Regulation of anoikis by mitochondrial activity and its ablation duringoncogenesis

锚定缺失过程中抑制TFAM转录活性的机制：不依赖于锚定的生长和存活（AIG/S）是癌细胞转移的先决条件。在正常上皮细胞中，所谓的细胞死亡是在锚定缺失的情况下诱导的。为了深入了解anoikis和/或AIG/S的分子机制，我们在本研究中重点研究了线粒体转录因子A （TFAM）的转录调控。在人乳腺上皮细胞（HMEC）中，锚定缺失会下调TFAM的表达，但在转移性乳腺癌细胞中则不会下调。值得注意的是，TFAM的持续表达是人类乳腺癌细胞转移所必需的。我们首先使用TFAM上游区域进行报告性分析，以确定导致TFAM下调的因素。我们发现含有保守序列SBS的上游区域介导了这种下调，这表明锌指转录因子ZNF143在识别SBS的过程中发挥了重要作用。与TFAM类似，ZNF143的另一个靶基因SKP2也在锚定缺失过程中下调，支持ZNF143在转录应答中的作用。Western blot和real-time RT-PCR分析显示，锚定丢失后，ZNF143蛋白水平表达降低。综上所述，这些结果表明TFAM被ZNF143转录下调，在失去锚定后表达降低。线粒体呼吸链活性的降低促进了疾病的发生。我们之前证明了转录因子CHOP-10可被线粒体功能障碍诱导并介导细胞死亡。这一发现促使我们研究CHOP-10在线粒体活性下降的锚定丧失的细胞反应中的作用。然而，我们观察到在锚固缺失的情况下没有诱导CHOP-10。另一方面，cDNA微阵列分析显示，在这种条件下，tnf相关的凋亡诱导配体（TRAIL）被显著诱导。我们在mRNA和蛋白水平上证实了诱导作用。有趣的是，当RNAi抑制TRAIL及其受体DR4而非DR5的表达时，锚定丢失下的细胞死亡率降低。综上所述，这些结果表明，在锚定丢失期间，TRAIL上调，通过DR4激活死亡信号并诱导死亡。少

A mechanism underlying repression of TFAM transcriptional activity during anchorage lossAnchorage-independent growth and survival (AIG/S) are prerequisite for cancer cell metastasis. In normal epithelial cells, cell death so-called anoikis is induced under loss of anchorage. To get insights into the molecular mechanisms of anoikis and/or AIG/S, we focused on transcriptional regulation of mitochondrial transcriptional factor A (TFAM) in this study. The expression of TFAM is downregulated in response to anchorage loss in human mammary epithelial cells (HMEC) but not in metastatic breast cancer cells. Of note, sustained expression of TFAM was required for metastasis in human breast cancer cells. We first performed reporter assay using a TFAM upstream region to identify factors contributing to the downregulation of TFAM. We found that the upstream region containing well-conserved sequence called SBS mediated the downregulation, suggesting a role of zinc finger transcription factor ZNF143 t … More hat recognized SBS. Similar to TFAM, SKP2, another target gene of ZNF143, was also downregulated during anchorage loss, supporting a role of ZNF143 in the transcriptional response. Western blot and real-time RT-PCR analysis showed that the expression of ZNF143 was decreased at a protein level in response to anchorage loss. Taken together, these results suggest that TFAM was transcriptionally downregulated by ZNF143, which expression was reduced upon loss of anchorage.Anoikis promoted by reduction of mitochondrial respiratory chain activityWe previously demonstrated that a transcription factor CHOP-10 was induced by mitochondrial dysfunction and mediated cell death. The finding prompted us to examine the involvement to CHOP-10 in the cellular responses to anchorage loss under which mitochondrial activity was decreased. However, we observed no induction of CHOP-10 under loss of anchorage. On the other hand, cDNA microarray analysis revealed that TNF-related apoptosis-inducing ligand (TRAIL) was remarkably induced under the conditions. We confirmed the induction at mRNA and protein levels. Interestingly, when the expression of TRAIL and its receptor, DR4 but not DR5, was repressed by RNAi, cell death rate under anchorage loss was decreased. Collectively, these results suggest that TRAIL is upregulated during anchorage loss, leading to the activation of death signaling through DR4 and induction of anoikis. Less