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Abstract

BACKGROUND:

The cancerous inhibitor of protein phosphatase 2A (CIP2A) is an oncoprotein which involves in the progression of several human malignancies. Development of cisplatin (DDP) resistance is the obstacle to an effective control of gastric cancer (GC) clinically.

OBJECTIVE:

We thus assessed whether CIP2A expression is associated with sensitivity of GC to DDP.

METHODS:

Real-time quantitative PCR, immunohistochemical analysis, or western blotting was performed to detect CIP2A expression in GC patients’ tissues. SGC7901/DDP cells were transfected with CIP2A siRNA. MTT assay was used to determine the DDP-sensitivity of cells. Flow cytometry was used to measure cell apoptosis.

RESULTS:

CIP2A has higher expression in DDP-resistant GC patients. DDP-resistant GC patients with high CIP2A expression presented with poorer overall survival rates than those with low CIP2A expression. CIP2A knockdown in DDP-resistant GC cells resulted in attenuated proliferative abilities and increased apoptosis level. CIP2A depletion sensitizes DDP-resistant cells to DDP and CIP2A overexpression antagonizes DDP-sensitive cells to DDP. CIP2A influences the expression of multidrug resistance-related proteins in GC cells.

CONCLUSIONS:

Our results suggested that CIP2A oncoprotein plays an important role in DDP resistance of GC and could serve as a novel therapeutic target for the treatment of GC patients with DDP resistance.

Keywords

CIP2A gastric cancer DDP chemoresistance apoptosis

1. Introduction

Gastric cancer (GC) represents one of the most common cancers in Eastern Asia (including China, Japan, and South Korea) and Eastern Europe [1]. The incidence and mortality of GC have declined dramatically over the past half century in most developed countries, but it remains the second most common cause of cancer-related death in the world [2]. Despite the substantial improvements made in chemotherapy, radiotherapy and surgical techniques for GC over the past few decades, most tumors eventually develop a drug resistant relapse selected during the course of therapy. Cisplatin (DDP) has been commonly used in the treatment of GC [3]. Several DDP resistance common mechanisms were reported, including failure of apoptosis, change in drug export, change in drug metabolism, and failure of DNA repair [4, 5]. And the driving factors underlying DDP resistance remain poorly defined and better understanding of drug resistance mechanisms is required for the development of rational strategies for the prevention and treatment of GC recurrence.

In 2002 Soo Hoo and group reported cloning and characterization of a novel 90 kDa ‘companion’ auto-antigen of p62 overexpressed in hepatocellular carcinoma [6]. Cancerous inhibitor of protein phosphatase 2A (CIP2A) was initially identified as an endogenous physiological inhibitor of tumor suppressor protein phosphatase 2A (PP2A) phosphatase [7]. CIP2A binds to the PP2A complex, and inhibition of CIP2A has been shown to increase the catalytic phosphatase activity of the PP2A complex in several different human cancer cell types [8, 9]. Recently, various independent studies have validated CIP2A’s role in promoting tumor growth and resistance to apoptosis. CIP2A promotes the proliferation and aggressiveness of several cancer types including head and neck squamous cell carcinoma, oral squamous cell carcinoma, oesophageal squamous cell carcinoma, colon, breast, prostate, tongue, lung, cervical cancer, acute myeloid leukemia and GC. Notably, high CIP2A expression predicts poor patient prognosis in several human cancer types [7, 10, 11, 12, 13]. However, the role of CIP2A in drug resistance has less reported. In 2011, Yeon A. Choi et al. have proven that CIP2A expression is associated with doxorubicin resistance [14]. In 2015, Zhang et al. have proven that CIP2A expression is associated with DDP resistance in ovarian cancer [15]. The effect of CIP2A on DDP-resistant GC cells has not been previously evaluated.

Here we analyzed the expression of CIP2A in DDP-sensitive and DDP-resistant specimens by immunohistochemistry, real-time quantitative PCR (QPCR) and western blotting. We also investigated the expression of CIP2A in DDP-sensitive GC cell line SGC7901 and DDP-resistant GC cell line SGC7901/DDP cells. To address the possible mechanisms, the overexpression and knockdown of CIP2A in SGC7901 and SGC7901/DDP cells was investigated, which may lead to the development of novel treatment strategies for chemoresistant GC.

