TCF21 inhibits proliferation and chemoresistance through the AKT pathway in human gastric cancer

Accepted Manuscript TCF21 inhibits proliferation and chemoresistance through the AKT pathway in human gastric cancer

Zhi Yang, Xiaodi Jiang, Deming Li, Qianze Dong, Haiying Zhao, Xiaofeng Jiang PII: DOI: Reference:

S0378-1119(18)31036-9 doi:10.1016/j.gene.2018.10.011 GENE 43267

To appear in:

Gene

Received date: Revised date: Accepted date:

25 June 2018 12 September 2018 4 October 2018

Please cite this article as: Zhi Yang, Xiaodi Jiang, Deming Li, Qianze Dong, Haiying Zhao, Xiaofeng Jiang , TCF21 inhibits proliferation and chemoresistance through the AKT pathway in human gastric cancer. Gene (2018), doi:10.1016/j.gene.2018.10.011

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ACCEPTED MANUSCRIPT TCF21 inhibits proliferation and chemoresistance through the AKT pathway in human gastric cancer

Department of General Surgery, The Fourth Affiliated Hospital of China Medical University,

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Zhi Yanga; Xiaodi Jiangb; Deming Lic; Qianze Dongd; Haiying Zhaoa*; Xiaofeng Jianga*

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Shenyang, China

Department of Infectious Diseases, The Shengjing Hospital of China Medical University,

Department of Anesthesiology, The Fourth Affiliated Hospital of China Medical University,

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Shenyang, China

Shenyang, China

Department of Pathology, First Affiliated Hospital and College of Basic Medical Science, China

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Medical University, Shenyang, China

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*Corresponding author: Xiaofeng Jiang, Department of General Surgery, The Fourth Affiliated

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Hospital of China Medical University, Chongshan East Road 4, Shenyang, 110032, Liaoning, China. E-mail: [email protected]; Haiying Zhao, Department of General Surgery, The

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Fourth Affiliated Hospital of China Medical University, Chongshan East Road 4, Shenyang, 110032, Liaoning, China. E-mail: [email protected]

Abstract In a previous study, we showed that transcription factor 21 (TCF21) is methylated and downregulated in human gastric cancer samples and serves as an independent prognostic factor. However, its biological role and potential mechanism in gastric cancer cells remain unexplored. 1

ACCEPTED MANUSCRIPT In the current study, we examined TCF21 expression in 6 gastric cancer cell lines. The BGC-823 and SGC-7901 cell lines were selected for small interfering RNA and plasmid transfection, respectively. The results of the Cell Counting Kit-8 assay demonstrated that TCF21 inhibited gastric cancer cell proliferation. Cell cycle analysis suggested that TCF21 inhibited cell cycle

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progression in gastric cancer cells. The Matrigel invasion assay demonstrated that TCF21

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negatively regulated invasion. The cell adhesion assay showed that TCF21 increased cell

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adhesion. Gastric cancer cells were treated with cisplatin to explore the role of TCF21 in chemoresistance. Cell Counting Kit-8 assay and AnnexinV/propidium iodide analyses showed

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that TCF21 overexpression sensitized SGC-7901 cells to cisplatin, whereas its depletion reduced

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sensitivity in BGC-823 cells. JC-1 staining was performed to measure the effect of TCF21 on mitochondrial potential. TCF21 downregulated mitochondrial membrane potential after

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treatment with cisplatin. Western blot analysis showed that TCF21 overexpression negatively

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regulated Bcl-xL, phosphorylated extracellular signal regulated kinase, and phosphorylated AKT expression and induced caspase 3 cleavage. LY294002, an AKT inhibitor, blocked the effect of

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TCF21 on Bcl-xL, caspase 3 and CDDP-induced apoptosis. Nude mice experiments

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demonstrated that TCF21 inhibited gastric cancer growth in vivo. In conclusion, our results suggest that TCF21 inhibits gastric cancer growth and chemoresistance possibly through the

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AKT signaling pathway.

Keywords: TCF21; Gastric cancer; AKT; Proliferation; Invasion; Chemoresistance

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ACCEPTED MANUSCRIPT 1 Introduction Although the incidence of gastric cancer has decreased, it remains a common disease and the second most frequent cause of cancer death worldwide [1]. The prognosis of advanced gastric cancer remains poor even after curative resection [2,3]. The mechanism underlying gastric

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cancer progression is complex and involves various genetic and epigenetic changes [4–6].

