探討MTHFD2在神經母細胞瘤發展進程扮演的角色

Abstract

神經母細胞瘤 (Neuroblastoma) 是胚胎在發育的過程中交感神經脊分化失敗形成惡性腫瘤的一種兒童癌症，臨床研究發現轉錄因子MYCN基因擴增的現象和神經母細胞瘤的不良預後及惡化程度具有關聯性，但目前對於MYCN如何去調控腫瘤的進程機制仍然尚未明瞭。在本篇研究中我們先從基因表達綜合數據庫 (Gene expression Omnibus, GEO) 分別將高風險 (年齡大於18個月，第四期) 的神經母細胞瘤病人分成MYCN多倍數與非多倍數去做微陣列基因顯著性分析驗證 (significance analysis of microarrays, SAM)，發現在MYCN多倍數的神經母細胞瘤病人中其亞甲基四氫葉酸脫氫酶2 (MTHFD2) 也會顯著地被表現，兩者間具有高度正相關 (P < 0.001)。此外在我們染色質免疫沉澱–測序（ChIP-Seq）的結果中也發現，MYCN會在亞甲基四氫葉酸脫氫酶2的轉錄起始點附近區域有很強的結合 (P = 3.7e-18)。MTHFD2在胚胎發育時期扮演單碳代謝途徑的重要酵素，目前研究指出MTHFD2的表現和乳癌的低存活率有關，在我們神經母細胞瘤病人的存活率分析中也看到了一樣的情形 (P < 2.4e-13)，由上述的資料分析，我們推測MYCN會調控MTHFD2的表現而影響神經母細胞瘤的進程。為了確定他們之間的相關性，我們分別在神經母細胞瘤細胞株中抑制或增加MYCN的表現量，發現MTHFD2的表現量也會與之有相同趨勢的改變。更進一步，啟動子冷光偵測結果顯示MTHFD2是一個受MYCN直接轉錄調控的基因。接著我們於MYCN高表現的SK-N-DZ細胞株中利用小髮夾核醣核酸 (shRNA) 抑制MTHFD2，亦或是在MYCN低表現的SK-N-AS細胞株中大量表現MTHFD2，由結果發現MTHFD2會促使神經母細胞瘤細胞株的生長。這項研究結果指出MTHFD2在神經母細胞瘤的進程中或許扮演重要的角色，也許是未來治療神經母細胞瘤的一項潛力。

Neuroblastoma (NBL) is a pediatric cancer derived from the sympathetic lineage of the neural crest with improper differentiation during development. The amplification of V-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN) is a clinical feature associated with the poor prognosis of NBL while the underlying mechanism still remains elusive. In this study, we first integrated several expression datasets from Gene Expression Omnibus (GEO) and performed significance analysis of microarrays (SAM) in high-risk NBL of patients with or without MYCN amplification. We found that MTHFD2 (methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 2, methenyltetrahydrofolate cyclohydrolase), a mitochondrial enzyme required for one-carbon metabolism and purine synthesis, was differentially up-regulated in NBL with MYCN amplification (P < 0.001). Additionally, in our chromatin immunoprecipitation (ChIP) - sequencing assay, we observed that MYCN strongly bound to promoter region nearing by MTHFD2 transcriptional start site (P = 3.7e-18). MTHFD2 is essential to embryonic development and has been shown to relate to the poor survival in breast cancer. It was also found that high MYCN and MTHFD2 expression correlated with poor survival (P < 2.4e-13) from our survival analysis in NBL patients. Based on data analyses above, we speculated that MYCN and MTHFD2 may play related role in NBL. To confirm this, we reduced and overexpressed MYCN expression, respectively, and observed MTHFD2 expression levels concomitantly changing in NBL cell lines. Moreover, promoter assay revealed that MTHFD2 is a direct transcriptional target of MYCN. To this end, we also conducted shRNA-mediated knockdown experiments in SK-N-DZ cells and overexpressed MTHFD2 in SK-N-AS cells. Interestingly, MTHFD2 promotes cell proliferation and colony formation of NBL cell lines. Together, our results suggest that MTHFD2 may play an important role in tumor aggressiveness as a potential therapeutic target for NBL.