肿瘤耐药性是目前肿瘤治疗的难题。肿瘤细胞在常规疗法作用下进入“治疗选择性沉默”状态，重组能量代谢通路，依靠加强的线粒体呼吸功能维持其生存，从而逐步产生耐药性。因此，靶向阻断线粒体呼吸功能是一个有希望打破肿瘤耐药性的治疗策略。前列腺癌在荷尔蒙疗法治疗一段时间后会逐渐成为去势抵抗性前列腺癌（CRPC），目前无有效治疗药物。我们的前期工作发现化合物SMIP004能够有效的抑制线粒体呼吸作用，引起氧化应激压力，促进活性氧产生，激活非折叠蛋白效应，诱发CRPC细胞凋亡。同时，SMIP004抑制CRPC移植小鼠的肿瘤生长且无显著毒性。本项目将在此基础上，重点研究SMIP004的作用机制，确认其作用靶点，通过系统毒理学实验，评价其毒性。同时，我们将通过蛋白组学，代谢组学方法，明确SMIP004敏感群体和生物标记物。本项目旨在评价线粒体抑制剂作为抗肿瘤药物的的可行性并提供有潜力的候选化合物。

Cancer resistance is a hurdle for effective cancer therapies. Common therapies such as radiation and genotoxic chemotherapy trigger selection of therapy resistant quiescent or slow-cycling cells, a process referred to as “Therapy-Selected Quiescence”. Quiescent cells reprogram the energy metabolism pathway, increase glucose uptake and mitochondrial respiration, therefore escaping from cyto-toxicity and inducing resistance. Standard care for advanced prostate cancer is androgen deprivation therapy (ADT). While most patients initially respond to ADT, virtually all progress to Castration-resistant prostate cancer (CRPC) after 18 to 24 months. On the basis that CRPC continues to depend on androgen receptor function, new anti-androgens and androgen synthesis blockers were introduced to the clinic with substantial therapeutic success. However, rapid development of resistance indicates a strong unmet need for alternative therapies. As mentioned above, recent recognition that therapy resistance coincides with upregulation of mitochondrial respiration in a variety of tumor types led to the hypothesis that mitochondrial inhibition may be a promising novel approach to target a specific bioenergetic liability of CRPCs failing hormonal therapy. The lab has previously discovered compound SMIP004, a novel inducer of apoptosis selectively in CRPC cells. Chemogenomic and proteomic profiling revealed that the compound induces a pro-apoptotic pathway, which initiates with disruption of mitochondrial respiration leading to oxidative stress. Oxidative stress activates pro-apoptotic signaling through the unfolded protein response pathway. SMIP004 potently inhibited the growth of prostate cancer xenografts in mice with no obvious toxicity. The main objective of this exploratory research is to assess the potential of SMIP004 as a novel therapy for CRPC. Moreover, we will identify biomarkers that predict sensitivity to SMIP004 by molecular profiling studies. Our project provides insights into molecular mechanisms of inhibition of CRPC growth by SMIP004, and also provides promising mitochondria inhibitors as candidate compounds for CRPC treatment.

SMIP004-7(4-丁基-2-甲基苯胺)是由申请人发现的，作为前列腺癌细胞死亡的选择性小分子诱导物。随后的作用机制研究显示SMIP004-7抑制线粒体呼吸复合物I(NADH:泛氧化还原酶)，导致氧化应激和激活非折叠蛋白反应(UPR)信号通路，最终导致细胞死亡。这一探索性研究的主要目的是评估SMIP004-7作为一种治疗耐药的去势抵抗性前列腺癌(CRPC)的新疗法。我们之前的研究结果发现并非所有肿瘤细胞系对SMIP004-7具有相似的敏感性。因此，我们在该课题中进一步探讨了SMIP004-7的作用机制和影响细胞对SMIP004-7的敏感性机理。 为了解开SMIP004-7的细胞毒性之谜，我们主要研究了下面列举的三个方面内容：1） 首先在药物作用压力下，对SMIP004-7不敏感的细胞表现出降低的活性氧簇积累和谷胱甘肽的表达量。这个现象部分解释了这些耐药细胞增强的抗氧化活性。2） 其次我们通过定量蛋白组学技术分析了对SMIP004-7敏感的细胞蛋白表达谱，结果发现一系列线粒体相关蛋白的表达的增多，包括我们前期确定的化合物的靶点，呼吸链复合体一。3） SMIP004-7敏感的细胞系表现出相当程度的对呼吸链复合体一酶活力的快速抑制，同时表现出在药物压力下基因表达的应激调整。这些数据说明对化合物SMIP004-7敏感的细胞可能通过增加线粒体内膜上的转运蛋白的表达，增加了SMIP004-7在线粒体基质上的浓度，从而表现出对SMIP004-7的敏感性。在接下来的研究化合物对细胞的毒性机理实验中，我们利用更接近于肿瘤干细胞（cancer stem cells）生存微环境的3维细胞培养体系，并通过改变细胞培养基中的营养成分，例如葡萄糖等，培养肿瘤细胞，发现细胞对SMIP004-7的敏感性增加。值得一提的是，对SMIP004-7耐药的CRPC细胞22rv1在3维培养体系中对化合物的敏感度大大增加。同时我们也发现CRPC细胞对雄性激素有一定的依赖作用。申请人一系列的实验结果说明肿瘤细胞对SMIP004-7的选择性与细胞对过氧化物的耐受有密切关系，也进一步证实了我们前期的研究结果，是我们对线粒体抑制剂作用于耐药性肿瘤细胞的作用机理有了更深入的了解。