2. Materials and methods

2.1 Patients

Two independent GC cohorts tissue microarray (TMA) were utilized in this study. The training cohort TMA was purchased from Wuhan Iwill Biological Technology Co., Ltd. (Wuhan, China). It included tumor tissues of DDP-sensitive patients ( $n=$ 15) and DDP-resistant patients ( $n=$ 12). The array dot diameter was 1.5 mm, and each dot represented a tissue spot from one individual specimen that was selected and pathologically confirmed. Immunohistochemical analysis as well as the scoring of immunoreactivity was performed using the mouse polyclonal anti-CIP2A antibody. The staining and number of CIPA positive cells was quantified by a light microscope (BX53; Olympus Corp., Tokyo, Japan) and Image pro-plus 6.0 analysis system. The intensity of CIP2A staining was scored as 0 (no signal), 1 (weak), 2 (moderate), and 3 (marked). Percentage scores were assigned as 1, 10–30%; 2, 30–60%; and 3, 60–100%. The scores of each tumor sample were multiplied to give a final score of 1–3, and the tumors were finally determined as negative (-), score $=$ 0; lower expression (+), score $=$ 1; moderate expression (++), score $=$ 2; and high expression (+++), score $=$ 3. Tumor sample scored (++) to (+++) were considered positive (overexpression).

Five pairs of DDP-sensitive patients and DDP-resistant patient’s tumor tissues were collected immediately after surgical resection and stored in liquid nitrogen until further use. For western blotting assay, tissue specimens were ground in liquid nitrogen cooled mortar, tissue powder was suspended in lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, complete protease inhibitor cocktail) and cleared by centrifugation. The samples were used by the approval of the Institutional Review Board of Hubei University of Medicine and Dongfeng General Hospital affiliated to Hubei University of Medicine. All tissue samples were obtained with written informed consent from patients at the Dongfeng General Hospital.

2.2 Cell culture

Human GC lines SGC7901 cell line was purchased from the American Tissue Culture Collection (ATCC; Manassas, VA, USA). Human DDP-resistant GC cell line SGC7901/DDP were purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). SGC7901 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 10% fetal bovine serum (FBS; HyClone; GE Healthcare Life Sciences, Chalfont, UK), 100 U/ml penicillin (Amresco, Cleveland, OH, USA), 100 mg/ml streptomycin (Amresco, Cleveland, OH, USA), and incubated in a humidified atmosphere with CO ${}_{2}$ at 37 ${}^{\circ}$ C. SGC7901/DDP cells were grown in RPMI 1640 (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) with FBS, 500 ng/ml DDP, 100 U/ml penicillin, 100 mg/ml streptomycin, and incubated in a humidified atmosphere with CO ${}_{2}$ at 37 ${}^{\circ}$ C.

2.3 Real-time quantitative PCR (QPCR)

Expression of the CIP2A, MRP1, MDR1, and HIF-1 $\alpha$ gene was examined by real-time quantitative PCR (QPCR) normalized to expression of GAPDH. Total RNA was extracted from cells using Trizol reagent (Invitrogen; Thermo Fisher Scientifc, Inc., Waltham, MA, USA) according to the manufacturer’s protocol. QPCR analysis of CIP2A, MRP1, MDR1, and HIF-1 $\alpha$ gene was performed with 2 $\mu$ g of total RNA and ReverTra Ace qPCR RT Kit (Toyobo Co., Ltd. Life Science Department, Osaka Japan). Mixed 2 $\mu$ g RNA, 4 $\mu$ l 5 $\times$ RT Buffer, 1 $\mu$ l RT Enzyme Mix, 1 $\mu$ l Primer Mix, and Nuclease-free Water up to 20 $\mu$ l volume. The reverse transcription step as follow: 37 ${}^{\circ}$ C for 15 min; 98 ${}^{\circ}$ C for 5 min, then stored at $-$ 20 ${}^{\circ}$ C. QPCR was performed in an ABI StepOnePlus ${}^{\rm TM}$ Real-Time PCR System (ABI; Thermo Fisher Scientifc, Inc., Waltham, MA, USA) using SYBR ${}^{\@setsize{\scriptsize}{8pt}{\viipt}{\@viipt}\textregistered}$ Green Realtime PCR Master Mix (Toyobo Co., Ltd. Life Science Department, Osaka Japan). Mixed SYBR Green PCR Master Mix 10 $\mu$ l, forward and reverse primers 200 nM, cDNA template 100 ng, and ddH ${}_{2}$ O up to 20 $\mu$ l volume. PCR conditions consisted of the following: 95 ${}^{\circ}$ C for 3 min for denaturation; 95 ${}^{\circ}$ C for 15 sec for annealing; and 60 ${}^{\circ}$ C for 1 min for extension, for 40 cycles. The threshold cycle for each sample was selected from the linear range and converted to a starting quantity by interpolation from a standard curve generated on the same plate for each set of primers (Table 1). The CIP2A, MRP1, MDR1, and HIF-1 $\alpha$ mRNA levels were normalized for each well to the GAPDH mRNA levels using the 2 ${}^{-\Delta\Delta Cq}$ method [16]. Each experiment was repeated three times.

