Increased ABCC2 expression predicts cisplatin resistance in non‐small cell lung cancer

Long‐term use of platinum‐based drugs can cause non‐small cell lung cancer (NSCLC) to develop extremely strong drug resistance. Increasing the drug dosage does not have better treatment effects and could lead to serious complications. High levels of drug resistance are considered to be characteristic of human tumours and are usually mediated by genes related to multidrug resistance. Multidrug resistance‐associated protein 2 (ABCC2), an ATP‐binding cassette multidrug resistance transporter, was found to be overexpressed in various human cancers. In this study, we found that ABCC2 was also upregulated in cisplatin (DDP)‐resistant A549 cells (A549/DDP). Functional studies demonstrated that ABCC2 knockdown reversed DDP resistance and promoted G1 phase arrest in A549/DDP cells, and PARP and caspase‐3 were activated in A549/DDP cells following ABCC2 knockdown. In vivo, ABCC2 knockdown enhanced the cytotoxicity of DDP to subcutaneous A549 tumours. Together, these results suggest that ABCC2 may be a potential therapeutic strategy for overcoming DDP resistance in NSCLC patients. Significance of the study In this study, we investigated the role of ABCC2 in cisplatin resistance of NSCLC cells. Our data show that ABCC2 expression was associated with resistance to cisplatin and that knockdown ABCC2 could reverse cisplatin resistance in NSCLC cells. Taken together, our study suggests that reducing the expression of ABCC2 could become an important strategy for enhancing the sensitivity of NSCLC cells to cisplatin.

expressed in various human cancers. In this study, we found that ABCC2 was also upregulated in cisplatin (DDP)-resistant A549 cells (A549/DDP). Functional studies demonstrated that ABCC2 knockdown reversed DDP resistance and promoted G1 phase arrest in A549/DDP cells, and PARP and caspase-3 were activated in A549/DDP cells following ABCC2 knockdown. In vivo, ABCC2 knockdown enhanced the cytotoxicity of DDP to subcutaneous A549 tumours. Together, these results suggest that ABCC2 may be a potential therapeutic strategy for overcoming DDP resistance in NSCLC patients.
Significance of the study: In this study, we investigated the role of ABCC2 in cisplatin resistance of NSCLC cells. Our data show that ABCC2 expression was associated with resistance to cisplatin and that knockdown ABCC2 could reverse cisplatin resistance in NSCLC cells. Taken together, our study suggests that reducing the expression of ABCC2 could become an important strategy for enhancing the sensitivity of NSCLC cells to cisplatin. Many studies have found that various members of the ABC superfamily play a role in mediating resistance to platinum compounds. 4 The ABC transport system is a huge family, which includes seven subfamilies, namely ABCA, ABCB, ABCC, ABCD, ABCE, ABCF and ABCG. [5][6][7] Some of them have been proven to be closely related to multidrug resistance, such as multidrug resistance proteins (ABCB1), multidrug resistance-associated proteins (ABCC), and breast cancer resistance protein (ABCG2). 8 The ABCC subfamily contains 11 subgroups encompassing ABCC1 to ABCC11. As a member of the ABCC subfamily, ABCC2 plays an important role in transporting endogenous and exogenous substances and impressing drug absorption, distribution and excretion. 9 It has a protective effect on the body, and at the same time, the expression of ABCC2 is also closely related to the effect of chemotherapy on malignant tumours. ABCC2 has important clinical value in clinical chemotherapy and the evaluation of the effect of chemotherapy. For patients diagnosed with urothelial carcinoma of the urinary bladder, upon DDP treatment, DDP-induced CCAAT/enhancer-binding protein delta expression activated ABCB1 and ABCC2, and the high expression of ABCB1 and ABCC2 resulted in a decline in the effect of DDP. 10 In ovarian cancer, upregulation of miR-490-3p enhanced the sensitivity of ovarian cancer cells to DDP by downregulating ABCC2 expression. 11 In addition, upregulation of ABCC2 and ABCG2 transporters was observed in head and neck squamous cell carcinoma cell lines with cytoplasmically sequestered mutant p53, and these cell lines were frequently found to be more resistant to DDP. 12,13 ABCC2 inhibition of head and neck squamous cell carcinoma cells with MK571 markedly enhanced the sensitivity of HNSCC cells to DDP. 14-16 Therefore, we found that the high expression of ABCC2 in various tumour cell lines is closely related to the resistance of these tumour cells to platinum drugs.
Our study compared normal A549 cells and DDP-resistant cells (A549/DDP) using RNA sequencing. The results revealed that in A549/DDP cells, the expression levels of several ABCC family members were changed. ABCC2 and ABCC4 were upregulated, with ABCC2 exhibiting the most obvious change. After knocking down ABCC2 expression, the sensitivity of A549/DDP cells to DDP increased, and these effects were valid both in vivo and in vitro. Our study suggests that ABCC2 has potential as a therapeutic target for the treatment of patients with DDP-resistant lung cancer.

