Background
Wider use of personalized targeted and endocrine-based therapies has significantly improved outcomes of Her2 amplificated and hormonal receptor (HR)-positive (HR +) breast cancer subtypes. However, triple negative breast cancer (TNBC) is still associated with high recurrence and short survival. Because TNBC lacks biological targets, it is mainly treated with chemotherapy. However, chemosensitivity in TNBC is limited and urgently needed to be improved.
Epithelial-to-mesenchymal transition (EMT) is a part of tumor metastasis, which is characterized by decreased epithelial marker E-cadherin and increased mesenchymal marker vimentin, is a subprocess of both tumor metastasis and drug resistance development [
1]. Increased vimentin expression has been related to taxane residues in ovary and breast cancer cells which shows drug resistance property [
2,
3]. EMT can also induce anthracycline resistance in cancer cells [
4]. As most of TNBC cells have a mesenchymal phenotype, EMT might be the major cause of TNBC multidrug resistance. Determining the EMT regulatory pathways and reversing the EMT process might thus improve TNBC chemosensitivity.
Tamoxifen (TAM) is commonly used in HR
+ breast cancer with more than 50% effectiveness [
5], whereas the effectiveness of TAM in estrogen receptor negative (ER
−) breast cancer is reportedly about 7% [
6]. Recently, TAM was shown to exert an antitumor effect in ER
− cancers, including gastric cancer, colon cancer and cholangiocarcinoma [
7‐
9]. Although the antitumor mechanism of TAM is considered to be competition with estrogen to block ER transcription, researchers have found a non-estrogen-dependent mechanism of TAM in ER
− cancers by activating the apoptosis pathway to induce apoptosis. However, the apoptotic mechanism does not explain TAM activity in all types of ER
− cancers. Other non-estrogen-dependent mechanisms for TAM has been suggested, such as the mediation of protein kinase C (PKC), transforming growth factor-β (TGF-β), oncogene
c-myc and mitogen-activated protein kinase (MAPK) [
10,
11]. The relationship between EMT and TAM has been established in ER
+ breast and endometrial cancers [
12]. Most researchers indicated that long-time use of TAM might induce TAM resistance, which could induce EMT in ER
+ cancers [
1]. However, the relationship between TAM and EMT in ER
− cancers, especially TNBC, is unclear.
In this report, our result revealed that TAM could reverse EMT characteristics in mesenchymal TNBC cells, but not epithelial breast cancer cells. Further study indicated that reversing EMT enhanced chemosensitivity. These results imply a possible clinical indication for TAM in TNBC.
Methods
Cell lines and reagents
Human breast cancer cell lines MCF-7 (TCHu74) and MDA-MB-231 (TCHu227) were obtained from the Cell Bank of the Chinese Academy of Sciences (China). MCF-7/ADR cells derived from MCF-7 and cultured with 1 μg/mL adriamycin for at least 1 year and possessed adriamycin-resistance [
13]. Human breast cancer cell lines MCF-7 and MCF-7/ADR cells were cultured in RPMI 1640 medium (GIBCO, Grand Island, NY), MDA-MB-231 cells were cultured in Leibovitz’s L-15 medium (GIBCO, Grand Island, NY, USA). Both RPMI 1640 and L15 medium were supplemented with 10% fetal calf serum (Gibco by Life Technologies, Cergy Pontoise, France), and penicillin (100 U/mL) and streptomycin (100 μg/mL). For MCF-7 cells, the medium additionally contained human-recombinant insulin (10 μg/mL) to maintain the endocrine dependency. For MCF-7/ADR cells, the medium additionally contained 1 μg/mL adriamycin to maintain the drug resistance property. The cells were cultured at 37 °C in a humidified atmosphere under 5% CO
2. The cells were subcultured every 2–4 days and harvested in the logarithmic phase of growth.
Reagents and antibodies
Tamoxifen (4-hydroxytamoxifen, 4-OHT/TAM) and 5-aza (5-aza-2′-deoxycytidine, 5-aza-dC) were purchased from Sigma-Aldrich (St Louis, Missouri, USA). Indicated cells were treated with 5 μmol/L TAM for 48 h. E-cadherin, vimentin, ER-α, PR, and Her-2 antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Actin, p-gp, DNMT1 and DNMT3a antibodies were purchased from Santa Cruz Biotechnology (USA).