Table 1
Primer sequences for QPCR

Gene	Primer sequence
CIP2A	F: TGCGGCACTTGGAGGTAATTTC
	R: AGCTCTACAAGGCAACTCAAGC
MDR1	F: TTGCGGCTCCGATACATGG
	R: CCAGTGGTGTTTTTAGGGTC
MRP1	F: GGAGGACACGTCGGAACAA
	R: CCGGCTGCTTCCTAGTCTTG
HIF-1 $\alpha$	F: CGTTCCTTCGATCAGTTGTC
	R: TCAGTGGTGGCAGTGGTAGT
GAPDH	F: TGTTGCCATCAATGACCCCTT
	R: CTCCACGACGTACTCAGCG

F, forward; R, reverse.

2.4 Western blotting

Cell pellets were lysed in RIPA buffer containing 50 mM Tris pH 8.0, 150 mM NaCl, 0.1% SDS, 0.5% deoxycholate, 1% NP-40, 1 mM DTT, 1 mM NaF, 1 mM sodium vanadate, 1 mM PMSF (Merck Millipore, Darmstadt, Germany), and 1% protease inhibitors cocktail (Merck, Millipore). Lysates were normalized for total protein (25 $\mu$ g) and loaded on 8% to 12% sodium dodecyl sulfate polyacrylamide gel, electrophoresed, and transferred to a PVDF membrane (Millipore, Kenilworth, NJ, USA), followed by blocking with 5% skimmed milk at room temperature for 1 h. The membrane was incubated with primary antibodies overnight at 4 ${}^{\circ}$ C and rinsed with Tris-buffered saline with Tween 20. The primary antibodies used were anti-MRP1 (1: 500 dilation; catalog no. sc-13960), anti-CIP2A (1: 1000 dilation; catalog no. sc-80662), anti-phospho-Akt (S473) (1: 500 dilation; catalog no. sc-7985), anti-Akt (1: 500 dilation; catalog no. sc-8312) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-caspase-3 (1: 1000 dilation; catalog no. 9662), anti-PARP (1: 1000 dilation; catalog no. 9542), anti-HIF-1 $\alpha$ (1: 1000 dilation; catalog no. 5537) (Cell Signaling Technology, Danvers, MA, USA), anti-PCNA (1: 3000 dilation; catalog no. 10205-2-AP) (Proteintech), anti-P-gp (1: 2000 dilation; catalog no. ab170904) (Abcam, Cambridge, UK), and anti-GAPDH (1: 5000 dilation; catalog no. M20006; Abmart, Shanghai, China) antibodies. The blots were then washed, and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1: 10000 dilation; catalog no. E030120-01 and E030110-01; EarthOx, LLC, San Francisco, CA, USA) for 1.5 h at room temperature. Detection was performed by using a SuperSignal ${}^{\@setsize{\scriptsize}{8pt}{\viipt}{\@viipt}{\textregistered}}$ West Pico Trial kit (catalog no. QA210131; Pierce Biotechnology, Inc., Rockford, IL, USA) [17]. The defined sections of the film were scanned for image capture and quantification using Adobe Photoshop software (CS4, Adobe Systems Incorporated, USA) and Image J software (National Institutes of Health, Bethesda, MD, USA).

