The Warburg effect dictates the mechanism of butyrate-mediated histone acetylation and cell proliferation

Abstract

Widespread changes in gene expression drive tumorigenesis, yet our knowledge of how aberrant epigenomic and transcriptome profiles arise in cancer cells is poorly understood. Here, we demonstrate that metabolic transformation plays an important role. Butyrate is the primary energy source of normal colonocytes and is metabolized to acetyl-CoA, which was shown to be important not only for energetics but also for HAT activity. Due to the Warburg effect, cancerous colonocytes rely on glucose as their primary energy source, so butyrate accumulated and functioned as an HDAC inhibitor. Although both mechanisms increased histone acetylation, different target genes were upregulated. Consequently, butyrate stimulated the proliferation of normal colonocytes and cancerous colonocytes when the Warburg effect was prevented from occurring, whereas it inhibited the proliferation of cancerous colonocytes undergoing the Warburg effect. These findings link a common metabolite to epigenetic mechanisms that are differentially utilized by normal and cancerous cells because of their inherent metabolic differences.

Figure 1. Prevention of the Warburg Effect in Colon Carcinoma Cells

(A and B) Glucose-6-phosphate (A) and lactate (B) levels in HCT116 colon cancer cells grown in low or high glucose conditions and in noncancerous fetal human colonocyte (FHC) epithelial cells. Results are from 3 independent experiments and are presented as mean ± SE with significant differences indicated (***p < 0.001). (C) Confocal microscopic images of colonocytes showing MitoTracker Red as a measure of oxidative metabolism (top row), MitoTracker Green as a measure of total mitochondria (middle row), and both MitoTracker probes merged (bottom row). The first four columns correspond to HCT116 cells grown in low or high glucose as indicated. The final three columns are two positive controls consisting of noncancerous human and mouse colonocytes followed by a negative control where the mouse colonocytes were treated with sodium azide to block oxidative metabolism. (D) Quantification of oxidative metabolism. Results are based on 20 cells per condition from 3 independent experiments and represent the mean ± SE with significant differences indicated (**p<0.01; n.s., not significant). (E) Oxygen consumption rates (OCR) for HCT116 cells grown in low or high glucose conditions and in FHC epithelial cells over a 1 hr period. (F) Western blot analysis of lactate dehydrogenase A (LDHA) levels in HCT116 cells at two timepoints (48h and 72h) following RNAi (siLDHA) and in control cells (siMock). Tubulin (lower panel) serves as a loading control. (G) Lactate levels in siLDHA and siMock HCT116 cells. Results are from 3 independent experiments and are presented as mean ± SE with significant differences indicated (***p < 0.001).

Figure 2. Butyrate-Induced Changes in Cell Proliferation and Apoptosis

(A) Butyrate levels in the lumen of proximal, medial, and distal segments of mouse colons. (B) A model showing two butyrate gradients in the mammalian colon. The proximal-to-distal luminal gradient arises because most bacterial fermentation occurs in the proximal colon and butyrate that is not absorbed by proximal colonocytes moves distally with luminal contents due to peristalsis. The luminal-to-crypt gradient arises because of peristalsis and the upward flow of mucous produced by goblet cells as it is sloughed away from the epithelium into the lumen. (C and D) Changes in HCT116 cell number (C) and BrdU incorporation (D) in response to 3 days of butyrate or TSA treatment relative to untreated control cells. Cells were grown in high glucose to facilitate the Warburg effect or either low glucose or siLDHA conditions (but in high glucose) to prevent the Warburg effect from occurring. In panel A, the histograms at the far left represent the growth of untreated control cells over the 3-day period (set at 100%), and the other histograms with SE bars represent the corresponding changes in cell growth relative to these controls. The histograms at the right that show negative values in panel A have decreased numbers of cells compared to the start of the 3-day culture period. Results are from 3 independent experiments. (E) Changes in BrdU incorporation in response to 3 days of butyrate in FHC epithelial cells grown in high glucose. Results are presented as mean ± SE from 3 independent experiments with significant differences indicated (*p < 0.05). (F) Flow cytometry displaying annexin V (x-axis) and propidium iodide (y-axis) levels in HCT116 cells grown in the presence (top row) or absence (bottom row) of the Warburg effect as indicated at the left. Cells were untreated or treated with either butyrate or TSA (from left to right, as indicated at the top). The percentage of cells positive or negative for annexin V and PI are indicated within the quadrants. Results are representative of 3 independent experiments.

