犬尿氨酸-3-單氧化脢異常表達在細胞膜上促進三陰性乳癌的細胞遷移

Abstract

犬尿氨酸-3-單氧化脢(Kynurenine 3-monooxygenase, KMO)是犬尿氨酸途徑(Kynurenine pathway, KP)中的次要關鍵酶。目前研究已證實KMO的失調會導致神經退行性疾病發生，但是在癌症相關的研究中鮮少提及它。我們先前的研究首次闡述KMO在犬乳腺腫瘤中過度表達並且與患者預後不良有關。此外，正常細胞的KMO表達在細胞質中，我們卻發現KMO異常表達在犬乳腺腫瘤細胞膜上。由於犬乳腺腫瘤與人類乳癌在致癌機制上具有高度相似性，本研究重點在探討人類乳癌細胞是否具有同樣的現象；假設確實如此，將進一步研究膜上KMO在腫瘤進程中扮演的角色，且是否和預後較差的人類三陰性乳癌(Triple-negative breast cancer, TNBC)進程有關，結果顯示KMO表現量與乳癌患者的總體存活率顯著負相關。在人類乳癌組織微陣列中，乳癌組織的KMO總表現量和膜上KMO表現量均顯著高於正常乳腺組織。而利用流式細胞儀分析乳癌細胞株HCC-1954、T47D、MDA-MB-231、MDA-MB-453、MDA-MB-468、Hs578T、HCC-1937和BT549，分別有7~86%不等之細胞表現膜上KMO。透過免疫螢光測定和免疫電子顯微鏡也證實三陰性乳癌細胞株MDA-MB-231表達膜上KMO。接著我們使用多種抗人類KMO抗體和Protter蛋白質結構預測軟體分析膜上KMO的結構，揭示它具有兩個跨膜區且N端與C端朝向細胞外。我們進一步製備特異性針對膜上KMO的多株抗體，將此抗體遏阻MDA-MB-231細胞的膜上KMO後，顯著抑制癌細胞的遷移。綜合上述結果，這項研究首次闡明KMO在乳癌臨床組織和細胞株的異常表達在細胞膜上，而且膜上KMO與癌症惡性程度相關並促進乳癌細胞的遷移能力。未來將進一步研究以剖析膜上KMO的致癌機制並評估其作為癌症治療靶點的可行性。

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.