Genome-Wide Study of Hypomethylated and Induced Genes in Patients with Liver Cancer Unravels Novel Anticancer Targets

Published OnlineFirst April 24, 2014; DOI: 10.1158/1078-0432.CCR-13-0283

Clinical Cancer Human Cancer Biology Research

Barbara Stefanska1,5, David Cheishvili1, Matthew Suderman1, Ani Arakelian2, Jian Huang6, Michael Hallett3, Ze-Guang Han6, Mamun Al-Mahtab7, Sheikh Mohammad Fazle Akbar9, Wasif Ali Khan8, Rubhana Raqib8, Imrana Tanvir10, Haseeb Ahmed Khan10, Shafaat A. Rabbani2, and Moshe Szyf1,4

Abstract Purpose: We utilized whole-genome mapping of promoters that are activated by DNA hypomethylation in hepatocellular carcinoma (HCC) clinical samples to shortlist novel targets for anticancer therapeutics. We provide a proof of principle of this approach by testing six genes short-listed in our screen for their essential role in cancer growth and invasiveness. Experimental Design: We used siRNA- or shRNA-mediated depletion to determine whether inhibition of these genes would reduce human tumor xenograft growth in mice as well as cell viability, anchorage- independent growth, invasive capacities, and state of activity of nodal signaling pathways in liver, breast, and bladder cancer cell lines. Results: Depletion of EXOSC4, RNMT, SENP6, WBSCR22, RASAL2, and NENF effectively and speciﬁcally inhibits cancer cell growth and cell invasive capacities in different types of cancer, but, remarkably, there is no effect on normal cell growth, suggesting a ubiquitous causal role for these genes in driving cancer growth and metastasis. Depletion of RASAL2 and NENF in vitro reduces their growth as explants in vivo in mice. RASAL2 and NENF depletion interferes with AKT, WNT, and MAPK signaling pathways as well as regulation of epigenetic proteins that were previously demonstrated to drive cancer growth and metastasis. Conclusion: Our results prove that genes that are hypomethylated and induced in tumors are candidate targets for anticancer therapeutics in multiple cancer cell types. Because these genes are particularly activated in cancer, they constitute a group of targets for speciﬁc pharmacologic inhibitors of cancer and cancer metastasis. Clin Cancer Res; 1–15. 2014 AACR.

Introduction epigenetic alterations leading to activation of oncogenes Cancer initiation and progression are driven by concur- and prometastatic genes, silencing of tumor suppressor rent changes in expression of multiple genes via genetic and genes and to genome rearrangements and instability (1– 3). As extensive research revealed methylation-mediated silencing of tumor suppressor genes to be common in Authors' Afﬁliations: Departments of 1Pharmacology and Therapeutics and cancer, therapeutic strategies have been developed with the 2 3 Medicine, McGill University Health Centre, Montreal; McGill Centre for goal of decreasing DNA methylation using inhibitors of Bioinformatics; and 4Sackler program for Psychobiology and Epigenetics at McGill University, Montreal, Quebec, Canada; 5Department of Nutrition DNA methyltransferases (DNMT), enzymes catalyzing Science, Purdue University, West Lafayette, Indiana; 6Shanghai-MOST Key DNA methylation reaction (1, 4–7). Laboratory for Disease and Health Genomics, Chinese National Human Genome Center at Shanghai, Shanghai, China; 7Department of Hepatology, However, in contrast with the overall focus of the ﬁeld on Bangabandhu Sheikh Mujib Medical University; 8International Centre for hypermethylation both from the therapeutic and diagnostic Diarrhoeal Diseases Research, Bangladesh (icddr,b), Dhaka, Dhaka District, perspectives, genome-wide high-resolution studies have Bangladesh; 9Department of Medical Sciences, Toshiba General Hospital, Tokyo, Kanto, Japan; and 10Department of Pathology, Fatima Memorial shown that DNA hypomethylation is as frequent and per- Hospital College of Medicine and Dentistry Lahore, Pakistan sistent as hypermethylation in cancer and it targets not just Note: Supplementary data for this article are available at Clinical Cancer repetitive sequences as has been previously well documen- Research Online (http://clincancerres.aacrjournals.org/). ted (8) but also promoters of genes involved in cell invasion and metastasis (2, 9–12). A recent comprehensive study B. Stefanska and D. Cheishvili contributed equally to this work. demonstrated that zebularine, a DNA methylation inhibi- Corresponding Author: Moshe Szyf, Department of Pharmacology and Therapeutics, McGill University Medical School, 3655 Sir William Osler tor, exhibited a dual mode of action on hepatocellular Promenade #1309, Montreal, Que., Canada H3G 1Y6. Phone: 1-514-398- carcinoma (HCC) cells depending on the cellular context 7107; Fax: 1-514-398-6690; E-mail: [email protected] (7). In zebularine-sensitive cells, the drug caused demeth- doi: 10.1158/1078-0432.CCR-13-0283 ylation and activation of tumor suppressor genes as well as 2014 American Association for Cancer Research. genes involved in regulation of apoptosis and cell-cycle

