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Published OnlineFirst October 3, 2014; DOI: 10.1158/1940-6207.CAPR-14-0233 Cancer Prevention Research Article Research Crucial Role of c-Jun Phosphorylation at Ser63/73 Mediated by PHLPP Protein Degradation in the Cheliensisin A Inhibition of Cell Transformation Junlan Zhu1,2, Jingjie Zhang1, Haishan Huang1,2, Jingxia Li1, Yonghui Yu1, Honglei Jin1,2, Yang Li1,2, Xu Deng3, Jimin Gao2, Qinshi Zhao3, and Chuanshu Huang1 Abstract Cheliensisin A (Chel A), as a novel styryl-lactone isolated from Goniothalamus cheliensis Hu, has been demonstrated to have an inhibition of EGF-induced Cl41 cell transformation via stabilizing p53 protein in a Chk1-dependent manner, suggesting its chemopreventive activity in our previous studies. However, its underlying molecular mechanisms have not been fully characterized yet. In the current study, we found that Chel A treatment could increase c-Jun protein phosphorylation and activation, whereas the inhibition of c-Jun phosphorylation, by ectopic expression of a dominant-negative mutant of c-Jun, TAM67, reversed the Chel A inhibition of EGF-induced cell transformation and impaired Chel A induction of p53 protein and apoptosis. Moreover, our results indicated that Chel A treatment led to a PHLPP downregulation by promoting PHLPP protein degradation. We also found that PHLPP could interact with and bind to c-Jun protein, whereas ectopic PHLPP expression blocked c-Jun activation, p53 protein and apoptotic induction by Chel A, and further reversed the Chel A inhibition of EGF-induced cell transformation. With the findings, we have demonstrated that Chel A treatment promotes a PHLPP protein degradation, which can bind to c- Jun and mediates c-Jun phosphorylation, and further leading to p53 protein induction, apoptotic responses, subsequently resulting in cell transformation inhibition and chemopreventive activity of Chel A. Cancer Prev Res; 7(12); 1–12. Ó2014 AACR. Introduction growth (5). However, the following studies have found that Cheliensisin A (Chel A), as a novel styryl-lactone isolated PHLPP could also act as a tumor suppressor in several types from Goniothalamus cheliensis Hu, has been reported to of cancer due to its ability to block growth factor–induced possess the potent chemoprevention effect (1–3). Our signaling in cancer cells (5, 6). Most recently, the studies published studies have demonstrated that chemopreventive from our group have indicated that PHLPP1 downregula- activity is mediated by its induction of apoptosis via trig- tion serves as cell apoptosis controller by promoting p53 gering p53 protein expression and activation (4). The protein translation via activation of Akt/p70S6K cascade pleckstrin homology domain leucine-rich repeat protein (7). We found here that PHLPP was downregulated in cells phosphatase (PHLPP), including PHLPP1 and PHLPP2, are treated with Chel A, which mediated chemopreventive protein phosphatases, which have been demonstrated to activity of Chel A. specifically dephosphorylate the hydrophobic motif of Akt, c-Jun, a member of the basic region leucine zipper subsequently triggering apoptosis and suppressing tumor protein family of transcription factors, in combination with itself or other proteins such as c-Fos, forms the transcription factor activator protein 1 (AP-1). c-Jun pro- 1Nelson Institute of Environmental Medicine, New York University School tein consists of a C-terminal DNA-binding domain and of Medicine, Tuxedo, New York. 2Zhejiang Provincial Key Laboratory for an N-terminal transactivation domain. The transcriptional Technology and Application of Model Organisms, School of Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, China. 3State Key Lab- activity of c-Jun is increased by phosphorylation of serines oratory of Phytochemistry and Plant Resources in West China and 63 and 73 in the transactivation domain (8, 9). c-Jun Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, phosphorylation at Ser 63 and Ser 73 could be mediated China. by activation of JNKs upon a large variety of external or J. Zhu, J. Zhang, and H. Huang contributed equally to this article. internal stimulations (10–12) or the inhibition of its phos- Corresponding Authors: Chuanshu Huang, Nelson Institute of Environ- phatase. However, to the best of our knowledge, phospha- mental Medicine, New York University School of Medicine, 57 Old Forge Road, Tuxedo, NY 10987. Phone: 845-731-3519; Fax: 845-351-2320; tase that targets phosphorylated c-Jun protein has not been E-mail: [email protected]; Qinshi Zhao, E-mail: identified yet. Upon activation, c-Jun exerts various biologic [email protected]; and Jimin Gao, E-mail: [email protected] effects on cell proliferation, differentiation, cellular trans- doi: 10.1158/1940-6207.CAPR-14-0233 formation, and apoptosis (10–12). It has been reported Ó2014 American Association for Cancer Research. that inhibition of c-Jun activation by expressing a c-Jun www.aacrjournals.org OF1 Downloaded from cancerpreventionresearch.aacrjournals.org