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Activation of AKT/AP1/Foxm1 Signaling Confers Sorafenib Resistance to Liver Cancer Cells

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Activation of AKT/AP1/Foxm1 Signaling Confers Sorafenib Resistance to Liver Cancer Cells ONCOLOGY REPORTS 42: 785-796, 2019 Activation of AKT/AP1/FoxM1 signaling confers sorafenib resistance to liver cancer cells DONGJING YAN1*, XIAOJING YAN2*, XUFANG DAI3,4*, LINGXI CHEN2, LIANGBO SUN2, TAO LI2, FENGTIAN HE2, JIQIN LIAN2 and WANGWEI CAI1 1Department of Biochemistry and Molecular Biology, Hainan Medical College, Haikou, Hainan 571199; 2Department of Biochemistry and Molecular Biology, Army Medical University, Chongqing 400038; 3Department of Educational Science College, Chongqing Normal University; 4Chongqing Key Laboratory of Psychological Diagnosis and Educational Technology for Children with Special Needs, Chongqing 400047, P.R. China Received December 5, 2018; Accepted June 6, 2019 DOI: 10.3892/or.2019.7192 Abstract. Sorafenib is the first‑line drug used in the treatment the AKT/AP1/FoxM1 signaling axis is an important determi- of liver cancer; however, drug resistance seriously limits the nant of sorafenib tolerance. clinical response to sorafenib. The present study investigated the molecular mechanisms of sorafenib resistance in liver Introduction cancer cells. The data indicated that forkhead box M1 (FoxM1) was significantly overexpressed in sorafenib‑resistant cells, at Liver cancer, the most common primary liver tumor, is the third the mRNA and protein levels. Knockdown of FoxM1 rendered leading cause of cancer mortality globally (1). Surgical resec- drug‑tolerant cells sensitive to sorafenib. Furthermore, FoxM1 tion, thermal ablation and liver transplantation are the current was upregulated at the transcriptional level. Overexpression treatments for early‑stage liver cancer (2). However, liver cancer of c‑jun was associated with the upregulation of FoxM1. The is often diagnosed at an advanced stage with poor prognosis. results of a reporter gene assay, electrophoretic mobility shift Sorafenib, a multikinase inhibitor of mitogen‑ activated protein assay and chromatin immunoprecipitation assay demonstrated kinase/ERK, vascular endothelial growth factor (VEGF) that there is an activator protein‑1 (AP1) binding site in the receptor and platelet‑derived growth factor, is the only approved promoter of FoxM1, located at ‑608 to ‑618. Knockdown of drug available for the treatment of advance liver cancer (3). c‑jun significantly decreased the levels of FoxM1, accompa- Sorafenib has a good tolerability profile and treatment leads nied by enhanced cell sensitivity to sorafenib. Furthermore, to a longer median survival time (3). Unfortunately, its activity the activation of AKT contributed to the upregulation of is limited by primary and acquired drug resistance (4). Thus c‑jun and FoxM1. Inhibition of AKT using BEZ‑235 mark- far, the mechanisms of sorafenib resistance are not well edly suppressed the upregulation of c‑jun and FoxM1, and established; therefore, it is crucial to determine the mecha- increased the sensitivity of drug‑resistant cells to sorafenib nisms associated with resistance and to develop additional in vitro and in vivo. The data indicated that the activation of therapeutic agents to enhance the effects of the drug. Forkhead box M1 (FoxM1) is a member of the forkhead box transcription factor family. Previous studies have revealed that FoxM1 has an important role in the pathogenesis and progression of human cancers, including liver cancer (5,6). Suppression of FoxM1 expression can lead to chromosome Correspondence to: Dr Jiqin Lian, Department of Biochemistry and Molecular Biology, Army Medical University, 30 Gaotanyan disaggregation, mitotic spindle defects, mitotic catastrophe Street, Chongqing 400038, P.R. China and cell cycle arrest (7). As a key regulator of the G1/S and E‑mail: [email protected] G2/M transition, FoxM1 regulates the expression of several cell cycle‑associated factors, including p21Cip1, cyclin B, Dr Wangwei Cai, Department of Biochemistry and Molecular p27Kip1, Aurora B kinase, survivin, CDC25B and polo‑like Biology, Hainan Medical College, 3 Xueyuan Road, Haikou, Hainan 571199, P.R. China kinase‑1 (8,9). Additionally, the phosphorylation of FoxM1 E‑mail: [email protected] promotes its nuclear translocation and enhances its tran- scriptional activity during G2/M (10). Previous research has *Contributed equally revealed that FoxM1 is a credible target for arresting cancer growth and progression. In tumor‑bearing mice, knockdown Key words: AKT, AP1, forkhead box M1, liver cancer, sorafenib, of FoxM1 can significantly reduce tumor growth (11). By resistance contrast, the overexpression of FoxM1 can markedly increase the size of tumors (11). FoxM1 can also promote tumor angio- genesis by enhancing VEGF expression at the transcriptional 786 YAN et al: AKT/AP1/FoxM1 AXIS PROMOTES SORAFENIB RESISTANCE level (12) and suppression of FoxM1 inhibited angiogenesis blot analysis was performed as previously described (19). in liver cancer (13). Additionally, senescence was induced in Anti‑FoxM1 (dilution 1:1,000; cat. no. ab180710) and c‑jun FoxM1‑knockout embryonic fibroblasts, while H2O2‑induced (dilution 