摘要:惡性神經膠質瘤是最常見的原發性腦瘤,即使以手術、放射線治療、及化學治療等多方面的治療方法,惡性神經膠質瘤病人的預後還是不好,因此,充分了解神經膠質瘤的生成、生長及侵襲的病態生理及作用機轉,進而發展出有效的治療方法是必要的。TFG (tropomyosin-related kinase fused gene,TRK融合基因)蛋白質位於內質網(endoplasmic reticulum, ER)出口處,負責調節蛋白質的分泌及物質從ER釋出的功能,對於蛋白質在細胞內的運輸非常重要。此外,TFG屬於細胞大小的調控系統,與細胞凋亡及分裂的調控有關,當TFG與receptor tyrosine kinase (RTK)結合,會產生前向回饋機制(feed-forwardmechanism),刺激tyrosine kinase活性,造成腫瘤生成,而且,RTK及TFG二者都是細胞變形(transformation)所必須的。文獻上並無TFG在神經膠質瘤的角色的報告,但是有幾種融合蛋白質曾被發現在神經膠質瘤扮演重要角色,且亦有一些研究發現能與TFG結合的蛋白質(TFG fusion partners)與神經膠質瘤的惡性度有關,因此TFG及TFG-RTK融合蛋白質在神經膠質瘤的腫瘤生成可能扮演重要角色。在我們的初步研究發現三種人的及一種大鼠的神經膠質瘤細胞都表現TFG,且四種細胞的TFG免疫沉澱物中都有TFG融合蛋白質包括ALK (anaplastic lymphoma receptor tyrosine kinase)、TRKA(tropomyosin-related kinase A receptor)、TANK (tumor necrosis factor receptor-associated factor-associatedNF-kB activator)、VEGFR1 (vascular endothelial growth factor receptor 1)及NLK (NEMO-like kinase)等,但是在各個細胞株的TFG免疫沉澱物中的不同的TFG融合蛋白質的表現程度各不相同;此外只有二種細胞株有EGFR (epidermal growth factor receptor)的表現。以TFG siRNA抑制TFG的表現可將A172神經膠質瘤細胞的存活率降低至55.3 ± 3.9%,這表示TFG在神經膠質瘤細胞可能扮演重要角色。因此,在此三年計畫中,我們將探討TFG在神經膠質瘤的腫瘤生成的角色。首先,除了TFG在神經膠質瘤細胞的表現外,我們將研究四種細胞的TFG免疫沉澱物的RTK及各種TFG融合蛋白質的表現,經由這些實驗,我們將可知道神經膠質瘤細胞的最重要的TFG融合蛋白質,而這些蛋白質及TFG將會是後續實驗的重點。接著,我們將探討以TFG siRNA抑制TFG對於神經膠質瘤細胞的生物行為包括形狀、聚集(aggregation)、黏著(adhesion)、增殖分裂、凋亡、形成球體的活力(sphere-forming activity)、侵襲力等的影響。然後以array screening來研究與TFG相關的凋亡因子,並以western blot及real time PCR來確認。同時我們也將探討TFG相關的訊息通路、腫瘤抑制蛋白質(tumor suppressor protein)及腫瘤促進蛋白質(oncoprotein)的表現、及其上下游的通路。此外我們也將研究TFG過度表現(overexpression)對於神經膠質瘤細胞的生物行為的影響,探討同時抑制TFG及其融合蛋白質時,神經膠質瘤細胞的生物行為的改變,並與單獨抑制時的結果做比較。最後我們將進行動物實驗,探討以TFG siRNA抑制TFG對於神經膠質瘤細胞在大鼠皮下及腦部的腫瘤生成的影響,也將研究TFG及TFG融合蛋白質在腦瘤組織的表現。本計畫的結果將幫助我們了解TFG及TFG融合蛋白質在神經膠質瘤的腫瘤生成的角色,將會為神經膠質瘤的治療提供一新的途徑。
Abstract: Malignant glioma is the most common primary brain tumor. Patients with malignant gliomas have poorprognoses, even using multidisciplinary treatment strategies including surgery, radiotherapy, andchemotherapy, Therefore, understanding the pathophysiology and mechanisms of tumor formation, growthand invasion; and developing a better therapeutic strategy for malignant brain tumors are mandatory.TFG (tropomyosin-receptor kinase fused gene, TRK-fused gene) protein, a conserved regulator of proteinsecretion that localizes in the ER exit sites and controls the export of materials from the ER, is important forintracellular trafficking of protein secretion. It belongs to the cell-size-control systems, and is involved in theapoptotic- and cell-proliferation-regulatory mechanisms.When TFG is fused with the receptor tyrosinekinase (RTK), a feed-forward mechanism may occur to activate tyrosine kinase activity and lead tooncogenesis. Further, both the RTK portion and the TFG portion of the oncogenic-fusion proteins areessential for the transforming activity. The role of TFG in gliomas has not been reported in the literature;however, several fusion proteins have been identified to play a role in gliomas and there are reports revealingthe expression of fusion partners of TFG is correlated with the degree of malignancy of gliomas, whichsuggest