on September 30, 2021. © 2014 American Association for Cancer Research. Published OnlineFirst October 3, 2014; DOI: 10.1158/1940-6207.CAPR-14-0233 Zhu et al. dominant-negative mutant TAM67 inhibits apoptosis due Reagent (SignaGen Laboratories) following the manufac- to survival signal withdrawal (11). In the current study, we turer’s instructions. Their stable transfectants were estab- revealed that Chel A treatment resulted in PHLPP protein lished by G418-resistant selection. PW cells were transfected degradation, which further mediated c-Jun phosphoryla- with TAM67 or its corresponding vector control by using the tion at Ser 63 and 73 through JNK-independent manner. same method as described above, and the stable transfec- Moreover, we found the downregulation of PHLPP and its tants were selected by G418. mediated activation of c-Jun were essential for the induction of apoptosis as well as the inhibition of cell transformation Anchorage-independent growth in soft agar induced by EGF. Soft agar colony formation assay was conducted as described previously (4, 15, 16, 19). Briefly, 2.5 mL of Materials and Methods 0.5% agar in basal modified Eagle medium (BMEM) sup- Reagents and plasmids plemented with 10% FBS and 20 ng/mL EGF, as well as Chel Chel A was isolated from Goniothalamus cheliensis by the A at indicated concentrations, was layered onto each well of Kunming Institute of Botany, Chinese Academy of Sciences 6-well tissue culture plates. A total of 1 Â 104 Cl41 cells, and (Kunming, Yunnan, China) as previously described (1, 3). their stable transfectants, were mixed with 1 mL of 0.33% The chemicals cycloheximide and MG132 were purchased agar BMEM (supplemented with 10% FBS with or without from Calbiochem. Luciferase assay substrate and EGF 20 ng/mL EGF, as well as with or without Chel A), and were from Promega. The antibodies specific against c-Jun, layered on top of the 0.5% agar layer. The plates were c-Jun(D), p-c-Jun Ser63, p-c-Jun Ser73, p-AKT Ser473, p- incubated at 37 Cin5%CO2 for 3 weeks. The colonies AKT Thr308, AKT, p-Erk1/2, Erk1/2, p-p38, p38, p-JNK1/2, were then counted under inverse microscopy. Colonies with JNK1/2, PARP, cleaved PARP, caspase-3, cleaved caspase-3, more than 32 cells were scored. Each experiment was done p53, p-p53 Ser15, GFP, and GAPDH were purchased from at least three independent times. The results were presented Cell Signaling Technology. HA antibody was obtained from as colonies/104 seeded cells. Covance Inc.. Antibodies specific against PHLPP1 and PHLPP2 were purchased from Bethyl Laboratories. Anti- Flow cytometry assay bodies against b-actin and a-tubulin were bought from Flow cytometry assay was conducted as described previ- Sigma. The plasmid, HA-PHLPP1 and HA-PHLPP2 were ously (4, 16, 20). Cl41 cells and their stable transfectants from Addgene. The plasmids, AP-1-luciferase reporter, were cultured in 6-well plates until they reached 70% to dominant-negative c-Jun–mutant plasmid TAM67, and 80% confluence. Cell culture medium was replaced with GFP-c-Jun were used and are described in our previous 0.1% FBS medium for 36 hours. The cells were then treated studies (13–15). with EGF (20 ng/mL) with or without Chel A at indicated concentrations in the medium containing 0.1% FBS. Cells Cell culture and transfection were harvested and fixed in ice-cold 70% ethanol. The cells Normal mouse epidermal Cl41 cells, which have been were stained with propidium iodide (PI) for 15 minutes and previously described (4, 16, 17), and their stable trans- then subjected to flow cytometry (Beckman Coulter) for fectants were maintained in 5% FBS Eagle’s minimum apoptotic analysis. essential medium (MEM), supplemented with 1% peni- cillin/streptomycin and 2 mmol/L L-glutamine(Life Tech- Western blotting nologies) at 37 Cin5%CO2 incubator that have been Cells were cultured using the same method described in described previously (4, 16, 17). PW cells have been flow cytometry assay, followed by pretreated with Chel A for described previously (18), and 293T cells and their stable 30 minutes, and afterwards exposed to EGF as indicated. transfectants were cultured in DMEM with 10% FBS. The The cells were subsequently washed on ice-cold PBS, and human colon cancer cell lines HCT116 cells and their then extracted with lysis buffer (10 mmol/L Tris–HCl, stable transfectants were cultured in McCoy’s 5A medium pH 7.4, 1% SDS, 1 mmol/L Na3VO4, and proteasome (Invitrogen), supplemented with 10% FBS. Cl41 cells inhibitor). The cell extracts were subjected to the Western stably transfected with AP-1 transactivation luciferase blot analysis and the protein bands specifically bound to reporter, TAM67, and their corresponding control vector antibodies were detected using alkaline phosphatase– have been established in our previous studies (15). These linked secondary antibody and ECF Western blotting sys- cells are all authenticated; the ATCC number of Cl41 cell tem as described previously (4, 16).

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