A Small-Molecule Modulator of the Tumor-Suppressor Mir34a Inhibits the Growth of Hepatocellular Carcinoma

Published OnlineFirst September 12, 2014; DOI: 10.1158/0008-5472.CAN-14-0855

Cancer Therapeutics, Targets, and Chemical Biology Research

A Small-Molecule Modulator of the Tumor-Suppressor miR34a Inhibits the Growth of Hepatocellular Carcinoma

Zhangang Xiao1, Chi Han Li1, Stephen L. Chan2, Feiyue Xu1, Lu Feng1, Yan Wang3, Jian-Dong Jiang3, Joseph J.Y. Sung4, Christopher H.K. Cheng1, and Yangchao Chen1,4,5

Abstract Small molecules that restore the expression of growth-inhibitory microRNAs (miRNA) downregulated in tumors may have potential as anticancer agents. miR34a functions as a tumor suppressor and is downregulated or silenced commonly in a variety of human cancers, including hepatocellular carcinoma (HCC). In this study, we used an HCC cell–based miR34a luciferase reporter system to screen for miR34a modulators that could exert anticancer activity. One compound identified as a lead candidate, termed Rubone, was identified through its ability to specifically upregulate miR34a in HCC cells. Rubone activated miR34a expression in HCC cells with wild- type or mutated p53 but not in cells with p53 deletions. Notably, Rubone lacked growth-inhibitory effects on nontumorigenic human hepatocytes. In a mouse xenograft model of HCC, Rubone dramatically inhibited tumor growth, exhibiting stronger anti-HCC activity than sorafenib both in vitro and in vivo. Mechanistic investigations showed that Rubone decreased expression of cyclin D1, Bcl-2, and other miR34a target genes and that it enhanced the occupancy of p53 on the miR34a promoter. Taken together, our results offer a preclinical proof of concept for Rubone as a lead candidate for further investigation as a new class of HCC therapeutic based on restoration of miR34a tumor-suppressor function. Cancer Res; 74(21); 1–12. 2014 AACR.

Introduction almost all aspects of cancer biology, such as proliferation, Hepatocellular carcinoma (HCC) remains to be one of the apoptosis, invasion/metastasis, and angiogenesis (6). Recently, most common malignancies and is a leading cause of cancer- a miR122 inhibitor has been evaluated in phase IIa clinical trial related deaths due to the high mortality and unsatisfactory for treating hepatitis C virus (HCV) infection. This builds a treatment options available (1). It is of urgent significance to bright prospect for developing modulators of miRNAs for develop more effective therapeutics for HCC. microRNAs disease treatment (7). (miRNA) are a class of single-stranded noncoding RNAs (2), miR34 family constitutes three members: miR34a, miR34b, and it is estimated that 25% to 70% of human genes are and miR34c. All miR34 family members are direct targets of regulated by miRNAs (3, 4). The patterns of miRNAs expression p53. miR34b and miR34c share the same primary transcript were proved to be correlated with cancer types, stages, while miR34a resides in another one (8). miR34a has been and other clinical variables (5). Therefore, the expression of well documented as a tumor suppressor in previous studies – miRNAs could be applied as a tool for cancer diagnosis and (9 13). Upregulation of miR34a could decrease the expres- prognosis. In cancer cells, miRNAs could function as onco- sion of a series of targets such as B-cell lymphoma 2 (Bcl-2), genes or tumor suppressors. Their roles are manifested in cyclin D1, cyclin-dependent kinase 6 (CDK6), Forkhead box protein P1 (FOXP1), Notch 1, and Sirtuin 1 (SIRT1). Thereby, miR34a could induce cell apoptosis, senescence, and inhibit 1School of Biomedical Sciences, Faculty of Medicine, The Chinese Uni- cellular differentiation and proliferation (14–16). miR34a versity of Hong Kong, Shatin, NT, Hong Kong. 2Department of Clinical Oncology, Prince of Wales Hospital, The Chinese University of Hong Kong, expression was dramatically downregulated or silenced in Shatin, NT, Hong Kong. 3State Key Laboratory of Bioactive Substance and various cancers including human HCC. Ectopic expression of Function of Natural Medicines, Institute of Materia Medica, Chinese Acad- miR34a inhibited HCC cell migration and invasion (17). emy of Medical Sciences and Peking Union Medical College, Beijing, China. 4State Key Laboratory of Digestive Disease, Institute of Digestive Thus, restoration of miR34a might represent a potential Disease, The Chinese University of Hong Kong, Shatin, NT, Hong Kong. therapeutic for HCC. 5 Shenzhen Research Institute, The Chinese University of Hong Kong, Few studies have focused on small-molecule modulators of Shenzhen, China. miRNAs so far. Previously, the small-molecule modulators of Note: Supplementary data for this article are available at Cancer Research miR21 and miR122 have been identified with potent biologic Online (http://cancerres.aacrjournals.org/). activities (18, 19). However, the small-molecule modulators of Corresponding Author: Yangchao Chen, School of Biomedical Sciences, fi Faculty of Medicine, Chinese University of Hong Kong, Shatin, NT, miR34a have not been identi ed yet. In this study, we devel- Hong Kong. Phone: 852-39431100; Fax: 852-26035123; E-mail: oped a luciferase reporter system for the screening of small- [email protected] molecule modulators of miR34a from a natural product library. doi: 10.1158/0008-5472.CAN-14-0855 After library screening, one hit compound named Rubone was 2014 American Association for Cancer Research. found to activate miR34a expression in HCC cells. We further

