| Budget Amount *help |
¥4,420,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥1,020,000)
Fiscal Year 2012: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
Fiscal Year 2011: ¥2,730,000 (Direct Cost: ¥2,100,000、Indirect Cost: ¥630,000)
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| Research Abstract |
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
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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
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