A Human ABC Transporter ABCC4 Gene SNP (rs11568658, 559 G > T, G187W) Reduces ABCC4-Dependent Drug Resistance

Broad-spectrum drug resistance is a major obstacle in cancer treatment, which is often caused by overexpression of ABC transporters the levels of which vary between individuals due to single-nucleotide polymorphisms (SNPs) in their genes. In the present study, we focused on the human ABC transporter ABCC4 and one major non-synonymous SNP variant of the ABCC4 gene in the Japanese population (rs11568658, 559 G > T, G187W) whose allele frequency is 12.5%. Cells expressing ABCC4 (G187W) were established using the Flp-In™ system based on Flp recombinase-mediated transfection to quantitatively evaluate the impacts of this non-synonymous SNP on drug resistance profiles of the cells. Cells expressing ABCC4 (WT) or (G187W) showed comparable ABCC4 mRNA levels. 3-(4,5-Dimethyl-2-thiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay indicated that the EC50 value of the anticancer drug, SN-38, against cells expressing ABCC4 (G187W) was 1.84-fold lower than that against cells expressing ABCC4 (WT). Both azathioprine and 6-mercaptopurine showed comparable EC50 values against cells expressing ABCC4 (G187W) and those expressing ABCC4 (WT). These results indicate that the substitution of Gly at position 187 of ABCC4 to Trp resulted in reduced SN-38 resistance.


Introduction
Broad-spectrum drug resistance, which is often due to ABC transporters, is a major obstacle in cancer treatment. The level of drug resistance of cancer cells is usually related to the expression levels of ABC transporters and is complicated by single-nucleotide polymorphisms (SNPs) in their genes. In fact, various quantitative analyses of ABC transporter function (WT or SNPs) showed that SNPs in ABC transporter genes modify their expression levels, transport function, and/or intracellular distribution [1][2][3][4][5][6][7][8][9][10]. Previous quantitative studies revealed the impacts of SNPs in ABC transporter genes on drug sensitivity of the cells [7][8][9][10][11].

Preparation of Plasmids Carrying the ABCC4 (G187W) Variant cDNA
The pcDNA5/FRT expression vector carrying ABCC4 (G187W) cDNA ( Figure 2A) was prepared from pcDNA5/FRT/ABCC4 (WT) vectors that we prepared previously [10]. The nucleic acid sequence of the ABCC4 (G187W) gene was obtained from the NCBI dbSNP database. Briefly, non-synonymous SNP variants of ABCC4 were generated by site-directed mutagenesis using the PrimeSTAR ® Max DNA Polymerase (Takara Bio Inc., Otsu, Japan) with the following primers: forward, 5 -cttagtaacatggccatgtggaagacaaccacaggc-3 ; reverse, 5 -gcctgtggttgtcttccacatggccatgttactaag-3 . After polymerase chain reaction (PCR) (10 s at 98 • C, 12 cycles of 10 s at 98 • C, 15 s at 55 • C and 10 min at 72 • C), the reaction mixture was treated with DpnI endonuclease for 1 h at 37 • C to degrade the original template plasmid pcDNA5/FRT/ABCC4 (WT) vectors. The nucleic acid sequence of ABCC4 (G187W) cDNA in the resulting amplicons was confirmed by sequencing with Applied Biosystems 3130 and 3130xl genetic analyzers (Applied Biosystems, Foster City, CA, USA) as shown in Figure 2B.

Preparation of Total RNA and Synthesis of First-Strand cDNA
Cells were seeded into 35-mm dishes (TrueLine) at a density of 1 × 10 6 cells/well and pre-cultured for 3 days. The cells were then collected by pipetting with culture medium into 1.5-mL tubes followed by centrifugation at 300× g for 5 min at 4 • C. The resulting cell pellets were rinsed twice with 1 mL of PBS(−) and suspended in 600 µL of lysis buffer prepared from Lysis/binding buffer (Roche Diagnostics, Mannheim, Germany) and stored at −80 • C until use for preparation of total RNA.
Total RNA was extracted from the cell lysates using a High Pure RNA Isolation Kit (Roche Diagnostics) according to the manufacturer's instructions. The concentration and quality of total RNA in each extract were measured spectrophotometrically and evaluated by calculating the A260/A280 values, respectively (DU640; Beckman Coulter, Fullerton, CA, USA). Thereafter, the total RNA was reverse transcribed using a High-Capacity cDNA Reverse-Transcription Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA) and random hexamers as a primer according to the manufacturer's instructions.

