Regulation of anoikis by mitochondrial activity and its ablation duringoncogenesis

锚定丧失时抑制TFAM转录活性的机制锚定非依赖性生长和存活(AIG/S)是癌细胞转移的先决条件。在正常的上皮细胞中，在失去锚定的情况下会诱导细胞死亡，即所谓的失巢凋亡。为了深入了解失巢凋亡和/或AIG/S的分子机制，本研究重点研究了线粒体转录因子A的转录调控。在人乳腺上皮细胞(HMEC)中，TFAM的表达随着锚定的丧失而下调，但在转移性乳腺癌细胞中不表达。值得注意的是，TFAM的持续表达是人乳腺癌细胞转移所必需的。我们首先使用TFAM上游区域进行了报告分析，以确定导致TFAM下调的因素。我们发现，含有保守序列的上游区域SBS介导了下调，提示锌指转录因子ZNF143t…的作用更多的HAT识别了SBS。与TFAM类似，ZNF143的另一个靶基因Skp2也在锚定丧失过程中下调，支持ZNF143在转录反应中的作用。Western印迹和实时定量RT-PCR分析表明，ZNF143的表达在蛋白水平上受到锚定丧失的影响。综上所述，这些结果表明，ZNF143在转录水平上下调了TFAM的表达，并在失去锚定后表达减少。Anoikis通过降低线粒体呼吸链的活性来促进Anoikis。我们之前已经证明，转录因子CHOP-10是由线粒体功能障碍诱导的，并介导了细胞死亡。这一发现促使我们研究了CHOP-10参与细胞对锚定丢失的反应，在这种情况下，线粒体的活性降低。然而，在失去锚定的情况下，我们没有观察到CHOP-10的诱导。另一方面，基因芯片分析显示，在此条件下，肿瘤坏死因子相关的凋亡诱导配体(TRAIL)被显著诱导。我们从信使核糖核酸和蛋白质水平证实了诱导。有趣的是，当TRAIL及其受体DR4而不是DR5的表达被RNAi抑制时，锚定丧失时的细胞死亡率降低。综上所述，这些结果表明，TRAIL在锚定丧失过程中上调，导致通过DR4激活死亡信号并诱导失巢。较少

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