Osterix在乳腺癌血管生成中的作用及分子机制研究

Osterix (Osx) is a key transcription factor required for osteoblast differentiation and bone formation. Our previous studies have revealed that Osx was significantly over-expressed in primary breast cancer tissues and metastatic breast cancer cells. Moreover, overexpression of Osx increased the expression of S100A4, an important factor involved in angiogenesis, and down-regulation of Osx led to a decrease in S100A4 protein level in breast cancer cells by mass spectrometric screening. That S100A4 is a downstream gene of Osx was further confirmed by Real-time PCR and Western blot assay. Besides, bioinformatics analyses indicated that there are five potential binding sites of Osx in the promoter of S100A4. Based on our findings and data from the literatures, we postulate that Osx induces angiogenesis, and thus promotes metastasis by regulating S100A4 expression in breast cancer. In this project, we will test this hypothesis using cell lines and mouse models as well as clinical investigations. In addition, the molecular mechanism by which Osx regulates S100A4 will be explored by using dual-luciferase reporter assay, electrophoretic mobility shift assay (EMSA), etc. This project will not only provide new insight into the role of transcription factor Osx, but also suggest its potential as a new target for the clinical intervention in patients with breast cancer.

Osterix（Osx）是成骨细胞分化和骨形成中的关键转录因子。我们前期研究发现Osx在乳腺癌组织和高转移潜能的乳腺癌细胞中显著高表达。通过质谱筛选，我们发现乳腺癌细胞中过表达Osx后，促血管生成因子S100A4蛋白表达水平升高；下调Osx的表达后，S100A4蛋白表达水平随之降低。Real-time PCR和Western blot检测结果进一步证实S100A4表达受Osx的调控。生物信息学分析结果显示S100A4基因的启动子区有5个潜在的Osx结合位点。由此我们推测：Osx通过调控S100A4诱导乳腺癌血管生成，促进乳腺癌的转移。本项目拟通过细胞和动物水平实验以及临床标本检测对此假说进行验证；并运用荧光素酶报告基因实验、凝胶迁移实验等阐明Osx调控S100A4的分子机制。本项目研究结果不仅可以丰富对Osx这一重要转录因子生物学功能的认识，而且可以为临床乳腺癌的预防和治疗提供新的靶点。

淋巴结与血管转移是乳腺癌患者死亡的主要原因。1971年Folkman首次提出实体瘤的生长和转移依赖于新生血管，并预言切断肿瘤血供可对肿瘤进行治疗。Osterix（Osx）是成骨细胞分化和骨形成中必需的转录因子。本研究中，我们发现Osx在乳腺癌的迁移和血管生成中发挥着重要作用。首先，我们通过体内、体外实验证实Osx通过上调S100A4的表达促进乳腺癌的迁移和血管生成，我们还进一步提出S100A4可能部分通过调节CD44和VEGF的表达水平调控细胞的迁移和血管生成。其次，我们利用乳腺癌原位模型对Osx调控S100A4介导乳腺癌迁移和血管生成进行验证。最后，我们通过临床标本检测证实乳腺癌组织中Osx与S100A4的表达呈正相关。总之，我们的数据表明Osx通过上调S100A4介导乳腺癌的迁移和血管生成。.大约70%的乳腺癌晚期病人会发生骨转移。然而，人们对乳腺癌骨转移的分子机制还没有全面深入的了解。在发现Osx在乳腺癌迁移和血管生成中的重要作用的同时，我们还发现Osx在乳腺癌的骨转移中发挥着重要作用。我们通过体内、体外实验证实Osx通过上调MMP9的表达促进乳腺癌细胞的侵袭和转移。通过荧光素酶报告基因实验、EMSA实验，我们证实了MMP9为Osx的直接靶基因。重要的是，我们通过胫骨注射模型，在小鼠体内观察到Osx过表达促进乳腺癌溶骨性骨转移，敲低Osx表达则减轻溶骨性骨转移。Osx过表达组裸鼠胫骨中破骨细胞活性增强，破骨细胞活化促进因子IL-8、PTHrP表达水平明显升高；而敲低Osx组裸鼠胫骨中破骨细胞活性减弱，IL-8、PTHrP表达水平明显降低。总之，我们的数据表明Osx通过上调包括MMP9在内的多个关键因子的表达促进乳腺癌骨转移的发生。综上所述，我们前期的研究结果提示Osx可能是临床上乳腺癌治疗的有效靶点。