2.5 Transfection of DNA

The pOTENT-1-CIP2A expression plasmid was purchased from Youbio Co. Ltd. (Changsha, China). Transfection of the pOTENT-1-CIP2A plasmid into GC cells were carried out using Lipofectamine ${}^{\@setsize{\scriptsize}{8pt}{\viipt}{\@viipt}\textregistered}$ 3000 transfection reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol.

2.6 Transfection of siRNA

Two siRNAs targeting CIP2A were designed and synthesized by Shanghai GenePharma Co. (Shanghai, China), referred to as siRNA1 and siRNA2. The siRNA sequences were as follows:

5’-CUGUGGUUGUGUUUGCACUTT-3’ (CIP2A siRNA1), 5’-ACCAUUGAUAUCCUUAGAATT-3’ (CIP2A siRNA2), 5’-UUCUCCGAACGUGUCACGU TT-3’ [negative control (NC) siRNA].

Using Lipofectamine ${}^{\@setsize{\scriptsize}{8pt}{\viipt}{\@viipt}\textregistered}$ 2000 (Invitrogen) according to the manufacturer’s protocol, cells were transfected with 100 nM siRNA. And 48 h after transfection the cells were then harvested for Western blotting, cell viability, and flow cytometric assays [18].

2.7 RNA preparation and reverse transcription PCR

The total RNA of the cells was isolated using the TRIZOL Reagent (Invitrogen) and the phenol-chloroform extraction method, according to the manufacturer’s instruction. Total RNA (2 $\mu$ g) was annealed with random primers at 65 ${}^{\circ}$ C for 5 min. The cDNA was synthesized using a 1st-STRAND cDNA Synthesis Kit (Fermentas, Hanover, MD, USA). For PCR amplification, primers are as follows: GAPDH, forward primer: 5’-TCACCAGGGCTGCTTTTA-3’, reverse primer: 5’-AAGGTCATCCCTGAGCTGAA-3’; CIP2A, forward primer: 5’-CCATATGCTCACTCAG ATGATGT-3’, reverse primer: 5’-GTGTATCATCTCC ACAGAGAGTT-3’.

2.8 Cytotoxic assay

Cells were seeded into 96-well plate and pre-cultur-ed for 24 h, then treated with DDP for 24. Cell cytotoxicity was determined by MTT assay. The absorbance was measured at 570 nm by automated microplated reader (Bio-Tek, VT, USA), and the cell death rate was calculated as followed: death rate (%) $=$ (average A ${}_{570}$ of the control group – average A ${}_{570}$ of the experimental group)/(average A ${}_{570}$ of the control group – average A ${}_{570}$ of the blank group) $\times$ 100% [19].

2.9 Flow cytometric assays for Annexin-V (AV)

Cell apoptosis was evaluated by AV detection using an AV-FITC kit (BD Biosciences, San Jose, CA, USA), according to the manufacturer’s instructions [20].

2.10 Statistical analysis

All experiments were repeated at least three times and the data were presented as the mean $\pm$ SD unless noted otherwise. Differences between data groups were evaluated for significance using Student’s t-test of unpaired data or one way analysis of variance and Bonferroni post-test. $P$ values less than 0.05 indicate statistical significance.

Figure 1.

CIP2A expression in GC and is associated with DDP resistance. (A) Schematic images of immunohistochemistry staining intensities for CIP2A expression in DDP-resistant GC patients’ tissues. (B) Scores of immunohistochemistry staining for CIP2A expression in DDP-sensitive (DDP ${}^{S}$ ) and DDP-resistant (DDP ${}^{R}$ ). * $P<$ 0.05. (C) Scores of immunohistochemistry staining for CIP2A expression in patients with TNM stage I-II and III-IV ${}^{*}P<$ 0.05. (D) Survival curves of GC patients with low expression versus high expression of CIP2A in DDP-resistant patients ( $P<$ 0.05). (E) Survival curves of GC patients with TNM stage I-II and III-IV ( $P<$ 0.05). (F) CIP2A relative expression in patient samples (P1–P9) determined by QPCR (compared to GAPDH, respectively). GC cell line SGC7901 was used as a positive control. * $P<$ 0.05. (G) CIP2A expression in patient samples (P1–P9) determined by western blotting. GC cell line SGC7901 was used as a positive control. (H) CIP2A relative expression in GC cell lines SGC7901 and SGC7901/DDP was determined by QPCR (compared to GAPDH, respectively). * $P<$ 0.05. (I) CIP2A expression in GC cell lines SGC7901 and SGC7901/DDP was determined by western blotting.

