miR-190 enhances endocrine therapy sensitivity by regulating SOX9 expression in breast cancer
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.
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.
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.
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.
KeywordsBreast cancer Endocrine therapy Wnt/β-catenin signaling miR-190 SOX9 ZEB1
cancer stem cell
reverse transcription quantitative polymerase chain reaction
small interfering RNA
sry-related high motility group box 9
transforming growth factor-β
zinc-finger E-box binding homeobox 1
Breast cancer is the most frequently diagnosed malignancy in women worldwide . It is the most common malignant tumor, and the third largest cause of cancer-related deaths in China. Although the incidence of this disease is increasing, the number of deaths caused by it is decreasing . Approximately 70% of breast cancers are hormone receptor-positive and express estrogen receptor-α (ERα) or/and progesterone receptor. ERα is a nuclear receptor and is a key regulator of breast cancer development and progression. Therapies targeting ERα have been successfully applied in patients with ERα+ breast cancer . However, intrinsic or acquired resistance to anti-estrogen therapy presents a major challenge. Thus, an improved understanding of the ERα-related regulation network may reveal new strategies for breast cancer endocrine therapy.
miRNAs are a class of small, endogenous, non-coding RNAs that negatively regulate the expression of a wide variety of genes by binding to complementary sequences in the 3′-untranslated regions (UTRs) of target mRNAs [4, 5]. A large number of studies have shown that miRNA alteration or dysfunction is involved in cancer development and progression by regulating cancer cell proliferation, differentiation, apoptosis, angiogenesis, metastasis, and metabolism [6, 7]. Dysregulated miRNAs are involved in breast cancer carcinogenesis and progression and function as oncogenes or tumor suppressors, as well as useful biomarkers in the diagnosis and prognosis of breast cancer [8, 9]. miR-190 is located in the intron region of the talin2 (TLN2) gene on chromosome 15q22.2. Previous studies have shown that the expression of miR-190 is reduced in aggressive neuroblastomas, and its overexpression leads to repression of tumor growth and prolonged dormancy periods in fast-growing tumors . miR-190 suppresses the migration, invasion, and angiogenesis abilities of hepatocellular carcinoma cells through inhibition of epithelial–mesenchymal transition (EMT) phenotype . In contrast, miR-190 expression is elevated in gastric cancer tissues and contributes to gastric cancer progression , suggesting that miR-190 may play a different role in different stages of tumor development and different tumor environments. Our previous study indicated that miR-190 suppresses breast cancer metastasis by regulation of transforming growth factor-β (TGF-β)-induced EMT . The expression of circulating miR-190 is lower in breast cancer patients with early relapse compared to those without early relapse . miR-190 is also involved in ER signaling, causing inhibition of breast cancer metastasis . Thus, we speculated that miR-190 is involved in the ERα-related regulation network in breast cancer.
In this study, we investigated the effect of miR-190 on endocrine therapy resistance in breast cancer. miR-190 decreases the stemness and the activation of Wnt signaling, resulting in enhancement of endocrine therapy sensitivity by targeting SRY-related high mobility group box 9 (SOX9). We further demonstrated a mechanism for zinc-finger E-box binding homeobox 1 (ZEB1)-miR-190-SOX9 axis-mediated resistance to endocrine therapy in breast cancer. ZEB1 binds to the miR-190 promoter region, competitively inhibiting ERα binding, and resulting in resistance to endocrine therapy. Therefore, our study revealed a novel mechanism of Wnt signaling pathway-mediated resistance to endocrine therapy in breast cancer.
Materials and methods
Antibodies, reagents, plasmids, miRNA, and small interfering RNA (siRNA)
The antibodies, reagents, plasmids, miRNAs, and siRNAs used in this study are listed in the Additional file 1: Supplementary Materials and Methods.
Cell culture and clinical samples
The human breast cancer cell lines MCF7, T47D, MDA-MB-453, MDA-MB-468, MDA-MB-231, and MDA-MB-435 were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Culture conditions have been described in the Additional file 1: Supplementary Materials and Methods.
Breast cancer specimens were obtained from Tianjin Medical University Cancer Institute and Hospital (TMUCIH). Thirty primary breast cancer tissue samples were used for this study. All tumor samples were obtained from patients newly diagnosed with breast cancer and who had received no therapy before sample collection. This study was approved by the Institutional Review Board of TMUCIH and written consent was obtained from all participants.
