ETS1 is associated with cisplatin resistance through IKKα/NF-κB pathway in cell line MDA-MB-231

The TNBC cell line 231/wt was bought from the cell resource center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. The DDP-resistant human TNBC cell line 231/DDP was obtained by stimulating 231/wt cell lines with different DDP concentrations, as described in our previous work [8]. These cell lines were recovered in the medium without DDP, then was cultured in the medium with DDP (1.5 μg/mL) on the next day in the atmosphere of 5% CO₂ at 37 °C.

The 1 μg/mL DDP was added in both of the 2 cell lines at the logarithmic phase, when the cell inhibitory concentration (IC₅₀) value was “0”. After 48 h pretreatment, the supernatant was abandoned, and the cells were washed in PBS for 3 times, and then collected by cell scraper. Thereafter, cells were suspended in lysis buffer containing concentrated nitric acid, and incubated at 60 °C for 20 min. Then, intracellular DDP concentration was measured. The splitting cells were transferred into a 1.8 mL EP centrifugal tube, and then put these into liquid nitrogen and water bath for 1 min, respectively. After repeating for 3 times, the DDP concentration was measured based on the inductively coupled plasma mass spectrometry (ICP-MS) method [9].

MTT assay was used to measure the drug resistance of DDP-resistant cell line. In brief, cells were inoculated in the 96-well plate, 5000 cells per well. After 24 h incubation, if all cells were attached, which could be separately treated with four drugs as DDP, doxorubicin (DOX), paclitaxel and cyclophosphamide (CTX). The specific operation steps is that DDP was dissolved in amide,n,n-dimethyl-formicaci (DMF) (2 mg/mL, 6.67 mM) then diluted by phosphate buffer solution (PBS). DOX was dissolved in dimethyl sulfoxide (DMSO) (2 mg/mL, 3.45 mM) then diluted by PBS. CTX was dissolved in DMSO (2 mg/mL, 7.17 mM) then diluted by PBS. Paclitaxel was dissolved in DMSO (2 mg/mL, 2.34 mM) then diluted by PBS. After 48 h treatment, the cells were incubated in MTT for 4 h, and then DMSO was added and shocked for 10 min. OD value of the survival cells were determined under 490 nm wave, with the reference under 630 nm. On this basis [10], the IC₅₀ value in each group was calculated.

Extent of apoptosis was measured by Annexin V-FITC apoptosis detection kit (Invitrogen Corporation, California, USA) according to supplier’s instruction. Briefly, cells were treated with 1 μg/mL DDP for 48 h, collected and stained with Annexin V-FITC, then analyzed by using FACScan flow cytometer (Becton–Dickinson, Newjersey, USA).

There were six samples in the following GeneChip probe array analysis: DDP-resistant (231/DDP, n = 3) and non-resistant breast cancer cell lines (231/wt, n = 3). First, total RNAs in each sample were extracted by the TRIzol method (Invitrogen), and then examined by the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Next, the RNAs were amplified and labeled using the GeneChip WT PLUS reagent kit (Affymetrix, Santa Clara, CA, USA) according to the manufacturer’s instruction. Afterwards, the labeled RNAs were hybridized to Affymetrix GeneChip Human Transcriptome Array 2.0 in hybridization oven (Affymetrix model 640) for 16 h at 45 °C [11, 12].

As aforementioned, total RNA was extracted by the TRIzol method (Invitrogen), and was then reversely transcribed into cDNA according to the manufacturer’s instruction of the TaqMan reverse transcription kit (Applied Biosystems) (Logan et al. 2006). Following, the quantitative real-time reverse transcription-PCR (qRT-PCR) was performed to calculate gene expressions, with GAPDH as the internal reference. Primer sequences of the genes are listed in Table 1. Rotor-Gene6 software was used to calculate the Ct value, and gene expressions were calculated by 2^−ΔΔCt method [13].

