大腸直腸癌是全世界普遍發生且高致死率的癌症，而大腸直腸癌的發生大部分都跟基因異常表現有關。過去實驗室透過生物資訊的方式，從大腸直腸癌細胞中找到新穎致癌基因TYRO3，也證實TYRO3能促使細胞增生、轉移以及整個腫瘤的進程。TYRO3是一種酪胺酸激酶，也是TAM (TYRO3、AXL、MER)家族的成員。根據大腸直腸腫瘤切片的免疫組織染色結果，發現TYRO3不僅在細胞膜表現外，在細胞核內也能觀察到TYRO3的存在，而且細胞核內TYRO3的表現程度跟腫瘤的進程是呈現正相關。因此，我們認為細胞核內TYRO3或許在腫瘤進程中扮演重要的角色，在此我們致力於探討大腸直腸癌細胞中細胞核內TYRO3的功能。我們從生物資訊工具比對出TYRO3擁有核定位訊號 (NLS)及出核訊號 (NES)的序列，為了探討細胞核內TYRO3的重要性，我們將TYRO3上的核定位序列突變使得TYRO3無法進入細胞核，接著進行細胞的功能測定，例如：細胞凋亡及細胞增生。實驗結果發現TYRO3無法入核的情況下，細胞凋亡的情形增加且能偵測到較多凋亡蛋白酶3 (caspase-3)表現，和正常的TYRO3功能相反。因此，我們得知細胞核內TYRO3的存在對於細胞存活是重要的。BrdU標定實驗結果顯示當大腸癌細胞過度表現NLS突變的TYRO3，其細胞增生受到抑制。我們進一步探討細胞核內TYRO3的功能，我們使TYRO3上的NES序列突變讓TYRO3不被送出細胞核外進而保留在細胞核內，再進行上述的功能測定。從BrdU標定實驗的結果得知，NES突變的TYRO3和正常的TYRO3皆能促進細胞增生。因此細胞核內TYRO3不僅和細胞存活有關，也能促進大腸癌細胞的增生。酪胺酸激酶受體大部分是藉由本身的激酶活性去磷酸化下游的受質，進而執行訊息的傳遞；為了釐清TYRO3激酶活性對於核內TYRO3執行功能是否重要，我們將TYRO3的激酶活性區進行突變，接著執行上述所提到的細胞凋亡及增生情形的偵測。實驗結果顯示細胞核內TYRO3的功能表現是需要激酶活性來驅動。另外，TYRO3的入核機制也是我們進一步要去探討的問題，讓細胞過度表現TYRO3後，利用三種辨認TYRO3上不同抗原的抗體(N端、中間區域、C端)進行免疫螢光染色，發現在細胞核內只有辨認C端的抗體能偵測到TYRO3，而N端則辨認到分布在細胞膜上的蛋白。根據上述染色結果，我們認為TYRO3有可能透過蛋白酶切割成片段後，C端的TYRO3才進入細胞核內。我們利用生物資訊工具進行預測，預測到在TYRO3序列上有類似基質金屬蛋白酶2(MMP-2)的切位，我們收集過度表現TYRO3的細胞且進行細胞外MMP-2降解的實驗，結果發現TYRO3的確能被MMP-2所切割成片段；而在TYRO3上突變MMP-2切割位置，螢光免疫染色結果發現細胞核內沒有TYRO3的表現。根據以上的實驗結果，TYRO3首次被證實能透過MMP-2切割而進入到細胞核，進而執行抗細胞凋亡及促進細胞增生的功能，因此，未來核內TYRO3能作為一個標靶分子以成為另一個對抗癌症的策略。

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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