MTT assay
The effects of different agents on cell proliferation were measured using the MTT assay [
14]. Briefly, indicated cells were seeded at 3 × 10
4 per well in 96-well plates in quadruplicate and incubated overnight. Different concentrations of the test agents were then added and incubated for 5 days. Thereafter, 25 μL of MTT solution (5 mg/mL) was added to each well and the cells were incubated for another 4 h at 37 °C. After the incubation, the supernatants were removed carefully, and 200 μL of DMSO was added to each well. The cells were then lightly shaked for 10 min. Absorbance was measured at 570 nm in a Microplate Reader (Bio-Rad, CA, USA). Analysis of the obtained results was done using GraphPad Prism 5 computer program to evaluate cell proliferation rate and cytostatic rate. Untreated cells were used as controls.
Transwell migration assay
Cells were washed in serum-free medium twice. The chemoinvasion assay was conducted using the 24-well chemotaxis chambers with 8 μm pores (Corning, NY, USA) according to the manufacturer’s instruction. Briefly, indicated cells were pretreated with or without TAM (5 μmol/L) for 48 h. Then, 2 × 104cells were resuspended in fresh serum-free media and seeded into the upper chamber of a 24-well plate, while the lower chamber contained fresh culture media with 10% FBS as a chemo-attractant. The cells were allowed to invade for 24 h at 37 °C and the chambers were then washed with PBS. The cells on lower surface of the chamber were stained with 0.1% Giemsa stain solution for 2 h and counted in four different random fields at ×10 magnifications under electron microscope. Each experiment was performed at least three times.
Western blot assay
Cells were washed twice with ice-cold PBS and solubilized in 1% Triton lysis buffer (50 mmol/L Tris-HCl, pH 7.4, 10 mmol/L EDTA, 100 mmol/L NaF, 150 mmol/L NaCl, 1% Triton X-100, 1 mmol/L PMSF 1 mmol/L Na
3VO
4, and 2 μg/mL aprotinin) on ice, then sonication and incubation at 4 °C for 30 min, followed by centrifugation at 12,000 g at 4 °C for 20 min. Then proteins were quantified according to BCA (Beyotime, China) method. Total proteins were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and electronically transferred to nitrocellulose membranes (Immobilon-P, Millipore, Bredford, MA, USA). After blocking with 5% skim milk in TBST (10 mmol/L Tris, pH 7.4, 150 mmol/L NaCl and 0.1% Tween-20) for 1 h, the bands were incubated in the indicated primary antibodies at 4 °C overnight, followed by secondary antibodies incubated for 30 min at room temperature. After washing with TBST, the proteins were detected using an enhanced chemiluminescence reagent (SuperSignal Western Pico Chemiluminescent Substrate, Pierce, USA) and visualized with an ECL detection system (DNR Bio-Imaging Systems, Jerusalem, Israel) [
15].
Immunofluorescence
The cells were seeded in Lab-Tek chamber slides (Nunc S/A, Polylabo, Strasbourg, France). The cells were treated with or without TAM (5 μmol/L) for 72 h and fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.2% Triton X-100 for 5 min, blocked with 5% bovine serum albumin (BSA) in 1× PBS for 1 h at room temperature and then incubated with E-cadherin and vimentin antibodies for 1 h. Then Alexa Fluor 546-conjugated goat anti-rabbit IgG or Alexa Fluor 488-conjugated goat anti-rabbit IgG (Molecular Probes) were added in blocking solution for 1 h at room temperature in the dark. 4′6-diamidino-2- phenylindole was used to stain nuclei for 5 min. After mounted with the Slow Fade Antifade Kit (Molecular Probes, Eugene, OR, USA), the cells were visualized by fluorescence microscopy (BX61, Olympus, Japan) [
16].
