Original Article Knockdown of SALL4 overcomes cisplatin-resistance through AKT/mTOR signaling in lung cancer cells

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1 Int J Clin Exp Pathol 2018;11(2): /ISSN: /IJCEP Original Article Knockdown of SALL4 overcomes cisplatin-resistance through AKT/mTOR signaling in lung cancer cells Gang Jiang 1, Chen-Tao Liu 2 1 Department of Respiration, Hunan Provincial People s Hospital, No 61 Jiefang West Road, Changsha , Hunan Province, China; 2 Department of Pediatrics, Xiang Ya Hospital, Central South University, No 87 Xiangya Road, Changsha , Hunan Province, China Received April 10, 2017; Accepted June 27, 2017; Epub February 1, 2018; Published February 15, 2018 Abstract: Drug resistance is an important factor for the poor prognosis of non-small cell lung cancer (NSCLC). Sal-like protein 4 (Sall4) is a stem cell marker, and plays a role in maintaining self-renewal. Previous studies have demonstrated that Sall4 may be a candidate for use as support in the diagnosis of lung cancer, and may also represent a therapeutic target. However, the role of Sall4 on drug resistance of lung cancer cells and the mechanism by which Sall4 regulates the sensitivity of lung cancer cells to cisplatin (DDP) remains unknown. In this study, we aim to investigate whether knockdown of Sall4 by sirna can enhance the apoptosis induced by cisplatin in lung cancer cells. We here reported that the expression of Sall4 was dramatically upregulated in cisplatin-resistant A549 cells compared with the parental cells. Knockdown of Sall4 by sirna in cisplatin-resistant A549 cells reduced the IC 50 compared with the parental cells. In addition, knockdown of Sall4 significantly inhibited cell proliferation, induced apoptosis and invasion cisplatin-resistant A549 cells through AKT/mTOR signaling. Our findings demonstrate that Sall4 is an essential regulator in cisplatin-induced apoptosis, and knockdown of Sall4 may restore cisplatin sensitivity in acquired resistant cells. Thus, our study provides an effective therapeutic strategy for NSCLC treatment. Keywords: Non-small cell lung cancer, Sal-like protein 4, drug resistance, cisplatin, AKT/mTOR Introduction Lung cancer is the most common human cancer, leading to the most cause of cancer-related death worldwide, and most of lung cancers are non-small cell lung cancer (NSCLC) [1]. The incidence and mortality rates of NSCLC have been at a high level in both the western countries and the developing countries, including China [2]. Since the drug resistance is an important factor for the poor prognosis of NSCLC, finding novel targets to overcome required resistance may help develop effective strategies for the treatment of this disease [3]. Sal-like protein 4 (Sall4) is a stem cell marker, and plays a role in maintaining self-renewal. Upregulation of Sall4 is found in hepatocellular carcinoma (HCC), breast cancer and endometrial cancer, predicting a poor prognosis. Sall4 expression was also highly upregulated in lung cancer tissues [4]. Previous study showed that in primary lung adenocarcinomas, the stronger expression of Sall4 was inversely correlated with tumor differentiations; and in primary lung squamous cell carcinomas, the expression of Sall4 is inversely correlated with the tumor stage [5]. As a diagnostic marker, the sensitivity and specificity of SALL4 reached 88% and 100%, respectively [4]. Kobayashi D et al found that downregulation of Sall4 revealed catastrophic growth inhibition of lung cancer cells by inducing cell cycle arrest at the G1/early S phase. In addition, the period until recurrence was shorter in the patients showing high Sall4 expression [6]. Therefore, Sall4 may be a candidate for use as support in the diagnosis of lung cancer, and may also represent a therapeutic target. However, the role of Sall4 on drug resistance of lung cancer cells and the mechanism by which Sall4 regulates the sensitivity of lung cancer cells to cisplatin (DDP) remains unknown. Thus, in this study, we aim to investigate whether knockdown of Sall4 by sirna can overcome cisplatin-resistance in lung cancer cells.

