Special AT-rich sequence binding protein 1 promotes multidrug resistance in gastric cancer by regulation of Ezrin to alter subcellular localization of ATP-binding cassette transporters

Abstract

Multidrug resistance is a primary factor in the poor response to chemotherapy and subsequent death in gastric cancer patients. However, the molecular mechanisms involved remain unclear. In this study, the high expression of special AT-rich sequence binding protein 1 (SATB1) in gastric cancer was found to be associated with reduced sensitivity to various chemotherapy drugs. Our results demonstrate that SATB1 can promote chemotherapy resistance in gastric cancer in vitro and in vivo. SATB1 exerts its effect by enhancing the activity of multiple ATP-binding cassette (ABC) transporters (P-glycoprotein, multidrug resistance-associated protein, and breast cancer resistance protein) in gastric cancer cell lines. We also found that SATB1 affects ABC transporters by altering the subcellular localization of the ABC transporter rather than its expression. Subsequently, we confirmed that Ezrin binds to various ABC transporters and affects their subcellular localization. In addition, we found that SATB1 can also bind to the Ezrin promoter and regulate its expression. In the present study, we elucidate the mechanism of SATB1-mediated multidrug resistance in gastric cancer, providing a basis for SATB1 as a potential target for reversal of resistance.

Abbreviations
• ABC
• ATP-binding cassette
• DDP
• cisplatin.
• ERM
• ezrin–radixin–moesin
• MDR
• Multidrug resistance
• P-gp
• P-glycoprotein
• SATB1
• Special AT-rich sequence binding protein 1
• VCR
• vincristine

INTRODUCTION

Gastric cancer is one of the main causes of cancer morbidity and mortality worldwide, with a particularly high disease incidence rate in East Asia.¹ Chemotherapy for advanced patients undergoing surgical resection can help prevent cancer metastasis and recurrence.² Chemotherapy is the most important treatment for patients who cannot undergo surgical resection or who have advanced metastasis.³ However, due to inherent or acquired drug resistance, a poor or nonresponse to chemotherapy is often observed, which becomes the most common cause of treatment failure for gastric cancer.⁴ Multidrug resistance (MDR) is an important mechanism of drug resistance in cancer, which directly affects the chemotherapy response.⁵ P-glycoprotein (P-gp) is a key factor in the process of MDR,⁶ but the underlying mechanism of MDR has yet to be confirmed. The exploration of how MDR manifests in gastric cancer may lead to new treatment strategies to overcome MDR and improve the prognosis of patients.

Special AT-rich sequence binding protein 1 (SATB1) is a higher-order chromatin organizer that can bind to specialized AT-rich genomic regions to regulate chromatin.⁷ SATB1 acts as a docking site for many chromatin modifiers and nucleosome remodelers to regulate target gene expression.^8,9 SATB1 has been studied in various cancers and is believed to play an important role in tumor development.^10–13 Notably, SATB1 has been reported in many studies to contribute to MDR.^14–16 Our previous studies have also confirmed that SATB1 is involved in promoting MDR in gastric cancer,¹⁷ but its specific mechanism for regulating MDR in gastric cancer remains to be explored.

ATP-binding cassette (ABC) transporter refers to a type of membrane protein that transports substrates across cell membranes in an ATP-dependent manner.¹⁸ The ABC transporters act as a pump to discharge many substrates, including chemotherapy drugs across membranes, reducing drug accumulation in the cancer cells, thus contributing to MDR.¹⁹ P-gp was the first member of a family of ABC transporters to be identified, and many articles have reported its important role in MDR.²⁰ In addition to P-gp, ABCC1/MRP1 and ABCG2/BCRP—members of ABC transporters—have been extensively studied for their prominent role in MDR.^21–23 ABC transporters need to translocate to the cell membrane to perform their normal functions,²⁴ and studies have reported that ezrin–radixin–moesin (ERM) proteins may have a binding affinity for them.²⁵ The ERM family of proteins act as cytoskeleton linkers, which can connect many proteins with the cytoskeleton to regulate their intracellular localization.²⁶ The function of ABC transporters depends not only on their expression but also their subcellular location.²⁷ Ezrin is a member of the ERM family, which can promote tumor progression.²⁸ In addition, studies have reported that it can promote MDR of human osteosarcoma by connecting P-gp with the cytoskeleton.²⁹ Since the role of Ezrin in gastric cancer is unclear, we sought to determine whether Ezrin also binds P-gp or other ABC transporters in gastric cancer cells to regulate their subcellular localization.

