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Introduction

α-Smooth muscle actin (α-SMA) is encoded by the ACTA2 gene and contributes to tumor cell migration and invasion through the suppression of E-cadherin (1–3). The abnormal induction of α-SMA expression was found to be associated with shorter disease-free survival in lung, colorectal and pancreatic cancers (4–6). Recently, we reported that breast cancer patients with high α-SMA and HER2 levels had a poorer prognosis than patients with low α-SMA and HER2 levels (2). The level of α-SMA expression is significantly increased during epithelial-mesenchymal transition (EMT) and is regulated by various stimuli such as IL-1β, IL-6 and TGF-β1 (7,8).

The TP53 gene encoding p53 is the single most frequently inactivated gene in human cancers, with somatic missense mutations being present in approximately 50% of all invasive tumors (9). These mutation frequencies of TP53 are lower in breast cancers than in other solid tumors (10). The level of TP53 protein expression is stabilized and activated in response to a wide variety of cellular stresses such as DNA damage, ultraviolet irradiation, hypoxia and nucleotide depletion (11–13). TP53 works as a transcriptional activator and regulates cell cycle arrest, senescence, apoptosis, DNA replication and repair through the expression of its downstream target genes such as p21, Bax, NOXA and p53R2 (14,15).

In the present study, the clinical significance of α-SMA expression was investigated in estrogen receptor-positive [(ER(+)] breast cancer patients and the effect of TP53 on α-SMA expression was evaluated in tamoxifen-resistant breast cancer cells. In this study, it was found that α-SMA expression is associated with the survival of ER(+) breast cancer patients. In addition, we observed that wild-type TP53 upregulates α-SMA expression in tamoxifen-resistant breast cancer cells.

Materials and methods

Reagents

Dulbecco's modified Eagle's medium (DMEM) was purchased from Thermo Fisher Scientific, Inc. (Hemel Hempstead, UK). Fetal bovine serum (FBS) was purchased from HyClone/GE Healthcare Life Sciences (Logan, UT, USA). Phenol red-free DMEM, penicillin (100 U/ml), and 100 mg/ml streptomycin were purchased from Life Technologies (Rockville, MD, USA). 4-Hydroxytamoxifen (4-OHT) was purchased from Sigma-Aldrich;Merck KGaA (Darmstadt, Germany). Proteome Profiler Human Phospho-Kinase Antibody Array Kits were purchased from R&D Systems (Minneapolis, MN, USA). Nutlin3 (TP53 activator, Nut3) and pifithrin-α (TP53 inhibitor, PFT-α) were purchased from Selleck Chemicals (Houston, TX, USA). Anti-p53 (1:1,000 dilution; cat. no. sc-126) and mouse monoclonal anti-β-actin (1:1,000 dilution; cat. no. sc-47778) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Anti-α-SMA (1:20,000 dilution; cat. no. ab124964) antibody was purchased from Abcam (Cambridge, UK). ECL Western Blotting Detection Reagents (West-Q Chemiluminescent Substrate Plus kit) were obtained from GenDepot (Barker, TX, USA).

Cell culture

Tamoxifen-sensitive and -resistant MCF-7 (TP53 wild-type), MDA-MB-231 (TP53-mutant R280K), and Hs578T (TP53-mutant V157F) human breast cancer cells were grown in a humidified atmosphere of 95% air and 5% CO2 at 37°C in DMEM supplemented with 10% FBS, 2 mM glutamine, 100 IU/ml penicillin and 100 µg/ml streptomycin. Each cell line was maintained in culture medium without FBS for 24 h before the experiments.

Establishment of tamoxifen-resistant MCF-7 breast cancer cells

Tamoxifen-sensitive (TamS) and -resistant (TamR) breast cancer cell lines were kindly provided by Professor Keon Wook Kang (Seoul National University, Seoul, Korea). The TamR cells were established using methodology as previously reported (16,17). Briefly, MCF-7 cells were washed with PBS, and the culture medium was replaced with phenol red-free DMEM containing 10% charcoal-stripped steroid-depleted FBS and 0.1 µM 4-OHT. The cells were continuously exposed to this treatment regimen for two weeks, and the 4-OHT concentration was increased gradually up to 3 µM over a 9-month period. Initially, cell growth rates were depressed. However, after exposure to the medium for 9 months, the rate of cell growth increased gradually, indicating the establishment of tamoxifen-resistant cells.

