2016-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/644839摘要：神經母細胞瘤（neuroblastoma）是兒童顱外最常見的固態腫瘤，也是最為致命的兒科疾病之一。在神經母細胞瘤腫瘤中充滿異質性的癌細胞族群中，有一小群腫瘤起始細胞（tumorinitiating cells）具有幹細胞特性、強大的自我再生能力、上皮間葉轉型（epithelial-mesenchymaltransition）及對化療及放射治療具有抗藥性。因此，若能開發針對腫瘤起始細胞的標靶治療，可作為許多癌症的有效策略。最近的研究發現，癌症表觀基因學（cancer epigenetics）方面的多種異常可能與腫瘤起始有關。其中，KDM5B（又名JARID1B/PLU1）蛋白，為高度專一性的組蛋白離胺酸去甲機酵素（H3K4me3 histone demethylase），在許多癌症中有過度表現情形，也被認為是腫瘤起始細胞的標記。我們初步的研究顯示KDM5B 蛋白在神經母細胞瘤腫瘤球體(tumor sphere)的形成及抗藥性，均扮演相當重要的角色。同時KDM5B mRNA 在腫瘤的高表現，也預測了較差的預後。進一步使用KDM5B 抑制劑也可抑制神經母細胞瘤腫瘤球體的形成。由此可見KDM5B 在神經母細胞瘤腫瘤生成應扮演了相當關鍵的角色，同時也可能成為治療標的。因此本計劃的主要目的係希望利用三年的時間，完成以下三項工作，來探討KDM5B 在神經母細胞瘤腫瘤起始細胞、腫瘤行為、及治療中的角色：(1) 確認KDM5B 對神經母細胞行為的影響及機制：神經母細胞在過度表達或抑制KDM5B後細胞行為的改變，包括分化、生長、移行、腫瘤球體形成及抗藥性、以及侵犯能力等將加以記錄。之後並用這些細胞株建立小鼠腫瘤模型，以檢測KDM5B 對腫瘤生長的影響，KDM5B不同表現量的細胞株將以微陣列基因晶片或二維蛋白質電泳檢測差異分子表現，以尋找KDM5B 對神經母細胞行為影響的可能機制；(2) KDM5B 對臨床神經母細胞瘤腫瘤行為及腫瘤起始細胞的影響：以免疫組織染色及西方墨點檢測病患腫瘤檢體中KDM5B 表現量，並分析其表現量和病患其他預後因子及預後的相關性。同時利用腫瘤檢體培養腫瘤起始細胞，並分析其KDM5B 表現量及細胞在培養皿中及動物模式中的行為以及藥物敏感性的相關性；(3) 測試 KDM5B 抑制劑對神經母細胞瘤細胞與動物模型的治療效果：將測試KDM5B 抑制劑(Destruxin B 及 PBIT) KDM5B 不同表現量的細胞株，以及病患的腫瘤起始細胞行為的影響。同時使用KDM5B 不同表現量的神經母細胞瘤細胞株以及病患的腫瘤起始細胞建立的異體裸鼠以及TH-MYCN 基因改造小鼠等動物模型，來確認藥物對腫瘤大小的抑制效果。本計劃的重要性在於將進一步確認KDM5B 在神經母細胞瘤腫瘤起始細胞、腫瘤行為、及治療中的角色的重要性，並測試KDM5B 能否作為新穎標靶治療的策略。研究結果未來可轉譯為可執行的臨床試驗設計，期待最終能提昇病患的治療成績。本計畫對社會、經濟、及學術發展可能之影響。神經母細胞瘤是兒童顱外最常見的固態惡性腫瘤，其5 年存活率僅約30％，且目前其病理成因不清，更使得其治療成績停滯不前。KDM5B 在神經母細胞瘤腫瘤起始細胞扮演重要角色，透過本研究不僅有助於開啟神經母細胞瘤病理成因之謎，而KDM5B 抑制劑更可望發展為有效的標靶治療藥物，以期大幅提升病童的存活率、減少經濟損失、並提高我國兒童癌症之學術研究水準。<br> Abstract: Neuroblastoma (NB) is the most common and lethal extracranial solid tumor in children.Among the heterogeneous cell populations within a NB tumor, a subset of cells, termed tumorinitiating cells (TICs), exhibits stem cell properties and demonstrates a robust capacity ofself-renewal, enhanced epithelial-mesenchymal transistion, and resistance to chemotherapy andradiation therapy. Thus, targeting and elimination of TICs may offer a more effective way for cancermanagement. KDM5B (also known as JARID1B/PLU1) is a highly specific histone lysinedemethylase whose overexpression has been found in a variety of cancers and a marker of TICs.Our preliminary data reveals that KDM5B is critical for the tumor sphere formation as well as drugresistance of NB. High expression of KDM5B mRNA in tumor samples predicts a poor prognosis.Furthermore, suppression of KDM5B by inhibitors may suppress the tumor sphere formation. Theselines of evidence suggest the critical potential of KDM5B in the tumoregenisis and therapeuticapplication of NB. Here we would like to propose a 3-year project with the following three majoraims to further elucidate the role of KDM5B in the TICs, tumor behavior, and treatment of NB:Aim Ⅰ: Clarifying the effects and mechanisms of KDM5B expression on NB cell behavior.For further understanding the effects of KDM5B on NB cell behavior, NB cells with stableoverexpression or downexpression of KDM5B are to be selected. Then NB cell phenotype andbehavior changes including proliferation, migration, invation, drug-resistence, and tumor sphereformation after overexpression or downexpression of KDM5B are evaluated by in vitro assays. Inaddition tumor behavior will also be evaluated by a nude mice xenograft model. Cells with high orlow KDM5B expression levels are subjected to microarray or 2D proteomics analysis to definepossible mechanisms for KDM5B to affect tumor cell behavior;Aim Ⅱ: Clarifying the effects of KDM5B on the behavior of tumor and tumor initiating cells.To further confirm the prognostic value of KDM5B expression in NB patients, tumor samples froma large cohort of NB patients are subjected to IHC and Western blot analysis of protein levels andare compared to other prognostic factors and patient survival. Cell behavior including drugsensitivity in vitro and in xenograft animal models are compared in cells from primary tumorcultures including TICs with various KDM5B expression levels.Aim Ⅲ: Clarifying therapeutic effects of KDM5B inhibitors on NB in vitro and in vivo. Thetumor suppression effects of KDM5B inhibitors including Destruxin B and PBIT on NB will beevaluated on NB cell lines, primary TICs as well as in nude mice xenograft and MYCN transgenicmice models.Altogether, our studies will not only establish the functional role of KDM5B in NB TICs andtumors, but also test the feasibility of novel therapeutic strategies targeting KDM5B. Our results mightshed light to the oncogenesis of NB as well as target therapy of NB.神經母細胞瘤KDM5B腫瘤起始細胞基因轉殖鼠治療NeuroblastomaKDM5Btumor initiating cellstransgenic micetreatment