Med19-cyclins信号通路在骨肉瘤增殖中的作用研究

Osteosarcoma (OS) is the most common primary malignant tumor of bone. Nearly 30–40% of OS patients have a poor prognosis despite multimodal treatments. Because the carcinogenesis of OS remains unclear, the identification of new oncogenes that control the tumorigenesis and progression of OS is crucial for developing new therapies. Recently, some studies have shown that Med19 plays an important role in promoting the growth of some malignancies.However, the role of Med19 in OS is still unclear. Here, we found that the expression of Med19 was increased in OS samples from patients compared to normal bone tissues. Cyclin D1 and cyclin B1 are upregulated in Med19 positive OS tissues. Importantly, among 97 OS patients of Enneking stage IIB or IIIB, Med19 expression was correlated with metastasis and poor prognosis. Med19 knockdown significantly induced growth inhibition, reduced colony-forming ability and suppressed migration in the OS cell lines Saos-2 and U2OS, along with the downregulated expression of cyclin D1 and cyclin B1. Med19 knockdown also induced apoptosis in Saos-2 cells via induction of caspase-3 and poly ADP-ribose polymerase (PARP).In addition, Med19 knockdown significantly suppressed tumor growth in an OS xenograft nude mouse model via suppression of cyclin D1 and cyclin B1. Simultaneously, Med19 down-regulation decreased the expression of Ki67 and proliferating cell nuclear antigen (PCNA) in tumor samples from OS xenograft nude mice. Med19 depletion remarkably reduced tumor metastasis in a model of OS metastatic spreading. Taken together, our previous data suggest that Med19 acts as an oncogene in OS via a possible cyclin D1/cyclin B1 modulation pathway.Meanwhile，protein chip assay demonstrated that knockdown of Med19 induced suppression of Akt/pAkt, which is a upstream modulator of cyclinD1. This indicates that Med19 might modulate expression of Cyclins through Akt and its downstream signal pathway.This project is designed to investigate and testify relationship between Med19 and Akt in tumorigenesis of OS in order to find out a potential target for osteosarcoma treatment.

骨肉瘤是最常见的原发性骨恶性肿瘤，恶性程度极高，预后差。由于辅助化疗的开展，骨肉瘤的预后有了较大改善。然而，仍有近40%的患者由于化疗耐药无法治愈，短期内死于复发转移。目前，临床上骨肉瘤主要的治疗药物为MTX、IFO、ADM、DDP，尚无标准的二线化疗方案，故探索新的骨肉瘤治疗靶点及相应的治疗药物对改善骨肉瘤患者的预后有重要意义。Med19在人类编码的基因名为LCMR1（肺癌转移相关蛋白），参与多种恶性肿瘤的形成。而目前对于Med19在骨肉瘤发生发展中的作用尚不明确；前期研究提示Med19在骨肉瘤的增殖、转移中发挥重要作用，这一机制可能涉及Cyclins表达的调控。蛋白芯片结果提示Med19可能通过调节Akt进而调控cyclins。 本项目将在前期基础上，验证Med19和Akt的上下游调控关系，进而揭示Med19调节Cyclins所涉及的关键信号分子，为骨肉瘤治疗提供潜在治疗靶点。

骨肉瘤（osteosarcoma,OS）是最常见的原发性骨恶性肿瘤，恶性程度高，预后差，过去十多年中，由于辅助化疗的开展，化疗联合手术的综合治疗模式使骨肉瘤患者的预后有了明显的改善。然而，仍有相当一部分患者在经过积极治疗后仍出现局部复发或远处转移，预后差，如何对这部分患者进行治疗，改善预后成为目前骨肉瘤临床诊疗的难点。靶向治疗是目前肿瘤治疗的热门领域，但骨肉瘤目前尚未发现满意的治疗靶点，故研究探索新的骨肉瘤治疗靶点，进而研制新的骨肉瘤治疗药物对于改善骨肉瘤患者的预后具有极其重要的意义。本项目组的前期研究发现Med19在骨肉瘤的增殖中发挥重要作用，有希望成为骨肉瘤的潜在治疗靶点，但其参与调控骨肉瘤的具体机制尚不明确。Microarray及IPA分析提示二酰甘油激酶 ζ（DGKZ）基因为Med19潜在的下游调控分子，该基因在真核生物的脑、骨、心脏和免疫系统中表达，并与肿瘤的发生发展密切相关，但在骨肉瘤中的作用尚不明确。通过深入研究，我们发现DGKZ基因在骨肉瘤细胞株中的表达明显高于正常成骨细胞株，在骨肉瘤组织中的表达亦明显高于正常骨组织，并与骨肉瘤患者的不良预后相关。之后通过慢病毒载体携带DGKZ的shRNA，证实在多株骨肉瘤细胞株中，下调DGKZ的表达可以明显抑制骨肉瘤细胞的增殖，促进其凋亡，这一抑制增殖的作用在裸鼠动物模型上亦得到证实。随后进行的Microarray及IPA分析揭示DKGZ调控骨肉瘤增殖的作用涉及多个MYC通路的关键分子表达（CCND1, CDKN2B,CDK6,PCNA，EGR1，ERK1/2），并得到了western blot及Co-ip的证实。本项目的研究首次揭示了DGKZ在骨肉瘤增殖中的重要作用，且初步揭示该作用涉及MYC这一明星信号通路的调控，为进一步阐明骨肉瘤的分子机制，开发新的靶向药物提供了潜在的靶点，鉴于MYC已证实在多种常见恶性肿瘤中发挥重要作用，本项目发现的这一对MYC通路的调控机制如能进一步阐明，对于其他常见恶性肿瘤的潜在靶点开发也有一定的意义。