Transient and stable transfection of breast cancer cells
For transient transfection, miRNAs or siRNAs were transfected into different cell lines using FuGENE HD Transfection Reagent (Promega, Madison, WI, USA) and plasmids were transfected using TransFast Transfection Reagent (Promega) according to the manufacturer’s recommendations. To generate stable cells, lentiviruses (RiboBio, Shanghai, China) were used to infect MDA-MB-231 cells according the manufacturer’s recommendations.
Cell proliferation assay
MTT, plate colony formation, and EdU assays were performed to evaluate cell proliferation, as previously described . Experiments were carried our as described in the Additional file 1: Supplementary Materials and Methods.
Mammosphere forming assay
Single cells were plated at 10, 000 cells/ml on 6-well plate in serum-free DMEM/F12 supplemented with 20 ng/ml EGF, 4 mg/ml insulin, 5 μg/ml heparin (Sigma-Aldrich), 1 μg/ml hydrocortisone, 0.5% BSA (Sigma-Aldrich) and B27 (Sigma-Aldrich). Fresh medium was supplemented every three days. The mammosphere formation efficiency (shown as percentage) was calculated by dividing the number of mammospheres formed by the original number of single cells seeded.
Western blotting and immunofluorescence
Cells were lysed in protein lysis buffer [20 mM Tris-HCl (pH 7.4), 5 mM EDTA, 1% Triton X-100, 150 mM NaCl, and 1% DTT] containing a protease inhibitor cocktail tablet (Roche Molecular Biochemicals, Indianapolis, IN, USA). Proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA), and incubated with primary antibodies overnight at 4 °C, followed by incubation with horseradish peroxidase-conjugated secondary antibody. The blots were visualized with ECL reagent (Millipore).
For immunofluorescence analysis, cells were seeded onto glass coverslips in 24-well plates, washed with phosphate buffered saline (PBS), fixed in 4% formaldehyde solution for 30 min, and then permeabilized with 0.2% Triton X-100/PBS for 15 min. Cells were blocked with 2% bovine serum albumin in PBS for 30 min. Coverslips were incubated with primary antibodies overnight at 4 °C, followed by incubation with FITC-/TRITC-conjugated secondary antibodies for 1 h at room temperature and then stained with 4′,6-diamidino-2-phenylindole. Finally, coverslips were observed under a fluorescence microscope.
RNA extraction and reverse transcription quantitative polymerase chain reaction (RT-qPCR)
Total RNA of cultured cells, surgically resected fresh breast tissues, and formalin-fixed paraffin-embedded clinical specimens were extracted using mirVana PARIS kit (Life Technologies) according to the manufacturer’s recommendations. qPCR was performed to detect mRNA expression using GoTaq qPCR Master Mix (Promega). TaqMan RT-qPCR was performed to detect mature miRNA expression using TaqMan miRNA reverse transcription kit, has-RNU6B (U6, ABI Assay ID: 001093), and miR-190 (ABI Assay ID: 000489) according to the manufacturer’s protocol (Life Technologies). The sequences of PCR primers are listed in Additional file 1: Table S1.
Chromatin immunoprecipitation (ChIP) analysis
ChIP assay was performed according to the protocol of Upstate Biotechnology, as previously described . The primer sequences used for miR-190 promoter were 5′-GAATGGCTCATGGTCTTTG-3′ and 5′-GCAGCAACTCCGATAACTG-3′ (ZEB1), 5′-GACAGTTATCGGAGTTGCT-3′ and 5′-CGTGTTCTTTCCTGTTGCC-3′ (ERα).
Luciferase reporter assays
Luciferase assays were carried out using a dual luciferase assay kit according to the manufacturer’s recommendations, as previously described .
Stable miR-190-overexpressing MDA-MB-231 and control cells (1 × 106 cells) together with 100 μg of Matrigel (BD Biosciences, San Diego, CA, USA) were inoculated into the mammary fat pads of 5-week-old female BALB/c mice. Tumor development was allowed to reach a volume of ~ 100 mm3. The mice were then randomized into 2 groups, and placebo or tamoxifen pellets (5 mg/pellet) were subcutaneously embedded for another 3 weeks. Tumor growth was recorded once a week with a caliper-like instrument. Tumor volume was calculated according to the formula volume = (width2 × length)/2. Eight weeks after inoculation, mice were killed, and the final volume and weight of tumor tissues were determined. All animal experimental protocols were approved by the Animal Ethics Committee of TMUCIH.
