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. Author manuscript; available in PMC: 2014 Apr 10.
Published in final edited form as: Oncol Rep. 2011 Oct 4;27(1):10–16. doi: 10.3892/or.2011.1488

The histone deacetylase inhibitor trichostatin A alters microRNA expression profiles in apoptosis-resistant breast cancer cells

LYNDSAY V RHODES 1, ASHLEY M NITSCHKE 1, H CHRIS SEGAR 1, ELIZABETH C MARTIN 1, JENNIFER L DRIVER 1, STEVEN ELLIOTT 1, SEUNG YOON NAM 6, MENG LI 6, KENNETH P NEPHEW 6, MATTHEW E BUROW 1,2,4,5, BRIDGETTE M COLLINS-BUROW 1,4,5
PMCID: PMC3982613  NIHMSID: NIHMS377639  PMID: 21971930

Abstract

The development of drug resistance represents a major complication in the effective treatment of breast cancer. Epigenetic therapy, through the use of histone deacetylase inhibitors (HDACi) or demethylation agents, is an emerging area of therapeutic targeting in a number of ontological entities, particularly in the setting of aggressive therapy-resistant disease. Using the well-described HDAC inhibitor trichostatin A (TSA) we demonstrate the suppression of in vitro clonogenicity in the previously described apoptosis-resistant MCF-7TN-R breast carcinoma cell line. Additionally, recent work has demonstrated that these agents can alter the expression profile of microRNA signatures in malignant cells. Using an unbiased microRNA microarray analysis, changes in miRNA expression of MCF-7TN-R cells treated with TSA for 24 h were analyzed. We observed significant up-regulation of 22 miRNAs and down-regulation of 10 miRNAs in response to TSA treatment. Our results demonstrate that the HDACi, TSA, exerts anticancer activity in the apoptosis-resistant MCF-7TN-R breast carcinoma cell line. This activity is correlated with TSA alteration of microRNA expression profiles indicative of a less aggressive phenotype.

Keywords: microRNA, trichostatin A, histone deacetylase, MCF-7, breast cancer, drug resistance

Introduction

Despite significant advancement in the area of endocrine therapies and chemotherapeutics, nearly half of breast cancer patients exhibit de novo resistance, while the majority of remaining patients ultimately progress to drug resistance (1). Drug resistant breast cancer is associated with poor prognoses (2,3), highlighting the critical need to develop novel therapeutics that are effective against these more aggressive forms of the disease. Epigenetic alterations, including aberrant DNA methylation and histone deacetylation, participate in cancer development and progression (4). Epigenetic aberrations lead to breast cancer chemotherapy resistance (5,6); hence, their reversal by inhibitors of DNA methylation and histone deacetylases (DNMTi and HDACi) may overcome it and are at present undergoing clinical testing, either alone or in combination with conventional chemotherapies (7).

Histone deacetylases (HDACs) and histone acetyltransferases (HATs) have important roles in the maintenance and function of chromatin by regulating the acetylation of histones. In addition, these enzymes have recently been shown to regulate the acetylation of many non-histone targets and therefore may represent a key means of post-translational regulation beyond their established roles in transcriptional regulation. The use of HDACi in the clinical setting is currently FDA-approved only for the treatment of progressive or recurrent cutaneous T-cell lymphoma following two other systemic therapies (8). Biologically, HDACi induce growth arrest, differentiation and cell death in breast cancer cells, but the underlying mechanism warrants further investigation.

In addition to direct regulation of mRNA gene expression, HDACis have been shown to alter microRNA (miRNA) expression in several human carcinomas including pancreatic (9), colon (10,11), gastric (12) and breast (13). microRNAs are small non-coding RNAs (18-22 nt) which function as an additional layer of regulation of mRNA stability and translation through 3′-UTR targeting (14). Through their ability to target the 3′-UTR of multiple genes, individual miRNAs can exert vast effects on mRNA-protein expression in cells. In cancer, miRNAs can function as tumor suppressors or oncogenes (15). We examined the effects of the HDACi trichostatin A (TSA) on the survival of the apoptotic-resistant MCF-7TN-R breast carcinoma cell line, as well as its effects on miRNA expression.