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investigated the anticancer activity of miR34a modulator in each well of 96-well plates and incubated for 12 hours at 37C both cell culture and animal models. with 5% CO2. Then, Huh7 cells were cotransfected with miR34a reporter and Renilla luciferase pRL-SV40. The transfection Materials and Methods medium was replaced with complete DMEM after 4 hours Cell lines and drugs incubation. Compounds from natural products library (NPL; The nontumorigenic human hepatocyte cell line MIHA (20) NPL contained 640 compounds including natural products and was obtained from Dr. J.R. Chowdhury's laboratory at Albert their derivatives; TimTec) were added to the cells at a final Einstein College of Medicine (New York, NY). The human concentration of 10 mmol/L (0.1% DMSO at final concentra- HCC cell line Huh7 (kindly provided by Dr. H. Nakabayashi, tion). The complete DMEM containing 0.1% DMSO was used as Hokkaido University School of Medicine, Sapporo, Japan; a control. After 48 hours of incubation, the luciferase activity ref. 21) and Bel-7404 (Cell Bank of the Chinese Academy of was measured. Sciences; ref. 22) were authenticated with short-tandem repeat profiling by the vendors. The human HCC cell lines HepG2, Luciferase assay PLC/PRF/5 (PLC), and Hep3B (American Type Culture Col- miR34a reporter, miR34a promoter, and miR34a-mutant lection) were verified by short-tandem repeat profiling at the promoter were transfected into HCC cells 12 hours before GENEWIZ, Inc. within 6 months of use. All cell lines were compound treatment. The Renilla luciferase vector pRL-SV40 cultured under the condition as previously described (23). was cotransfected as an internal control. HCC cells were then Carboxymethylcellulose was provided by Unitech Chemicals. treated with compound for 48 to 72 hours. The luciferase Sorafenib was purchased from Bayer Healthcare. Dimethyl activity was measured by the Dual-Luciferase Reporter Assay sulfoxide (DMSO), Cisplatin (CDDP), 5-Fluorouracil (5-FU), (Promega) with a Wallac VICTOR3V luminometer. The ratio of and doxorubicin were purchased from Sigma. firefly luciferase to Renilla expression was calculated for each of the triplicates. Constructs The pmiR-REPORT luciferase plasmid (Ambion) was Total RNA and protein extraction sequentially digested with restriction enzyme SacI and HindIII Total RNA from cell cultures and HCC xenograft tumors (10 units each in 50 mL reaction, NEB) and was purified after gel were extracted using TRIzol (Invitrogen) according to the electrophoresis. DNA oligos containing the miR34a-binding manufacturer's protocols. Total RNAs were dissolved into 0 0 0 site (5 -CTG GCA GTG TCT TAG CTG GTT GTA-3 and 5 -AGC nuclease-free ddH2O. For protein extraction, HCC cells and TTA CAA CCA GCT AAG ACA CTG CCA GAG CT-30), and xenograft tumors were lysed in 1 RIPA buffer with 1 mmol/L scrambled miR34a-binding site (50-CTG TTT CGT TGG GCG phenylmethylsulfonylfluoride (PMSF) and 1 complete pro- TTT CGA AGA-30 and 50-AGC TTC TTC GAA ACG CCC AAC tease inhibitor cocktail (Roche). Both RNA and protein were GAA ACA GAG CT-30) were ordered from TechDragon. The stored at 80C. oligos were annealed at 95C and then cooled to 4C. The annealed DNA fragments were then ligated into pmiR-REPORT Quantitative real-time PCR analysis luciferase vector with T4 DNA ligase (200 U in 10-mL reaction, Total RNA was reversely transcribed into cDNAs using the NEB). The generated constructs containing miR34a-binding High Capacity cDNA Reverse Transcription Kit (Applied Bio- site or scrambled binding site were named as miR34a reporter systems). Quantitative real-time PCR (qRT-PCR) was per- or miR34a scr. The luciferase promoter constructs containing formed using SYBR Green PCR Master Mix (TaKaRa). GAPDH miR34a promoter or miR34a mutant promoter were kindly was used as an internal control. miR34a level was measured by provided by Prof. J.T. Mendell from Johns Hopkins University qRT-PCR using SYBR Green PCR Master Mixture (Invitrogen). (Baltimore, MD; ref. 16). U6 was measured as an internal control. Fold change was DD calculated by relative quantification (2 Ct). The sequences of Transfection the primers were listed in Supplementary Table S1. miR34a mimics duplex, single-stranded miR34a inhibitors and siRNAs targeting p53 were purchased from Shanghai Western blotting GenePharma Co. The sip53 sequence was as following: Protein concentration was measured with BCA protein sip53a: 50-CUA CUU CCU GAA AAC AAC G dTdT-30; sip53b: assay kit (Thermo Fisher Scientific). Of note, 25 to 50 mg GCA UGA ACC GGA GGC CCA U dTdT. The miR34a mimics, protein was separated by SDS-PAGE (sodium dodecyl sul- inhibitors, and siRNAs were transfected into HCC cells using fate–polyacrylamide gel electrophoresis) and transferred to DharmaFECT siRNA transfection reagent (Thermo Scientific) poly(vinylidene fluoride) (PVDF) membranes (Bio-Rad). Pri- according to the manufacturer's protocol. The plasmids men- mary antibodies used in this study included anti-cyclin D1 tioned in previous section were transfected into HCC cells (1:1,000 dilution; cat. no., 2922; Cell Signaling Technology), using Lipofectamin 2000 (Invitrogen) according to the manu- anti-Bcl-2 (1:1,000 dilution; cat. no., AM2209, ABZOOM), anti- facturer's protocol. b-actin (1:5,000; cat. no., 4967; Cell Signaling Technology), and anti-p53 (1:1,000; cat. no., Sc-126; Santa Cruz Biotechnology). Library screening with miR34a reporter Membranes were incubated with primary antibodies at 4C Huh7 cells were cultured in complete Dulbecco's Modified overnight, and then washed three times with TBST (Tris- Eagle Medium (DMEM). 5 103 Huh7 cells were seeded into buffered saline plus Tween-20). The membranes were then