Data are presented as mean ± standard deviation. The student’s t-test (2-tailed) was used to determine differences between the experimental and control groups. The level of significance was set to P < 0.05. Spearman’s correlation was used to test the significance of association between miR-190 and SOX9 expression. All calculations were performed with the SPSS for Windows statistical software package (SPSS Inc., Chicago, IL, USA).
miR-190 increases the anti-estrogen sensitivity of breast cancer cells in vitro
Overexpression of miR-190 increases the anti-estrogen sensitivity of breast cancer cells in vivo
SOX9 is a direct target of miR-190
miR-190 affects anti-estrogen sensitivity and stem cell content by suppressing SOX9
miR-190 inhibits Wnt/β-catenin signaling by down-regulation of SOX9
ERα and ZEB1 competitively bind to the miR-190 promoter and regulate its expression
miR-190 mediated the regulation of SOX9 expression via ERα and ZEB1
Development of resistance to anti-estrogen therapy remains to be one of the major barriers to the successful treatment of patients with breast cancer. Our work suggested an important role of miR-190 in anti-estrogen resistance in breast cancer. First, we demonstrated that miR-190 inhibits Wnt/β-catenin signaling and increases the anti-estrogen sensitivity of breast cancer cells both in vitro and in vivo. Second, SOX9 was found to be a direct target of miR-190. Third, ERα and ZEB1 were shown to competitively bind to the miR-190 promoter and regulate its expression. Therefore, our results revealed a novel mechanism of constitutive Wnt/β-catenin signaling activation in anti-estrogen resistance in breast cancer and demonstrated that miR-190 functions as a tumor-suppressive miRNA in breast cancer.
miRNAs are small, non-protein coding RNAs first identified over a decade ago, and their dysregulation has been implicated in cancer development, progression, and drug resistance. Numerous miRNAs have been shown to be involved in acquired resistance to anti-estrogen therapies through regulating ERα and its interactors, growth factor receptor signaling, cell cycle regulators, and EMT . miR-190 has been identified to be a tumor suppressor in several types of cancer, including neuroblastoma, prostate cancer, hepatocellular carcinoma, and breast cancer [10, 14, 23, 24]. Our previous study also indicated that miR-190 suppresses breast cancer metastasis and EMT phenotype through regulating TGF-β signaling . Here, we observed that miR-190 increases the anti-estrogen sensitivity of breast cancer cells both in vitro and in vivo. CSCs possess the capacity for self-renewal and have the ability to drive the continued expansion of a population of malignant cells with invasive and metastatic propensity . A growing body of evidence indicates that endocrine therapy-resistant breast cancer cells possess CSC characteristics . Consistent with this, we showed that miR-190-overexpressing ER− breast cancer cells have a decreased percentage of CD44high/CD24low CSC population, and therefore, they will be more sensitive to tamoxifen.
SOX9, one of the members of the SOX family of transcriptional factors, plays a critical role in regulating developmental processes, including sex determination, chondrogenesis, neurogenesis, and neural crest development [27, 28, 29, 30]. Many studies have demonstrated that SOX9 plays active roles during cancer tumorigenesis and progression in various types of cancer, including breast cancer . Gene expression profiling has identified SOX9 as one of the signature genes that defines the basal-like subtype of breast cancer . Furthermore, co-expression of SOX9 and Slug promotes the tumorigenic and metastasis-seeding capacities of breast cancer cells and is associated with unfavorable survival . Consistently, depletion of SOX9 suppresses the tumor-initiating and metastatic abilities of breast cancer cells . Recently, SOX9 was reported to be upregulated in tamoxifen-resistant breast cancer cells and drive breast cancer endocrine resistance [34, 35]. In our study, we identified SOX9 as a direct target of miR-190. More importantly, re-expression of SOX9 could reverse miR-190-induced increase in anti-estrogen sensitivity. These results indicated that SOX9 dysfunction contributes to the biological functions of miR-190 in breast cancer, especially anti-estrogen sensitivity.