MTT Assay
MTT assay was performed as previously described [10]. In facts, cells were seeded into 96-well plates (Thermo Fisher Scientific) at a density of 5 × 10 5 cells/well and pre-cultured for 24 h. The cells were then exposed to different concentrations of the anticancer drugs (azathioprine, 6-mercaptopurine and SN-38) for 72 h according to the results reported previously [10], where the highest final concentrations of these drugs were 100 nM (SN-38) or 100 µM (azathioprine and 6-mercaptopurine) and the lowest was 0 M (control). After 72 h of treatment with each drug, the cells were incubated in the presence of 500 µg/mL MTT for a further 3 h. Thereafter, 100 µL of 20% sodium dodecyl sulfate (SDS) was added to each well and the plates were incubated overnight at 37 • C in a humidified atmosphere of 5% (v/v) CO 2 in air. The absorbance of formazan, a metabolite of MTT, in each well of the resulting solution was measured photometrically at 570 nm and at a reference wavelength of 630 nm using a Thermo Lab systems Multiskan Jax (Thermo Fisher Scientific Inc., Waltham, MA, USA). Cell viability was expressed as a percentage relative to control. Cytotoxicity was assessed with reference to the EC 50 value, which was defined as the concentration required for 50% reduction of viability based on the survival curve.

Preparation of Cell Lysates for SDS-PAGE
Cell lysates for SDS-PAGE was prepared as previously described [10]. In facts, cells were seeded into 35-mm dishes (TrueLine, Baton Rouge, LA, USA) at a density of 1 × 10 6 cells/well and pre-cultured for 3 days. After pre-culture, the cells were collected by pipetting with culture medium into 1.5-mL tubes and centrifuged at 300× g for 5 min at 4 • C. The resulting cell pellets were rinsed twice with 1 mL of PBS(−) and suspended in lysis buffer (50 mM Tris-HCl, pH 7.6, 5 mM EDTA, pH 8.0, 120 mM NaCl, 1% Triton X-100, 1 mM DTT, protease inhibitor and phosphatase inhibitor). Thereafter, the cell lysates were centrifuged at 800× g for 10 min at 4 • C. The resulting supernatants were retained and the protein contents of the samples were determined by the Bradford method. After this assay, 50 µg of the supernatant protein was treated with PNGase F for 10 min at 37 • C.

SDS-PAGE and Western Blotting
SDS-PAGE was performed as previously described [10]. In facts, cell lysate samples were prepared independently from each group of cells in triplicate and aliquots of 5 µg were mixed. The mixtures were fractionated by SDS-polyacrylamide gel electrophoresis (PAGE) (7.5%) and the gels were transferred onto nitrocellulose membranes (GE Healthcare UK Ltd., Bucks, UK). The membranes were soaked in blocking buffer consisting of TBST (50 mM Tris-HCl, 150 mM NaCl and 0.05% (v/v) Tween 20) containing 5% (w/v) skim milk powder at room temperature for >1 h and left to stand overnight at 4 • C. The membranes were incubated with monoclonal anti-ABCC4 antibody (M4I-10; GeneTex Inc., Alton Parkway Irvine, CA, USA) or anti-GAPDH antibody (anti-GAPDH-Clone 6C5 mouse monoclonal, igG2b; American Research Products, Inc., Waltham, MA, USA) at 1:1000 dilution in TBST containing 5% (w/v) skim milk powder for 1 h at room temperature with shaking after rinsing with TBST. Thereafter, the membranes were washed with TBST, followed by incubation with appropriate secondary antibody for 1 h at room temperature with shaking. The secondary antibody for ABCC4 was rabbit anti-Rat IgG:HRP (Enzo Life Sciences, Inc., Farmingdale, NY, USA) and that for GAPDH was HRP-conjugated anti-mouse IgG antibody (Cell Signaling Technology, Inc., Danvers, MA, USA) at 1:1000 dilution in TBST containing 5% (w/v) skim milk powder. The blots were developed using Western Lighting Chemiluminescent Reagent Plus (PerkinElmer Life and Analytical Sciences, Boston, MA, USA) and detected with WSE-6100 LuminoGraph I (Atto Corp., Tokyo, Japan). The signal intensities derived from ABCC4 or GAPDH were determined using ImageJ (Wayne Rasband, Bethesda, MD, USA).

Statistical Analysis
Statistical analyses were performed using JSTAT version 20.0J (Masato Sato, Japan), which is a software for statistical One-way ANOVA and Tukey's honestly significant difference (HSD) test analysis. In all analyses, p < 0.01 was taken to indicate statistical significance.