2 Materials and methods Cell culture and reagents Human lung cancer cell line A549 and A549/ DDP (cisplatin-resistant cells) were obtained from the cancer hospital at the Chinese Academy of Medical Sciences (Beijing, China). Cells were cultured with RPMI1640 containing 10% fetal bovine serum (FBS, Gibco-BRL) and maintained in a humidified atmosphere of 5% CO 2 in air at 37 C. The A549/DDP cells medium additionally contained 2 μg/ml cisplatin to maintain the cells cisplatin resistance. The following antibodies were used in this study: β-actin (Santa Cruz Biotechnology, Dallas, TX, USA), Sall4, p-akt, AKT, p-mtor and mtor (Cell Signaling Tech), LRP and P-GP (Santa Cruz Biotechnology, SC-8007). Cisplatin was obtained from Sigma-Aldrich (St. Louis, MO, USA). RNA interference The lentivirus containing Sall4 sirna and scramble control were purchased from Genepharma Company (Beijing, China). The sequence targeting Sall4 was 5 -TTACTGTGGCTTC- ATCCTCAC-3. The A549/DDP cells were transfected with these lentivirus by using Lip3000 (Life technologies) according to the manufacturers instructions. Cell viability assay and colony formation assay Twenty four hours before the experiment, A549/DDP cells were plated in 96-well plates at a density of 1000 cells in 100 μl medium per well. The cells were treated with a range of cisplatin (0.01, 0.1, 1, 10, 100, 1000 μg/ml), and the cell viability was assessed by CKK-8 assay (Beyotime, Shanghai, China) according to the manufacturers instructions. For the colony formation assay, following treatment, adherent cells were trypsinised and 1000 viable cells were subcultured in six-well plates (in triplicate). Cells were allowed to adhere and colonise for two weeks. To visualise colonies, media were removed and cells were fixed in 96% ethanol for 10 min, and then stained with crystal violet staining solution. The colonies were captured and counted. Annexin V-FITC staining and flow cytometry Staining was performed by using Annexin V- FITC kit following the manufacturer s instructions (KeyGENBioTech, Nanjing, China). Briefly, A549/DDP cells were harvested by centrifugation at 1000 g for 5 min and resuspended in 100 μl binding buffer, followed by a 15 min incubation with 5 μl Annexin V-FITC in the dark at 37 C. After that, 10 μl PI staining was added with gentle shaking for 10 min incubation in the dark at 37 C. Flow cytometry (BD) analysis was employed for detecting apoptotic events. Western blotting Cells were lysed in cold RIPA buffer, and the protein was separated with 10% SDS-PAGE, which was then transferred to PVDF membrane (Thermo Fisher). After that, the membrane was incubated in PBS with 5% nonfat dried milk (Mengniu, Hohhot, China) for 3 hr at 4 C. Then, the membrane was incubated with primary antibodies (mouse monoclonal anti-β-actin, rabbit polyclonal anti-sall4, rabbit polyclonal anti-p-akt, rabbit polyclonal anti-akt, and mouse monoclonal anti-p-mtor and mtor, rabbit polyclonal anti-mrp1, rabbit polyclonal anti-lrp and rabbit polyclonal anti-p-gp) overnight at 4 C, and then with appropriate secondary antibody (Abcam) for 1 hr at 37 C. The immune complexes were detected using ECL Western Blotting Kit (Millipore). The relative protein expression was analyzed using Image- Pro plus software 6.0, and β-actin was used as the internal reference. Transwell assay The A549/DDP cells were starved for 24 h, and then resuspended in serum-free medium and added to the upper chamber. The lower chamber was filled with medium containing 10% FBS. Following 48 hours culture, cells attached to the bottom were fixed and stained with crystal violet for 45 min and dried in air. The optical density (OD) at 570 nm of crystal violet dissolved by 10% acetic acid was detected by an enzyme immunoassay analyzer (Synergy TM Mx; BioTek, Winooski, VT, USA). Statistical analysis In this study, all experiments were repeated at least three times, and all data are expressed as the mean ± S.E.M. SPSS 18.0 software package (SPSS, Chicago, IL, USA) was used to perform statistical analysis. Difference between two groups was compared by independent- 635 Int J Clin Exp Pathol 2018;11(2):

