| Summary: | 碩士 === 國立成功大學 === 生理學研究所 === 105 === Colorectal cancer is one of the most common death-related cancers worldwide. Aberrant expression of oncogenes is regarded as a predominant factor in colorectal cancer development. Our previous study discovered TYRO3, an oncogene overexpressed in colorectal cancer, promotes cell proliferation, metastasis, and tumor progression. TYRO3 belongs to TAM (TYRO3, AXL, and MER) family, which is a subfamily of receptor tyrosine kinases. According to data from immunohistochemistry staining, we found TYRO3 locates in nuclei and the levels of nuclear TYRO3 are positively associated with tumor progression. Herein, we aim to study the functions of nuclear TYRO3 in cancer development. By staining with three different antibodies recognizing N-terminus, central region, and C-terminus of TYRO3, respectively, we identified that central and C-terminus of TYRO3 are the fragments translocated into the nuclei. By bioinformatics tool prediction, we found there are a nuclear localization signal (NLS) and a nuclear export signal (NES) in TYRO3. These results suggest TYRO3 is probably transported into nucleus through proteolytic processing. To evaluate the importance of nuclear translocation of TYRO3, NLS of TYRO3 was mutated. Then mutated NLS of TYRO3 was used to perform several functional studies, such as apoptosis and cell proliferation. The results showed TYRO3 was only detected in cytosol but not in nucleus under NLS mutation, accompanied with increased numbers of apoptotic cells and activated-caspase 3 protein. These data suggest that nuclear translocation of TYRO3 is essential for cell survival. BrdU incorporation assay revealed that proliferation of cancer cells overexpressing mNLS-TYRO3 was suppressed as well. To further investigate the functions of nuclear TYRO3, we established NES mutation in TYRO3, which allows TYRO3 retaining in nucleus, and performed the above-mentioned assays. We found transfection with NES mutated TYRO3 also increased cell proliferation similar to that seen in cells transfected with wild-type TYRO3. Furthermore, we also proved that promoting cell growth by nuclear TYRO3 requires its kinase activity. These findings imply nuclear TYRO3 plays some roles in cell survival and proliferation in colon cancer. By sequencing analyzing using protease database, we found TYRO3 has a matrix metalloproteinase 2 (MMP2) cutting site at upstream of NLS sequence. The MMP2 in vitro digestion assay demonstrated that TYRO3 can be cleaved by MMP2. In addition, we observed TYRO3 did not locate in nucleus under MMP2 cleavage site mutation. Taken together, we demonstrated, for the first time, that TYRO3 is translocated into nucleus through MMP2 cleavage to exert anti-apoptotic and pro-proliferative functions, which provides an alternative strategy to target nuclear translocation of TYRO3 for colon cancer therapy.
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