Levels of ABCC4 mRNA and Protein in Cells Expressing ABCC4 (G187W)
We used Flp-In-293 cells corresponding to the Flp-In™ system to establish cells expressing ABCC4 (G187W) (Figure 1; Figure 2). Flp-In-293 cells were transfected with the ABCC4 (G187W) cDNA, which integrated into the FRT-tagged genomic DNA and then selected using hygromycin B. The resulting hygromycin B-resistant cells were analyzed by qPCR to determine the ABCC4 and GAPDH mRNA levels and compared with Flp-In-293/Mock and ABCC4 (WT) cells to confirm the proper functioning of the Flp-In™ system. In the present study, total RNA was prepared from Flp-In-293/Mock, ABCC4 (WT) and ABCC4 (G187W) cells and their qualities were evaluated by comparing the A260/A280 values of total RNA and the threshold cycles in qPCR among samples. Since those were comparable (data not shown), the levels of ABCC4 mRNA were subsequently normalized relative to those of GAPDH and compared. As shown in Figure 3, ABCC4 mRNA levels in cells transfected with ABCC4 cDNA were >23-fold higher than those in Flp-In-293/Mock cells, as we reported previously [10]. In contrast, the levels of ABCC4 mRNA were comparable between Flp-In-293/ABCC4 (WT) and (G187W) cells, indicating that the Flp-In™ system was functioning in the cells established in the present study. Since those were comparable (data not shown), the levels of ABCC4 mRNA were subsequently normalized relative to those of GAPDH and compared. As shown in Figure 3, ABCC4 mRNA levels in cells transfected with ABCC4 cDNA were >23-fold higher than those in Flp-In-293/Mock cells, as we reported previously [10]. In contrast, the levels of ABCC4 mRNA were comparable between Flp-In-293/ABCC4 (WT) and (G187W) cells, indicating that the Flp-In™ system was functioning in the cells established in the present study.    Since those were comparable (data not shown), the levels of ABCC4 mRNA were subsequently normalized relative to those of GAPDH and compared. As shown in Figure 3, ABCC4 mRNA levels in cells transfected with ABCC4 cDNA were >23-fold higher than those in Flp-In-293/Mock cells, as we reported previously [10]. In contrast, the levels of ABCC4 mRNA were comparable between Flp-In-293/ABCC4 (WT) and (G187W) cells, indicating that the Flp-In™ system was functioning in the cells established in the present study.   Since those were comparable (data not shown), the levels of ABCC4 mRNA were subsequently normalized relative to those of GAPDH and compared. As shown in Figure 3, ABCC4 mRNA levels in cells transfected with ABCC4 cDNA were >23-fold higher than those in Flp-In-293/Mock cells, as we reported previously [10]. In contrast, the levels of ABCC4 mRNA were comparable between Flp-In-293/ABCC4 (WT) and (G187W) cells, indicating that the Flp-In™ system was functioning in the cells established in the present study.    As qPCR clearly showed that the Flp-In™ system functioned in Flp-In-293/ABCC4 (G187W) cells, western blotting analysis was performed to evaluate ABCC4 and GAPDH expression in the cells after treatment with PNGase F to remove the glycomoieties on ABCC4. As shown in Figure 4, the level of ABCC4 expression corresponded to that of ABCC4 mRNA and the levels of ABCC4 in Flp-In-293/ABCC4 (WT) and (G187W) cells were much higher than that in Flp-In-293/Mock cells. In contrast, the levels of ABCC4 in cells expressing ABCC4 (G187W) were comparable to those in cells expressing ABCC4 (WT). As qPCR clearly showed that the Flp-In™ system functioned in Flp-In-293/ABCC4 (G187W) cells, western blotting analysis was performed to evaluate ABCC4 and GAPDH expression in the cells after treatment with PNGase F to remove the glycomoieties on ABCC4. As shown in Figure 4, the level of ABCC4 expression corresponded to that of ABCC4 mRNA and the levels of ABCC4 in Flp-In-293/ABCC4 (WT) and (G187W) cells were much higher than that in Flp-In-293/Mock cells. In contrast, the levels of ABCC4 in cells expressing ABCC4 (G187W) were comparable to those in cells expressing ABCC4 (WT).