In this study, we have revealed the relationship between SATB1, Ezrin, and ABC transporters (P-gp, MRP1, and BCRP) in MDR of gastric cancer and determined that SATB1 can affect the subcellular localization of multiple ABC transporters by regulating Ezrin expression to promote MDR in gastric cancer.

MATERIALS AND METHODS Chemotherapeutic response prediction

For the Cancer Genome Atlas (TCGA) database, we downloaded the tumor RNA-seq (FPKM) of gastric cancer from the Genomic Data Commons (GDC). FPKM data was converted to TPM, and the data normalized to log2 (TPM + 1) while keeping samples with associated clinical information. We predicted the chemotherapeutic response for each sample based on the largest publicly available pharmacogenomics database (the Genomics of Drug Sensitivity in Cancer [GDSC], https://www.cancerrxgene.org/). The prediction process was implemented using the R package “pRRophetic,” where the samples’ half-maximal inhibitory concentration (IC50) was estimated by ridge regression and the prediction accuracy. All parameters were set by the default values with the removal of the batch effect of “combat” and tissue type of “allSoldTumours”. Duplicate gene expression was summarized as the mean value. All the above analysis methods and R package were implemented using the R foundation for statistical computing (2020) version 4.0.3.

Cell culture

The human gastric adenocarcinoma cell line SGC-7901 and its corresponding vincristine (VCR)-resistant variant SGC7901/VCR and cisplatin (DDP)-resistant variant SGC7901/DDP, AGS and its corresponding vincristine (VCR)-resistant variant AGS/VCR and cisplatin (DDP)-resistant variant AGS/DDP (obtained from the Xiangya Center Laboratory, Changsha, Hunan, China) were cultured in DMEM medium supplemented with 10% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin in a 37°C incubator with 100% humidity and 5% CO₂.

Chemicals and reagents

We obtained 5(6)-Carboxy-2′,7′-dichlorofluorescein (CDF, a precursor of MRP substrate), MK571 sodium salt hydrate (an MRP inhibitor), Rhodamine 123 (R123, a P-gp substrate), verapamil (a P-gp inhibitor), Hoechst 33342 hydrochloride (H33342, a BCRP substrate), and Ko-143 (a BCRP inhibitor) from Sigma-Aldrich.

Plasmid construction and transfection

The coding region of SATB1 and Ezrin was subcloned into the mammalian expression vector pEX-M29 (GeneCopoeia) to generate the plasmid pEX-SATB1 and pEX-Ezrin. The recombinant construction was verified by DNA sequencing. pEX-SATB1 and pEX-Ezrin were prepared using a Fastfilter Endo-Free Plasmid Midi Kit (OMEGA) according to the manufacturer's specification and transfected into SGC7901 cells using lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. The cells were harvested 36 or 48 h later for the following experiments.

RNA interference

Three SATB1 siRNA oligonucleotides, three Ezrin siRNA oligonucleotides, and one negative control sequence were designed based on the SATB1 and Ezrin sequences and synthesized by Guangzhou RIBOBIO (Guangzhou, China). SGC7901 cells were seeded at 2 × 10⁵/well in six-well plates and cultured in serum-free medium. The next day when the cells grew to 70% confluence, 50 nM siRNA was transfected into the cells using lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Six hours later, the cells were cultured in DMEM medium supplemented with 10% FBS. The cells were harvested at indicated time points for the following experiments.

Quantitative RT-PCR

Total RNA was isolated from cultured cells using TRIzol reagent (TAKARA) according to the manufacturer's protocol. The first strand cDNA was synthesized using RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific), and SYBR Green PCR Master Mix (Monad, China) was used to perform fluorescent quantitative PCR. Finally, relative mRNA expression levels were analyzed by qRT-PCR using LightCycler 480 (Roche, Switzerland). The primers were as follows: SATB1: forward 5′-GTGGGTACGCGATCAACTGA-3′ and reverse 5′-TGTTAAAGCCAGTGCAA-3′. Ezrin: forward 5′-CGCTCTAAGGTTCTGCTCT-3′ and reverse 5′-TCCTGGGCA GACACCTTCTTA-3′. ABCC1: forward 5′-ATGTCACGTGGAATACCAGC-3′ and reverse 5′-GAAGACTGAACTCCCTTCCT-3′. ABCB1: forward 5′- CCCATCATTGCAATAGCAGG-3′ and reverse 5′- TGTTCAAACTTCTGCTCCTGA-3′. ABCG2: forward 5′-AGATGGGTTTCCAAGCGTTCAT-3′ and reverse 5′-CCAGTCCCAGTACGACTGTGACA-3′. GAPDH (internal control): forward 5′-TTGGTATCGTGGAAGGACTCA −3′ and reverse 5′-TGTCATCATATTTGGCAGG TT-3′.

