Regulation Mechanisms of Transcription Factor BACH1 by TANK Binding Kinase 1

概要

BTB and CNC homology 1 (BACH1) represses the expression of genes involved in the metabolism of iron, heme and reactive oxygen species. Heme directly binds to BACH1 to promote its degradation, but the link between BACH1 degradation and signaling pathways is not yet clear. In HEK293T cells, by overexpressing BACH1 and TBK1, a protein kinase which activates innate immune response, we found that TBK1 phosphorylated BACH1 and promoted its degradation. TBK1 promoted BACH1 degradation by two distinct mechanisms: one was dependent on its protein kinase activity and the other was independent of it. By using inhibitors of lysosomal and proteasome, autophagy-lysosomal and ubiquitin-proteasome pathways were found to be involved in the effects of TBK1.

BACH1 is known to promote metastasis of various cancers including pancreatic ductal adenocarcinoma (PDAC). In patients with PDAC, we found a strong positive correlation between BACH1 mRNA with TBK1 mRNA. TBK1 knockdown led to the reduction of BACH1 protein and mRNA levels in AsPC-1 human pancreatic cancer cells. Gene ontology enrichment analysis suggested the involvement of TBK1 and BACH1 in the regulation of iron homeostasis. Ferritin mRNAs were increased upon knockdown or knockout of BACH1 in AsPC-1 cells. FACS analysis showed that AsPC-1 cells with BACH1 knockout or knockdown contained lower labile iron, suggesting that BACH1 increased labile iron in AsPC-1 cells by repressing the expression of ferritin genes. Based on the above results, TBK1-BACH1 pathway involves in cancers metastasis by the regulation of iron metabolism. We next examined the changes in BACH1 mRNA and protein in response to fluctuations of iron by adding ferrous sulfate (FeSO4) or iron chelator deferasirox (DFX) in AsPC-1. BACH1 protein level increased with DFX and decreased with FeSO4, whereas FBXO22 was gradually decreased with DFX. Consistent with protein level, mRNA of BACH1 increased while that of FBXO22 decreased in response to DFX. When taken together, these results suggested that iron deficiency induces BACH1 at both transcriptional and post-translational steps.

TBK1 is suggested to regulate BACH1 by two different mechanisms. In HEK293T cells, TBK1 promotes the degradation of BACH1 protein through phosphorylation-dependent and -independent pathways. In AsPC-1 cells, TBK1 promotes BACH1 gene transcription and increases the amount of BACH1 ptotein, leading to reduction of genes such as FTH1 and FTL, and hence an induction of labile iron and E-cadherin, which maybe related to the promotion of metastasis by BACH1. Our findings expand knowledge on the regulation of BACH1 and provide novel clues to understand the function of BACH1.