Analysis of a public database

Expression data were downloaded from a public database [Kaplan-Meier plotter database (http://kmplot.com/breast)] (18). The clinical value of ACTA2 mRNA expression was analyzed by Kaplan-Meier survival plots in 209 ER(+) breast cancer patients with tamoxifen treatment (GSE2034) (19). The hazard ratios with 95% confidence intervals and log-rank P-values were calculated.

Human Phospho-Kinase Antibody Array

TamS and TamR cells were seeded at 1×106 cells/plate in two separate 100-mm dishes. Protein lysates from TamS and TamR cells were prepared using the supplied buffers and 300 µg of protein was hybridized to each array from the Proteome Profiler Human Phospho-Kinase Antibody Array (R&D Systems, Minneapolis, MN, USA). Further steps were performed based on the manufacturer's protocol.

Soft agar colony formation assay

TamR breast cancer cells were seeded at a density of 5×104 cells/well in 6-well plates in growth medium containing 0.7% agar (1.5 ml/well). The cells were seeded on top of a layer of growth medium containing 1.4% agar (2 ml/well). Next, growth medium (500 µl) with 10% FBS was added on top of the agar. In addition, 3 µM 4-OHT was added on top of the agar for some of the plates. The cells were plated and cultured in a 37°C incubator for two weeks. After two weeks, the viable colonies were stained with 0.01% crystal violet and observed using an Olympus CK40 inverted microscope at ×10 magnification (Olympus Corp., Tokyo, Japan).

Flow cytometric analysis (FACS)

As in a previous study, we performed an apoptosis assay using the Annexin V-Fluorescein Isothiocyanate (FITC) Apoptosis Kit-I (BD Pharmingen; BD Biosciences, San Diego, CA, USA) (3). Briefly, cells (1×106 cells/ml) were collected and washed twice with PBS, and then resuspended in 500 µl of staining solution containing 5 µl FITC-conjugated Annexin V and propidium iodide (PI). After incubation for 15 min at room temperature (RT) in the dark, the cells were immediately analyzed on a flow cytometer. Apoptotic cells were double-stained with Annexin V and PI and then they were analyzed using the FACS Vantage system (Becton-Dickinson, San Diego, CA, USA). The percentage of cells undergoing apoptosis was determined.

Western blotting

Cell lysates were prepared to detect α-SMA, TP53, and β-actin expression. Equal amounts of protein (50 µg) were boiled for 5 min in Laemmli sample buffer, and then electrophoresed on 10% sodium dodecyl-sulfate polyacrylamide (SDS-PAGE) gels. Separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes, and the membranes were blocked with 10% skim milk in Tris-buffered saline (TBS) containing 0.01% Tween-20 (TBS/T) for 15 min. Blots were washed three times in TBS/T and then incubated overnight with antibodies against α-SMA, TP53 and β-actin in TBS/T buffer at 4°C. The blots were then washed three times in TBS/T and subsequently incubated with secondary HRP-conjugated antibodies (1:2,000 dilution; cat. nos. sc-2004 and sc-2005; Santa Cruz Biotechnology) in TBS/T buffer. After 1 h of incubation at room temperature (RT), the blots were washed three times in TBS/T and positive immunoreactive proteins were detected using the West-Q Chemiluminescent Substrate Plus kit.

Real-time PCR

Total RNA was extracted from the cells using Invitrogen™ TRIzol reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Isolated RNA samples were then used for RT-PCR. Samples of total RNA (1 µg) were reverse transcribed into cDNA in 20 µl reaction volumes using a first-strand cDNA synthesis kit for RT-PCR according to the manufacturer's instructions (MBI Fermentas, Hanover, MD, USA). Gene expression levels were quantified by real-time PCR using a SensiMix SYBR kit (Bioline Ltd., London, UK) and 100 ng of cDNA per reaction. The primer sequences used for this analysis were as follows: human TP53 (forward, 5′-GGCCCACTTCACCGTACTAA-3′ and reverse, 5′-AAGCGAGACCCAGTCTCAAA-3′); human α-SMA (forward, 5′-AGACATCAGGGGGTGATGGT-3′ and reverse, 5′-CATGGCTGGGACATTGAAAG-3′), and GAPDH was used as an internal control (forward, 5′-ATTGTTGCCATCAATGACCC-3′ and reverse, 5′-AGTAGAGGCAGGGATGATGT-3′). An annealing temperature of 60°C was used for all the primers. PCR was performed in a standard 384-well plate format with an ABI 7900HT Real-Time PCR Detection System (Thermo Fisher Scientific, Inc.). For data analysis, the raw threshold cycle (CT) value was first normalized to the housekeeping gene for each sample to obtain a ΔCT. The normalized ΔCT was then calibrated to the control cell samples and to obtain the ΔΔCq values (20).

