促前列腺癌增殖蛋白MS4A8B调节钙池操纵的钙通道（SOC）的分子机制及其靶向性单克隆抗体相关研究

Our study has revealed that up-regulation of MS4A8B is closely associated with prostate cancer progression after radical prostatectomy, and transcription of MS4A8B gene is regulated by androgen receptor (AR) signaling to promote the proliferation of prostate cancer cells. However，the mechanisms for the tumor-promoting roles of MS4A8B are yet unclear and the targeted theraputics still under development. Our previous data also indicated that MS4A8B-targeted RNA interference(RNAi) and administration of anti-MS4A8B monoclonal antibody could significantly block the calcium entry mediated by store-operated calcium channels(SOC). Therefore, we hypothesize that MS4A8B might promote cell proliferation through up-regulating SOC activity. To test this hypothesis, we plan to combinedly employ mutiple approaches，including calcium imaging, patch clamp, RNAi/overexpression, mass spectrometric analysis, Co-immunoprecipitation and ChIP assays，to investigate the potential mechanism for the tumor-promoting function of MS4A8B and the involvement of SOC in cell, tissue or animal models. Meanwhile, we are also committed to screening for the best monoclonal antibody targeting the second extracellular loop of MS4A8B protein that may effectively antagonizing the function of MS4A8B. This work, if funded, would not only bring mechanistic insight into prostate cancer progression, but also facilitate the development of targeted monoclonal antibody-based therapy for precise treament of prostate cancer.

我们的前期研究表明，MS4A8B基因受雄激素受体转录调控、促进细胞增殖并与前列腺癌进展密切相关。然而其促进增殖的分子机制以及对应的治疗策略都尚未明了。同时，我们的初步研究也显示针对MS4A8B的RNA干扰和单克隆抗体均可显著阻断由前列腺癌细胞膜上钙池操纵的钙通道(SOC)激活导致的钙内流，但作用机制未明。我们提出假说：MS4A8B可能通过调节SOC从而促进前列腺癌细胞增殖。为此，我们将采用钙成像、膜片钳、RNA干扰/过表达、免疫共沉淀、质谱分析及ChIP等手段验证此假说；并针对MS4A8B蛋白第二个胞外区作为抗原抗体结合区设计抗原表位，筛选制备亲和力和专一性最强的单克隆抗体并开展靶向性MS4A8B治疗前列腺癌前期研究。我们的项目将从分子、细胞和动物水平深入探索MS4A8B促进前列腺癌细胞增殖的分子机制，揭示前列腺癌发生发展的机理，为发展靶向性的前列腺癌单克隆抗体治疗提供理论依据和实验基础。

背景:我们的前期研究表明，MS4A8B基因受雄激素受体转录调控、促进细胞增殖并与前列腺癌进展密切相关。然而其促进增殖的分子机制以及对应的治疗策略都尚未明了。同时，我们的初步研究也显示针对MS4A8B的RNA干扰和单克隆抗体均可显著阻断由前列腺癌细胞膜上钙池操纵的钙通道(SOC)激活导致的钙内流，但作用机制未明。.主要研究内容: MS4A8B在前列腺癌组织中的表达以及与临床病理特征的相关性分析(PSA水平、临床分期、Gleason评分、神经侵犯、术后生化复发等)；在体外分别在LNCaP细胞中下调MS4A8B表达、在RWPE-1细胞中上调MS4A8B表达后，观察到细胞增殖、细胞周期G1/S调控点、平板克隆形成及细胞周期相关蛋白发生相应的变化；MS4A8B可通过SOC调节胞内钙离子浓度变化；针对MS4A8B第二个胞外区域制备并筛选高选择性单克隆抗体。MatInspector及Jaspar预测可能转录调控MS4A8B的分子并以Luciferase及CHIP实验加以验证，IP联合质谱分析探究与MS4A8B 互相作用的分子，PEX100磷酸化芯片筛选MS4A8B促前列腺癌增殖的下游通路。.重要结果:Luciferase及CHIP实验显示MS4A8B受AR转录调控；IP联合质谱发现MS4A8B与14-3-3相互作用，共聚焦显示MS4A8B与14-3-3存在共定位且这种共定位可被比卡鲁胺减弱；过表达MS4A8B后发现p-AKT Ser473 显著上调，提示MS4A8B促前列腺癌增殖效应与AKT通路密切相关。进一步细胞功能实验（CCK-8、平板克隆、细胞周期、细胞凋亡等）验证了AR拮抗剂比卡鲁胺或AKT抑制剂Perifosine可显著降低MS4A8B的促前列腺癌增殖与抗凋亡效应，进一步说明了MS4A8B上游受AR调控，与14-3-3相互作用，下游激活通过Ser473位点激活AKT从而达到促前列腺癌增殖效应。.关键数据及其科学意义: 本项目发现MS4A8B促前列腺癌增殖效应是受AR调控的，且通过与14-3-3相互作用，帮助激活AKT Ser473位点。本研究完善了MS4A8B促前列腺癌增殖的机制，此外，MS4A8B还可通过SOC调节胞内钙离子浓度变化。针对MS4A8B设计的单克隆抗体期望能给临床治疗前列腺癌带来帮助。