Abstract 544: Protein phosphatase 1 regulatory subunit 1A (PPP1R1A) promotes tumor growth and metastasis via inhibition of protein phosphatase 1 in Ewing sarcoma

Abstract

Abstract Ewing sarcoma is a malignant pediatric bone and soft tissue tumor. Children with metastatic Ewing sarcoma have a cure rate of less than 30% (De loris MA et al, 2013). The major hurdle of curing Ewing sarcoma is the lack of specific and druggable targets. EWS/FLI is the master regulator of Ewing sarcoma and functions as an aberrant transcription factor to deregulate downstream targets which eventually leads to cancer phenotypes. By comparing genes deregulated by EWS/FLI in RNA-sequencing analyses across multiple model systems, we identified protein phosphatase 1 regulatory subunit 1A (PPP1R1A), a potent protein phosphatase 1 (PP1) inhibitor, as one of the core targets of EWS/FLI (Niedan S et al, 2014; Sankar S et al, 2013; Tirode F et al, 2007). Public data mining and western blotting analysis has demonstrated that PPP1R1A is highly expressed at both RNA and protein levels in Ewing sarcoma, but not the putative cell of origin, mesenchymal stem cells, or other sarcomas. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis showed that PPP1R1A is up-regulated by EWS/FLI. Further data mining with published chromatin immunoprecipitation (ChIP)-Seq, Encyclopedia of DNA Elements (ENCODE), and formaldehyde-assisted isolation of regulatory elements (FAIRE) data suggested, and subsequent luciferase reporter assay validated, that PPP1R1A is directly targeted by EWS/FLI via a GGAA microsatellite enhancer element. We next studied the role of PPP1R1A in Ewing sarcoma by functional assays in vitro and in vivo. Intriguingly, depletion of PPP1R1A caused a loss of oncogenic transformation of patient-derived Ewing sarcoma cells in soft agar, and limited xenograft tumor growth in NOD-SCID mice (p = 0.0001). Furthermore, reduction of PPP1R1A resulted in decreased cell migration in wound healing and Boyden chamber assays, and limited lung metastasis in an orthotopic mouse model. Further studies demonstrated that activation and phosphorylation at Thr35 of PPP1R1A by protein kinase A (PKA), is required for PPP1R1A to promote tumorigenesis and metastasis, as evidenced by rescuing of PPP1R1A-knockdown induced phenotypes by constitutively active (T35D) but not wild-type PPP1R1A. Consistently, we found that Ewing sarcoma but not HEK293 cells were sensitive to the treatment of PKA inhibitors (H89 and PKI 5-24). Finally, we found that further mutating of the PP1 binding site (R8A/K9A) in T35D PPP1R1A rendered this constitutively active mutant unable to rescue the knockdown phenotype, indicating that PP1 binding and inhibition by activated PPP1R1A is indispensable for PPP1R1A function. Collectively, our data suggest that PPP1R1A and related pathway plays a positive role in tumorigenesis and metastasis in Ewing sarcoma. Therefore, targeting PKA/PPP1R1A/PP1 pathway has potential therapeutic value in the treatment of both primary and metastatic Ewing sarcoma. Citation Format: Wen Luo, Changxin Xu, Janet Ayello, Filemon Dela Cruz, Jeremy Rosenblum, Stephen Lessnick, Mitchell S. Cairo. Protein phosphatase 1 regulatory subunit 1A (PPP1R1A) promotes tumor growth and metastasis via inhibition of protein phosphatase 1 in Ewing sarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 544.