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Identification of new biomarkers and elucidation of the underlying molecular mechanism,

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especially with respect to chemoresistance, are crucial for the development of new therapeutic

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strategies [7,8].

Transcription factor 21 (TCF21) was originally identified as a transcription factor that

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regulates mesenchymal-epithelial transition during embryonic development [9]. TCF21 is downregulated in several cancers through promoter methylation. TCF21 is methylated and

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silenced in head and neck carcinomas and non-small cell lung cancers [10]. TCF21 mRNA levels

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are downregulated in breast cancer tissues and cell lines [11]. Our previous study demonstrated

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that TCF21 is methylated and downregulated at the protein level in human gastric cancer samples. Loss of TCF21 could serve as an independent prognostic factor in gastric cancer

unexplored.

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patients [12]. However, the biological roles of TCF21 and the underlying mechanism remain

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In the present study, we examined the function of TCF21 in gastric cancer both in vitro and in vivo and provided evidence that TCF21 inhibits gastric cancer proliferation, invasion, cell cycle, and chemoresistance and increases adhesion. Investigation of the underlying mechanisms showed that TCF21 regulates mitochondrial membrane potential possibly through the modulation of AKT/Bcl-xL signaling.

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ACCEPTED MANUSCRIPT 2 Materials and methods 2.1 Cell culture and transfection The BGC-823, SGC-7901, HGC-27, NCI-N87, MGC-803, and AGS cell lines were obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai,

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China). Cells were cultured in PRMI-1640 with 10% bovine serum. Gastric cancer cells were

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cultured on sterilized bottles under the conditions of 37C and 5% CO2, and were passaged every 2 days with 0.25% trypsin (Invitrogen, USA).

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pCMV6 empty vector and pCMV6-TCF21 were obtained from Origene (Origene, USA).

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Plasmid transfection was performed using the Attractene reagent (Qiagen, Germany). TCF21 small interfering RNA (siRNA) and negative control siRNA were obtained from Dharmacon/GE

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Healthcare. Dharmafect1 was used for siRNA transfection (GE Healthcare, USA). The target

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sequence of siRNA was GAUCCUGGCUAACGACAAA. The shRNA target sequence was GATCCTGGCTAACGACAAA. The shRNA was cloned into the pGLV-h1-GFP-puro lentiviral

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2.2 Western blotting

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vector.

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Total protein was separated by SDS-PAGE, transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA), and incubated overnight at 4C with antibodies against TCF21 (1:800; Proteintech, Chicago, IL, USA), phosphorylated extracellular signal regulated kinase (pERK), phosphorylated AKT (p-AKT), ERK, AKT, caspase 3, cleaved caspase 3, Bcl-xL (1:900; Cell Signaling Technology, USA), and GAPDH (1:2000; Santa Cruz, USA). After incubation with HRP-coupled anti-mouse/rabbit IgG (1:2000, Cell Signaling Technology, Boston, MA,

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ACCEPTED MANUSCRIPT USA) at 37C for 2 h, bound proteins were visualized using ECL (Pierce, Rockford, IL, USA) and detected using BioImaging Systems (UVP Inc., Upland, CA, USA). 2.3 Cell Counting Kit-8 (CCK-8) assays

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For the CCK-8 assay, gastric cancer cells were plated in 96-well plates (3000 cells per well)

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and cultured for 5 days. Twenty microliters of CCK-8 solution were added and the plate was

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incubated for 2–4 h, followed by measurement of absorbance at 490 nm.

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2.4 Apoptosis detection

An Annexin V/propidium iodide (PI) staining kit from BD bioscience was used to determine

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the rate of apoptosis according to the manufacturer’s protocol. Flow cytometry was performed

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using a BD FACSCalibur flow cytometer (Becton Dickinson, USA).

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2.5 Matrigel invasion assay

Cell invasion assays were performed using a 24-well Transwell chamber coated with 20 µL

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Matrigel (1:6 dilution, BD). Cell suspension with serum-free medium was transferred to the

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upper Matrigel chamber. Medium supplemented with 10% fetal bovine serum was added to the lower chamber. After 18 h, invading cells that passed through were fixed and stained with

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hematoxylin.

