Single-Cell RNA Analysis of Murine Osteosarcoma Uncovers Skp2 Function in Metastasis, Genomic Instability, and Immune Activation and Reveals Additional Target Pathways.

Abstract

UNLABELLED: Osteosarcoma is the most common primary pediatric bone malignancy. One promising new target is SKP2, encoding a substrate recognition factor of the SCF E3 ubiquitin ligase that targets p27 for proteasomal degradation, driving cellular proliferation. Knockout (KO) of Skp2 in an immunocompetent transgenic mouse model of osteosarcoma improved survival, drove apoptosis, and induced antitumor immunity. In this study, we applied single-cell RNA-sequencing (scRNA-seq) to primary osteosarcoma tumors from Osx-Cre conditional Rb1/Trp53 KO mice. We further compared with models of Skp2 disruption: Skp2 KO or disruption of the Skp2-p27 interaction (resulting in p27 overexpression). We report that murine osteosarcoma models recapitulate the tumor heterogeneity and microenvironment complexity observed in patient tumors. Skp2 disruption led to reduction of T-cell exhaustion and upregulation of interferon (IFN) signaling, as well as induction of cell type-specific replicative and endoplasmic reticulum stress, which we validated with proteomics analysis. Furthermore, we showed that IFN induction was correlated with improved survival in patients with osteosarcoma. Additionally, our scRNA-seq analysis uncovered decreased expression of metastasis-related gene signatures in Skp2-disrupted osteosarcoma, which we validated by a strong reduction in lung metastasis in the Skp2 KO mice. Finally, we report several mechanisms potentially used by osteosarcoma to escape from Skp2 targeting, including upregulation of Myc targets, induction of genomic instability, overexpression of alternative E3 ligases, and lineage plasticity. These mechanistic insights into osteosarcoma tumor biology and Skp2 function suggest novel targets for new, synergistic therapies, whereas the data and our comprehensive analysis may serve as a public resource for further big data-driven osteosarcoma research. SIGNIFICANCE: Our single-cell study of murine osteosarcoma models uncovers Skp2 function in metastasis, genomic instability, and immune activation and reveals additional target pathways to overcome resistance to Skp2 disruptions.