Background
Breast cancer is one of the most common cancers in the world. Abnormal expression of claudins (CLDNs) has been interpreted as a mechanism of the malignant progression of cancer [
1]. We cloned and identified the claudin-6 (CLDN6) gene, one of 27 members in the CLDN family, low levels of CLDN6 expression was observed in both human and rat mammary cancer [
2,
3]. Besides, we have found that CLDN6 function as a tumor suppressor in breast cancer cells, inhibiting the malignant phenotype of breast cancer cells, such as growth, migration, and invasion via p38/MAPK pathway [
4]. And we also reported that CLDN6 induced human breast cancer cells apoptosis via ASK1 signaling [
5]. These observations indicate that CLDN6 functions as a tumor suppressor in breast cancer. However, we also found that CLDN6 was highly expressed in multidrug resistant breast cancer cell line MCF-7/MDR that derived from human MCF-7 cell line. Besides, studies demonstrated that other CLDNs expression were associated with chemoresistance in cancers [
6‐
8]. We therefore hypothesized that CLDN6 may also play a role in conferring chemoresistance on breast cancer cells.
Resistance to chemotherapeutic drugs is a significant obstacle in the treatment of patients with breast cancer. The acquisition of a multidrug resistant phenotype in metastatic breast cancer is primarily responsible for the failure of current treatment measures. As the development of effective therapies against chemoresistant tumors is a high priority in breast cancer, identification of chemoresistance associated genes is critical for the successful treatment of breast cancer. Chemoresistance correlates with many different biochemical changes, including decreased influx and increased efflux of the cytotoxic drugs as well as altered expression of genes that control cell cycle and apoptosis [
9]. Cytostatic drugs can also be detoxified by enzymes, for example glutathione transferases (GSTs) can be induced during chemotherapy. As numerous previous studies have shown that cells selected in vitro for resistance to various types of agents, increased levels of GSTs were often associated with the emergence of resistance to some agents in drug-selected cell lines [
10]. The GSTs are multifunctional enzymes that are associated with cellular detoxification, and because they catalyze the conjugation of reduced glutathione to hydrophobic electrophilic compounds, it is believed that GSTs affect mutagenesis and carcinogenesis. Among the 4 GST isozymes, glutanthione S-transferase-p1 (GSTP1) has been reported to play an important role in the resistance of cancer cells to alkylating agents [
11]. In ADM resistance breast cancer cell line MCF-7/ADR, upregulation of GSTP1 confers resistance to ADM [
12]. In several types of cancers, overexpression of GSTP1 was associated with decreased treatment response and survival. GSTP1 expression was closely associated with the response to chemotherapy and the clinical outcome of breast cancer [
13‐
16]. While it has been reported that there was no significant association between high GSTP1 expression and outcome with improved adjuvant chemotherapy in early breast cancer [
10], GSTP1 positive expression showed a significant correlation with a lower histological grade carcinoma in invasive breast cancer [
17]. In ER-negative breast cancer, GSTP1 expression predicted a poor pathological response to neoadjuvant chemotherapy [
14]. Furthermore, in osteosarcoma and prostate cancer, GSTP1 expression was associated with the chemosensitivity [
17,
18]. Whether GSTP1 and CLDNs playing significant roles in different biological characteristics of cancers is not clear, we hypothesize that there may be a close correlation in activity/function between CLDNs and GSTP1, which may provide a possible direction for cancer chemotherapy. Recently a study confirmed CLDN23 as a potential biomarker for the diagnosis and prognosis of gastric cancer, and it was further demonstrated that CLDN23 expression was positively correlated with GSTP1 activity in gastric cancer [
19]. Abnormal high CLDN6 expression was found in MCF-7/MDR multidrug resistant breast cancer cells, however whether CLDN6 confers resistance to various anti-cancer drugs in this cell line need to be explored. In the current study, we investigated the effects of CLDN6 expression in response to various anti-cancer drugs in breast cancer, and explore the role of GSTP1 in CLDN6 mediated chemoresistance in human breast cancer cells.
