Published OnlineFirst March 4, 2019; DOI: 10.1158/0008-5472.CAN-18-2676 Cancer Molecular Cell Biology Research Epigenetic Regulation of the PTEN–AKT–RAC1 Axis by G9a Is Critical for Tumor Growth in Alveolar Rhabdomyosarcoma Akshay V. Bhat1, Monica Palanichamy Kala1, Vinay Kumar Rao1, Luca Pignata2, Huey Jin Lim3, Sudha Suriyamurthy1, Kenneth T.Chang4,Victor K. Lee3, Ernesto Guccione2, and Reshma Taneja1 Abstract Alveolar rhabdomyosarcoma (ARMS) is an aggressive suppressor PTEN was a direct target gene of G9a. G9a pediatric cancer with poor prognosis. As transient and stable repressed PTEN expression in a methyltransferase activity– modifications to chromatin have emerged as critical dependent manner, resulting in increased AKT and RAC1 mechanisms in oncogenic signaling, efforts to target epige- activity. Re-expression of constitutively active RAC1 in G9a- netic modifiers as a therapeutic strategy have accelerated in deficient tumor cells restored oncogenic phenotypes, demon- recent years. To identify chromatin modifiers that sustain strating its critical functions downstream of G9a. Collectively, tumor growth, we performed an epigenetic screen and our study provides evidence for a G9a-dependent epigenetic found that inhibition of lysine methyltransferase G9a sig- program that regulates tumor growth and suggests targeting nificantly affected the viability of ARMS cell lines. Targeting G9a as a therapeutic strategy in ARMS. expression or activity of G9a reduced cellular proliferation and motility in vitro and tumor growth in vivo.Transcrip- Significance: These findings demonstrate that RAC1 is an tome and chromatin immunoprecipitation–sequencing effector of G9a oncogenic functions and highlight the poten- analysis provided mechanistic evidence that the tumor- tial of G9a inhibitors in the treatment of ARMS. Introduction the expression of PAX3-FOXO1 and PAX7-FOXO1 fusion pro- teins in 60% and 20% cases, respectively (2, 3). PAX3-FOXO1 is Rhabdomyosarcoma (RMS) is a highly malignant soft-tissue believed to be critical for oncogenesis by control of genes required sarcoma of childhood, which accounts for approximately 5% to for proliferation, survival, and metastasis (4–8). ARMS expressing 8% of pediatric cancers, occurring mostly in children below 10 either of these translocations is termed fusion positive, whereas a years of age. Despite expression of the myogenic proteins MyoD small percentage of ARMS tumors (20%) are devoid of these and myogenin, RMS cells fail to undergo myogenic differentiation chromosomal translocations and are termed fusion negative. and are morphologically similar to premature mesenchymal Fusion-negative ARMS is similar to ERMS in terms of prognosis, cells (1). The most common forms of RMS are embryonal rhab- event-free survival, frequency of metastases, and distribution of domyosarcoma (ERMS) and alveolar rhabdomyosarcoma site signifying the importance of PAX3/PAX7-FOXO1 in fusion- (ARMS). ARMS, the most aggressive subtype, is associated with positive ARMS tumors (9). In addition to the presence of fusion frequent metastasis at the time of diagnosis and exhibits limited oncoproteins, comprehensive analysis of the genomic landscape response to treatment, resulting in poor survival rates. ARMS is in ARMS has shown deregulation of growth factor signaling distinguished from the other subtypes of RMS by frequent trans- including insulin-like growth factor (IGF)/insulin-like growth locations t(2; 13) (q35; q14) and t(1; 13) (p36; q14), resulting in factor receptor 1, mutations in PIK3CA, and amplification of CDK4 (10–12). Elevated growth factor levels stimulate the PI3K pathway, resulting in phosphorylation of phosphatidylinositol- 1Department of Physiology, Yong Loo Lin School of Medicine, National University 4,5-bisphosphate (PIP2) to produce phosphatidylinositol-3,4,5- of Singapore, Singapore, Singapore. 2Institute of Molecular and Cell Biology bisphosphate (PIP3). This facilitates recruitment of AKT to the (IMCB), Agency for Science, Technology and Research (AÃSTAR), Singapore, plasma membrane. Subsequent phosphorylation of AKT at thre- 3 Singapore. Department of Pathology, Yong Loo Lin School of Medicine, onine 308 (Thr308) by phosphoinositide-dependent kinase National University of Singapore, Singapore, Singapore. 