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cell proliferation and differentiation, cell-cycle progression, Translational Relevance signal transduction, transcriptional regulation, DNA and Our study provides evidence that aberrant DNA pro- RNA modifications. In this study, we selected 6 genes from moter hypomethylation in cancer leads to activation of this list, Ras-GTPase-activating protein (RASAL2), neuron- genes that are required for continued cancer cell prolif- derived neurotrophic factor (NENF, neudesin), Williams– eration and invasiveness. These sets of genes are candi- Beuren syndrome chromosome region 22 (WBSCR22), date targets for anticancer therapy. We show, for the first SUMO1/sentrin-specific peptidase 6 (SENP6), exosome time, that silencing of RASAL2, NENF, EXOSC4, SENP6, complex component RRP41 (EXOSC4), RNA (guanine-7-) and WBSCR22 in human liver cancer cell lines inhibits methyltransferase (RNMT); and tested whether their deple- cell growth and invasive properties. Depletion of tion with selective siRNA attenuates cell growth and inva- RASAL2 and NENF reduces anchorage-independent siveness of HCC cancer cell line HepG2. RASAL2 is a protein growth in vitro and human subcutaneous tumor xeno- containing the GAP-related domain (GRD) that is a char- graft growth in mice. We establish the critical role of acteristic domain of GTPase-activating proteins (GAP) that RASAL2 and NENF in multiple human cancer cell lines were shown to be crucial for AKT activity and bind to AKT and provide insights into the mechanism of action. Our inducing its phosphorylation via integrin-linked kinase studies describe a general workflow for identifying novel (13). RASAL2 was recently shown to interact with ECT2, anticancer therapeutic targets by selecting genes hypo- a guanine nucleotide exchange factor that is overexpressed methylated in cancer. in primary astrocytomas, activating promigratory RHO family members (14). Knockdown of RASAL2 decreases ECT2 activity toward RHO in astrocytoma cells and leads to mesenchymal–amoeboid transition, altering the invasive progression. This was concomitant with inhibition of pro- properties of the cells. NENF is a secreted protein with liferation and induction of apoptosis. However, the same neurotrophic activity expressed abundantly in the develop- drug led to demethylation and activation of oncogenes and ing brain and spinal cord (15). It was shown to induce oncogenic pathways in resistant cells. This differential phosphorylation of ERK1/2 and AKT in primary cultured action was independent of the fact that DNMT1 inhibition mouse neurons and MCF-7 breast cancer cells (15). NENF is was observed in both sensitive and resistant cells (7). Genes also highly expressed in several cancers and its overexpres- that are activated in cancer and are required for cancer sion in MCF-7 breast cancer cells increases their tumorige- growth and cancer metastasis are likely targets for novel nicity and invasiveness (16). WBSCR22 that contains a drug development that could also overcome resistance to methyltransferase domain and is part of the chromosomal existing chemotherapeutics. Moreover, genome-wide anal- region deleted in Williams syndrome, a multisystem devel- yses might reveal genes that are commonly hypomethylated opmental disorder (17), was shown to be overexpressed in in many cancers and are thus candidates for novel broad- invasive breast cancer. Ectopic expression of WBSCR22 in spectrum anticancer and antimetastatic agents. Thus, DNA nonmetastatic cells enhances metastasis formation without hypomethylation analysis should unravel novel targets for affecting cell growth and motility and its knockdown in anticancer therapeutics that were unrealized to date. tumor cells reverses metastasis in breast cancer cells (18). We tested this hypothesis by examining a dataset of 230 SENP6 is member of a family of sumoylation proteases (19) genes that were hypomethylated and induced in HCC in that were shown to be elevated in several kinds of tumors comparison with normal adjacent tissue (1.5 fold change and contribute to cancer through altering the balance of in promoter methylation and in expression, P 10E3) as sumoylation of important transcription regulators (20). For we have recently delineated (2). Within these 230 hypo- SENP6 however, a few reports show downregulation in methylated genes, we focused on 111 genes with promoters breast cancer (20). EXOSC4, is a noncatalytic member of of high CpG density as they showed overall the most robust RNA exosome complex and is involved in RNA degradation induction in expression in tumors (2-fold, P 10E4; with no documented role to our knowledge in cancer (21). ref. 2). We used GO, KEGG, and NCBI databases in order to RNMT is involved in methylating mRNA cap structures establish biologic processes, functions, and pathways that (22). mRNA cap methylation is rate limiting for translation these genes are involved in. We found a large body of of the c-MYC oncogene in epithelial cells (23). literature that documented the involvement of 91 of these We showed for the first time that depletion of 5 of the 6 genes in biologic processes, functions, and/or pathways genes (excluding RNMT) in liver cancer cell lines caused characteristic of cancer and carcinogenesis. The role of the attenuation of cancer growth and invasiveness, suggesting remaining 20 genes in cancer has been unknown. Although that the genome-wide approach we have undertaken should some of the genes have unknown biologic functions, hypo- point to potentially cancer critical genes with high efficien- methylation and induction of these genes in tumors com- cy. We further focused on 2 of these genes, RASAL2 and pared with normal tissue suggest their potential role as NENF, as several reports suggest their involvement in reg- novel anticancer targets and warranted further validation ulation of key oncogenic signal transduction pathways, and elaboration on their effects. Pathway analysis of these MAPK and AKT (13, 15). Our findings show that depletion 20 new candidates showed possible involvement in func- of RASAL2 or NENF decreases tumorigenic properties of tional networks, which might be crucial to cancer such as different cancer cells, including liver, bladder, and breast

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cancer, and reduces the ability of liver cancer cells to form Viability, invasion, and anchorage-independent tumor xenografts in mice. This was associated with reduced growth assays phosphorylation of key members of MAPK, AKT/mTOR, Cell viability was determined by the Trypan blue (Sigma- and WNT oncogenic signaling cascades and downregula- Aldrich) exclusion test. Cells were harvested after transfection of DNMT1 and MBD2, proteins that were shown to be tion with siRNA on day 3, 6, and 9. Following 3 to 5 minute involved in tumorigenesis and metastasis (4, 5). Our pres- incubation with Trypan blue, the viable cells were counted ent work provides proof of principle that hypomethylated under the microscope. genes in tumors are broad-spectrum candidate anticancer The ability of cells treated with siCtrl and siRASAL2 or targets and describes a method for identifying these targets siNENF to invade through extracellular matrix was evalu- using genome-wide DNA methylation and transcriptome ated by the Cell Invasion Assay Kit (Chemicon Int.). The kit screens in human tumors. utilizes a reconstituted basement membrane matrix of proteins derived from Engelbreth–Holm–Swarm (EHS) mouse Materials and Methods tumor. Briefly, 50,000 cells resuspended in serum-free Tissue samples, cell lines, and transfection with siRNA media were added to the inserts dipped in the lower cham- Cancerous and normal adjacent tissue samples were ber containing complete media. Following 24-hour incu- obtained from 11 patients with HCC in Chinese National bation at 37C, invasive cells were stained and counted Human Genome Center at Shanghai, China (Dr. Z.-G. under the microscope. Han). All patients provided written informed consent, and The effects of transfection with siRNA are transient. the Ethics Committee from Chinese National Human To measure long-term impact of depletion of these genes, Genome Center at Shanghai approved all aspects of this we generated RASAL2-orNENF-depleted stable HepG2, study. Human HCC HepG2, HCC Hep3B, adenocarcinoma SkHep1, and NorHep cell lines with shRNA-expressing SkHep1, bladder cancer T24, and breast cancer MDA-MB- lentivirus (see Supplementary Materials and Methods for 231 cells were purchased from ATCC (HB8065, HB8064, details on lentivirus production and cell transduction and HTB52, HTB4, and HTB26, respectively), whereas human Supplementary Table S1D for shRNA sequences). To deter- untransformed hepatocytes (normal hepatocytes, NorHep) mine anchorage-independent growth on soft agar, an in were obtained from Celprogen (33003-02). HepG2, vitro measure of oncogenesis (24), 6,000 to 12,000 live cells Hep3B, SkHep1, and MDA-MB-231 cells were maintained with stable knockdown of RASAL2 or NENF were seeded in MEM medium (Gibco) and T24 cells in McCoy’s 5A into soft agar and plated in triplicate in a 6-well plate for 21 medium, supplemented with 2 mmol/L glutamine (Sigma- days as previously described (25). The number of colonies Aldrich), 10% fetal bovine serum (FBS; Gibco), 1 U/mL (>10 cells/colony) in 5 random fields (40) per well, penicillin, and 1 mg/mL streptomycin (Gibco). NorHep throughout all planes of the triplicate wells, was counted cells were maintained in human hepatocyte cell culture under the microscope. complete medium (Celprogen). Cells were grown in a humidified atmosphere of 5% carbon dioxide at 37C. RNA extraction and quantitative real-time PCR Twenty-four hours before siRNA treatment, cells were plat- Total RNA was isolated using TRizol (Invitrogen, Life ed at a density of 3 to 6 105 per a 10-cm tissue culture dish. Technologies) according to the manufacturer’s protocol. The following siRNA, obtained from Dharmacon, were One micrograms of total RNA served as a template for cDNA used in this study (see Supplementary Table S1A for the synthesis using 20 U of AMV reverse transcriptase (Roche sequences): control siRNA (siCtrl), human RASAL2 siRNA Diagnostics), as recommended by the manufacturer. The (siRASAL2, M-009411-02-0020, and siRASAL2-1), human quantitative real-time PCR (QPCR) reaction was carried out NENF siRNA (siNENF, M-016079-00-0020, siNENF-1), in Light Cycler 480 machine (Roche) using 2 mL of cDNA, human EXOSC4 siRNA (siEXOSC4, M-013760-01-0020), 400 nmol/L forward and reverse primers listed in Supple- human RNMT siRNA (siRNMT, M-019525-00-0020), mentary Table S1B and 10 mL of Light Cycler 480 SybrGreen human SENP6 siRNA (siSENP6, M-006044-01-0020), I Master (Roche) in a final volume of 20 mL. Amplification human WBSCR22 siRNA (siWBSCR22, M-009383-00- was performed using the following conditions: denatur- 0020). The cells were transfected with siRNA using lipofec- ation at 95C for 10 minutes, amplification for 60 cycles at tamine RNAiMAX (Invitrogen) in serum-free Opti-MEM. 95C for 10 seconds, annealing temperature for 10 seconds, Fifteen microliters of lipofectamine were incubated in 500 72C for 10 seconds, and final extension at 72C for 10 mL of Opti-MEM for 45 minutes at room temperature. minutes. Quantification was performed using a standard siRNA was added to the Opti-MEM-lipofectamine solution curve and analyzed by the Roche LightCycler 480 software. to a final concentration of 56 nmol/L. The mixture was incubated for 15 minutes at the same conditions; Opti- Pyrosequencing MEM was added to a final volume of 5 mL and was then Bisulfite conversion was performed as previously added onto the plates. The transfection solution was described (26). Specific bisulfite converted promoter removed from the cells and replaced with standard medium sequences were amplified with HotStar Taq DNA polymer- after 4 hours. The cells were split 1:2 after 48 hours and ase (Qiagen) using biotinylated primers listed in Supple- transfected again 24 hours later. The transfection sequence mentary Table S1C. The biotinylated DNA strands were was repeated 3 times. pyrosequenced in the PyroMarkTMQ24 instrument