Adenovirus induction

Adenovirus expressing Lac Z and human TP53 cDNA (Ad-TP53) was a gift from Dr Hyunil Ha (Korean Institute of Oriental Medicine, Daejeon, Korea). Recombinant adenovirus expressing human TP53 was reproduced in 293A cells. The expression of this construct was confirmed by western blot analysis. Each construct was transfected into Hs578T and BT549 cells for 24 h and incubated for 24 h in fresh culture media. Ad-TP53-overexpressing MDA-MB-231 and Hs578T cells were further incubated for 24 h in serum-free culture media. The cell lysates and culture medium were then harvested for analysis of α-SMA and TP53 expression.

TP53 siRNA transfection

TP53 siRNA was purchased from Bioneer Corporation (Daejeon, Korea). A synthetic siRNA against two different TP53 mRNAs was used to inhibit endogenous TP53 gene expression. The duplex sequences of TGFBRI and TGFBRII siRNA used for this experiment are as follows: Human #1 TP53 siRNA [sense, CACUACAACUACAUGUGUA (dTdT) and antisense, UACAUAUGUAGUUGUAGUG (dTdT)]; and human #2 TP53 siRNA [sense, GAGGUUGGCUCUGACUGUA (dTdT) and antisense, UACAGUCAGAGCCAACCUC (dTdT)]. It was found that the optimal siRNA knockdown conditions involved transfecting TamR cells. Effectene (Qiagen, Inc., Valencia, CA, USA) was used for transfections with siRNA following protocols provided by the manufacturer. After the 72 h transfection, the levels of TP53 and α-SMA mRNA expression were analyzed by real-time PCR.

Statistical analysis

Statistical significance was determined using Student's t-test. The results are presented as the mean ± SEM. All the quoted P-values are two-tailed and the differences were considered statistically significant when the P-value was <0.05. Statistical analyses were performed using the Microsoft Excel 2016.

Results

α-SMA expression is associated with a poor prognosis in estrogen receptor-positive breast cancer patients

In a previous study, we reported that the levels of mesenchymal marker proteins such as fibronectin and α-SMA were significantly increased in TamR cells (3). In the present study, we investigated the clinical significance and the regulatory mechanism of α-SMA in breast cancer models. We found that the levels of α-SMA mRNA expression were associated with a poor prognosis in 209 ER(+) breast cancer patients using the GSE2034 dataset (Fig. 1A). ER(+) breast cancer patients with a high expression of α-SMA exhibited poorer relapse-free survival compared to the patients with low expression (P=0.0058, Fig. 1A).

Figure 1. α-SMA expression is associated with a poor prognosis in estrogen receptor-positive [ER(+)] breast cancer patients. (A) The clinical value of α-SMA mRNA expression was analyzed in ER(+) breast cancer patients using a public database [Kaplan-Meier plotter database (http://kmplot.com/breast)]. (B) Established TamS and TamR cells were seeded in a 6-well soft agar plate with or without 3 µM 4-OHT for 2 weeks for establishment of the colony formation. (C) Established TamS and TamR cells were seeded with or without 3 µM 4-OHT for 24 h. Apoptotic cells were analyzed by flow cytometry. (D and E) Levels of α-SMA protein and mRNA expression were analyzed by western blot analysis (D) and real-time PCR (E), respectively. The results are representative of three independent experiments. Values shown are the mean ± SEM. *P<0.05, **P<0.01 vs. the control; φP<0.05 vs. 4-OHT treated TamS. α-SMA, α smooth muscle actin; TamS, tamoxifen-sensitive cells; TamR, tamoxifen-resistant cells; Con, control.

Next, we analyzed the effect of tamoxifen on TamS and TamR cells. As shown in Fig. 1B and C, the anchorage-independent growth and apoptotic cell death of TamS were completely inhibited by 4-OHT treatment. However, cell growth and the death of TamR cells were not affected by 4-OHT. Furthermore, we examined the level of α-SMA expression in the TamS and TamR cells. α-SMA expression was significantly increased in the TamR cells (Fig. 1D). Under the same condition, the level of α-SMA mRNA expression was increased by 71.8-fold compared with the TamS cells (Fig. 1E). Therefore, we demonstrated that α-SMA expression was significantly increased in TamR cells and was associated with survival in ER(+) breast cancer patients.