Establishment of Human ABCC4 (G187W)-Expressing Cells Using the Flp-In™ System
The drug resistance of cancer cells and the individual differences in their levels are usually due to the overexpression of ABC transporters and SNPs in their genes, respectively [8]. We found that the drug sensitivities of cells expressing ABCC4 were modified by the first (rs2274407 (K304N)) and second (rs3765534 (E757K)) most common SNPs in the ABCC4 gene present in the Japanese population (Table 1) [10]. In the present study, we established cells expressing the third most common ABCC4 gene SNP in Japanese population (rs11568658 (G187W)) ( Table 1) based on the Flp-In™ system, which can be sued to integrate a single copy of a cDNA into the FRT site prepared in the telomeric region of the short arm on one copy of chromosome 12 in Flp-In-293 cells [7] and quantitatively evaluated the impacts of this SNP on drug resistance of the cells. Although the integration site and the ABCC4 cDNA copy number were not determined in the present study, the levels of ABCC4 mRNA expression were comparable between Flp-In-293/ABCC4 (WT) and (G187W) cells ( Figure 3). These results suggested that the transfected ABCC4 cDNA was integrated into the designated site in the chromosome of the cells and that the integrated ABCC4 cDNA copy number was the same in Flp-In-293/ABCC4 (WT) and (G187W) cells.

Establishment of Human ABCC4 (G187W)-Expressing Cells Using the Flp-In™ System
The drug resistance of cancer cells and the individual differences in their levels are usually due to the overexpression of ABC transporters and SNPs in their genes, respectively [8]. We found that the drug sensitivities of cells expressing ABCC4 were modified by the first (rs2274407 (K304N)) and second (rs3765534 (E757K)) most common SNPs in the ABCC4 gene present in the Japanese population (Table 1) [10]. In the present study, we established cells expressing the third most common ABCC4 gene SNP in Japanese population (rs11568658 (G187W)) ( Table 1) based on the Flp-In™ system, which can be sued to integrate a single copy of a cDNA into the FRT site prepared in the telomeric region of the short arm on one copy of chromosome 12 in Flp-In-293 cells [7] and quantitatively evaluated the impacts of this SNP on drug resistance of the cells. Although the integration site and the ABCC4 cDNA copy number were not determined in the present study, the levels of ABCC4 mRNA expression were comparable between Flp-In-293/ABCC4 (WT) and (G187W) cells (Figure 3). These results suggested that the transfected ABCC4 cDNA was integrated into the designated site in the chromosome of the cells and that the integrated ABCC4 cDNA copy number was the same in Flp-In-293/ABCC4 (WT) and (G187W) cells.
The drug resistance profiles of cells expressing ABCC4 (G187W) have not been examined previously, although Banerjee et al. (2016) established cells expressing ABCC4 (G187W). They prepared cells expressing ABCC4 (G187W) using a traditional transfection system with pcDNA3.1 vector, where the copy number and integration site of the cDNA into the chromosome were not controlled and the resulting cells were not suitable to examine the effects of ABCC4 gene SNPs on ABCC4-mediated drug resistance in cells [20]. On the other hand, we established cells expressing the wild-type (WT) or SNP variants of human ABCC4 with controlled copy number and site of cDNA integration into the chromosome and showed that the drug resistance profiles of cells expressing the SNP variants of ABCC4 were significantly different from those of cells expressing ABCC4 (WT) [10]. The drug resistance profiles of cells were reported to be affected by the levels of ABC transporter expression [29,30]. Our previous studies showed that non-synonymous SNPs in the ABCG2 gene can affect drug resistance of cells by altering their substrate specificity, intracellular localization, or intracellular stability under conditions where the mRNA levels are comparable [4,7,9]. Banerjee et al. reported that ABCC4 (G187W) altered the affinity of ABCC4 for MMA(GS)2 in a protein-based assay but did not affect intracellular localization [20]. In the present study, the expression levels of ABCC4 (G187W) in Flp-In-293/ABCC4 (G187W) cells were comparable to those in Flp-In-293/ABCC4 (WT) cells. Thus, ABCC4 (G187W) would increase intracellular accumulation levels of test drugs by altering the substrate specificity of ABCC4. It is important to measure the levels of intracellular accumulation of test drugs to understand the molecular mechanisms by which substitution of Gly187 to Trp in ABCC4 alters the drug resistance profiles of cancer cells. Such studies are currently in progress in our laboratory.
In summary, we quantitatively evaluated the impact of rs11568658 (G187W) on the drug resistance profiles of cells using our previously proposed easy-to-use and quantitative approach [10]. The results presented here will improve our understanding of the effects of non-synonymous SNPs in the ABCC4 gene on cancer chemotherapy and could contribute to the development of novel therapeutic strategies as well as diagnostic and therapeutic approaches for cancer chemotherapy.

Conflicts of Interest:
The authors declare no conflict of interest.