Nrf2 status affects tumor growth, HDAC3 gene promoter associations, and the response to sulforaphane in the colon

Abstract

Background: The dietary agent sulforaphane (SFN) has been reported to induce nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2)-dependent pathways as well as inhibiting histone deacetylase (HDAC) activity. The current investigation sought to examine the relationships between Nrf2 status and HDAC expression in preclinical and translational studies.

Results: Wild type (WT) and Nrf2-deficient (Nrf2(-/+)) mice were treated with the colon carcinogen 1,2-dimethylhydrazine (DMH) followed by 400 ppm SFN in the diet (n = 35 mice/group). WT mice were more susceptible than Nrf2(-/+) mice to tumor induction in the colon. Tumors from WT mice had higher HDAC levels globally and locally on genes such as cyclin-dependant kinase inhibitor 2a (Cdkn2a/p16) that were dysregulated during tumor development. The average tumor burden was reduced by SFN from 62.7 to 26.0 mm(3) in WT mice and from 14.6 to 11.7 mm(3) in Nrf2(-/+) mice. The decreased antitumor activity of SFN in Nrf2(-/+) mice coincided with attenuated Cdkn2a promoter interactions involving HDAC3. HDAC3 knockdown in human colon cancer cells recapitulated the effects of SFN on p16 induction. Human subjects given a broccoli sprout extract supplement (200 μmol SFN equivalents), or reporting more than five cruciferous vegetable servings per week, had increased p16 expression that was inversely associated with HDAC3 in circulating peripheral blood mononuclear cells (PBMCs) and in biopsies obtained during screening colonoscopy.

Conclusions: Nrf2 expression varies widely in both normal human colon and human colon cancers and likely contributes to the overall rate of tumor growth in the large intestine. It remains to be determined whether this influences global HDAC protein expression levels, as well as local HDAC interactions on genes dysregulated during human colon tumor development. If corroborated in future studies, Nrf2 status might serve as a biomarker of HDAC inhibitor efficacy in clinical trials using single agent or combination modalities to slow, halt, or regress the progression to later stages of solid tumors and hematological malignancies.

Keywords: Broccoli; Colon cancer; HDAC3; Nrf2; Sulforaphane; p16.

Figures

Fig. 1

Continuous and alternating dosing schedules for dietary SFN in the DMH model. a Male ICR mice were injected i.p. with DMH (20 mg/kg), once per week for 10 weeks (arrows). One week after completing the DMH treatment, mice were continued on standard AIN93 diet (no SFN) or given AIN93 diet containing 400 ppm SFN either continuously (daily SFN, green box) or on alternate days (Alt SFN, stripes). b Tumor multiplicity and c total tumor burden, i.e., the sum of individual tumor volumes, were determined for each animal at 25 weeks, and mean values were calculated (dotted line). *P < 0.05 vs. no SFN controls

Fig. 2

SFN decreased HDAC activity and HDAC3 protein expression in mouse colon tumors. a HDAC activity in colon tumor lysates was determined as described in “Methods” section. Mean values were calculated in each group (dotted line). *P < 0.05 vs. no SFN controls. b HDAC expression and histone acetylation determined by immunoblotting. c Densitometry data for HDACs were normalized to β-actin, whereas acetylated histone H4K12 (AcH4K12) was normalized to histone H4. Data = mean ± SD (n = 3); *P < 0.05 vs. no SFN controls

Fig. 3

Antitumor activity of dietary SFN in Nrf2+/+ and Nrf2−/+ mice. a Nrf2+/+ (WT) or Nrf2−/+ mice were injected i.p. with DMH (20 mg/kg), once per week for 8 weeks (arrows). One week after completing the carcinogen treatment, mice were continued on standard AIN93 diet (−SFN) or AIN93 diet containing 400 ppm SFN (+SFN). b Total tumor burden/mouse indicates the sum of individual tumor volumes after 35 weeks, plotted for each animal; dotted line represents the mean value in each group; P < 0.05 as indicated by the asterisk. c Relative mRNA expression levels of Nrf2 normalized to the β-actin gene, Actb. Data = mean ± SD (n = 3)

