Background
The problem of drug-resistance is a major clinical concern for the successful management of cancer patients. Estrogen receptor (ER) is expressed in 75 % of breast cancers [
1] and for such breast cancers, tamoxifen is one of the important drug of choice for targeted personalized therapy. Tamoxifen can significantly lower the chances of developing recurrent breast cancer and can be very effective in women who initially present with metastatic disease. It remains the primary therapeutic agent for the management of ER and/or progesterone receptor (PR)-expressing breast cancers, particularly in premenopausal women without or with conventional chemotherapeutics. However, many ER-positive cancers that initially respond to tamoxifen, eventually develop resistance with the continued administration of the drug [
2]. Acquired resistance to tamoxifen is seen in 30–40 % of breast cancer patients treated with tamoxifen for 5 years [
3], which clearly indicates that this is a major clinical problem. The tumors that have acquired drug resistance are usually far more aggressive and difficult to treat with conventional therapeutics. They are invariably linked to poor prognosis as well as overall poor survival.
There is an emerging interest in microRNAs (miRNAs) as therapeutic targets in drug-resistant cancers [
4]. These short non-coding RNAs have been implicated in multiple stages of cancer progression and metastasis, and reports in the last few years have indicated the involvement of miRNAs in tamoxifen resistance as well [
5‐
11]. The miRNAs directly or indirectly implicated in tamoxifen resistance in breast cancer models include miR-221/222 [
5,
6], miR-15a/16 [
7], miR-342 [
8], miR-375 [
9], miR-200 s [
10], miR-126/miR-10a [
11] and miR-519a [
12]. We designed the current study to investigate miRNA-regulation of tamoxifen resistance, and used paired cell lines – parental MCF-7 and tamoxifen resistant MCF-7 (MCF7TR) as our model. Tamoxifen resistance has been linked to epithelial-mesenchymal transition (EMT) through an involvement of miR-375 [
9], and EMT-regulating miRNAs such as miR-200 s [
13,
14] and let7s [
15] have been reported to play a role in resistance to tamoxifen [
10]. In our model, we observed increased invasion of MCF7TR cells, a phenomenon which has been linked to EMT [
16], which prompted us to investigate the miRNAs that have been linked to invasion and EMT characteristics of breast cancer cells. We observed a significant over-expression of miR-10b in MCF7TR cells which correlated with acquired tamoxifen resistance. Mechanistically, we identified HDAC4 as a target of miR-10b which mediated the miR-10b action. Our results provide the first evidence in support of such action of miR-10b and HDAC4 and further highlight the importance of miRNA-regulation in drug resistance phenotype.
Methods
Cell lines and reagents
MCF-7 and T47D breast cancer cells were purchased from ATCC and maintained in DMEM and RPMI mediam (Invitrogen, Carlsbad, CA), respectively, with 10 % fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin in a 5 % CO2 atmosphere at 37 °C. The tamoxifen resistant MCF-7 derivatives, MCFTR cells, were generated by culturing parental MCF-7 cells in DMEM medium supplemented with 5 % FBS, antibiotics and 10−6 M 4-hydroxy tamoxifen. Concentration of tamoxifen was gradually increased until the final concentration was 10−6 M. Cells were continuously exposed to tamoxifen for 6 months during which time the medium was replaced every 3 to 4 days. The cell lines have been tested and authenticated in the core facility (Applied Genomics Technology Center at Wayne State University) by short tandem repeat profiling using the PowerPlex 16 System from Promega. Antibodies were purchased from following sources – HDAC4 (Cell Signaling) and β-actin (Sigma-Aldrich).
Western blot analysis
For Western blot analysis, cells were lysed in RIPA buffer containing complete mini EDTA-free protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktails 1 and 2 (Sigma-Aldrich). After resolution on 12 % polyacrylamide gels under denaturing conditions, proteins were transferred to nitrocellulose membranes, incubated with appropriate primary/horseradish peroxidase-conjugated secondary antibodies and visualized using chemiluminescence detection system (Pierce).
