Silencing of NRAGE induces autophagy via AMPK/Ulk1/Atg13 signaling pathway in NSCLC cells

709676TUB0010.1177/1010428317709676Tumor BiologyZhou et al. research-article2017 Original Article Silencing of NRAGE induces autophagy via AMPK/Ulk1/Atg13 signaling pathway in NSCLC cells Tumor Biology June 2017: 1 8 The Author(s) 2017 Reprints and permissions: sagepub.co.uk/journalspermissions.nav https://doi.org/10.1177/1010428317709676 DOI: journals.sagepub.com/home/tub Yiyang Zhou 1, Nan Huang 2, Jianchun Wu 1, Ni Zhen 2, Ning Li 3, Yan Li 1 and Yong-Xin Li 1,3 Abstract NRAGE has been reported to be overexpressed in cancer cells, especially in lung cancer cells. To determine the role of NRAGE in non-small-cell lung cancer cells, we investigated the effects of NRAGE on autophagy in non-small-cell lung cancer cells. Human A549 and H1299 cells were transfected with NRAGE-specific small-interfering RNA. The Cell Counting Kit-8 and plate clone assay showed that downregulation of NRAGE could induce the proliferation in A549 and H1299 cells. In addition, our data suggested that downregulation of NRAGE enhances autophagosome formation by immunofluorescence. We found that knockdown of NRAGE induced autophagy, together with downregulation of P62 and upregulation of LC3-II protein. Furthermore, to elucidate the mechanism of NRAGE in suppressing autophagy, the protein expressions of AMPK, Ulk1, and Atg13 were assessed. Collectively, these results demonstrate the effective anti-autophagic of NRAGE in non-small-cell lung cancer cells through AMPK/Ulk1/Atg13 autophagy signaling pathways. Therefore, NRAGE could be used as a potential therapeutic target for lung cancer. Keywords NRAGE, proliferation, autophagy, AMPK/Ulk1/Atg13 signaling pathway, non-small-cell lung cancer Date received: 31 March 2017; accepted: 22 April 2017 Introduction Neurotrophin receptor interacting melanoma antigen encoding gene homolog (NRAGE), also known as MAGE-D1 or Dlxin-1, is a member of the MAGE family of proteins. It has recently been reported that NRAGE is overexpressed in lung cancer, 1 pancreatic cancer, 2 breast cancer cells, 3 gastric cancer, 4 and esophageal carcinomas 5 and play pivotal roles in regulating various cellular functions such as regulation of apoptosis, cell cycle, and cell proliferation. 6 In recent decades, efforts have been made to dissect the relationship between NRAGE and tumorigenesis. An increasing number of studies show that NRAGE plays a crucial role in regulating the tumorigenesis and metastasis. Downregulation of NRAGE may be associated with the formation and metastasis in a variety of tumor cells, such as hepatocarcinoma 7 and breast cancer. 3 Furthermore, the overexpression of NRAGE has been reported to inhibit angiogenesis in vitro and in vivo. 8 All the research above indicated that NRAGE played a tumor suppressor role. Autophagy is a general term for the process by which cytoplasmic material is delivered to lysosomes for degradation. More than 30 genes have been involved in 1 Department of Oncology, Shanghai Municipal Hospital of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, China 2 Department of Clinical Laboratory Medicine, Shanghai Tenth People s Hospital of Tongji University, Shanghai, China 3 Central Laboratory, Shanghai Municipal Hospital of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, China Corresponding authors: Yan Li, Department of Oncology, Shanghai Municipal Hospital of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 200071, China. Email: 18916767226@163.com Yong-Xin Li, Department of Oncology, Shanghai Municipal Hospital of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 200071, China. Email: lyxycg@hotmail.com Creative Commons Non Commercial CC BY-NC: This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 License (http://www.creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage).