Western blotting

Cells were lysed with RIPA lysis buffer (Santa Cruz Biotechnology), and the protein concentration of the cell lysate was quantitated using the BCA method. An equal amount of protein was loaded and separated by 10% SDS-PAGE, then transferred to nitrocellulose membranes (Millipore). The membranes were blocked in 5% non-fat milk for 2 h and then incubated with SATB1 antibody (1:1000 dilution, Epitomics), Ezrin antibody (1:1000 dilution, Cell Signaling Technology), MRP1 antibody (1:1000 dilution, Cell Signaling Technology, USA), P-gp antibody (1:1000 dilution, Cell Signaling Technology), BCRP antibody (1:1000 dilution, Cell Signaling Technology), and β-actin antibody (1:2000 dilution, Sigma) at 4°C overnight. After washing three times with TBST, the membranes were incubated with HRP-conjugated secondary antibodies (Boster) for 4 h at room temperature. The membranes were developed using an ECL kit (Santa Cruz Biotechnology) and exposed to X-ray film. β-actin was used as a loading control.

In vitro drug sensitivity assay

The sensitivity of cells to VCR/DDP was evaluated using MTT assay as described previously.³⁰ Briefly, cells in logarithmic phase were seeded in 96-well plates at a density of 10⁴ cells/well. The next day, the cells were cultured in serum-containing medium supplemented with different concentrations of VCR/DDP. Seventy-two hours later, 50 μL MTT (Sigma) was added to each well. After incubation for 4 h, the supernatant was replaced with 150 μL dimethyl sulfoxide (Sigma). The optical density was read at 490 nm on a spectrophotometer (MPR-2100, Syntron). The relative inhibitory rate of cell growth by different concentrations of VCR/DDP was calculated according to the formula R = (V1–V2)/V2, where R stands for the relative inhibitory rate, V1 is the absorbance value of control cells without VCR treatment, and V2 is the absorbance value in the presence of VCR/DDP.

Xenograft tumor assay

All experiment procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Renmin Hospital of Wuhan University. An in vivo chemosensitivity experiment was performed using the subcutaneous transplantation of cells into BALB/c nude mice. BALB/c nude mice (n = 10; 5–6 weeks old) were divided into two groups (five mice/group) and injected subcutaneously into the left flank with either SGC7901-control or SGC7901-siSATB1 (10⁷ cells in 100 μL PBS). From 10 days post-injection, VCR (0.5 mg/kg) was intraperitoneally administered every 3 days for 18 days. The mice were then killed, and tumor weights were measured. The tumor volume (V) was calculated using the formula V = (4π/3) × (D/2)³, where D is the diameter of the tumor.

Transporter activity assays

Transporter activities were assayed according to previously described methods.³¹ Briefly, CDF (10 μM), R123 (50 μM), and H33342 (10 μM), with or without their respective inhibitors (50 μM MK571, 50 μM verapamil, and 10 μM Ko-143), were added 30 min before the SGC7901 cells were treated with siRNA for 48 h. After a 1 h incubation for CDF and H33342 or a 2 h-incubation for R123, the cells were washed twice with PBS. The cells’ fluorescence intensity was then measured using a Multimode Plate Reader (EnSight, PerkinElmer) at Ex/Em wavelengths of 495/530 nm for CDF, 480/530 nm for R123, and 355/460 nm for H33342.

Confocal immunofluorescence

Confocal immunofluorescence microscopy was performed on SGC7901 cells. Briefly, cells were fixed and incubated with rabbit monoclonal anti-MRP1/P-gp/BCRP antibody at 4°C overnight and then incubated with secondary antibodies (Cell Signaling Technology) for 1 h. Finally, the cells were incubated with DAPI (Sigma-Aldrich) for 5 min and viewed with a Fluoview FV1200 microscope (Olympus).