3. Results

3.1 CIP2A is associated with the development of DDP resistance

To investigate whether CIP2A involves the development of DDP resistance in GC tissue, we examined CIP2A protein levels in the GC specimens of DDP-sensitive patients ( $n=$ 15) and DDP-resistant patients ( $n=$ 12) by immunohistochemical staining. We divided the GC patients into CIP2A high (+++), moderate (++), and low expression (+) groups by immunohistochemical assay. The scoring of immunoreactivity was performed as described [21]. As a result, significantly elevated of CIP2A was observed in the cancer tissues of DDP-resistant patients comparing to DDP-sensitive patients (Fig. 1A and B). Furthermore, we analyzed the relationship between DDP resistance and CIP2A expression or, clinicopathological characteristics. As shown in Table 1, DDP resistance and gender, age, or differentiation showed no statistically significant correlations ( $P>$ 0.05). Nevertheless, both TNM stage ( $P=$ 0.038) and CIP2A score ( $P=$ 0.017) show significant correlations with DDP resistance. Besides, we analyzed the relationship between CIP2A score and TNM stage and found that TNM stage shows significant correlations with CIP2A expression, suggesting the critical role of CIP2A in GC progression (Fig. 1C). Survival analysis revealed that DDP-resistant patients with high CIP2A expression had poorer overall survival than those with low CIP2A expression ( $P<$ 0.05; Fig. 1D). Besides, patients with TNM stage III-IV had poorer overall survival than those with TNM stage I-II ( $P<$ 0.05; Fig. 1E). We analyzed the CIP2A mRNA and protein level in 5 available DDP-resistant specimens and 4 DDP-sensitive specimens using QPCR and western blotting approach. Consistent with the immunohistochemical results, CIP2A was higher expression in the cancer tissues of DDP-resistant patients (Fig. 1F and G). Moreover, to verify this differential expression of CIP2A, we detected the CIP2A expression in DDP-resistant cells SGC7901/DDP, which was developed from the parental SGC7901 cells. Consistent with the results in GC tissues, CIP2A was overexpressed in DDP-resistant cells SGC7901/DDP (Fig. 1H and I). These results indicated that there is a correlation between high levels of CIP2A and DDP resistance.

Figure 2.

Effect of CIP2A expression on the GC cell proliferation and apoptosis. (A) SGC7901 cells were transfected with CIP2A expression plasmid, and total RNA was isolated 24 h after transfection and then subjected to reverse transcription PCR (RT-PCR) analysis, total protein was isolated and then followed by subjected to western blotting (WB) analysis. OE, overexpression (B) SGC7901 cells were transfected with CIP2A expression plasmid, and then MTT was used to detect proliferation 24 h after transfection. * $P<$ 0.05. (C) SGC7901/DDP cells were transfected with CIP2A siRNA, and total protein was isolated 48 h after transfection and then subjected to western blotting analysis. (D) SGC7901/DDP cells were transfected with CIP2A siRNA, and then MTT was used to detect proliferation 48 h after transfection. * $P<$ 0.05. (E) SGC7901/DDP cells were transfected with CIP2A siRNA, and total protein was isolated 48 h after transfection and then subjected to western blotting analysis.

