Published OnlineFirst November 14, 2012; DOI: 10.1158/0008-5472.CAN-12-2038 Cancer Therapeutics, Targets, and Chemical Biology Research Targeting Truncated Retinoid X Receptor-a by CF31 Induces TNF-a–Dependent Apoptosis Guang-Hui Wang1, Fu-Quan Jiang1, Ying-Hui Duan2, Zhi-Ping Zeng1, Fan Chen1, Yi Dai2, Jie-Bo Chen3, Jin-Xing Liu1, Jie Liu1, Hu Zhou1,3, Hai-Feng Chen1, Jin-Zhang Zeng1, Ying Su3, Xin-Sheng Yao2, and Xiao-Kun Zhang1,3 Abstract A truncated version of retinoid X receptor-a, tRXR-a, promotes cancer cell survival by activating the phosphoinositide 3-kinase (PI3K)/AKT pathway. However, targeting the tRXR-a–mediated survival pathway for cancer treatment remains to be explored. We report here our identification of a new natural product molecule, CF31, a xanthone isolated from Cratoxylum formosum ssp. pruniflorum, and the biologic evaluation of its regulation of the tRXR-a–mediated PI3K/AKT pathway. CF31 binds RXR-a and its binding results in inhibition of RXR-a transactivation. Through RXR-a mutational analysis and computational studies, we show that Arg316 of RXR-a, known to form salt bridges with certain RXR-a ligands, such as 9-cis-retinoic acid (9-cis-RA), is not required for the antagonist effect of CF31, showing a distinct binding mode. Evaluation of several CF31 analogs suggests that the antagonist effect is mainly attributed to an interference with Leu451 of helix H12 in RXR-a. CF31 is a potent inhibitor of AKT activation in various cancer cell lines. When combined with TNF-a, it suppresses TNF- a activation of AKT by inhibiting TNF-a–induced tRXR-a interaction with the p85a regulatory subunit of PI3K. CF31 inhibition of TNF-a activation of AKT also results in TNF-a–dependent activation of caspase-8 and apoptosis. Together, our results show that CF31 is an effective converter of TNF-a signaling from survival to death by targeting tRXR-a in a unique mode and suggest that identification of a natural product that targets an RXR- mediated cell survival pathway that regulates PI3K/AKT may offer a new therapeutic strategy to kill cancer cells. Cancer Res; 73(1); 307–18. Ó2012 AACR. Introduction with the development of human liver cancer (7) and colon a Retinoid X receptor (RXR)-a, a unique member of the cancer (8). Levels of RXR- protein are often reduced in – nuclear receptor superfamily, regulates diverse biologic pro- cancer cells and tumor tissues (9 14), suggesting that a cesses, including growth, differentiation, apoptosis, and diminished RXR- expression is associated with the devel- immune response, and ligands for RXR-a show promise as opment of certain malignancies. Numerous studies have a therapeutic agents for many diseases, such as cancer (1–4). shown that proteolytic cleavage of RXR- protein is primar- Alterations in the expression and function of RXR-a are ily responsible for its diminished levels in tumor cells and implicated in the development of a number of diseases and represents an important mechanism controlling the func- a – cancer. Targeted disruption of RXR-a gene leads to prostatic tion and activities of RXR- (13 18). Recent studies have a a preneoplastic lesions (5) and skin abnormalities (6). Dysre- shown that RXR- binding to PML/RAR- is essential for the gulation of RXR-a function by phosphorylation is associated development of acute promeylocytic leukemia (19, 20), fur- ther showing the oncogenic potential of this protein when it acts inappropriately. fi a Authors' Affiliations: 1School of Pharmaceutical Science, Xiamen Univer- Although signi cant progress has been made, how RXR- sity, Xiamen; 2Institutes of Traditional Chinese Medicine and Natural regulates cancer cell growth and how its ligands suppress 3 Products, Jinan University, Guangzhou, China; and Cancer center, San- tumorigenesis is still poorly understood. Like other nuclear ford-Burnham Medical Research Institute, La Jolla, California receptors, RXR-a interacts with DNA to regulate the transcrip- Note: Supplementary data for this article are available at Cancer Research tion of target genes in a ligand-dependent manner (1–4). Aside Online (http://cancerres.aacrjournals.org/). from its role in DNA binding and transactivation, accumulating G.-H. Wang and F.-Q. Jiang contributed equally to this work. evidence indicates that RXR-a also has extranuclear nonge- Corresponding Authors: Xiao-Kun Zhang, Sanford-Burnham Medical nomic actions (15, 21–24). RXR-a resides in the cytoplasm at Research Institute, 10901 N. Torrey Pines Road, La Jolla, CA 92037. Phone: certain stages during development (25, 26). It migrates from 858-646-3141; Fax: 858-646-3195; E-mail: [email protected]; and Xin-Sheng Yao, Institutes of Traditional Chinese Medicine and the nucleus to the cytoplasm in response to differentiation (23), Natural Products, Jinan University, Guangzhou 510632, China. E-mail: apoptosis (21), and inflammation (22, 24). Cytoplasmic local- [email protected] ization of RXR-a facilitates nuclear export of its