ARL1(ADP核糖基化因子相似蛋白1)在骨肉瘤中的作用及其机制研究

The superfamily of Ras small GTPases serves as a signal transducer to regulate a diverse array of cellular processes, including cell cycle progression, differentiation, cell polarity and movement. The ectopic expression or mutations frequently comes along with tumorigenesis and tumor progression. Therefore, it is extremely important to investigate the role of Ras superfamily members in cancer research. In order to rule this out, we established a scaled Ras small GTPases screening system which is based on CRISPR-Cas9 knockout library. Some novel candidates, as well as the Ras proteins with known functions in tumor growth, have been investigated in our screening, and ARL1 is one of the candidates with our interests. As shown in our previous data, the ARL1 knockdown tumor cells are along with decreased cell viability and increased migration and invasion ability. In the other hand, we applied the protein microarray, basically targeting the signaling pathway proteins, to screen the effectors of ARL1 by comparing the knockdown cells with control cells. The results are currently being analyzed, and appropriate signaling pathways or proteins will be the candidates that we are going to work on to investigate the molecular mechanism. In this project, we are taking advantage of osteosarcoma as a model to study the function and molecular mechanism of ARL1 in tumor growth and metastasis, both in cellular level and animal models. Besides this, we plan to investigate the relativity of ARL1 and its effectors in clinical samples. The accomplishment of this project will reveal the functions and mechanisms of ARL1 in growth and metastasis of osteosarcoma, and shed a light on its diagnoses and treatments.

Ras超家族是一类小GTPase蛋白，参与调控细胞增殖、分化、运动等生理功能，其异常表达或突变是多种肿瘤中的常见事件。因而很多Ras超家族成员的作用及其调控在肿瘤中尤为重要。最近我们团队构建了针对Ras超家族所有成员的CRISPR-Cas9基因敲除文库，并成功筛选出了一些新的调控肿瘤生物行为的Ras超家族成员，ARL1（ADP核糖基化因子相似蛋白1）就是其中之一。预实验表明：肿瘤细胞中敲低ARL1，可降低其细胞活力，增强迁移和侵袭能力。同时，对这些敲低ARL1的肿瘤细胞进行了蛋白质芯片（主要信号通路蛋白的磷酸化水平）检测，结果正在分析中。本项目拟以骨肉瘤为模型，在细胞水平和动物模型中明确ARL1在肿瘤生长、转移中的作用及机制；在骨肉瘤临床标本中分析ARL1及其相关机制分子之间的相关性、临床意义。本项目的完成将阐明ARL1调控骨肉瘤生长、转移的分子机制，可能为骨肉瘤的综合诊治提供新策略。

Ras超家族成员是一类小GTPase蛋白（20-25kDa），其参与调控细胞增殖及存活、细胞骨架、细胞极性及运动、囊泡运输等过程。Ras超家族成员的异位表达及突变是肿瘤中的常见事件，因而Ras超家族成员在肿瘤中的作用及其调控研究尤为重要。为此，我们建立了一套基于CRISPR-Cas9敲除技术并靶向Ras超家族所有成员的文库，系统研究了Ras超家族成员对骨肉瘤细胞活力的作用。通过筛选，我们得到了一批新的调控肿瘤生物行为的Ras超家族成员，ARL1就是其中之一。在本课题研究中，我们报道了ARL1可促进骨肉瘤等肿瘤细胞增殖及克隆形成能力。与此同时，ARL1可抑制骨肉瘤细胞迁移及侵袭过程。通过分析临床数据，我们发现在骨肉瘤肿瘤标本中，ARL1高表达与肿瘤转移及不良预后正相关，可作为预后标志物。此外，通过转录组测序，我们发现PCOLCE在骨肉瘤中高表达，PCOLCE可促进肿瘤转移，其受到TWIST1正向转录调控。本课题首次发现了ARL1、PCOLCE等在骨肉瘤中的功能及临床意义，并提示该分子可作为不良预后标志物。我们预计靶向ARL1,PCOLCE等分子，将有可能为抑制骨肉瘤转移提供新的思路。