Phosphoinositide 3-kinase-related overgrowth: cellular phenotype and future therapeutic options.
Phosphoinositide 3-kinase-related overgrowth: cellular phenotype and future therapeutic options.
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DOI：
10.1016/s0140-6736(15)60392-0
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发表时间：
2015-02-26
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影响因子：
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通讯作者：
Semple, Robert K
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作者：
Parker, Victoria E R;Knox, Rachel G;Semple, Robert K
背景技术背景：编码磷酸肌醇-3-激酶（PI 3 K）p110 α催化亚基的PIK 3CA的体细胞激活突变经常在癌症中发现，并已在一系列镶嵌性过度生长疾病中发现，范围从孤立的手指肿大到身体、大脑或血管系统的更广泛过度生长。我们的目的是研究受影响的真皮成纤维细胞，以期告知治疗研究，并观察癌症相关的突变isolation.METHODS：我们测量PIP 3浓度与内源性PIK 3CA突变的真皮成纤维细胞和野生型成纤维细胞使用质谱，我们测量下游信号与ELISA和免疫印迹事件。用5-溴-2 '-脱氧尿苷掺入评价细胞增殖，并通过荧光激活细胞分选（FACS）评估细胞大小。用细胞外通量分析仪（Seahorse Bioscience，Billerica，MA，USA）进行糖酵解和线粒体测试，并通过基于FACS的JC 1染色测量线粒体电位。结果：突变体成纤维细胞的基础PIP 3浓度比野生型成纤维细胞高两倍（p= 0.0017），伴随着下游的AKT和p70 S6激活。在低血清条件下，突变细胞的细胞增殖率较高，但中值细胞大小无统计学差异。糖酵解能力是相似的突变体和野生型成纤维细胞，但线粒体功能的细微差异检测与钝化反应解偶联剂和降低膜电位。依维莫司逆转异常AKT（ser 473）和p70 S6信号转导，减缓细胞增殖，逆转线粒体异常，但相关的，矛盾的是，增加PIP 3 concentration.Interpretation：这些实验表明活化的PI 3 K-AKT通路在受影响的成纤维细胞增殖增加，但没有肥大。此外，我们确定了线粒体功能的变化，这与已知的AKT调节瓦尔堡效应元素的倾向一致。这些结果表明，哺乳动物雷帕霉素靶蛋白（mTOR）的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。更有针对性的治疗与p110 α抑制剂是一个诱人的未来选择。基金：惠康信托基金，萨克勒基金，国家卫生研究所。
BACKGROUND: Somatic activating mutations in PIK3CA, which encodes the p110alpha 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.METHODS: 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.FINDINGS: 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.INTERPRETATION: 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 p110alpha inhibitors is an enticing future option.FUNDING: Wellcome Trust, Sackler Fund, National Instititute for Health Research.