RSK2催化SOX9调控软骨肉瘤恶性生物学行为的机制研究

Chondrosarcoma is a primary malignant bone tumor with high incidence rate. High malignance, fast growing, strong invasion,and early metastasis are the most characteristic features, but there is a lack of clear molecular mechanism and effective therapeutic targets about it. Our team find that an important protein RSK2 is highly expressed in chondrosarcoma. We have constructed ACAN loci-based chondrocytes RSK2 overexpression transgenic mice and find that RSK2 induce proliferation and bone destruction of chondrosarcoma. Moreover we find that RSK2 might bind and phosphorylate SOX9, and further regulate its protein stability and transcription activity. This process may be the key mechanism about generation and progression of chondrosarcoma. We plan to explore the specific sites where RSK2 phosphorylate SOX9 and the effect on chondrosarcoma cells after the phosphorylation of SOX9, which aims to clarify the mechanism of RSK2 mediating malignant biological behavior of chondrosarcoma by phosphorylating SOX9 in molecular and cellular levels. We also plan to explore the impact of RSK2 phosphorylating SOX9 in the generation and malignant behavior of human chondrosarcoma with animal models. In this study we can provide the new theory of the molecular mechanism in the generation and malignant behavior of human chondrosarcoma, and further provide new direction and medicine targets for the cure of chondrosarcoma.

软骨肉瘤是一种高发的原发恶性骨肿瘤，恶性度高、侵袭力强且易转移是其最显著的特点，目前尚缺乏明确的分子机制研究以及有效的治疗靶点。我们在前期研究中发现，一种重要的肿瘤调控蛋白RSK2在软骨肉瘤中高表达。通过构建基于ACAN基因座的软骨细胞高表达RSK2的转基因鼠，我们发现RSK2诱导软骨细胞过度增殖及骨破坏。同时我们发现RSK2可能通过催化软骨特异蛋白SOX9磷酸化来调节其稳定性及转录活性，这可能是软骨肉瘤发生及恶性转化的关键分子机制。本项目拟通过探索RSK2催化SOX9的磷酸化位点以及对软骨肉瘤肿瘤学行为的影响，从分子和细胞水平确定RSK2通过SOX9调控软骨肉瘤恶性生物学行为的过程；通过转基因模型在体实验，在动物水平明确RSK2调控SOX9对软骨肉瘤的发生、成瘤、转移的影响。本项目不仅可以为软骨肉瘤病因学研究提供重要的分子机制基础，还可以为软骨肉瘤的治疗提供新的理论依据及药物靶点。

背景：软骨肉瘤是最常见的恶性骨肿瘤之一，具有恶性程度高、生长迅速、侵袭性强、易发生肺转移等特点，可造成剧烈疼痛、病理性骨折、进行性神经功能障碍，导致患者瘫痪，甚至威及患者生命。目前软骨肉瘤发生及生物学行为的机制尚不清楚，这已成为软骨肉瘤基础研究与临床治疗的瓶颈。.研究内容与结果：我们总结并分析了98例脊柱软骨肉瘤患者的临床资料，我们发现脊柱软骨肉瘤整体预后较差，术后复发率、远处转移率、死亡率均较高。相关结果发表在肿瘤外科年鉴上（Ann Surg Oncol. 2014 Oct;21(11):3572-8）。.我们通过基因芯片分析发现RSK2在软骨肉瘤中表达升高，临床样本的qRT-PCR以及Western blot检测进一步发现软骨肉瘤中的RSK2表达量较对照组织升高了20倍。上调软骨肉瘤细胞中的RSK2表达，我们发现RSK2升高可显著增强软骨肉瘤细胞的增殖及侵袭能力。课题组利用免疫沉淀-酶解串联质谱检测联合蛋白质N-端测序技术、质谱分析发现RSK2可催化一种在软骨肉瘤高表达的因子SOX9的磷酸化并可使SOX9表达量明显增高。.我们利用TALEN技术构建了过表达RSK2的稳定细胞株（OE-RSK2），并证实OE-RSK2中RSK2的表达量较对照组提升了10倍。相对于对照组，OE-RSK2组软骨肉瘤细胞的增殖能力能力均显著增强。我们应用野生型、OE-RSK2构建软骨肉瘤小鼠模型，我们发现相对于野生型对照组软骨肉瘤模型，OE-RSK2组小鼠肿瘤体积大、骨破坏多、肺转移比例高。.结论：我们认为RSK2 作为一种促癌激酶在软骨肉瘤的发生中起到关键作用，其通过催化调控SOX9 实现对软骨肉瘤发生、成瘤、转移的影响。研究成果阐述了软骨肉瘤发生及恶性生物学行为的部分机制，为软骨肉瘤的治疗提供新的理论依据及药物靶点，对于改善软骨肉瘤患者的治疗以及预后具有积极的意义。