去泛素化酶USP4调节骨微环境在肺癌骨转移中的作用与机制研究

Bone metastasis from lung cancer possesses a high incidence, and often results in serious impair to health. Until now, the mechanism about formation of bone metastasis from lung cancer remains unclear, and there is no specific molecular targeting the process of bone metastasis formation. According to recent studies, the regulation of microenvironment is an important step in the process of tumor metastasis. Bone metastasis of lung cancer is closely correlated to the regulation of bone microenvironment, and the regulatory mechanism may be a key to reveal the mechanism of bone metastasis from lung cancer. The applicant has successfully established a mouse model with highly bone metastasis from lung cancer previously. Microarray analysis demonstrated that the expression level of deubiquitinating enzyme USP4 was significantly up-regulated in osteoblasts after co-cultured with a highly metastatic lung cancer cell line which was obtained from the above mouse model, and this up-regulation tendency was then proved by Western blot and qPCR test. Furthermore, the study on the cell differentiation model of USP4 gene knockout mice in vivo and in vitro indicated that USP4 can influence bone microenvironment remodeling by regulating differentiation of osteoblasts and osteoclast. These above results suggest that bone microenvironment regulation by USP4 may play an important role in the formation of bone metastasis from lung cancer. The present project intends to a further study in the levels of animal model in vivo, cellular in vitro and clinical data, aiming to illustrate the effect of USP4 regulating bone microenvironment in the process of lung cancer cells metastasizing to bone, and uncover the regulation molecular mechanism, and then interpret the mechanism of bone metastasis of lung cancer from a new perspective, which will benefit to finding specific molecular for diagnosis and target therapy on bone microenvironment.

肺癌骨转移具有高发病率与严重危害，其发生机制未明，尚缺乏针对骨转移的分子诊治靶点。近期研究显示，微环境调节是肿瘤转移的重要环节，肺癌易于发生骨转移与骨微环境调节密切关联，其调控机理很可能是揭示肺癌骨转移发生机制的关键突破点。申请人前期成功建立肺癌骨高转移小鼠模型，利用亲骨转移的肺癌细胞系与骨细胞共培养，Microarray检测发现去泛素化酶USP4表达在成骨细胞中明显上调，并获得Western和qPCR证实；应用USP4基因敲除小鼠体内和体外细胞分化模型研究发现，USP4可通过调控成骨细胞和破骨细胞分化影响骨微环境重塑。上述结果提示USP4调控骨微环境很可能在肺癌骨转移中发挥重要作用。本项目拟在临床样本、细胞分子以及动物模型水平进一步研究，以阐明USP4调控骨微环境在肺癌骨转移中的作用，揭示其分子机理，从新的角度诠释肺癌骨转移发生机制，为肺癌骨转移分子诊断和靶向药物研发提供潜在靶点。

本项目研究阐明USP4如何调节肺癌骨转移过程中骨微环境破骨细胞与成骨细胞功能及其具体分子机制与临床意义，为临床肺癌骨转移靶向治疗提供理论依据，发现潜在的新分子靶标，为抗肿瘤药物研发提供新的线索。本研究完成预期理论成果，我们发现USP4是体外和体内骨量的负调控因子，通过BMP-Smad4介导的转录抑制，USP4在成骨细胞中的表达下调。机制上，USP4通过与Smurf1直接相互作用，诱导Smurf1去泛素化并稳定成骨细胞活性。此外，药物抑制USP4的去泛素酶活性可显著降低Smurf1蛋白水平，提高体外成骨细胞活性。研究结果证明了USP4在骨稳态中的重要作用，这表明抑制USP4可能是一种潜在的治疗骨微环境紊乱治疗方法。