DAB2IP调节雄激素-AR 信号通路在去势抵抗型前列腺癌进展中的作用研究

Castration resistant prostate cancer (CRPC) is a lethal disease with fewer available therapeutic options and results in mortality of patients. Clinical and experimental data suggest that androgen/androgen receptor (AR) signaling becomes re-activated by a castration-resistant mechanism after medical or surgical castration. Increased local intracrine synthesis of androgens and enhanced AR activities facilitate the AR signaling activation in CRPC despite low serum testosterone levels. DAB2IP is a novel RAS-GTPase activating protein capable of maintaining prostate epithelial cell homeostasis through its different functional motifs, and its loss promotes prostate cancer (PCa) cell proliferation, survival, metastasis and radioresistance. In our recent study, we have identified DAB2IP as a novel AR antagonist which could inhibit AR phosphorylation, translocation and transcription activity induced by dihydrotestosterone treatment. We also observed that it could abrogate AR transcrption activity or the expression of several target genes (i.e., prostate-specific antigen, PSA) in PCa cells after androgen ablation, however, the underlying mechanisms have not been elucidated. Here, we suspect that DAB2IP loss could potentiate the activation of signal transducer and activator of transcription 3 (STAT3) signaling pathway, which may initiate the expression of several steroidogenic enzymes and subsequently increase the intratumoral de novo steroid synthesis. In this study, we plan to utilize two PCa cell lines (androgen-independent C4-2 and androgen-dependent LAPC-4) stably manipulated with different DAB2IP expression levels and their xenografts to investigate the difference of cell proliferation, colony formation, subcutaneous tumor growth, TMPRSS2 or PSA expression, and intracellular or intratumoral testosterone levels after androgen ablation in vitro and in vivo. Mechanically, we will apply Affymetrix cDNA microarray to screen out the potential steroidogenic enzymes (i.e., AKR1C3) regulated by DAB2IP in these two cell models. After that, we plan to use gain of function or knock-down strategies to show how DAB2IP regulates the STAT3 signaling activation, AKR1C3 expression, androgen metabolism, castration-resistant tumor growth and PSA expression in vitro and in vivo. In addition, we will use DAB2IP knock-out or wild-type mice (DAB2IP-/- or DAB2IP+/+) and CRPC tissue or serum samples as study models to confirm the phenomenon and mechanisms described above. Taken together, this study will provide a novel mechanism that loss of a tumor suppressor gene, such as DAB2IP, drives or enhances intratumoral de novo androgen synthesis leading to CRPC progression following castration.

去势抵抗型前列腺癌（CRPC）已成为威胁我国老年男性健康的重要疾病，雄激素-AR信号通路的再激活是CRPC发生、发展的重要机制，其中PCa细胞内新的雄激素合成和AR活性增强是关键调节因素。DAB2IP是新近发现的维系前列腺上皮内环境稳定的重要基因，我们前期研究证实：DAB2IP可抑制CRPC细胞AR本身的活性，同样会影响去势条件下PCa雄激素靶基因的表达。经过预实验，我们推测可能与DAB2IP缺失后PCa细胞内雄激素代谢酶谱和代谢途径的改变有关。本项目拟利用PCa细胞或移植瘤、DAB2IP基因敲除小鼠和CRPC临床标本等多种研究模型，以受STAT3调控、调节睾酮合成的重要代谢酶AKR1C3为切入点，应用现代分子生物学的基因转染或shRNA 封闭技术，探讨DAB2IP通过STAT3调节AKR1C3的表达，影响PCa雄激素代谢和去势后的抵抗性生长，为揭示CRPC新的分子机制奠定理论和实验基础。

去势抵抗型前列腺癌（CRPC）生物学行为复杂，本项目主要研究新型抑癌基因DAB2IP在CRPC进展中的作用，特别是其对雄激素-雄激素受体（AR）信号通路的调控机制。先后发现：一、DAB2IP通过双重机制影响CRPC中多种可能途径激活的AR功能。实验结果：1.DAB2IP通过其PR结构域结合Src并抑制其磷酸化，继而抑制雄激素诱导的AR非基因组信号，影响PCa细胞体外增殖。2.DAB2IP通过PP2A抑制多种途径介导的AR靶基因表达、AR磷酸化和核转位。3.DAB2IP-/-小鼠前列腺不良增生，AR活性增加；DAB2IP在高级别PCa标本中表达降低，与AR胞核染色负相关。二、DAB2IP缺失诱导Egr1/Clusterin过表达参与CRPC后Docetaxel耐药表型的获得。实验结果：1.DAB2IP改变多种PCa细胞对Docetaxel等化疗药物敏感性。2.DAB2IP直接调控CRPC关键耐药基因Clusterin的转录。3.DAB2IP干扰Wnt/β-catenin和IGF-I/IGF-IR信号通路对话直接调节Egr-1和Clusterin表达。4.CRPC标本中DAB2IP染色降低，与Egr-1胞核染色、Clusterin胞浆染色负相关。三、DAB2IP缺失异常活化AR旁路信号参与CRPC去势抵抗表型的获得。实验结果：1.DAB2IP调控PCa细胞在去势后的体外生长、克隆形成和皮下移植瘤形成。2.DAB2IP调控PCa细胞在去势后内源性凋亡。3.DAB2IP通过PR结构域与STAT3直接结合，抑制其磷酸化和转录活性，继而调节Survivin和Bcl-2等基因表达。4.DAB2IP-/-小鼠前列腺STAT3胞核定位和Survivin表达增高，去势后细胞凋亡减少。5.人PCa移植瘤及CRPC标本中DAB2IP表达降低，STAT3胞核定位及Survivin表达增高。四、DAB2IP缺失可增加PCa细胞内雄激素新生限速酶AKR1C3表达，继而调控肿瘤内雄激素合成和AR剪接突变体稳定性。实验结果：1.DAB2IP直接影响包括AKR1C3在内多种雄激素新生限速酶基因的表达，继而维系肿瘤细胞内睾酮和DHT水平。2.ARK1C3与AR剪接突变体直接结合并影响其蛋白稳定性，从而参与维系AR本身信号通路的持续活化。这些研究结果将为揭示CRPC复杂的分子机制提供实验依据。