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incubated with 1:3,000 peroxidase-conjugated secondary anti- calculated by counting TUNEL-positive cells from three ran- body (Santa Cruz Biotechnology) for 1 hour at room temper- dom fields per slide. ature, followed by three washings in TBST. Protein expressions were detected by autoradiography film using Amersham ECL Chromatin immunoprecipitation assay Plus Western Blotting Detection Reagents (GE Healthcare). Chromatin immunoprecipitation (ChIP) assay was performed as described previously (25). Cross-linked chromatin Cell viability assay were incubated overnight with anti-H3 (cat. no., 4620; Cell Cell viability was measured by the 3-(4, 5-dimethylthiazol-2- Signaling Technology), IgG (cat. no., 2729; Cell Signaling yl)-2, 5-diphenyltetrazolium bromide (MTT) assay as described Technology), p53 (cat. no., sc-126; Santa Cruz Biotechnology). previously (24). Briefly, 1 103 cells were seeded in 96-well The precipitated DNA were quantitated absolutely by real-time plates in triplicate and treated with compounds at indicated PCR and normalized by respective 2% input. concentrations for indicated time points. At each time point, a set of cells was incubated with 0.5 mg/mL MTT diluted in 1 Statistical analysis PBS. After 2 to 4 hours of incubation, the MTT solution was GraphPad Prism 5 (GraphPad Software) was used for removed. The insoluble MTT was dissolved in DMSO. Absor- statistical analysis. Four-parameter logistic model was t bance at 570 nm was measured using a Benchmark Plus applied to calculate IC50. The two-tailed Student test was microplate reader (Bio-Rad). applied for paired data analysis. All data are expressed as mean SD from three separate experiments performed in In vivo HCC xenograft mouse model triplicate. P values less than 0.05 were considered statisti- A total of 5 106 HepG2 cells were suspended in 100 mL cally significant. serum-free medium and subcutaneously (s.c.) inoculated into the dorsal flanks of nude mice. When tumors reached Results 5 to 10 mm in diameter, mice were randomly divided into Identification of small-molecule modulator of miR34a by four groups (n ¼ 5). Rubone and sorafenib were suspended library screening in a carboxymethylcellulose vehicle formulation, which con- We first established a luciferase reporter by cloning the tained 0.4% carboxymethylcellulose sodium and 0.9% NaCl. complementary sequence of miR34a into pmiR-Reporter vec- Tumor-bearing mice were treated with 20 or 50 mg/kg tor (Fig. 1A). The scrambled miR34a-binding sequence was Rubone in 200 mL vehicle by gavage once every 2 days. used as a control. Theoretically, this miR34a reporter could be Vehicle alone and sorafenib were used as negative and used for screening miR34a modulators by measuring luciferase positive control, respectively. The starting date of treatment signals (Fig 1B). The endogenous miR34a level in HepG2 cells was defined as day 0. Tumor volumes and body weight were was significantly higher (22.29-fold; P ¼ 0.0017) than that of measured once every 2 days. Tumor volume was calculated Huh7 cells as measured by qRT-PCR (Supplementary Fig. S1A). as [(length width height)/2]. Treatment was continued We transfected miR34a reporter into HepG2 and Huh7 cells for 24 days. Finally, mice were sacrificed and tumors were respectively and measured the luciferase activities. The lucif- excised. Tumor weight was recorded. Tumor tissues were erase activities in Huh7 cells was significantly higher (15.66- collected for subsequent RNA extraction or tissue section. fold, P ¼ 0.044) than that of HepG2 cells (Supplementary All animal experiments were approved by the Animal Exper- Fig. S1B). Cotransfection of miR34a mimics and miR34a report- imental Ethics Committee of the Chinese University of er decreased the luciferase signal in Huh7 cells (Supplementary Hong Kong (Shatin, Hong Kong). Fig. S1C). Conversely, cotransfection of miR34a inhibitor and miR34a reporter increased the luciferase signal in HepG2 cells Immunohistochemical staining (Supplementary Fig. S1D). No luciferase signal was changed in Immunohistochemical staining was performed on xeno- control group where miR34a reporter was replaced with grafted tumor tissues with proliferating cell nuclear antigen miR34a scr. These results indicated that miR34a reporter was (PCNA; 1:400 dilution; cat. no., SC-56; Santa Cruz Biotechnol- responsive to miR34a level. ogy), p16 (1:200 dilution; cat. no., BM0174, ABZOOM), and p21 Using the miR34a reporter system, we screened the NPL (1:250 dilution; cat. no., SC-397; Santa Cruz Biotechnology)- for miR34a modulators. The screening process was depicted specific antibodies. Subcutaneous tumor tissue sections were in Fig 1C. After a primary screening of 640 compounds in Huh7 incubated with antibodies overnight at 4C. Mean positive cells cells, one hit compound named Rubone was found to be a were calculated by averaging positive cells from three random potential miR34a modulator (The full library screening dataset fields per slide. was listed in Supplementary Table S2). Rubone was re-assayed in triplicate with miR34a reporter system. Figure 1D showed TUNEL assay Rubone inhibited the luciferase activity in a dose-dependent m Analysis of apoptotic cells in xenografted tumor tissues was manner, with an IC50 value at 3.8 mol/L. Rubone was a performed by terminal deoxynucleotidyl transferase–mediat- chemically synthesized plant chalcone derivative, which was 0 0 0 ed dUTP nick-end labeling (TUNEL) staining. The in situ cell also named 2 -hydroxy-2,4,4 ,5,6 -pentamethoxychalcone. Its death detection kit was used by following the manufacturer's molecular weight was 374.39 and its chemical structure was protocol (Roche Diagnostics). Images of the sections were shown in Fig. 1D. Rubone has not been previously reported to taken by a fluorescent microscope. Mean positive cells were have any known biologic activities.

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Figure 1. Identification of miR34a modulator through library screening. A, schematic illustration of miR34a reporter system. The complementary sequences including mature miR34a and the scrambled miR34a sequence were cloned into pMIR-REPORT miRNA Reporter Vector (Ambion) to establish miR34a reporter and miR34a. B, working mechanism of the miR34a reporter. The miR34a reporter could detect the presence of functional mature miR34a as reflected by the rate of luciferase activity repression. C, the process of library screening for the identification of miR34a modulators. D, a small-molecule compound named Rubone significantly inhibited the luciferase activity of miR34a reporter at a dose-dependent manner. Huh7 cells were transfected with miR34a reporter and then treated with indicated concentrations of Rubone for 48 hours. Cell lysates were subjected to luciferase assay. Results are expressed as luciferase activity values. Error bars represent mean SD of three independent experiments.