3.2 Effect of CIP2A expression on the GC cell proliferation and apoptosis

SGC7901 cells were first transfected with CIP2A expression plasmid (7901/CIP2A), and the expression of CIP2A was confirmed using RT-PCR and Western blotting analysis (Fig. 2A). CIP2A was highly expressed at both the mRNA and protein levels. The overexpression of CIP2A resulted in a significant increase in the proliferation of GC cells (Fig. 2B) and the expression of proliferating cell nuclear antigen (PCNA) (Fig. 2A). We further examined the effect of CIP2A knockdown in SGC7901/DDP cells. SGC7901/DDP cells were transfected with CIP2A siRNA, and the expression of CIP2A was examined using western blotting analysis. The level of CIP2A was markedly decreased by the CIP2A siRNA treatment and the depletion of CIP2A resulted in a significant decrease in the proliferation (Fig. 2D) and the expression of PCNA (Fig. 2C). To determine which cell death pathway was induced by CIP2A depletion, western blotting was used to detect apoptosis proteins expression. As presented in Fig. 2E, the results showed that PARP cleavage, as well as cleaved-caspase-3 (downregulation of pro-caspase-3), were detected in CIP2A depleted SGC7901/DDP cells. These results imply that the level of CIP2A expression plays a critical role in proliferation and anti-apoptosis of DDP-resistant GC cells.

3.3 Effect of CIP2A expression on the DDP-mediated inhibition of GC cell proliferation and apoptosis

Furthermore, we examined whether the difference in CIP2A expression was associated with sensitivity against the DDP-induced inhibition of cell proliferation, the cell proliferation levels of both cells were measured after DDP treatment. SGC7901 cells were more susceptible to DDP-mediated inhibition of cell proliferation than SGC7901/CIP2A cells (Fig. 3A); and CIP2A depleted-SGC7901/DDP cells were more susceptible to DDP-mediated inhibition of cell proliferation than SGC7901/DDP cells (Fig. 3B). Next, flow cytometric assay was used to detect apoptosis. As presented in Fig. 3C, CIP2A high expression resisted DDP-induced apoptosis effect and CIP2A silencing promoted DDP-induced apoptosis effect (Fig. 3D). These data demonstrate that the level of CIP2A expression antagonize sensitivity to DDP of DDP-resistant GC cells.

Figure 3.

Effect of CIP2A expression on the DDP-mediated inhibition of GC cell proliferation and apoptosis. (A) SGC7901 cells were transfected with or without CIP2A expression plasmid. Twenty-four hours after transfection, MTT was used to detect the cytotoxicity of DDP. (B) SGC7901/DDP cells were transfected with 100 nM CIP2A or negative control (NC) siRNA. And, 48 hours after transfection the cells were then subjected to MTT to detect the cytotoxicity of DDP. (C) SGC7901 cells were transfected with or without CIP2A expression plasmid. Twenty-four hours after transfection, cells were treated with DDP for 24 h then subjected to flow cytometry to detect apoptosis. (D) SGC7901/DDP cells were transfected with 100 nM CIP2A or NC siRNA. And forty-eight hours after transfection the cells were treated with DDP for 24 h and then subjected to flow cytometry to detect apoptosis. ** $P<$ 0.01.

Table 2

Characteristics of CIP2A expression in DDP-sensitive or resistant GC patients

Characteristics		DDP		$P$ value†
	All ( $n=$ 27)	Sensitive ( $n=$ 15)	Resistant ( $n=$ 12)
	No.	No.	No.
Age
$<$ 60	14	9	5	0.288
$\geqslant$ 60	13	6	7
Gender
Male	18	10	8	0.657
Female	9	5	4
TNM stage
I–II	13	10	3	0.038
III–IV	14	5	9
Differentiation
Well, moderatelly	12	9	3	0.076
Poorly, undifferentiated	15	6	9
CIP2A score
Negative and low	14	11	3	0.017
high	13	4	9

†Chi-sqaure test.

Table 3

Relationship between CIP2A expression and MDR-related proteins in GC patients

MDR-related proteins		CIP2A expression		$P$ valve†
	All ( $n=$ 27)	Low ( $n=$ 10)	High ( $n=$ 17)
	No.	No.	No.
P-gp
Low	9	6	3	0.034
High	18	4	14
MRP1
Low	14	6	8	0.402
High	13	4	9
HIF-1 $\alpha$
Low	11	7	4	0.024
High	16	3	13

†Chi-sqaure test.

Figure 4.