Figure 3. The Warburg Effect Influences Energetic and Epigenetic Mechanisms that Regulate Cell Proliferation and Histone Acetylation

(A and B) Changes in BrdU incorporation in HCT116 cells in response to butyrate or TSA treatment relative to untreated control cells in the absence (A) or presence (B) of the Warburg effect. The effect of etomoxir and vehicle on BrdU incorporation was evaluated under each condition. Results in each panel are from 3 independent experiments and are presented as mean ± SE with significant differences indicated (*p < 0.05; **p < 0.01 compared to no-butyrate control; n.s., not significant). (C) Western blot analysis showing global H3ac levels (top panels) in HCT116 cells in the presence (left panel) or absence (right panel) of the Warburg effect. Cells were either untreated or treated with butyrate as indicated at the bottom. Total H3 (middle panels) and β-actin (bottom panels) served as loading controls. (D) Western blot analysis of H3ac (top panel), total H3 (middle panel), and β-actin loading control (bottom panel) in fetal FHC epithelial cells that were untreated or treated with butyrate as indicated. (E) Western blot analysis of acetyl-p53 (Ac-p53) and acetyl-tubulin (Ac-Tubulin) in HCT116 cells in response to butyrate in the presence or absence of the Warburg effect. p53, β-actin, and tubulin were analyzed as loading controls. The p53 experiments were performed on HCT116 cells following exposure to ionizing radiation. (F) ¹³C₄-butyrate concentration within nuclei isolated from HCT116 cells that were untreated (0.0 at bottom) or treated with ¹³C₄-butyrate (at 0.5- or 5-mM as indicated at the bottom). Cells were grown in the presence or absence of the Warburg effect as indicated. Measurements were based on analysis of LC-MS spectra from three independent experiments and are presented as mean ± SE. The limit of detection for ¹³C₄-butyrate was 0.5 µM.

Figure 4. Butyrate Increases Histone Acetylation in an ACL-Dependent Manner

(A) A model for two butyrate-induced histone acetylation mechanisms. In addition to acting as an HDAC inhibitor (depicted by the horizontal line at the bottom), butyrate can act as an acetyl-CoA donor and stimulate HAT activity (depicted by the vertical line pointing downward at the right). See text for a detailed description. (B) Total ACL protein levels in HCT116 cells grown in the presence (+) or absence (−) of the Warburg effect for 3 days. ACL levels were normalized to β-actin and are presented as mean relative units ± SE (*p < 0.05) based on 3 independent experiments. (C) Western blot analysis for ACL (top panels), β-actin loading control, H3ac, and total H3 (bottom panels) in siMock (left) and siACL (right) HCT116 cells that were untreated or treated with butyrate as indicated at the bottom. (D) Quantification of the ACL dependence versus independence of butyrate-induced H3ac from western blots. H3ac levels were normalized using β-actin. Each butyrate treatment had the untreated value subtracted, and the siACL/siMock ratio was computed. Results are from 3 independent experiments. (E) Western blot analysis of ACL phosphorylated at Ser454 (top panel), β-actin loading control (middle panel), and H3ac (bottom panel) in primary colonocytes freshly isolated from C57BL/6 mice. Colonocytes were treated with butyrate and/or radicicol (as indicated at the bottom) as they were being harvested over a 60-minute period. (F) Western blot analysis for ACL (top panel), β-actin loading control (middle panel), and H3ac (bottom panel) in siMock and siACL HCT116 cells that were treated with vehicle or TSA as indicated at the bottom.