CoImmunoprecipitation

Cells were harvested and lysed for 30 min at 4°C in 500 μL IP lysis buffer containing protease inhibitor. Supernatant was collected by centrifugation and pre-cleared by adding 20 μL protein G agarose beads (Thermo Fisher Scientific) at 4°C for 30 min. Then primary antibody was added and incubated overnight at 4°C with rotation. Samples were then incubated at 4°C with rotation for 4 h with 20 μL protein G agarose beads washed with TBS the next day. Protein G agarose beads were collected after centrifuging at 3000 g for 1 min, mixed with 100 μL IP lysis buffer and analyzed by western blot, as described above. Input panels were used as the negative control.

ChIP Assay

For SATB1 binding sites analysis, the JASPAR tool (http://jaspar.genereg.net/) was used. Promoter sequences (−2000/+100) of Ezrin were collected in The Eukaryotic Promoter Database (https://epd.epfl.ch/). The three high-score binding sites of SATB1 on the Ezrin promoter were selected. The ChIP kit was purchased from Abcam. Briefly, SGC7901 (6.0 × 10⁶) cells were fixed with 1% of formaldehyde. Genomic DNA was sheared to lengths ranging from 200 to 1000 bp with a Sonic Dismembrator (Thermo Fisher Scientific): Ampl 80%, 3 s on, 10 s off, for 10 cycles. One percent of the cell extract was taken as “input,” and the rest of the extract was incubated with anti-SATB1 or control IgG overnight at 4°C, followed by precipitation with protein A agarose beads. The immunoprecipitates were sequentially washed with a low salt buffer, a high salt buffer, a LiCl buffer, and TE buffer. The DNA-protein complex was eluted and proteins were then digested with proteinase K. The DNA was detected by qRT-PCR analysis. qRT-PCR was performed using three different sets of primers: P1: forward 5′- ACCATTCTCCTGCCTCAGCTTC-3′ and reverse 5′-GAGGCAGGTGGAACACCTGAG-3′. P2: forward 5′-TCGGCCTCCCAAAGTGCTGG-3′ and reverse 5′-TGCTCACCTCGGCCTCCCAAAG-3′. P3: forward 5′-CGAGGTGAGCAGATCACCTGAGGT-3′ and reverse 5′- TCCTGCCTCAGCCTCTTGAGT-3′.

Dual-luciferase reporter assay

The predicted binding site fragments between the Ezrin promoter and SATB1 and mutant fragments were cloned into the luciferase reporter vector as reporter plasmids Ezrin promoter-WT and Ezrin promoter-MUT. The reporter plasmids were then co-transfected with oe-NC and oe-SATB1 into 293 T cells. After 24 h, the cells were lysed and centrifuged at 12,000 g for 1 min, with the supernatant harvested. The luciferase activity was determined using the Dual-Luciferase Reporter Assay System (Promega). The relative luciferase activity was calculated as the ratio of relative luciferase activity of Firefly luciferase to that of Renilla luciferase.

Statistical analysis

All experimental data are presented as mean ± SEM of at least three independent replicates. Statistical analyses were carried out in GraphPad Prism v 8.0. Pairwise comparisons were performed using Student's t-test. p < 0.05 was considered statistically significant.

RESULTS Special AT-rich sequence binding protein 1 promotes multidrug resistance of gastric cancer in vivo and in vitro

To determine the association between SATB1 and MDR, the GDSC database and The Cancer Genome Atlas (TCGA) database were exploited to analyze the relationship between the drug sensitivity in gastric cancer and the expression of SATB1. We analyzed six chemotherapy drugs commonly used in gastric cancer treatment to predict the IC50 of each sample (cisplatin, 5-fluorouracil, paclitaxel, vinblastine, doxorubicin, and gemcitabine). The results revealed that the IC50 values for all drugs were elevated in the SATB1 high expression group (Figure 1). These results confirmed that SATB1 was related to MDR to chemotherapy for gastric cancer. We then compared SATB1 expression in SGC7901/VCR cells, SGC7901/DDP cells, and parent SGC7901 cells. We used qRT-PCR and western blot analysis to assess the expression level of SATB1. Our results showed that SATB1 expression was higher in SGC7901/VCR cells and SGC7901/DDP cells than in SGC7901 cells at both the mRNA and protein levels (Figure 2A, B). Then SATB1 was overexpressed in SGC7901 cells and knocked down in SGC7901/VCR and SGC7901/DDP cells; qRT-PCR verified the overexpression or knockdown of SATB1 (Figure 2C). MTT assay was used to examine the sensitivity of these cell lines to VCR or DDP. Compared with SGC7901-control cells, SGC7901-oeSATB1 cells exhibited a significantly increased IC50 value for VCR and DDP. Conversely, the IC50 value was reduced in SATB1 knockdown cells compared to parental drug-resistant cells (Figure 2D–G). We then validated it in another gastric cancer cell line, AGS, and found similar results (Figure 2H–N). Moreover, in vivo experiments using SATB1 knockdown in gastric cancer cells were performed in nude mice, demonstrating that SATB1 knockdown significantly reduced tumor volume and weight compared with controls (Figure 2O–Q). Overall, these results suggested that upregulation of SATB1 was correlated with the MDR of gastric cancer.