Total proteins were extracted and protein concentrations were evaluated using Bradford assay (Pierce Biotechnology Inc., Rock-ford, USA). Western blotting assay was performed as described previously [3]. The antibodies against ETS1(ab26096), MRP2(ab3373), BRCP(ab3380), P-gp(ab103477), P38(ab31828), p-P38(ab45381), IKKα(ab32041), IKKβ(ab32135), and NF-κB (ab16502) were purchased from Abcam.

PHY-310-GFP plasmid expressing human ETS1 was provided by Hanyi Biotechnology Shanghai. For the knockdown experiment, the ETS1 shRNA (5′-CGCUAUACCUCGGAUUACU-3′) was cloned into PHY-31-GFP vector. The 231/DDP cells were subcultured in a 6-well plate for 24 h, with a concentration of 70–80%. 231/DDP was transfected stably PHY-310-GFP-shETS1 by using Lipofectamine™ 2000 Reagent (Invitrogen, USA), followed by selection in 2-μg/mL puromycin. Stable ETS1 knockdown cells were used in the subsequent study.

Cells were placed on a glass slide at the density of 1.0 × 10⁵ cells/mL. Cultured for 48 h, the cells washed for with PBS and then fixed with 4% paraformaldehyde for 20 min at 4 °C. Cells were permeabilized using 0.3% Triton X-100/PBS for 20 min and washed with PBS. The cells were then incubated for 30 min in the 0.5% BSA. Diluted primary antibody (1:50) was applied to each coverslip and incubate overnight at 4 °C. After washing in PBS, the cells were incubated with rabt antibody at a 1:200 dilution for 1 h at room temperature in the dark and followed by DAPI nuclear staining (1 μg/mL) for 5 min. After washing, the images were taken using a Zeiss Laser Scanning Confocal Microscope (LSM7 DUO).

To construct the IKKα promoter, we amplified the promoter region from − 217 to − 2216 from transcription start site by PCR using hot-start DNA polymerase (New England Biolabs, Ipswich, Massachusetts, USA). A 3-kb fragment containing KpnI and XhoI sites was then ligated into the PCR2 vector by using a Plasmid minni Kit I (200) (OMEGA). The promoter fragment was excised from IKKαby digesting it with KpnI and XhoI and then clone in PGL3-Basic vector (IKKα-NC) and IKKα-luciferase vector (IKKα-OE) to produce promoter luciferase report.

Sub-confluent cells cultured in 6-well plates were transiently co-transfected with luciferase reporter and internal control plasmids, using the DNAfectin Transfection Reagent according to the manufacture’s protocol. Cells were lysed in passive lysis buffer, and luciferase assay was performed using dual-luciferase reporter assay kit in 48 h post transfection. Renilla luciferase activity was used as control for transfection was performed in triplicate.

ChIP was performed using ChIP assay kit (Cell Signaling Technology, CST, USA) according to the manufacture`s instruction. The main step is that cells in a 10-cm culture plate were crosslinked for 10 min by 1% formaldehyde. Crosslinking was neutralized by 0.2 M glycine. Cells were collected and suspended in lysis buffer (25 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, 1% SDS and 5% glycerol). Genomic fragments were sonicated to a proper length. Protein–DNA complexes were precipitated with ETS1 antibody or immunoglobulin G overnight at 4 °C. The complexes were purified by protein A/G magnetic beads and the crosslinks were reversed at 68 °C. Quantitative PCR was performed, after DNA fragments were purified. Primer sequences used in ChIP-qPCR are as follows: IKKa 5′-GTGGTTCCGTTCAGCCCT-3′ (Forward), 5′-TGCTCGCGCGTCTTTG-3′ (Reverse); U2 5′-ATCGCTTCTCGGCCTTTTGG-3′ (Forward), 5′-AGGTCGATGCGTGGAGTGGA-3′ (Reverse).

