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JQ-1 inhibits colon cancer proliferation via suppressing Wnt/#-catenin signaling and miR-21 Yan Zhang, Suli Tian, Jidong Xiong, Yongxu Zhou, Hongyu Song, and Chang Liu Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.7b00346 • Publication Date (Web): 30 Mar 2018 Downloaded from http://pubs.acs.org on March 31, 2018

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Chemical Research in Toxicology

JQ-1 inhibits colon cancer proliferation via suppressing Wnt/β-catenin signaling and miR21 Yan Zhang, Suli Tian, Jidong Xiong, Yongxu Zhou, Hongyu Song, Chang Liu*

Department of General Surgery, The Forth Hospital Affiliated to Harbin Medical University, No. 37 Yiyuan Road, Harbin 150001, China

*Corresponding author: Chang Liu, Department of General Surgery, The Forth Hospital Affiliated to Harbin Medical University, No. 37 Yiyuan Road, Harbin 150001, China E-mail: [email protected]

Keywords: JQ-1; colon cancer; miR-21; Wnt signaling

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Abstract Bromodoamin and extraterminal (BET) protein inhibitors are a novel class of cancer therapeutics. Here we aim to investigate the efficacy and mechanism of JQ-1, a potent BET inhibitor, in colon cancer therapy. JQ-1 was used to treat SW480 colon cancer mouse xenografts. Tumor size and mouse survival were recorded. Cell apoptosis was evaluated by Annex V-FIC/PI flow cytometry. ChIP-q-PCR analysis was used to assess the H3K27 trimethylation (H3K27m3) of the p16 promoter. Wnt signaling was evaluated by Nkd2 and β-catenin levels. RT-PCR was used to evaluate the level of miR-21. MiR-21 was overexpressed with a lentiviral system was used to evaluate the relationship between miR-21 and JQ-1. JQ-1 significantly reduced tumor growth, improved mouse survival and induced apoptosis. JQ-1 epigenetically inhibited the H3K27me3 promoter activity, promoting p16 expression. Nkd2 and β-catenin were upregulated and downregulated by JQ-1, respectively. MiR-21 was downregulated by JQ-1. MiR-21 overexpression compensated proliferation inhibition by JQ-1. Nkd2 levels were also downregulated by miR-21 overexpression. JQ-1 is effective in inhibiting colon cancer. We revealed that the mechanism of JQ-1 action is associated with its regulation of Wnt/β-catenin signaling and miR-21 levels.

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Introduction Colon cancer is one of the leading causes of cancer-related death worldwide.1 The high mortality rate of colon cancer is attributed to the fact that, most colon cancers are diagnosed at late stages and are characterized by resistance to chemotherapies.2 There is an urgent need to explore novel chemotherapy drugs to combat colon cancer resistance and prolong patient survival. In recent years, a growing body of evidences demonstrated the therapeutic potential of the epigenetic regulation of cancers.3-5 Epigenetic changes significantly affect the onset, progression and metastasis of cancer, which has stimulated interest in manipulation of transcription as a mode of cancer therapy. Histone lysine acetylation is one of the putative mechanisms of protein posttranslational modification that modulates the expression of a large array of onco-proteins in tumorigenesis and proliferation.6 In colon cancer, aberrant histone acetylation, resulted from aberrant recruitment of histidine modifying proteins, is characteristic of colon cancer with poor clinical outcomes.7 Bromodomain and extraterminal (BET) proteins are a class of histidine modifying proteins.8 They bind to acetylated lysine (Kac) and read lysine acetylation state, orchestrating with histone acetylation to promote cancer progression. JQ-1, a novel thienotriazolo-1, 4-diazepine, has been used as an inhibitor of BET proteins by competitive binding to acetyl-lysine recognition motifs. JQ-1 has been broadly used for regulating immune responses and a number of BET-positive cancers. The use of JQ-1 to reverse aberrant gene expression has benefited a wide range of cancers, such as pancreatic cancer,9 breast cancer,10 and ovarian cancer.11 This has spurred interests in elucidating cancer gene pathways affected by JQ-1. Wnt signaling pathway is a putative cancer-promoting mechanism, in which the activation of Wnt protein induces modification and nuclear translocation of β-catenin, leading to the expression of key proteins that