FIGURE 1. The effect of special AT-rich sequence binding protein 1 (SATB1) expression on gastric cancer chemotherapy response. The box plots of the estimated IC50 of SATB1 at high and low expression in gastric cancer are shown in (A) for cisplatin, (B) for 5-fluorouracil, (C) for paclitaxel, (D) for vinblastine, (E) for doxorubicin, and (F) for gemcitabine.

FIGURE 2. Special AT-rich sequence binding protein 1 (SATB1) affects gastric cancer multidrug resistance (MDR) in vitro and in vivo. (A) mRNA levels of SATB1 were compared in SGC7901, SGC7901/ vincristine (VCR), and SGC7901/DDP by quantitative RT-PCR (qRT-PCR). (B) Protein expression of SATB1 was compared in SGC7901, SGC7901/VCR, and SGC7901/DDP by western blot. (C) The mRNA level of SATB1 was detected after overexpression or knockdown of SATB1 by qRT-PCR in SGC7901. (H) mRNA levels of SATB1 were compared in AGS, AGS/VCR, and AGS/DDP by qRT-PCR. (I) Protein expression of SATB1 was compared in SGC7901, AGS, AGS/VCR, and AGS/DDP by western blot. (H) The mRNA level of SATB1 was detected after overexpression or knockdown of SATB1 by qRT-PCR in AGS. (D, F, K, M) MTT assay revealed the inhibition of cell proliferation by VCR and DDP in different SGC7901/AGS-derived cell lines. The relative inhibition rate was calculated using the formula described in the Materials and Methods. IC50 values of VCR (E, L) and DDP (F, M) were calculated based on the MTT assay. The data are shown as the mean ± SEM from three independent experiments. The image (O), tumor volume (P), and tumor weight (Q) of subcutaneous tumors. *p [less than] 0.05, **p [less than] 0.01, ***p [less than] 0.001, ****p [less than] 0.0001.

Special AT-rich sequence binding protein 1 enhances the function of multiple ATP-binding cassette transporters in gastric cancer cells

To clarify the effects of SATB1 on the transport activity of ABC transporters (MRP, P-gp, and BCRP), we used three ABC transporter-specific inhibitors while overexpressing SATB1 in SGC7901 cells, and the IC50 of the two chemotherapy drugs was examined. All three inhibitors partially reversed the elevated IC50 caused by SATB1 overexpression (Figure 3A, B). We knocked down SATB1 and overexpressed three ABC transporters in SGC7901 cells, and then examined the IC50 of the two chemotherapy drugs. Overexpression of all three transporters partially reversed the elevated IC50 caused by SATB1 knockdown, except that BCRP failed to reverse VCR (Figure 3C, D). We then determined the intracellular fluorescence intensity of transporter substrates in SGC7901 cells. Three ABC transporter-specific inhibitors were used as positive controls. SATB1 knockdown significantly increased intracellular accumulation of CDF, Rho123, and H33342 (Figure 3E–G). These results indicated that SATB1 could enhance the activity of multiple ABC transporters.

FIGURE 3. Special AT-rich sequence binding protein 1 (SATB1) regulates the function of multiple ATP-binding cassette (ABC) transporters. IC50 values of vincristine (VCR) (A) and DDP (B) after SATB1 overexpression and treatment with three inhibitors in SGC7901. #p [less than] 0.05 vs control, *p [less than] 0.05, **p [less than] 0.01, ***p [less than] 0.001, vs oeSATB1. SATB1 regulates the function of multiple ABC transporters. IC50 values of VCR (C) and DDP (D) after SATB1 knockdown and overexpression of MRP1/P-gp/BCRP in SGC7901. #p [less than] 0.05 vs control, *p [less than] 0.05, **p [less than] 0.01, ***p [less than] 0.001, vs siSATB1. Accumulation of 5(6)-Carboxy-2′,7′-dichlorofluorescein (CDF) (E), R123 (F), and H33342 (G) (MRP1, P-gp, and BCRP substrates, respectively) in SGC7901-siSATB1 cells. *p [less than] 0.05, **p [less than] 0.01, ***p [less than] 0.001. The data are shown as the mean ± SEM from three independent experiments.

