AKT Inhibition Promotes Non-autonomous Cancer Cell Survival [Microvesicles small RNA-Seq]

Small-molecule inhibitors of AKT signaling are being in evaluated in patients with various cancer types, but have so far proven therapeutically disappointing for reasons that remain unclear. Here, we treat cancer cells with sub-therapeutic doses of Akti-1/2, an allosteric small molecule AKT inhibitor, in order to experimentally model pharmacologic inhibition of AKT signaling in vitro. We then apply a combined RNA, protein, and metabolite profiling approach to develop an integrated, multi-scale, molecular snapshot of this “AKTlow” cancer cell state. We find that AKT-inhibited cancer cells suppress thousands of mRNA transcripts, and proteins related to the cell cycle, ribosome, and protein translation. Surprisingly, however, these AKT-inhibited cells simultaneously up-regulate a host of other proteins and metabolites post-transcriptionally, reflecting activation of their endo-vesiculo-membrane system, secretion of inflammatory proteins, and elaboration of extracellular microvesicles. Importantly, these microvesicles enable rapidly proliferating cancer cells of various types to better withstand different stress conditions, including serum deprivation, hypoxia, or cytotoxic chemotherapy in vitro and xenografting in vivo. These findings suggest a model whereby cancer cells experiencing a partial inhibition of AKT signaling may actually promote the survival of neighbors through non-cell autonomous communication. Microvesicle content profiles of MCF7 and HCT116 Akti-1/2 treated cells and MCF7 and HCT116 vehicle (i.e. DMSO) treated cells were generated, using technical replicates, using small RNA-Seq.

针对AKT信号通路的小分子抑制剂已在多种癌症患者中开展临床试验，但迄今临床疗效未达预期，其背后机制仍未阐明。本研究使用亚治疗剂量的别构小分子AKT抑制剂Akti-1/2处理癌细胞，以在体外构建AKT信号通路药物抑制的实验模型。随后我们采用整合RNA、蛋白质及代谢物组学的分析策略，对此类“AKT低活性（AKTlow）”癌细胞状态生成整合性多尺度分子快照。研究发现，AKT受抑制的癌细胞会抑制数千个mRNA转录本以及与细胞周期、核糖体及蛋白质翻译相关的蛋白质表达。但令人意外的是，此类AKT受抑制细胞同时在转录后层面上调了大量其他蛋白质与代谢物，这反映出其胞内囊泡膜系统被激活、炎性蛋白质分泌增强以及细胞外微泡（extracellular microvesicles）生成增加。值得注意的是，这些微泡可使多种类型的快速增殖癌细胞更好地耐受多种应激条件，包括体外实验中的血清剥夺、缺氧或细胞毒性化疗，以及体内异种移植模型中的应激。本研究结果提示了一种全新模型：部分AKT信号通路受抑制的癌细胞，可通过非细胞自主通讯机制促进邻近癌细胞的存活。本研究通过小RNA测序（small RNA-Seq）并设置技术重复，构建了经Akti-1/2处理的MCF7、HCT116细胞，以及经赋形剂（即二甲基亚砜DMSO）处理的MCF7、HCT116细胞的微泡内容物谱。