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drive cancer progression.12, 13 Previous evidences have implicated the potential of JQ-1 to inhibit Wnt signaling in mesenchymal stem cells.14, 15 We hypothesize that JQ-1 inhibits colon cancer progression partly through the regulation of Wnt signaling. Besides, high miR-21 has been reported as an emerging biomarker in colon cancer.16 It post-transcriptionally regulates a number of oncogenes, thus promoting the invasion, migration and chemoresistance. MiR-21 expression was also found to be closely correlated to Wnt signaling.17 It is worth investigating if JQ-1 modulates miR-21 expression in colon cancer. Herein, we aimed to test the efficacy of JQ-1 in colon cancer suppression and elucidate the mechanism of JQ-1 in colon cancer, by investigating its regulation of Wnt signaling pathway and miR-21 expression. We aimed to investigate the role of JQ-1 in inducing colon cancer regression and cell apoptosis.

Experimental Procedures Cells and animal handling SW480 cells were acquired from American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in RPMI-1640 medium supplemented with Pen/strep and 10% fetal bovine serum (FBS). Cells were maintained in an incubator kept at 37 °C and 5% CO2. We treated cell with 0.033% DMSO in medium or 300 µM JQ-1 dissolved in 0.033% DMSO in medium for 24 h and the cells were harvested for the further study. This dose is determined based on a previously published protocol18. Balb/c nude mice were purchased from SLAC company (Shanghai, China). All animal experiments were performed in compliances of protocols approved by the ethic committee of The Forth Hospital Affiliated to Harbin Medical University. To initiate colon cancer xenografts,

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SW480 cells (106 cells) in 100 µL PBS were injected subcutaneously to the armpit of mice. Treatment was performed at one week after tumor inoculation using 50 mg/kg JQ-1 (0.033% DMSO in PBS) or equivalent volumes of DMSO solution (0.033% in PBS) intravenously. This dose is determined based on a previously published protocol18. Treatment lasted for 70 days, during which tumor volume was recorded. Tumor volume = width2×length/2. Survival data of the mice were also recorded and presented as Kaplan-Meier curves. Flow cytometry and CCK8 viability assay Cells were stained with Annexin V-FITC and propidium iodide (PI) using the Annexin-V-FITC & FITC Apoptosis Kit (Thermofisher Scientific, Waltham, MA, USA) according to manufacturer’s recommendations. Stained cells were analyzed with flow cytometry using fluorescence emission at 530 nm and >575 nm. Apoptotic cells were identified to have both green and red fluorescence. Viability assay was performed using the Cell Counting Kit-8 (CCK8) according to manufacturer’s recommendations. Viability was quantified using the absorbance of treatment group and control groups. Chromatin immunoprecipitation (ChIP) qPCR Levels of trimethylation of histone at lysine-27 (H3K27me3) and wild-type H3 under JQ-1 treatment were analyzed by ChIP assay according to a previous protocol 18. The cells treated with DMSO was used as control. Briefly, chromatin of cells was extracted after cell lysis. 10 µL chromatin was used as the input, the remaining was incubated with agarose beads with H3K27me3 antibody or H3 antibody (Abcam, Cambridge, MA, USA). DNA was isolated from immunoprecipitates using IPure Kit (Belgium). qPCR was performed in LC480 system (Roche, Penzberg, Upper Bavaria, Germany) according to standard protocols. A set of primers for the ChIP (Figure S1A and Table S1) were designed based on a previous publication19. These

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primers were under the activation of Wnt signal by LiCl. Primer5 (P5) was used for the further study since it had the most fold change in the activation of Nkd2 expression. Lentiviral transfection Ectopic expression of miR-21 was realized using lentiviral transfection of hsa-miR-21expressing vector. The vector was constructed by cloning the pre-miRNA of miR-21 into a BLOCK-iT Pol II miR RNAi expression vector. This vector and packaging vectors were transfected into 294T packaging cells. Harvested virus was used to transfect SW480 cells. Statistical analysis Data were expressed as mean ± standard deviation (SD). Data analyses were performed by student t test and one or two-way ANOVA analysis using SPSS 18.0 software, where appropriate. Difference of data was considered statistically significant if p

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