CIP2A influenced the expression of multidrug resistance-related proteins. (A) SGC7901/DDP cells were transfected with CIP2A siRNA, and then QPCR was used to detect indicated gene mRNA level expression 48 h after transfection. (B) SGC7901 cells were transfected with or without CIP2A expression plasmid. Twenty-four hours after transfection, QPCR was used to detect indicated gene mRNA level expression. (C) SGC7901/DDP cells were transfected with CIP2A siRNA, and 48 h after transfection, western blotting was performed using antibodies indicated. (D) SGC7901 cells were transfected with or without CIP2A expression plasmid. Twenty-four hours after transfection, western blotting was performed using antibodies indicated. * $P<$ 0.05. (E) Schematic images of immunohistochemistry staining intensities for CIP2A, P-gp, MRPI, and HIF-1 $\alpha$ expression in DDP-resistant GC patients’ tissues.

3.4 CIP2A influenced the expression of multidrug resistance-related proteins

Multidrug resistance (MDR) to cancer chemotherapy was known to be the major obstacle to successful treatment of GC [22]. To investigate the mechanism by which CIP2A enhance the DDP resistance of GC cells, we detected the expression levels of several MDR-related proteins [P-glycoprotein (P-gp, coded by MDR1), multidrug resistance-associated protein 1 (MRP1), and hypoxia inducible factor-1 $\alpha$ , (HIF-1 $\alpha$ ) by QPCR. The mRNA level of MDR1, MRP1, and HIF-1 $\alpha$ in SGC7901/DDP cells transfected CIP2A siRNA was significantly lower than that in control. In addition, we transfected SGC7901 cells with CIP2A expression plasmid and found that the mRNA level of MDR1, MRP1, and HIF-1 $\alpha$ is significantly upregulated in CIP2A overexpressed SGC7901 cells. (Fig. 4A and B). To better understand the function of CIP2A, we detected the protein levels of P-gp, MRP1, and HIF-1 $\alpha$ with western blotting. We found that the protein level of P-gp, MRP1 and HIF-1 $\alpha$ increased in CIP2A knockdown SGC7901/DDP cells and decreased in CIP2A overexpressed SGC7901 cells (Fig. 4C and D). Phosphorylated Akt is a cancer MDR locus [23]. We next investigated the activity of Akt in SGC7901 and SGC7901/DDP cells. As shown in Fig. 4C and D, CIP2A depletion decreased Akt phosphorylation and CIP2A overexpression increased Akt phosphorylation. Furthermore, we analyzed the relationship between MDR-related proteins (P-gp, MRP1 and HIF-1 $\alpha$ ) and CIP2A expression by immunohistochemical analysis of TMAs. As shown in Table 3 and Fig. 4E, CIP2A expression and MRP1 showed no statistically significant correlations ( $P>$ 0.05). Nevertheless, both P-gp ( $P=$ 0.038) and HIF-1 $\alpha$ score ( $P=$ 0.017) show significant correlations with CIP2A expression. These results indicate that CIP2A influenced the expression of MDR-related proteins.

4. Discussion

In China, GC has become the second most frequently diagnosed cancer and the third leading cause of cancer death and Chinese GC patients have a worse outcome than US GC patients [24]. Despite improvements resulting from the current standard of care, surgery followed by chemotherapy and adjuvant DDP, DDP resistance remains the main cause of treatment failure and death in GC patients [25]. Several mechanisms are proposed to explain the reason of chemoresistance, including increased expression of ATP-dependent drug efflux pumps and decreased influx, decreased apoptosis, superior DNA damage repair, decreased apoptosis, autophagy, cancer stem cells (CSCs), long non-coding RNA, and epithelial-mesenchymal transition (EMT) [18, 26, 27]. Although tumor suppressors or oncogenes have been identified to participate in the development and progression of GC, it’s still poor for the prognosis of chemoresistant GC. Therefore, identification of critical players in chemoresistance will help to discover new therapeutic targets for GC.