RNA extraction and quantitative real-time PCR (qRT-PCR)
The cells were cultured and harvested at the indicated times. Total RNA was extracted from cells using the RNeasy mini kit (Qiagen, Carlsbad, CA, USA). For miRNAs, The One Step PrimeScript
® miRNA cDNA Synthesis Kit (Takara, Japan) was used for RNA reverse transcription. Relative expression of miRNAs was calculated via the comparative cycle threshold (Ct) method, and the expression of small nuclear RNA U6 was used as reference. The sequence-specific forward primers for mature miR-200c was: 5′-ACACTCCAGCTGGGTAATACTGCCGGGTAA-3′ and for U6 internal control was forward (5′-GCTTCGGCAGCACATATACTAAAAT-3′) and reverse (5′-CGCTTCACGAATTTGCGTGTCAT-3′), respectively. The Uni-miR qPCR Primer was included in the kit. SYBR
® Premix Ex Taq™ II (Perfect Real Time) (Takara, Japan) was used for monitoring the amount of miRNA. The PCR conditions were 30s at 95 °C, followed by 45 cycles at 95 °C for 5 s and 58 °C for 25 s. The relative amount of the target RNA was calculating by 2
-ΔΔCt method. The detailed method was described in our previous studies [
16].
MicroRNA microarray analysis
MCF-7 and MCF-7/ADR cells were cultured without insulin or adriamycin for 3 days. The expression levels of miRNAs were quantified using GeneChip miRNA Array (Affymetrix, Santa Clara, CA, USA) according to the manufacturer’s instructions by Gene Tech Biotechnology Company (Shanghai, China) [
16]. In brief, total RNA (1 μg) was extracted with miRNeasy Mini Kit (Qiagen, Germany) and labeled with a FlashTag Biotin RNA Labeling kit (Genisphere, Hatfield, PA, USA). Then the labeled RNA was injected onto the microarrays and incubated at 48 °C for 16 h. After washing and staining, the signals were obtained using a GeneChip Scanner 3000 7G (Affymetrix, Santa Clara, CA). Data was normalized using the RMA algorithm. The PCA and unsupervised clustering of microarray analysis were shown in Additional files
1 and
2. QC report and raw data of microarray analysis were shown in Additional files
3 and
4. The microarray result was uploaded to GEO database. Data from this microarray is available at GSE96821.
Transfection
The si-DNMT1 and si-DNMT3a and corresponding negative control were designed and synthesized by RiboBio (Guangzhou, China) and stored at −80 °C before use. Cells were transiently transfected with si-DNMT1 and si-DNMT3a and corresponding negative control using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s protocol.
Xenograft study in nude mice
5-week-old female Balb/c nude mice were from SLAC Laboratory Animal Co., Ltd. (Shanghai, China). Mice were randomly allocated to two groups (n = 6 per group). 1 × 106 MDA-MB-231 cells were injected into the tail vein of the control group. 1 × 106 MDA-MB-231 T cells (MDA-MB-231 cells pretreated with 5 μmol/l TAM for 5 d) were injected into the tail vein of the treatment group and given with TAM orally every other day for 2 months. Mice were killed by cervical dislocation according to the protocol filed with the Guidance of Institutional Animal Care and Use Committee of China Medical University, and lung tissue was taken out for HE staining to ensure metastasis focals. Experimental research on mice complied with the Guidance of Institutional Animal Care and Use Committee of China Medical University, and had been approved by the ethics committee of China Medical University.
CpG island predictor
The CpG island was predicted by the MethPrimer software. MethPrimer accepts a DNA sequence as input, performs a digital bisulfite conversion of the input sequence, and then picks primers on the converted sequence. Results of primer selection are delivered through a Web browser in text and graphic views (
http://www.urogene.org/cgi-bin/methprimer/methprimer.cgi) [
17]. The miR-200c promoter region used an island size of 3000 nucleotides, a GC percentage of at least 50% and an observation/expectation CpG ratio of more than 0.6.
Statistical analysis
All the presented data were expressed as the mean ± SD and the representative results were confirmed in at least three independent experiments. Statistical comparisons were calculated by Student’s t-test. P < 0.05 was considered statistically significant. IC50 values were calculated by nonlinear regression analysis using GraphPad Prism 5 software.
Discussion
TNBC is an aggressive breast cancer subtype that metastasizes early and is associated with poor overall survival. Preventing metastasis is hampered by limited treatment options. TAM is an ER antagonist, mainly used in ER
+ breast cancers through ER-dependent mechanism with definitive efficacy [
9‐
11,
19,
26‐
31], and is thus much less efficacious in ER
− breast cancers, such as TNBC [
32]. Recently, TAM showed broad-spectrum antitumor properties in ER
− cancers, such as gastric cancer, colorectal cancer and cholangiocarcinoma [
33]. In gastric cancer cells, TAM could inhibit the PI3K/Akt signaling pathway [
7]. TAM could also decreased P-gp expression, to reverse multidrug resistance in QBC939/ADM cholangiocarcinoma cells [
9]. However, in our results, P-gp expression barely changed in TNBC cells after TAM treatment, which implied that their increased chemosensitivity occurred through EMT changes.
