2015-08-012024-05-17https://scholars.lib.ntu.edu.tw/handle/123456789/683670摘要：本計劃主要目的是要探討NF-kB activating protein (NKAP)對神經母細胞瘤(NB)的腫瘤生物特 性、病理變化、病人預後的影響及其中的分子機制。爾源自於胚胎時期發育為人體交感神經系統的 神經脊細胞（neural crest cells)，也是幼兒期最常見的惡性腫瘤。大約60%的NB病患在診斷時已經 是第四期，預後非常差，五年存活率大約只有40%。此腫瘤的特徵在於其多樣性，此特性亦表現於 其病理組織檢查的型態學上。在NB中，往往可看到分化程度不同的腫瘤細胞組織同時存在。診斷時， 組織病理上的分化程度和病人的預後也有相關，分化程度愈好者，預後愈好。雖然其病理成因仍然 不明，但很可能是胚胎神經母細胞在發育過程中無法正常分化而造成。Notch訊息傳導路徑會調節神經脊細胞的發育，也跟NB細胞的分化有相關性。我們之前的研究 發現Notchl基因的表現和NB的腫瘤分化程度有關，而且是病人較差存活的獨立預後因子，但調節 Notch訊息傳導路徑活性的上游機制並不清楚。NKAP是一個新發現會調節NF-kB蛋白活化的分子， 它也會抑制Notch訊息傳導路徑的轉譯活性，因此可能會跟Notch訊息傳導路徑協同作用，進而影響 NB的腫瘤生成。在我們初步的研究結果中顯示，NKAP在NB腫瘤組織的表現量與Notch1的表現 量呈現負相關，NKAP在組織分化程度較成熟的NB腫瘤表現量較高，且其高表現量可預測病人較佳 的存活狀況。這樣的發現，讓我們想進一步去探討NKAP的表現在NB中，對其Notch訊息傳導路 徑的活性、腫瘤細胞的分化及惡性程度、乃至於對病人預後的影響。因此我們主要的目標為：1.確認NKAP的表現與NB分化程度及病人預後的相關性。我們會以免疫染色的方式在台大醫院的病人腫瘤組織中（約150位），確認NKAP的表現與腫瘤組織分化程度的相關性，同時比對病人的臨床 表徵，包括Notch1的表現量，和腫瘤組織NKAP的表現量比對後，探討NKAP的表現量與NB病人 預後的相關性。並以NB細胞株的實驗進一步確認這樣的關係。2.確認NKAP的表現會影響NB細胞的惡性程度及Notch訊息傳導路徑的活性，並探討影響NKAP表現的表關遺傳(Epigenetic)機轉。我們將會建立NKAP穩定表現的NB細胞株，在和NKAP不表現的細胞株比較後，觀察細胞生物特性、惡性程度的變化，及其Notch訊息傳導路徑活性的差別。另 外也將探討，在病人腫瘤組織中NKAP的表現是否與其啟動子的DNA曱基化有關。3.在活體實驗中確認NKAP的表現對NB腫瘤生長、轉移及動物存活的影響。我們將以小鼠的動物 實驗模式，將NKAP穩定表現及不表現的NB細胞株分別種在小鼠身上，來探討NKAP的表現在活 體動物中，對NB腫瘤生長、轉移及動物存活的影響。本研究完成後，我們將確認NKAP的表現對NB病人預後的重要性，了解NKAP的表現在NB 中對腫瘤的分化、惡性程度、Notch訊息傳導路徑活性的影響，及NKAP的表現是否與其啟動子的 DNA曱基化有關。藉由這樣的了解，希望未來可以進一步對這個預後極不好的腫瘤設計出新的治療 藥物，增進治療的成绩，改善病人的預後。<br> Abstract: The objective of this proposal is to investigate the biological and pathological role of NF-kB activating protein (NKAP) in neuroblastoma (NB). NB is a childhood tumor derived from sympathoadrenal lineage of the neural crest progenitor cells. It is quite a heterogeneous tumor and presents a broad clinical and biologic spectrum ranging from highly undifferentiated tumors with very poor outcomes to the most differentiated benign ganglioneuroma or NB with favorable prognosis.Notch signaling pathway can regulate neural crest cells development and is related to NB cell differentiation. Our previous study revealed that Notch1 expression was correlated with differentiation status of NB and predicted advanced tumor stages and unfavorable outcomes. The molecular mechanism responsible for neuronal differentiation of NB cells regulated by Notch signaling is mediated by a downstream JNK-calreticulin-dependent pathway. However, the upstream mechanism which regulates the activity of Notch signaling pathway and in turn the tumorigenesis of NB is not elucidated.NKAP is a newly identified nuclear protein that promotes NF-kB activation. NKAP is also a transcriptional repressor of Notch signaling and therefore may act concordantly with Notch signaling pathway in the tumorigenesis of NB. Our preliminary data showed that NKAP expression inversely correlates with Notch1 expression in NB tumors and NKAP expression was higher in NB tumors with more mature differentiation histology. In addition, positive NKAP expression in NB tumors was significantly associated with favorable prognostic factors and predicted a significantly better survival. These preliminary results provide the basis for addressing the correlation between the NKAP expression and the differentiation, behaviors, activity of Notch signaling pathway in NB cells, as well as prognosis of NB patients.Comprehensive studies of the molecular mechanism underlying such relationship could shed light on the development of novel therapeutics for the treatment of NB. Therefore our specific aims are:1.Verify the association of NKAP expression and Notchl expression, NB tumor differentiation and the prognostic roles of NKAP protein expression in NB patients. We will exam the association of NKAP expression and NB tumor differentiation by IHC staining in a cohort of NB patients in our hospital and further define the prognostic roles of NKAP protein expression in NB patients by analyzing correlations between NKAP expression and clinicopathologic and biologic variables of NB tumors, including Notch1 expressions. We also will verify the association of NKAP expression and NB cells differentiation in vitro.2.Determine the effects of NKAP expression on behaviors and the Notch signaling activity of NB cells, as well as the epigenetic regulations by which affect the NKAP expression in NB tumors. We will establish NKAP stable transfectants and these NKAP stable transfectants will be compared the malignant behaviors of cancer cells with mock transfectants. The downstream target genes expression of Notch signaling pathway will also be compared between NKAP stable transfectants and mock transfectant NB cells. In addition, since NKAP expression was detected in a part of NB tumors (41%) in our preliminary results and the regressive mechanisms of NKAP expression is unclear, we will investigate the methylation status of promoter region on NKAP in primary NB tumors to test our hypothesis that hypermethylation of promoter region may be responsible for depress of NKAP expression.3.Determine the effect of NKAP expression on NB tumor growth, survival and metastasis in vivo. Wewill establish the NB xenograft model in SCID mice and compare the NB tumor growth, survival and tumor metastasis in SCID mice bearing NAKP stable transfectants with mock transfectants.神經母細胞瘤NKAPNotch訊息傳遞路徑NeuroblastomaNF-kB activating proteinNotch signaling