Figure 5. Butyrate is a Carbon Donor for Acetyl-CoA and Histone Acetylation

(A and B) Nuclear acetyl-CoA levels (A) and HAT activity (B) in siMock and siACL (as indicated at the top) HCT116 cells. Cells were untreated or treated with butyrate as indicated at the bottom. Results are from 3 independent experiments and are presented as mean ± SE with significant differences indicated (*p < 0.05; **p < 0.01 compared to no-butyrate control; n.s., not significant). (C) LC-MS chromatograms of hydrolyzed histones from HCT116 cells treated with 0.5 mM of uniformly labeled ¹³C₄-butyrate for 3 days. The ¹³C₂-acetate peak is larger in siMock cells (top spectrum) than siACL cells (bottom spectrum) as indicated by the numbers, which correspond to the area under the peaks. (D) Quantification of ¹³C₂-acetate levels from hydrolyzed histones isolated from untreated or ¹³C₄-butyrate-treated siMock and siACL HCT116 cells. The results are normalized to histone levels (as indicated on the y axis) and are presented as the mean ± SE from 3 independent experiments.

Figure 6. Butyrate Regulates Gene Expression Using ACL-Dependent and -Independent Mechanisms

(A) Western blot analyses of HCT116 cells used for transcriptome profiling. ACL (top panel) and β-actin loading control (bottom panel) levels are shown in siMock and siACL cells in response to different butyrate treatments. (B) Venn diagram showing the overlap between genes upregulated by butyrate at doses of 0.5, 2, and 5 mM. (C) Pie charts showing the percentage of genes upregulated by 0.5-mM (left), 2-mM (middle), and 5-mM (right) butyrate in an ACL-dependent (white) and -independent (gray) manner. Beneath the 2 mM and 5 mM pie charts are two additional pie charts. The chart shown on the left corresponds to genes that were also upregulated at the previous dose (shared). The chart shown on the right corresponds to genes that were not upregulated at the previous dose (newly induced).

Figure 7. Butyrate Induces Histone Acetylation by Dual Mechanisms

(A, B) Quantitative ChIP assays showing H3ac enrichment at the promoters of genes in HCT116 cells that were untreated or treated with butyrate as indicated. In panel A, the most-highly induced cell proliferation (left) and apoptotic (right) genes are shown. In panel B, the assays were performed under siMock and siACL conditions. The first 4 panels correspond to genes (SLC22A17, SLC36A1, GPRIN1, ADPRHL1) whose expression switched from ACL dependence at 0.5 mM to ACL independence at 5 mM. The final gene (GPATCH8) was expressed in an ACL-dependent manner at both doses. qPCR results for each ChIP were normalized to input, and the values of the untreated samples (0-mM butyrate) were set at 1.0. The 0.5- and 5-mM butyrate-treated samples are presented as histograms that show H3ac enrichment relative to the untreated controls. Each histogram represents the mean ± SE for 3 independent experiments. (C) A model depicting how butyrate might regulate histone acetylation and cellular turnover in the colonic epithelium. Butyrate is produced by microbiota in the proximal lumen, and mucous flow and peristalsis result in butyrate concentration gradients shown as two shaded bars. Near the crypt base, where butyrate concentrations are relatively low (0.5 mM), most of the butyrate molecules are metabolized and contribute to histone acetylation by the acetyl-CoA/HAT mechanism. This is associated with the proliferation of mitotically-active colonocytes. Near the lumen, butyrate concentrations are relatively high (5 mM) and exceed the concentration (1–2 mM) that can be metabolized efficiently. As a result, butyrate accumulates in the nucleus and acts as an HDAC inhibitor (HDACi). These colonocytes are not mitotically active, undergo apoptosis, and exfoliate into the lumen. Due to the Warburg effect, cancerous colonocytes (labeled with a star) metabolize relatively little butyrate regardless of their position within the epithelium. Consequently, the HDAC inhibition mechanism predominates, and butyrate is associated with decreased proliferation and increased apoptosis.