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標題: | Notch 訊息傳遞路徑對於神經母細胞瘤細胞分化的機制及其與病人預後關係之研究 The role of Notch signaling pathway in neuronal differentiation of neuroblastoma and prognosis of patients with neuroblastoma |
作者: | Hsiu-Hao Chang 張修豪 |
指導教授: | 林凱信(Kai-Hsin Lin),謝豐舟(Fon-Jou Hsieh) |
關鍵字: | 神經母細胞瘤,Notch訊息傳遞路徑,分化治療, neuroblastoma,Notch signaling pathway,differentiation therapy, |
出版年 : | 2010 |
學位: | 博士 |
摘要: | 神經母細胞瘤源自於胚胎時期將來會發育為人體交感神經系統的神經脊細胞(neural crest cells),腫瘤可發生在交感神經系統分佈的任一部位,但最常發生在後腹腔,尤其一半以上在腎上腺的位置。在兒童癌症中,它是除了腦瘤、淋巴瘤之外,最常見的固態腫瘤,也是兒童在嬰兒時期(小於一歲)最常見的惡性腫瘤,大約每7000位新生兒就會有一個發病。神經母細胞瘤很少發生在成人,百分之九十八的病人發病時的年齡小於10歲。整體而言,這個疾病約佔兒童癌症的百分之六到八左右,但在兒童因癌症而死亡的案例中卻佔了約百分之十五。
神經母細胞瘤本身臨床變化多端,在不同的病童身上其表現可由近乎良性到極惡性。這個多樣性來自於腫瘤本身就有複雜的基因型別和病理組織的變化,而且這些變化和病人的預後也有相關。神經母細胞瘤是一個很好的腫瘤分化的範例,而臨床上使用藥物促進其腫瘤分化的治療,也已證明是神經母細胞瘤有效的治療方式。 之前文獻研究顯示,Notch訊息傳遞路徑可以影響神經母細胞瘤的腫瘤生成與細胞分化,但事實上其調控的分子機制並不清楚。在近幾年發現,當神經母細胞瘤細胞中的Notch訊息傳遞路徑被抑制後,細胞走向分化成熟時,不僅在型態學上表現神經突起的生長 (neurite outgrowth),細胞本身也表現出許多神經細胞分化成熟的標記蛋白,如GAP-43及calreticulin (CRT)。其中CRT這個分子,目前被發現與神經母細胞瘤的分化有極為密切的關係,CRT不僅可以促進神經母細胞瘤的細胞分化,甚至可以影響神經組織的發育。 因此,本研究的假說和目的就是要探討Notch訊息傳遞路徑的活性是否跟神經母細胞瘤病人的預後有關係,是不是神經母細胞瘤中的Notch訊息傳遞路徑的活性越高的,其腫瘤在病理上就較不分化,因此病人的預後比較不好。此外,我們以不同的神經母細胞瘤的細胞株,來探討Notch訊息傳遞路徑調控神經母細胞瘤分化的分子機制,其中是否藉由CRT或其他機制來影響整個神經母細胞瘤的分化成熟。最後,我們建立了神經母細胞瘤的老鼠模式,來研究當以γ-secretase inhibitor當作藥物,注射到老鼠體內,來阻斷老鼠身上神經母細胞瘤的Notch訊息傳遞路徑時,是否可以促進神經母細胞瘤的分化進而抑制腫瘤的生長,以確定Notch訊息傳遞路徑在神經母細胞瘤中可否可能是治療的標靶 (Therapeutic target),以及γ-secretase inhibitor可以當作治療神經母細胞瘤的新一代藥物。 我們的研究結果顯示,在我們85位神經母細胞瘤的病人中,有46位病人的Notch1染色結果為陽性 (54.1%),這些病人的腫瘤其病理組織的分化程度大部分都是不好的,統計起來,Notch1染色結果和病人神經母細胞瘤的分化程度是反比的 (P<0.001,chi-quare test)。Notch1的表現量與一些不好的臨床預後因子有相關性,神經母細胞瘤Notch1染色陽性多見於大於一歲以上發病的病患(P=0.004)、臨床高期別的病患(期別3、4) (P<0.001)、MYCN amplification的病患(P=0.001),以及CRT染色陰性的病患(P<0.001)。當我們以Kaplan-Meier存活分析來看這85位病人的預後時,發現Notch1染色陽性的病人其5年存活率只有28.5%,而Notch1染色陰性的病人其5年存活率則高達85% (P<0.001),Notch1染色陽性的病人其預後明顯較差。若在進一步以多變項方式分析,結果顯示Notch1染色陽性和臨床高期別(期別3、4)及MYCN amplification一樣,都是獨立的預後因子,也能夠預測病人較差的預後。另外在神經母細胞瘤細胞株的實驗中,我們也證實在Notch訊息傳遞路徑阻斷後所誘發細胞分化的分子機制,是先經由JNK的活化,再調控CRT的啟動子,使得CRT表現量增加後所造成的。最後,在神經母細胞瘤的老鼠模式中,我們發現注射Jia142 (γ-secretase inhibitor) 的那組老鼠,從第4天開始平均腫瘤大小跟注射DMSO組的老鼠比起來,有明顯的縮小 (p < 0.05 by student's t test)。到了第14天時,Jia142組的老鼠跟注射DMSO組的老鼠比起來,腫瘤平均小了48.9%,而注射retinoic acid組的老鼠跟注射DMSO組的老鼠比起來,腫瘤平均則是小了24.7%,若將注射Jia142組的老鼠和注射retinoic acid組的老鼠相比,腫瘤大小的變化也有統計上的差別 (p < 0.05 by student's t test)。 總之,在我們的這個研究中,我們證實了Notch 1蛋白的表現量在神經母細胞瘤的病人中是一個新的預後因子,可以預測病人較差的預後,它的效度是獨立的,而且不受其他預後因子的影響。另外,從細胞株的實驗中,我們知道神經母細胞瘤的細胞,在Notch訊息傳遞路徑阻斷後所誘發細胞分化的分子機制,是先經由JNK蛋白的活化,再調控CRT的啟動子,使得CRT表現量增加後所造成的,表示Notch-JNK-CRT訊息傳遞路徑可主導神經母細胞瘤的細胞分化過程。而在我們的神經母細胞瘤小鼠模式中,γ-secretase inhibitor的治療確實阻斷了Notch訊息傳遞路徑,並且跟給與DMSO的對照組小鼠比起來,神經母細胞瘤的腫瘤生長受到明顯的抑制,同時腫瘤組織也明顯走向分化成熟的方向。我們的研究結果顯示,γ-secretase inhibitor抑制神經母細胞瘤成長的效果,甚至比現有的口服維他命A酸 (13-cis retinoic acid) 還要有效。總結來說,我們的研究不僅證實Notch訊息傳遞路徑可以調控神經母細胞瘤的細胞分化,並清楚呈現Notch訊息傳遞路徑可以當作神經母細胞瘤新的治療標靶 (therapeutic target),而且γ-secretase inhibitor有可能做為神經母細胞瘤分化治療的新一代藥物。 