1:1,000; cat. no. ab31419) primary antibodies were senescence was reversed by FoxM1 overexpression (14). purchased from Abcam. Phosphorylated (p)‑JNK (dilution Recent studies have indicated that the overexpression of 1:1,000; cat. no. 9255), p‑AKT (dilution 1:1,000; cat. no. 4060), FoxM1 may accelerate the development of acquired drug p‑ERK (dilution 1:1,000; cat. no. 9101) antibodies were resistance (15‑17). However, whether the overexpression of obtained from Cell Signaling Technology, Inc. GAPDH FoxM1 contributes to sorafenib resistance in liver cancer (dilution 1:5,000; cat. no. sc‑137179) antibody was obtained requires further investigation. from Santa Cruz Biotechnology, Inc. In the present study, FoxM1 was significantly upregu- lated in sorafenib‑resistant liver cancer cells. Knockdown of Reverse transcription‑quantitative PCR (RT‑qPCR). Cells FoxM1 increased the sorafenib sensitivity of drug‑tolerant were lysed and total RNA was extracted with TRIzol reagent cells. Furthermore, a signaling axis, the AKT/activator (Thermo Fisher) as previously described (19), and the protein‑1 (AP1)/FoxM1 signaling pathway, was revealed first‑strand cDNA was synthesized using M‑MLV transcrip- to contribute to sorafenib resistance in liver cancer cells. tase (Invitrogen; Thermo Fisher Scientific, Inc.). qPCR was Suppression of this axis using AKT inhibitor BEZ‑235 increased performed to detect the level of mRNA using SYBR qPCR the sensitivity of drug‑tolerant cells to sorafenib in vitro and master mix (Takara Biotechnology Co., Ltd.) in a 20‑µl in vivo. The data indicated that the AKT/AP1/FoxM1 signaling volume according to the manufacturer's instructions. FOXM1 axis is a critical determinant of sorafenib tolerance. was amplified using the forward primer, 5'‑GCC ATC AAC AGC ACT GAG AG‑3' and reverse primer, 5'‑ TGG GGT GAA Materials and methods TGG TCC AGA AG‑3'. JUN was amplified using the forward primer, 5'‑GAG CTG GAG CGC CTG ATA AT‑3' and reverse Cell culture. Human liver cancer cell lines HepG2 and primer. 5'‑CCC TCC TGC TCA TCT GTC A C ‑ 3 ' . Huh7 cells were purchased from the American Type Culture Collection (ATCC), and cultured in high‑glucose DMEM with Plasmid construction. To construct reporter and mutant 10% FBS, in a humid incubator with 5% CO2. The two lines reporter plasmids, FoxM1 genomic DNA was extracted were originally tested by ATCC and passaged <6 months in from HepG2 cells. The promoter region of human FoxM1 the laboratory of Biochemistry and Molecular Biology of (‑2,000 to +100) containing the AP1 binding site was ampli- Hainan Medical College. fied by PCR using the PrimeSTAR HS DNA polymerase (Takara Biotechnology Co., Ltd.), and then inserted into Establishment of sorafenib‑resistant cells. The IC50 value of the pGL3‑Basic vector (Promega Corp.). The recombinant liver cancer cells to sorafenib was initially determined by plasmid was termed pGL3‑FoxM1. The mutant plasmid the 4PL method as previously described (18). The equation is containing the promoter without the AP1 binding site expressed as follows: ; where Y is the response, and was constructed using an overlap primer and termed X is the concentration. The variable ‘a’ is the bottom of the pGL3‑FoxM1‑mu. curve, and ‘d’ is the top of the curve. The variable ‘b’ is the slope factor, and ‘c’ is the concentration corresponding to the Transfection assay. The small interfering RNAs against response midway between ‘a’ and ‘d’. The cells were cultured FoxM1, c‑Jun and control siRNA were obtained from Santa in 6‑well plates at 1x10 4 cells/well and incubated with sorafenib Cruz Biotechnology, Inc. The cells were separately transfected at a concentration just below their respective IC50 values. The with FoxM1 or c‑jun siRNAs and control siRNA (Shanghai concentration of sorafenib was slowly increased by 0.25 µmol/l GenePharma Co., Ltd.) at 50 nM using Lipofectamine® 2000 per week. After 6‑7 months, two sorafenib‑resistant cell (Invitrogen; Thermo Fisher Scientific, Inc.) according to the lines, termed ‘HepG2‑SR’ and ‘Huh7‑SR’, were obtained and manufacturer's instructions. were continuously maintained by culture in the presence of sorafenib. Luciferase reporter assay. HepG2 cells were seeded in 48‑well plates and cultured to 70‑80% confluence. Then, the cells were Cell viability assay. Cell viability was assayed using a Cell co‑transfected with pGL3‑FoxM1/or pGL3‑FoxM1‑mu and Counting Kit‑8 (CCK‑8; Dojindo Molecular Technologies, monitor plasmid RL‑PTK. After 36 h, the cells were lysed, Inc.). Briefly, the cells were seeded in triplicate in 96‑well and the Firefly andRenilla luciferase activities were measured plates and underwent different treatments (including sorafenib using the Dual‑Luciferase Reporter System (Promega Corp.) at 5, 10, 15 and 20 µmol/l dose, sorafenib combined FoxM1 according to the manufacturer's instructions. The transfection siRNA, sorafenib combined c‑jun siRNA and sorafenib experiments were performed at least three times in triplicate. combined BEZ‑235) for 48 h, and then the optical density Data are presented as the fold induction after normalizing the value at 450 nm was detected according to the manufacturer's luciferase activity of the tested sample to that of the corre- instructions.
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