We next examined whether Rubone modulated miR34a on pri-miR34a or miR34a expression in HCC cells (Supple- expression in HCC cells. As shown in Fig 2, both primary and mentary Fig. S2D). These results indicated that Rubone pref- mature miR34a levels significantly increased after treatment erentially modulated miR34a expression in HCC cells. Cyclin with Rubone in Huh7 and HepG2 cells. However, Rubone D1 and Bcl-2 were two of the most well-studied miR34a targets caused no change in the expression of primary and mature with important roles in HCC. We examined the expression miR34a in Hep3B cells (Fig. 2). To note, mutant p53 and wild- levels of cyclin D1 and Bcl-2 after Rubone treatment. Rubone type p53 was expressed in Huh7 and HepG2 cells, respectively, significantly reduced the mRNA and protein levels of cyclin D1 while p53 was deleted in Hep3B cells. These results indicated and Bcl-2 in Huh7 and HepG2 cells but not in Hep3B cells that Rubone modulated miR34a expression in HCC cells (Fig. 3). Meanwhile, we measured the expression levels of other expressing either wild-type or mutant p53 but not in HCC miR34a targets including CDK6, FOXP, Notch 1, and SIRT as cells with p53 deletion. well as p53 targets including p21 and Puma. Similarly, the To exclude the possibility of nonspecific modulation on expression levels of these miR34a targets were decreased after miR34a expressions by Rubone, we measured the global Rubone treatment in Huh7 and HepG2 but not in Hep3B cells human miRNAs expression after Rubone treatment. The global (Supplementary Fig. S2E), although there were no change human miRNA profiling showed that Rubone preferentially on the expression of p53 targets in these cells (Supplementary activated the expression of miR34a (Supplementary Fig. S1A). Fig S2F). We further demonstrated that other chemotherapeutic drugs such as CDDP, 50-FU, doxorubicin and sorafenib significantly miR34a modulator inhibited HCC cells growth in vitro inhibited HCC cell growth (Supplementary Fig. S2B). Among We next examined the anticancer activity of Rubone in vitro. these drugs, doxorubicin could induce the expressions of p53 Five HCC cell lines and MIHA cell line were treated with and its downstream targets including p21 and Puma (Supple- different concentrations of Rubone at indicated time points. mentary Fig. S2C). However, we found these drugs had no effect Cell viability was measured by the MTT assay. Rubone

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Figure 2. Rubone increased miR34a expression levels in HCC cells. Huh7, HepG2, and Hep3B cells were treated with Rubone at the concentration of 10 mmol/L or vehicle control for 48 hours. Then, RNA samples were prepared and the fold change of primary and mature miR34a levels were measured by qRT-PCR. Error bars represent mean SD of three independent experiments. inhibited the growth of HepG2, Bel-7404, Huh7, and PLC in a attenuated the growth-inhibitory effect of Rubone. These dose-dependent manner. There was little growth inhibition on results indicated that Rubone inhibited HCC cell growth by MIHA after Rubone treatment, suggesting no cytotoxicity of modulating miR34a expression. Rubone to nontumorigenic human hepatocytes (Fig. 4A). Similarly, Rubone caused no growth inhibition on Hep3B cells miR34a modulator inhibited hepatocellular tumor in which p53 was deleted. growth in vivo We also compared the growth inhibition effect of Rubone We next examined the in vivo anticancer activities of Rubone and sorafenib on HCC cells. The results revealed that in HepG2 xenografted nude mice model. The tumor-bearing Rubone exerted stronger growth inhibition on most HCC mice were gavaged with Rubone. The anti-HCC agent sorafenib cell lines including Bel-7404, HepG2, and Huh7, though was used for efﬁcacy comparison. As shown in Fig 5A and sorafenib exhibited stronger growth inhibition on PLC cells Supplementary Table S3, the tumors in vehicle control showed at 24 hours. Rubone showed little growth inhibition on a fast and stable growth. HepG2 xenografts were sensitive to Hep3B and MIHA cells, whereas sorafenib exhibited signif- sorafenib with a tumor growth inhibition rate at 84.22% (P ¼ icant growth inhibition on these two cell lines at different 0.0013 vs. vehicle control) at the dose of 50 mg/kg. Rubone time points (Fig. 4B). delayed the growth of tumors by 78.27% (P ¼ 0.0070 vs. vehicle We asked whether Rubone inhibited HCC cell growth control) and 89.64% (P ¼ 0.0010 vs. vehicle control) at the dose through modulation of miR34a. HepG2, Bel-7404, and PLC of 20 and 50 mg/kg, respectively. Rubone exhibited a stronger cells were transfected with miR34a mimics or inhibitors and tumor growth inhibition than sorafenib at the same dosage of then treated with Rubone. Figure 4C showed that miR34a 50 mg/kg (P ¼ 0.047 vs. sorafenib-treated group; Supplemen- mimics showed concomitant effect of growth inhibition with tary Table S3). When comparing the anticancer activities of Rubone to all HCC cell lines. Conversely, miR34a inhibitors Rubone and sorafenib during the whole treatment process,

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Figure 3. Rubone decreased the expression of miR34a targets. Huh7, HepG2, and Hep3B cells were treated with Rubone at the concentration of 10 mmol/L or vehicle control for 48 or 72 hours. RNA samples collected at 48 hours were applied for measuring miR34a targets cyclin D1 and Bcl2 by qRT-PCR. The cell lysates collected at 72 hours were subjected to Western blotting. Speciﬁc antibodies against cyclin D1 (1:2,500 dilution), Bcl-2 (1:2,500 dilution), and b-actin (1:5,000 dilution) were used. Error bars represent mean SD of three independent experiments.

Rubone exhibited higher anticancer activities than that of miR34a modulator inhibited proliferation and induced sorafenib (Fig. 5B). Another xenografted nude mice model apoptosis in HepG2 xenografts was established by using Huh7 cells. In this model, we also TUNEL staining was performed to assess the in vivo apo- found that Rubone significantly inhibited tumor growth ptosis induction by Rubone in HepG2 xenografts. The average when compared with the vehicle control. The tumors from number of apoptotic cells increased from 13/area in the control Rubone-treated group were smaller and lighter when com- group to 109/area (P ¼ 0.0033) in the Rubone-treated group pared with the sorafenib-treated group (Supplementary Fig. (Fig. 6A). The in vivo antiproliferative effect of Rubone treat- S3A–C). ment on HCC xenografts was investigated by PCNA immu- The expression levels of pri-miR34a, miR34a, cyclin nostaining. Qualitative analysis showed a significant decrease D1, and Bcl-2 in the xenografted tumors were measured by in the average number of PCNA-positive cells after Rubone qRT-PCR. In Rubone-treated tumors, both pri-miR34a and treatment (P ¼ 0.011 vs. vehicle control; Fig. 6B). In addition, miR34a levels were upregulated, while miR34a targets cyclin p21 and p16 stainings were performed to assess cell senescence D1 and Bcl-2 were downregulated (Fig. 5C and Supplemen- in HCC xenografts. There were no significant change in the tary Fig. S4A). Sorafenib treatment caused no change in the number of p21 and p16-stained positive cells between control expression of miR34a and its targets (Fig. 5C and Supple- and Rubone-treated groups (Fig. 6C and D). Triggering prolif- mentary Fig. S4A). These results could also be validated eration inhibition and apoptosis are important hallmarks for in Huh7-xenografted nude mice model (Supplementary an effective anticancer chemotherapeutic agent. These results Fig. S3D). Moreover, there was no obvious body weight loss indicated Rubone inhibited HCC growth by inhibiting cell in the treated mice compared with vehicle control (Supple- proliferation and elevating level of apoptosis but not inducing mentary Table S3). Meanwhile, we also treated Balb/C mice cell senescence. with Rubone (200 mg/kg) for 24 days. No obvious side effect We also found that the subcutaneous tumors treated with was observed at the end of the experiment (Supplementary Rubone were visibly less vascularized compared with the Fig. S4D). These results indicated that Rubone was an control group. Tumors were then stained with endothelial effective and safe anti-HCC agent in animal. marker CD31. The new blood vessels were highly vascularized