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(Biotage, Qiagen) as previously described (27). Data were translated. HepG2 and SkHep1 cells were treated with 56 analyzed using PyroMarkTMQ24 software. nmol/L siRASAL2, siNENF, or siCtrl as described above. After 48 hours, the transfection was repeated and the cells were Western blot analyses harvested and counted 24 hours later. A small pool of 105 Total protein extract was obtained by dissolving in RIPA cells was saved for QPCR analysis in order to confirm buffer (1 PBS, 1% NP-40, 0.5% sodium deoxycholate, knockdown of the target genes. Viable cells in amount of 0.1% SDS, and 1 complete protease inhibitors; Roche 2 106 of HepG2 and 1 106 of SkHep1 cells were injected Diagnostics). The total protein yield was determined using subcutaneously into the mice in 50% matrigel. The next steps Bradford reagent (Bio-Rad). A total of 50 to 100 mgof were identical to the protocol with stable cell lines. proteins were loaded on a 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were immuno- Statistical analysis blotted with anti-RASAL2 (Bethyl Laboratories Inc., A302- Statistical analysis was performed using an unpaired t test. 109A) or anti-NENF (Abcam, ab74474) antibody at 1:1,000 Each value represents the mean SD of 3 determinations in and 1:500 dilution, respectively, followed by a secondary either 2 or 3 independent experiments. The results were anti-rabbit (Santa-Cruz Biotechnology; sc-2004) IgG anti- considered statistically significant when P < 0.05. body at 1:5,000 or 1:4,000 dilution, respectively. The membranes were blotted with an anti-b-actin antibody as Results a loading control (Sigma-Aldrich). Biologic functions of novel liver cancer candidate genes hypomethylated and induced in HCC Determination of kinase phosphorylation We reasoned that genes that are potentially critical for Relative levels of phosphorylation of a set of kinases were cancer growth and metastasis but not for normal tissue determined using Human Phospho-MAPK Array Kit survival and physiology would be epigenetically repro- (ARY002B; R&D Systems, Inc.). The kit utilizes control and grammed and activated in tumors in comparison with nor- capture antibodies carefully selected and spotted in dupli- mal noncancerous tissue. Our previous study of the land- cate on nitrocellulose membrane. Briefly, cell lysates were scape of DNA methylation in tumors from patients with liver diluted, mixed with a cocktail of biotinylated detection cancer revealed a group of 230 genes that were induced and antibodies, and incubated overnight with the Proteome whose promoters were hypomethylated in HCC in compar- Profiler Human Phospho-MAPK array membrane. The ison with the matched adjacent tissue (2). We shortlisted 20 membranes were washed to remove unbound material. genes that were not previously shown to be anticancer targets Streptavidin-horseradish peroxidase and chemilumines- (Table 1). The analysis of their functions based on publicly cent detection reagents were then applied and a signal was available datasets such as Gene Ontology (GO), KEGG, and produced at each capture spot corresponding to the amount NCBI revealed biologic processes and pathways that are of phosphorylated protein bound. The densitometric anal- crucial to carcinogenesis (Table 1). The Pharmacogenomics ysis of the spot intensity on developed film was performed Knowledgebase (PharmGKB) and Therapeutic Target Data- using ImageJ software. A signal from a clear area of the array base were used to verify whether any of these 20 genes is was used as a background value. An average background already a target of a known drug or acts within a known drug signal was subtracted from each positive spot and the pathway. We did not find any information about drug resulting value constituted a relative phosphorylation level. development for any of the candidates listed in Table 1. To define the functional role of the identified novel putative Human tumor xenografts in NOD/SCID mice cancer targets in cellular transformation, we used siRNA/ For xenograft studies, 4 to 6 weeks old male NOD/SCID shRNA-mediated depletion in liver cancer cell lines as a mice (The Jackson Laboratory) were divided randomly into 6 tractable cellular model for human liver tumors where these groups of 6 animals each. We used stable RASAL2 or NENF- genes were found to be hypomethylated and activated. We depleted HepG2 and SkHep1 cell lines in the in vivo study to first focused on 2 genes, RASAL2 and NENF because previous verify long-term knockdown (see Supplementary Materials literature data suggested their involvement in MAPK and/or and Methods for details on lentivirus production and cell AKT signal transduction pathways and their likely important transduction). Two million viable cells transduced with sh- role in carcinogenesis and metastasis (14, 16). Scr (nontargeting negative control shRNA), shRASAL2 (sequence No. 7), or shNENF (sequence No. 2) expressing RASAL2 and NENF are regulated by DNA promoter lentivirus were injected subcutaneously into the mice in 50% methylation in HCC tumors and liver cancer cell lines matrigel. Tumor volume was measured at weekly intervals and are overexpressed in several types of human cancer starting at week 2 using the formula: tumor volume ¼ (length Promoter methylation microarrays and Affymetrix width2)/2 until the mice were sacrificed. The experimental expression microarray data indicated that RASAL2 and animal protocol was in accordance with the McGill Univer- NENF promoters are hypomethylated and that their expres- sity Animal Care Committee guidelines. We also repeated the sion is induced in HCC samples compared with adjacent study with transiently transfected cells to measure the long- normal tissue (Fig. 1A–C; see Supplementary Materials and term impact of transient depletion of these genes, which Methods for details on microarray analyses). Statistically would mimic a pharmacologic treatment if these data are significant hypomethylation of several CG sites in the