Cell growth inhibition studies by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
Cells were seeded at a density of 5 x 103 cells per well in 96-well culture plates. After overnight incubation, liquid medium was removed and replaced with a fresh medium containing DMSO (vehicle control) or different concentrations of tamoxifen, as indicated. After 48 h, 25 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (5 mg/ml in phosphate-buffered saline, PBS) was added to each well and incubated further for 2 h at 37 °C. Upon termination, the supernatant was aspirated and the MTT formazan, formed by metabolically viable cells, was dissolved in DMSO (100 μl) by mixing for 30 min on a gyratory shaker. The absorbance was measured at 595 nm on Ultra Multifunctional Microplate Reader (TECAN, Durham, NC).
Cell viability studies by Trypan Blue assay
Cells were seeded in 6-well culture plates and appropriately treated. Upon completion of incubation, culture medium (with floating dead cells) was collected and pooled with the adherent cells removed from the plate by trypsinization. The cells were briefly spun and re-suspended in the normal culture medium. Cell viability was assessed by adding 50 μl of Trypan Blue solution (0.4 % in PBS) to 50 μl of the cell suspension. After 2 min, the number of living cells, which did not retain the dye was counted using a hemocytometer, and was compared to the total number of cells (living + dead) to calculate the viability percentage.
Histone/DNA ELISA for detection of apoptosis
The Cell Death Detection ELISA Kit (Roche) was used to detect apoptosis. Cells were treated, as indicated for individual experiments. After treatment, the cytoplasmic histone/DNA fragments from these cells were extracted and incubated in the microtiter plate modules coated with anti-histone antibody. Subsequently, the peroxidase-conjugated anti-DNA antibody was used for the detection of immobilized histone/DNA fragments followed by color development with ABTS substrate for peroxidase. The spectrophotometric absorbance of the samples was determined by using Ultra Multifunctional Microplate Reader (TECAN) at 405 nm.
miRNA transfections
Transfections of pre/anti-miR-10b were done using methodology previously described [
13]. Briefly, cells were seeded (2.5 × 10
5 cells per well) in six well plates and transfected with pre/anti-miR-10b or non-specific pre/anti-miRNA controls (Life Technologies) at a final concentration of 200 nM, using DharmaFECT transfection reagent (Dharmacon). After 48 h of transfection, cells were passaged and transfected once again before being used in the experiment.
Real-time RT-PCR
Real-Time RT-PCR analyses were done as described previously [
13]. Total RNA was isolated using the mirVana miRNA isolation kit (Life Technologies). The levels of miRNAs were determined using miRNA-specific Taqman probes from the Taqman MicroRNA Assay (Life Technologies). The relative amounts of miRNA were normalized to RNU48.
Cell migration and invasion assays
Cell migration and invasion assays were performed using 24 well transwell permeable supports with 8 μM pores (Corning) [
13]. After transfections with pre/anti-miR-10b or the non-specific controls, as described above, cells were suspended in serum free medium and seeded into the transwell inserts. For invasion assays, the transwell inserts were coated with growth factor reduced Matrigel (BD Biosciences). Bottom wells were filled with complete media. After 24 h, cells were stained with 4 μg/ml calcein AM (Life Technologies) in PBS at 37 °C for 1 h. Cells were detached from inserts by trypsinization and fluorescence of the invaded cells was read in ULTRA Multifunctional Microplate Reader (TECAN, San Jose, CA).
Luciferase assay
For luciferase reporter assays, MCF-7 cells were co-transfected with HDAC4 3′UTR luciferase vector (GeneCopoeia, Catalog # HmiT023167-MT05) and pre-miR-10b or miRNA negative control, using DharmaFECT Duo Transfection Reagent (Dharmacon). The vector has HDAC4 3′ UTR sequence inserted downstream of the secreted Gaussia luciferase (GLuc) reporter gene system, driven by SV40 promoter for expression in mammalian cells. A secreted Alkaline Phosphatase (SEAP) reporter, driven by a CMV promoter, is also cloned into the same vector (pEZX-MT05) and serves as the internal control. 48 h post- transfection, Gluc and SEAP luciferase activities were assayed using Secrete-Pair™Dual Luminescence Assay Kit (GeneCopoeia), following exactly the same procedure as described in the vendor’s protocol.