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Figure 4. Rubone inhibited HCC cell growth in vitro. A, Rubone inhibited the growth of HCC cells in a dose-dependant manner. HCC cell lines HepG2, Huh7, Hep3B, Bel-7404, PLC, and MIHA were treated with Rubone at designated concentrations ranging from 1 to 20 mmol/L for 48 hours. B, the comparison between the inhibition effect of Rubone and sorafenib on HCC cell growth at different time points. HepG2, Huh7, Hep3B, Bel-7404, PLC, and MIHA cell lines were treated with 10 mmol/L Rubone or sorafenib for 0 to 72 hours. The differences between Rubone- and sorafenib-treated group was considered as significantly when P < 0.05 (, P < 0.05; , P < 0.01; and , P < 0.001 vs. sorafenib-treated group, n ¼ 4). C, Rubone inhibited HCC cell growth by modulating miR34a expression. HepG2, Bel-7404, and PLC cells were treated with Rubone at the concentration of 10 mmol/L alone or in combination with 2 mmol/L miR34a mimics or inhibitors for 72 hours. All cell viabilities were measured by the MTT assay. Error bars represent mean SD of three independent experiments. in the control group, while tumors treated with Rubone had p53 mutant, Hep3B: p53 deletion) were used for promoter significantly reduced microvessels (Supplementary Fig. S4B). activity assay. These three cell lines were first transfected with We next found that Rubone caused a dose-dependent inhibi- miR34a promoter or miR34a promoter without p53-binding tion on tube formation of HUVECs in vitro (Supplementary site (miR34a-mutant promoter), and then were treated with Fig. S4C). These results suggested that antiangiogenesis may be Rubone. No luciferase activity change was observed in miR34a- another potential molecular mechanism by which Rubone mutant promoter transfected HCC cell lines. miR34a promoter exerts its in vivo anti-HCC effect. activities were both increased in HepG2 and Bel-7404 cells but not in Hep3B cells (Fig. 7A). These results suggested a potential miR34a modulator increased miR34a promoter role of p53 in Rubone modulation of miR34a promoter activity. activities and p53 occupancy on miR34a promoter We first found there were no significant change in Because both primary and mature miR34a were increased p53 expression level after Rubone treatment in HepG2 and after treatment with Rubone, we next examined whether Bel-7404 cells (Supplementary Fig. S5A). Then, we used two Rubone could modulate miR34a promoter activity. HCC cell independent siRNAs targeting p53 (sip53a and sip53b) to lines with different p53 status (HepG2: p53 wild-type, Bel-7404: knockdown both wild-type and mutant p53 (Supplementary

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Figure 5. Rubone inhibited HepG2 xenografted tumor growth in nude mice. A, Rubone inhibited hepatocellular tumor growth in vivo in a dose-dependent manner. Photographs of representative tumors excised from nude mice at day 24. Scale bars, 1 cm. B, the comparison between the hepatocellular tumor inhibition effect of Rubone and sorafenib in vivo. C, modulation of miR34a and its targets by Rubone in vivo. RNA was extracted from the excised tumors. miR34a, cyclin D1, and Bcl-2 levels were measured by qRT-PCR. Error bars represent mean SD of three independent experiments.

Fig. S5B and S5D), and followed by Rubone treatment in HepG2 cardiac diseases (27–29), and malignancy. miR34a was dra- and Bel-7404 cells. qRT-PCR results showed that miR34a matically reduced or silenced in a large portion of clinical level decreased after p53 knockdown (Fig. 7B). Moreover, samples from patients with HCC (30–32). Because miR34a the increased expression level of miR34a by Rubone was functions as an effective suppressor of malignant properties in significantly attenuated by p53 knockdown (Fig. 7B). Mean- cancers (9–13), reexpression of miR34a in tumors represents a while, pri-miR34a expressions exhibited similar change with potential approach for HCC treatment. Indeed, such potential that of miR34a in HepG2 and Bel-7404 cells (Supplementary has been illustrated in numerous studies. It is showed that Fig. S5C and S5E). The MTT assay further revealed that the systemic miR34a delivery suppressed tumor growth in xeno- growth-inhibitory effect of Rubone on HCC cells was also graft or genetically engineered mouse models (33). Further- significantly reversed by knocking down p53 (Fig. 7C and more, overexpression of miR34a in HCC cells significantly Supplementary Fig. S5F). These results suggested that p53 inhibited cell growth and induced apoptosis (17, 34, 35). played important role in the biologic activity of Rubone. We Despite the need for the development of miR34a-based ther- next used ChIP assay to examine whether Rubone could apeutic treatment, few strategies are developed that are lim- modulate p53 activities. The results showed that Rubone ited to direct introduction of miR34a into human cells such as treatment significantly increased p53 occupancy on miR34a systemic delivery of miR34a molecules or viruses. Here, we promoter in both HepG2 and Bel-7404 cells. The effect of identified a small molecule Rubone that acted as the modula- Rubone on p53 was specific to miR34a promoter, as we showed tor of miR34a level. Rubone induced miR34a in HCC cells, but that Rubone failed to increase p53 occupancy on p21 promoter not the nontumor MIHA cells. In contrast, systemic delivery of that was a reported p53-binding region. (Fig. 7D). miR34a will lead to uptake of miR34a by normal cells that induce undesirable miR34a gene silencing. The discovery of Rubone as the agent against HCC can Discussion improve the current therapeutic strategies. The efficacy of miR34a is well known to play important roles in the path- available chemotherapeutic drugs for HCC is low (36). Sora- ogenesis of human diseases, such as metabolic diseases (26), fenib is a forefront therapeutic agent for HCC treatment, which