Materials and methods

Cell generation and culture

The apoptotic-resistant MCF-7TN-R cells were derived from MCF-7 cells grown in increasing concentrations of tumor necrosis factor α (TNFα) until resistance was established. Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) (10% DMEM). The detailed methods are described by Weldon et al (16).

Clonogenicity assay

Colony survival assays were performed as previously published (17). Briefly, cells were cultured in 10% FBS-DMEM media. Cells were seeded at a density of 2,000 cells/well in 2 ml of media in 6-well plates. Cells were allowed to adhere overnight at 37°C and treated on the following day with vehicle (DMSO) or TSA (0.1, 1, 10 μM). After 10 days, media was removed and the cells were fixed with gluteraldehyde. Cells were stained with crystal violet (0.1% in 20% methanol) for visualization. Colonies >50 cells were manually counted and treatments normalized to vehicle control. Assays were run in quadruplicate with internal duplicates.

microRNA microarray analysis

MCF-7TN-R cells were plated at a density of 2×106 cells in 25 cm2 flasks in normal culture media (DMEM media supplemented with 5% FBS, 1% penicillin/streptomycin, 1% essential amino acids, 1% non-essential amino acids and 1% sodium pyruvate) and allowed to adhere overnight at 37°C, 5% CO2 and air. The following day the media was changed to phenol red-free media (supplemented as above) and 5% charcoal-stripped serum was substituted for the 5% FBS. Cells were treated with TSA (10 μM) or DMSO for 24 h. Cells were harvested in PBS, collected by centrifugation, and total RNA extracted using the miRNeasy kit (Qiagen) according to manufacturer’s protocol. Enrichment for miRNA was not performed. Quantity and quality of RNA was determined by absorbance (260, 280 nm), and 5 μg total RNA was used for microarray analysis. Microarray analysis was performed as we have previously described (18). Briefly, a custom microarray (19) was used to determine miRNA expression, using three biological replicates for each condition (± TSA). Low intensity probes (signal <100 in more than half samples) were excluded from the analysis. Raw data was log-transformed and normalized by IQR. Clustering of miRNA expression data was performed using CLUSTER (20), with filtering to remove inconsistencies between replicates. For clustering, we first log-transformed the data and median-centered the array and genes, followed by average linkage clustering. Clustering results were visualized by TreeView (http://rana.lbl.gov/EisenSoftware.htm). Full array data is shown in Table I.

Table I.

microRNA microarray results for MCF-7TN-R cells treated with TSA (10 μM) for 24 h.