The Wnt/β-catenin signaling pathway plays a critical role in adult-tissue homeostasis, stem cell self-renewal, and somatic cell reprogramming [36, 37]. Furthermore, this pathway regulates various processes that are important for cancer progression, including tumor initiation, tumor growth, cell senescence, cell death, differentiation, and metastasis, and the agents which can alter Wnt/β-catenin signaling have been used for clinical trials in preclinical models . Emerging evidences have shown a close interaction between Wnt/β-catenin and ERα signaling [39, 40]. β-catenin has been shown to be associated with redistribution of ERα . In addition, activation of Wnt/β-catenin signaling has been shown to be responsible for maintaining the stem cell-like characteristics of breast cancer cells, resulting in resistance to drugs, including tamoxifen [42, 43]. SOX9 has been identified to drive Wnt/β-catenin signaling pathway activation and cancer progression [19, 20, 44]. In line with these findings, we demonstrated that overexpression of miR-190 prevents the nuclear translocation of β-catenin and inhibits the activity of Wnt/β-catenin signaling. More importantly, re-expression of SOX9 could reverse miR-190-induced suppression of Wnt/β-catenin signaling. These results indicated that miR-190 inhibits the activity of Wnt/β-catenin signaling by targeting SOX9. ZEB1, a crucial member of the zinc finger homeodomain transcription factor family, is overexpressed in breast cancer cells and promotes breast tumorigenesis and cancer progression . Recently, ZEB1 has been reported to repress ERα expression and attenuate cell growth inhibition by anti-estrogens . Interestingly, we found a half ERE and two E-box binding sites on the miR-190 promoter. Furthermore, ERα and ZEB1 were found to competitively bind to the miR-190 promoter and regulate its expression. Taken together, our results showed that overexpression of ZEB1 could transcriptionally suppress the expression of ERα and miR-190 and led to SOX9 elevation and activation of Wnt/β-catenin signaling, resulting in anti-estrogen resistance therapies in breast cancer.
In summary, we demonstrated that miR-190 inhibits Wnt/β-catenin signaling and increases anti-estrogen sensitivity by targeting SOX9. Furthermore, miR-190 is a transcriptional target of both ZEB1 and ERα. ERα and ZEB1 were found to competitively bind to the miR-190 promoter and regulate its expression. Based on the findings from this study and others, we propose a model that highlights the role of miR-190 in regulating Wnt/β-catenin signaling during breast cancer anti-estrogen resistance (Fig. 7d). The uncovering of the ZEB1/ERα-miR-190-SOX9 axis provides a better understanding of the Wnt/β-catenin network complexity. miR-190 may serve as a new option to target Wnt/β-catenin signaling for breast cancer intervention.
This study was supported by the National Natural Science Foundation of China (No. 81502518, No. 81372843 and No. 81702624), and the Natural Science Foundation of Tianjin City (No. 17JCQNJC10400).
Availability of data and materials
All data generated or analyzed during this study are included in this published article and its supplementary information files.
YY and CXC designed the study; YY, YW, YZJ, ZJY, CJR performed the experiments; YY and CJR performed the statistical analysis; GJ participated in the clinical specimens detection; YY and CXC wrote and revised the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
This study was approved by the Institutional Review Board of TMUCIH and written consent was obtained from all participants.
Consent for publication
The authors declare that they have no competing interests.
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- 15.Chu HW, Cheng CW, Chou WC, Hu LY, Wang HW, Hsiung CN, Hsu HM, Wu PE, Hou MF, Shen CY, Yu JC. A novel estrogen receptor-microRNA 190a-PAR-1-pathway regulates breast cancer progression, a finding initially suggested by genome-wide analysis of loci associated with lymph-node metastasis. Hum Mol Genet. 2014;23:355–67.CrossRefGoogle Scholar
- 16.Yu Y, Wang XY, Sun L, Wang YL, Wan YF, Li XQ, Feng YM. Inhibition of KIF22 suppresses cancer cell proliferation by delaying mitotic exit through upregulating CDC25C expression. Carcinogenesis. 2014;35:1416–25.Google Scholar
- 27.Chaboissier MC, Kobayashi A, Vidal VI, Lutzkendorf S, van de Kant HJ, Wegner M, de Rooij DG, Behringer RR, Schedl A. Functional analysis of Sox8 and Sox9 during sex determination in the mouse. Development. 2004;131:1891–901.Google Scholar
- 35.Xue Y, Lai L, Lian W, Tu X, Zhou J, Dong P, Su D, Wang X, Cao X, Chen Y, Wang Q. SOX9/FXYD3/Src axis is critical for ER+ breast cancer stem cell function. Mol Cancer Res. 2018;17:238–49.Google Scholar
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