2.6 Wound healing assay Gastric cancer cells were seeded in culture plates as a monolayer. Then, the cell monolayer was scratched with a pipette tip. Images of the cell monolayer were taken using a microscope at 0 and 24 h. The gap distance was measured.

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ACCEPTED MANUSCRIPT 2.7 Flow cytometry for cell cycle analysis Cells were seeded into 6 cm tissue culture dishes. At 48 h after transfection, cells were harvested, fixed in 1% paraformaldehyde, washed with PBS, and stained with 5 mg/mL propidium iodide in PBS supplemented with RNase A (Roche, Indianapolis, IN) for 30 min at

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room temperature. Cells in each individual phase of the cell cycle were determined based on

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their DNA ploidy profile using ACEA Flow Cytometer and NovoExpress software. 2.8 Cell adhesion assay

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The cell adhesion assay was carried out using fibronectin-coated 12-well plates. Cells after

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transfection were plated at a density of 105 cells/well, and incubated for 1 h at 37C to allow adherence. The non-adherent cells were washed away using PBS. The remaining cells were fixed

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and stained with hematoxylin.

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2.9 Tumorigenicity assay in vivo

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The Experimental Animal Ethics Committee of China Medical University approved the use of laboratory animals. All animal experiments followed the NIH Guide for the Care and Use of

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Laboratory Animals. A total of 12 BALB/c athymic nude mice (4 weeks old) were purchased

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from Shanghai Slac Laboratory Animals Ltd. (Shanghai, China) and randomized into four groups, including negative shRNA, TCF21 shRNA, empty plasmid, and TCF21 plasmid. After 7 days, mice were injected subcutaneously with 5×106 of the indicated cells into the flanks. Tumor size was measured every week. Tumor volume was calculated according to the following formula: tumor volume=length×(width)2/2. After 5 weeks, the mice were sacrificed, and the xenograft tumor tissues were retrieved for further analysis.

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ACCEPTED MANUSCRIPT 3.0 Statistical analysis SPSS version 16 was used for all analyses. Student’s t-test was carried out to compare data between control and experimental groups. A value of p < 0.05 was regarded as statistically significant.

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3 Results

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3.1 TCF21 inhibits cell proliferation, invasion, migration, and cell cycle and increases adhesion

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TCF21 protein expression was analyzed in 6 gastric cancer cell lines including BGC-823, SGC-7901, HGC-27, NCIN87, MGC-803, and AGS. As shown in Fig S1A, endogenous TCF21

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was expressed at low levels in the SGC-7901 cell line, whereas BGC-823 cells expressed relatively high levels of TCF21. SGC7901 cells were therefore selected for plasmid transfection

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and BGC-823 cells were used for TCF21 siRNA transfection. As shown in Fig S1B, TCF21

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siRNA significantly downregulated protein expression in BGC-823 cells, whereas plasmid

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transfection upregulated the TCF21 protein in SGC-7901 cells. The CCK-8 assay was performed to investigate the effect of TCF21 on the proliferation of

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gastric cancer cells. The results showed that the proliferation rate of SGC-7901 cells decreased significantly in response to TCF21 overexpression, whereas TCF21 depletion promoted BGC-

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823 cell proliferation (Fig 1A). To determine whether TCF21 downregulation contributes to the invasion of gastric cancer cells, we performed Matrigel invasion assays. As shown in Fig 1B, the number of invading cells decreased after TCF21 overexpression in SGC-7901 cells. TCF21 depletion increased the number of invading BGC-823 cells. To determine the effect of TCF21 on cell migration, a wound healing assay was performed. TCF21 overexpression inhibited cell

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ACCEPTED MANUSCRIPT migration in SGC-7901 cells, whereas TCF21 knockdown promoted BGC-823 cell migration. The relative migration distances are shown in Fig 1C. Cell cycle analysis was performed to examine the effect of TCF21 on cell cycle progression. As shown in Fig 2A, TCF21 depletion increased the number of cells in the S phase and

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decreased the number of cells in G1 phase,, whereas TCF21 plasmid transfection decreased the S

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percentage and increased the G1 phase percentage, suggesting that TCF21 inhibited cell cycle

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progression in gastric cancer cells. Cell adhesion assays showed that TCF21 depletion decreased cell adhesion in BGC-823 cells, whereas TCF21 overexpression increased cell adhesion (Fig

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2B).