Analysis of kinase activities between TamS and TamR cells

To characterize α-SMA expression-related protein kinases, we analyzed the phosphorylated levels of various protein kinases in TamS and TamR cells using the Proteome Profiler Human Phospho-Kinase Array. Protein lysates were loaded to the Proteome Profiler Human Phospho-Kinase Array Kit membranes. A schematic model of the membrane is indicated in Fig. 2A. In the present study, we found that the phosphorylation levels of Akt and TP53 were significantly increased in TamR cells when compared with TamS cells (Fig. 2B, red square). To confirm the results obtained by the Phospho-Kinase array, we examined the levels of pS392-p53 and pS46-p53 by western blot analysis. As expected, our results showed that the levels of pS392-p53 and pS46-p53 were markedly increased in the TamR cells (Fig. 2C). In addition, we observed that the level of TP53 protein (Fig. 2C) and mRNA (Fig. 2D) expression were slightly increased in the TamR cells. However, the phosphorylation level of p70 S6 kinase (T421/S424) was observably decreased in the TamR cells (Fig. 2B, green square). Based on these results, it was theorized that TP53 activity may be involved with α-SMA expression in breast cancer cells.

Figure 2. Analysis of kinase activities between TamS and TamR cells. (A) Schematic model of the kinase array membrane. (B) Protein lysates of TamS and TamR cells were subjected to a human phospho-kinase array. (C) Levels of p-TP53 (S392 and S46), t-TP53 and β-actin protein expression were analyzed by western blot analysis. (D) Levels of TP53 mRNA expression were analyzed by real-time PCR. The results are representative of three independent experiments. TamS, tamoxifen-sensitive cells; TamR, tamoxifen-resistant cells.

TP53 activator, nutlin3, upregulates α-SMA expression in breast cancer cells with wild-type TP53

To verify the role of TP53 on α-SMA expression, activator of TP53, Nut3, was administered to block the interaction of MDM2 and TP53 in TamS cells with wild-type TP53 for 24 h. After 24 h, we harvested whole cell lysates for the detection of α-SMA and TP53 protein and mRNA expression. As shown in Fig. 3A, the level of α-SMA expression was observably increased by Nut3 treatment. Under the same conditions, we analyzed the levels of α-SMA and TP53 mRNA expression. As expected, the level of α-SMA mRNA expression increased by 63.5±14.3-fold with Nut3 relative to the control (Fig. 3B). However, TP53 mRNA levels were not affected by Nut3 treatment, although the basal level of TP53 expression was slightly increased (Fig. 3A and B).

Figure 3. TP53 activator, Nutlin3 (Nut3), upregulates α-SMA expression in breast cancer cells with wild-type TP53. (A and C) TamS, MDA-MB-231 (MDA231) and Hs578T cells were treated with 5 µM Nut3 for 24 h. Levels of α-SMA, TP53 and β-actin protein expression were analyzed by western blot analysis. (B and D) Levels of α-SMA and TP53 mRNA expression were analyzed by real-time PCR. The results are representative of three independent experiments. Values shown are the mean ± SEM. *P<0.05 vs. control. α-SMA, α smooth muscle actin; Con, control; Nut3, nutlin3; TamS, tamoxifen-sensitive cells.

Next, we examined the effect of Nut3 on α-SMA expression in TP53-mutant breast cancer cells. The TP53 status of breast cancer cell lines was determined from the database http://p53.free.fr/Database/Cancer_cell_lines/Breast_cancer.html, which revealed the TP53-mutant breast cancer cells (MDA-MB-231 and Hs578T). As shown in Fig. 3C, the levels of α-SMA and TP53 protein expression were not observably altered by Nut3 treatment in MDA-MB-231 and Hs579T cells. In addition, the levels of α-SMA and TP53 mRNA expression were also markedly changed by Nut3 (Fig. 3D). Therefore, we demonstrated that the TP53 status plays an important role on α-SMA expression in breast cancer cells.

