Oncotarget 8:108181-108194(2017) 

SREBP-2-driven transcriptional activation of human SND1 oncogene

María José Martínez


摘要:

Upregulation of Staphylococcal nuclease and tudor domain containing 1 (SND1) is linked to cancer progression and metastatic spread. Increasing evidence indicates that SND1 plays a role in lipid homeostasis. Recently, it has been shown that SND1-overexpressing hepatocellular carcinoma cells present an increased de novo cholesterol synthesis and cholesteryl ester accumulation. Here we reveal that SND1 oncogene is a novel target for SREBPs. Exposure of HepG2 cells to the cholesterol-lowering drug simvastatin or to a lipoprotein-deficient medium triggers SREBP-2 activation and increases SND1 promoter activity and transcript levels. Similar increases in SND1 promoter activity and mRNA are mimicked by overexpressing nuclear SREBP-2 through expression vector transfection. Conversely, SREBP-2 suppression with specific siRNA or the addition of cholesterol/25-hydroxycholesterol to cell culture medium reduces transcriptional activity of SND1 promoter and SND1 mRNA abundance. Chromatin immunoprecipitation assays and site-directed mutagenesis show that SREBP-2 binds to the SND1 proximal promoter in a region containing one SRE and one E-box motif which are critical for maximal transcriptional activity under basal conditions. SREBP-1, in contrast, binds exclusively to the SRE element. Remarkably, while ectopic expression of SREBP-1c or -1a reduces SND1 promoter activity, knocking-down of SREBP-1 enhances SND1 mRNA and protein levels but failed to affect SND1 promoter activity. These findings reveal that SREBP-2 and SREBP-1 bind to specific sites in SND1 promoter and regulate SND1 transcription in opposite ways; it is induced by SREBP-2 activating conditions and repressed by SREBP-1 overexpression. We anticipate the contribution of a SREBPs/SND1 pathway to lipid metabolism reprogramming of human hepatoma cells.To determine whether SND1 expression was regulated by SREBP-2, human HepG2 cells were cultured under different cellular conditions that modulate SREBP-2 pathway. Sterol starvation triggers the activation of SREBPs pathway and the cleavage of SREBP-2 to the active form. Thus, cells were treated with the cholesterol lowering drug simvastatin or were grown in a culture medium with lipoprotein deficient serum (LPDS) in order to activate SREBP-2. Simvastatin is an inhibitor of hydroxymethylglutaryl-coenzyme A reductase (HMGCR), the enzyme catalysing the rate limiting step in cholesterol biosynthesis, whereby the mevalonate pathway is inhibited and the intracellular cholesterol levels diminish. We confirmed that HepG2 treatment with 10 μM simvastatin or the cell culture in LPDS medium resulted in the induction of SREBP-2 mRNA and protein expression, concomitant with the increase of transcripts of downstream target genes HMGCR and LDL receptor (LDLR) and unchanged SREBP-1 mRNA content (Figure 1D and 1E). In parallel, we found that simvastatin or LPDS significantly up-regulated (3-fold or 1.6-fold) the expression of SND1 mRNA in human hepatoma cells and that SND1 protein accumulated in both the nucleus and the cytoplasm of simvastatin-treated cells (Figure 1A), though it was not significantly altered by LPDS (Figure 1B).To asses the response of SND1 gene expression to a sterol rich condition, HepG2 cells were incubated during 24 h in the presence of 10 μg/ml cholesterol plus 1 μg/ml 25-hydroxycholesterol. These sterols bind to SCAP and Insig, respectively, impairing the ER-to-Golgi transfer of SREBPs and their travelling to the nucleus, thus inhibiting SREBP-2 and SREBP-1 pathways. As shown in Figure 1C, a significant decrease of about 50% was noticed in the SND1 transcript level, although SND1 protein remained invariable. The transcript level of expression of SREBP-2 and its target genes was unmodified by sterols (Figure 1D and 1E).Next, we investigated whether the SND1 promoter transcriptional activity changed accordingly to the changes in SND1 mRNA expression and SREBP-2 activity. We assayed HepG2 cells transfected with 5’-deletion fragments of the SND1 promoter in the presence or absence of simvastatin, LPDS or exogenous sterols. We firstly constructed plasmids that contained SND1 promoter sequences covering the regions from nucleotide -112 or -274 or -416 to +221 (relative to the transcriptional start site) ahead from the luciferase coding region into pGL3-Basic as detailed in previous work [44]. A consistent increase in the reporter activity of the SND1 deletion fragments was observed in the transfected HepG2 cells following exposure to simvastatin (Figure 2A) or LPDS medium (Figure 2B). Conversely, exogenous sterols not only reduced luciferase activity (Figure 2C, upper panel) but also counteracted the LPDS-mediated activation of SND1 promoter activity (Figure 2C, lower panel). Transfection assays of human embryonic kidney HEK293 cells, used as a non-hepatic non-tumoral cell line model, exhibited similar SND1 promoter activation by simvastatin and LPDS, with the exception of the lack of response of fragment 416 to simvastatin (Figure 2A and 2B). Also of note is the absence of promoter response to exogenous sterols in HEK293 cells (Figure 2C). Altogether these results are consistent with the concept that there is a sterol-sensitive mechanism of transcriptional regulation operating for the SND1 gene in human hepatoma cells which seems to be somewhat less operative in HEK293 cells. Both, the activation of the SND1 proximal promoter and the upregulation of SND1 expression upon sterol deprivation, point to SND1 as a novel SREBP-2 inducible gene.Human hepatocellular carcinoma HepG2 cells (ATCC) and human embryonic kidney HEK293 cells (ATCC) were grown in EMEM (ATCC) supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin (Sigma-Aldrich) and 10% (v/v) foetal bovine serum (ATCC) at 37°C and 5% CO2. In some experiments foetal bovine serum was replaced by 10% lipoprotein deficient serum (LPDS) (Sigma). For RNA or protein determination 7×105 cells were seeded in 6 well plates; and for measurement of SND1 transcriptional activity 10×103 cells/well in 96 well plates were used. When indicated, cells were treated for 24 h with 10 μM simvastatin or with a mixture of 10 μg/ml cholesterol plus 1 μg/ml 25-hydroxycholesterol; same volume of solvent, DMSO or ethanol respectively, was added to control cells.The authors thank Mr. José Antonio López for his technical help with cell cultures. Technical and human support provided by SGIker (UPV/EHU, MICINN, GV/EJ, ERDF and ESF) is gratefully acknowledged.
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