heterodimer- doi: 10.1158/0008-5472.CAN-12-2038 ization partner Nur77, leading to Bcl-2 conversion and apo- Ó2012 American Association for Cancer Research. ptosis (21, 27, 28). www.aacrjournals.org 307 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2013 American Association for Cancer Research. Published OnlineFirst November 14, 2012; DOI: 10.1158/0008-5472.CAN-12-2038 Wang et al. We showed recently that proteolytic cleavage of RXR-a Ligand-binding assay results in production of truncated RXR-a protein, tRXR-a, RXR-a LBD was incubated with [3H]-9-cis-RA in the pres- which acquires new function that is different from the full- ence or absence of unlabeled 9-cis-retinoic acid (9-cis-RA) or length RXR-a (29). Unlike the full-length RXR-a that resides in CF31. Bound [3H]-9-cis-RA in RXR-a. LBD protein was deter- the nucleus, tRXR-a is cytoplasmic and interacts with the p85a mined in a scintillation counter (29). subunit of phosphoinositide 3-kinase (PI3K) in a TNFa–depen- dent manner, leading to activation of the PI3K/AKT pathway, a Transient transfection and reporter assays major survival pathway important for uncontrolled growth of CV-1 green monkey kidney cells were grown in Dulbecco's tumor and as drug resistance (29). Activation of the PI3K/AKT Modified Eagle's Medium supplemented with 10% FBS. For pathway by tRXR-a contributes significantly to anchorage- chloramphenicol acetyltransferase (CAT) reporter assays, cells independent growth of cancer cells in vitro and tumor growth were seeded at 5 Â 104 cells per well in 24-well plates and in animals (29), offering an opportunity to suppress cancer cell transfected with 50 ng TREpal-tk-CAT reporter plasmid (37), growth by targeting the tRXR-a–mediated PI3K/AKT pathway. 20 ng b-galactosidase expression vector (pCH 110; Amersham), Epidemiologic studies have shown that dietary phytochem- 20 ng RXR-a expression vectors using Lipofectamine 2000 icals provide beneficial effects for cancer prevention, and (Invitrogen). Cells were then treated with CF31 at different among them, xanthones are of great interest as cancer che- dose for 20 hours. CAT activity was normalized with b-galac- mopreventive agents because of their potent antioxidative and tosidase activity. For luciferase reporter assay, 24 hours after anticancer activity (30–33). We previously purified a series of transfected with pGL5 luciferase reporter vector (40 ng/well) xanthones from a medicinal plant, Cratoxylum formosum ssp. and pGAL4-RXR-a-LBD expression vector (40 ng/well), cells pruniflorum, which belongs to the Clusiaceae family and is were incubated with varied concentrations of compounds for widely distributed in several Southeast Asian countries (34, 35). 12 hours. Luciferase activities were measured using the Dual- Here, we report our identification of one of the xanthones, Luciferase Assay System Kit (Promega). CF31, which suppresses tRXR-a–dependent AKT activation through its unique RXR-a binding. CF31, when combined with Molecular docking and molecular dynamics simulations TNF-a inhibits TNF-a–induced tRXR-a–p85a interaction and AutoDock version 4.0 was used for the docking screening. AKT activation. Moreover, the CF31 and TNF-a combination The Lamarckian genetic algorithm was selected for ligand results in synergistic induction of TNF-a–dependent caspase-8 conformational searching. Docking parameters were as fol- activation and apoptosis in cancer cells. Thus, CF31 represents lows: grid box of 40 Å Â 40 Å Â 40 Å, xyz-coordinates of a new modulator of the tRXR-a survival pathway and a potent gridcenter: 49.192 63.991 À7.523, population size of 150, ran- converter of TNF-a signaling from survival to death. dom starting position and conformation, translation step ranges of 2 Å, rotation step ranges of 50, elitism of 1, mutation rate of 0.02, cross-over rate of 0.8, local search rate of 0.06, and Materials and Methods 2.5 million energy evaluations. Molecular dynamics simula- Isolation of natural products tions were adopted to sample possible configurations of each Compounds CF31, also called cochinchinone B or 1,3,6,7- RXR-a LBD complex using the AMBER (version 7.0) program tetrahydroxy-2-(3-methyl-2-butenyl)-5-(3,7-dimethyl-2,6-octadie- (38). To set up each molecular dynamics simulation, the nyl)xanthone (36), CF13 (1,3,5-trihydroxy-4-geranylxanthone), electrostatic potentials of the ligand was computed by using and CF26 (pruniflorone Q) were isolated from the stems of the Gaussian 98 package at the HF/6-31GÃ level. Atom-cen- Cratoxylum formosum ssp. pruniflorum as described (35). tered partial charges were derived by using the Restrained Electrostatic potential method implemented in the AMBER Plasmids package. The pGAL4-RXR-a-LBD plasmid was obtained by inserting RXR-a ligand-binding domain (LBD) sequence (amino acids Immunofluorescence microscopy 198–462) in-frame with the Gal4 DBD coding sequence in the HepG2 cells mounted on glass slides were permeabilized pBIND vector (Promega).