Targeting Androgen Receptor (AR)!IL12A Signal Enhances Efficacy
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Published OnlineFirst March 3, 2016; DOI: 10.1158/1535-7163.MCT-15-0706 Cancer Biology and Signal Transduction Molecular Cancer Therapeutics Targeting Androgen Receptor (AR)!IL12A Signal Enhances Efficacy of Sorafenib plus NK Cells Immunotherapy to Better Suppress HCC Progression Liang Shi1,2, Hui Lin1, Gonghui Li1, Ren-An Jin1, Junjie Xu1,2, Yin Sun2, Wen-Lung Ma3, Shuyuan Yeh2, Xiujun Cai1, and Chawnshang Chang2,3 Abstract Gender disparity has long been considered as a key to fully matin immunoprecipitation assay were applied for mechanism understand hepatocellular carcinoma (HCC) development. At dissection. IHC was performed for sample staining. Our results the same time, immunotherapy related to IL12 still need more showed AR could suppress IL12A expression at the transcrip- investigation before being applied in clinical settings. The aim tional level via direct binding to the IL12A promoter region that of this study is to investigate the influence of the androgen resulted in repressing efficacy of NK cell cytotoxicity against receptor (AR) on natural killer (NK) cell–related innate HCC, and sorafenib treatment could enhance IL12A signals via immune surveillance in liver cancer, and provide a novel suppressing AR signals. These results not only help to explain therapeutic approach to suppress HCC via altering IL12A. By the AR roles in the gender disparity of HCC but also provide a using in vitro cell cytotoxicity test and in vivo liver orthotopic potential new therapy to better suppress HCC via combining xenograft mouse model, we identifiedtheroleofARinmod- sorafenib with NK cell–related immunotherapy. Mol Cancer Ther; ulating NK cell cytotoxicity. Luciferase report assay and chro- 15(4); 731–42. Ó2016 AACR. Introduction AR in selective cells, Wu and colleagues and Ma and colleagues found the dual roles of AR in HCC. They found that AR might Hepatocellular carcinoma (HCC), the major liver cancer, is the enhance HCC initiation and early development, but suppress second leading cause for cancer-related death in men (1). The HCC metastasis at the later stages of the disease (8–10). etiology of HCC could be attributed to many liver diseases Recent studies also indicated that various immune cells in the including viral hepatitis, cirrhosis, and other chronic liver damage tumor microenvironment might play important roles to influence (2, 3). Importantly, gender disparity involving the androgen the HCC progression (11–13) and the natural killer (NK) cells receptor (AR) during HCC initiation and progression has also have been implicated to be effective for immunotherapy in HCC been well documented but detailed mechanisms remain to be (14, 15). The detailed mechanisms of how NK cells suppress HCC further elucidated (4, 5). and their potential linkage to AR function, however, remain Although the androgen deprivation as therapy for HCC unclear. Furthermore, early studies indicated that IL12, also called remains controversial (6, 7), using mice with knocked out AR in the NK cell stimulatory factor, might play important roles to hepatocytes and the AR degradation enhancer, ASC-J9, to degrade þ activate and mediate the cytotoxicity of NK cells and CD8 cytotoxic T cells (16, 17), and suggested that IL12 gene therapy 1Department of General Surgery, Chawnshang Chang Liver Cancer might represent an effective approach to suppress HCC progres- Center, Sir Run-Run Shaw Hospital, Zhejiang University, Hangzhou, sion (18–21). However, the development of lethal toxicity shown 2 China. Departments of Pathology, Urology, and Radiation Oncology; during phase II clinical trials prevented further study (22, 23). The George Whipple Lab for Cancer Research; and the Wilmot Cancer Center, University of Rochester Medical Center, Rochester, New York. Here we revealed the relationship between cytotoxicity of NK 3Sex Hormone Research Center, China Medical University/Hospital, cells in HCC and AR by demonstrating that AR could suppress Taichung, Taiwan. IL12A, one of the two subunits of IL12. Importantly, we demon- Note: Supplementary data for this article are available at Molecular Cancer strated that sorafenib, the only drug approved by FDA for Therapeutics Online (http://mct.aacrjournals.org/). advanced HCC, could enhance the cytotoxicity of NK cells by L. Shi, H. Lin, and G. Li contributed equally to this article. elevating the IL12A levels through inhibition of AR. Corresponding Authors: Chawnshang Chang, University of Rochester Medical Center, 601 Elmwood Avenue, Box 626, Rochester, NY 14642. Phone: 585-275- 9994; Fax: 585-756-4133; E-mail: [email protected]; and Xiujun Cai, Materials and Methods Zhejiang University, General Surgery, Sir Run Run Shaw Hospital, 3 East Cell culture and transfection Qingchun Road, Hangzhou, 310016 China. E-mail: [email protected] The human HCC cells were maintained in DMEM (Invitrogen) doi: 10.1158/1535-7163.MCT-15-0706 