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3.2 TCF21 sensitizes gastric cancer cells to cisplatin (CDDP) treatment

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To examine the effect of TCF21 on chemoresistance in gastric cancer cell lines, cells were

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treated with 5 μM CDDP, and cell viability was determined using the CCK-8 assay. As shown in Fig 3A, TCF21 depletion significantly enhanced cell viability in BGC-823 cells at 24 and 48 h of

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CDDP treatment, whereas TCF21 plasmid transfection impaired CDDP resistance and decreased

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cell viability at 24 and 48 h of treatment. Annexin V/PI analysis was used to detect the level of apoptosis after 24 h of CDDP treatment

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(Fig 3B). TCF21 significantly enhanced the rate of apoptosis in SGC-7901 cells, whereas TCF21 depletion downregulated CDDP-induced apoptosis in BGC-823 cells. 3.3 TCF21 regulates mitochondrial membrane potential Next, we examined the effect of TCF21 on the mitochondrial membrane potential (Δψm). For this purpose, cells were exposed to JC-1 staining, which enters mitochondria with normal potential and shows red fluorescence (Fig 3C). JC-1 shows green fluorescence in cells with low

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ACCEPTED MANUSCRIPT Δψm. Red fluorescence was decreased in SGC-7901 cells transfected with TCF21 compared with control cells, whereas red fluorescence was increased in BGC-823 cells transfected with TCF21 siRNA. These results indicated that TCF21 negatively regulates mitochondrial membrane potential.

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3.4 TCF21 regulates AKT/Bcl-xL

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Several proteins were screened in TCF21 overexpressing and depleted cells. Western blot analysis showed that knockdown of TCF21 upregulated Bcl-xL, p-AKT, and p-ERK and

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inhibited caspase 3 cleavage in BGC-823 cells (Fig 4A). TCF21 overexpression in SGC-7901 cells inhibited Bcl-xL, p-AKT, and p-ERK and increased the level of caspase 3 cleavage.

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To validate the role of AKT during TCF21 induced Bcl-xL downregulation, BGC-823 cells

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were treated with the AKT inhibitor LY294002. As shown in Fig 4B and Fig S2, AKT inhibition

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significantly downregulated p-AKT and Bcl-xL. LY294002 also promoted caspase 3 cleavage. In cells treated with LY294002, TCF21 depletion failed to induce Bcl-xL, inhibit cleaved

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caspase 3 and reduce CDDP-induced apoptosis, suggesting that AKT activation is involved in

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the downstream effects of TCF21.

3.5 TCF21 inhibits gastric cancer cell growth in vivo

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To examine the effect of TCF21 on tumor growth in vivo, we established TCF21 overexpression and shRNA stable cell lines by G418 selection. The effects of shRNA and stable transfection are shown in Supplementary Fig S1C. Nude mice experiments showed that the size of TCF21 overexpressing SGC-7901 tumors was larger than that of control cell tumors (Fig 5). The rate of tumor growth of the TCF21-depleted stable BGC-823 cell line was higher than that

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ACCEPTED MANUSCRIPT of the negative shRNA-transfected cells. TCF21-stably overexpressing SGC-7901 cells showed inhibited growth rate compared with empty plasmid transfected cells. 4 Discussion Methylation and downregulation of TCF21 have been reported in many cancers, including

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head and neck carcinomas, non-small cell lung cancer, breast cancer, and colorectal cancer

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[10,11,13,14]. Previously, we have demonstrated that TCF21 is methylated and downregulated in human gastric cancers, and loss of TCF21 serves as an independent prognostic factor [12].

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Several reports indicate that restoration of TCF21 inhibits cancer cell growth and invasion [11,13].

