Abstract 3648: Protein phosphatase 1 regulatory subunit 1A regulates cell cycle progression in Ewing sarcoma

Abstract

Abstract Ewing sarcoma (ES) is a highly malignant pediatric bone and soft tissue tumor characterized by the expression of chimeric fusions of EWS and ETS family transcription factors, mostly EWS/FLI, as a consequence of chromosomal translocation. The dominant role of EWS/FLI in ES indicates that insights into the biology of this aberrant transcription factor and its downstream targets in ES initiation and progression could lead to the discovery of novel therapeutic strategies. We recently identified protein phosphatase 1 regulatory subunit 1A (PPP1R1A), a potent protein phosphatase 1 (PP1) inhibitor, as one of the core EWS/FLI targets and a potential specific therapeutic target for primary and metastatic ES. Small molecule compound inhibition of PPP1R1A and the associated PKA/PPP1R1A/PP1 signaling pathway impaired tumor growth and metastasis in ES xenograft mouse model. In the current study, we seek to identify the underlying mechanisms of PPP1R1A mediated tumorigenesis and metastasis and define additional role of PPP1R1A in ES pathogenesis to facilitate discovery of additional therapeutic targets. We performed shRNA induced knockdown (iR1A) and CRISPR knockout (R1A-KO) of PPP1R1A in ES cells and found that iR1A and R1A-KO cells proliferated much slower than the control cells. Cell cycle analysis showed that PPP1R1A depleted cells were arrested in G1 to S phase transition which could be rescued by re-expression of 35D, a constitutively active PPP1R1A. We found that Rb, whose phosphorylation releases cell cycle arrest in G1 phase, was hypo-phosphorylated upon PPP1R1A knockdown and hyper-phosphorylated after 35D rescue. Furthermore, depletion of PPP1R1A increased expression levels of cell cycle inhibitors p21 and p27, but not other G1 phase associated proteins. Results from RNA-seq and qRT-PCR analysis showed that PPP1R1A knockdown decreased the level of total mRNA but increased that of poly-adenylated mRNA of a subset of replication-dependent histone genes, suggesting a compensation mechanism of the loss of normal pre-mRNA processing of the histone genes in G1-arrested PPP1R1A knockdown cells. Our findings demonstrate that PPP1R1A promotes cell cycle progression through G1 to S phase by down-regulating cell cycle inhibitors p21 and p27 which leads to Rb hyper-phosphorylation and de-repression of cell cycle. Since PPP1R1A is specifically highly expressed in ES but not its putative cell of origin, mesenchymal stem cells (MSCs), our results indicate that PPP1R1A serves as an ES specific cell cycle modulator. Recently, IGF-1R inhibitors have shown synergistic effect with cell cycle modulators such as CDK4/6 inhibitors. Thus, combination of PPP1R1A inhibition with IGF-1R inhibitor may be explored for synergistic specific and effective tumor control in ES. Further studies for identification of the molecular mechanisms by which PPP1R1A regulates p21 and p27 may reveal more therapeutic targets for ES treatment. Citation Format: Wen Luo, Marcela Laukova, Sarah Phillips, Janet Ayello, Mitchell S. Cairo. Protein phosphatase 1 regulatory subunit 1A regulates cell cycle progression in Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3648.