Special AT-rich sequence binding protein 1 enhances plasma membrane localization of multiple ATP-binding cassette transporters in gastric cancer cells

To further elucidate the underlying mechanism by which SATB1 regulated ABC transporters, we first measured the expression of three ABC transporters in SGC7901 cells with SATB1 knockdown or overexpression. The results showed that the expression of SATB1 did not affect the expression level of ABC transporters (Figure 4A, B). Since the function of an ABC transporter depends not only on its expression but also on its localization, we sought to explore whether SATB1 is associated with ABC transporter localization. Confocal laser scanning microscopy was used to detect the subcellular localization of ABC transporters. We observed that the three ABC transporters were more localized to the plasma membrane in SGC7901-control cells than in SGC7901-siSATB1 cells (Figure 4C–E). Based on the above results, we determined that SATB1 affected the activities of multiple ABC transporters by altering their subcellular localization rather than their expression.

FIGURE 4. Special AT-rich sequence binding protein 1 (SATB1) affects the subcellular localization of multiple ATP-binding cassette (ABC) transporters. (A) The mRNA level of three ABC transporters was detected after overexpression or knockdown of SATB1 by quantitative RT-PCR (qRT-PCR). (B) Protein expressions of three ABC transporters were compared after overexpression or knockdown of SATB1 by western blot. Cellular localization of MRP1 (C), P-gp (D), or BCRP (E) was analyzed by confocal microscopy. The data are shown as the mean ± SEM from three independent experiments. No comparison between groups was statistically significant.

Ezrin can bind to multiple ATP-binding cassette transporters to maintain their plasma membrane localization in gastric cancer cells

To clarify the role of Ezrin in drug resistance, we knocked down Ezrin in SGC7901 cells and in drug resistance SGC7901 cells induced by overexpression of SATB1. The results show that knockdown Ezrin in SGC7901 cells can increase the sensitivity of VCR and DDP, while knockdown Ezrin can reverse VCR and DDP resistance in SGC7901 cells overexpressing SATB1-induced VCR and DDP resistance (Figure 5A–D). Ezrin has been reported to be involved in the intracellular localization of ABC transporters. To illustrate the connection between Ezrin and ABC transporters in gastric cancer, we first discovered using CoImmunoprecipitation (CoIP) assays that Ezrin could bind to three ABC transporters (Figure 5E). Either knockdown or overexpression of Ezrin in SGC7901 cells did not alter the expression of three ABC transporters (Figure 5F–H). Subsequently, the relationship between Ezrin and ABC transporter localization was also explored. Knockdown of Ezrin led to reduced plasma membrane localization of the three ABC transporters. Moreover, overexpression of Ezrin in SGC7901-siSATB1 cells restored the plasma membrane localization of the three ABC transporters (Figure 5I–K). These results clarified that Ezrin could bind to multiple ABC transporters in gastric cancer cells to regulate their intracellular localization, and the regulation of SATB1 was mediated through Ezrin.

FIGURE 5. MTT assay revealed the inhibition of cell proliferation by vincristine (VCR) (A) and DDP (C) in different SGC7901 cell lines. The relative inhibition rate was calculated using the formula described in the Materials and Methods. IC50 values of VCR (B) and DDP (D) were calculated based on the MTT assay. Ezrin regulates subcellular localization of multiple ABC transporters by binding to them. (E) CoImmunoprecipitation (CoIP) validation of protein interactions between Ezrin and three ABC transporters. (F) The mRNA level of Ezrin was detected after overexpression or knockdown of Ezrin by quantitative RT-PCR (qRT-PCR). (G) The mRNA level of three ABC transporters was detected after overexpression or knockdown of Ezrin by qRT-PCR. (H) Protein expressions of three ABC transporters were compared after overexpression or knockdown of Ezrin by western blot. Cellular localization of MRP1 (I), P-gp (J), or BCRP (K) was analyzed by confocal microscopy. The data are shown as the mean ± SEM from three independent experiments.