Methods
Cell culture and reagents
Human breast cancer cell lines MCF-7, MDAMB231, Hs578t, and human embryonic kidney (HEK) 293T cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Human breast cancer multidrug resistance cell MCF-7/MDR was purchased from the Cell Bank of Xiangya Medical College, Central South University. Cells were cultured in Dulbecco’s Modified Eagle’s Medium (Gibco, California, USA) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Beijing, China), 10 mmol/l HEPES, at 37 °C and 5% CO2. Adriamycin (ADM), 5-fluorouracil (5-FU), and cisplatin (DDP) were purchased from sigma (Sigma, MO, USA).
Plasmid
CLDN6 shRNA plasmid, i.e. pGCsilencer-U6/Neo/GFP was constructed which also expresses Green Fluorescent Protein (GFP) (KeyGEN Biotech, Nanjing, China). Target sequence is GGCAAGGTGTACGACTCA. A functional non-targeting siRNA was used as a control.
A CLDN6 overexpression plasmid was generated by sub-cloning human CLDN6 cDNA (NCBI RefSeq record: NM_021195) into the mammalian expression plasmid EX-LV201 (GeneCopoeia, Guangzhou, China) with FLAGs SV40-eGFP-IRES-puromycin.
Lentivirus production and stable knockdown of the GSTP1 genes shRNA plasmid (sh-GSTP1–21:5′-GCCCTACACCGTGGTCTATTT-3′,sh-GSTP1–22:5′-AGGACCTCCGCTGCAAATACA-3′ and sh-GSTP1–23:5′-GGCAAGGATGACTATGTGAAG-3′) and negative-shRNA as shRNA-control (sh-GSTP1-CT) were purchased from GeneCopoeia (GeneCopoeia, Guangzhou, China). Silencer negative control with no significant homology to any known human genes was used as a negative control shRNA with FLAGs U6-mCherry-Hygromycin.
Lentivirus production and stable knockdown of the TP53 gene shRNA interference (shRNAi) plasmids were inserted into the psi-LVRU6MH vector downstream of the U6 promoter with FLAGs U6-mCherry-Hygromycin (GeneCopoeia, Guangzhou, China). Plasmids sh-TP53–32 (5′-GGAAATTTGCGTGTGGAGTAT-3′, sh-TP53–33 (5′-CCACTACAACTACATGTGTAA-3′) and sh-TP53–34 (5′-GGACTTCCATTTGCTTTGTCC-3′) were used to knockdown the TP53 gene and a functional non-targeting shRNA (sh-TP53-CT) was served as a negative control clone. For stable RNAi, lentiviral particles were produced.
A GSTP1 overexpression plasmid was generated by subcloning human GSTP1 cDNA (NCBI RefSeq record: NM_000852) into the mammalian expression plasmid EX-LV206 with FLAGs SV40-mCherry-IRES-puromycin (GeneCopoeia, Guangzhou, China).
Lentiviral packaging plasmids expressing gag-pol, rev, and pVSVG genes were obtained from Institute of Biochemistry and Cell Biology of Shanghai Life Science Research Institute, Chinese Academy of Sciences. These vectors were transfected into 293T cells by FuGene HD (Roche Applied Science, Basel, Switzerland). Viral supernatants were harvested at 48 h and 72 h after transfection and concentrated by ultracentrifugation. Viruses were transduced in the presence of 5 μg/mL polybrene.
Reverse-transcription PCR (RT-PCR) and quantitative RT-PCR (qRT-PCR) analysis
Total RNA was isolated from 5 × 106 cells using TRIzol reagent (Invitrogen, California, USA), according to the manufacturer’s instructions. Total RNA concentration and purity were analyzed in duplicate samples using a Nanodrop ND-2000 spectrophotometer (Thermo Fisher Scientific, MA, USA). cDNA was synthesized from the qualified RNA using an RT-PCR reverse transcription kit (TransGen Biotech, Beijing, China). 1000 ng of total RNA was reverse transcribed into cDNA under the condition: 25 °C 10 min, 42 °C 30 min, and 85 °C 5 s, as manufacturer’s recommendations. Then the cDNA was stored at −20 °C until use. The PCR was performed using a PCR kit (TransGen Biotech, Beijing, China). The PCR product was electrophoresed on 1.5% agarose gels. Primers were synthesized by Sangon (Sangon, Shanghai, China). Quantitative PCR was carried out with either Taq-Man or SYBR Green PCR reagents on an ABI Prism 7300 detection system (all from Applied Biosystems, Foster City, CA). The reaction program was consisted of 95 °C for 3 min followed by 40 cycles of 95 °C 30 s, 55 °C 20 s, and 72 °C 15 s. GAPDH gene was served as internal control and the relative mRNA levels were calculated by 2−ΔΔCt. Primers designed were synthesized by Sangon (Shanghai, China).