Suppression of TP53 downregulates α-SMA expression in tamoxifen-resistant cells

Conversely, we investigated the effect of a TP53 inhibitor, pifithrin-α (PFT-α), on α-SMA expression in TamR cells. We treated the TamR cells with the indicated concentrations (10 and 30 µM) of PFT-α for 72 h. As shown in Fig. 4A, the basal levels of α-SMA protein expression were dose-dependently decreased by PFT-α treatment. In addition, the levels of α-SMA mRNA expression were also decreased by PFT-α (Fig. 4B). The reduction of α-SMA mRNA expression by PFT-α was decreased to 0.62 and 0.41-fold relative to the control (Fig. 4B). However, the MDA-MB-231 and Hs578T cells with mutant TP53 were not altered by PFT-α treatment (Fig. 4C). In a previous study, TPA stimulated the ubiquitination and degradation of TP53 through the downregulation of PKC-d and the suppression of TP53 transcriptional activity (21,22). Therefore, we treated the TamS cells with 10 nM TPA for 24 h with wild-type TP53. As expected, the level of α-SMA mRNA expression was decreased to 0.31-fold relative to the control (Fig. 4D). In addition, the level of TP53 mRNA expression decreased 0.54-fold relative to the control (Fig. 4D). Based on these results, we demonstrated that the level of wild-type TP53 plays an important role in the α-SMA expression in breast cancer cells.

Figure 4. Suppression of TP53 downregulates α-SMA expression in TamR cells. (A-C) TamR, MDA-MB-231 (MDA231) and Hs578T cells were treated with the indicated doses of PFT-α for 72 h. (D) TamS cells were treated with 10 nM TPA for 24 h. Levels of α-SMA and TP53 protein and mRNA expression were analyzed by western blot analysis and real-time PCR, respectively. The results are representative of three independent experiments. Values shown are the mean ± SEM. *P<0.05 vs. control. α-SMA, α smooth muscle actin; TamS, tamoxifen-sensitive cells; TamR, tamoxifen-resistant cells; Con, control; PFT-α, pifithrin-α (TP53 inhibitor).

Wild-type TP53 upregulates α-SMA expression in breast cancer cells

We investigated whether TP53 upregulates α-SMA expression in breast cancer cells. In the present study, breast cancer cells were transfected with adenoviral LacZ and TP53 genes for 48 h and further incubated for 24 h in fresh serum-free media. As expected, our results showed that the level of α-SMA protein expression was significantly increased by TP53 overexpression (Fig. 5A). Under the same conditions, the level of α-SMA mRNA expression was increased by TP53 overexpression (Fig. 5B). The level of α-SMA mRNA expression increased 3.11±0.23-fold relative to the Vec alone (Fig. 5B). As shown in Fig. 3C and D, triple-negative breast cancer (TNBC) cells with mutant TP53 did not exhibit changes in the levels of α-SMA expression by Nut3 treatment. However, these cells also exhibited significantly increased levels of α-SMA expression following wild-type TP53 overexpression (Fig. 5C). In contrast, we examined the effects of TP53 knockdown by two different TP53 siRNAs on α-SMA expression in TamR cells. As expected, our results showed that the downregulation of TP53 by TP53 siRNA decreased the levels of α-SMA mRNA expression in TamR cells (Fig. 5D). Therefore, we demonstrated that wild-type TP53 expression upregulates the level of α-SMA expression in breast cancer cells.

Figure 5. Wild-type TP53 upregulates α-SMA expression in breast cancer cells. (A and C) After Ad-Lac Z and Ad-TP53 infection for 48 h using whole cell lysates, levels of α-SMA, TP53 and β-actin protein expression were measured by western blot analysis. (B) Levels of α-SMA and TP53 mRNA expression were analyzed by real-time PCR. (D) TamR cells were transfected with two different TP53 siRNA, respectively, for 72 h. The levels of TP53 and α-SMA mRNA expression were analyzed by real-time PCR. The results are representative of three independent experiments. Values shown are the mean ± SEM. *P<0.05, **P<0.01 vs. control (vector transfected or scrambled cells). α-SMA, α smooth muscle actin; Con, control; TamR, tamoxifen-resistant cells.

