鈣網蛋白與血管內皮生長因子於神經母細胞瘤分化調控之研究

Abstract

神經母細胞瘤是幼兒期最常見的惡性腫瘤，其腫瘤形成的分子機制依然不清楚，有可能是胚胎交感神經系統發育異常使神經母細胞無法分化或無法凋亡而造成。我們過去的文獻回顧發現內質網伴護蛋白包括鈣網蛋白(Calreticulin, CRT)與葡萄糖調節蛋白(GRP)為神經系統的胚胎發過育過程所必需。我們的斑馬魚研究亦顯示CRT在神經系統的胚胎發育過程極具重要性。過去的研究發現CRT是神經母細胞瘤一項重要的預後指標，CRT的大量表現與神經母細胞瘤的分化程度呈正相關性，病人並有較佳的預後表現。因此，CRT也在神經母細胞瘤的分化上扮演了重要的角色。 血管內皮生長因子(VEGF-A)與其引導的血管新生現象亦被證實在神經母細胞瘤的形成具有重要的角色。而在胃癌研究發現CRT與VEGF-A具有相關性。因此，本研究希望藉由細胞實驗與動物實驗探討VEGF-A是否參與在CRT對於神經母細胞瘤的分化調控，並進一步探討VEGF-A對於人類神經母細胞瘤的分化調控與臨床意義。我們過去的研究結果發現在三種不同的神經母細胞瘤細胞株實驗，CRT的大量表現皆會正向調控VEGF-A與其上游調控分子缺氧誘導因子HIF-1a的表現量，並增加VEGF-A的蛋白質分泌。反之，利用shRNA抑制CRT的表現亦會造成VEGF-A及HIF-1a的表現下降。而在本研究中，我們進一步發現CRT的大量表現不會影響細胞凋亡，但會促進細胞分化並抑制細胞增生。此外，我們利用VEGF接受器抗體去抑制VEGF-A的作用，則神經母細胞瘤的神經分化指標包括GAP43、NSE、NFH及TrkA亦會受到抑制。以上結果顯示VEGF-A的確在CRT誘導的神經母細胞分化扮演重要角色。然而，我們利用神經母細胞瘤細胞株進行實驗發現CRT大量表現會促進細胞之神經分化無法增生，因此經由四環黴素誘導CRT表現之stNB-V1神經母細胞瘤細胞株來進行動物實驗。我們接著利用腫瘤異體移植實驗進一步證實CRT對於VEGF-A及HIF-1a的正向調控，實驗亦發現誘導CRT的表現可以顯著抑制腫瘤體積並促進腫瘤分化。除此之外，我們發現在病人腫瘤裡CRT的表現和VEGF-A的表現呈現顯著正相關。更重要的是，從病患的病理切片染色發現具有VEGF-A表現的病患其預後亦較佳。VEGF-A的表現與腫瘤的分化程度呈正相關，與MYCN的表現呈負相關，但與內皮血管形成無相關性，顯示VEGF-A可能透過與內皮血管新生無關之機轉來調控神經母細胞瘤的形成與分化。 總之，本研究證實在神經母細胞瘤中，CRT可正向調控VEGF-A表現並促進腫瘤的神經分化，且VEGF-A確實參與此神經分化調控。我們亦首次證實VEGF-A是神經母細胞瘤的一個獨立預後因子，具有VEGF-A表現的病患其預後亦較佳。我們的研究為神經母細胞瘤的腫瘤形成開啟嶄新的機轉，同時也有助於對神經母細胞瘤新治療的發展。

Neuroblastoma (NB) is the most common malignant tumor of infancy. The tumorigenesis of NB could be a divergence of the embryonic development of sympathetic nervous system. ER chaperones including calreticulin (CRT) and GRP78 are suggested to participate during embryonic development in our previous review. Our present study in zebrafish also revealed that CRT is essential for embryonic and neuronal development. Previous study has identified CRT as an independent favorable prognostic factor which is related to differentiated histologies in NB. Taken together, CRT could play an important role in neuronal differentiation of NB. Recently evidence has suggested that vascular endothelial growth factor (VEGF)-A, a key regulator of physiological and pathologic angiogenesis, participates in the behavior of NB. Furthermore, recent studies have found a correlation between CRT and VEGF-A in gastric cancers. In the present study, we aimed to determine whether the CRT expression in NB was associated with the VEGF-A pathway and to determine the role of VEGF-A in regulating NB behavior focusing on angiogenesis and neuronal differentiation in vitro and in vivo. Our previous study clearly demonstrated that in different NB cell lines, CRT over-expression increases the expression and secretion of VEGF-A and HIF-1a, a major positive regulator of VEGF-A. In contrast, knockdown of CRT decreases VEGF-A and HIF-1a expression. In the present study, we further demonstrated that NB cell apoptosis was not affected by CRT over-expression in stNB-V1 cells. Nevertheless, over-expression of CRT suppressed cell proliferation and enhanced cell differentiation in stNB-V1 cells, whereas blockage of VEGFR-1 markedly suppressed the expression of neuron specific markers including GAP43, NSE and NFH as well as TrkA, a molecular marker indicative of NB cell differentiation. These results indicate an essential role of VEGF-A in CRT-related neuronal differentiation in NB. However, constitutive over-expression of CRT led to NB cell differentiation without proliferation. Thus, we used an inducible-CRT stNB-V1 cell line by a tetracycline-regulated gene system for further animal experiments. The mice xenograft models further confirmed the positive regulation of CRT on VEGF-A and HIF-1a, as well as the role of CRT in enhancing neuronal differentiation and suppressing tumor growth in NB. Furthermore, we have demonstrated a significantly positive correlation between CRT and VEGF-A expression in human NB tumors. Most important of all, we verified that VEGF-A expression predicts a favorable outcome in NB patients and are associated with differentiated histology and normal MYCN expression, both of which are favorable prognostic factors. On the other hand, there was no correlation between the expression of VEGF-A and CD34, a marker of endothelial cells, suggesting a novel mechanism of VEGF-A participating in NB formation through angiogenesis-independent pathway. In conclusion, our study indicated that CRT-dependent VEGF-A up-regulation is critical for NB differentiation and VEGF-A is involved in CRT-related neuronal differentiation in NB. For the first time, we have demonstrated that VEGF-A is an independent prognostic factors and predicts favorable outcomes in NB patients with tight relationship with differentiated histology and MYCN status. Our findings also delineate a novel mechanism of VEGF-A in the biology of NB. This study provides important information that is needed for deciphering the crucial role of CRT and VEGF on the regulation of NB differentiation. Furthermore, our findings will shed light to a novel therapeutic strategy to improve the outcome of NB patients in the future.