Discussion
The major conclusions from our present study are a) endogenous levels of miR-10b are significantly higher in MCF7TR cells, the tamoxifen-resistant derivatives of MCF-7 cells; b) induced expression of miR-10b in MCF-7 cells, by pre-miR-10b oligonucleotides, was correlated with increased invasion and resistance to tamoxifen-induced apoptosis while reduced expression of miR-10b in MCF7TR cells, by anti-miR-10b oligonucleotides, inhibited invasion along with reduced resistance to tamoxifen; c) HDAC4 appears to be an important target of miR-10b; its expression was found to correlate inversely with miR-10b levels and its levels modulated by altered miR-10b levels; and d) functional significance of HDAC4 regulation by miR-10b was suggested by the observation that over-expression of HDAC4 reversed tamoxifen resistance induced by ectopic expression of miR-10b in MCF cells, and silencing of HDAC4 attenuated the effects of anti-miR-10b transfections in MCF7TR cells.
Most targeted therapies are known to work initially but with the passage of time and continued administration, patients eventually develop resistance to the therapeutic agent, and this process is called extrinsic (acquired) drug resistance. While intrinsic (
de novo) drug resistance characterized by resistance to therapy right from the beginning is itself clinically challenging, the phenomenon of acquired drug resistance is equally a big concern. Tamoxifen is an ER-targeting drug which is used for the successful management of ER-driven breast cancers. Acquired resistance to tamoxifen [
20] is a major clinical concern and a survey of literature suggests that the major mechanisms currently under investigation include EMT and the cancer stem cells (CSCs). Multiple studies have provided direct as well as indirect evidence supporting this notion. In support of a mechanistic role of EMT in tamoxifen resistance of breast cancer cells, over-expression of Pin-1 [
21], AKT [
22], Nicastrin and Notch4 [
23], FoxM1 [
24], brachyury [
25] as well as modulation of several microRNAs [
9,
10] has been reported. Involvement of CSCs in tamoxifen resistance of breast cancer cells has been reported, which appears to be mechanistically linked with higher expression of CXCR4 [
26], STAT3 [
27], Sox2 [
28], EZH2 [
29], and lower expression of CD24 [
30,
31].
Here we report a novel role of miR-10b in tamoxifen resistance of breast cancer cells. Tamoxifen-resistant breast cancer cells exhibit increased invasive potential, a phenomenon that is well established for high miR-10b expressing breast cancer cells [
17,
32]. A recent report [
33] has identified critical role of miR-10b in TGF-β1-induced EMT. In this work, miR-10b was found to be a downstream target of TGF-β1, essential for TGF-β1-induced down-regulation of epithelial marker E-cadherin and up-regulation of mesenchymal marker vimentin. Inhibition of miR-10b in metastatic breast cancer MDA-MB-231 and MDA-MB-435 cells significantly reversed the TGF-β1 effects. Further, a role of miR-10b in proliferation and growth of CSCs,
in vitro as well as
in vivo, has also been reported [
34]. Thus, it appears that miR-10b is functionally involved in the induction of EMT and CSCs phenotypes, which would explain its role in drug resistance phenotype, such as tamoxifen resistance as observed in our study. While our work is the first report on mechanistic involvement of miR-10b in drug resistance of breast cancer cells, such role of miR-10b in other cancer models has been reported. miR-10b was observed to be consistently high in all the cisplatin resistant sublines derived from parental cisplatin-sensitive germ cell tumor cell lines [
35], and it was reported to confer resistance to 5-fluorouracil in colorectal cancer cells [
36]. Clearly, there is evidence in support of miR-10b-mediated induction of drug resistance which is in direct agreement with our findings.