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Table 1. Functional analysis of 20 genes whose promoters were signiﬁcantly hypomethylated and induced in liver cancer samples compared with matched adjacent normal tissue as assessed by genome-wide promoter microarray and Affymetrix gene expression array

Gene Pathway/function/ symbol Gene name Chromosome biologic process CASD1 CAS1 domain containing 1, O-acetyltransferase 7 Not known, integral to membrane CCDC138 Coiled-coil domain containing 138 2 Not known CSPP1 Centrosome and spindle pole–associated protein 1 8 Positive regulation of cell division, cell-cycle–dependent microtubule organization EXOSC4 Exosome complex component RRP41 8 mRNA metabolic process, positive regulation of cell growth FAM83D Family with sequence similarity 83, member D 20 Chromosome congression and alignment during mitosis, cell division GDPD1 Glycerophosphodiester phosphodiesterase 17 Lipid metabolic process, glycerol metabolic domain containing 1 process IFT81 Intraflagellar transport 81 homolog (Chlamydomonas), 12 Development of the testis and carnitine deficiency-associated gene expressed spermatogenesis, cell differentiation, in ventricle 1 cardiac hypertrophy caused by carnitine deficiency JPH3 Junctophilin 3, trinucleotide repeat-containing 16 Formation of junctional membrane gene 22 protein complexes, cross-talk between the cell surface and intracellular calcium release channels, regulation of neuronal synaptic plasticity KCTD2 Potassium channel tetramerization domain containing 2 17 Protein homooligomerization, voltage-gated potassium channel activity NEIL3 Nei endonuclease VIII-like 3 (E. coli) 4 DNA N-glycosylase activity, base-excision repair, damaged DNA binding NENF Neudesin neurotrophic factor 1 Activation of MAPK and AKT signaling pathways, growth factor activity PAQR4 Progestin and adipoQ receptor family member IV 16 Not known, integral to membrane, putative receptor activity PFKFB2 6-Phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 2 1 Carbohydrate metabolic process RASAL2 RAS protein activator like 2 1 Regulation of small GTPase-mediated signal transduction, GTPase activator activity, inhibitory regulator of the Ras/cyclic-AMP pathway, putative activator of the AKT pathway, somatic mutations identified in colorectal cancer RNMT RNA (guanine-7-) methyltransferase 18 mRNA-capping methyltransferase, hepatocyte growth factor receptor (HGFR), putative proto-oncogenic receptor tyrosine kinase SENP6 SUMO1/sentrin specific peptidase 6 6 Regulation of transcription, deconjugation of SUMO1, SUMO2 and SUMO3 from targeted proteins SMYD5 SET and MYND domain-containing protein 5, 2 Not known, metal ion binding retinoic acid–induced protein 15 SRRT Serrate RNA effector molecule homolog, arsenite 7 Cell proliferation, regulation of transcription, resistance protein RNA-mediated gene silencing by miRNAs TMX2 Thioredoxin-related transmembrane protein 2, cell 11 Not known, cell redox homeostasis proliferation–inducing gene 26 protein WBSCR22Williams–Beuren syndrome chromosome region 22, 7 Methyltransferase activity putatively Williams–Beuren candidate region putative acting on DNA methyltransferase

NOTE: Functional analyses were performed using GO, KEGG, and NCBI databases.

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promoters of these genes in the tumors group in compar- human liver cell culture model, a cancer cell type where the ison with the control tissue group was confirmed by pyr- epigenetic activation of these genes was first discovered (Fig. osequencing of DNA samples from all 11 patients as shown 1A and B). We depleted RASAL2 or NENF mRNA in HepG2 in Fig. 1D and E (see Supplementary Fig. S1 for extensive HCC and SkHep1 liver adenocarcinoma cell lines, as well as methylation analysis). Interestingly, RASAL2 and NENF in non-transformed normal hepatocytes (NorHep) to test were also upregulated in HepG2 HCC and SkHep1 liver whether the effects are cancer specific. For long-term studies adenocarcinoma cell lines as compared with nontrans- such as soft-agar assay, stable HepG2, SkHep1, and NorHep formed normal hepatocytes (NorHep; Fig. 1F) and their cell lines with RASAL2 or NENF depletion were established promoters were hypomethylated at the same CpG sites as and used rather than siRNA-transfected cells (see Supple- in patients with HCC (Fig. 1G and H; see Supplementary mentary Materials and Methods for details on lentivirus Fig. S1 for extensive methylation analysis). The cell lines production and cell transduction). Depletion of either were chosen for further experiments as a suitable model RASAL2 or NENF expression in the liver cancer cell lines to study the functional role of these genes in cellular (Fig. 2A and E and Supplementary Fig. S4 and S5C, S5D, transformation and invasiveness using siRNA/shRNA- S5G, and S5H) resulted in inhibition of the rate of cancer mediated depletion. cell growth (Fig. 2B and F and Supplementary Fig. S5A and To determine causal relationship between promoter S5E), cell invasive capacities (Fig. 2C and G and Supple- hypomethylation and activation of these genes in HCC, mentary Fig. S5B and S5F) and anchorage independent we tested whether the DNA methylation inhibitor 5-aza- growth as compared with control nontreated cells (Fig. 20-deoxycytidine (5-azaCdR) would induce these genes in 2D and H). Importantly, using other sets of siRNAs (siR- HepG2 liver cancer cells as well as in NorHep cell culture. ASAL-1 and siNENF-1) or shRNAs (shRASAL-9 and NorHep were included in this experiment because of shNENF-1), we observed similar effects on cell growth and high-methylation state of promoters of the tested genes. metastatic properties in both HepG2 and SkHep1 cells Both RASAL2 and NENF were induced upon treatment of (Supplementary Figs. S3 and S5). These data demonstrate HepG2 cells with 1.0 mmol/L 5-azaCdR for 3 days, which that the results we present are not an idiosyncrasy of the was accompanied with strong hypomethylation of their siRNA sequence used and provide high confidence beyond a promoters (Fig. 1I; see Supplementary Fig. S1 for exten- reasonable statistical doubt that we are measuring effects of sive methylation analysis). In NorHep, hypomethylation RASAL2 and NENF depletion. We also confirmed the role of of NENF promoter was associated with its upregulation RASAL2 and NENF in HCC by examining the consequences just after 3-day treatment, whereas hypomethylation of knockdown of these genes in a different HCC cell line that and induction of RASAL2 were observed after 20-day bears an integrated hepatitis B virus (HBV) genome, Hep3B exposure to 5-azaCdR (Supplementary Fig. S2; see Sup- (Supplementary Fig. S6A). Depletion of RASAL2 and NENF plementary Fig. S1 for extensive methylation analysis, with siRNAs in Hep3B cells significantly inhibited cell note no cytotoxic effect of the treatment in NorHep in growth and invasive properties. Supplementary Fig. S2A). These results support regula- The effects of siRNA/shRNA-mediated depletion of the tion of transcription of these candidates by DNA tested candidates on cell growth, anchorage-independent methylation. growth, and invasiveness differentiate between cancer and We reasoned that hypomethylation should target genes normal cell lines. NorHep cells that were treated with that are fundamental to the transformation process and siRNAs/shRNAs targeting RASAL2 or NENF proliferated at that, therefore, there should be a common set of genes that almost the same rate as control cells with a slight decrease is hypomethylated in several cancer types. Consistent with only after triple siNENF treatment (9 days; Fig. 2B and F and this hypothesis we show that overexpression of RASAL2 and Supplementary Fig. S5A and S5E). There was no significant NENF is not specific only to liver cancer. Using publicly effect on invasiveness or anchorage independent growth in available expression microarray data (Oncomine), we dem- either of the treatments (Fig. 2 and Supplementary Fig. S5). onstrate that the genes show increased expression in lym- These data confirm the cancer-specific requirement for phoma, breast, colorectal, lung (NENF), pancreatic upregulation of these genes and that targeting these genes (RASAL2), and an independent set of HCC tumors (differ- for inhibition has a profound anticancer effect demonstrat- ent from the set analyzed by our group; Supplementary Fig. ing their potential as targets for anticancer and antimeta- S3A). The data are consistent with a general role for these static drugs. proteins in many different types of cancer. This is consistent We then evaluated whether 4 other genes from the list of with the hypothesis that these proteins may play a funda- 20 novel putative cancer candidates (Table 1) such as mental role in either cancer progression or metastasis and EXOSC4, RNMT, SENP6, and WBSCR22 have a functional with recent data, suggesting a role for RASAL2 and NENF in role in liver cancer and are potential targets of anticancer cancer (14, 16). therapies. Our previous genome-wide studies revealed hypomethylation of promoters of these genes and induction Invasiveness of liver cancer is dependent on RASAL2, of their expression in HCC tumors in comparison with NENF, EXOSC4, SENP6,orWBSCR22 expression adjacent normal tissue (Fig. 3A and B). The genes were To test whether RASAL2 and NENF play a causal role in depleted in HepG2 HCC cells using siRNA as described in cellular transformation and invasiveness, we first used a Materials and Methods (Fig. 3C) followed by assessing the