Special AT-rich sequence binding protein 1 upregulates the expression of Ezrin in gastric cancer cells

To evaluate the potential relationship between Ezrin and SATB1 expression in gastric cancer cells, we first examined the expression of Ezrin in two drug-resistant gastric cancer cell lines, which were also upregulated in line with SATB1 (Figure 6A, B). We found that the expression of Ezrin was downregulated when SATB1 was knocked down in SGC7901 cells (Figure 6C, D). We then predicted three high-score binding sites for SATB1 on the Ezrin promoter through the JASPAR database (Figure 6E). ChIP assay was applied to determine whether SATB1 was bound to these sequences directly. The results showed that the sequences at the predicted binding site 2 were amplified to a greater extent following immunoprecipitation with an anti-SATB1 antibody than with the non-specific IgG control (Figure 6F). Furthermore, the dual luciferase reporter assay showed that knockdown of SATB1 significantly reduced the luciferase activity in cells transfected with reporter plasmid containing Ezrin promoter-wild type (WT). However, knockdown of SATB1 failed to reduce the luciferase activity in cells transfected with reporter plasmids containing Ezrin promoter mutant type (MUT), which predicted binding site 2 was mutated (Figure 6G). These data suggest that SATB1 could bind directly to the predicted binding site 2 of the Ezrin promoter and regulate its transcription.

FIGURE 6. Special AT-rich sequence binding protein 1 (SATB1) binds to the Ezrin promoter to regulate its expression. (A) mRNA levels of Ezrin were compared in SGC7901, SGC7901/vincristine (VCR), and SGC7901/DDP by quantitative RT-PCR (qRT-PCR). (B) Protein expressions of Ezrin were compared in SGC7901, SGC7901/VCR, and SGC7901/DDP by western blot. (C) mRNA levels of Ezrin were detected after knockdown of SATB1 by qRT-PCR. (D) Protein expression of Ezrin was detected after knockdown of SATB1 by western blot. (E) Schematic diagram on three high-score transcription regulatory binding sites and ChIP-PCR (F) to verify them. (G) The interaction between the SATB1 and Ezrin promoter region was detected by dual reporter gene assay. The data are shown as the mean ± SEM from three independent experiments.

DISCUSSION

Our studies have identified SATB1 as a crucial protein that can inhibit the function of ABC transporters from promoting MDR of gastric cancer, which is achieved by regulating Ezrin and subsequent modulation of the subcellular localization of ABC transporters.

Special AT-rich sequence binding protein 1 (SATB1), a matrix attachment region (MAR)-binding protein, can act as a global regulator of cell function. SATB1 was originally found to have a prominent role in breast cancer, which serves to reprogram chromatin organization and alter the transcriptional profiles of breast tumors.³² Subsequently, more and more studies have reported that SATB1 plays an important role in various tumors.^33,34 Some studies have reported that SATB1 promotes the proliferation and metastasis of gastric cancer and is an independent prognostic factor for gastric cancer.^35–37 The MDR-promoting effect of SATB1 on gastric cancer has been confirmed by our previous research.¹⁷ In our study, we determined the role of SATB1 in promoting MDR in gastric cancer by predicting the chemotherapeutic efficacy of multiple chemotherapeutic drugs in TCGA samples. It is expected that SATB1 may become a clinically relevant biomarker for predicting the efficacy of chemotherapy for the treatment of gastric cancer. In the follow-up, we will verify the predictive effect of SATB1 on the efficacy of chemotherapy using clinical samples from our own center.

Some studies have reported the important role of SATB1 in tumor chemotherapy resistance, but the specific underlying mechanism has not yet been elucidated. Li et al reported that SATB1 was able to upregulate P-gp associated and suppress drug-induced apoptosis in breast cancer. They also found that the expression of SATB1 affects P-gp-non-related drugs, and our study verified this with VCR (P-gp-related drug) and DDP (P-gp-non-related drug) chemotherapy-resistant cell lines. It has also been reported that the mRNA levels of ABCC1 and ABCG2 were significantly reduced after silencing SATB1 in osteosarcoma and reversed chemotherapy resistance.¹⁶ We speculate that SATB1 exerts its function through multiple ABC transporters in gastric cancer. In the following experiments, we determined that SATB1 can simultaneously regulate the function of multiple ABC transporters through specific substrates and inhibitors of the three ABC transporters (P-gp, MRP1, and BCRP) in gastric cancer. However, we did not find changes in the expression of the three ABC transporters after overexpression or knockdown of SATB1 in SGC7901 cells. This may be due to tissue and cell specificity. The function of SATB1 also depends on its phosphorylation, acetylation, and other epigenetic regulation states.³⁸ The function of SATB1 in different cells may be different, chromatin accessibility may be different, or SATB1 does not directly regulate ABC transporters. The mechanism of differences still needs to be further explored. SATB1 regulates the function of the ABC transporter not by modulating its expression in gastric cancer but by other means.