3 Figure 1. The expression of Sall4 in cisplatin-resistant A549 cells. A. Representative images of cells after cisplatin treatment. B. CKK-8 assay was used to determine the cell viability. The IC 50 of parental A549 cells (1.5 μg/ml) was significantly lower than those of in cisplatin-resistant A549 cells (A549/DDP) (14 μg/ml). C. Western blot analysis for MRP1, LRP and P-gp in A549 and A549/DDP cells. DDP, cisplatin. D. Western blot analysis for Sall4 in A549 and A549/DDP cells. DDP, cisplatin. resistant A549 cells by increasingly treating cisplatin. As shown in Figure 1A, the parental cells and resistant cells were treated with 14 μg/ml cisplatin. We observed that the cell number of parental cells was significantly decreased compared with cisplatin-resistant A549 cells. And we treated the cells with a range of concentrations of cisplatin to measure the half maximal inhibitory concentration (IC 50 ). We found that the IC 50 of parental cells (1.5 μg/ ml) was significantly lower than those of in cisplatinresistant A549 cells (14 μg/ ml) (Figure 1C). Total protein was isolated from the cells, and western blot was performed to examine the expression levels of drug resistant markers, including MRP1, LRP, P-gp. These proteins were significantly increased in A549/DDP cells (Figure 1C). Our data also showed that the expression of Sall4 was dramatically upregulated in cisplatin-resistant A549 cells compared with the parental cells (Figure 1D), indicating that upregulation of Sall4 may be involved in lung cancer cells resistance. Figure 2. Knockdown of Sall4 overcomes cisplatin resistance in A549/DDP cells. A. Western blot analysis for Sall4 in A549/DDP cells after transfection with Sall4 sirna or scramble control. B. CKK-8 assay was used to determine the cell viability. The IC 50 of scramble control cells was significantly lower than those of in cells knocking down Sall4. samples t test. The P value less than 0.05 were considered statistically significant. Results Sall4 is upregulated in cisplatin-resistant A549 cells Downregulation of Sall4 by sirna sensitizes A549 cells to cisplatin We further investigated whether alteration of Sall4 ex- To investigate role of Sall4 in cisplatin-resistant A549 cells, we generated the cisplatin- pression contributed to cispl- atin resistance of lung cancer cells. We knocked down the expression of Sall4 by sirna in cisplatin-resistant A549 cells (Figure 2A). We observed that downregulation of Sall4 significantly reduced the IC 50 compared with the parental cells (1.2 vs. 13 μg/ml, Figure 2B). Furthermore, we performed Transwell assay to evaluate the role of Sall4 on A549/DDP cell invasion. We found that knockdown of Sall4 in A549/DDP cells resulted in significant reduce 636 Int J Clin Exp Pathol 2018;11(2):

4 Figure 3. Downregulation of Sall4 reduces chemoresistance in A549/DDP cells. A. A549/DDP cells were transfected with Sall4 sirna or scramble, the effect of Sall4 on cell invasion was determined by Transwell assay. B. Colony formation assay showed the numbers of colonies of A549/DDP cells transfected with Sall4 sirna in the presence or absence of cisplatin (10 μg/ml). C. A549/DDP cells were transfected with Sall4 sirna in the presence or absence of cisplatin (10 μg/ml), cell apoptosis was determinded by flow cytometry. Data are presented as means ± SD. *P<0.05. of the ability of invasion compared with scramble control (Figure 3A). Moreover, to investigate whether the knockdown of Sall4 in A549/DDP could influence the cisplatin resistance, we performed colony formation assay. In absence of cisplatin, Inhibition Sall4 showed a decreased rate of colony formation. In present of cisplatin (10 μg/ml), knockdown of Sall4 sensitized A549/DDP cells to cisplatin, as indicated by significantly decreased rate of colony formation (Figure 3B). To further determine whether decreased cisplatin resistance of A549/DDP reflected cell apoptosis, the flow cytometry assays was performed. The results showed that both of the cisplatin treated or non-treated in Sall4 knocked down A549/DDP had higher apoptotic rates in comparison with the control group, and the apoptosis rate of Sall4 sirna 637 Int J Clin Exp Pathol 2018;11(2):