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miR34a Modulator Inhibits the Growth of Hepatocellular Carcinoma

Figure 6. Rubone inhibited proliferation and induced apoptosis in HepG2 xenografts. A, cell apoptosis was detected by TUNEL assay on the HepG2 xenografts after treatment with Rubone. B, cell proliferation was examined using immunohistochemistry with PCNA antibody. C and D, cell senescence was examined using immunohistochemistry with p16 and p21 antibodies. All scale bars, 50 mm. Error bars represent mean SD of three independent experiments.

showed certain efficacy for patients with HCC by inhibiting potential combined treatment of Rubone and sorafenib in Raf-1 and vascular endothelial growth factor (VEGF) pathways HCC. Meanwhile, given previous studies demonstrated (37). However, sorafenib-treated patients have mild overall increased miR34a in setting of heart disease (27–29), a thor- survival benefits from the treatment and a significant number ough assessment of Rubone on cardiac function in future of them experiences disease progressions (38). Moreover, studies will be required in the future. sorafenib shows toxicity to normal cells (Fig. 4B) and elicits Our study suggested that the action of Rubone to induce serious side effect such as hand-foot skin reaction (39) and miR34a expression is p53-dependent. Previous study has eruptive melanocytic lesions (33). Other agents such as suni- revealed p53 directly and positively regulated the expression tinib and brivanib fail to show superiority to sorafenib (40, 41). of miR34a (8). More than 50% of human tumors contain Therefore, it is still of urgent clinical significance to develop mutation or deletion of p53 gene (43). However, there is still novel therapeutics with better efficacy and less side effect. no report on the relationship between miR34a and mutant p53. Our results showed that Rubone exhibited similar or even Our study showed that Rubone required wild-type or mutant higher anti-HCC potency compared with sorafenib. Regarding p53 to effectively reactivate miR34a expression, suggested for the unspecific toxicity, Rubone did not show any toxicity to the first time that mutant p53 could also modulate miR34a nontumor hepatocytes MIHA cells. In addition, Rubone treat- expression. Many p53 mutations possess gain-of-function ment showed no impairment of the tissues of major organs in effect, but not inactivation of p53 function. For example, the BALB/C mice (Supplementary Fig. S4D). Therefore, Rubone A220G mutation in huh7 cells lowers the melting temperature warrants further investigation as a potential effective anti-HCC and stability of p53 in its DNA-binding domain, causing the agent. Besides, miR34a was reported to enhance the sensitivity denaturation of p53 (44). Study also showed that alterations of anti-HCC effect induced by sorafenib (42), which suggested a induced by p53 mutation can be reversible (45). Another HCC

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Figure 7. Rubone activated miR34a expression by increasing p53 activities. A, Rubone modulated miR34a promoter activity. The arrow above construct P indicates the position of the transcription start site. Filled circles show the position of the p53-binding site. The miR34a promoter reporter constructs were transfected into HepG2, Hep3B, and Bel-7404 cells, respectively. The luciferase activities were measured after 10 mmol/L Rubone treatment. B and C, knockdown of p53 reversed miR34a expression and cell growth inhibition induced by Rubone in HepG2 and Bel-7404 cells. HepG2 and Bel-7404 cells were transfected with 2 mmol/L siRNAs targeting p53 and then treated with 10 mmol/L Rubone. miR34a level was measured by qRT-PCR. Cell viability was measured by the MTT assay. D, Rubone increased p53 occupancy on miR34a promoter. ChIP assay was conducted in HepG2 and Bel-7404 cells treated with 10 mmol/L Rubone. qPCR was used to quantify the p53 occupancy on miR34a and p21 promoter regions. Error bars represent mean SD of three independent experiments.

cell line PLC that was responsive to Rubone expressed G249T- different mechanism compared with other chemotherapeutic mutated p53. It is showed that a designed peptide can rescue drugs. the structural distortion of G249T-mutated p53 (46). In these Practically, the use of Rubone as therapeutic treatment is p53-mutant–expressing cells, the function of p53 is diminished feasible in HCC. We showed that Rubone could induce but not abolished and evidence suggested that the function of miR34a expression with wild-type or mutant p53, but not in p53 mutant could be modulated. It is possible that Rubone acts p53-deleted cells. In HCC, the most common type of p53 as a modulator during the p53-mediated transactivation of alteration is point mutation. Over 50% of human aflatoxin- miR34a. Besides, we believed that the multiple molecular role induced HCC harbor G249T mutation (47), while non-aflatox- of p53 determined the specificity of Rubone to induce miR34a in-induced, HBV-related and HCV-induced HCC have low expression. Rubone may restore or enhance a particular mutation rate (48, 49). More importantly, deletion of p53 is molecular function of p53, such as DNA-binding ability or rare in human HCC (50). Therefore, Rubone is a suitable agent cofactor recruiting ability, which is either in a normal state in to treat HCC that retains p53 in cancer cells. wild-type p53 or suboptimal state in mutant p53. This function is indispensible for the activation of miR34a expression. In this Disclosure of Potential Conflicts of Interest respect, it is understandable that Rubone failed to reactivate No potential conflicts of interest were disclosed. miR34a in Hep3B cells with p53 deletion. Yet, the effect of Rubone to p53 is still unknown, which requires further inves- Authors' Contributions tigation. Our studies showed different p53 response induced by Conception and design: Y. Chen Development of methodology: Z. Xiao Rubone compared with other chemotherapeutic drugs. We Acquisition of data (provided animals, acquired and managed patients, speculate that the reason may be that Rubone worked through provided facilities, etc.): Z. Xiao, S.L. Chan, F. Xu, L. Feng, J.J.Y. Sung

OF10 Cancer Res; 74(21) November 1, 2014 Cancer Research

miR34a Modulator Inhibits the Growth of Hepatocellular Carcinoma

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, Grant Support computational analysis): Z. Xiao, J.J.Y. Sung, C.H.K. Cheng, Y. Chen The work described in this article was supported by grants from the Writing, review, and/or revision of the manuscript: Z. Xiao, C.H. Li, Research Grants Council-General Research Fund of Hong Kong Special S.L. Chan, L. Feng, C.H.K. Cheng, Y. Chen Administrative Region, China (CUHK462109 and CUHK462211), Health and Administrative, technical, or material support (i.e., reporting or orga- Medical Research Fund (11100452) from the Food and Health Bureau, nizing data, constructing databases): C.H. Li, C.H.K. Cheng, Y. Chen Government of Hong Kong Special Administrative Region, National Natural Study supervision: Y. Chen Science Foundation of China (81101888), Shenzhen Basic Research Program Other (helped to obtain data and comment on the article): Y. Wang, (JC201105201092A), and Direct Grant from CUHK to Y. Chen. J.-D. Jiang The costs of publication of this article were defrayed in part by the payment of Other (discussion on future studies): C.H.K. Cheng page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Acknowledgments The authors thank Prof. J.T. Mendell from Johns Hopkins University for Received March 28, 2014; revised August 21, 2014; accepted August 27, 2014; providing the luciferase reporter vector containing miR34a promoter. published OnlineFirst September 12, 2014.