Western blot analysis
When cells reach 80% confluence, cells were harvested and washed with PBS. Total protein was extracted with 200 μL RIPA Lysis Buffer (Beyotime, Shanghai, China) with 1 mM phenylmethylsulfonyl fluoride (PMSF) (Sigma, MO, USA) and 1 mM Phosphatase inhibitor (Solarbio, Beijing, China), followed with centrifugation 12,000 g for 20 min. The protein concentration was determined using a BCA Protein Assay Kit (Beyotime, Shanghai, China). 50 μg of denatured protein was applied to 12% SDS-PAGE gels. After electrophoresis the proteins on the gel were then transferred onto a nitrocellulose membrane (Millipore, California, USA). The membrane was blocked with 5% defatted milk at 37 °C for 1 h and incubated overnight at 4 °C with the primary antibodies. The membrane was incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. Finally the immunoreactive bands were visualized using an ECL western blotting system (Beyotime, Shanghai, China). The following antibodies were used: a monoclonal mouse anti-β-actin antibody (Santa Cruz Biotechnology, California, USA), a polyclonal rabbit anti-CLDN6 antibody (Santa Cruz Biotechnology, California, USA), a polyclonal rabbit anti-cleaved-caspase-9 antibody (Cell Signaling Technology, MA, USA), a polyclonal rabbit anti-cleaved-PARP antibody (Cell Signaling Technology, MA, USA), a polyclonal rabbit anti-γH2AX antibody (Cell Signaling Technology, MA, USA), a polyclonal rabbit anti-p-Ser15-p53 antibody (Cell Signaling Technology, MA, USA), and a polyclonal mouse anti-GSTP1 antibody (Cell Signaling Technology, MA, USA).
In vitro drug sensitivity assay
In vitro drug cytotoxicity was measured by Cell Counting Kit-8 (CCK-8) assay (Dojindo, Kumamoto, Japan). The cells were seeded into 96-well plates (3 × 103 cells/well) and then treated for 48 h in 100 μL of medium with anticancer drugs. The cells incubated without drugs (i.e. control wells) were set at 100% survival and were utilized to calculate the concentration of each cytostatic drug lethal to 50% of the cells (IC50). CCK-8 reagent was then added and then incubated at 37 °C for 2 h. The optical density (OD) of each well at 450 nm was recorded on a Microplate Reader (Thermo, Schwerte, Germany). The cell viability (% of control) is expressed as the percentage of (ODtest − ODblank)/(ODcontrol − ODblank). The assay was conducted in three replicate wells for each sample and three parallel experiments were performed.
Apoptosis assay
4′,6-diamidino-2-phenylindole (DAPI) staining was used to detect apoptosis in vitro. Cells were harvested when grown to 60-80% confluency, and treated with ADM for 48 h, then fixed with 4% paraformaldehyde, stained with the 1 mg/mL DAPI (Sigma, MO, USA) for 15 min and examined by fluorescence microscopy to determine the fraction of apoptotic cells. Apoptotic cells were recognized as chromatin condensed, punctate nuclear ghosts with stained, degraded nuclei when examined by fluorescence microscopy. The incidence of apoptosis was analyzed by counting nuclear deep dyeing cells with condensed chromatin, and determining the percentage of apoptotic cells.
GST activity assay
GST activity was measured using a GST activity kit (Solarbio, Beijing, China) according to the manufacturer’s protocol. It was defined as the amount of enzyme that was required to reflect the ability to reduce GSH and 1-chloro-2, 4-dinitrobenzene (CDNB). The changes in absorbance of the GSH and CDNB were recorded at 340 nm for 10 s and 310 s respectively. GST activity was expressed as nmol per min per mg of total protein concentration.
Nuclear/cytosol fractionation
To monitor the nuclear and cytosol p53 protein level after CLDN6 overexpression, nuclear/cytosol fractionation along with immunoblotting analysis were performed. 1 × 106 cells were needed. Nuclear/Cytosol Fractionation Kit (TransGen Biotech, Beijing, China) was applied to isolate nucleus and cytosol protein according to the manufacturer’s instructions.