The function of ABC transporters, including P-gp, depends not only on the expression level but also on, for instance, their intracellular localization and post-translational modifications.^39,40 We tried to determine whether SATB1 can regulate the localization of ABC transporters. Through immunofluorescence and confocal microscopy, we observed that the expression of SATB1 affects the membrane localization of ABC transporters.⁴¹ Previously, it was reported that ERM proteins might play an important role in the intracellular localization of ABC transporters.²⁵ In lung cancer cells, Ezrin and moesin were associated with BCRP and P-gp function, while in renal cancer cells, radixin was associated with BCRP function, indicating which ERM protein regulating the function of ABC transporter is organ-specific.⁴² Ezrin has been reported to be associated with poor prognosis in gastric cancer. The inhibition of ezrin expression increased sensitivity to camptothecin-induced apoptosis.⁴³ Ezrin has also been reported to maintain cell membrane localization of P-gp in osteosarcoma.²⁹ In view of the similar structure of ABC transporters, we speculate that Ezrin can bind a variety of ABC transporters in gastric cancer to anchor them to the cell membrane, and SATB1 can regulate Ezrin. We used CoIP and fluorescence immunoassay to confirm that Ezrin can bind to the three ABC transporters anchoring them to the cell membrane. Following knockdown of SATB1, we found that the expression of Ezrin was downregulated, confirming that SATB1 can also regulate Ezrin expression. The ABC transporter was relocated to the plasma membrane by Ezrin complementation in the SGC7901-siSATB1 cell line. We confirmed that the effect of SATB1 on ABC transporters is through Ezrin. Finally, we predicted three promoter binding sites for SATB1 and Ezrin through the Jaspar database, verified by ChIP-PCR, and determined that P2 was the binding site of SATB1.

In contrast to previous reports that the expression of Ezrin in liver cancer can affect the expression of P-gp,⁴⁴ Ezrin did not affect the expression of ABC transporters in gastric cancer cells in our experiments. In our study, we focused on the regulation of ABC transporter localization by SATB1 through Ezrin expression. Whether SATB1 can regulate other ERM proteins and whether other ERM proteins also regulate ABC transporters in gastric cancer remains to be studied. Radixin has been reported to regulate MRP2 expression and function selectively.⁴⁵ The post-translational modification of ERM proteins also affects their function; for example, phosphorylation can activate the ERM proteins to function.²⁶ It remains to be elucidated if SATB1 can alter the phosphorylation of Ezrin and other ERM proteins. It has also been reported that silencing of SATB1 can upregulate SLC22A18 to reverse temozolomide resistance in human glioblastoma cells.⁴⁶ The solute Carrier family also belongs to the family of ABC transporters; the functional regulation of the solute Carrier family by ERM proteins has not yet been explored. In addition, whether SATB1 can regulate multiple ERM proteins in different ways and whether ERM proteins can regulate additional transporters other than ABC transporters may be future directions for research. In summary, we clarified the mechanism of SATB1-mediated promotion of MDR in gastric cancer and provided a new theoretical basis and molecular target for reversing MDR in gastric cancer.

AUTHOR CONTRIBUTIONS

Qiang Tong designed the study, interpreted the results, and wrote the manuscript. Jiajun Luo, Yu Yang, Yue Jiang, Junfeng Yan and Jingwen Yuan performed the experiments. All authors read and approved the final manuscript. Jiajun Luo and Jingwen Yuan contributed equally to this work.

ACKNOWLEDGMENTS

This work was supported by the National Natural Science Foundation of China (No. 81172186) (QT), by the Natural Science Foundation of Hubei Province (No. 2018CFB504) (QT), and by the Guidance Foundation of Renmin Hospital of Wuhan University (No. RMYD2018M67) (QT).

CONFLICT OF INTEREST

There are no conflicts of interest for all the authors.

DATA AVAILABILITY STATEMENT

The datasets used and analyzed during the current study are available from the corresponding authors upon reasonable request.

ETHICS STATEMENT

Approval of the research protocol by an Institutional Reviewer Board: N/A.

Informed Consent: N/A.

Registry and the Registration No. of the study/trial: N/A.

Animal Studies: All experiment procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Renmin Hospital of Wuhan University (ethics approval number: WDRY2018-K055).