芳香烴接受器調控神經母細胞瘤MYCN基因表現之研究

Abstract

神經母細胞瘤是一種目前最常見的小兒顱外固態惡性腫瘤。MYCN是神經母細胞瘤一項重要的預後診斷因子，MYCN基因的大量重覆表現往往代表腫瘤的高度惡化及不佳的預後表現。於此研究，我們希望能尋找出MYCN相關之基因並研究其對於神經母細胞瘤之影響。首先透過基因微陣列技術分析十個MYCN重複表現病患與另十個MYCN正常表現之病患腫瘤中基因表現之差異，再利用IPA訊息傳遞路徑分析找出可能與MYCN調控相關之基因，我們發現在病人腫瘤裡芳香烴接受器(AHR，戴奧辛接受器)的表現和MYCN的表現呈現明顯負相關。此現象亦透過另外的14個病患腫瘤檢體加以驗證。除此之外，從病患的病理切片染色也發現，AHR蛋白質的表現量與腫瘤細胞的分化程度成正相關，且具有AHR表現的病患其預後亦較佳。利用神經母細胞瘤細胞株進行實驗亦發現AHR大量表達會促進細胞之神經分化且可以透過抑制MYCN基因之啟動子之活性，造成負向調控MYCN基因的表現。另外，由於E2F1的大量表達可以補償AHR表現造成MYCN基因轉錄之抑制作用，故E2F1被認為參與在AHR調控MYCN表現之訊息傳遞鏈。反之，利用shRNA抑制AHR之表現後亦造成E2F1及MYCN之表現上升。根據以上研究結果顯示，AHR確實為MYCN的上游調控者，其可透過E2F1調控MYCN的表現，進而影響神經母細胞瘤之分化。為了進一步證實AHR對於神經母細胞瘤發生之影響，我們利用新發現的AHR受體”犬尿酸”(Kynurenine, Kyn)進行in vivo的神經母細胞瘤治療實驗。實驗發現Kyn成功的延長了TH-MYCN基因轉殖鼠之壽命，於腫瘤異體移植實驗亦發現Kyn可以有效地抑制神經母細胞瘤之轉移。此動物實驗更加驗證AHR於神經母細胞瘤病情發展之重要性。

Neuroblastoma (NB) is the most common malignant disease of infancy. MYCN amplification is a prognostic factor for NB and is a sign of highly malignant disease and poor patient prognosis. In this study, we aimed to investigate novel MYCN-related genes and assess how they affect NB cell behavior. The different gene expression found in 10 MYCN amplification NB tumors and 10 tumors with normal MYCN copy number were analyzed using tissue oligonucleotide microarrays. Ingenuity Pathway Analysis was subsequently performed to identify the potential genes involved in MYCN regulation pathways. Aryl hydrocarbon receptor (AHR), a receptor for dioxin-like compounds, was found to be inversely correlated with MYCN expression in NB tissues. This correlation was confirmed in a further 14 human NB samples. Moreover, positive AHR expression by immunostaining of NB tumors was found to correlate well with histological grade of differentiation and predicted a favorable prognosis. In vitro studies revealed that AHR overexpression in NB cells induced spontaneous cell differentiation. In addition, it was found that ectopic expression of AHR suppressed MYCN promoter activity, resulting in downregulation of MYCN expression. The suppression effect of AHR on the transcription of MYCN was compensated for by E2F1 overexpression, indicating that E2F1 is involved in the AHR-regulated MYCN pathway. Furthermore, AHR shRNA promotes the expression of E2F1 and MYCN in NB cells. These findings suggest that AHR is one of the upstream regulators of MYCN. Through the modulation of E2F1, AHR regulates MYCN gene expression, which may in turn affect NB differentiation. In order to further confirm the effect of AHR on the tumorigenesis of NB, the novel endogenous ligand of AHR, Kynurenine (Kyn), a tryptophan catabolite, was employed in the in vivo NB therapy experiments. It was found that Kyn could significantly prolong the survival of TH-MYCN transgenic mice and suppress metastasis of NB in the xenograft mice models. This result further suggests the important role of AHR in NB tumor progression.