Characteristics of TNBC cells are similar to cells that undergo EMT. Their migratory and invasive properties increase while their adhesive properties decrease as epithelial cells transform to mesenchymal cells. The relationship between TAM and EMT was reported in some TAM-resistant breast cancers: after acquiring endocrine resistance, most of breast cancer cells grew as loosely packed colonies, underwent EMT morphological changes and altered their growth rate and increased aggressive behavior [
34]. However, the relationship between TAM and EMT is not restricted to breast cancer. Similar researches were reported in ER
− endometrial cancer. After long exposure to TAM, epithelial-like endometrial cancer cells underwent EMT, and their ER status changed from ER
+ to ER
− [
12]. This research demonstrated that long exposure to TAM by epithelial cells might induce phenotypical changes, through a mechanism unrelated to ER status. However, the process of EMT was reversible (mesenchymal–epithelial transition; MET), although no reports are available on whether TAM could reverse EMT in mesenchymal breast cancer cells. Our study showed that TAM could change the mesenchymal phenotype of breast cancer cells by reversing EMT. EMT is closely associated with metastasis, drug resistance and progression of malignant tumors [
35‐
38]. Our study provides the first evidence to suggest that TAM could reverse EMT and decrease migratory ability in vitro and vivo, while concomitantly increasing chemosensitivity in mesenchymal TNBC cells.
To explore the mechanism of MET induced by TAM on mesenchymal TNBC cells, we compared miRNA expression between mesenchymal and epithelial breast cancer cells, using microarray analysis. Among the most significant miRNAs, we focused on the regulation of miRNAs that affect the EMT process. The reported role of the miR-200 family as EMT inhibitors, to reduce tumor cell migration was confirmed by our microarray analysis [
39]. MiR-200c could promote breast cancer cell epithelial identity, and repress related genes that regulate E-cadherin and cell polarity [
40], and reportedly regulates the EMT process directly, thus affecting chemosensitivity [
17]. Reciprocally, the process of EMT could also regulate miR-200c expression [
17]. Therefore, the study we report here is based on the known ability of miRNA-200s family to reverse the process of EMT.
However, to our knowledge, this is the first report of the mechanism of how TAM regulates miR-200c. MiRNAs are highly conserved sequences with promoters of about 2000 base pairs [
41]. As DNA methylation can change promoter activity [
42], we used online software to predict the methylation status of miR-200c promoters. We also verified the existence of CpG islands in miR-200c promoters, and high expression of DNMTs in mesenchymal TNBC cells. Expression and methylation status of miR-200 might be useful as markers for EMT in breast cancer [
43]. The result was consistence with the research of Vrba, et al. who also showed the important role of DNA methylation in regulating miR-200c expression and the control of phenotypic conversions in cancer cells [
44]. After TAM treatment, DNMT expression decreased and miR-200c expression increased in TNBC cells. These results imply that TAM regulate miR-200c expression by downregulating DNMT expression. As similar results were seen with the demethylation agent 5-AZA, we considered that 5-AZA could also downregulate DNMT expression and reactivate the gene function turned off by hypermethylation [
45,
46]. To test our hypothesis, we knocked down
DNMT1 and
DNMT3a using siRNAs. Our result showed that double knockdown of
DNMT1 and
DNMT3a also upregulated miR-200c, which confirmed the relationship between miRNA and hypermethylation. Therefore, TAM can apparently reverse EMT by downregulating DNMTs, thus increasing miR-200c expression.
Conclusions
In conclusion, we verified that TAM up-regulates miR-200c expression in mesenchymal TNBC cell lines MDA-MB-231 and MCF-7/ADR, by downregulating DNMT expression, thus attenuating cell migratory capacities in vivo and in vitro. The MET process increased chemosensitivity of mesenchymal TNBC cells in vivo and vitro. The present study expands the effect of TAM, and may explain why some ER− breast cancers respond to TAM. Our results suggest that TAM could be a DNMT inhibitor, thus indicating a wider range of both research in, and clinical use for, TAM in TNBC.