2 Tumor Biology autophagy called ATG for autophagy-related. ATG13 is a target of the target of rapamycin (TOR) kinase signaling pathway that regulates autophagy through phosphorylation of ATG13 and ULK1 and the regulation of the ATG13/ ULK1 complex. 9 Ulk1/atg13 complex plays a role in the regulation and function of the kinase activity of cell proliferation in macroautophagy. The adenosine monophosphate activated protein kinase (AMPK) regulates autophagy by phosphorylating the autophagy-associated kinases ULK1. There is evidence to support a role for AMPK in autophagy induction in response to various cellular stresses, including glucose starvation. 10 Up to now, there is no data demonstrating the role of NRAGE in autophagy. In this study, we elucidate the effect of NRAGE on autophagy in non-small-cell lung cancer (NSCLC). We demonstrated that downregulation of NRAGE significantly induced proliferation and autophagy via AMPK/Ulk1/Atg13 signaling pathway in NSCLC cells, suggesting that NRAGE is a critical regulator of tumorigenesis through regulating these cellular processes. Therefore, understanding how NRAGE regulates autophagy involved in NSCLC development required strategies for the treatment of lung cancer. Materials and methods Antibodies and reagents The following primary antibodies were used in this study: anti-ampk antibody (#2532S; Cell Signaling Technology), anti-p-ampk antibody (#2535S; Cell Signaling Technology), anti-lc3 I/II (#4108S; Cell Signaling Technology), anti-p62/sqstm1 (#8025S; Cell Signaling Technology), anti-beclin1 (ab62557; Abcam), anti- NRAGE (sc-136552; Santa Cruz Biotechnology), anti- GAPDH (#5174; Cell Signaling Technology), anti-ulk1 (#2707773; Millipore), anti-p-ulk1 (Ser758; #2571270; Millipore), anti-atg13 (#13468S; Cell Signaling Technology), anti-p-atg13 (S355; #43533S; Cell Signaling Technology), anti-pi3k (#4263S; Cell Signaling Technology), and anti-mtor (#2643610; Millipore). Secondary antibodies used for western blotting were as follows: 800CW goat anti-mouse and 800CW goat antirabbit, purchased from LI-COR Biosciences. Hydroxychloroquine (HCQ), purchased from Sigma, was diluted in phosphate-buffered saline (PBS, ph 8.0) at a stock concentration 5 M. Cell culture The NSCLC cell lines (A549 and H1299) purchased from American Type Culture Collection (ATCC) were all maintained in complete Dulbecco s Modified Eagle Medium (DMEM; Gibco), supplemented with 10% fetal bovine serum (FBS; Gibco), 100 units/ml penicillin, and 100 µg/ml streptomycin at 37 C in a humidified incubator with 5% CO 2. Small-interfering RNA and cell transfection The small-interfering RNAs (sirnas) against non-specific sequence (sicontrol) or different sequences of NRAGE (sinrage #1 and #2) were purchased from Sigma. Transfection of sirnas and establishment of stable cell lines were all performed according to our previous experimental procedures. 5,11 Cell Counting Kit-8 assay The cell proliferation was determined using Cell Counting Kit-8 (CCK-8) kit and carried out according to our previous protocols. 11 In short, the stably transfected A549 and H1299 cells with sicontrol or sinrage#1 and #2 (equal concentrations) were plated at a density of 1 10 4 cells/well in 96-well multiplates. After 24 h, 10 µl of CCK-8 solution was added to each well and further incubated for 2 h. Then, the absorbance values were detected at a wavelength of 450 nm using a Bio- Rad microplate reader. The cell viability was calculated by the optical density (OD) values of treated groups/od values of control groups 100%. Plate clone assay To find the effect of NRAGE silencing on the colony formation of non-small lung cancer cells, A549 and H1299 cells were seeded into six-well plates at a density of 500 cells/well after transfection with sicontrol or sin- RAGE#1 and #2 (equal concentrations). The medium was changed every 3 days until 2 weeks of culture. The cells were fixed with 4% paraformaldehyde (PFA) and then stained with freshly prepared and diluted Crystal violet stain for 20 min. After rinsing with distilled water, the colonies formed in each well were counted under light/fluorescence microscopy. Autophagic flux assay The autophagic lentiviruses expressing Stub-RFPSens- GFP-LC3 wild-type (GFP-RFP-LC3 WT) deficiency of glycine at 120 site were commercially purchased from GeneChem (Shanghai, China). According to the manufacture s protocol, A549 cells were infected with the lentiviruses for 36 h and then selected with 2 µg/ml puromycin (Sigma) for 72 h. Finally, the efficiency was confirmed by observing the expression of GFP and RFP under the inverted fluorescence microscope (Leica). Then, the stable A549 GFP-RFP-LC3 WT cells were seeded into the 24-well plates. Afterwards, the cells on the coverslips were fixed with 4% PFA, perforated with 0.5% Triton X-100, and stained with 4,6-diamidino-2-phenylindole (DAPI) for 2 min at room temperature away from light. Finally, the slides were sealed and pictured under the inverted confocal fluorescence microscope (Zeiss).