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Figure 1. RASAL2 and NENF are hypomethylated and activated in patients with HCC and liver cancer cell lines. A and B, box plots of log2 differential methylation (A) and expression (B) in patients with HCC (log2 [cancer-normal]) as measured by promoter methylation and expression microarrays. Negative differential methylation indicates hypomethylation, whereas positive differential expression is a marker of overexpression in tumor samples compared with matched adjacent tissue. C, a heatmap showing log2 differential methylation and expression for RASAL2 and NENF in every patient with HCC samples tested by genome-wide microarrays. D and E, average methylation state of CpG sites as determined by pyrosequencing in the tested promoter fragment of RASAL2 (D) and NENF (E) in patients with HCC and normal liver along with a map of the tested promoter fragment ﬂanking the transcription start site (see Supplementary Fig. S1 for extensive methylation analysis). The CpG sites that were chosen for pyrosequencing are circled and numbered. The putative transcription factor binding sites are indicated as predicted by TransFac. F, expression of RASAL2 and NENF in HepG2, SkHep1, and nontransformed NorHep cells as determined by QPCR. G and H, average methylation state of CpG sites as determined by pyrosequencing in the tested promoter fragment of RASAL2 (G) and NENF (H) in HepG2 and SkHep1 cancer cell lines and in nontransformed NorHep cells (see Supplementary Fig. S1, for extensive methylation analysis). I, expression and methylation levels of RASAL2 and NENF in HCC HepG2 cells after 1 mmol/L 5-azaCdR treatment for 3 days (3d). Relative expression is expressed as a percentage of expression in control nontreated cells. Average methylation state of CpG sites was determined by pyrosequencing in control and treated cells. All results represent mean SD of 2 or 3 independent experiments; , P < 0.001; , P < 0.01; , P < 0.05. functional effects. Cell growth was impaired by 55% to 74% strongly reduced by 80% to 90% (Fig. 3E). Interestingly, on day 6 of treatment with siEXOSC4, siSENP6, and we did not observe any changes after depletion of RNMT, siWBSCR22 as compared with siCtrl (Fig. 3D). The num- which was previously shown to have a putative oncogenic ber of cells invaded through extracellular matrix was role (23). Taken together, downregulation of 5 of the 6

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Figure 2. RASAL2 or NENF depletion decreases cancer cell growth and invasive capacities in vitro with selectivity for cancer versus nontransformed cells. A and E, expression of the depleted genes, RASAL2 (A) and NENF (E), quantiﬁed by QPCR after ﬁrst (I), second (II), and third (III) transfection and by Western blot analysis after second transfection of HepG2, SkHep1, and NorHep with scrambled siRNA (siCtrl) and siRNA directed to RASAL2 (siRASAL2) or NENF (siNENF). (Continued on the following page.)

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Figure 3. A–E, regulation of HepG2 HCC cancer cell growth and invasion by EXOSC4, RNMT, SENP6,andWBSCR22 that were found to be hypomethylated and activated in patients with HCC and were not previously assigned a role in liver cancer; F–H, the impact of RASAL2 or NENF depletion on cell growth and invasive capacities in bladder and breast cancer cells. A and B, box plots of log2 differential methylation (A) and expression (B) in patients with HCC (log2 [cancer-normal]) for the candidate genes. Negative differential methylation indicates hypomethylation whereas positive differential expression is a marker of overexpression in tumor samples compared with matched adjacent tissue. C, the candidate genes were depleted in HepG2 cells using siRNA. Expression of the depleted genes after second transfection was quantified by QPCR in HepG2 cells transfected with scrambled siRNA (siCtrl) and siRNAs directed to target genes. D, effect on cell growth after first (day 3), second (day 6), and third (day 9) transfection with siRNAs. E, effect on invasive capacities as measured by Boyden chamber invasion assay after second transfection with siRNAs. F, expression of the depleted genes, RASAL2 and NENF, quantified by QPCR after second (II) transfection of bladder cancer T24 and breast cancer MDA-MB-231 cells with scrambled siRNA (siCtrl) and siRNA directed to RASAL2 (siRASAL2) or NENF (siNENF). G, effect on cell growth after first (day 3), second (day 6), and third (day 9) transfection of T24 and MDA-MB-231 cells with siRASAL2 or siNENF. H, effect on cell invasive capacities as measured by Boyden chamber invasive assay after second transfection of T24 and MDA-MB-231 cells with siRASAL2 or siNENF. All results represent mean SD of 3 determinations in either 2 or 3 independent experiments; , P < 0.001; , P < 0.01; , P < 0.05. genes selected for further testing from the list of hypo- and MDA-MB-231 breast cancer cells (Fig. 3F) exerted methylated and induced genes had an anticancer effect, robust effects on cancer cell growth and invasiveness. suggesting high efficacy of this selection method of anti- The number of viable cells was reduced to 30% and cancer targets. 8% in T24 and to 20% and 40% in MDA-MB-231 cells after siNENF or siRASAL2 treatment, respectively, as com- Functional consequences of RASAL2 or NENF depletion pared with siCtrl after double siRNA treatment (Fig. 3G). in bladder and breast cancer cells The invasiveness of viable cells was impaired by 90% and We then showed that targeting NENF and RASAL2 has an 70% in MDA-MB-231 after either NENF or RASAL2 effect beyond liver cancer confirming previous data (14, 16). knockdown, respectively, and almost completely sup- Depletion of RASAL2 or NENF in invasive T24 bladder pressed in T24 cells (Fig. 3H).