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To date, there are no functional studies investigating the biological function of TCF21 in

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gastric cancer. In the present study, we have shown that TCF21 inhibited gastric cancer cell

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proliferation in vitro and in vivo. TCF21 overexpression inhibited cell migration and invasion and increased cell adhesion, whereas TCF21 depletion promoted cell migration and invasion and

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inhibited cell adhesion. These data are consistent with previous reports indicating that TCF21

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serves as an important tumor suppressor in gastric cancers [12]. The basic biological characteristic of malignant tumors is unlimited proliferation, which may

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be due to alterations in cell cycle progression. Study of TCF21 in human adrenocortical tumor cells confirmed that the expression of TCF21 is inversely correlated with cyclin dependent kinase 1 [15], which is essential for G1/S phase transitions of eukaryotic cell cycle [16]. A recent study also reported that sumoylation of TCF21 could decrease the proportion of S-phase cells in the cell cycle [17]. Consistent with these findings, we found that TCF21 overexpression in gastric cancer cells caused an increase in the cell population at G1 phase and a decrease in S

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ACCEPTED MANUSCRIPT phase. These results confirmed the role of TCF21 in the inhibition of cell cycle progression in cancer cells. The role of TCF21 in cancer cell chemoresistance and drug-induced apoptosis has not been reported to date [18]. Therefore, we assessed cell viability in response to CDDP treatment and

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examined the CDDP-induced apoptosis rate in gastric cancer cells. The results of the CCK-8

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assay showed that TCF21 suppressed chemoresistance, and Annexin V/PI analysis demonstrated

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that TCF21 overexpression increased the apoptosis rate. Consistently, TCF21 overexpression increased caspase 3 cleavage after CDDP treatment, which further confirmed that TCF21

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overexpression could enhance CDDP-induced apoptosis. Chemotherapeutic drugs, especially

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platinum-containing agents, often induce apoptosis via the mitochondrial apoptosis pathway [19]. We therefore investigated the effect of TCF21 on mitochondrial membrane potential using

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JC-1 staining. The results showed that TCF21 decreased the membrane potential, whereas

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TCF21 depletion maintained the membrane potential. Loss of mitochondrial membrane potential triggers mitochondrial pathway apoptosis by increasing membrane permeability and cytochrome

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c expression. The present data identified TCF21 as a negative regulator of mitochondrial

cancer cells.

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membrane potential, resulting in decreased resistance to CDDP induced apoptosis in gastric

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Further analysis demonstrated that TCF21 overexpression downregulated Bcl-xL, p-ERK, and p-AKT. The ERK pathway induces matrix metalloproteinase 2 transcription through the activation of activator protein 1 [20]. The association between ERK signaling and gastric cancer invasion was demonstrated in several studies [21,22]. Therefore, it is possible that TCF21 modulates gastric cancer invasion partly through the ERK signaling pathway. Bcl-xL is a Bcl-2 family transmembrane protein located in mitochondria. It functions as an anti-apoptotic protein

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ACCEPTED MANUSCRIPT by maintaining mitochondrial membrane potential, preventing cytochrome c release and caspase activation [23,24]. Bcl-xL is more functional than Bcl-2 when induced by chemotherapy drugs. Bcl-xL is also a target protein of AKT signaling [25–27]. The present results showed that TCF21 inhibited AKT phosphorylation, suggesting that TCF21 promoted CDDP-induced apoptosis by

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modulating AKT/Bcl-xL signaling. To further validate this, the cells were treated with the AKT

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inhibitor LY294002, which abolished the effect of TCF21 depletion on Bcl-xL, caspase 3 and

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CDDP-induced apoptosis. These results supported a link between TCF21, AKT/Bcl-xL signaling, and CDDP-induced apoptosis.

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The underlying mechanism of resistance to chemotherapy is complex. The poor response of

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cancer to anticancer drugs is usually due to a combination of mechanisms of chemoresistance, which include a reduction in drug uptake, increased drug efflux from cancer cells, a reduced

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proportion of active agents in cancer cells due to a reduction in pro-drug activation or an

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enhancement in drug inactivation, changes in the expression/function of the molecular targets of anticancer drugs, enhanced repair of chemotherapy-induced DNA damage, and decreased

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expression/function of pro-apoptotic factors or up-regulation of anti-apoptotic genes [28,29]. Our

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present study had some limitations. Although TCF21 expression enhanced the sensitivity to CDDP, it is unclear whether TCF21-induced reduced chemoresistance is specific and hence may

