| Summary: | 碩士 === 國立臺灣大學 === 獸醫學研究所 === 107 === Kynurenine 3-monooxygenase (KMO) is the secondary enzyme in kynurenine pathway and locates on the mitochondrial outer membrane. The dysregulation of KMO has been proved to lead to various neurodegenerative diseases; however, it is rarely mentioned in cancer progression. Our previous study has firstly shown KMO overexpression in canine mammary gland tumor (cMGT) was associated with the poor prognosis in cMGT patients. Surprisingly, we also identified that KMO can aberrantly locate on the cell membrane of cMGT cells, not just like normal cells with KMO expressing only within cytosol. Based on the similar morphology and pathogenesis between cMGT and human breast cancer, this study intends to focus on investigating if surface KMO can also be detected in human breast cancer, and if it does, what is the role of surface KMO in the tumor development, especially for human triple-negative breast cancer (TNBC). The correlation between KMO expressions and the malignancy or outcome of clinical breast cancer cases was analyzed in TCGA/UCSC databases and tissue microarray. It was revealed that higher KMO expression significantly correlated with the poor overall survival rate in breast cancer patients. Moreover, using human breast cancer tissue microarray, we found both total and surface KMO expressions were significantly elevated in breast cancer tissues comparing to those of normal breast tissues. We further demonstrated that 7 to 86% of KMO could be detected on the cell membrane in breast cancer cell lines HCC-1954, T47D, MDA-MB-231, MDA-MB-453, MDA-MB-468, Hs578T, HCC-1937 and BT549 by flow cytometry. These results of the aberrant surface expression of KMO were also confirmed by immunofluorescence assay (IFA) and immune electron microscopy. The topology of surface KMO was probed by epitope mapping using arrays of anti-human KMO antibodies and amino acid (a.a.) sequences analyzer by Protter algorithm to disclose it was a periplasmic N-termini (Nout orientation) and C-termini (Cout orientation) protein with two membrane-spanning domains. Treating MDA-MB-231 cells with anti-KMO polysera produced specifically against surface KMO was found to significantly inhibit migration of tumor cells. Taken together, this study has shown for the first time KMO aberrantly and highly expressed on the cell membrane of breast cancer tissues and cell lines. Additionally, surface KMO associated with cancer malignancy and played the role in promoting cell migration of breast cancer cells. Further investigations are needed to dissect the tumorigenic mechanisms of surface KMO and evaluate its feasibility as the target of cancer treatment.
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