HDGF促进Ewing肉瘤侵袭转移的作用及机制

Poor prognosis of Ewing sarcoma is associated with tumor metastasis. Therefore, it is of clinical relevance and urgency to explore the molecular mechanisms of the metastasis of Ewing sarcoma. Our previous study showed that HDGF was frequently up-regulated in Ewing sarcoma and exhibited oncogenic properties. Meanwhile, HDGF was significantly associated with overall survival rate and was an independent prognostic factor for patients with Ewing sarcoma [J Pathol. 2013]. Our preliminary study further indicated that HDGF promoted pro-metastatic phenotype of Ewing sarcoma cells in vitro, including invasion and migration. Furthermore, high HDGF expression was significantly associated with an increased incidence of distant metastasis and poor metastasis-free survival. Such effects might be achieved via up-regulation of a cohort of metastasis-promoting genes encoding proteins involved in the process of cell adhesion, cytoskeletal remodeling, and angiogenesis. In this project, we will further clarify the functions of HDGF in Ewing sarcoma progression by in vitro and in vivo assays. In addition, high-throughput genomic technologies such as the Chip-sequencing technique and gene expression profiling analysis will be used to identify HDGF targets which govern the process of invasion and metastasis in Ewing sarcoma. Finally, we will detect the expression of HDGF and its targets in clinical specimens and analyze whether they could be candidate metastatic markers for Ewing sarcoma. Together, this project will contribute to elucidate the mechanism of Ewing sarcoma metastasis and identify a novel therapeutic target for metastatic Ewing sarcoma.

转移是Ewing肉瘤患者预后差的关键因素，因此探寻Ewing肉瘤转移的核心机制是临床的迫切需求。我们前期研究发现：HDGF在Ewing肉瘤中呈高水平表达并发挥致瘤基因的作用，且与患者总生存率显著相关，是Ewing肉瘤患者的独立预后因子[J Pathol. 2013]。近期的预实验结果进一步发现：HDGF促进了Ewing肉瘤细胞体外侵袭及迁移并且与Ewing肉瘤患者的远处转移及无转移生存率低显著相关；上述作用可能是通过调控一组促转移基因而实现的。本项目拟采用Chip-sequencing、luciferase等技术深入探究HDGF调控Ewing肉瘤侵袭转移的关键靶基因和具体分子机制，并通过体外、体内实验及临床标本分析进一步探讨HDGF及其靶基因作为Ewing肉瘤转移标志物的可能性和临床应用前景。本项目将有助于阐明Ewing肉瘤的侵袭转移机制，并为Ewing肉瘤转移患者的靶向治疗提供新的思路。

Ewing肉瘤是好发于儿童及青少年的骨及软组织的高度侵袭性肿瘤，转移是Ewing肉瘤患者最不利的预后因素，然而，发生转移的分子机制迄今未明。我们前期研究发现：核转录因子HDGF在Ewing肉瘤中常常高表达，且是重要的致瘤基因。在本研究中，我们的数据显示，HDGF可促进Ewing肉瘤细胞体外转移相关性能的上调，包括诱导肌动蛋白细胞骨架重构和黏着斑形成，增加细胞与基质间的粘附能力，从而促进Ewing肉瘤细胞体外运动、侵袭及迁移；在实验性转移模型中，HDGF促进了Ewing肉瘤细胞的体内转移。为探索HDGF促进Ewing肉瘤细胞侵袭及转移的潜在机制，我们使用Chip-sequencing技术于全基因组范围内探寻HDGF的结合位点，并分析人Ewing肉瘤细胞中HDGF表达敲低后的基因表达谱，对两组数据进行整合、比对，从而鉴定HDGF的靶基因及下游信号网络。我们发现，HDGF调控了一组转移相关基因，这些基因或参与细胞骨架重构，或参与黏着斑形成，使用Real-time PCR技术验证HDGF基因敲除后，这些转移相关基因表达水平的变化，其中，ALCAM的表达变化最为显著，当HDGF基因敲除后，ALCAM基因的表达水平约上调了4倍，Chip-qPCR检测结果进一步证实HDGF靶向结合ALCAM基因的启动子区域，而敲低ALCAM基因表达的Ewing肉瘤细胞则呈现出更多间充质细胞来源的特征—形成更多的黏着斑和肌动蛋白应力纤维，并具有更强的基质粘附能力，因而，ALCAM可能是HDGF下游关键的效应分子，介导了HDGF对Ewing肉瘤细胞侵袭及转移过程的调控。此外，在临床标本中，HDGF核内高表达的Ewing肉瘤患者，其远处转移发生率显著增高，无转移生存时间显著缩短。因而，我们的研究结果强有力地支持HDGF有作为Ewing肉瘤转移标记物和预后因子的潜在应用前景，并可为Ewing肉瘤转移患者的靶向治疗提供新的思路。