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Title: | 鑑定TIMP3作為神經母細胞瘤轉移之調節因子 Identification of TIMP3 as a regulator for metastasis in neuroblastoma |
Authors: | Mati Kargren 卡爾根馬緹 |
Advisor: | 李心予(Hsinyu Lee) |
Keyword: | 神經母細胞瘤,轉移,JQ1,CL-387,785,TIMP3, Neuroblastoma,Metastasis,JQ1,CL-387,785,TIMP3, |
Publication Year : | 2019 |
Degree: | 碩士 |
Abstract: | 神經母細胞瘤 (Neuroblastoma, NB) 是一種好發於兒童的交感神經顱外實體腫瘤。儘管可用的治療方案有所改善,高風險神經母細胞瘤患者的預後因為轉移性擴散仍然不是十分看好。在我們的研究中,我們測試了JQ1(一種Bromodomain和Extra-Terminal(BET)的抑制劑)和CL-387,785(EGFR / ErbB2受體酪氨酸磷酸激酶雙重抑制劑)阻斷神經母細胞瘤細胞轉移能力。 BET抑制劑主要的作用目標為癌基因MYCN,而該基因的擴增和高風險神經母細胞瘤有高度相關。另外,CL-387,785 的作用目標EGFR和ErbB2均在神經母細胞瘤腫瘤組織中有高度表達,並且之前研究已證明這兩個受體酪氨酸磷酸激酶的功能與其他類型腫瘤的轉移有高度相關。利用wound healing和transwell assay兩種檢測腫瘤細胞移動能力的方法,我們證明這兩種化合物都能顯著減少神經母細胞瘤細胞的遷移和侵入組織的能力。接著以colony formation assays檢測腫瘤細胞可懸浮生長形成聚落的特性,更證明這兩種化合物皆能顯著地抑制神經母細胞瘤細胞的腫瘤聚落生成的能力。使用人類腫瘤基因微陣列分析這兩種藥物是否可共同影響一個可控制神經母細胞瘤擴散能力的調控基因時,我們發現金屬蛋白酶組織抑制因子3(TIMP3),在被JQ1或CL-387,785處理過的神經母細胞瘤細胞中,TIMP3基因表現量比對照組有明顯上升的現象。這一結果我們也已利用 RT-qPCR、西方點墨法、和細胞免疫螢光染色法再次證實,JQ1和CL-387,785的確可引發神經母細胞瘤細胞TIMP3基因的高度表現。當我們使用網路R2生物資訊共享平台的神經母細胞瘤患者數據做進一步分析時更顯示,高度表現TIMP3的腫瘤患者其總體存活率(Overall Survival Probability)和無事件存活率(Event-free Survival Probability)均有顯著增加的現象。依此,我們使用台灣神經母細胞瘤患者的腫瘤樣本做獨立驗證分析時,也同樣發現TIMP3在分化程度高的神經母細胞瘤腫瘤中基因表現量與蛋白質表現量都比分化程度較低的腫瘤更高。綜合這些研究結果,我們發現JQ1和CL-387,785都能夠藉由促進TIMP3基因的高度表現後,顯著地抑制神經母細胞瘤的腫瘤轉移能力。這個現象也與在預後良好的神經母細胞瘤中TIMP3的表現量較高有一致的趨勢。我們的研究結果提供了初步且重要的證據,可支持將JQ1和CL-387,785進一步發展成為治療高風險神經母細胞瘤的藥物,也同時驗證了TIMP3在神經母細胞瘤中的預後價值及可開發為新穎的神經母細胞瘤藥物標的。 Neuroblastoma is a tumor disease of the sympathetic nervous system and it is the most common type of extra-cranial solid tumors in children. Despite improvements in available treatment options, prognosis for high-risk neuroblastoma patients remains unfavorable, with metastatic spread being one of the reasons behind it. In our study we tested the ability of JQ1, a Bromodomain and Extra-Terminal (BET) inhibitor, and CL-387,785, an EGFR/ErbB2 dual inhibitor, to block metastatic properties of neuroblastoma cells. BET inhibitors target MYCN, an oncogene whose amplification is associated with high-risk neuroblastoma. Additionally, both EGFR and ErbB2 were shown to be highly expressed in NB cells and tissues, and have been implicated in metastasis of various tumors. Using wound healing and transwell assays, we were able to show that both of the compounds significantly reduce migration and invasion of neuroblastoma cells. Consistently, colony formation assays showed that both compounds are able to inhibit anchorage-independent growth of neuroblastoma cells. Using a human tumor gene microarray, we later identified tissue inhibitor of metalloproteinases 3 (TIMP3) as one of the significantly upregulated genes in neuroblastoma cells treated with JQ1 or CL-387,785. These results were confirmed by quantitative real-time PCR, Western blotting and immunocytochemistry. Furthermore, analysis of neuroblastoma patient data from the web-based R2 platform revealed that patients with tumors expressing high levels of TIMP3 have a significantly increased overall and event-free survival probability. Lastly, quantitative real-time PCR, Western blotting and immunohistochemical analysis of tumor samples from a Taiwanese cohort of neuroblastoma patients showed that TIMP3 is expressed at higher levels in more differentiated neuroblastoma tumors compared to less differentiated ones. In summary, our results show that JQ1 and CL-387,785 are able to significantly inhibit molecular processes involved in metastasis of NB, and that expression of TIMP3 is higher in cells treated by these compounds, as well as tumor samples from NB patients with favorable prognoses. Our findings provide the proof-of-principle evidence that JQ1 and CL-387,785 could be further developed to become the target therapies for high-risk neuroblastoma, and also verify the prognostic merit of TIMP3 in neuroblastoma. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/72885 |
DOI: | 10.6342/NTU201901345 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 生命科學系 |
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