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. 2016 Apr;49(4):238-43.
doi: 10.5483/bmbrep.2016.49.4.031.

SETDB1 Mediated FosB Expression Increases the Cell Proliferation Rate During Anticancer Drug Therapy

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Free PMC article

SETDB1 Mediated FosB Expression Increases the Cell Proliferation Rate During Anticancer Drug Therapy

Han-Heom Na et al. BMB Rep. .
Free PMC article

Abstract

The efficacy of anticancer drugs depends on a variety of signaling pathways, which can be positively or negatively regulated. In this study, we show that SETDB1 HMTase is down-regulated at the transcriptional level by several anticancer drugs, due to its inherent instability. Using RNA sequence analysis, we identified FosB as being regulated by SETDB1 during anticancer drug therapy. FosB expression was increased by treatment with doxorubicin, taxol and siSETDB1. Moreover, FosB was associated with an increased rate of proliferation. Combinatory transfection of siFosB and siSETDB1 was slightly increased compared to transfection of siFosB. Furthermore, FosB was regulated by multiple kinase pathways. ChIP analysis showed that SETDB1 and H3K9me3 interact with a specific region of the FosB promoter. These results suggest that SETDB1- mediated FosB expression is a common molecular phenomenon, and might be a novel pathway responsible for the increase in cell proliferation that frequently occurs during anticancer drug therapy. [BMB Reports 2016; 49(4): 238-243].

Figures

Fig. 1.
Fig. 1.. SETDB1 is commonly regulated by various anticancer drugs. (A) A549 cells were treated with various anticancer drugs including taxol (0.5 μM), cisplatin (2 μM), 5-FU (5 μM), and doxorubicin (2 μM). SETDB1 protein was down-regulated, whereas p53 protein was up-regulated in these treatments. (B) Luciferase assay was performed following anticancer drug treatment after pGL3-SETDB1-p-Luc transfection. (C) Western blots were done following doxorubicin or taxol treatment. SETDB1 was decreased, p53 was increased, but EZH2 or SUV39H1 displayed no change in protein levels. (D) Doxorubicin was treated in a dose- or time-dependent manner in A549 cells. H3K9me3 levels decreased, consistent with SETDB1 down-regulation.
Fig. 2.
Fig. 2.. SETDB1 is a relatively unstable protein. (A) Cycloheximide (CHX), a protein synthesis inhibitor, was pretreated for 12 hr prior to doxorubicin or taxol treatment. Anticancer drug was treated for 12 hr. SETDB1 down-regulation was aggravated in the combination treatment, whereas other HMTases were not. (B) SETDB1 protein was decreased in the treatment of the RNA synthesis inhibitor, actinomycin D. Other HMTases were not changed in the same treatment.
Fig. 3.
Fig. 3.. Nine genes were identified as commonly regulated genes through RNA sequence analysis. (A) Two siSETDB1 were prepared and transfected into A549 cells. SETDB1 protein level was downregulated by siSETDB1 transfection. (B) Up-regulated genes were analyzed after doxorubicin, taxol, and siSETDB1 treatment. (C) Nine genes were identified as common target genes for doxorubicin, taxol, and SETDB1. Among them, FosB gene expression showed the highest change. (D) RT-PCR analysis was performed to check the expression of FosB and EGR2. (E) Western blot showed that doxorubicin treatment increased the FosB gene expression in a dose- or time-dependent manner. (F) Treatment of taxol or etoposide increased the FosB gene expression in a dose-dependent manner.
Fig. 4.
Fig. 4.. SETDB1 mediated FosB expression changes the cell proliferation rate. (A) A549 cells were transfected with pCDNA3-FosB plasmid in 60 mm diameter dishes, after which they were seeded at a density of 2,000 cells/well in 96-well plates. The MTT assay was performed after doxorubicin treatment. (B) A549 cells were transfected with siRNA, and were treated with doxorubicin after seeding in 96-well plates for the MTT assay. Cell proliferation rate decreased with siFosB, but increased marginally in the combinatory transfection of siSETDB1and siFosB. (*P < 0.05) (C) A549 cells were treated with various kinase inhibitors, along with doxorubicin. Western blot showed that most kinase inhibitors regulated the increased FosB gene expression by doxorubicin. (D) ChIP assay was performed with three primer sets for FosB promoter region, in the absence or presence of doxorubicin treatment. SETDB1 binding was detected in the specific regions (−267 to −115). H3K9me3 occupancy was consistent in these regions.

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