15-LOX-1 transcription suppression through the NuRD complex in colon cancer cells - PubMed (original) (raw)

15-LOX-1 transcription suppression through the NuRD complex in colon cancer cells

X Zuo et al. Oncogene. 2009.

Abstract

15-Lipoxygenase-1 (15-LOX-1) is transcriptionally silenced in cancer cells, and its transcription reactivation (for example, through histone deacetylase inhibitors (HDACIs)) restores apoptosis to cancer cells. However, the exact mechanism underlying 15-LOX-1 transcription reactivation in cancer cells is still undefined. Therefore, we evaluated the critical mechanisms required for 15-LOX-1 transcription reactivation in colon cancer cells. Specific HDAC1 and HDAC2 inhibition activated 15-LOX-1 transcription. 15-LOX-1 transcription was repressed through transcription repressor complex recruitment in the region of -120 to -391 of the 15-LOX-1 promoter. The nucleosome remodeling and histone deacetylase (NuRD) repression complex was recruited to this region. Depsipeptide significantly reduced the recruitment of NuRD key components (for example, metastasis-associated protein 1 (MTA1) and HDAC1) to the 15-LOX-1 promoter before 15-LOX-1 transcriptional activation. Knock down of NuRD key components (for example, MTA1 and HDAC1) by small interfering RNA (siRNA) activated 15-LOX-1 transcription, as measured by luciferase reporter assays in stably transfected SW480 cells with the 15-LOX-1 promoter construct of the -391, but not the -120 region. Relative to expression in normal tissue, MTA1 expression in colorectal cancer mucosa from colorectal cancer patients was negatively related to 15-LOX-1 expression. Thus, our results show that NuRD contributes to 15-LOX-1 transcription suppression in colon cancer cells and that HDACIs can inhibit NuRD recruitment to a promoter to activate gene transcription, as in the case of 15-LOX-1.

PubMed Disclaimer

Figures

Fig. 1

HDAC1 and HDAC2 inhibition and 15-LOX-1 transcription activation. (A and B) Depsipeptide effects on 15-LOX-1 mRNA expression in Caco-2 colon cancer cells. Caco-2 cells were treated with depsipeptide at various concentrations (0-20 nM) for 24 hours (A) and for different time periods (B) at a concentration of 5 nM, and 15-LOX-1 mRNA expression was measured with real-time PCR. The relative expression levels were calculated as the values relative to that of the calibrator sample (time point or concentration 0). Values are the means ± SDs of triplicate experiments. (C and D) Depsipeptide induction of 15-LOX-1 protein expression in Caco-2 cancer cells. Caco-2 cells were treated for various times (at 5 nM depsipeptide concentration, Fig. 1C) and with various concentrations (for 24 h. Fig. 1D) as indicated and processed for 15-LOX-1 Western blotting. + indicates positive control (HCT-116 cells transfected with a 15-LOX-1 expression vector). (E) Effects of 15-LOX-1 enzymatic activity on depsipeptide-induced apoptosis in Caco-2 cancer cells. Caco-2 cells treated with various depsipeptide concentrations, as indicated, were cultured with or without caffeic acid (CAF) at the 2.2 μM concentration that specifically inhibits 15-LOX-1 enzymatic activity. Apoptosis was assessed by measuring caspase 3 activity levels. Values are the means ± SDs of triplicate measurements. F-I. Effects of 15-LOX-1 downregulation on depsipeptide- and SAHA- induced survival inhibition in colon cancer cells. Caco-2 colon cancer cells were transfected with a pool of four siRNA duplexes for 15-LOX-1 or nonspecific siRNA sequence (NS siRNA). 24 h later, they were treated with either (F and G) depsipeptide (5 nM) or (H and I) SAHA (1μM). Control indicates vehicle-solvent treated cells. 48 h later, 15-LOX-1 mRNA was measured by real-time PCR (depsipeptide [F], SAHA treatment [H]). G and I. Cell survival was assessed by SRB assays 72 h after treatment. Values are presented as the survival percentages relative to control (solvent)-treated cells transfected with nonspecific siRNA. Values shown are the means ± SDs of triplicate measurements. * P< 0.0001; ** P = 0.0005.

Fig. 2

Effects of HDAC1–3 siRNAs on 15-LOX-1 expression. (A–C) Effects of HDAC1–3 siRNA transfection on HDAC expression. Caco-2 cells were transfected with pools of four siRNA duplexes, each designed specifically against HDAC1, HDAC2, or HDAC3 or with a nonspecific siRNA sequence. Cells were harvested 24 h later. HDAC1 (A), HDAC2 (B), and HDAC3 (C) mRNA levels were measured by real-time PCR. The relative expression levels were calculated as the values relative to those of the calibrator samples (nonspecific siRNA). Values shown are the means ± SDs of triplicate experiments. (D) Effects of HDAC1–3 downregulation on 15-LOX-1 expression. Caco-2 cells were transfected as in panel A-C. Cells were harvested 24, 48, and 72 h after transfection and processed for 15-LOX-1 measurements by real-time PCR. Values shown are the means ± SDs of triplicate experiments. * P < 0.0001.