ID Serial no. Average (TSA) Average (DMSO) p-value logFC
hsa-miR-215 990 498.33 323.67 0.001 1.46
hsa-miR-657 725 206.33 573.67 0.002 −1.13
hsa-miR-139 524 886.33 783.67 0.003 0.88
hsa-miR-155 544 192.67 176.33 0.005 0.73
hsa-miR-146b 1501 182.67 178.00 0.005 0.66
hsa-miR-645 1681 181.33 519.67 0.006 −1.27
hsa-miR-544 127 374.67 305.67 0.006 1.01
hsa-miR-194 1541 362.67 319.67 0.007 0.95
hsa-miR-628 696 190.67 371.67 0.007 −0.60
hsa-miR-144 1498 148.00 115.33 0.008 0.94
hsa-miR-144 530 154.67 110.67 0.008 1.10
hsa-miR-559 1112 128.67 111.67 0.008 0.74
hsa-miR-128b 511 868.00 905.00 0.009 0.62
hsa-miR-143 529 204.67 143.67 0.009 1.13
hsa-miR-568 1121 145.67 130.33 0.009 0.74
hsa-miR-769-5p 744 230.33 502.33 0.010 −0.69
hsa-miR-1 1461 226.00 130.33 0.010 1.48
hsa-miR-497 645 296.67 639.67 0.011 −0.66
hsa-miR-632 1668 217.33 603.00 0.012 −1.12
hsa-miR-767-5p 1708 178.00 392.67 0.012 −0.73
hsa-miR-512-3p 660 155.33 362.67 0.013 −0.82
hsa-miR-191* 1537 206.67 138.33 0.014 1.25
hsa-miR-191* 569 285.33 155.33 0.015 1.65
hsa-miR-155 1512 163.67 150.67 0.015 0.70
hsa-miR-613 1649 246.00 624.33 0.016 −0.93
hsa-miR-202* 977 479.33 378.00 0.016 1.05
hsa-miR-636 704 226.67 705.33 0.018 −1.20
hsa-miR-486 1600 192.00 129.67 0.019 1.21
hsa-miR-622 1658 181.33 401.67 0.019 −0.79
hsa-miR-215 22 469.00 289.00 0.020 1.61
hsa-miR-22 27 1007.33 568.33 0.021 1.82
hsa-miR-875-5p 903 139.67 103.33 0.024 0.99
hsa-miR-620 1656 210.00 127.33 0.024 1.36
hsa-miR-370 581 1265.67 1152.67 0.026 0.88
hsa-miR-200c 7 272.67 278.00 0.026 0.60
hsa-miR-638 706 1096.67 1021.00 0.026 0.85
hsa-miR-630 1666 140.33 129.00 0.028 0.71
hsa-miR-208 14 191.33 168.67 0.028 0.74
hsa-miR-526c 121 321.33 215.33 0.028 1.27
hsa-miR-643 711 138.33 268.33 0.029 −0.61
hsa-miR-651 719 142.33 332.67 0.029 −0.84
hsa-miR-149 537 733.67 711.00 0.031 0.71
hsa-miR-589 1880 150.67 137.00 0.032 0.77
hsa-miR-768-3p 1709 994.33 1006.00 0.033 0.79
hsa-miR-22 995 935.67 635.00 0.035 1.65
hsa-miR-627 1663 133.67 102.00 0.035 0.96
hsa-miR-450 1588 270.67 571.33 0.036 −0.63
hsa-miR-620 688 198.33 136.67 0.036 1.17
hsa-miR-211 987 618.67 578.33 0.037 0.83
hsa-miR-346 84 625.33 604.67 0.041 0.77
hsa-miR-1 493 256.00 184.67 0.041 1.11
hsa-miR-607 191 171.33 155.33 0.041 0.70
hsa-miR-153 1509 145.67 124.00 0.041 0.85
hsa-miR-621 1657 277.00 196.00 0.042 1.10
hsa-miR-668 1700 233.33 538.67 0.043 −0.74
hsa-miR-607 1159 105.33 254.00 0.043 −1.04
hsa-miR-129 1480 533.33 495.00 0.045 0.82
hsa-miR-335 76 175.67 134.00 0.045 0.96
hsa-miR-640 1676 188.67 447.00 0.047 −0.80
hsa-miR-524* 116 256.00 243.00 0.048 0.61
hsa-miR-663 731 681.33 757.67 0.049 0.60
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Statistical analyses

Colony assays were analyzed by one-way ANOVA with the Tukey’s post-test (Graph Pad Prism V.4); p<0.05 was considered to indicate statistically significant differences. The Student’s t-test was performed to evaluate the statistical significance of the cluster selection. For microRNA microarray data, the Welch’s t-test was performed for each probe using their normalized signals, with p-values <0.05 considered significant as previously described (21).

Results

TSA inhibits drug-resistant breast cancer cell clonogenic survival

We have previously described the generation of the MCF-7TN-R cells (16) which have acquired resistance to TNFα- and TRAIL-induced cell death. These cells have been characterized as highly aggressive and metastatic, and have been developed as a model system of chemoresistant breast carcinoma (22,23). To determine the effects of HDACi on apoptotic and clonogenic survival, MCF-7TN-R cells were treated with varying concentrations of TSA (0.1, 1 and 10 μM) or vehicle control (DMSO) for 10 days. MCF-7TN-R cells treated with TSA at 10 μM for 10 days demonstrated a decrease (p<0.01) in colony formation compared to vehicle-treated cells (62.37±6.45%, Fig. 1).