| Cell culture
To establish the DDP-resistant A549 cell subline, A549 cells were initially cultured in Dulbecco's modified Eagle's medium (DMEM) with an increasing dose of DDP for the first month (0.5, 1.0, 2.0 and 4.0 μM DDP increasing each week). In the following months, A549 cells were cultured in DMEM with DDP concentrations ranging from 4 to 10 μM. Ten μM DDP was then used to culture A549 cells for the following 2 months. After long-term exposure, a subline resistant to DDP (referred to as A549/DDP) was successfully constructed. A549/ DDP cells were maintained in DMEM supplemented with 1% penicillin/streptomycin and 10 μM DDP. The human non-small cancer cell line A549 was purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China). A549 cells were cultured with 10% foetal bovine serum (FBS) and 1% penicillin/streptomycin.

| Flow cytometric analysis of cell cycle distribution
A549 and A549/DDP cells (2 × 10 5 cells/well) were seeded in 6-well plates. For cell cycle distribution analysis, the cells were collected using centrifugation at 1500 rpm for 5 minutes, and the cell precipitate was washed with 70% alcohol and stored overnight in 70% alcohol. The next day, the cell precipitation was centrifuged at 1500 rpm for 5 minutes, and the supernatant was removed. Cells were stained using a Cell Cycle Staining Kit (Beyotime, China) for 30 minutes in the dark and then analysed using flow cytometry.

| RNA isolation and quantitative real-time polymerase chain reaction
Total RNA was extracted from the cells using TRIzol reagent (Invitrogen). cDNA was obtained using a reverse transcription kit (Takara). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using SYBR Premix Ex Taq (Takara, Japan). GAPDH was used as the standard control. All fold changes were calculated using the relative quantification method (2 −ΔΔCt ). The following primers were used: ABCC2: forward 5 0 -CTCACTTCAGCGAGACCG-3 0 ,

| shRNA transfection and establishment of stable cell lines
sh-negative control, sh1-ABCC2 and sh2-ABCC2 were purchased from GenePharma (Shanghai, China). Each shRNA was packaged in 293 T cells for 24 hours and collected according to the manufacturer's instructions. All transfections were conducted using Lipofectamine 3000 Transfection Reagent (Invitrogen, Thermo Fisher, Carlsbad, California). Forty-eight hours after transfection, puromycin was added to select stable cell lines for 1 month.

| Animal experiment
Male BALB/c nude mice (4 weeks old) were purchased from the Beijing Vital River Laboratory Animal Technology Co. Mice were subcutaneously implanted with 5 × 106 A549 and A549/DDP cells

| Immunohistochemistry
To detect ABCC2 expression, mouse subcutaneous tumours were fixed with paraffin. Tissues were dewaxed and hydrated using xylene and ethanol After that, the antigens were recovered. Antigen retrieval was performed at 95 C for 20 minutes. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide (H 2 O 2 ). After incubating F I G U R E 1 Upregulation of ABCC2 is related to lung cancer prognosis. Association between ABCC2 expression and OS, A, and PFS, B. OS, overall survival; PFS, progression-free survival the primary and secondary antibodies, haematoxylin was used for counterstaining. Finally, the samples were dehydrated and made transparent, and the slides were blocked using resin. The main antibody, Ki-67 (1.5 μg/mL, ab238020), was purchased from Abcam. The number of Ki-67 positive cells was counted using an Olympus inverted microscope and cells were scored in eight randomly chosen fields under a magnification of ×200 per sample.

| Statistical analysis
Data from the three independent experiments are expressed as the mean ± SD. Student's t test or one-way analysis of variance (ANOVA) was used to compare means. The relevant data were analysed using GraphPad 6.0. Differences between samples were considered significant at P < .05.

