Abstract 2008: Targeting SKP2 by p27 Inhibits stemness and prolong the survival in osteosarcoma
Authors: Wang Jichuan , Aldahamsheh Osama , Ferrena Alexander , Singh Swapnil , Singla Amit , Borjihan Hasibagan , Yaguare Simon , Viscarret Valentina , Tingling Janet , Zi Xiaolin , Lo Yungtai , Gorlick Richard , Zheng Deyou , Schwartz Edward L. , Zhao Hongling , Yang Rui , Geller David S. , Hoang Bang H. 
Published: July 2021

Abstract
Objective: Osteosarcoma is a highly aggressive malignancy for which treatment has remained essentially unchanged for years. Our previous studies found that the F-box protein SKP2 is overexpressed in osteosarcoma, acting as a proto-oncogene. p27Kip1 (p27) is an inhibitor of cyclin-dependent kinases and a downstream substrate of SKP2. The present study focused on the role of SCF-SKP2-mediated ubiquitination and degradation of p27 in osteosarcoma (OS) tumorigenesis.
Methods: We generated a genetically engineered mouse model with double-knockout of Rb1 and Trp53 within cells of the osteoblastic lineage using the Osterix1-Cre (Osx1-Cre;Rb1lox/lox;Trp53lox/lox, DKO). To block the interaction between Skp2 and p27, we cross DKO mice into the p27T187A knock-in mutation (p27:187site Thr to Ala) background to create Osx1-Cre;Rb1lox/lox;Trp53lox/lox;p27T187A/T187A (DKOAA) animals. Mice of both genotypes were monitored for overall survival and tumor growth. Early passage osteosarcoma cells were harvested from mice tumors and used for in vitro analysis. Annexin V staining and TUNEL assay were used for apoptosis analysis. RNA-seq and RT-qPCR were used to compare transcriptional differences. Further, a small-molecule SKP2/CKS1 pocket inhibitor (C1) and a neddylation inhibitor, Pevenodistat, were tested both in vitro using adherent cells and organoid culture, and in vivo using xenograft.
Results: All genotypes were born at the expected ratios, and mutant animals are viable, fertile, and developmentally normal. The p27T187A mutation significantly prolonged the overall survival of DKO osteosarcoma mice. Western and IHC staining showed an accumulation of p27 in DKOAA, accompanied by an increased TUNEL staining level and apoptotic subpopulation determined by Annexin V staining. Hence, DKOAA tumors proliferate slower than DKO both in vivo and in vitro organoid. RNA-seq based enrichment analysis further confirmed an increase of apoptosis and cell cycle arrest in DKOAA tumors than DKO. RNA-seq also revealed a significant downregulation of multiple cancer stemness markers in DKOAA tumors. Further GESA enrichment analysis, ALDH activity assay, and sphere-formation assay consistently demonstrated a decreased stemness status in DKOAA tumors. Finally, SKP2 inhibitors, both C1 and Pevenodistat, showed selective inhibition in DKO than DKOAA in adherent culture, osteosarcoma organoid, and xenograft model.
Conclusion: Blocking p27 degradation by SKP2 significantly delayed osteosarcoma tumorigenesis and prolonged survival, promoted apoptosis, and reduced tumor-initiating properties in OS model. Given that RB1 and TP53 co-inactivation is common in osteosarcoma, our study suggests that inhibiting the SKP2-p27 axis may represent a desirable therapeutic strategy for this cancer.
Citation Format: Jichuan Wang, Osama Aldahamsheh, Alexander Ferrena, Swapnil Singh, Amit Singla, Hasibagan Borjihan, Simon Yaguare, Valentina Viscarret, Janet Tingling, Xiaolin Zi, Yungtai Lo, Richard Gorlick, Deyou Zheng, Edward L. Schwartz, Hongling Zhao, Rui Yang, David S. Geller, Bang H. Hoang. Targeting SKP2 by p27 Inhibits stemness and prolong the survival in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2008.