with 10% FBS, 1% glutamine, and 1% penicillin/streptomycin. Ó2016 American Association for Cancer Research. NK-92MI cells were obtained from ATCC on December 3, 2010, www.aacrjournals.org 731 Downloaded from mct.aacrjournals.org on September 28, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst March 3, 2016; DOI: 10.1158/1535-7163.MCT-15-0706 Shi et al. and 25 ampules (2  106 cell/mL) were frozen in liquid N2. ratio at 1:1 and incubated for another 24 hours then performed Resuscitated cells were used within 3 months. NK-92MI cells were TUNEL staining. Apoptotic cell death was determined using maintained in a-MEM (Invitrogen) with 0.2 mmol/L inositol, TUNEL staining with an In Situ Cell Death Detection Kit (Roche 0.1 mmol/L 2-mercaptoethanol, 0.02 mmol/L folic acid, horse Molecular Biochemicals), following the manufacturer's protocol. serum to a final concentration of 12.5%, and FBS to a final TUNEL-positive cells were calculated as the number of positive concentration of 12.5% based on ATCC guidelines. Human HCC cells  100% divided by the total number of cells per field in 10 SKAR3, SKAR7, and SK-pBABE cells were established and described random fields at  100 magnification. The apoptosis rate was previously (9). SK-Hep1 and HepG2 cells were obtained from calculated on the basis of numbers of positive cells and total cells. ATCC and authenticated by a professional biotechnology compa- ny in 2015. SNU-423 cells were bought from ATCC on April 4, ELISA 2014, and all the experiments were performed within 3 months of Serum-starved NK-92MI cells were activated by conditioned resuscitation. HA22T cell (BCRC No. 60168) was a gift from Prof. media (CM) collected from HCC cells with different AR expres- Yuh-Shan Jou, Academia Sinica, Taiwan (24). All cell lines were sion levels. After activation, the media from NK cells were col- fi cultured in a 5% (v/v) CO2 humidi ed incubator at 37 C. lected for detection of IFNg using an ELISA Kit (Thermo Scientific). The HepG2 (ATCC) and HA22T AR–stable transfectants were The standard curve was made to determine the IFNg concentra- established on the basis of a previous procedure (25). To generate tion. IL12p35 was detected in culture media collected from HCC AR knocked-down stable clones of SK-Hep1 (ATCC) and SNU423 cells with an ELISA Kit (Thermo Scientific). All the procedures cells (ATCC), HEK-293T cells were transfected with lentiviral were performed according to the manufacturer's instructions. vectors, pLKO1-AR-si/pLKO1-scr, with the psAX2 packaging plas- mid, and pMD2G envelope plasmid, then transfected into 293T Quantitative real-time PCR analysis cells for 48 hours to get the lentivirus soup, which was frozen at Total RNAs were isolated using TRIzol reagent (Invitrogen). À 80 C for later use for infection of cells to produce stable clones. One microgram of total RNA was subjected to reverse transcrip- For the luciferase reporter assay, cells were transfected using tion using Superscript III transcriptase (Invitrogen). qRT-PCR was Lipofectamine 3000 (Invitrogen) reverse transfection protocol, conducted using a Bio-Rad CFX96 system (Bio-Rad) with SYBR according to the manufacturer's instructions. See Supplementary green to determine the mRNA expression level of a gene of Table for detailed reporter plasmid sequences. interest. Expression levels were normalized to the expression of GAPDH RNA (see Supplementary Table for detailed sequence). MTT cell viability assay HCC cells were placed in 24-well plates at a density ranging from 0.4  105 to 1.0  105 cells/well based on the cell size. NK cells were Western blot analysis coincubated at an effector-to-target (E:T) ratio at 2:1 for 6 hours. Cells were lysed in RIPA buffer and proteins (60 mg) were Then, NK cells were washed away and 0.5 mL MTT (0.5 mg/mL) per separated on 8% to 10% SDS/PAGE gel and then transferred onto fl well was added and incubated for another 2 hours. The absorbance polyvinylidene di uoride membranes (Millipore). After blocking at 570/630 nm was detected. The percentage of survival was membranes, they were incubated with appropriate dilutions of specific primary antibodies, the blots were incubated with HRP- calculated using the following formula: survival (%) ¼ ODE/ODC conjugated secondary antibodies, and visualized using ECL sys-  100% (ODE is the OD value of the NK cells added cell group; and tem (Thermo Fisher Scientific). Anti-GAPDH (1:1,000, 6c5) and ODC is the OD value of the HCC cells without NK cells added). anti-AR (1:1,000, N20) antibodies were purchased from Santa Cellular cytotoxicity assay Cruz Biotechnology. Anti-IL12A (Anti-IL12p35, 1:500, R12- NK cell cytotoxicity against HCC cells was analyzed using a 2200) antibody was purchased from Assay Biotechnology Com- standard lactate dehydrogenase (LDH) release assay. NK92-MI cells pany. Human IL12 polyclonal antibody (PeproTech) was used to were starved in serum-free media for 24 hours and target cells were test both IL12A and IL12B (1:500).