DACH1通过调控HIF-1α信号通路抑制肾癌血管生成的作用及机制研究

Highly active tumor angiogenesis is a characteristic manifestation of renal cell carcinoma(RCC), which contributing to tumor metastasis and treatment resistance. Despite advances in anti-angiogenic therapy, the efficiency of current treatment remains transient and insufficient requiring more in-depth study of mechanisms and new effective targets..Dachshund is a key component of the Retinal Determination Gene Network (RDGN) governing the normal initiation of the morphogenetic furrow. Recent studies have demonstrated an important role for human dachshund (DACH1)in tumorigenesis, in particular, breast, prostate and ovarian cancer. However, the exact role of DACH1 in RCC has not been reported. Our most recently studies have found that the immunohistochemical staining of DACH1 was either negative or weak in cancer tissues of RCC. Ectopic expression of DACH1 in RCC cell lines CAKI and ACHN inhibited DNA synthesis, S phase progression and colony formation, which prompted that DACH1 may be the key regulator of the development of RCC. More importantly,we discovered a clear tumor boundary with very few vessels in DACH1 expression group ,whereas an unclear boundary with a more intensive blood vessels in control group, suggesting DACH1 may inhibit tumor angiogenesis. Luciferase reporter gene assay indicated that DACH1 suppressed transcriptional activity of HIF-1α. All of the above results suggest that DACH1 may regulate angiogenesis process through HIF-1α signaling pathway in RCC but the underlying molecular and cellular mechanism is still elusive..This project intends to test the hypothesis that DACH1 suppresses angiogenesis and malignant phenotype via inhibition of HIF-1α signaling in RCC. To this end, the relationship between DACH1 and HIF-1α, as well as other related markers and clinicopathologic features in RCC patients will be evaluated. Moreover, the changes of HIF-1α and some key molecules of angiogenesis will be measured after DACH1 intervention in RCC cells. The migration and tube formation of endothelial cell will be tested after stimulation by the conditioned medium derived from RCC cells exposed to DACH1 intervention using a co-culture system. Furthermore, subcutaneous mouse model will be utilized to evaluate the results ulteriorly in vivo. Through the implementation of this project, we will reveal the role of DACH1 in the regulation of HIF-1α signaling and angiogenesis process in RCC, as well as the the possible underlying mechanisms, providing a theoretic and experimental basis for development of novel targeted therapies in RCC.

肿瘤血管生成活跃是肾癌的标志性特征，抗血管生成是目前肾癌分子靶向治疗的主要靶点。申请者前期研究发现，在肾癌标本中细胞命运决定因子DACH1表达明显下降，裸鼠移植瘤实验发现DACH1表达组肿瘤血管生成受抑，基因表达数据分析显示DACH1与血管生成标志的表达成反比，进一步荧光素酶报告基因结果提示DACH1抑制缺氧诱导因子HIF-1α的转录活性。结合HIF-1α信号通路是调节肿瘤血管生成的核心通路之一，我们推测DACH1可能通过HIF-1α信号途径调控肾癌血管生成。DACH1表达下降与多种肿瘤的发生发展有关，但其在肿瘤血管生成中的作用尚未见相关报道。本项目拟利用肾癌细胞系、裸鼠移植瘤模型和肾癌患者标本，采用免疫组织化学、RNA干扰、荧光素酶报告基因、抗体芯片等技术和方法，深入研究DACH1通过调控HIF-1α信号通路抑制肾癌血管生成的作用和分子机制，为肾癌靶向治疗提供新的靶点和实验依据。

抗血管生成是目前肾癌分子靶向治疗的主要靶点。申请者前期的研究发现，DACH1对肾癌细胞恶性表型的调控发挥关键作用，并提示HIF-1通路可能参与介导DACH1对肿瘤血管生成的调控作用。本课题探讨DACH1调控HIF-1α抑制肾癌血管生成和恶性表型的作用和分子机制。73例肾癌标本DACH与HIF-1α表达负相关，DACH1高表达者生存明显优于低表达者，DACH1低表达而HIF1α高表达者预后最差。组织分级、DACH1和HIF-1α表达情况均为肾癌的独立预后因素。体外研究中，DACH1高表达组HIF-1α的mRNA表达下降，血管标记PFKB1、Ang2和VEGF的mRNA表达也显著下调，其中，调控血管生成的下游关键因子VEGFA的表达在蛋白和mRNA水平均有下降。上调HIF-1α表达后，DACH1对血管生成调控作用显著受抑。荧光素酶报告基因结果显示DACH1抑制HIF-1α的转录活性。DACH1抑制肾癌细胞增殖、凋亡和迁移，上调HIF-1α，DACH1抑制细胞增殖作用显著减弱。裸鼠皮下移植瘤模型显示DACH1表达抑制肿瘤生长。通过本项研究我们进一步明确了DACH1与HIF-1α在肾癌组织中的表达以及与预后的关系，表明了DACH1和HIF-1α对肾癌恶性表型和血管生成的重要调控作用，为设计以DACH1和HIF-1α信号通路为靶点的治疗策略提供理论基础和实验依据。