Preliminary Study On Molecular Mechanisms For TTRAP’s Function On Tumor Cell Growth And Etoposide Chemosensitivity

The repair of DNA damage is related to the occurrence and development of tumors. The key molecules and signal pathways in this process will be important targets for the development of new anti-cancer agents. TTRAP (TRAF and TNF receptor-associated protein) is a newly defined5’tyrosyl-DNA phosphodiesterase. Upon DNA damage, it will cleave the5’-phosphotyrosyl bonds, helping to release the free5’-phosphate termini and to repair the double-strand DNA breaks. The object of our research will focus on the effects of TTRAP on the growth of tumor cells and their chemosensitivity for anti-cancer drugs.In order to know if TTRAP is related to tumor occurrence, we surveyed the expression of TTRAP in Gene Expression Omnibus. The expression of TTRAP was relatively lower in normal-appearing colonic mucosa of early onset colorectal cancer patients and in metastatic prostate cancer tumors that refractory to hormones. We also evaluated the endogenous expression of TTRAP protein with Western blot assay in different tumor cell lines, including lung cancer, breast cancer, prostate cancer and osteosarcoma cell lines. The results indicated that TTRAP ubiquitously expressed in all cell lines, but its expression profile was different depending on individual cell line.U2OS is a human osteosarcoma cell line with relatively lower expression of TTRAP protein. Both in transient transfection assay and in lentivirus transduction process, TTRAP significantly inhibited the colony formation in U2OS cells. The stable transfectant of wild type TTRAP (TTRAPwt) gene showed decreased growth speed when cultured continuously for6days. Two dominant negative mutations for TTRAP’s tyrosyl DNA phosphodiesterase activity, TTRAP (E152A) and TTRAP (D262A), could partially block this inhibition. These two dominant negative mutants of TTRAP also rescued the inhibition of wild TTRAP on the transactivation activity of NF-κB. TTRAP also inhibited the migration of U2OS cells in a cell wound healing assay, but these two mutants could not. These results indicated that TTRAP could inhibit the growth of tumor cells, which depends on its5’tyrosyl-DNA phosphodiesterase activity. This inhibitory effect might be related to the NF-κB signaling pathway.In the DNA damage response, TP53is a key mediator for DNA repair, cell apoptosis and senescence. In order to find TTRAP’s effects on signal pathways relating to tumor cell growth, we employed yeast two hybridization assay to detect the direct interaction between TTRAP and TP53. Deletion analysis revealed that the DNA-binding motif of TP53is responsible for the interaction with TTRAP. TTRAP could be co-immunoprecipitated with TP53in293T cells. The fusion protein EGFP-p53and TTRAP-DsRed could also be co-localized in the nucleus, indicating the possibility of interaction of TTRAP and TP53in space. With luciferase-based reporter assay, we found that the transactivation activity of TP53could be weakly promoted by TTRAP when the expression level of TP53was relatively low.Etoposide is a wide-employed anti-tumor drug in the clinic. It can increase topoisemerase2-mediated DNA breakage primarily by inhibiting the ability of the enzyme to religate cleaved nucleic acid molecules and result in cell death. With in vitro chemosnsitivity assay, we detected that the overexpression of TTRAPwt increased the survival of cell colonies than vector control in U2OS cells. When the endogeneous TTRAP was knock down by SiRNA, the number of survived colonies decreased. The cytotoxicity of etoposide in TTRAPwt overexpressed U2OS cells was lower than the vector control in a cell proliferation assay, while in cells expressing the two mutants, TTRAP (E152A) and TTRAP (D262A), the cytotoxicity was higher. These results indicated that the expression of TTRAP could influent the etoposide chemosensitivity of U2OS cells.We further tested the time-dependent profile of the transcription and translation of TTRAP after the application of TTRAP. There was no significant up-regulation of TTRAP transcription1-6hours later after etoposide treatment, but the protein level of TTRAP increased. Cycloheximide could inhibit new protein synthesis. TTRAP protein was degraded slowly upon cycloheximide treatment. When cells were treated with cycloheximide and etoposide together, the degradation of TTRAP protein speed down. The stability of a protein could be influenced by post-translational modifications (PTM), such as ubiquitinylation and sumoylation, we presumed that the effects of etoposide on the stability of TTRAP might relate to these two PMT mechanisms. It needed to be tested by appropriate experiments. Non-covalent SUMO interaction is also one kind of PMT. The expression vector for TTRAP(SIMm), in which the SUMO-interacting motif was mutated, was constructed to test the effects of SUMO-binding on TTRAP’s stability. As compared with wild-type TTRAP, the expression level of TTRAP (SIMm)-DsRed was reduced significantly. The mutant’s localization in the cell also changed from the classical dot-like profile of PML-NBs to irregular distribution in the nucleus. This result suggested that SUMO interaction is quite important for the stability and localization of TTRAP protein.The preliminary study here indicated for the first time that TTRAP had inhibitory effects on tumor cell growth, and etoposide chemosensitivity of U2OS cells was correlated negatively with the expression of TTRAP. These results supplied information for the potential application of TTRAP in the adjuvant therapy for solid tumors.