| Summary: | 博士 === 國立臺灣大學 === 生命科學系 === 103 === 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.
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