Fig. 3

Transcription activity of 15-LOX-1 promoter deletion constructs. (A) SW480 cells and (B) Caco-2 cells were transiently transfected with PGL4.16 luciferase reporter vectors containing the −120 to +18 bp (−120), −391 to +18 bp (−391), or −3500 to +18 bp (−3500) regions of the 15-LOX-1 promoter and treated with either depsipeptide or the control vehicle. Transcription activity was measured 24 h after treatment. (C and D) PGL4.16 vectors containing −120, −391, or −729 to +18 bp (−729) regions of the 15-LOX-1 promoter were stably transfected into SW480 cells. (C) Basal transcription levels (measured as luciferase reporter activity) for representative stably transfected clones with −120, −391, and −729 region vectors are shown. Values are the means ± SDs of triplicate experiments. (D) Depsipeptide effects on transcription activation for the −120 and −391 regions of the 15-LOX-1 promoter. SW480 stably transfected clones with −120 or −391 region vectors (as described in panel C) were treated with either depsipeptide or DMSO only (control). Luciferase activity was measured 24 h later. Values are the ratios of depsipeptide- to control-treated cells (means ± SDs of triplicate experiments). * P < 0.0001.

Fig. 4

Dissociation of the NuRD complex from the 15-LOX-1 promoter and 15-LOX-1 transcription activation. (A and B) Inhibition of NuRD complex recruitment to the 15-LOX-1 promoter during 15-LOX-1 transcription activation. (A) Caco-2 cells were treated with depsipeptide (5 nM) or DMSO for 15 min. Cells were subjected to ChIP/real-time PCR assays using HDAC1, HDAC2, HDAC3, Mi2, MTA1, or MTA2 antibodies. Data are given as percentages of the respective input genomic DNA for the 15-LOX-1 promoter. Values are the means ± SDs of triplicate measurements. (B) SW480 cells were treated with depsipeptide (5 nM) or DMSO for 3.5 h. Cells were subjected to the same assays described in A, and data are presented in the same way. (C) Effects of HDAC1–3 and MTA1 downregulation on 15-LOX-1 expression. SW480 cells stably transfected with PGL4.16 luciferase reporter vector containing the −120 or −391 bp region of the 15-LOX-1 promoter were transfected with pools of four siRNA duplexes. Each pool was designed specifically against HDAC1, HDAC2, HDAC3, or MTA1 or a nonspecific siRNA sequence (control). Luciferase activity was measured 72 h later. Relative luciferase activity represents the ratios to that of the mock experiment (transfection medium alone). Values shown are the means ± SDs of triplicate experiments.

Fig. 5

MTA1 and 15-LOX-1 expression in colorectal cancer. (A) MTA1 mRNA expression levels in cancerous and normal colorectal mucosa. MTA1 was measured by real-time PCR in paired colorectal cancer and normal tissue samples from 12 patients. Values shown are the means ± SDs of duplicate measurements. (B) 15-LOX-1 mRNA expression levels in cancerous and normal colorectal mucosa. 15-LOX-1 was measured by real-time PCR from the same paired colorectal cancer and normal tissue samples used for MTA1 measurements in Fig. 5A. Values shown are the means ± SDs of triplicate measurements.

References

1. Ariel A, Serhan CN. Resolvins and protectins in the termination program of acute inflammation. Trends Immunol. 2007;28:176–83. - PubMed
1. Baer AN, Costello PB, Green FA. In vivo activation of an omega-6 oxygenase in human skin. Biochem Biophys Res Commun. 1991;180:98–104. - PubMed
1. Balasenthil S, Broaddus RR, Kumar R. Expression of metastasis-associated protein 1 (MTA1) in benign endometrium and endometrial adenocarcinomas. Human Pathology. 2006;37:656–661. - PubMed
1. Bolden JE, Peart MJ, Johnstone RW. Anticancer activities of histone deacetylase inhibitors. Nat Rev Drug Discov. 2006;5:769–84. - PubMed
1. Brash AR, Boeglin WE, Chang MS. Discovery of a second 15S-lipoxygenase in humans. Proc Natl Acad Sci U S A. 1997;94:6148–52. - PMC - PubMed

15-LOX-1 transcription suppression through the NuRD complex in colon cancer cells - PubMed (original) (raw)