References 1. Kima HY, Park JW. Clinical trials of combined molecular targeted 18. Gumireddy K, Young DD, Xiong X, Hogenesch JB, Huang Q, Deiters A. therapy and locoregional therapy in hepatocellular carcinoma: past, Small-molecule inhibitors of microrna miR-21 function. Angew Chem present, and future. Liver Cancer 2014;3:9–17. Int Ed Engl 2008;47:7482–4. 2. Carthew RW. Gene regulation by microRNAs. Curr Opin Genet Dev 19. Young DD, Connelly CM, Grohmann C, Deiters A. Small molecule 2006;16:203–8. modifiers of microRNA miR-122 function for the treatment of hepatitis 3. Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often flanked C virus infection and hepatocellular carcinoma. J Am Chem Soc by adenosines, indicates that thousands of human genes are micro- 2010;132:7976–81. RNA targets. Cell 2005;120:15–20. 20. Nakabayashi H, Taketa K, Miyano K, Yamane T, Sato J. Growth of 4. Selbach M, Schwanhausser B, Thierfelder N, Fang Z, Khanin R, human hepatoma cell lines with differentiated functions in chemically Rajewsky N. Widespread changes in protein synthesis induced by defined medium. Cancer Res 1982;42:3858–63. microRNAs. Nature 2008;455:58–63. 21. Brown JJ, Parashar B, Moshage H, Tanaka KE, Engelhardt D, Rabbani 5. Ha TY. MicroRNAs in human diseases: from cancer to cardiovascular E, et al. A long-term hepatitis B viremia model generated by transplant- disease. Immune Netw 2011;11:135–54. ing nontumorigenic immortalized human hepatocytes in Rag-2-defi- 6. Hwang HW, Mendell JT. MicroRNAs in cell proliferation, cell death, and cient mice. Hepatology 2000;31:173–81. tumorigenesis. Br J Cancer 2006;94:776–80. 22. Chen R, Zhu D, Ye X, Shen D, Lu R. Establishment of three human liver 7. Janssen HL, Reesink HW, Lawitz EJ, Zeuzem S, Rodriguez-Torres M, carcinoma cell lines and some of their biological characteristics in vitro. Patel K, et al. Treatment of HCV infection by targeting microRNA. Sci Sin 1980;23:236–47. N Engl J Med 2013;368:1685–94. 23. Chen Y, Lin MC, Yao H, Wang H, Zhang AQ, Yu J, et al. Lentivirus- 8. He L, He X, Lim LP, Stanchina ED, Xuan Z, Liang Y, et al. MicroRNA mediated RNA interference targeting enhancer of zeste homolog 2 component of the p53 tumor suppressor network. Nature 2003;447: inhibits hepatocellular carcinoma growth through down-regulation of 1130–4. stathmin. Hepatology 2007;46:200–8. 9. Welch C, Chen Y, Stallings RL. MicroRNA-34a functions as a potential 24. Xiao Z, Chow SC, Li CH, Tang CS, Tsui SK, Lin Z, et al. Role of tumor suppressor by inducing apoptosis in neuroblastoma cells. microRNA-95 in the anticancer activity of Brucein D in hepatocellular Oncogene 2007;26:5017–22. carcinoma. Eur J Pharmacol 2014;728:141–50. 10. Bommer GT, Gerin I, Feng Y, Kaczorowski AJ, Kuick R, Love RE. P53- 25. Li CH, To KF, Tong JH, Xiao Z, Xia T, Lai PB, et al. Enhancer of zeste mediated activation of miRNA-34 candicate tumor-suppressor genes. homolog 2 silences miR-218 in human pancreatic ductal adenocar- Curr Biol 2007;17:1298–307. cinoma cells by inducing formation of heterochromatin. Gastroenter- 11. Yamakuchi M, Ferlito M, Lowenstein CJ. MiR-34a repression of SIRT1 ology 2013;144:1086–97. regulates apoptosis. Proc Natl Acad Sci U S A 2008;105:13421–6. 26. Li S, Chen X, Zhang H, Liang X, Xiang Y, Yu C, et al. Differential 12. Tarasov V, Jung P, Verdoodt B, Lodygin D, Epanchintsev A, Menssen expression of microRNAs in mouse liver under aberrant energy met- A, et al. Differential regulation of microRNAs by p53 revealed by abolic status. J Lipid Res 2009;50:1756–65. massively parallel sequencing: miR-34a is a p53 target that induces 27. Boon RA, Iekushi K, Lechner S, Seeger T, Fischer A, Heydt S, et al. apoptosis and G1-arrest. Cell Cycle 2007;6:1586–93. MicroRNA-34a regulates cardiac ageing and function. Nature 13. Tazawa H, Tsuchiya N, Izumiya M, Nakagama H. Tumor-suppressive 2013;495:107–10. miR-34a induces senescence-like growth arrest through modulation of 28. Bernardo BC, Gao XM, Winbanks CE, Boey EJ, Tham YK, Kiriazis H, the E2F pathway in human colon cancer cells. Proc Natl Acad Sci U S A et al. Therapeutic inhibition of the miR-34 family attenuates patholog- 2007;104:15472–7. ical cardiac remodeling and improves heart function. Proc Natl Acad 14. Toyota M, Suzuki H, Sasaki Y, Maruyama R, Imai K, Shinomura Y, et al. Sci U S A 2012;109:17615–20. Epigenetic silencing of microRNA-34b/c and B-cell translocation gene 29. Bernardo BC, Gao XM, Tham YK, Kiriazis H, Winbanks CE, Ooi JY, 4 is associated with CpG island methylation in colorectal cancer. et al. Silencing of miR-34a attenuates cardiac dysfunction in a setting Cancer Res 2008;68:4123–32. of moderate, but not severe, hypertrophic cardiomyopathy. PLoS ONE 15. Hashimi ST, Fulcher JA, Chang MH, Gov L, Wang S, Lee B. MicroRNA 2014;9:e90337. profiling identifies miR-34a and miR-21 and their target genes JAG1 30. Chiu LY, Kishnani PS, Chuang TP, Tang CY, Liu CY, Bali D, et al. and WNT1 in the coordinate regulation of dendritic cell differentiation. Identification of differentially expressed microRNAs in human hepa- Blood 2009;114:404–14. tocellular adenoma associated with type I glycogen storage disease: a 16. Chang TC, Wentzel EA, Kent OA, Ramachandran K, Mullendore M, potential utility as biomarkers. J Gastroenterol 2013;49:1274–84. Lee KH, et al. Transactivation of miR-34a by p53 broadly influences 31. Mizuguchi Y, Mishima T, Yokomuro S, Arima Y, Kawahigashi Y, gene expression and promotes apoptosis. Mol Cell 2007;26: Shigehara K, et al. Sequencing and bioinformatics-based analyses of 745–52. the microRNA transcriptome in hepatitis B-related hepatocellular 17. Li N, Fu H, Tie Y, Hu Z, Kong W, Wu Y, et al. miR-34a inhibits migration carcinoma. PLoS ONE 2011;6:e15304. and invasion by down-regulation of c-Met expression in human hepa- 32. Bader AG. miR-34—a microRNA replacement therapy is headed to the tocellular carcinoma cells. Cancer Lett 2009;275:44–53. clinic. Front Genet 2012;3:120.

