新因子hARAP3在AR介导基因转录调控及前列腺癌中的作用及机制

AR is a member of nuclear receptor superfamily and induces target gene transcription in a ligand-dependent manner. In this process, AR recruits a series of co-regulators to modulate AR-mediated transactivation and exerts its biological functions. AR and its co-regulators individually play important roles in Androgen dependent (ADPCa) and Androgen independent/castration resistant prostate cancers (AIPCa/CRPC). By AR-position effect variegation (AR-PEV) Drosophila experimental model, we selected a series of new co-regulators involved in AR-mediated transactivation and named as of AR associated proteins (ARAPs). These co-regulators include DNA damage checkpoint factors (ARAP1 and ARAP2), Ser/Thr protein kinase (ARAP3), RNA binding protein (ARAP4) and a factor with unknown function. Based on the preliminary experiments, we selected one of them (ARAP3) for further study in this grant. Both ARAP3 and its human homologue (hARAP3) belong to casein kinase 1 (CK1) protein family and contain S/T Kc domain, which phosphorylates many kinds of proteins. In the previous studies, we examined hARAP3 significantly enhanced AR-mediated transactivation. We also found that sihARAP3 decreased the level of histone H3S10 phosphorylation (H3S10ph) and H3K9 acetylation (H3K9Ac). Furthermore, hARAP3 is overexpressed in human prostate cancer tissues. We then performed western blotting experiments with 5 prostate cancer cell lines originally from ADPCa and AIPCa/CRPC and revealed that hARAP3 were differently expressed in them. The expression levels of hARAP3 were higher in 2 CRPC cell lines (DU145,Vcap) than in ADPCa cell ines (LNCaP, 22RV1). These results suggest that hARAP3 may play crucial roles in the progression of prostate cancer, specially in CRPC oncogenic transformation. In the future, we will analyze the epigenetic mechanism of hARAP3 involved in the regulation of transcription. And to further study its function in proliferation, metastasis and castration resistance of prostate cancer to clarify its molecular mechanism. Thus, these studies will provide the experimental data and new target to prevent and therapy this disease.

AR是核受体超家族成员，以配体依赖方式介导基因转录，并招募一系列辅调节因子调控其转录，发挥其生物学功能。AR及其辅调节因子分别在雄激素依赖和去势抵抗前列腺癌（CRPC）中扮演着重要角色。本项目利用AR-PEV果蝇模型筛选出AR新辅调节因子hARAP3。该因子属CK1蛋白激酶家族，含有对多种蛋白有磷酸化作用的丝/苏氨酸蛋白激酶结构域。前期工作中，我们验证了hARAP3对AR介导基因转录显著上调；还发现干扰hARAP3蛋白表达减少了组蛋白H3S10ph及H3K9Ac水平；此外，hARAP3在PC肿瘤组织中随分化程度降低呈高表达趋势，并在2种CRPC细胞中高表达，提示hARAP3可能在PC，特别是CRPC转化过程中起关键作用。我们将深入解析hARAP3参与转录调控的表观遗传学机制，研究其在PC生长、侵袭转移及去势抵抗等方面的作用，试图分析其分子机制，为该疾病的预防和治疗提供理论依据和新的靶点。

雄激素受体（androgen receptor，AR）是核受体超家族成员之一。AR及其辅转录调节因子在前列腺癌发生发展等过程中起重要作用，因此，鉴定和解析AR辅调节因子在AR介导基因转录中的调控机制及在前列腺癌治疗中的作用越来越受到关注。. 本研究中，我们通过果蝇系统筛选出调控AR介导基因转录的辅调节因子Gish。首先，我们构建了Gish人同源基因hARAP3(即ARAP3)全长及其截短的表达质粒，并对ARAP3在哺乳动物细胞内对AR介导基因转录的调节作用进行了验证。我们通过多种实验方法证实了在哺乳动物细胞内ARAP3与AR存在相互作用，并确定二者在细胞内共定位；在培养的细胞内证实ARAP3对AR介导的基因转录活性起上调作用，并确定ARAP3发挥作用的关键结构域位于N端的蛋白激酶域；证实了敲除ARAP3能够降低内源AR下游靶基因的转录。这些结果提示在哺乳动物细胞内ARAP3与AR相互作用，是新的AR辅激活因子。然后，为了明确ARAP3调控AR介导基因转录的表观遗传学机制，我们利用染色质免疫共沉淀（ChIP assay）实验证实ARAP3与AR 能够共同招募到AR靶基因启动子区，且敲低ARAP3的表达减少了AR靶基因启动子区的组蛋白H3S10ph水平，提示ARAP3上调AR介导的基因转录的机制可能与ARAP3对AR靶基因启动子区域组蛋白H3S10磷酸化有关。之后，为了检测ARAP3在前列腺癌中的生物学功能，我们利用细胞克隆形成实验、细胞计数、细胞周期检测实验发现ARAP3稳定沉默可以抑制CWR22Rv1前列腺癌细胞系的增殖，而ARAP3过表达能够促进CWR22Rv1前列腺癌细胞系的增殖。结果提示ARAP3促进前列腺癌的生长。进一步地，在临床来源的前列腺癌病例组织样本中，利用免疫组化实验发现ARAP3在前列腺癌中的表达水平高于正常前列腺组织；在前列腺癌细胞系中， ARAP3呈现出随着前列腺癌细胞系恶性程度升高而升高的趋势，且ARAP3在前列腺癌细胞系中的表达量与H3S10磷酸化及H3K9乙酰化水平相关，提示ARAP3可能与H3S10ph和H3K9Ac共同作为前列腺癌诊断的表观遗传学指标。此外，我们还检测到ARAP3对AR也有磷酸化作用。综上，ARAP3是新发现的AR共激活因子，参与调控AR靶基因启动子区组蛋白H3S10ph水平，在促进前列腺癌进程中发挥重要作用。