Molecular Cell Biology Epigenetic Regulation of the PTEN–AKT–RAC1 Axis by G9a Is Critical for Tumor Growth in Alveolar Rhabdomyosarcoma Akshay V. Bhat 1 , Monica Palanichamy Kala 1 , Vinay Kumar Rao 1 , Luca Pignata 2 , Huey Jin Lim 3 , Sudha Suriyamurthy 1 , Kenneth T. Chang 4 ,Victor K. Lee 3 , Ernesto Guccione 2 , and Reshma Taneja 1 Abstract Alveolar rhabdomyosarcoma (ARMS) is an aggressive pediatric cancer with poor prognosis. As transient and stable modifications to chromatin have emerged as critical mechanisms in oncogenic signaling, efforts to target epige- netic modifiers as a therapeutic strategy have accelerated in recent years. To identify chromatin modifiers that sustain tumor growth, we performed an epigenetic screen and found that inhibition of lysine methyltransferase G9a sig- nificantly affected the viability of ARMS cell lines. Targeting expression or activity of G9a reduced cellular proliferation and motility in vitro and tumor growth in vivo. Transcrip- tome and chromatin immunoprecipitation–sequencing analysis provided mechanistic evidence that the tumor- suppressor PTEN was a direct target gene of G9a. G9a repressed PTEN expression in a methyltransferase activity– dependent manner, resulting in increased AKT and RAC1 activity. Re-expression of constitutively active RAC1 in G9a- deficient tumor cells restored oncogenic phenotypes, demon- strating its critical functions downstream of G9a. Collectively, our study provides evidence for a G9a-dependent epigenetic program that regulates tumor growth and suggests targeting G9a as a therapeutic strategy in ARMS. Significance: These findings demonstrate that RAC1 is an effector of G9a oncogenic functions and highlight the poten- tial of G9a inhibitors in the treatment of ARMS. Introduction Rhabdomyosarcoma (RMS) is a highly malignant soft-tissue sarcoma of childhood, which accounts for approximately 5% to 8% of pediatric cancers, occurring mostly in children below 10 years of age. Despite expression of the myogenic proteins MyoD and myogenin, RMS cells fail to undergo myogenic differentiation and are morphologically similar to premature mesenchymal cells (1). The most common forms of RMS are embryonal rhab- domyosarcoma (ERMS) and alveolar rhabdomyosarcoma (ARMS). ARMS, the most aggressive subtype, is associated with frequent metastasis at the time of diagnosis and exhibits limited response to treatment, resulting in poor survival rates. ARMS is distinguished from the other subtypes of RMS by frequent trans- locations t(2; 13) (q35; q14) and t(1; 13) (p36; q14), resulting in the expression of PAX3-FOXO1 and PAX7-FOXO1 fusion pro- teins in 60% and 20% cases, respectively (2, 3). PAX3-FOXO1 is believed to be critical for oncogenesis by control of genes required for proliferation, survival, and metastasis (4–8). ARMS expressing either of these translocations is termed fusion positive, whereas a small percentage of ARMS tumors (20%) are devoid of these chromosomal translocations and are termed fusion negative. Fusion-negative ARMS is similar to ERMS in terms of prognosis, event-free survival, frequency of metastases, and distribution of site signifying the importance of PAX3/PAX7-FOXO1 in fusion- positive ARMS tumors (9). In addition to the presence of fusion oncoproteins, comprehensive analysis of the genomic landscape in ARMS has shown deregulation of growth factor signaling including insulin-like growth factor (IGF)/insulin-like growth 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- 4,5-bisphosphate (PIP2) to produce phosphatidylinositol-3,4,5- bisphosphate (PIP3). This facilitates recruitment of AKT to the plasma membrane. Subsequent phosphorylation of AKT at thre- onine 308 (Thr308) by phosphoinositide-dependent kinase (PDK1) and at serine 473 (Ser473) by the mTORC-2 leads to its activation and affects several cellular processes such as prolifer- ation, growth, survival, migration, and metabolism (13). The guanine nucleotide exchange factors are also activated by PI3K/ AKT signaling, resulting in increased Ras-related C3 botulinum toxin substrate 1 (RAC1) activity (14). Elevated RAC1 activity is associated with aggressive cancers (15) and has multiple effects on growth, proliferation, and cell invasion. The tumor-suppressor PTEN dephosphorylates PIP3 and thus negatively regulates the 1 Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore. 2 Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A  STAR), Singapore, Singapore. 3 Department of Pathology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore. 4 Department of Pathol- ogy, KK Women and Children's Hospital, Singapore, Singapore. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Author: Reshma Taneja, National University of Singapore, Block MD9, 2 Medical Drive, Singapore 117593, Singapore. Phone: 65-6516-3236; Fax: 65-6778-8161; E-mail: phsrt@nus.edu.sg doi: 10.1158/0008-5472.CAN-18-2676 Ó2019 American Association for Cancer Research. Cancer Research Cancer Res; 79(9) May 1, 2019 2232 on July 4, 2020. © 2019 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst March 4, 2019; DOI: 10.1158/0008-5472.CAN-18-2676