Neuroblastoma (NB) is a childhood tumor derived from sympathoadrenal lineage of the neural crest progenitor cells. It is one of the most common pediatric cancers with an incidence of 8.0 per million per year, and 96% of cases occur before the age of 10 years. The disease is remarkable for its broad spectrum of clinical manifestations. The heterogeneity of NB tumors is largely due to the diverse biological characteristics that link to the prognosis of the patients. The differentiation status of tumor histology is also an important prognostic factor in NB, and differentiating therapy has been proven to improve the outcomes of NB patients. Notch signaling has been implicated to play a critical role in the tumorigenesis of neuroblastoma (NB), and can modulate calreticulin (CRT) expression that strongly correlates with tumor differentiation and favorable prognosis of NB. We thus sought to determine how Notch regulates CRT expression and affects NB tumor behavior, and the prognostic role of Notch signaling in patients with NB. We examined Notch1 protein expression in 85 NB tumors by immunohistochemical staining and correlated its expression levels with the clinicopathologic/biologic characters of the patients. The molecular mechanism underlying the Notch-dependent regulation of neuronal differentiation was determined by using cultured NB cells. The Notch-dependent regulation of CRT expression in cultured NB cells was analyzed by confocal microscopy and Western blotting. The effects of attenuated Notch signaling on NB tumor growth were examined by a xenograft mouse model of NB. NB tumors with elevated Notch1 protein expression were strongly correlated with advanced tumor stages, MYCN amplification, an undifferentiated histology, as well as a low CRT expression level. Using survival analysis, we demonstrated that patients with positive Notch1 expression on NB tumors exhibit a five-year overall survival rate of 28.5 % comparing to 85 % (P < 0.001, log-rank test) among those with negative Notch1 expression. Most importantly, the opposing effect between Notch1 and CRT could reciprocally affect NB patient survival. Consistently, high levels of Notch1 protein independently predicted poor prognosis of NB patients in multivariate analysis. We further demonstrated that CRT is essential for the neuronal differentiation of NB cells elicited by inhibition of Notch signaling. This effect was mediated by a JNK-dependent pathway. To substantiate the role of Notch signaling in the progression of NB, the administration of a γ-secretase inhibitor (GSI) into a xenograft mouse model of NB resulted in a significant suppression in NB tumor growth, concomitant with a prominent differentiation in xenograft tumors. Our findings provide the evidence for the first time that a JNK-CRT-dependent pathway is essential for the neuronal differentiation elicited by Notch signaling blockade and that Notch1 and CRT can synergistically predict the clinical outcomes of NB patients. The present data suggest that Notch signaling could be a therapeutic target for NB and that GSI could be the basis for the development of novel drugs for the treatment of NB. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/48621 |
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