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Background <p>The upregulation of DBF4 is correlated with a poor prognosis in patients with clear cell renal cell carcinoma (ccRCC), yet the mechanisms governing the expression and protein stability of DBF4 remain unknown. Ubiquitin-specific proteases (USPs), known regulators of protein stability in diverse biological contexts, have been implicated in ccRCC, although their specific functions have not been fully elucidated.</p> Methods <p>In this study, 40 USPs were screened to identify potential stabilizers of DBF4. Following the selection of USP48, knockdown experiments were performed, and various assays, including CCK-8, colony formation, apoptosis, cell cycle, and migration assays, were carried out to investigate the impact of USP48 on ccRCC cells. The expression levels and correlation of USP48 and DBF4 in clinical sample were analyzed using data from The Cancer Genome Atlas (TCGA) and the Genotype Tissue Expression (GTEx) databases. Their protein expression was then further validated by immunohistochemistry (IHC) data from the Human Protein Atlas (HPA) database and our previously established ccRCC cohort. Additionally, coimmunoprecipitation (co-IP) and deubiquitination assays were performed to confirm the interaction between USP48 and the DBF4 protein. A rescue experiment involving the overexpression of DBF4 in ccRCC cells was conducted. Furthermore, the effect of USP48 on the growth of ccRCC cells in vivo was assessed through subcutaneous injection of ccRCC cells into mice.</p> Results <p>We initially screened 40 ubiquitin-specific proteases (USPs) and found that USP48 exhibited the highest luciferase activity in assays using luciferase-tagged DBF4. Correspondingly, knockdown of USP48 reduced DBF4 expression, which subsequently inhibited ccRCC cell proliferation, migration, and invasion, while also inducing cell cycle arrest and apoptosis. Clinical sample analysis revealed that both USP48 and DBF4 were significantly upregulated in ccRCC tissues, and their expression levels showed a significant positive correlation (<i>R</i> = 0.3504, <i>p</i> &lt; 0.0001). The results of the co-IP and ubiquitination assays demonstrated that USP48 can interact with and stabilize DBF4 by modulating its ubiquitination. Overexpression of DBF4 in USP48-deficient ccRCC cells effectively reversed the deficiencies resulting from USP48 knockdown. Additionally, the xenograft assay results showed that USP48 deficiency hinders the development of ccRCC in vivo.</p> Conclusion <p>Our research reveals that USP48 facilitates ccRCC progression by increasing the stability of DBF4, suggesting that USP48 is a promising therapeutic target for ccRCC.</p>

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USP48 promotes tumour progression through the deubiquitination and stabilization of DBF4 in clear cell renal carcinoma

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年5月20日

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  • Lingfeng Zhu,
  • Liuyan Chen,
  • Lvying Wu,
  • Na Lin,
  • Wenyi Deng,
  • Kuanyin Wang,
  • Yaoyao Liang,
  • Jianan Zhang,
  • Jin Chen

摘要

Background

The upregulation of DBF4 is correlated with a poor prognosis in patients with clear cell renal cell carcinoma (ccRCC), yet the mechanisms governing the expression and protein stability of DBF4 remain unknown. Ubiquitin-specific proteases (USPs), known regulators of protein stability in diverse biological contexts, have been implicated in ccRCC, although their specific functions have not been fully elucidated.

Methods

In this study, 40 USPs were screened to identify potential stabilizers of DBF4. Following the selection of USP48, knockdown experiments were performed, and various assays, including CCK-8, colony formation, apoptosis, cell cycle, and migration assays, were carried out to investigate the impact of USP48 on ccRCC cells. The expression levels and correlation of USP48 and DBF4 in clinical sample were analyzed using data from The Cancer Genome Atlas (TCGA) and the Genotype Tissue Expression (GTEx) databases. Their protein expression was then further validated by immunohistochemistry (IHC) data from the Human Protein Atlas (HPA) database and our previously established ccRCC cohort. Additionally, coimmunoprecipitation (co-IP) and deubiquitination assays were performed to confirm the interaction between USP48 and the DBF4 protein. A rescue experiment involving the overexpression of DBF4 in ccRCC cells was conducted. Furthermore, the effect of USP48 on the growth of ccRCC cells in vivo was assessed through subcutaneous injection of ccRCC cells into mice.

Results

We initially screened 40 ubiquitin-specific proteases (USPs) and found that USP48 exhibited the highest luciferase activity in assays using luciferase-tagged DBF4. Correspondingly, knockdown of USP48 reduced DBF4 expression, which subsequently inhibited ccRCC cell proliferation, migration, and invasion, while also inducing cell cycle arrest and apoptosis. Clinical sample analysis revealed that both USP48 and DBF4 were significantly upregulated in ccRCC tissues, and their expression levels showed a significant positive correlation (R = 0.3504, p < 0.0001). The results of the co-IP and ubiquitination assays demonstrated that USP48 can interact with and stabilize DBF4 by modulating its ubiquitination. Overexpression of DBF4 in USP48-deficient ccRCC cells effectively reversed the deficiencies resulting from USP48 knockdown. Additionally, the xenograft assay results showed that USP48 deficiency hinders the development of ccRCC in vivo.

Conclusion

Our research reveals that USP48 facilitates ccRCC progression by increasing the stability of DBF4, suggesting that USP48 is a promising therapeutic target for ccRCC.