RNF2 Promotes Chondrosarcoma Progression by Regulating Ubiquitination of CBX7

Academic Background

Chondrosarcoma (CHS) is a malignant tumor composed of hyaline cartilage matrix and chondrocytes, ranking as the second most common primary bone tumor after osteosarcoma, accounting for 20%-27% of all primary malignant bone tumors. Although the 5-year survival rate for low-grade chondrosarcoma is relatively high (83%), the rates for high-grade and dedifferentiated chondrosarcoma are only 53% and 7%-24%, respectively. Due to the high resistance of chondrosarcoma to conventional chemotherapy and radiotherapy, especially in advanced stages, current treatment options are very limited. Therefore, exploring the molecular mechanisms of chondrosarcoma and developing new therapeutic strategies have become a focus of current research.

Ubiquitination, as a crucial post-translational modification, plays an important role in normal physiology and diseases. It regulates protein degradation or non-degradative signaling by covalently attaching ubiquitin to target proteins. In cancer, ubiquitination influences tumor initiation and progression by modulating tumor-suppressing and tumor-promoting pathways. RNF2 (Ring Finger Protein 2) is an E3 ubiquitin ligase involved in the monoubiquitination of histone H2A and is highly expressed in various tumors, closely related to their clinical features. However, the role of RNF2 in chondrosarcoma has not been fully investigated.

Additionally, CBX7 (Chromobox 7) is a key component of the Polycomb Repressive Complex 1 (PRC1), participating in chromatin remodeling and gene silencing. Although CBX7 has been shown to suppress tumor progression in bladder and breast cancers, its function in chondrosarcoma remains unexplored. Based on this background, this study aims to investigate the role and regulatory mechanism of the RNF2/CBX7 axis in chondrosarcoma, providing new insights for molecularly targeted therapies.

Source of the Paper

This paper was co-authored by Yue Wu, Zheng Huang, Ping Luo, and others, affiliated with the Department of Orthopedics at Beijing Chaoyang Hospital, Union Shenzhen Hospital of Huazhong University of Science and Technology, and the Department of Spinal Surgery at the Fourth Hospital of Changsha. The paper was published in 2024 in the journal Cancer & Metabolism, with the DOI 10.1186/s40170-024-00359-x.

Research Process and Results

1. High Expression of RNF2 in Chondrosarcoma

The study first detected the expression levels of RNF2 in chondrosarcoma tissues and cells using RT-qPCR, Western blot, and immunohistochemistry (IHC). The results showed that RNF2 was significantly overexpressed in chondrosarcoma tissues and cells, particularly in the JJ012 cell line. This finding suggests that RNF2 may play an important role in the initiation and progression of chondrosarcoma.

2. RNF2 Knockdown Inhibits Proliferation, Migration, and Angiogenesis in Chondrosarcoma Cells

To further validate the function of RNF2, the research team knocked down RNF2 expression in JJ012 cells through cell transfection. The results showed that RNF2 knockdown significantly inhibited the proliferation, migration, and angiogenesis of JJ012 cells while promoting apoptosis. Specifically, CCK-8 and EdU assays indicated that RNF2 knockdown markedly reduced cell proliferation; flow cytometry revealed that RNF2 knockdown increased the apoptosis rate; scratch and angiogenesis assays further confirmed that RNF2 knockdown suppressed cell migration and angiogenesis.

3. RNF2 Overexpression Promotes Malignant Behaviors in Chondrosarcoma Cells

To further confirm the role of RNF2, the research team overexpressed RNF2 in SW1353 cells, which have relatively low RNF2 expression. The results showed that RNF2 overexpression significantly promoted the proliferation, migration, and angiogenesis of SW1353 cells while inhibiting apoptosis. These findings further support the tumor-promoting role of RNF2 in chondrosarcoma.

4. RNF2 Degrades CBX7 via Ubiquitination

Using the online tool Ubibrowser, CBX7 was predicted to be a downstream ubiquitination substrate of RNF2. Western blot results showed that CBX7 expression was significantly lower in chondrosarcoma tissues compared to normal cartilage tissues, and RNF2 expression was negatively correlated with CBX7. Further experiments demonstrated that RNF2 knockdown upregulated CBX7 protein levels, while RNF2 overexpression reduced CBX7 protein levels. Additionally, RNF2 had no effect on CBX7 mRNA expression, indicating that RNF2 regulates CBX7 degradation through the ubiquitination pathway.

5. RNF2 Promotes CBX7 Ubiquitination

To verify whether RNF2 degrades CBX7 through ubiquitination, the research team conducted CHX (cycloheximide) chase experiments and in vivo ubiquitination assays. The results showed that RNF2 knockdown significantly prolonged the half-life of CBX7, while RNF2 overexpression accelerated CBX7 degradation. Furthermore, RNF2 promoted CBX7 polyubiquitination through Lys48-linked ubiquitin chains, thereby accelerating its degradation.

6. RNF2 Promotes Chondrosarcoma Progression by Inhibiting CBX7

To validate the role of the RNF2/CBX7 axis in chondrosarcoma, the research team performed rescue experiments. The results showed that CBX7 knockdown partially reversed the inhibitory effects of RNF2 knockdown on chondrosarcoma cell proliferation, migration, and angiogenesis, while CBX7 overexpression partially counteracted the tumor-promoting effects of RNF2 overexpression. These findings suggest that RNF2 promotes chondrosarcoma progression by downregulating CBX7 expression.

7. Animal Models Validate the Tumor-Promoting Role of RNF2

Finally, the research team validated the role of RNF2 in vivo using a nude mouse xenograft model. The results showed that RNF2 knockdown significantly inhibited tumor growth and lung metastasis while reducing the number of Ki-67-positive cells. These results further confirm the tumor-promoting role of RNF2 in chondrosarcoma.

Conclusions and Significance

This study is the first to reveal the molecular mechanism by which RNF2 promotes chondrosarcoma progression through ubiquitination-mediated degradation of CBX7. The high expression of RNF2 accelerates CBX7 degradation, promoting chondrosarcoma cell proliferation, migration, and angiogenesis while inhibiting apoptosis. This discovery provides new insights for molecularly targeted therapies in chondrosarcoma, particularly highlighting the potential clinical value of targeting the RNF2/CBX7 axis.

Research Highlights

1. Innovative Discovery: First to reveal that RNF2 promotes chondrosarcoma progression by degrading CBX7 through ubiquitination.
2. Multi-Level Experimental Validation: Comprehensive validation of the RNF2/CBX7 axis in chondrosarcoma, from cellular experiments to animal models.
3. Potential Therapeutic Target: The RNF2/CBX7 axis offers a new direction for molecularly targeted therapies in chondrosarcoma.

Other Valuable Information

The study also mentions that future research could further explore the upstream regulators of RNF2 and other ubiquitination substrates, as well as its role in other epigenetic modifications in chondrosarcoma. Additionally, targeted protein degradation technologies based on the ubiquitin-proteasome system (PROTAC) may become important tools for future chondrosarcoma treatment.