Comprehensive single cell transcriptomics analysis of murine osteosarcoma uncovers Skp2 function in metastasis,
Alexander Ferrena1,2, Ranxin Zhang3,4, Jichuan Wang3,4
1Institute for Clinical and Translational Research, Albert Einstein College of Medicine, Bronx, NY, USA.
Abstract:
Osteosarcoma (OS) is the most common primary pediatric bone malignancy. One promising new therapeutic target is SKP2, encoding a substrate recognition factor of the SCF E3 ubiquitin ligase responsible for ubiquitination and proteasome degradation of substrate p27, thus driving cellular proliferation. We have shown previously that knockout of Skp2 in an immunocompetent transgenic mouse model of OS improved survival, drove apoptosis, and induced tumor inflammation. Here, we applied single-cell RNA-sequencing (scRNA-seq) to study primary OS tumors derived from Osx-Cre driven conditional knockout of Rb1 and Trp53. We showed that murine OS models recapitulate the tumor heterogeneity and microenvironment complexity observed in patient tumors. We further compared this model with OS models with functional disruption of Skp2: one with Skp2 knockout and the other with the Skp2-p27 interaction disrupted (resulting in p27 overexpression). We found reduction of T cell exhaustion and upregulation of interferon activation, along with evidence of replicative and endoplasmic reticulum-related stress in the Skp2 disruption models, and showed that interferon induction was correlated with improved survival in OS patients. Additionally, our scRNA-seq analysis uncovered decreased activities of metastasis-related gene signatures in the Skp2-disrupted OS, which we validated by observation of a strong reduction in lung metastasis in the Skp2 knockout mice. Finally, we report several potential mechanisms of escape from targeting Skp2 in OS, including upregulation of Myc targets, DNA copy number amplification and overexpression of alternative E3 ligase genes, and potential alternative lineage activation. These mechanistic insights into OS tumor biology and Skp2 function suggest novel targets for new, synergistic therapies, while the data and our comprehensive analysis may serve as a public resource for further big data-driven OS research.
Insights
Targeting SKP2 in osteosarcoma (OS) reduces tumor growth and metastasis. Disrupting SKP2 enhances anti-tumor immunity and reveals potential resistance mechanisms, suggesting new therapeutic strategies for this pediatric bone cancer.
Area of Science:
- Oncology
- Molecular Biology
- Immunology
Background:
- Osteosarcoma (OS) is a common pediatric bone cancer with limited therapeutic options.
- SKP2 is a key regulator of p27 degradation, promoting cellular proliferation and identified as a potential therapeutic target in OS.
- Previous studies demonstrated that SKP2 knockout in a mouse model of OS improved survival and induced anti-tumor inflammation.
Purpose of the Study:
- To investigate the effects of SKP2 disruption on tumor heterogeneity, microenvironment, and therapeutic response in OS using single-cell RNA-sequencing (scRNA-seq).
- To compare OS models with SKP2 knockout versus disrupted SKP2-p27 interaction.
- To identify mechanisms of therapeutic escape and potential synergistic targets for OS treatment.
Main Methods:
- Generation of conditional knockout mouse models for Rb1 and Trp53, followed by SKP2 disruption (knockout or SKP2-p27 interaction inhibition).
- Application of scRNA-seq to analyze primary OS tumors from these models.
- Validation of scRNA-seq findings, including metastasis reduction and analysis of resistance mechanisms.
Main Results:
- Murine OS models recapitulated human tumor complexity.
- SKP2 disruption led to reduced T cell exhaustion, increased interferon activation, and endoplasmic reticulum stress.
- SKP2-disrupted OS showed decreased metastasis-related gene signatures and significantly reduced lung metastasis; potential escape mechanisms identified.
Conclusions:
- SKP2 targeting in OS demonstrates therapeutic potential by enhancing anti-tumor immunity and reducing metastasis.
- Interferon induction correlates with improved survival in OS patients.
- Understanding resistance mechanisms like Myc activation and alternative E3 ligase overexpression is crucial for developing novel, synergistic therapies.
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