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affect the response to any pro-apoptotic drugs. The specific mechanisms whereby TCF21 inhibiting chemoresistance remain largely undefined. Further detailed studies are currently underway to explore the participation of TCF21 in this process, to provide more comprehensive information about the functions of TCF21 in cancer. In conclusion, the present study shows that TCF21 functions as a tumor suppressor in gastric cancer cell lines. TCF21 inhibited proliferation, invasion, migration, and cell cycle progression,

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ACCEPTED MANUSCRIPT and increased cell adhesion in vitro and in vivo. TCF21 also promoted CDDP-induced apoptosis through the modulation of mitochondrial membrane potential, possibly through the AKT/Bcl-xL signaling pathway. Acknowledgments

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This research was supported by the National Natural Science Foundation of China (No.

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81502073, No. 81672877). The funders had no role in study design, data collection and analysis,

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decision to publish, or preparation of the manuscript. Conflict of interest

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We declare that we have no conflict of interest.

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ACCEPTED MANUSCRIPT Figure legends Fig. 1 TCF21 regulates gastric cancer cell proliferation, invasion, and migration A. The CCK-8 assay showed that TCF21 overexpression inhibited proliferation in SGC-7901 cells. TCF21 knockdown promoted proliferation in BGC-823 cells. B. Matrigel invasion assay

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showed that TCF21 overexpression decreased invading cell numbers, whereas TCF21

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knockdown increased invading cell numbers. C. The wound healing assay showed that TCF21

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transfection inhibited cell migration in SGC-7901 cells, whereas TCF21 knockdown promoted

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Fig. 2 TCF21 regulates cell cycle and adhesion

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BGC-823 cell migration. * p < 0.05.

A. TCF21 depletion increased the number of cells in S phase and decreased the number of cells

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in G1 phase, whereas TCF21 plasmid transfection decreased the S percentage and increased the

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G1 phase percentage. B. TCF21 depletion decreased cell adhesion in BGC-823 cells, whereas

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TCF21 overexpression increased cell adhesion. * p < 0.05.

cancer cells

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Fig. 3 TCF21 regulates CDDP resistance and mitochondrial membrane potential in gastric

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A. TCF21 significantly decreased SGC-7901 cell viability at 24 and 48 h of CDDP treatment. TCF21 siRNA showed the opposite effect by increasing cell viability. B. Annexin V/PI analysis showed that TCF21 overexpression increased the apoptosis rate induced by CDDP. TCF21 siRNA decreased CDDP-induced apoptosis rate in BGC-823 cells. C. JC-1 staining/flow cytometry demonstrated that TCF21 decreased mitochondrial membrane potential, whereas TCF21 siRNA increased mitochondrial membrane potential in cells treated with CDDP. * p < 0.05. 17

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Fig. 4 TCF21 regulates AKT signaling A. Western blot analysis showed that knockdown of TCF21 upregulated Bcl-xL, p-AKT, and pERK and downregulated cleaved caspase 3 in BGC-823 cells. TCF21 overexpression in SGC-

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7901 cells inhibited Bcl-xL, p-AKT, and p-ERK and increased the level of cleaved caspase 3. B.

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The AKT inhibitor LY294002 downregulated p-AKT and Bcl-xL and upregulated cleaved

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caspase 3. In BGC-823 cells treated with LY294002, TCF21 depletion failed to upregulate Bcl-

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xL.

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Fig. 5 TCF21 inhibits gastric cancer cell growth in vivo

The tumor growth rate of TCF21 overexpressing SGC-7901 cells was lower than that of the

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empty plasmid-transfected cell line. The BGC-823 TCF21 shRNA cell line exhibited a higher

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growth rate than that of control shRNA. * p < 0.05.

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ACCEPTED MANUSCRIPT Abbreviations list

TCF21 - transcription factor 21 siRNA - small interfering RNA p-ERK - phosphorylated extracellular signal regulated kinase

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p-AKT - phosphorylated AKT CCK-8 - Cell Counting Kit-8

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PI - propidium iodide

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CDDP - cisplatin

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ACCEPTED MANUSCRIPT Highlights

1. TCF21 inhibits the malignant phenotype of gastric cancer. 2. TCF21 promotes CDDP-induced apoptosis.

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3. TCF21 inhibits chemoresistance through the AKT signaling pathway.

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