E3 ubiquitin ligase APC/CCdh1 regulates SLC35F2 protein turnover and inhibits cancer progression in HeLa cells

Jencia Carminha Colaco; Arun Pandian Chandrasekaran; Janardhan Keshav Karapurkar; D A Ayush Gowda; Girish Birappa; Sripriya Rajkumar; Bharathi Suresh; Nare Ko; Seok-Ho Hong; Seung Jun Oh; Kye-Seong Kim; Suresh Ramakrishna

doi:10.1016/j.bbagen.2023.130454

E3 ubiquitin ligase APC/C^Cdh1 regulates SLC35F2 protein turnover and inhibits cancer progression in HeLa cells

Biochim Biophys Acta Gen Subj. 2023 Nov;1867(11):130454. doi: 10.1016/j.bbagen.2023.130454. Epub 2023 Sep 7.

Affiliations

¹ Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul 04763, South Korea.
² Biomedical Research Center, Asan Institute for Life Sciences, Seoul 05505, Republic of Korea; Department of Nuclear Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul 05505, Republic of Korea.
³ Department of Internal Medicine, School of Medicine, Kangwon National University, Chuncheon, South Korea.
⁴ Department of Nuclear Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul 05505, Republic of Korea. Electronic address: sjoh@amc.seoul.kr.
⁵ Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul 04763, South Korea; College of Medicine, Hanyang University, Seoul, 04763, South Korea. Electronic address: ks66kim@hanyang.ac.kr.
⁶ Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul 04763, South Korea; College of Medicine, Hanyang University, Seoul, 04763, South Korea. Electronic address: suri28@hanyang.ac.kr.

PMID: 37689217
DOI: 10.1016/j.bbagen.2023.130454

Abstract

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.

Methods: The immunoprecipitation and Duolink proximity ligation assays (PLA) were used to determine the interaction between APC/C^Cdh1 and SLC35F2 proteins. A CRISPR/Cas9-mediated knockdown and rescue experiment were used to validate the functional significance of APC/C^Cdh1 on SLC35F2 protein stabilization. The ubiquitination function of APC/C^Cdh1 on SLC35F2 protein was validated using in vitro ubiquitination assay and half-life analysis. The role of APC/C^Cdh1 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/C^Cdh1 interacts with and reduces SLC35F2 protein level. APC/C^Cdh1 promotes SLC35F2 ubiquitination and decreases the half-life of SLC35F2 protein. On the other hand, the CRISPR/Cas9-mediated depletion of APC/C^Cdh1 increased SLC35F2 protein levels. The mRNA expression analysis revealed a negative correlation between APC/C^Cdh1 and SLC35F2 across a panel of cancer cell lines tested. Additionally, we demonstrated that depletion in APC/C^Cdh1 promotes SLC35F2-mediated cell proliferation, colony formation, migration, and invasion in HeLa cells.

Conclusion: Our study highlights that APC/C^Cdh1 is a critical regulator of SLC35F2 protein turnover and depletion of APC/C^Cdh1 promotes SLC35F2-mediated tumorigenesis. Thus, we envision that APC/C^Cdh1-SLC35F2 axis might be a therapeutic target in cancer.

Keywords: Cervical cancer; Destabilization; Post-translational modifications; Ubiquitin-proteasome system; Wound healing.