Immunoprecipitation–western blots
The cells were lysed in IP lysis buffer (Beyotime, Shanghai, China) for 30 min on ice, vortex for 10 s interval of 5 min, then transferred to a 1.5 mL microcentrifuge tube and centrifuged for 20 min at 14,000 g to remove cellular debris. The supernatants were analyzed for total protein content, and 300 μg of total protein was incubated with 25 μL of agarose-immobilized goat polyclonal anti-rabbit antibody in a final volume of 500 μL, adjusted with lysis buffer. Immunoprecipitation was carried out with gentle rocking, overnight at 4 °C. The agarose beads were pelleted by centrifugation at 3000 rpm for 5 min, and then washed 3 times with 1 mL lysis buffer, with each wash followed by a 3 min centrifugation at 3000 rpm. After the final wash, 24 μL lysis buffer and 6 μL of 5× SDS sample buffer was added to the beads, the samples were boiled and then loaded onto 12% SDS-PAGE gels. Following protein transfer to PVDF membrane (Millipore, California, USA), p53 and CLDN6 expression were detected by western blotting as described earlier.
Immunohistochemistry
Immunohistochemistry of tumor tissues collected from human patients breast cancer samples were performed as we described elsewhere [
2]. 40 patients with breast cancer at the department of pathology of the second hospital of Jilin university who had not been treated with any chemotherapy and those received neoadjuvant chemotherapy for relapsed disease after initial biopsy either for organ preservation or for unresectable disease. Formalin-fixed, paraffin-embedded biopsy tissues were available. Immunohistochemistry was performed as described elsewhere. Tissue sections were immunostained with CLDN6 antibody (Abcam, MA, USA) and GSTP1 antibody (Cell Signaling Technology, MA, USA). Diaminobenzidine (DAB) was used for color development. CLDN6 expression is indicated in brown and is expressed in the membrane of breast cancer cells and GSTP1 is indicated in brown and expressed in the nuclear of breast cancer cells.
Immunoreactivity was evaluated independently by two observers without specific knowledge of the patients. The staining was graded for intensity (0-negative, 1-weak, 2-moderate, and 3-strong) and percentage of positive cells as follows: 0, <5% positive tumor cells; 1, 5%–25% positive tumor cells; 2, 26%–50% positive tumor cells; 3, 51%–75% positive tumor cells; 4, >75% positive tumor cells. The grades were multiplied to determine an H-score. The H-scores for tumors with multiple scores were averaged. Protein expression was then defined as negative (H-score = 0), weakly positive (H-score = 1–4), moderately positive (H-score = 5–8), and strong (H-score = 9–12). Positive or negative reactions were determined in five random fields of each sample with image processing software Image-Pro Plus 6.0.
Statistical analysis
All experiments were performed in triplicate and data was reported as means ± SD. Statistical analysis was performed with SPSS 19.0 software. Statistical significance was determined by Student’s t-test or one-way analysis. Correlation between CLDN6 and GSTP1 was evaluated using Spearman’s test. P < 0.05 was considered statistically significant.
Discussion
Breast cancer is one of leading cause of premature death in developed and developing countries for women. Although significant advances have been achieved, chemoresistance remains a major clinical obstacle in successful treatment of breast cancer. Our pervious studies have confirmed that CLDN6 is significantly downregulated in breast invasive ductal carcinoma which correlates with lymphatic metastasis [
3]. CLDN6 is therefore suggested to function as a tumor suppressor by inhibiting malignant phenotype of breast cancer cells through various signaling pathways such as p38-MAPK, JAKs-STATs, ASK1-JNK, and other pathways [
4,
5]. As chemoresistance is a big problem facing the successful treatment of patients with breast cancer, and CLDN6 is expressed at a higher level in chemoresistant MCF-7/MDR cells as compared to parental MCF-7 cells, whether CLDN6 plays any role in conferring chemoresistance to breast cancer cells is unknown. In the current study, we explored the role of CLDN6 in breast cancer chemoresistance and the underlying mechanism, and found that high level expression of CLDN6 conferred chemoresistance on MCF-7 and MCF-7/MDR cells to ADM, 5-FU, and DDP through GSTP1. TNBCs are claudin-low breast cancers with the poorest prognosis [
20]. We have also shown that overexpression of CLDN6 in TNBC cell line Hs578t increased ADM chemoresistance, accompanying with upregulated expression and enzyme activity of GSTP1. Inhibition of GSTP1 in Hs578t/CLDN6 cells decreased IC
50 of ADM.