The miRNA-mediated regulation of tamoxifen resistance has been studied in breast cancer models for many years where miR-221 and miR-222 are the most well characterized microRNAs [
6,
5,
37,
38]. These oncogenic miRNAs confer resistance to tamoxifen through down-regulation of tumor suppressors p27Kip1 [
6,
38] and TIMP3 [
37]. Another oncogenic miRNA, miR-519a induces tamoxifen resistance via regulation of several tumor suppressor genes in PI3K pathway [
12]. Not all miRNAs that are functionally involved in tamoxifen resistance are oncogenic. Tumor suppressors miR-15a and miR-16 regulate tamoxifen sensitivity by targeting Bcl-2 [
7], miR-451 targets 14-3-3ζ [
39], let7s target ER-α36 [
15], miR-375 targets metadherin [
9] and miR-200b/c target ZEB1 [
10]. Also, an elevated expression of miR-126 and miR-10a has been linked to better prognosis and longer relapse-free time in breast cancer patients treated with tamoxifen [
11]. Thus, the regulation of sensitivity to tamoxifen is influenced by both oncogenic and tumor suppressive miRNAs. Our results are suggestive of an oncogenic role of miR-10b. We used multiple bioinformatics-based methodologies to find a functionally viable target of miR-10b in our model system. Using IPA and online tools, we identified HDAC4 as a target of miR-10b, which was correlated with tamoxifen resistance/sensitivity, as determined by over-expression/silencing studies.
HDAC4 is a member of class IIa histone deacetylases and our results support an inverse relationship between HDAC4 expression and tamoxifen resistance. This is surprising, given the focus on HDAC inhibitors as anticancer agents. Consistent with the many reports on tumor-progressing role of HDACs, HDAC4 has been reported to be tumorigenic in different human cancers [
40,
41]. Indicative of a tumor suppressor function of HDAC4 is the observation that HDAC4 was down-regulated in 15 of 18 urothelial cancer cell lines [
42]. The paradox of HDAC4 activity also extends to its involvement in drug resistance. A number of reports present a positive correlation between HDAC4 expression and drug resistance. For instance, HDAC4 was shown to activate STAT1 leading to platinum resistance in ovarian cancer patients-derived cell lines [
43] and resistance to etoposide in lung cancer cells [
44]. HDAC4 also induced resistance to 5-fluorouracil in breast cancer cells [
45] and inhibited docetaxel-related cytotoxicity in gastric cancer cells [
46]. A careful review of the literature revealed that the only miRNA that has been associated with HDAC4, in the context of drug resistance, is miR-140 [
47]. Interestingly, this study found a very similar function of HDAC4, as observed by us in the current study. Performed in colon and osteosarcoma cells, this study reported higher miR-140 expression in colon CSCs with increased resistance to 5-fluorouracil. HDAC4 inhibition was proposed as the mechanism of miR-140-induced chemoresistance. Thus, the only published work that investigated miRNA regulation of HDAC4 in resistant cells documented similar findings consistent with our results. In an earlier published work [
48], we demonstrated that inhibitors of HDACs, such as Trichostatin A (TSA) and Suberoylanilide hydroxamic acid (SAHA), induced EMT in prostate cancer cells, as evidenced by up-regulated markers of mesenchymal phenotype. Further, TSA treatment resulted in increased expression of Sox2 and nonog indicating an enrichment of CSCs. Thus, antagonizing HDACs made the cancer cells more invasive, which is in agreement with our current findings, and, moreover, we provide here a mechanism through the novel involvement of miR-10b. It is tempting to suggest that such EMT/CSC-inducing activity of HDAC inhibitors might be a factor for their disappointing progress in clinical trials. Combined with the results from this study where low levels of HDAC4 correlated with drug resistance, it is important that the mechanistic involvement of HDACs in EMT, CSCs and drug resistance be evaluated in-depth before their selective targeting in clinics.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
AA participated in the design of the study, performed experiments, analyzed data and drafted the manuscript. KRG helped with the immunoblot analysis. SY generated the tamoxifen resistant cells. ABF helped with the online database analyses. KBR provided resources for generation of tamoxifen resistant cells and helped with the manuscript draft. FHS conceived of the study, participated in its design and coordination, and finalized the draft. All authors read and approved the final manuscript.