(Continued.) B and F, effect on cell growth after ﬁrst (day 3), second (day 6), and third (day 9) transfection with siRASAL2 (B) or siNENF (F). C and G, effect on cell invasion as measured by Boyden chamber invasion assay after second transfection with siRASAL2 (C) or siNENF (G). For SkHep1 cells transfected with siRASAL2 or siNENF and for HepG2 cells transfected with siNENF, evaluation of expression on mRNA and protein levels after third transfection was not possible because of the reduced cell number after 3 transfections. Therefore, all the comparisons and invasive assays measurements were performed after the second transfection for all 3 cell lines. D and H, effect on anchorage independent growth as measured by soft-agar assay in stable cell lines with RASAL2 (C) or NENF (G) depletion (see Supplementary Materials and Methods for details on lentivirus production and cell transduction). All results represent mean SD of 3 determinations in either 2 or 3 independent experiments; , P < 0.001; , P < 0.01; , P < 0.05.

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Depletion of RASAL2 or NENF causes impairment of cells to grow as explants in vivo, we established stable MAPK, WNT/b-catenin, and PI3K/AKT/mTOR signaling RASAL2-orNENF-depleted HepG2 and SkHep1 cell lines pathways by alterations in phosphorylation of their using lentiviral transduction in vitro, as described in Sup- effectors plementary Materials and Methods (Supplementary Fig. Because RASAL2 and NENF were previously implicated S5), and then injected the cells subcutaneously into in cellular signaling pathways (14–16), we measured NOD/SCID mice (The Jackson Laboratory). Tumor growth phosphorylation levels of a set of kinases in invasive was monitored and the results are presented in Fig. 4B. SkHep1 cells treated with siCtrl and either siRASAL2 or Depletion in vitro of RASAL2 or NENF had a significant siNENF to determine whether depletion of these proteins effect on the ability of the cells to grow as explant tumors in affects PI3K/AKT/mTOR and MAPK pathways in cancer vivo. The ability of both HepG2 and SkHep1 cells to form cells. Significant changes exceeding 10% are demonstrat- tumors in the mice was blocked after RASAL2 and NENF ed in Table 2, whereas all observed alterations are listed in depletion with slightly greater extent of reduction after Supplementary Table S2. RASAL2 and NENF depletion RASAL2 knockdown (Fig. 4B). Similar results were obtained reduced phosphorylation of several kinases downstream when transiently depleted cells were injected into mice to PI3K; AKT 1, 2, and 3 and p70S6 kinase whereas mTOR (Supplementary Fig. S6B), suggesting a long-term effect of phosphorylation was slightly affected (22% reduction) transient depletion of these genes through possibly reversal only by NENF depletion. of the transformed phenotype of the cells. NENF and RASAL2 depletion reduces phosphorylation of RSK1 and RSK2, which are downstream to both PI3K and Discussion MAPK and in turn regulate transcription of several genes via The programmatic changes in gene expression that trigger phosphorylation of transcription factors such as CREB, cancer growth and metastasis involve both activation and CREBP, NF-kB, c-FOS, and ERa (ref. 28; Fig. 4A). Kinases suppression of gene expression. The focus in the field to date of RSK p90 subfamily were also shown to phosphorylate has been on targeting hypermethylated and silenced genes GSK3 and activate WNT signaling (28), a critical pathway in cancer. It is extremely difficult to specifically activate a for oncogenesis. Phosphorylation of GSK3b at serine 9 gene silenced in cancer. Pharmacologic agents are usually inactivates its enzymatic activity and results in induction more effective in inhibiting protein function than activating of the WNT/b-catenin pathway. Upon treatment with siR- proteins that are poorly expressed, particularly when gene- ASAL2 or siNENF, GSK3b phosphorylation is attenuated by silencing results in almost complete deficiency of a protein. 15% to 20% that is presumably a result of AKT and RSK1/2 Therefore, different strategies were used to develop inhibi- decreased activities, which may lead to downregulation of tors that target the epigenetic mechanisms that control gene WNT signal transduction. silencing such as histone methyltransferases, DNMTs and The MAPK signaling pathway includes 3 main branches of histone deacetylases rather than targeting the particular kinase cascades: MEK(1,2)/ERK(1,2), MKK(3,6)/p38, and silenced genes and their products (5). However, these MKK(4,7)/JNK (Fig. 4A; ref. 29). Depletion of RASAL2 or methods are inherently nonspecific as each of these NENF affects phosphorylation of JNK and MKK6 (Table 2 enzymes controls numerous gene expression programs, and Fig. 4A) without any effect on ERKs (Supplementary including potentially those that are essential for cellular Table S2). The most relevant 40% to 80% reduction in differentiation and cell physiology. phosphorylation was seen for JNK3 and MKK6 in response Pharmacologic inhibitors could be developed to target to either RASAL2 or NENF depletion. Depletion of NENF had proteins that are specifically and persistently activated in a more robust action on MAPK signaling than RASAL2 cancer. The challenge is identifying targets that are quali- depletion, resulting in 15% to 35% attenuation of p38b and tatively selective for cancer cells rather than normal tissue. MSK2 phosphorylation. MSK2 is required for MAPK-induced DNA methylation is a stable mark of epigenetic program- phosphorylation and activation of CREB and ATF transcrip- ming that participates in defining the differentiated identity tion factors (30) and for phosphorylation of histone H3, of cells (32). Genes that are methylated in differentiated which is important for the rapid induction of immediate- normal cell types encompass a repertoire that is not required early genes (31). In summary, depletion of NENF and for their normal physiology and are therefore stably RASAL2 affects the nodal PI3K and MAPK pathways at dif- silenced. Epigenetic reprogramming and activation of these ferent points as well as the WNT/b-catenin pathway. Because genes by DNA hypomethylation should result in a stable these signaling pathways converge on regulatory circuitries repertoire of cancer-specific genes that are likely candidates that control cell-cycle progression, apoptosis, and metastasis, to be particularly required for the cancer state and therefore these data provide an explanation for the anticancer effect of serve as ideal candidates for specific inhibitors that act as NENF and RASAL2 depletion in cancer cells (Fig. 4A). anticancer agents. Therefore, we hypothesized that genes that are activated by DNA hypomethylation in cancer and Diminished expression of RASAL2 or NENF causes silenced by methylation in normal cells should be prime reduction of human liver tumor xenograft growth in targets for inhibition in cancer. We tested this hypothesis by NOD/SCID mice delineating the landscape of hypomethylation in liver can- To address the question of whether depletion in vitro of cer (2). Using transcription and promoter methylation RASAL2 and NENF would block the ability of liver cancer genome-wide microarrays we unraveled a shortlist of genes