Previous studies have also showed that CLDN1, CLDN3, CLDN4, and CLDN7 expression are related with drug resistance to chemotherapy [
8,
21‐
24]. CLDN3 and CLDN4, which are
Clostridium perfringens enterotoxin (CPE) receptors, affected chemoresistance mediated by CPE in ovarian cancers [
22,
23], it was also reported that CLDN3 and CLDN4 modulated the sensitivity to cisplatin partially through the copper and cisplatin influx transporter CTR1 [
25]. CLDN7 increased chemosensitivity through the caspase pathway in human lung cancer cells [
26]. One mechanism of CLDNs and chemoresistance acquisition may be related to cancer stem cells [
27]. However, the mechanism of CLDN6 mediated chemoresistance is still unclear.
To explore the mechanism of CLDN6 mediated chemoresistance, using next generation sequencing techniques, we found that CLDN6-overexpressing MCF-7 cells (referred as MCF-7/CLDN6 cells) (Additional file
5: Figure S5) also expressed high level of GSTP1. Cytostatic drugs can be detoxified by enzymes, for example glutathione transferases, and GSTs can be induced during chemotherapy. Recent evidence indicated that GSTP1 directly involved in chemoresistance in multiple cancer cells [
12,
13,
28,
29], we believed that GSTP1 may be an important gene in regulating chemoresistance in breast cancer MCF-7 cells. GSTP1 is often overexpressed in tumors and confer resistance against many types of drugs, including ADM and DDP. Similarly, a large numbers of studies also comfirmed the role of GSTP1 in chemoresistance in breast cancer [
28,
30‐
32], overexpression of GSTP1 is linked to chemoresistance. Apart from P-gp, which is considered to be the most important multidrug resistance associated protein [
33], is also overexpressed in the multidrug resistant cell line MCF-7/MDR. In consistence with our hypothesis, we found that, in addition to CLDN6 overexpression, GSTP1 expression and its enzyme activity were also increased in MCF-7/MDR cells as compared with the parental MCF-7 cells. Furthermore, silencing CLDN6 in MCF-7/MDR cells resulted in a decrease of both the expression and enzyme activity of GSTP1. To further demonstrated the cause relationship between CLDN6 and GSTP1, GSTP1 expression in MCF-7/CLDN6 cells was suppressed by siRNA (referred as MCF-7/CLDN6-sh-GSTP1–23 cells), both apoptosis and DNA damage were increased in MCF-7/CLDN6-sh-GSTP1–23 cells after ADM treatment, whereas overexpression of GSTP1 in MCF-7/MDR-sh-CLDN6 cells inhibited ADM induced apoptosis. These observations suggest that GSTP1 plays a crucial role in CLDN6 conferring chemoresistance on breast cancer MCF-7 cells.
In addition to glutathionylation and detoxification functions, GSTP1 has been shown to possess chaperone functions, regulation of nitric oxide pathways, and control over various kinases signaling pathways [
34‐
37]. Furthermore, overexpression of GSTP1 is closely correlated with p53 in caner, and wtp53 positively regulates the expression of GSTP1 [
38]. Accordingly, results presented in current study showed that the expression of p53 was positively correlated with GSTP1 in MCF-7 cells. Similarly, in cells with overexpressed CLDN6, p53 and GSTP1 were upregulated, shRNA inhibition of p53 expression significantly downregulated the expression of GSTP1 at both mRNA and the protein levels and its enzyme activity as well. In addition, inhibition of p53 also decreased the IC
50 of ADM in MCF-7/CLDN6 cells. In accordance with above observations, silencing CLDN6 in MCF-7/MDR cells decreased both p53 and GSTP1 expressions. Therefore, upregulation of GSTP1 expression by CLDN6 in breast cancer MCF-7 cells is dependent upon p53. As a transmembrane protein, cytoplasmic tail of CLDN6 is the key domain responsible for their association with other binding protein [
39]. Our results showed that cytosol p53 protein level was increased whereas nuclear p53 was decreased in cells with overexpressed CLDN6. IP experiment results showed CLDN6 interacted with p53 structurally, but how does CLDN6 interact with p53 still needs to be further studied.