SKP2 knockout in Rb1/p53 deficient mouse models of osteosarcoma induces immune infiltration and drives a transcriptional program with a favorable prognosis

PURPOSE: Osteosarcoma (OS) is an aggressive bone malignancy with a poor prognosis. One putative proto-oncogene in OS is SKP2, encoding a substrate recognition factor of the SCF E3 ubiquitin ligase. We previously demonstrated that SKP2 knockout in murine OS improved survival and delayed tumorigenesis. Here we aim to define the SKP2 drives transcriptional program and its clinical implication in OS. EXPERIMENTAL DESIGN: We performed RNA-sequencing (RNA-seq) on tumors from a transgenic OS mouse model with conditional Trp53 and Rb1 knockouts in the osteoblast lineage (“DKO”: Osx1-Cre;Rb1(lox/lox);p53(lox/lox)) and a triple-knockout model with additional Skp2 germline knockout (“TKO”: Osx1-Cre;Rb1(lox/lox);p53(lox/lox);SKP2(−/−)). We validated our RNA-seq findings using qPCR and immunohistochemistry. To investigate the clinical implications of our results, we analyzed a human OS patient cohort (“NCI-TARGET OS”) with RNA-seq and clinical data. RESULTS: We found large differences in gene expression after SKP2 knockout. Strikingly, we observed increased expression of genes related to immune microenvironment infiltration in TKO tumors. We observed significant increases in signature genes for macrophages and to a lesser extent, T cells, B cells and vascular cells. We also uncovered a set of relevant transcription factors that may mediate the changes. In OS patient cohorts, high expression of genes upregulated in TKO was correlated with favorable overall survival, which was largely explained by the macrophage gene signatures. This relationship was further supported by our finding that SKP2 expression was negatively correlated with macrophage infiltration in the NCI-TARGET OS and the TCGA Sarcoma cohort. CONCLUSION: Our findings indicate that SKP2 may mediate immune exclusion from the OS tumor microenvironment, suggesting that SKP2 modulation in OS may induce anti-tumor immune activation.