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Figure 4. Impact of RASAL2 and NENF depletion on phosphorylation state of effectors of the MAPK and PI3K/AKT signaling pathways and on xenograft tumor volume in NOD/SCID mice in vivo. A, parallel determination of the relative levels of phosphorylation of MAPK and other serine/threonine kinases was performed in SkHep1 cells using Human Phospho-MAPK Array Kit (ARY002B; R&D Systems, Inc.). Red squares indicate kinases whose phosphorylation was signiﬁcantly attenuated upon siRASAL2 and siNENF treatments as compared with siCtrl-treated cells, except p38, MSK2, and mTOR kinases that were affected only by siNENF treatment. B, stable RASAL2 or NENF depleted HepG2 and SkHep1 cell lines were established using lentiviral transduction (see Supplementary Materials and methods for details on lentivirus production and cell transduction), and then injected subcutaneously into the NOD/SCID mice as described in the Materials and Methods. Each group was composed of 6 male mice 4 to 6 weeks old. Differences in tumor volume were monitored starting from postinjection week 2 until the mice were sacriﬁced and are demonstrated in charts. Results represent the mean SEM for each group of animals; , P < 0.001; , P < 0.01; , P < 0.05.

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Table 2. A list of kinases whose phosphorylation was signiﬁcantly attenuated in SkHep1 liver cancer cells after RASAL2 or NENF depletion

Name Signaling pathway siRASAL2 (% control) siNENF (% control) AKT1 PI3K/AKT/mTOR, WNT 69.9 54.7 AKT2 PI3K/AKT/mTOR, WNT 58.0 52.4 AKT3 PI3K/AKT/mTOR, WNT 42.9 46.2 AKT pan PI3K/AKT/mTOR, WNT 41.6 37.4 GSK3a, b PI3K/AKT, WNT 84.9 80.6 GSK3b PI3K/AKT, WNT 107.4 78.3 JNK1 MAPK 106.5 67.4 JNK3 MAPK 65.4 48.3 MKK3 MAPK 94.6 88.5 MKK6 MAPK 20.9 53.4 MSK2 MAPK 87.1 66.7 p38b MAPK 104.0 84.0 p70S6 kinase PI3K/AKT/mTOR 56.4 36.2 RSK1 PI3K/AKT/mTOR, WNT 47.4 78.2 RSK2 PI3K/AKT/mTOR, WNT 58.6 66.7 mTOR PI3K/AKT/mTOR 95.5 77.7

NOTE: The level of phosphorylation is expressed as a percentage of phosphorylation in the control—cells transfected with scrambled siRNA (siCtrl). Phosphorylation of a panel of 26 kinases was assessed using Human Phospho-MAPK Array Kit (ARY002B; R&D Systems Inc.).

that are induced and hypomethylated in HCC tumors with death or inhibition of proliferation as the number of cells no or little data on their implications in cancer (Fig. 1A– was corrected for the number of viable cells and it is well C, Fig. 3A and B, Table 1). We determined using publicly known that proliferation and invasiveness are controlled available datasets such as GO, KEGG, and NCBI that bio- by different pathways (33). Thus, our approach focusing logic processes and pathways that play a key role in carci- on genes activated by hypomethylation in tumors nogenesis are enriched within this group of genes and resulted in a high yield of candidate anticancer drug several of these genes were indeed just recently shown to targets whose pharmacologic inhibition could block cel- play different roles in cancer metastasis such as NENF (16), lular transformation and invasiveness. RASAL2 (14), and WBSCR22 (ref. 18; Table 1). Because Surprisingly, we did not observe any anticancer effects by none of the genes was found among targets of known drugs depleting RNMT that was previously shown to enhance to date, such genes could be considered as new targets for human mammary epithelial cell transformation (23). cancer drug development. In this study, we tested using RNMT increased the number and size of colonies only in siRNA/shRNA-mediated gene depletion whether inhibition combination with c-Myc. It is possible that RNMT may be of these targets that were identified in samples from patients critical for cancer only in specific cancer subsets that express with liver cancer would have anticancer and anti-invasive particular genomic programs. We then focused on 2 of the effects in 2 liver cancer cell lines in vitro and whether these genes RASAL2 and NENF that were shown previously to effects would be specific to liver cancer cells or whether they trigger oncogenic signal transduction pathways, MAPK and would also affect nontransformed liver cells (Fig. 1B and F AKT (13, 15). Both genes were hypomethylated and and Fig. 3B). induced in HCC tumors and liver cancer cell lines as Our data provide supports for the approach we have compared with normal tissue and normal nontransformed taken. We show that a genome-wide analysis of hypo- hepatocyte culture (NorHep), respectively, providing an in methylated and activated genes in cancer could yield new vitro model to test whether blocking these targets will have a anticancer drug targets with high efficiency. We have specific anticancer effect (Fig. 1). We examined whether tested 6 candidates, EXOSC4, RNMT, SENP6, WBSCR22, expression of these genes could differentiate HCC from RASAL2,andNENF that are likely to be involved in signal chronic hepatitis B infection. Although the results were transduction, DNA methylation, and regulation of tran- inconclusive because of a small number of patients, they scription. We demonstrated for the first time that deple- warrant further investigation on higher number of samples tion of 5 of the 6 tested candidates reduced cancer cell in order to determine whether these genes could serve as growth and invasive capacities in liver cancer (Figs. 2 predictive markers in chronic hepatitis B samples (Supple- and 3 and Supplementary Fig. S5). The effects on cell mentary Fig. S3H). Please note that RASAL2 possesses 2 invasion cannot be just explained as a consequence of cell isoforms with their transcription start sites more than 100

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Hypomethylated and Induced Genes as Anticancer Drug Targets