that TFG and TFG-RTK fusion proteins might be important in the tumorigeneiss of gliomas. In ourpreliminary study, we found that TFG was noted to express in three human glioma cell lines and one ratglioma cell line. By immunoprecipitation (IP), these four cell lines showed ALK (anaplastic lymphomareceptor tyrosine kinase), TRKA (tropomyosin-related kinase A receptor), TANK (tumor necrosis factorreceptor-associated factor-associated NF-kB activator), VEGFR1 (vascular endothelial growth factor receptor1) and NLK (NEMO-like kinase) in the TFG IP products, although their expressions were variable in thesecell lines. Further, only two cell lines had EGFR (epidermal growth factor receptor) in the TFG IP products.In addition, the effect of the suppression of TFG by siRNA on the cell viability of the A172 glioma cells wasstudied.We found that the inhibition of TFG decreased the glioma cell survival to 55.3 ± 3.9%. The datasuggested TFG might play a role in gliomas. Thus, in this three-year project, we intend to study the role ofTFG in the tumorigenesis of gliomas. Firstly, we will investigate the expression of TFG protein in these 3human glioma cell lines and 1 rat glioma cell line. Then the expression of various fusion partners of TFG inthe immunoprecipitation products of TFG in the glioma cells will be explored (including the importanttyrosine kinase receptors in gliomas and the fusion partners of TFG noted in different kinds of cancer cells inthe literature). From these experiments, the prominent fusion partners of TFG in these glioma cells will beknown and will be the targets of subsequent experiments, in addition to TFG. Next, the effects of siRNAblockade of TFG on the biological behaviors including the morphology, aggregation, adhesion, proliferation,apoptosis, sphere-forming activity and invasion of the glioma cells will be studied. The possible TFG-relatedapoptotic factors in the glioma cells will be studied by array screening. The data from the array screening willbe further confirmed, and the TFG-related signaling pathways, tumor suppressor proteins andoncoproteins/growth factors will be investigated by western blot/real time polymerase chain reaction, etc.From these data, the upstream and downstream pathways will be further analyzed. Further, the effects of theoverexpression of TFG on the biological behaviors of the glioma cells will be explored too. In addition, theeffects of double blockade of TFG and its prominent fusion partners on the biological behaviors of the gliomacells will be studied, and the data will be compared with the effects induced by single blockade. Finally, wewill study the effects of siRNA blockade on the tumorigenesis of the glioma cells in the subcutaneous tissueand brain of the rats. In addition, the expression of TFG and TFG fusion partners in the tumor tissues will bestudied too. The results in this project will help us to understand the role of TFG and its fusion partners in thetumorigenesis of gliomas. It might open a new way to the treatment of gliomas.