Sun Wenyue, Zhou Shaoyu, Chang Steven S, McFate Thomas, Verma Ajay, Califano Joseph A
Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins Medical Institutions, Baltimore, Maryland 21287, USA.
Clin Cancer Res. 2009 Jan 15;15(2):476-84. doi: 10.1158/1078-0432.CCR-08-0930.
Mitochondrial mutations have been identified in head and neck squamous cell carcinoma (HNSCC), but the pathways by which phenotypic effects of these mutations are exerted remain unclear. Previously, we found that mitochondrial ND2 mutations in primary HNSCC increased reactive oxygen species (ROS) and conferred an aerobic, glycolytic phenotype with HIF1alpha accumulation and increased cell growth. The purpose of the present study was to examine the pathways relating these alterations.
Mitochondrial mutant and wild-type ND2 constructs were transfected into oral keratinocyte immortal cell line OKF6 and head and neck cancer cell line JHU-O19 and established transfectants. The protein levels of HIF1alpha, pyruvate dehydrogenease (PDH), phosphorylated PDH, and pyruvate dehydrogenease kinase 2 (PDK2), together with ROS generation, were compared between the mutant and the wild type. Meanwhile, the effects of small molecule inhibitors targeting PDK2 and mitochondria-targeted catalase were evaluated on the ND2 mutant transfectants.
We determined that ND2 mutant down-regulated PDH expression via up-regulated PDK2, with an increase in phosphorylated PDH. Inhibition of PDK2 with dichloroacetate decreased HIF1alpha accumulation and reduced cell growth. Extracellular treatment with hydrogen peroxide, a ROS mimic, increased PDK2 expression and HIF1alpha expression, and introduction of mitochondria-targeted catalase decreased mitochondrial mutation-mediated PDK2 and HIF1alpha expression and suppressed cell growth.
Our findings suggest that mitochondrial ND2 mutation contributes to HIF1alpha accumulation via increased ROS production, up-regulation of PDK2, attenuating PDH activity, thereby increasing pyruvate, resulting in HIF1alpha stabilization. This may provide insight into a potential mechanism, by which mitochondrial mutations contribute to HNSCC development.
已在头颈部鳞状细胞癌（HNSCC）中鉴定出线粒体突变，但这些突变发挥表型效应的途径仍不清楚。此前，我们发现原发性HNSCC中的线粒体ND2突变会增加活性氧（ROS），并赋予有氧糖酵解表型，伴有HIF1α积累和细胞生长增加。本研究的目的是研究与这些改变相关的途径。
将线粒体突变型和野生型ND2构建体转染到口腔角质形成细胞永生细胞系OKF6和头颈部癌细胞系JHU - O19中，并建立转染子。比较突变型和野生型之间HIF1α、丙酮酸脱氢酶（PDH）、磷酸化PDH和丙酮酸脱氢酶激酶2（PDK2）的蛋白水平以及ROS生成情况。同时，评估靶向PDK2的小分子抑制剂和线粒体靶向过氧化氢酶对ND2突变型转染子的作用。
我们确定ND2突变型通过上调PDK2下调PDH表达，同时磷酸化PDH增加。用二氯乙酸抑制PDK2可减少HIF1α积累并降低细胞生长。用过氧化氢（一种ROS模拟物）进行细胞外处理可增加PDK2表达和HIF1α表达，而引入线粒体靶向过氧化氢酶可降低线粒体突变介导的PDK2和HIF1α表达并抑制细胞生长。
我们的研究结果表明，线粒体ND2突变通过增加ROS产生、上调PDK2、减弱PDH活性，从而增加丙酮酸，导致HIF1α稳定，进而促进HIF1α积累。这可能为线粒体突变促进HNSCC发展的潜在机制提供见解。