Figure 1.

Figure 1

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TSA suppression of MCF-7TN-R cell clonogenic survival. MCF-7TN-R cells were plated (2,000 cells/well) in 10% DMEM in 6-well plates and allowed to adhere overnight. Twenty-four hours later the cells were treated with vehicle (DMSO) or TSA (0.1, 1, 10 μM) for 10 days. Colonies of ≥50 cells were counted as positive. Bars represent mean percentage clonogenic survival normalized to DMSO control cells ± SEM. (**p<0.01).

TSA induces microRNA expression in MCF-7TN-R cells indicative of tumor suppressive and anti-metastatic effects

In human breast cancer, we and others have shown that specific miRNAs are significantly altered, as compared with normal breast tissue (21,24-26). Altered expression of specific miRNAs has been associated with poor prognosis (27), as well as breast cancer initiation, invasion and metastasis (28-31). The importance of miRNAs in these advanced breast cancer phenotypes raises the question of their further involvement in apoptotic resistance. Furthermore, based on the above result demonstrating MCF-7TN-R growth-inhibition by TSA, we performed microRNA microarray analysis of MCF-7TN-R cells after treatment with TSA (10 μM for 24 h). As shown in Fig. 2, a number of microRNA expression changes were observed. In addition, the three biological replicates from vehicle-treated MCF-7TN-R clustered together and separately from TSA-treated cells, demonstrating high reproducibility between biological repeats as well as differential microRNA expression induced by TSA. Of the microRNAs significantly altered by HDACi treatment, 22 were up-regulated (Table II) and 10 were down-regulated (Table III). Their predicted (TargetScan and miRanda) or confirmed gene targets are provided.

Figure 2.

Figure 2

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TSA regulation of microRNA expression in MCF-7TN-R. Heatmap of microRNA changes induced by treatment with TSA (10 μM) after 24 h in MCF-7TN-R cells. microRNAs demonstrating statistically significant changes in expression are shown (p<0.01). Green indicates down-regulated expression and red indicates up-regulated expression of microRNAs. Individual samples are represented in columns while specific microRNAs are represented by rows as labeled.

Table II.

Up-regulated miRNA following TSA treatment.

microRNA Mean fold change p-value Description Gene targets (ref.)
hsa-miR-1 2.44 <0.05 Tumor suppressor cMET (39), TAGLN2 (40,41)
hsa-miR-22 2.69 <0.05 Anti-migration, cell cycle arrest MYCBP, MAX
hsa-miR-139 1.61 <0.05 Anti-metastatic, tumor suppressor ROCK2 (29), CDK6*, HOXB2*
hsa-miR-143 1.94 <0.05 Tumor suppressor KRAS (42), BCL2 (43)
hsa-miR-144 1.97 <0.05 Tumor suppressor Notch1 (44)
hsa-miR-153 1.70 <0.05 Tumor suppressor BCL2 (45)
hsa-miR-155 1.53 <0.01 OncomiR FOXO3A (46), SOCS1 (47),
FADD and IKKE (48)
hsa-miR-191* 2.51 <0.05 Tumor suppressor SPEN*
hsa-miR-194 1.70 <0.01 Tumor suppressor, anti-metastatic CDH2, HBEGF, RAC1 and IGF1R (49)
hsa-miR-215 2.26 <0.01 Anti-EMT, cell cycle arrest ZEB1/2 (50)
hsa-miR-202* 1.82 <0.05 Tumor suppressor TGFBR2*, ROCK1*
hsa-miR-335 1.83 <0.05 Tumor suppressor, cell cycle arrest,
metastasis suppressor		 RB1/p105 (51), SOX4 and TNC (28)
hsa-miR-486 1.97 <0.05 Tumor suppressor CD40 (52)
hsa-miR-526c 2.14 <0.05
hsa-miR-544 1.74 <0.01
hsa-miR-559 1.61 <0.05 Tumor suppressor ERBB2 (53)
hsa-miR-568 1.59 <0.05
hsa-miR-620 2.31 <0.05
hsa-miR-627 1.93 <0.05
hsa-miR-638 1.55 <0.01
hsa-miR-641 2.02 <0.05
hsa-miR-888 1.76 <0.05
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*

Putative targets as indicated by TargetScan and miRanda.

