Abstract 5817: Protein phosphatase 1 regulatory subunit 1A promotes tumorigenesis and metastasis in Ewing sarcoma

Abstract

Abstract Background: Ewing sarcoma (ES) is a highly invasive and metastatic pediatric soft tissue and bone tumor. Children with metastatic ES have a cure rate of less than 30% (De loris MA et al, 2013). Novel and specific therapeutic targets are urgently needed. ES is characterized by the oncogenic fusions, mostly EWS/FLI, which functions as an aberrant transcription factor to deregulate downstream targets and mediate ES pathogenesis. By comparing genes dysregulated by EWS/FLI across multiple model systems (Niedan S et al, 2014; Sankar S et al, 2013; Tirode F et al, 2007), we identified protein phosphatase 1 regulatory subunit 1A (PPP1R1A), a potent protein phosphatase 1 (PP1) inhibitor upon PKA phosphorylation, as one of the core EWS/FLI targets (Luo W et al, 2016). Objective: In the current study, we seek to define the role of PPP1R1A in ES pathogenesis. Methods: Quantitative reverse transcription polymerase chain reaction and luciferase reporter assays were performed to investigate transcriptional regulation of PPP1R1A by EWS/FLI. In vitro functional assays and orthotopic injections in immune-deficient mice were conducted to investigate the effect of PPP1R1A on ES oncogenic transformation and cell migration, and tumorigenesis and metastasis, respectively. High-throughput sequencing and functional annotation were utilized to identify PPP1R1A regulated genes and cellular functions. Drug treatment was performed to test the effect of PKA inhibitors on ES cell proliferation and tumor development. Results: We found that PPP1R1A is directly up-regulated by EWS/FLI via a GGAA microsatellite enhancer element. Depletion of PPP1R1A caused a significant decrease in oncogenic transformation (p<0.05) and cell migration (p=0.009) in vitro and limited xenograft tumor growth (p=0.0009) and metastasis (p=0.009) in vivo. We also discovered that PPP1R1A regulates genes involved in various cellular processes including acting binding, cell adhesion, and differentiation. Interestingly, PPP1R1A regulated gene set significantly overlap with that of ZEB2 (p=2.67246E-33) and EWS (p=1.2559E-45), which regulates metastasis and neuronal differentiation in ES, respectively. Further, we demonstrated that PKA phosphorylation and activation of PPP1R1A, and subsequent PP1 binding and inhibition by activated PPP1R1A, is required for PPP1R1A mediated ES pathogenesis, likely by increasing the phosphorylation level of various PP1 substrates, such as RB and CREB, which are critical for fundamental cellular functions including proliferation and differentiation. Consistently, we found that PKA inhibitors impaired ES cell proliferation and xenograft tumor growth and metastasis. Conclusion: Collectively, we identified an essential kinase and phosphatase pathway, PKA/PPP1R1A/PP1, that plays a critical role in ES tumorigenesis and metastasis, and thus is a potential therapeutic target in the treatment of primary and metastatic ES. Citation Format: Wen Luo, Changxin Xu, Janet Ayello, Filemon De La Cruz, Jeremy Rosenblum, Stephen L. Lessnick, Mitchell S. Cairo. Protein phosphatase 1 regulatory subunit 1A promotes tumorigenesis and metastasis in Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5817. doi:10.1158/1538-7445.AM2017-5817