Four-week old female BALB/C nude mice, weight of 15–16 g, were obtained from Shanghai SLAC Laboratory Animal Company (Shanghai China). The mice were housed specific pathogen-free conditions at 22 ± 2 °C, at 70% relative humidity and under a 12-h light/dark cycle. Six-week old female nude mice, approximately 20 g, were transfected 231/DDP cells (sh-ETS1 and empty vector) were harvested at a concentration of 1.0 × 10⁷ cells/mL and suspended cell (0.2 mL) subcutaneously implanted into the left second breast fat pad of each mouse. Tumor growth was examined every 3 days, and tumor volume was calculated using the equation V = D × d²/2 (V, volume; D, long diameter; d, short diameter). When the average tumor size reached approximately 60 mm³, DDP was administered 2 times per week by intraperitoneal injection at a dose of 4.2 mg/kg. A month after the injection, mice were killed and the subcutaneous growth of each tumor was examined.

The SPSS 19.0 software was used to perform the statistical analysis. P values less than 0.05 were considered significant. All the data were presented using “mean ± SD”, each experiment was repeated for three times [14].

To verify whether 231/DDP [8] had the resistant properties to DDP and multidrug, MTT method was used to detect cell inhibitory rate, namely IC₅₀ of the aforementioned drugs (DDP, DOX, paclitaxel and cyclophosphamide). As presented in Fig. 1a, after 48 h treatment with DDP, the IC₅₀ of the four drugs in 231/DDP cells were dramatically increased compared with MB-MDA-231 cells, especially the IC₅₀ of DDP (Table 2). Annexin V/PI assay indicated that after 48 h cultivation with different concentration of DDP, the apoptosis ratio was significantly lower in 231/DDP group, than 231/wt group (Fig. 1b, c, P < 0.05).

Based on ICP-MS detection, we found intracellular DDP concentration was 0.51 ng/mg in the 231/DDP cell line after 24 h cultivation with 1 μg/mL DDP complete medium, significantly lower than that in the 231/wt cells (0.87 ng/mg, P < 0.01, Fig. 1d). To further verify the drug resistance, western blot and qRT-PCR was conducted to detect the ABC transporters expressions of MRP2, P-gP and BCRP, three known proteins related to drug resistance on membrane, in both of 231/wt cells and 231/DDP cells. As expected, mRNA and protein expressions of them were obviously increased in 231/DDP cells, compared with 231/wt cells (Fig. 1e, f).

Based on the aforementioned selection criteria (P ≤ 0.05 and FC > 1), a set of 895 DEGs were identified between 231/wt cell line and 231/DDP cell line, which could well distinguish the two kinds of cell lines (Fig. 2a). Based on information in the STRING database, a PPI network for the DEGs was constructed. A node in the PPI network represents a protein product of a DEG, and an edge denotes an interaction between two genes. Degree of a node represents the interplayed protein numbers with this gene. Among the PPI network, several nodes had relatively high degrees, such as RPL6 (degree = 2), RBBP8 (degree = 3), ETS1 (degree = 7), BIRC2 (degree = 5), PIK3A (degree = 4) and RARS (degree = 4) (Fig. 2b). Integrating information in the PPI network with DEGs, the highlighted gene was used as a key word to search drug-resistant genes in databases such as the PubMed, in combination with words of “multidrug resistance (MDR)” or “drug resistance” OR “chemotherapy resistance”. The above six predominant nodes in PPI network have been reported in the literature, thus their expressions were validated in vitro.

Results of qRT-PCR revealed that gene expressions of ETS1, RPL6, BIRC3, RBBP8, RARS and PIK3A in 231/DDP cells were all increased compared to 231/wt cells, especially the expression of ETS1 (Fig. 3a). Protein expression via western blot indicated that ETS1 protein was dramatically increased in 231/DDP cell line, compared with 231/wt cell line (Fig. 3b), suggesting this gene’s alteration at both mRNA and protein level was tied up with drug resistance to DDP in 231/DDP cell line.

