探討胞內乙醯葡萄醣胺與神經母細胞瘤病患預後因子相關性及其累積有效抑制神經母細胞瘤之惡性行為

The pattern of O-GlcNAcylation correlates with neuroblastoma prognosis and its accumulation ameliorates malignant phenotypes in NB cells

謝佳燁(Chia-Yeh Hsieh)

指導教授 ： 林能裕

國立臺灣大學/醫學院/解剖學暨細胞生物學研究所/碩士(2018年)

https://doi.org/10.6342/NTU201804145

摘要

神經母細胞瘤為兒童最常見的顱外固態腫瘤，佔總體兒童癌症死亡率達15%。神經母細胞瘤起源於交感神經系統，為神經脊前驅細胞發育受阻所致，因具有高度異質性而難以根治。神經母細胞瘤高風險群病患的長期存活率不佳，即使切除或接受高強度治療後仍無法改善病況且有復發的風險存在。提出更多預後因子幫助臨床分析是目前研究的趨勢。我們的研究發現，較不分化、較惡性的神經母細胞瘤病患的腫瘤，其表現較少的O-GlcNAc。另外，我們的小鼠模型，TH-MYCN誘發的神經母細胞瘤腫瘤中胞內蛋白O-GlcNAcylation也較正常小鼠腎上腺胞內少，此趨勢與病患腫瘤分析一致。結合臨床統計分析發現158位病患中，總體O-GlcNAc陽性的病患存活率較高外；而從臨床早、晚期與MYCN無擴增病患的存活率分析，O-GlcNAc陽性者存活率也較高，且O-GlcNAc是除了年齡外的獨立預後因子，O-GlcNAcylation將有機會提供臨床上新的治療方向。O-GlcNAc（乙醯葡萄醣胺）為修飾細胞內蛋白質的醣類結構，目前發現受到兩種酵素專一性的調控胞內蛋白質的加醣與去醣作用（乙醯葡萄醣胺轉移酶（OGT）負責加醣；乙醯葡萄醣胺水解酶（OGA）負責去醣）。O-GlcNAcylation作用於胞內蛋白的絲胺酸和蘇胺酸上氫氧基。O-GlcNAcylation涉及細胞內眾多生理機制，失調後會引起許多疾病產生。我們想調查O-GlcNAcylation在神經母細胞瘤的角色。我們的研究發現，以13-cis Retinoic Acid（13-cis RA）治療神經母細胞瘤細胞株後，胞內O-GlcNAc的表現隨分化而提升；以藥物Thiamet G抑制OGA蛋白活性使胞內O-GlcNAcylation蛋白累積後也能改善神經母細胞瘤惡性行為與刺激細胞分化並同時降低致癌因子N-myc蛋白表現。在裸鼠異種移植實驗中發現用sh-OGT lentivirus敲除神經母細胞瘤ogt基因後會使腫瘤生長惡化。綜合以上結論，我們的研究結果指出，以藥物促使細胞內O-GlcNAc上升將能幫助神經母細胞瘤分化並減緩神經母細胞瘤的惡性行為及抑制致癌因子N-myc表現。臨床上分析顯示O-GlcNAc陽性能夠作為獨立預後因子，提供未來治療新方向。O-GlcNAcylation是如何調控神經母細胞瘤相關分化基因的表現，並且抑制致癌基因表現是我們未來的研究重點。

關鍵字

神經母細胞瘤 ； 乙醯葡萄醣胺 ； 乙醯葡萄醣胺轉移酶 ； 乙醯葡萄醣胺水解酶 ； Thiamet G ； 細胞分化

並列摘要

Neuroblastoma is the most common malignant extracranial solid tumor of childhood and accounts for 15% cancer death in children. Neuroblastoma arises from sympathetic nervous system due to the aberrant developmental differentiation of early neural crest precursor cells. The disease is remarkable for its broad spectrum of heterogeneous behaviour. Further identify novel prognosis factors is the aim of clinical research. Here we present evidence that hypo-O-GlcNAc expression in undifferentiation, malignant NB patient tumors similar to the result of TH-MYCN mice tumor IHC experiments. According to clinical statistical analyses, NB patients with positive O-GlcNAc expression has higher long-term survival than those O-GlcNAc negative. Moreover, based on Survival-rate analyses, patients with O-GlcNAc-positive in early or late stage as well as MYCN-nonamplification has higher long-term survival. Multivariate analyses revealed that positive O-GlcNAc expression in tumor tissues predicted a favorable prognosis in NB patients independent of other prognostic markers except age. Intracellular O-GlcNAcylation could proivde new clinical strategy.O-GlcNAcylation is a dynamic modification of serine or threonine hydroxyl moieties nucleocytoplasmic proteins by UDP-GlcNAc. The dynamic and inducible cycling of the modification is governed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which add and remove the O-GlcNAc moiety respectively. The O-GlcNAcylation involes in many important cellular pathways and unbalance of O-GlcNAcylation has implicated in many kinds of diseases. We are going to investigate the role of O-GlcNAcylation in Neuroblatoma.The differentiation of NB cell induced by 13 cis-Retinoic Acid caused O-GlcNAc accumulation and N-myc decreased in NB cell, simultaneously. Furthermore, using Thiamet G to inhibit OGA activity result in cellular O-GlcNAc level increased, also ameliorates malignant phenotypes, induces differentiation and decreases N-myc protein expression in NB cells.Above all, our findings suggest that O-GlcNAcylation regulates malignant phenotypes of NB cells through the differentiation. Moreover, clinical analysis also indicates that O-GlcNAc-positive could be an independent prognosis factor. These results regarding O-GlcNAcylation may also provide an alternative approach for cancer therapy by means of modulating oncogene-specific O-GlcNAcylation.

並列關鍵字

Neuroblastoma ； O-GlcNAc ； O-GlcNAc transferase ； O-GlcNAcase ； Thiamet G ； differentiation

參考文獻

1. Maris, J.M., et al., Neuroblastoma. The Lancet, 2007. 369(9579): p. 2106-2120.

2. Kamijo, T. and A. Nakagawara, Molecular and genetic bases of neuroblastoma. Int J Clin Oncol, 2012. 17(3): p. 190-5.

3. Park, J.R., A. Eggert, and H. Caron, Neuroblastoma: biology, prognosis, and treatment. Hematol Oncol Clin North Am, 2010. 24(1): p. 65-86.

4. W.B. London, R.P.C., K.K. Matthay, A.T. Look, R.C. Seeger, H. Shimada, P. Thorner, G. Brodeur, J.M. Maris, C.P. Reynolds, and S.L. Cohn Evidence for an Age Cutoff Greater Than 365 Days for Neuroblastoma Risk Group Stratification in the Children's Oncology Group. JOURNAL OF CLINICAL ONCOLOGY, 2005. 23.

5. Hsu, W.-M., The study of biologic prognostic factors of neuroblastoma-clinical significance and impact on surgical decision, in Graduate institute of Clinical Medicine College of Medicine. 2004, National Taiwan University.