Institute of Metabolic Science, Metabolic Research Laboratories, University of Cambridge, Cambridge, UK.
Institute of Metabolic Science, Metabolic Research Laboratories, University of Cambridge, Cambridge, UK.
Lancet. 2015 Feb 26;385 Suppl 1:S77. doi: 10.1016/S0140-6736(15)60392-0.
Somatic activating mutations in PIK3CA, which encodes the p110α catalytic subunit of phosphoinositide-3-kinase (PI3K) are frequently found in cancers and have been identified in a spectrum of mosaic overgrowth disorders ranging from isolated digit enlargement to more extensive overgrowth of the body, brain, or vasculature. We aimed to study affected dermal fibroblasts with a view to inform therapeutic studies, and to observe cancer-associated mutations in isolation.
We measured PIP3 concentrations in dermal fibroblasts with endogenous PIK3CA mutations and in wild type fibroblasts using mass spectrometry, and we measured downstream signalling events with ELISA and immunoblotting. Cellular proliferation was evaluated with 5-bromo-2'-deoxyuridine incorporation, and cell size assessed by fluorescence-activated cell sorting (FACS). Glycolysis and mitochondrial tests were performed with an extracellular flux analyser (Seahorse Bioscience, Billerica, MA, USA), and mitochondrial potential was measured by FACS-based JC1 staining. Experiments were repeated after exposure to 5 nmol everolimus for 72 h.
Mutant fibroblasts had two times higher basal PIP3 concentrations than wild-type fibroblasts (p=0·0017), with concomitant AKT and p70S6 activation downstream. The rate of cellular proliferation was higher in mutant cells under low serum conditions, but median cell size was not statistically different. Glycolytic capacity was similar between mutant and wild type fibroblasts, but subtle differences in mitochondrial function were detected with blunted responses to uncoupling agents and reduced membrane potentials. Treatment with everolimus reversed aberrant AKT(ser473) and p70S6 signalling, slowed cellular proliferation, and reversed mitochondrial abnormalities, but was associated, paradoxically, with increases in PIP3 concentrations.
These experiments demonstrate activation of the PI3K-AKT pathway in affected fibroblasts with increased proliferation, but no hypertrophy. Moreover, we identified changes in mitochondrial function in keeping with the known propensity of AKT to modulate elements of the Warburg effect. These results suggest that inhibitors of the mammalian target of rapamycin (mTOR) might be beneficial, but these inhibitors will require formal evaluation in clinical trials. More targeted therapy with p110α inhibitors is an enticing future option.
Wellcome Trust, Sackler Fund, National Instititute for Health Research.
PIK3CA 基因编码的磷酸肌醇-3-激酶（PI3K）的 p110α 催化亚基的体细胞激活突变经常发生在癌症中，并已在从孤立的数字增大到身体、大脑或脉管系统的更广泛过度生长的一系列镶嵌性过度生长障碍中被鉴定。我们旨在研究受影响的真皮成纤维细胞，以期为治疗研究提供信息，并观察孤立的癌症相关突变。
我们使用质谱法测量了具有内源性 PIK3CA 突变的真皮成纤维细胞和野生型成纤维细胞中的 PIP3 浓度，并使用 ELISA 和免疫印迹法测量了下游信号事件。通过 5-溴-2'-脱氧尿苷掺入评估细胞增殖，通过荧光激活细胞分选（FACS）评估细胞大小。使用细胞外通量分析仪（美国马萨诸塞州比勒瑞卡的 Seahorse Bioscience）进行糖酵解和线粒体测试，通过基于 FACS 的 JC1 染色测量线粒体电位。在暴露于 5nmol 依维莫司 72 小时后重复实验。
突变型成纤维细胞的基础 PIP3 浓度比野生型成纤维细胞高两倍（p=0·0017），同时下游 AKT 和 p70S6 激活。在低血清条件下，突变细胞的细胞增殖率更高，但中位数细胞大小无统计学差异。突变型和成纤维细胞的糖酵解能力相似，但检测到线粒体功能的细微差异，对解偶联剂的反应减弱，膜电位降低。依维莫司治疗逆转了异常的 AKT(ser473)和 p70S6 信号，减缓了细胞增殖，并逆转了线粒体异常，但矛盾的是，与 PIP3 浓度的增加有关。
这些实验表明，受影响的成纤维细胞中 PI3K-AKT 通路的激活与增殖增加有关，但没有肥大。此外，我们发现线粒体功能的变化与 AKT 调节沃伯格效应的某些元素一致。这些结果表明，哺乳动物雷帕霉素靶蛋白（mTOR）抑制剂可能是有益的，但这些抑制剂需要在临床试验中进行正式评估。使用 p110α 抑制剂进行更有针对性的治疗是一个诱人的未来选择。
英国惠康信托基金会、萨克勒基金会、英国国家卫生研究院。