4Department of Pathol- (PDK1) and at serine 473 (Ser473) by the mTORC-2 leads to its ogy, KK Women and Children's Hospital, Singapore, Singapore. activation and affects several cellular processes such as prolifer- Note: Supplementary data for this article are available at Cancer Research ation, growth, survival, migration, and metabolism (13). The Online (http://cancerres.aacrjournals.org/). guanine nucleotide exchange factors are also activated by PI3K/ Corresponding Author: Reshma Taneja, National University of Singapore, Block AKT signaling, resulting in increased Ras-related C3 botulinum MD9, 2 Medical Drive, Singapore 117593, Singapore. Phone: 65-6516-3236; toxin substrate 1 (RAC1) activity (14). Elevated RAC1 activity is Fax: 65-6778-8161; E-mail: [email protected] associated with aggressive cancers (15) and has multiple effects on doi: 10.1158/0008-5472.CAN-18-2676 growth, proliferation, and cell invasion. The tumor-suppressor Ó2019 American Association for Cancer Research. PTEN dephosphorylates PIP3 and thus negatively regulates the 2232 Cancer Res; 79(9) May 1, 2019 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst March 4, 2019; DOI: 10.1158/0008-5472.CAN-18-2676 G9a Regulates RAC1 Activity in ARMS PI3K–AKT pathway. The downregulation of PTEN expression in ma-Aldrich). For transient knockdown experiments, cells were ARMS contributes to elevated PI3K/AKT/RAC1 signaling that is transfected with 100 nmol/L of human G9a-specific siRNA (ON- linked to tumor cell survival (16, 17). However, the molecular TARGETplus EHMT2 siRNA SMARTpool, Dharmacon) contain- mechanisms that result in loss of PTEN expression are not under- ing a pool of three to five 19–25 nucleotide siRNAs. Control cells stood and could provide a tool to suppress oncogenic signaling were transfected with 100 nmol/L scrambled siRNA (ON-TAR- through the PI3K/AKT/RAC1 pathway. GETplus, nontargeting pool, Dharmacon) using Lipofectamine Recent advances in chromatin biology have revealed deregula- RNAiMax (Thermo Fisher Scientific). Cells were analyzed 48 tion of the epigenetic landscape in ARMS (18). The chromatin hours after transfection for all assays. For generating stable knock- modifiers CHD4, PCAF, and BRD4 have been shown to interact down cell lines, 293FT cells were transfected with packaging with and affect PAX3-FOXO1 activity (19–21). Nevertheless, the plasmids plP1 (5 mg) and plP2 (5 mg), envelope plasmid plP/ epigenetic network that is central to ARMS tumorigenesis remains VSV-G (5 mg; ViraPower Lentiviral Packaging Mix, Thermo Fisher to be identified. EHMT2/G9a and EHMT1/GLP are key SET domain Scientific), and 5 mg lentiviral expression constructs shRNA containing lysine methyltransferases that catalyze dimethylation of (pLKO.1, Mission shRNA DNA clone, Sigma-Aldrich) or shG9a histone H3 lysine 9 (H3K9me2), resulting in transcriptional repres- (pLKO.1) (#SHCLND-NM_025256 MISSION shRNA Plasmid sion of target genes (22). Deregulated expression and function of DNA). Sixteen hours after transfection, the cell supernatant was G9a, and to a lesser extent GLP, have been reported in cancers of replaced with basal DMEM. The cell supernatants were collected different origins (23–34). In addition to its canonical role in gene 48 and 72 hours after transfection, centrifuged, and the viral pellet repression, G9a can also activate genes through association with was resuspended in 200 mL of RPMI medium as stock solution. coactivator complexes in a context-dependent manner (23). How- RH30 cells at 40% to 50% confluency were transduced with ever, the signaling pathways that G9a regulates and its downstream shRNA control virus (control), or shG9a virus and 2 mL polybrene effectors are poorly understood. In the context of skeletal myogen- (8 mg/mL; Sigma-Aldrich) in RPMI 1640 basal medium. Six hours esis, we and others have shown that G9a inhibits muscle differen- after transduction, cell supernatants were replaced with RPMI tiation and promotes proliferation of myoblasts (35–38). We medium (10% FBS) for 24 hours. Transduced cells were selected therefore hypothesized that G9a expression may be deregulated with 1 mg/mL puromycin (Sigma-Aldrich) for 3 days. For rescue in ARMS and contribute to tumorigenesis. experiments, shcontrol and shG9a cells were transfected with 2 mg Here, we provide evidence that the G9a pathway is central to of Rac1-GFP plasmid (pcDNA3-EGFP-Rac1-Q61L, Addgene) using ARMS tumorigenesis. Using an epigenetic screen, we demonstrate Lipofectamine 2000 with PLUS reagent (Thermo Fisher Scientific). that G9a inhibitors potently affect viability of ARMS cell lines. Downregulation of G9a expression or pharmacologic inhibition Drug sensitivity of ARMS cells to inhibitors of of its activity not only reduces tumor cell proliferation and methyltransferases invasion, but also results in myogenic differentiation. In vivo, RH30 and RH4 cells were treated for 8 days with the indicated reduction of G9a function reduces tumor growth in xenograft compounds in 384 wells at three different concentrations (3, 1, mouse models. Transcriptomic and chromatin immunoprecipi- and 0.3 mmol/L). Viability at days 4 and 8 was scored by MTS assay tation (ChIP)-sequencing (ChIP-seq)