www.aacrjournals.org Cancer Res; 74(21) November 1, 2014 OF11

Xiao et al.

33. Kong HH, Sibaud V, Chanco Turner ML. Sorafenib-induced eruptive 42. Yang F, Li QJ, Gong ZB, Zhou L, You N, Wang S, et al. MicroRNA-34a melanocytic lesions Arch Dermatol 2008;144:820–2. targets Bcl-2 and sensitizes human hepatocellular carcinoma cells to 34. Lou W, Chen Q, Ma L, Liu J, Yang Z, Shen J, et al. Oncolytic adenovirus sorafenib treatment. Technol Cancer Res Treat 2014;13:77–86. co-expressing miRNA-34a and IL-24 induces superior antitumor activ- 43. Levine AJ. p53: the cellular gatekeeper for growth and division. Cell ity in experimental tumor model. J Mol Med 2013;91:715–25. 1997;88:323–31. 35. Yang P, Li QJ, Feng Y, Zhang Y, Markowitz GJ, Ning S, et al. TGF-beta- 44. Khoury K, Popowicz GM, Holak TA, Domling A. The p53-MDM2/ miR-34a-CCL22 signaling-induced Treg cell recruitment promotes MDMX axis—a chemotype perspective. Medchemcomm 2011;2: venous metastases of HBV-positive hepatocellular carcinoma. Cancer 246–60. Cell 2012;22:291–303. 45. Boeckler FM, Joerger AC, Jaggi G, Rutherford TJ, Veprintsev DB, 36. Lee J, Park JO, Kim WS, Park SH, Park KW, Choi MS, et al. Phase II Fersht AR. Targeted rescue of a destabilized mutant of p53 by an study of doxorubicin and cisplatin in patients with metastatic hepa- in silico screened drug. Proc Natl Acad Sci U S A 2008;105:10360–5. tocellular carcinoma. Cancer Chemother Pharmacol 2004;54:385–90. 46. Friedler A, DeDecker BS, Freund SM, Blair C, Rudiger S, Fersht AR. 37. Wilhelm SM, Adnane L, Newell P, Villanueva A, Llovet JM, Lynch M. Structural distortion of p53 by the mutation R249S and its rescue by a Preclinical overview of sorafenib, a multikinase inhibitor that targets designed peptide: implications for "mutant conformation". J Mol Biol both Raf and VEGF and PDGF receptor tyrosine kinase signaling. Mol 2004;336:187–96. Cancer Ther 2008;7:3129–40. 47. Bressac B, Kew M, Wands J, Ozturk M. Selective G to T mutations of 38. Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc JF, et al. p53 gene in hepatocellular carcinoma from southern Africa. Nature Sorafenib in advanced hepatocellular carcinoma. N Engl J Med 1991;350:429–31. 2008;359:378–90. 48. Dejean A, Bougueleret L, Grzeschik KH, Tiollais P. Hepatitis B virus 39. Lacouture ME, Reilly LM, Gerami P, Guitart J. Hand foot skin reaction in DNA integration in a sequence homologous to v-erb-A and steroid cancer patients treated with the multikinase inhibitors sorafenib and receptor genes in a hepatocellular carcinoma. Nature 1986;322: sunitinib. Ann Oncol 2008;19:1955–61. 70–2. 40. Huynh H, Choo SP, Toh HC, Tai WM, Chung AY, Chow PK, et al. 49. Wang J, Chenivesse X, Henglein B, Brechot C. Hepatitis B virus Comparing the efﬁcacy of sunitinib with sorafenib in xenograft models integration in a cyclin A gene in a hepatocellular carcinoma. Nature of human hepatocellular carcinoma: mechanistic explanation. Curr 1990;343:555–7. Cancer Drug Targets 2011;11:944–53. 50. Tornesello ML, Buonaguro L, Tatangelo F, Botti G, Izzo F, Buonaguro 41. Scarpitta SC, Harley NH. Adsorption and desorption of noble gases on FM. Mutations in TP53, CTNNB1 and PIK3CA genes in hepatocellular activated charcoal: II. 222Rn studies in a monolayer and packed bed. carcinoma associated with hepatitis B and hepatitis C virus infections. Health Phys 1990;59:393–404. Genomics 2013;102:74–83.

OF12 Cancer Res; 74(21) November 1, 2014 Cancer Research

A Small-Molecule Modulator of the Tumor-Suppressor miR34a Inhibits the Growth of Hepatocellular Carcinoma

Zhangang Xiao, Chi Han Li, Stephen L. Chan, et al.

Cancer Res Published OnlineFirst September 12, 2014.

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