Fig. 4

SFN decreased HDAC3 protein expression in colon tumors of Nrf2+/+ but not Nrf2−/+ mice. a HDAC expression was immunoblotted in tumor (T) and adjacent normal-looking colon (N) of WT mice, with densitometry data normalized to β-actin. b The corresponding data for Nrf2−/+ mice. Data are given as mean ± SD (n = 3); *P < 0.05, **P < 0.01 for control diet vs. SFN treatment. In addition to global reductions in HDAC3, marked loss of HDAC3 on gene targets was examined (see Fig. 6)

Fig. 5

Differential gene expression in mouse colon tumors and normal colon. a Heat map representing the expression of differentially expressed genes, relative to Actb. Each vertical column represents the gene expression profile of pooled colon tumors or adjacent normal colon (n = 6) of mice on standard AIN93 diet (−SFN) or AIN93 diet containing 400 ppm SFN (+SFN), according to the groups indicated. Each row in the heat map represents a single named gene. Red represents high expression, green designates low expression. b–e Scatter plots compared the normalized expression of genes in the array by plotting log10 transformed 2−ΔCt values between selected groups. The most significantly altered genes, with an arbitrary cutoff of fivefold in either direction (upregulated or downregulated), are identified by the name adjacent to the corresponding data point. The red arrow designates Cdkn2a (p16)

Fig. 6

Nrf2 status affects p16 expression and HDAC3 promoter interactions in mouse colon tumors. ap16 mRNA expression levels in mouse colon tumors were normalized to Actb. Data = mean ± SD (n = 3); *P < 0.05 for control diet vs. SFN treatment. b–e Representative immunohistochemical staining (×10) of p16 in mouse colon tumor and adjacent normal colon; tumor (T), normal (N). A magnified (×40) image is shown for each dotted box, with a 50-μm scale bar included in b, inset. f In vivo ChIP assays on mouse colon tumors, interrogating HDAC3 interactions on p16. Data = mean ± SD and are representative of three independent experiments. A region 10 Kb upstream of the p16 promoter, containing a Runx1 binding site, served as a negative control for the ChIP assays

Fig. 7

HDAC3 knockdown induces p16 expression in human colon cancer cells. a HCT116 cells were transfected with non-specific scrambled negative control siRNA (Neg siRNA), HDAC3 siRNA, or Keap1 siRNA for 48 h, followed by vehicle (−SFN) or 15-μM SFN treatment for 24 h. p16 mRNA levels were normalized to ACTB; data = mean ± SD (n = 3), *P < 0.05 compared to untreated Neg siRNA. b The corresponding whole cell lysates were immunoblotted for p16, HDAC3, HDAC6 and Nrf2. HDAC6 was unchanged and served as loading control. c The same samples were analyzed for changes in heme oxygenase-1 (HO1), a known Nrf2 target gene. d–e Confirmation of Keap1 and HDAC3 knockdown. Kelch-like ECH-associated protein 1 (Keap1)

Fig. 8

Human subjects given a broccoli sprout extract, or consuming high levels of cruciferous vegetables, have altered HDAC3 and p16 expression. a Human subjects (n = 5, each arm of the study) consumed a broccoli sprout extract supplement (BSE, 200 μmol SFN equivalent, IND #111736) or a placebo for 7 days, and blood was drawn at the times indicated. b Total levels of SFN and its metabolites in plasma (black symbols), and b, c HDAC3 and p16 protein expression changes in circulating PBMCs. Results are from a single volunteer and are representative of other subjects who took the BSE supplement. No such changes occurred after taking the placebo (data not shown). Individual SFN metabolites, noted in Additional file 3: Table S2, were determined as reported [27]. d Study design in screening colonoscopy patients. e Relative mRNA expression of p16 in subjects reporting low (0–1 servings/week) vs. high (>5 servings/week) cruciferous vegetable intake. f Immunoblotting of colon biopsies, arranged left to right according to cruciferous vegetable intake. g–i Associations between p16, HDAC3, AcH4K12, and Nrf2 normalized to β-actin (relative densitometry of proteins in panel f)

Fig. 9

Nrf2 and p16 are inversely associated in human colon tumors and normal colon. The Cancer Genome Atlas (TCGA, https://tcga-data.nci.nih.gov/tcga/) was mined for NFE2L2 (Nrf2) and CDKN2A (p16) mRNA expression data in colon tumors (black) and normal colon (red)