Background
The solute carrier family 35 F2 (SLC35F2), belongs to membrane-bound carrier proteins that control various physiological functions and are activated in several cancers. However, the molecular mechanism regulating SLC35F2 protein turnover and its implication in cancer progression remains unexplored. Therefore, screening for E3 ligases that promote SLC35F2 protein degradation is essential during cancer progression.
Conclusion
Our study highlights that APC/CCdh1 is a critical regulator of SLC35F2 protein turnover and depletion of APC/CCdh1 promotes SLC35F2-mediated tumorigenesis. Thus, we envision that APC/CCdh1-SLC35F2 axis might be a therapeutic target in cancer.
Methods
The immunoprecipitation and Duolink proximity ligation assays (PLA) were used to determine the interaction between APC/CCdh1 and SLC35F2 proteins. A CRISPR/Cas9-mediated knockdown and rescue experiment were used to validate the functional significance of APC/CCdh1 on SLC35F2 protein stabilization. The ubiquitination function of APC/CCdh1 on SLC35F2 protein was validated using in vitro ubiquitination assay and half-life analysis. The role of APC/CCdh1 regulating SLC35F2-mediated tumorigenesis was confirmed by in vitro oncogenic experiments in HeLa cells.
Results
Based on the E3 ligase screen and in vitro biochemical experiments, we identified that APC/CCdh1 interacts with and reduces SLC35F2 protein level. APC/CCdh1 promotes SLC35F2 ubiquitination and decreases the half-life of SLC35F2 protein. On the other hand, the CRISPR/Cas9-mediated depletion of APC/CCdh1 increased SLC35F2 protein levels. The mRNA expression analysis revealed a negative correlation between APC/CCdh1 and SLC35F2 across a panel of cancer cell lines tested. Additionally, we demonstrated that depletion in APC/CCdh1 promotes SLC35F2-mediated cell proliferation, colony formation, migration, and invasion in HeLa cells.