kb apart. Methylation differences between tumors and cycle progression, survival, migration, and metastatic normal liver tissue were detected by the microarray properties. RAS and RAF are mutated in many types of approach and validated by pyrosequencing for transcript cancers, including colon and pancreatic cancer, melano- variant 2. Thus, our data indicate that DNA methylation ma, and HCC that result in the constitutive activation of seems to regulate the activity of this transcript variant of the pathway and hyperproliferative state (34). Activation RASAL2. Interestingly, expression of these 2 isoforms is of PI3K/AKT is also observed in numerous human cancers upregulated upon treatment of HepG2 and NorHep cells and leads to stimulation of WNT and NF-kB signals with a demethylating agent, 5-azaCdR (Supplementary Fig. through regulating GSK3b and IKKb (34). The WNT S8). It suggests that both isoforms might be regulated by pathwayisfrequentlyswitchedonincancersasaresult demethylation. It is plausible that the fragment tested in of inactivation of its negative regulators such as APC and pyrosequencing that is located in intron 1 of variant 2 GSK3 (34). In normal cells, GSK3 in a complex with APC downstream of a CpG island (note that no differences were and axin promotes proteolytic degradation of b-catenin in detected within the CpG-rich region upstream of CpG 1; cytoplasm and prevents b-catenin nuclear translocation Supplementary Fig. S1), may act as a regulatory region for and activation of pro-proliferative genes. Depletion of both transcripts. This issue requires extensive studies and RASAL2 and NENF seems to simultaneously target key remains to be elucidated in future experiments. We dem- kinases of all these 3 pathways as well as downregulate onstrated that there is causal relationship between hypo- proteins involved with the DNA methylation machinery methylation of these genes and their activation. Treatment such as DNMT1 and MBD2 (Supplementary Fig. S7). of HepG2 liver cancer cells and normal hepatocytes with a DNMT1 expression was found to be upregulated in cancer demethylating agent 5-azaCdR increased expression of the (35), and ectopic expression of DNMT1 resulted in cel- genes and reduced DNA methylation in their regulatory lular transformation (36). Inhibition of DNMT1 reversed regions that is consistent with methylation-dependent reg- the growth of cancer cells (37) and had antitumorigenic ulation of their transcription (Fig. 1I and Supplementary effects in animal models (4, 5, 38, 39). MBD2 was shown Figs. S1 and S2). We then determined that both genes are to be involved in demethylation and activation of met- overexpressed in other human cancer types as shown in astatic genes in cancer (40, 41), such as MMP2, PLAU, Supplementary Fig. S3A, which suggested that inhibitors of S100A5,andNUPR1 (2, 5). Knockdown of MBD2 inhi- these proteins would have a broad spectrum of anticancer bits tumorigenicity (42) and Mbd2 depletion inhibits action. Our results confirmed this proposition and show colorectal neoplasia in mice (43). Knockdown of MBD2 the anticancer and anti-invasiveness activity of RASAL2 decreased cancer cell growth and invasiveness in breast, and NENF depletion in liver cancer as well as in bladder prostate, and liver cancer cells with concomitant suppres- and breast cancers (Fig. 2 and Fig. 3F–H and Supplemen- sion of genes implicated in carcinogenesis (2, 40, 41). tary Fig. S5). Stable inhibition of RASAL2 and NENF has Taken together, these effects on signaling pathways and an impact on tumorigenic properties of liver cancer cells DNA methylation enzymes provide a mechanism of as explants in NOD/SCID mice in vivo significantly reduc- action for the anticancer effects of RASAL2 and NENF ing the rate of tumor growth in vivo (Fig.4B).Further- inhibition positioning these genes as excellent anticancer more, depletion of RASAL2 or NENF in both HepG2 and drug targets. SkHep1 liver cancer cells led to changes in expression of Our study lays down a workflow for identifying drug several genes involved in the DNA methylation machin- target candidates for anticancer therapy. This workflow ery and implicated in cancer and cancer metastasis. As involves identifying hypomethylated and activated genes shown in Supplementary Fig. S7, siRNA-mediated knock- in tumor samples from several types of cancer that are down of RASAL2 or NENF diminished expression of methylated and silenced in normal tissue and have putative DNMT1 and MBD2 and changed DNMT3A and DNMT3B important roles in cancer and/or cancer metastasis. We levels. determined from the shortlist of these genes the functional A remarkable observation is the exquisite specificity of the role of RASAL2, NENF, and several other genes in cancer effects of RASAL2 or NENF depletion on cancer cells (Fig. 2 and provided evidence that these genes were potential and Supplementary Fig. S5). One possible explanation is targets for anticancer drug development as they are not that these genes when overexpressed affect transcription essential for normal cell growth but are required for con- and/or activity of downstream effectors with tumorigenic tinued cancer cell proliferation and invasiveness. Our properties and thereby their depletion attenuates cancer results established for the first time the role of RASAL2 and growth and invasiveness. Because RASAL2 and NENF are NENF in multiple cancers and defined the potential func- expressed at low levels in normal cells, their depletion tional role of DNA hypomethylation in activation of these would have no impact on the effectors. These data support genes. The fact that these genes were found to reduce cell the idea of focusing on gene targets that are epigenetically growth and invasiveness of cancers derived from vastly silenced in normal cells. different tissues suggested that these proteins may be We demonstrate that Ras/Raf/MAPK, PI3K/AKT/mTOR, involved in the most fundamental and common pathways and WNT/b-catenin signaling pathways are affected by that are required for cellular transformation and cancer depletion of RASAL2 or NENF. These pathways form a metastasis and are candidate targets for anticancer complex network that promotes cell proliferation, cell- inhibitors.

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Disclosure of Potential Conﬂicts of Interest Study supervision: M. Al-Mahtab, S.M.F. Akbar, R. Raqib, S.A. Rabbani, No potential conﬂicts of interest were disclosed. M. Szyf

Authors' Contributions Conception and design: B. Stefanska, S.M.F. Akbar, W.A. Khan, R. Raqib, Grant Support M. Szyf This research was supported by grants from the Ministere du Developpe- Development of methodology: D. Cheishvili, M. Al-Mahtab, I. Tanvir, ment Economique, de l’Innovation et de l’Exportation (MDEIE), a program H.A. Khan, S.A. Rabbani of the government of Quebec (No. 215004) and the Canadian Institute of Acquisition of data (provided animals, acquired and managed patients, Health Research and granted to M. Szyf. D. Cheishvili is funded by provided facilities, etc.): A. Arakelian, Z.-G. Han, M. Al-Mahtab, S.M.F. a postdoctoral fellowship from the Israel Cancer Research Fund (ICRF). Akbar, W.A. Khan, R. Raqib, I. Tanvir, H.A. Khan, S.A. Rabbani, M. Szyf M. Szyf is a fellow of the Canadian Institute for Advanced Research. Hep3B Analysis and interpretation of data (e.g., statistical analysis, biosta- cell line was a kind gift from Dr. O.M. Andrisani (Department of Basic tistics, computational analysis): B. Stefanska, M. Suderman, A. Arakelian, Medical Sciences, Purdue University, West Lafayette, IN). M. Al-Mahtab, I. Tanvir, H.A. Khan, S.A. Rabbani, M. Szyf The costs of publication of this article were defrayed in part by the Writing, review, and/or revision of the manuscript: B. Stefanska, payment of page charges. This article must therefore be hereby marked M. Suderman, M. Hallett, M. Al-Mahtab, S.M.F. Akbar, I. Tanvir, H.A. Khan, advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate M. Szyf this fact. Administrative, technical, or material support (i.e., reporting or orga- nizing data, constructing databases): B. Stefanska, J. Huang, M. Hallett, Received January 31, 2013; revised March 19, 2014; accepted April 14, S.M.F. Akbar, W.A. Khan, I. Tanvir, H.A. Khan, M. Szyf 2014; published OnlineFirst April 24, 2014.

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Genome-Wide Study of Hypomethylated and Induced Genes in Patients with Liver Cancer Unravels Novel Anticancer Targets

Barbara Stefanska, David Cheishvili, Matthew Suderman, et al.

Clin Cancer Res Published OnlineFirst April 24, 2014.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-13-0283

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