Discussion

Breast cancer is the most common cancer in women worldwide accounting for approximately 25% of all cancer cases and 15% of all cancer-related deaths (23). Although therapeutic strategies for breast cancer patients involve systemic therapy including chemotherapy, hormonal therapy and targeted therapy, their therapeutic efficacy is still limited by either intrinsic or acquired resistances (24,25). To date, the level of α-SMA expression has been known as a prognostic marker for a variety of cancers, including oral tongue squamous cell carcinoma and lung adenocarcinomas (4,26). Aberrant α-SMA expression is significantly associated with worse overall survival and metastasis-free survival rates in lung adenocarcinomas (4). Consistent with these reports, we also observed that α-SMA expression is associated with survival in breast cancer. ER-α(+) breast cancer patients with high expression of α-SMA exhibited decreased relapse-free survival compared to α-SMA-low expressing patients. In addition, we found that the levels of α-SMA mRNA and protein expression were increased in TamR cells when compared to TamS cells. Therefore, we focused on the underlying mechanism of α-SMA expression which is highly expressed in tamoxifen-resistant breast cancer cells.

The level of TP53 expression is also regulated by a wide variety of cellular stress, such as UV, hypoxia and phorbol ester (11,22,27). Our results showed that the basal level of TP53 expression decreased while α-SMA mRNA and protein expression were significantly increased through TPA treatment. In previous studies, the basal level of α-SMA expression was upregulated through focal adhesion kinase (FAK) as well as the JAK2/STAT1-dependent pathway in breast cancer cells and fibroblasts (2,28,29). In the present study, we analyzed kinase activities to identify the regulatory kinases on α-SMA expression in TamS and TamR cells. Our results showed that the activities of TP53 and Akt were significantly increased in TamR cells, but did not affect the activity of p70 S6 kinase. Therefore, we investigated the role of TP53 in the regulation of α-SMA expression in breast cancer cells.

Nutlin3 (Nut3), a cis-imidazoline analog, interferes with the binding between MDM2 and p53 as an antagonist of MDM2, an E3 ubiquitin ligase of protein of the TP53 family (30). Nut3 induces prominent p21WAF1 expression by upregulating the phosphorylation of Ser 46 on p53 (31). Although we did not assess the phosphorylation of TP53 by Nut3, our results showed that the basal level of TP53 expression was slightly increased through Nut3 treatment in breast cancer cells with wild-type TP53, but not in breast cancer cells with mutant TP53. In addition, α-SMA expression was significantly increased following Nut3 treatment in breast cancer cells with wild-type TP53. In contrast, the basal level of α-SMA mRNA and protein expression dose-dependently decreased with PFT-α in TamR cells that blocked the transcriptional activity of TP53. Therefore, we demonstrated that the activity and expression of wild-type TP53 plays an important role in the regulation of α-SMA expression in breast cancer cells.

The tumor-suppressor protein TP53 is a transcription factor that regulates anti-carcinogenesis programs associated with apoptosis, cell cycle arrest, and DNA repair in genotoxic and not-genotoxic cellular injuries (32,33). In recent studies, we reported that the level of fibronectin expression is associated with the status and expression of TP53 in breast cancer cells (34,35). Wu et al reported that Slug is transcriptionally induced by TP53 and then escapes from apoptosis by repressing the TP53-mediated transcription of Puma (36). We found for the first time that the levels of α-SMA expression are increased by wild-type TP53 overexpression in all breast cancer cells with wild-type or mutant TP53. Therefore, we demonstrated that wild-type TP53 is involved with α-SMA expression in breast cancer cells.

In conclusion, aberrant α-SMA expression is associated with the survival of ER(+) breast cancer patients. Nut3 significantly increased the levels of α-SMA mRNA and protein expression in TamS cells with wild-type TP53, but not in MDA-MB-231 and Hs578T cells with mutant TP53. In addition, the overexpression of wild-type TP53 augmented the level of α-SMA expression in all breast cancer cells with wild-type or mutant TP53. In contrast, we observed that PFT-α dose-dependently suppressed α-SMA expression in TamR cells. Based on these results, we demonstrated that wild-type TP53 expression augments the level of α-SMA expression in breast cancer cells. We also will investigate whether TP53 directly regulates α-SMA expression in breast cancer cells.

Acknowledgements

Not applicable.

Funding

The present study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2016R1D1A1B01010508) and by a National Research Foundation of Korea (NRF) grant funded by the Korean Government (MSIP) (2016R1A5A2945889).

Availability of data and materials

The datasets used during the present study are available from the corresponding author upon reasonable request.

Authors' contributions

SK and JEL designed the study, interpreted the data and wrote the manuscript. DY and YJ were responsible for the laboratory experiments such as real-time PCR and western blotting; JY, SWK and SJN analyzed the data and developed the prognostic signature. All authors read and approved the manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors state that they have no competing interests.

References