摘要:神經母細胞瘤(NB)是源自於胚胎時期交感神經系統神經脊細胞的腫瘤,其腫瘤形成的機制可能是胚胎神經母細胞無法分化或凋亡。神經生長因子 (Nerve growth factor, NGF)在胚胎發育過程調控交感神經細胞分化或凋亡最重要的因子。過去的研究發現 NGF在NB的分化扮演必要的角色。血管內皮生長因子(VEGF)亦被證實在NB的形成扮演重要角色,但過去研究多著眼其於血管生成的影響。然而,我們過去的研究發現鈣網蛋白(Calreticulin, CRT)會正向調控VEGF並促進NB的細胞分化。過去兩年我們在科技部計畫的支持下,發現無論在NB的細胞株、異體移植腫瘤與病人的腫瘤,VEGF與CRT的表現量及腫瘤的分化程度皆呈顯著正相關,但與血管的生成無相關性。我們亦發現若VEGF在病人腫瘤的表現量較高,病人的預後較佳。我們的研究首次支持VEGF在NB的分化扮演著重要角色。因此我們推論過去建議在NB所使用的抗VEGF藥劑治療或許不恰當。NGF在正常神經細胞會調控VEGF,我們亦推論VEGF應在NGF調控NB分化的過程扮演重要角色。最近研究亦發現NGF的訊號調控參與了13-cis-retinoic acid (13cRA)治療NB的機轉,因此,VEGF亦可能參與其中。我們在目前進行的科技部計畫中發現VEGF的確參與NGF調控NB細胞的分化,我們亦發現VEGF會直接造成NB細胞的分化,我們並建立了數種抑制CRT表現的NB細胞(不同的惡性度),以著手進一步的實驗。未來這三年計畫,我們將在惡性度不同的NB細胞株與動物實驗探討VEGF對於NB的神經分化調控及其與NGF和13cRA分化治療的關聯性。首先,我們將進一步探討NGF對於VEGF蛋白表現量、分泌量與神經分化表現在不同細胞株的影響。我們並藉由異體移植來分析NGF刺激與VEGF、血管生成、腫瘤分化之間的關聯。在第二年的計劃中,我們將探討CRT是否參與在此調控分化的過程中。我們並接著探討VEGF是否參與13cRA分化治療NB 細胞的機轉。在第三年的計劃中,我們將進行13cRA治療和抗VEGF治療在動物腫瘤的影響與療效。此外,我們這一年的實驗顯示VEGF會直接造成NB細胞的分化,我們將以RNA定序與次世代基因檢測在此過程中的相關基因變化,以找出VEGF在NB分化的可能機轉。本計劃的完成將有助於NB新治療的發展。
Abstract: Neuroblastoma (NB) is derived from the sympatho-adrenal lineage of embryonic neural crest cells with proposed pathogenesis due to failure of differentiation or apoptosis of precursor cells. The nerve growth factor (NGF) and its receptor, TrkA, constitute the most crucial signaling to control the differentiation and apoptosis of sympathetic neurons during development, as well as the tumorigenesis of NB. Recent evidence also suggested crucial roles of vascular endothelial growth factor (VEGF) in the behavior of NB while most previous studies focused on its role in tumor angiogenesis. Recent studies conducted clinical trials of Bevacizumab, an anti-VEGF antibody, in NB patients and the results showed no effects. However, our past works demonstrated involvement of VEGF in calreticulin (CRT), a molecular chaperon found to be essential for NB biology, -related neuronal differentiation in NB cells. In the past 2 years, under the grant support of MOST, we found positive correlation of VEGF expression with CRT expression and differentiated status in human NB tumors, NB mice xenografts and NB cells, while VEGF expression predicted a favorable prognosis in NB patients. Our work, for the first time, demonstrated that VEGF may affect NB behavior by playing roles in neuronal differentiation rather than angiogenesis. Previously suggested anti-VEGF agents may be inappropriate in NB and need further investigation. Because NGF can regulate VEGF in normal neurons, VEGF may also participate in NGF-related NB differentiation. Besides, recent studies found involvement of NGF/TrkA signaling in 13-cis-retinoic acid differentiation therapy of NB cells, suggesting VEGF may play important roles in retinoic acid-induced NB differentiation. In my MOST proposal this year, we have demonstrated that VEGF is upregulated in NGF-stimulated differentiation in NB cells and blockage of VEGF signaling suppresses the expression of neuronal differentiation. We also found VEGF can directly induce neuronal differentiation in NB cells. Furthermore, we have established several CRT-knockdown NB cells with different degree of malignancy for our further experiments. In the forthcoming 3-year proposal, we would like to investigate the role of VEGF in governing neuronal differentiation of NB focusing on its relationship with NGF signaling and differentiation therapy in vitro and in vivo. In the first year, we want to investigate VEGF expression in NGF-mediated differentiation in NB cells with different degree of malignancy. We will establish mice xenograft models for investigating NGF effects +/- anti-VEGF therapy on VEGF expression, angiogenesis and neuronal differentiation. In the second year, we will examine if VEGF mediates NGF-related differentiation of NB through CRT-dependent pathway using our CRT-knockdown NB cells. Besides, we will investigate the role of VEGF in 13-cis-retinoic acid therapy in NB cells. In the third year, we will use mice xenograft models to investigate the role of VEGF in 13-cis-retinoid acid therapy and the effects of combined anti-VEGF therapies. Because we have demonstrated VEGF can directly induce NB cells differentiation, we will investigate underlying mechanisms by studying the associated gene changes for further pathway studies. Altogether, our present studies would provide important information for developing a new therapeutic strategy to improve the outcome of NB patients.