5 Figure 4. Sall4 regulates key molecules of AKT/mTOR signaling. A. Western blot analysis for Sall4, p-akt, AKT, p- mtor, mtor, LRP and P-GP in A549/DDP cells after transfection with Sall4 sirna or scramble control, and quantification. B. Quantification of the relative expression based on western blot. Data are presented as means ± SD. *P<0.05. group was significantly in present of cisplatin (Figure 3C). Oncogenic role of Sall4 is involved in AKT/ mtor signaling Previous study showed that activation of AKT/ mtor signaling is a cytoprotective event to cisplatin-induced cell death. Here, we evaluated whether downregulation of Sall4 would trigger inhibition of AKT/mTOR signaling in lung cancer cells. A549/DDP cells were transfected with Sall4 sirna lentivirus to knock down the levels of Sall4 and the cells transfected with scramble sequence were used as control. We found that cisplatin resistant cells showed activated AKT/mTOR signaling. Importantly, knockdown of Sall4 reversed this activation of AKT/mTOR signaling (Figure 4). Furthermore, we treated the A549/DDP cells with IGF1 to activate Akt signaling (Figure 5A). As shown in Figure 5B and 5C, the results demonstrated that activation of Akt/mTOR signaling by IGF1 can reversed the inhibitory effects of Sall4 downregulation evaluated by cell apoptosis and colony formation in A549/DDP cells, which were treated with 10 μg/ml cisplatin for 24 h. Discussion In this study, we report that the expression of Sall4 was dramatically upregulated in cisplatinresistant A549 cells compared with the parental cells. Knockdown of Sall4 by sirna in cisplatin-resistant A549 cells reduced the IC 50 compared with the parental cells. In addition, knockdown of Sall4 can increase apoptosis rate, inhibit colony formation and invasion through AKT/mTOR signaling. Due to chemoresistance, the prognosis of NS- CLC so far is still little optimism [7]. It has also been demonstrated that stem cell markers such as Oct3, SOX2, BMI-1 and Sall4 are associated with patient s prognosis, pathological stages, cancer recurrence and therapy resistance [8-10]. It was demonstrated that Sall4 could specifically bind to the c-myc promoter region, and downregulation of Sall4 leads to a decreased expression of c-myc at both protein and mrna levels. And manipulation of Sall4 expression could affect drug sensitivity of endometrial cancer cells to carboplatin [11]. Jeong HW et al found that overexpression of Sall4 led to drug resistance in leukemia cell lines, cells with decreased Sall4 expression were more sensitive to doxorubicin treatments than the parental cells by inhibiting side population cells [12]. In the drug resistant breast cancer cell line, MCF-7/ADR, Sall4 was significantly up-regulated compared to the other breast cancer cell lines. Down-regulation of SALL4 inhibited the proliferation of MCF-7/ADR 638 Int J Clin Exp Pathol 2018;11(2):

6 Figure 5. Activation of AKT/mTOR signaling reverses the inhibitory effects of Sall4 downregulation on A549/DDP cells. (A) Western blot analysis for p-akt, AKT, p-mtor, mtor in A549/DDP cells after transfection with Sall4 sirna or scramble control, and quantification. The A549/DDP cells were treated with a final concentration of 10 μg/ml cisplatin for 24 h, flow cytometry was used to measured cell apoptosis (B) and colony formation assay was used to determine the cell growth after indicated treatment (C). Data are presented as means ± SD. *P<0.05. cells and induced the G1 phase arrest in cell cycle [13]. In line with these findings, we demonstrated that down-regulating Sall4 can resensitize A549/DDP cells to cisplatin and had potent synergy with cisplatin in A549/DDP cells. Depletion of Sall4 led to a decrease in 639 Int J Clin Exp Pathol 2018;11(2):

