Regulation of SLC35F2 protein ubiquitination by E3 ligases and its impact on cancer progression Jencia Carminha Colaco

Abstract

국문초록 Solute carrier family 35 F2 (SLC35F2)는 다양한 생리적 기능을 조절하는 막 결합 운반체 단백질로서, 여러 암에서 활성화되는 것으로 알려져 있다. 하지만 SLC35F2 단백질 분해를 조절하는 분자 메커니즘과 암 발생에 미치는 구체적인 기전은 아직 밝혀지지 않았다. 따라서 SLC35F2 단백질 분해를 촉진하는 E3 ligase를 발굴하는 것은 암의 진행을 억제할 수 있는 방법이 될 수 있다. 본 연구에서는 E3 ligase 스크리닝 및 in vitro 생화학적 실험을 통해, APC/CCdh1 및 βTrCP1이 SLC35F2 단백질과 상호작용하며, SLC35F2 단백질을 분해시키는 것으로 확인되었다. APC/CCdh1 및 βTrCP1은 SLC35F2 유비퀴틴화를 촉진하고, SLC35F2 단백질의 반감기를 감소시켰다. 이와 반대로, CRISPR/Cas9을 이용하여 APC/CCdh1 및 βTrCP1을 제거하였을 경우 SLC35F2 단백질은 증가하였다. 암 세포주의 mRNA 발현 분석 결과, APC/CCdh1과 SLC35F2 및 βTrCP1과 SLC35F2 사이에 음의 상관관계가 있는 것으로 나타났다. 또한 HeLa 세포에서 APC/CCdh1 및 βTrCP1를 제거하였을 경우, SLC35F2-매개 세포 증식, 콜로니 형성, 이동 및 침습이 촉진되었다. 이러한 결과를 통해, APC/CCdh1-SLC35F2 및 βTrCP1-SLC35F2 경로를 조절하면 암을 치료할 수 있는 새로운 방법이 될 수 있을 것으로 사료된다. |CHAPTER II - ABSTRACT SLC35F2 belongs to membrane-bound carrier proteins that are associated with multiple cancers. The main factor that determines cancer progression is the expression level of SLC35F2. Thus, identifying the E3 ligase that controls SLC35F2 protein abundance in cancer cells is critical. Here, I identified βTrCP1 interacting with and reducing the SLC35F2 protein level. βTrCP1 signals SLC35F2 protein ubiquitination and reduces SLC35F2 protein half-life. The mRNA expression pattern between βTrCP1 and SLC35F2 across a panel of cancer cell lines showed a negative correlation. Additionally, the depletion of βTrCP1 accumulated SLC35F2 protein and promoted SLC35F2-mediated cell growth, migration, invasion, and colony formation ability in HeLa cells. Overall, I demonstrate that βTrCP1 acts as a tumor suppressor by controlling SLC35F2 protein abundance in cancer cells. The depletion of βTrCP1 promotes SLC35F2-mediated carcinogenesis. Thus, I envision that βTrCP1 may be a potential target for cancer therapeutics. |GENERAL ABSTRACT 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. Based on the E3 ligase screen and in vitro biochemical experiments, APC/CCdh1 and βTrCP1 were identified to interact with and reduce SLC35F2 protein level. Both the E3 ligases, APC/CCdh1 and βTrCP1 promote SLC35F2 ubiquitination and decreases the half-life of SLC35F2 protein. On the contrary, the CRISPR/Cas9-mediated depletion of APC/CCdh1 and βTrCP1 increased SLC35F2 protein levels. The mRNA expression analysis revealed a negative correlation between APC/CCdh1 and SLC35F2 as well as βTrCP1 and SLC35F2 across a panel of cancer cell lines. Additionally, depletion in APC/CCdh1 and βTrCP1 promotes SLC35F2-mediated cell proliferation, colony formation, migration, and invasion in HeLa cells. Thus, APC/CCdh1-SLC35F2 and βTrCP1-SLC35F2 axis might be a potential therapeutic target for cancer.|CHAPTER I - ABSTRACT 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. 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. Based on the E3 ligase screen and in vitro biochemical experiments, I 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, I demonstrated that depletion in APC/CCdh1 promotes SLC35F2-mediated cell proliferation, colony formation, migration, and invasion in HeLa cells. My study highlights that APC/CCdh1 is a critical regulator of SLC35F2 protein turnover and depletion of APC/CCdh1 promotes SLC35F2-mediated tumorigenesis. Thus, I envision that APC/CCdh1-SLC35F2 axis might be a therapeutic target in cancer.