Zhou et al. 3 Figure 1. Effect of NRAGE knockdown on the viability of NSCLC. (a) and (b) Clonogenic survival analysis of stable A549 and H1299 cells, infected with sinrage #1 or sinrage #2. (c) Transfected A549 and H1299 cells were tested by CCK-8 assay at 24 h. Each value was expressed as the mean ± SD of triplicate experiments (*p < 0.05 and **p < 0.01 as compared with control groups). Western blot Western blotting was performed according to standard procedures. 12 A total of 80 µg protein was separated and transferred onto nitrocellulose membranes. Membranes were then blocked and incubated with primary antibodies at 4 C overnight. The membranes were then washed and probed with appropriate secondary antibodies. After the final wash, the membranes were visualized using the Odyssey LI-CDR system. All the gray-scale values in the study were obtained using WCIF ImageJ software. Statistical analysis All data are presented as means ± standard deviation (SD). The mean values were calculated based on the data taken from at least three independent experiments conducted on separate days using freshly prepared reagents. To determine the statistical significance, *p < 0.05 and **p < 0.01 were used. Results Effect of NRAGE knockdown on the viability of NSCLC cells To find out the role of NRAGE in NSCLC growth, we employed sirna to knock down NRAGE gene expression in A549 and H1299 cells. As shown in Figure 1(a), CCK-8 experiments demonstrated that downregulation of NRAGE enhances cell growth compared with control groups in both A549 and H1299 cell lines. Moreover, the results from colony formation assays further confirmed that NRAGE suppressed the growth of A549 and H1299 Cells (Figure 1(b) and (c)). These results indicated that knockdown of NRAGE could remarkably inhibit the viability of NSCLC cells. Knockdown of NRAGE regulated autophagy marker proteins in NSCLC cells Autophagy can act either as a tumor suppressor or as a survival mechanism for established tumors. 13 We previously reported that NRAGE promotes cancer cell proliferation and suggested that NRAGE could play a potential role in autophagy. 2 To explore the role of NRAGE on autophagy in NSCLC, we evaluated autophagy markers by western blotting and found that LC3-II turnover and SQSTM/p62 degradation were obviously increased compared to controls in A549 and H1299 cells (Figure 2). Our data suggested that knockdown of NRAGE could enhance autophagy in NSCLC cells. Knockdown of NRAGE increased accumulation of autophagosomes in A549 LC3-GFP-RFP WT cells To assess whether NRAGE inhibits autophagosome formation in lung cancer cells, autophagosomes were stained

4 Tumor Biology Figure 2. NRAGE deficiency increases accumulation of autophagosomes in NSCLC. Western blot analysis of LC3, P62, and Beclin1 protein levels in A549 (a) and H1299 (b) lung cancer cell lines after 24 h of transfection. GAPDH protein was used as an internal control. Each value was expressed as the mean ± SD of triplicate experiments (*p < 0.05 and **p < 0.01 as compared with control groups). with a specific GFP-RFP-LC3, and the presence of autophagosomes in A549 cells was determined by Confocal Laser Scanning Microscope. A549 cells transduced with empty vector were used as control. The number of cells with autophagosomes and autophagosomes per cell were increased upon HCQ treatment, suggesting that knockdown of NRAGE leads to accumulation of autophagosomes due to blocking of the fusion of autophagosomes with lysosomes, thereby inhibiting autophagy (Figure 3). The effect of NRAGE on autophagy signaling pathway related molecules Ulk1, Atg13, PI3k, and mtor in NSCLC The Akt/PI3k/mTOR signaling pathway is known to regulate several downstream processes including protein synthesis and autophagy. 14 And inhibition of mtor activity led to the activation of autophagy via the ULK1-ATG13-FIP200 complex. 