The clinical significance of CIP2A expression as clinical markers has already been reported. High tumor CIP2A expression is a poor prognostic factor in several cancer types [7, 10, 11, 12, 13]. Among them, high CIP2A is associated with poor prognosis and chemoresistance in ovarian and breast cancer [14, 15]. Ventala et al. reported that CIP2A is associated with radioresistance in head and neck squamous cell cancer [28]. Moreover, CIP2A is involved in regulating MDR of cervical adenocarcinoma upon chemotherapy by enhancing P-gp expression through E2F1 [29]. In the present study, CIP2A was overexpressed in DDP-resistant patients and cell line comparing to DDP-sensitive patients and cell line, and DDP-resistant patients with high CIP2A expression had poorer overall survival than those with low CIP2A expression, thereby suggesting that CIP2A has a positive role in DDP resistance of GC (Fig. 1).

Evading apoptosis is one of the hallmarks of drug resistance, and targeting apoptosis has become a cancer therapeutic strategy [30]. We next detected whether CIP2A can influence apoptosis to promote DDP resistance. The result showed that CIP2A depletion increased DDP-resistant GC cells apoptosis (Fig. 2E). Furthermore, we tested the function of CIP2A in DDP therapy. The results showed that the increase in CIP2A expression is associated with DDP resistance and decrease in CIP2A expression is associated with DDP sensitivity (Fig. 3A and B). Moreover, CIP2A knockdown promoted the effects of DDP-induced apoptosis (Fig. 3C and D). These results suggest that CIP2A may contribute to DDP-resistance in part, through inhibition of apoptosis.

MDR is largely responsible for ineffective chem-otherapy [22]. The complicated mechanisms of MDR in cancer cells include redistribution of intracellular drug accumulation, increased drug efflux, increased DNA damage repair, alterations in the drug’s target molecules, and suppression of drug-induced cell apoptosis [18]. There are at least two molecular pumps in tumor cell membranes, P-gp and MRP, to expel the chemotherapy drugs out of the cancer cells and attenuate the drug effect. Many tumor patients are found with higher overexpression of MDR-related protein in their tumor tissues compared to the normal tissues [22]. We detected the expression of MDR-related protein and found that depleted CIP2A expressions in SGC7901/DDP cells can downregulate the expression of MDR-related proteins (P-gp, MRP1, HIF-1 $\alpha$ , and pAkt). Moreover, CIP2A overexpression promoted the expression of MDR-related proteins (Fig. 4A and D). Furthermore, immunohistochemical analysis of TMAs showed that CIP2A expression has significant correlations with P-gp ( $P=$ 0.038) and HIF-1 $\alpha$ expression (Table 3 and Fig. 4E). The results showed that CIP2A influences the expression of MDR-related proteins to promote DDP resistance.

In conclusion, high expression of CIP2A is a recurrent event in DDP-resistant GC, where it serves as a marker of reduced overall survival, as previously reported in other tumors. Moreover, CIP2A promotes proliferation, antagonizes apoptosis of DDP-treated DDP-resistant GC cells and, promotes the expression of MDR-related proteins. Our results confirmed that CIP2A behaves as an oncoprotein in DDP resistance and could represent a novel therapeutic target in DDP-resistant GC.

Footnotes

Acknowledgments

This work was supported by grants from the National Natural Sciences Foundation of China (grant no., 81400157, 81372998, and 11647002); Natural Science Foundation of Hubei Province (grant no. 2016CFB528); the Foundation of Health and Family Planning Commission of Hubei Province (grant no., WJ2017F065 and WJ2017F067); the Foundation of Hubei University of Medicine (grant no. FDFR201605); and the National Training Program of Innovation and Entrepreneurship for Undergraduates (grant no. 201710929012 and 201710929013).

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Increase in CIP2A expression is associated with cisplatin chemoresistance in gastric cancer

Abstract

BACKGROUND:

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSIONS:

Keywords

1. Introduction

2. Materials and methods

2.1 Patients

2.2 Cell culture

2.3 Real-time quantitative PCR (QPCR)

Table 1 Primer sequences for QPCR

2.5 Transfection of DNA

2.6 Transfection of siRNA

2.7 RNA preparation and reverse transcription PCR

2.8 Cytotoxic assay

2.9 Flow cytometric assays for Annexin-V (AV)

2.10 Statistical analysis

3.1 CIP2A is associated with the development of DDP resistance

3.3 Effect of CIP2A expression on the DDP-mediated inhibition of GC cell proliferation and apoptosis

4. Discussion

Footnotes

Acknowledgments

References

Table 1
Primer sequences for QPCR