| Upregulation of ABCC2 is related to lung cancer prognosis
We analysed the relationship between ABCC2 expression level and survival rate for patients with lung cancer using an online Kaplan-Meier survival analysis tool (KMplot, http://kmplot.com/analysis/).
The results showed that higher expression levels of ABCC2 were significantly associated with poor overall survival (OS) (P < .01) in lung cancer (1925 samples) ( Figure 1A). Similarly, the increased expression of ABCC2 was significantly associated with progression-free survival (PFS) (P < .001) in lung cancer (982 samples) ( Figure 1B). These results suggest that higher levels of ABCC2 are associated with poor prognosis in lung cancer patients. respectively. The IC 50 of A549/DDP cells was more than 10-fold that of A549 cells. This demonstrated that we successfully constructed DDP-resistant A549 cells (Figure 2A,B). The RNA sequencing results indicated that the expression level of the cell membrane transporter family increased in A549/DDP cells, with the greatest increases recorded in the expression of ABCC2 (12.531 times), ABCG2 (8.386 times), and ABCA1 (7.054 times) compared with A549 cells ( Figure 2C,D). We then examined A549 and A549/DDP cells using PCR and found that the expression of ABCC2 (7.860 times) had increased significantly. Hence, the PCR results were consistent with the RNA sequencing data ( Figure 2E). In addition, western blot analysis also revealed an increase in ABCC2 protein expression levels ( Figure 2F,G). Therefore, in DDP-resistant A549 cell lines, we found that ABCC2 exhibited higher expression in terms of both mRNA and protein levels as compared with that in normal A549 cells.  3.4 | Knockdown of ABCC2 promotes A549/DDP cell apoptosis by activating the caspase-3 signalling pathway TUNEL staining showed that the apoptosis rate of A549/DDP cells in the sh1-ABCC2 group increased significantly ( Figure 4A). The apoptosis rate in sh1-ABCC2 cells was 9.5% ± 1.1%, while almost no apoptosis was observed in the other groups. PARP cleavage is considered an important indicator of apoptosis and an indicator of caspase-3 activation. A protease became cleaved caspase-3 when caspase-3 was activated. Increased expression of cleaved PARP and cleaved caspase-3 in the sh1-ABCC2 + DDP group indicated that the caspase apoptosis signalling pathway was activated ( Figure 4C,D).

| ABCC2 inhibits tumour growth in vivo
We have confirmed that DDP promotes apoptosis of A549/DDP cells transfected with sh1-ABCC2 in vitro. Therefore, we injected A549/ DDP cells into nude mice to establish a xenograft model. A549/DDP cells were transfected with sh-control and sh1-ABCC2. Nude mice were sacrificed on day 20, and the subcutaneous tumours were removed ( Figure 5A). Tumour volumes were measured from day 0 to day 20 after DDP treatment. The tumour size of the sh1-ABCC2 + DDP group was markedly smaller than that of the other groups ( Figure 5B). Tumours were weighed after 20 days of tumour formation in nude mice. Similar to the tumour size results, the sh1-ABCC2 + DDP group also had a lower tumour weight than the other groups ( Figure 5C). In addition, the expression of Ki-67 in the sh1-ABCC2 + DDP group was significantly lower than that in the other groups according to immunohistochemical staining. TUNEL staining showed that apoptosis in subcutaneous tumours in the sh1-ABCC2 + DDP group increased to a large extent ( Figure 5D). The results of these in vivo experiments demonstrate that A549/DDP cells are more sensitive to DDP after knockdown of ABCC2. Previous research has shown that ATP-binding cassette (ABC) transporters play important roles in drug chemoresistance in many cancers. 17 ABCC2, a member of the ABC family of transmembrane proteins, is also known as multidrug resistance-associated protein 2 (MRP2) or the canalicular multiple organic anion transporter (cMOAT). 7,18 ABCC2 is mainly present in the outer membrane surrounding liver cells, and within small amounts in the kidneys, intestines and placenta. [19][20][21][22] After the long-term use of platinum drugs in patients with ovarian cancer, lung cancer and colon cancer, the ABCC2 gene is often upregulated in some parts of these patients and upregulation of the ABCC2 gene usually causes patients to develop resistance to platinum drugs. 23,24 In addition, high ABCC2 expression is correlated with poor overall survival. ABCC2 has also been shown to increase DDP resistance and is controlled by let-7c in NSCLC. [25][26][27] In our study, we demonstrated that ABCC2 was upregulated in DDP-resistant cells (A549/DDP). RNA sequencing results showed that the expression levels of various proteins related to cell membrane transporters were upregulated, including ABCC2 and ABCC4, and the expression of ABCC2 showed the most significant increase.
Next, we performed PCR on A549/DDP and A549 cells, and changes in ABCC2 mRNA expression levels were consistent with the RNA sequencing data. The cell cycle results showed that normal A549 cells accumulated in the G1 phase after DDP treatment, which cau- Collectively, these results indicate that the reduced expression of ABCC2 is likely to restore DDP sensitivity in DDP-resistant NSCLC cancer cells. Our results highlight that targeting ABCC2 is considered as an approach to reverse DDP resistance in NSCLC patients.