Knock-Down of the TIM/TIPIN Complex Promotes Apoptosis in Melanoma Cells

Abstract

Background: The Timeless (TIM) and its interacting partner TIPIN protein complex is well known for its role in replication checkpoint and normal DNA replication processes. TIM appears to be an essential component of the DNA damage response. Recent studies revealed the involvement of TIM and TIPIN in human malignancies; however, no evidence is available regarding TIM/TIPIN protein complex expression and its potential role in melanoma. Therefore, we investigated the role of TIM as well as TIPIN in melanoma. Methods: To assess the role of TIM/TIPIN in melanoma, we analyzed TIM/TIPIN expression data from the publicly accessible TCGA online database, Western blot analysis, and RT-qPCR in a panel of melanoma cell lines. Lentivirus-mediated TIM/TIPIN knockdown in A375 melanoma cells was used to examine cell proliferation, colony formation, and apoptosis. A xenograft tumor formation assay was performed using shTIM, shTIPIN, and shMock cells. Findings: The TIM/TIPIN complex was frequently overexpressed in melanoma cells compared to normal melanocytes. We also discovered that the overexpression of TIM and TIPIN was significantly associated with poorer prognosis of melanoma patients. Furthermore, we observed that shRNA-mediated knockdown of TIM and TIPIN reduced cell viability and proliferation due to the induction of apoptosis and increased levels of γH2AX, a marker of DNA damage. In a xenograft tumor nude mouse model, shRNA-knockdown of TIM/TIPIN significantly reduced tumor growth. Interpretation: Our results suggest that TIM and TIPIN play an important role in tumorigenesis of melanoma, which might reveal novel approaches for the development of new melanoma therapies. Further mechanistic investigations are needed to determine this complex’s potential as a biomarker of melanoma susceptibility. Targeting TIM/TIPIN might be a potential therapeutic strategy against melanoma. Funding Statement: This work was supported by the Hormel Foundation (Z. Dong). Declaration of Interests: All authors have agreed to publish this manuscript and declare no competing interests. Ethics Approval Statement: All animal studies were performed following the guidelines approved by the University of Minnesota Institutional Animal Care and Use Committee (Protocol ID: 1803-35739A).