15 In an attempt to elucidate the mechanism of NRAGE in suppressing cellular growth and autophagy, the protein expressions of Ulk1, Atg13, PI3k, and mtor were assessed in A549 and H1299 cells. Our results showed that total protein expression of Ulk1 and Atg13 was upregulated upon knockdown NRAGE in both cell lines when compared with control cells (Figure 4), but the protein expression of PI3k and mtor were not altered in both cell lines. This result indicated that the inhibition effect of NRAGE on cellular autophagy was associated with Ulk1/Atg13 complex. The effect of NRAGE on the expression of phosphorylation of AMPK, Ulk1, and Atg13 in NSCLC Previous studies have shown that AMPK regulates autophagy through direct phosphorylation of Ulk1/Atg13 complex. 10 In this study, we examined the phosphorylation

Zhou et al. 5 Figure 3. RFP-GFP-LC3 distribution in A549 cells transfected with RFP-GFP-LC3 and NRAGE or control sirnas was analyzed by confocal microscopy. Cells were treated with 20 μmol/l HCQ for 4 h. (a) Cell morphology was observed by fluorescence microscopy. The bottom panel indicates quantification of autophagosomes or LC3 puncta numbers. The asterisks indicate significant statistical difference. (b) Efficiency of sirna transfection in A549 WT LC3-GFP-RFP cell line. (*p < 0.05 and **p < 0.01) as compared with control groups (scale bar = 5 μm). Figure 4. The effect of NRAGE on autophagy signaling pathway related proteins Ulk1, Atg13, PI3k, and mtor in NSCLC. A549 and H1299 cells were pre-transfected with specific sirna against NRAGE or control sinc at 30 nm into six-well plates at 24 h. Western blot analysis shows that total proteins of Ulk1 and Atg13 were upregulated after 24 h of transfection in A549 and H1299 lung cancer cell lines. GAPDH was used as a loading control. The values represented are relative to control. Each value was expressed as the mean ± SD of triplicate experiments (*p < 0.05 and **p < 0.01 as compared with control groups).

6 Tumor Biology Figure 5. The effect of NRAGE on the expression of phosphorylation of Ulk1 and Atg13 in NSCLC. Western blot analysis shows that phosphorylation of Ulk1 and Atg13 was upregulated after 24 h of transfection in A549 (a) and H1299 (b) lung cancer cell lines. The values represented are relative to control. Each value was expressed as the mean ± SD of triplicate experiments (*p < 0.05 and **p < 0.01 as compared with control groups). levels of AMPK, Ulk1, and Atg13 compared with 50 mm hydroxychloroquine (HCQ) treatment at 4 h in A549 and H1299 cells. Our results showed that knockdown of NRAGE activates Ulk1 through phosphorylation of Ser758 in both cell lines. Under HCQ treatment, the protein expressions of Ulk1 and Atg13 were higher than without HCQ treatment (Figure 5). This suggests that NRAGE may be directly involved in the regulation of autophagy via AMPK/Ulk1/Atg13 signaling pathway. Discussion Previous studies report that NRAGE plays a crucial role in regulating the tumorigenesis, proliferation, and apoptosis. 6

Zhou et al. 7 Up till now, there is no data about NRAGE in autophagy and proliferation in lung cancer. In this study, we demonstrated that silencing of NRAGE induces autophagy and inhibits proliferation in NSCLC cells and the AMPK/ ULk1/ATG13 pathway is required for this autophagy. In recent years, there is increasing evidence to suggest that NRAGE has anti-proliferative activity. When NRAGE acts as a tumor suppressor, it is mainly localized to the cytoplasm. However, when it promotes epithelial-to-mesenchymal transition, NRAGE is reported to transduce into the nucleus. 16 Our previous data demonstrated that NRAGE promotes proliferation in the nucleus of the esophageal tissues by enhancing the proliferating cell nuclear antigen (PCNA) protein. 