Table III.

Down-regulated miRNA following TSA treatment.

microRNA Mean fold change p-value Description (ref.) Gene targets (ref.)
hsa-miR-500 −1.90 <0.05 OncomiR (54)
hsa-miR-512-3p −1.59 <0.05 cFLIP (55)
hsa-miR-607 −1.68 <0.05
hsa-miR-613 −1.69 <0.05 LXR (56)
hsa-miR-622 −1.57 <0.05
hsa-miR-632 −1.86 <0.01
hsa-miR-636 −2.00 <0.05
hsa-miR-645 −2.03 <0.01 OncomiR (57)
hsa-miR-651 −1.60 <0.05
hsa-miR-657 −1.91 <0.01 IGF2R (58)
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Discussion

Drug resistance, acquired or de novo, remains a major obstacle in the treatment of cancer (1). Progression to resistance represents one of the hallmarks of aggressive carcinomas with limited treatment options and poor prognoses (2,3). Epigenetic therapies, including HDACi, provide a novel class of treatment for therapeutically-resistant cancer patients (32), including breast cancer (33). Here we demonstrate the ability of the HDACi TSA to suppress in vitro clonogenic survival of the apoptotically resistant MCF-7TN-R cells. This cell culture data indicates that in progressive drug resistance or recurrent breast carcinoma, the use of HDACis may exhibit greater inhibitory effects on tumor cell survival. Numerous studies have analyzed gene expression changes in response to HDACi, with the goal of defining specific mechanisms of their anticancer activity (34). Recent studies have also demonstrated the regulation of microRNA expression changes in breast and other cancer cells treated with HDACi alone (9,10,13,35), or in combination with DNMTi (11,36-38). Overall, these studies revealed microRNA expression profiles suggestive of a less aggressive and more tumor-suppressive phenotype after treatment with epigenetic therapies. Consistent with those studies, our microarray results revealed that many of the microRNAs that were significantly up-regulated (Table II) following TSA treatment (compared to control) have been characterized as having tumor suppressive (miR-1, miR-143, miR-144, miR-191*, miR-202*, miR-486, miR-559), anti-migration/anti-metastasis (miR-22, miR-139, miR-194, miR-335), or anti-EMT (miR-215) roles in cancer. Only one known oncomiR, miR-155, was found to be increased following TSA treatment, but with a mean fold change of 1.53 compared to control, this was the lowest change observed, although statistically significant. Of the microRNAs identified as significantly decreased (Table III) following TSA treatment (compared to control), two have been identified as oncomiRs (miR-500, miR-645) and three others have confirmed targets involved in tumorigenesis (miR-512-3p, miR-613, miR-657). Taken together, these data indicate that HDACi treatment may promote an anti-tumor microRNA expression profile in the apoptotically resistant cell line MCF-7TN-R, providing novel therapeutic targets for the treatment of drug resistant breast cancer.

Acknowledgements

This research was supported by The Department of Defense Breast Cancer Research Program BC085426 (B.M. Collins-Burow); The National Institutes of Health/National Center for Research Resources P20RR020152 (B.M. Collins-Burow); The Integrated Cancer Biology Program: Centers for Cancer Systems Biology NIH/NCI U54CA113001 (K. Nephew); and The Walther Cancer Foundation, Indianapolis, IN (S.Y. Nam). The funders did not have any involvement in study design; the collection, analysis, or interpretation of the data; the writing of the manuscript; or the decision to submit the manuscript for publication.

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