevated in gastric cancer For transient transfection, miRNAs or siRNAs were tissues and contributes to gastric cancer progression transfected into different cell lines using FuGENE HD [12], suggesting that miR-190 may play a different role Transfection Reagent (Promega, Madison, WI, USA) and in different stages of tumor development and different plasmids were transfected using TransFast Transfection tumor environments. Our previous study indicated that Reagent (Promega) according to the manufacturer’s recom- miR-190 suppresses breast cancer metastasis by regula- mendations. To generate stable cells, lentiviruses (RiboBio, tion of transforming growth factor-β (TGF-β)-induced Shanghai, China) were used to infect MDA-MB-231 cells EMT [13]. The expression of circulating miR-190 is according the manufacturer’s recommendations. lower in breast cancer patients with early relapse com- pared to those without early relapse [14]. miR-190 is also Cell proliferation assay involved in ER signaling, causing inhibition of breast MTT, plate colony formation, and EdU assays were cancer metastasis [15]. Thus, we speculated that performed to evaluate cell proliferation, as previously miR-190 is involved in the ERα-related regulation described [13]. Experiments were carried our as de- network in breast cancer. scribed in the Additional file 1: Supplementary Materials In this study, we investigated the effect of miR-190 on and Methods. endocrine therapy resistance in breast cancer. miR-190 decreases the stemness and the activation of Wnt Mammosphere forming assay signaling, resulting in enhancement of endocrine therapy Single cells were plated at 10, 000 cells/ml on 6-well sensitivity by targeting SRY-related high mobility group plate in serum-free DMEM/F12 supplemented with box 9 (SOX9). We further demonstrated a mechanism 20 ng/ml EGF, 4 mg/ml insulin, 5 μg/ml heparin for zinc-finger E-box binding homeobox 1 (ZEB1)-- (Sigma-Aldrich), 1 μg/ml hydrocortisone, 0.5% BSA miR-190-SOX9 axis-mediated resistance to endocrine (Sigma-Aldrich) and B27 (Sigma-Aldrich). Fresh medium therapy in breast cancer. ZEB1 binds to the miR-190 was supplemented every three days. The mammosphere promoter region, competitively inhibiting ERα binding, formation efficiency (shown as percentage) was calculated and resulting in resistance to endocrine therapy. by dividing the number of mammospheres formed by the Therefore, our study revealed a novel mechanism of original number of single cells seeded. Wnt signaling pathway-mediated resistance to endocrine therapy in breast cancer. Western blotting and immunofluorescence Cells were lysed in protein lysis buffer [20 mM Tris-HCl Materials and methods (pH 7.4), 5 mM EDTA, 1% Triton X-100, 150 mM NaCl, Antibodies, reagents, plasmids, miRNA, and small and 1% DTT] containing a protease inhibitor cocktail interfering RNA (siRNA) tablet (Roche Molecular Biochemicals, Indianapolis, IN, The antibodies, reagents, plasmids, miRNAs, and siRNAs USA). Proteins were resolved by sodium dodecyl used in this study are listed in the Additional file 1: sulfate-polyacrylamide gel electrophoresis, transferred Supplementary Materials and Methods. onto polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA), and incubated with primary anti- Cell culture and clinical samples bodies overnight at 4 °C, followed by incubation with The human breast cancer cell lines MCF7, T47D, horseradish peroxidase-conjugated secondary antibody. MDA-MB-453, MDA-MB-468, MDA-MB-231, and The blots were visualized with ECL reagent (Millipore). Yu et al. Journal of Experimental
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