11 Moreover, NRAGE showed significant anti-proliferative effects in the kidney epithelial cells (HEK293), 17 human hepatocellular carcinoma cells (HepG2), 18 and human breast cancer (MDA-MB-231 and MCF-7) 3 through p53-dependent pathway. In this study, CCK-8 and cell clone formation assay showed that knockdown of NRAGE could effectively promote the proliferation of A549 and H1299 lung cancer cells. NRAGE mainly localizes in the cytoplasm of the A549 lung cancer cell line, as demonstrated in Supplementary Figure 1S. Unfortunately, it is still unclear what mechanisms enable NRAGE to shuttle between the cytoplasm and nucleus, and further studies are needed to answer this question. AMPK is a tumor suppressor and sensor of cellular energy status. It plays an important role in proliferation and autophagy. 19 AMPK can be phosphorylated and activated by liver kinase B1, calcium-dependent protein kinase kinase-β, and some small molecules. Activated AMPK is closely related to tumor growth, invasion, and migration of cancer cells. 20 Therefore, AMPK is a positive regulator that stimulates autophagy in response to energy depletion. Recent studies suggest that AMPK directly regulates autophagy through phosphorylating and activating ULK1. In this study, the results obtained clearly indicated that knockdown of NRAGE activates AMPK in lung cancer cell lines A549 and H1299, thereby suppresses the lung cancer cell proliferation. Apoptosis and autophagy are both forms of programmed cell death. 21 Gump reported that the process of autophagy could control apoptosis by making it either more or less probable. Furthermore, the process of apoptosis could control autophagy. 22 The autophagy proteins p62 and Beclin1 are likely crucial molecular players, regulating and being regulated by pro- and anti-apoptotic molecules. 23 A large number of studies have indicated that NRAGE induced apoptosis through combining with the P75 nerve growth factor receptor (p75ntr) and XIAP-TAKITAB1, downregulating Che-1, and activating the nuclear factorκb (NF-κB) pathway. 6 These researches demonstrate that NRAGE also plays pivotal role on apoptosis in different kinds of cancer. However, there is no data known about NRAGE in autophagy. Therefore, in this study, we elucidated the effect of NRAGE on autophagy in NSCLC cells. Our results suggest that NRAGE may induce apoptosis by inhibiting autophagy. In summary, to investigate the probable mechanism of anti-proliferative efficacy of NRAGE in NSCLC, we examined the effect of NRAGE knockdown on autophagy. In this study, we introduce NRAGE as a new autophagy regulatory gene that acts as a tumor suppressor in lung cancer cells. Our results have shown that knockdown of NRAGE enhanced autophagy by the activation of autophagy-related protein AMPK, Ulk1, and Atg13 in A549 and H1299 lung cancer cells. The results provide a better understanding of the mechanisms for the role of NRAGE in tumor microenvironment. Therefore, knockdown of NRAGE could be developed as a promising cancer therapeutic target. Acknowledgements The first two authors (Y.Z. and N.H.) have contributed equally to this work. Declaration of conflicting interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Funding The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by Bureau-Level Scientific Research Projects Shanghai Municipal Commission of Health and Family Planning (20154Y0046) and Annual Research Budget of Shanghai University of Traditional Chinese Medicine (2015YSN55) and sponsored by Shanghai Pujiang Program (16PJ1409000). References 1. Lee DH, Lee GK, Yoon KA, et al. Pathway-based analysis using genome-wide association data from a Korean nonsmall cell lung cancer study. PLoS ONE 2013; 8(6): e65396. 2. Chu CS, Xue B, Tu C, et al. NRAGE suppresses metastasis of melanoma and pancreatic cancer in vitro and in vivo. Cancer Lett 2007; 250: 268 275. 3. Du Q, Zhang Y, Tian XX, et al. MAGE-D1 inhibits proliferation, migration and invasion of human breast cancer cells. Oncol Rep 2009; 22: 659 665. 4. Zhang Z, Yan A, Zhang Z, et al. 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