7 IC 50 for cisplatin and an inhibitory effect on the ability to form colonies and the ability of invasion in A549/DDP cells, which was consist with its role in endometrial cancer [11]. With Sall4 knockdown, the expression of p-akt, p-mtor, LRP and P-GP was significantly decreased. PI3K/AKT/mTOR pathway is altered in a variety of cancers including non small cell lung cancer [14]. Moreover, aberrant activation of PI3K/AKT/mTOR pathway is one of the mechanisms of acquired resistance to EGFR-TK inhibitors [15]. Several inhibitors including AKT inhibitors, rapamycin and rapalogs for mtor inhibition are undergoing evaluation in preclinical and clinical studies [16, 17]. For example, NVP- BEZ235, a dual inhibitor of PI3K/mTOR, effectively inhibited the growth and migration of lung cancer cells, H1975 cells in vivo as well as in vitro by attenuating the phosphorylation of PI3K/AKT/mTOR signaling pathway proteins [18]. Knockdown of Sall4 by sirna resulted in inactivation of PTEN/PI3K/Akt signaling and subsequent inhibition of cell migration and invasion in intrahepatic cholangiocarcinoma cells [19]. Thus, our findings indicate that downregulation of Sall4 mediated inhibition of cell proliferation and enhancement of cisplatin induced apoptosis through inactivating AKT/mTOR signaling. In conclusion, these findings demonstrate that Sall4 is an essential regulator in cisplatin-induced apoptosis, and knockdown of Sall4 may restore cisplatin sensitivity in acquired resistant cells. Thus, our study provides an effective therapeutic strategy for use in NSCLC treatment. Acknowledgements This work was supported in part by the Hunan Provincal Natural Science Foundation of China (2015JJ2091), the Scientific Research Foundation from Ministry Education of Hunan Province of China (15C0832) and the Scientific Research Foundation from health and family commission of Huan province of China (B ). Disclosure of conflict of interest None. Address correspondence to: Dr. Gang Jiang, Department of Respiration, Hunan Provincial People s Hospital, No 61 Jiefang West Road, Changsha , Hunan Province, China. Tel: ; Fax: ; hnjianggang2016@163.com References [1] Hirsch FR, Suda K, Wiens J, Bunn PJ. New and emerging targeted treatments in advanced non-small-cell lung cancer. Lancet 2016; 388: [2] Olaussen KA, Postel-Vinay S. Predictors of chemotherapy efficacy in non-small-cell lung cancer: a challenging landscape. Ann Oncol 2016; 27: [3] Romanidou O, Imbimbo M, Mountzios G, Abidin A, Morgillo F, Califano R. Therapies in the pipeline for small-cell lung cancer. Br Med Bull 2016; 119: [4] Gautam AK, Wang C, Zeng J, Wang J, Lu J, Wei J, Huang G, Mo B, Luo M, Mo B. Expression and clinical significance of SALL4 and LGR5 in patients with lung cancer. Oncol Lett 2015; 10: [5] Rodriguez E, Chen L, Ao MH, Geddes S, Gabrielson E, Askin F, Zhang H, Li QK. Expression of transcript factors SALL4 and OCT4 in a subset of non-small cell lung carcinomas (NSCLC). Transl Respir Med 2014; 2: 10. [6] Yanagihara N, Kobayashi D, Kuribayashi K, Tanaka M, Hasegawa T, Watanabe N. Significance of SALL4 as a drugresistant factor in lung cancer. Int J Oncol 2015; 46: [7] Hiley CT, Le Quesne J, Santis G, Sharpe R, de Castro DG, Middleton G, Swanton C. Challenges in molecular testing in non-small-cell lung cancer patients with advanced disease. Lancet 2016; 388: [8] Iida H, Suzuki M, Goitsuka R, Ueno H. Hypoxia induces CD133 expression in human lung cancer cells by up-regulation of OCT3/4 and SOX2. Int J Oncol 2012; 40: [9] Islam F, Gopalan V, Wahab R, Smith RA, Lam AK. Cancer stem cells in oesophageal squamous cell carcinoma: identification, prognostic and treatment perspectives. Crit Rev Oncol Hematol 2015; 96: [10] Kaufhold S, Garban H, Bonavida B. Yin Yang 1 is associated with cancer stem cell transcription factors (SOX2, OCT4, BMI1) and clinical implication. J Exp Clin Cancer Res 2016; 35: 84. [11] Li A, Jiao Y, Yong KJ, Wang F, Gao C, Yan B, Srivastava S, Lim GS, Tang P, Yang H, Tenen DG, Chai L. SALL4 is a new target in endometrial cancer. Oncogene 2015; 34: [12] Jeong HW, Cui W, Yang Y, Lu J, He J, Li A, Song D, Guo Y, Liu BH, Chai L. SALL4, a stem cell factor, affects the side population by regula- 640 Int J Clin Exp Pathol 2018;11(2):

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