K–M curve [15] showed that the median survival of breast cancer patients in ETS1 low-expression group was 8.3 months, significantly longer than that in high-expression group with 4.4 months (P = 0.0112, Fig. 3c). Besides, the HR of the ETS1 level conducted by univariate Cox proportional hazards regression method was 0.401 (CI 0.198, 0.812).

The downstream effector of P38/ETS1 signaling was identified by cisplatin resistanc. The translocation of NF-κB in 231/DDP cells was determined by using immunofluorescence staining (Fig. 3d). In addition, the expression of P38, phosphor-P38, ETS1, IκB kinase α (IKKα) and IκB kinase β (IKKβ) as well as NF-κB was determined. Cisplatin resistance induced the high expression of ETS1, IKKα, NF-κB and phosphor-P38 (Fig. 3b). Moreover, it is noting that cisplatin resistance by the high expression of IKKα and further affecting up-regulation expression of NF-κB, not IKKβ which IKKβ is considered as classic path way to activate NF-κB [16].

After ETS1 knocking down, the resistance to four drugs as DDP, DOX, paclitaxel and cyclophosphamide in 231/DDP cell was remarkably reduced; especially to DDP, whose IC₅₀ was significantly reduced from 19.62 to 2.92 (P < 0.001, Fig. 4a; Table 3). ICP-MS detection results showed that intracellular DDP concentration was significantly increased in 231/DDP-KD cell line, compared to that in the 231/DDP cell (P < 0.001, Fig. 4b). With different concentration of DDP, the apoptosis ratio was significantly higher in 231/DDP-KD cell lines than 231/DDP cell lines (Fig. 4c, d). These results suggest ETS1 inhibition enhances chemo-sensitivity to DDP in 231/DDP cell lines.

After ETS1 knocking down, protein expression of ETS1 was remarkably reduced in the 231/DDP cell line, compared to the blank control (NC) (Fig. 4e), indicating ETS1 expression was successfully inhibited. ABC transporters and IKKα as well as NF-κB were also decreased in 231/DDP cell with ETS1-KD (Fig. 4e), suggesting the 231/DDP cell line might be more sensitive to ETS1 alterations and ETS1 could be the upstream effector of IKKα and NF-κB.

Promoter analysis of IKKα, using PROMO TOOL V. 8.3 of TRANSFAC [17] (Beverly, MA, USA), indicated the presence of ETS1 transcription factor putative-binding sites that cluster into four main regions (Fig. 5a). To validate the elements for regulation in the IKKα promoter region by ETS1, we generated one pCDNA3.1-based luciferases constructs of IKKα containing clusters A, B, C, D (Fig. 5b). A luciferase reporter assay revealed that IKKα-Luc in 231/DDP-NC was higher than 231/DDP-KD. This results suggest that ETS1 has one or some clusters for regulatory promoter element for regulation of IKKα. ChIP assay was performed, In comparison with control IgG, the IKKα promoter had increased (3.3- to 7.8-fold) expression in 231/wt and 231/DDP. The result strongly induced ETS1 to bind to the IKKα promoter over time. No enrichment was observed in the control IgG group (Fig. 5c).

To further investigate the underlying roles of ETS1 in enhancing the chemosensitivity of TNBC cell to DDP, we used nude mice xenograft model. 231/DDP cells transfected with shETS1 or empty vector were subcutaneously injected into mice, and followed by treatment with DDP. A month after the initial DDP treatment, the body weight, volume and average weight of tumor xenograft was recorded (Fig. 6a–e). Immunofluorescence staining was used to detect localization and expression level of ETS1 in tumors tissue (Fig. 6f). As shown in Fig. 6d, the tumors formed from shETS1-transfected 231/DDP cells grew significantly more slowly than those from empty vector following DDP treatment. It was also observed that the down-regulation of ETS1 led to the inhibition of tumor growth. These results suggested that ETS1 low expression increased the in vivo chemosensitivity of TNBC cells to DDP.