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. 2017 Jun 13;8(24):38351-38366.
doi: 10.18632/oncotarget.16323.

Histone Hypoacetylation Contributes to CXCL12 Downregulation in Colon Cancer: Impact on Tumor Growth and Cell Migration

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Free PMC article

Histone Hypoacetylation Contributes to CXCL12 Downregulation in Colon Cancer: Impact on Tumor Growth and Cell Migration

Benoît Romain et al. Oncotarget. .
Free PMC article

Abstract

CXCL12 has been shown to be involved in colon cancer metastasis, but its expression level and molecular mechanisms regulating its expression remain controversial. We thus evaluated CXCL12 expression in a large cohort of colon adenomas and carcinomas, investigated for an epigenetic mechanism controlling its expression and evaluated the impact of CXCL12 levels on cell migration and tumor growth. CXCL12 expression was measured in human colon adenomas and carcinomas with transcriptome array and RT-qPCR. The promoter methylation was analyzed with whole-genome DNA methylation chips and protein expression by immunohistochemistry. We confirm a reduced expression of CXCL12 in 75% of MSS carcinomas and show that the decrease is an early event as already present in adenomas. The methylome analysis shows that the CXCL12 promoter is methylated in only 30% of microsatellite-stable tumors. In vitro, treatments with HDAC inhibitors, butyrate and valproate restored CXCL12 expression in three colon cell lines, increased acetylation of histone H3 within the CXCL12 promoter and inhibited cell migration. In vivo, valproate diminished (65%) the number of intestinal tumors in APC mutant mice, slowed down xenograft tumor growth concomitant to restored CXCL12 expression. Finally we identified loss of PCAF expression in tumor samples and showed that forced expression of PCAF in colon cancer cell lines restored CXCL12 expression. Thus, reduced PCAF expression may participate to CXCL12 promoter hypoacetylation and its subsequent loss of expression. Our study is of potential clinical interest because agents that promote or maintain histone acetylation through HDAC inhibition and/or HAT stimulation, may help to lower colon adenoma/carcinoma incidence, especially in high-risk families, or could be included in therapeutic protocols to treat advanced colon cancer.

Keywords: PCAF; acetylation; butyrate; chemokine; valproate.

Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare they have no competing interest.

Figures

Figure 1
Figure 1. CXCL12 mRNA expression distribution
(A) Boxplot of intensity values according to sample types (left) (47 adenomas, 75 MSI carcinomas, 444 MSS carcinomas, and 59 non-tumor tissues), colon cancer subtype (middle) (n = 566 MSS and MSI carcinomas of the discovery set) and CIMP status (right) (n = 496). (B) Distribution and discretization of CXCL12 expression established from 702 samples, including the 566 samples (19), 30 carcinomas from the GSE4183 data set, the 59 non-tumor tissues and 47 carcinomas of undefined phenotype. (Left) Density estimation of CXCL12 expression value with mclust approach defining two distinct distributions that correspond to a cut-off value of 7.8. (Right) Proportion of the defined discretization of CXCL12 expression within sample type, colon cancer subtype and CIMP status groups. Black boxes indicate % of samples not expressing CXCL12 mRNA; white boxes indicate the % of samples expressing CXCL12 mRNA.
Figure 2
Figure 2. CXCL12 protein and mRNA expression
Immunohistochemical expression in (AB) normal human colon mucosa, (C) an adenoma and a (D) carcinoma. (E–F) CXCL12 mRNA expression by RT-qPCR in human colon adenomas (E, n = 30) and carcinomas (F, n = 46). *Liver metastases. All quantifications were performed in duplicate in three independent experiments and normalized to the endogenous PDGF mRNA levels.
Figure 3
Figure 3
Immunohistochemical expression of CXCL12 in (A) normal mouse colon mucosa and (B) APCΔ14/+ mouse colon carcinoma.
Figure 4
Figure 4. Heatmap of methylation levels of CpG sites in the CXCL12 CpG island region
β-values of the 9 CpG sites in the CpG island associated with CXCL12 are represented in blue (low level of methylation) to yellow (high level of methylation). The values are ordered based on clusters separately defined by unsupervised hierarchical clustering in MSS and MSI tumors and by CpG site location distances from the transcription start site (from -1389 to 4556). The molecular annotations of the samples and their p-value of association with the cluster defined by Fisher's exact test are given at the top. At the bottom, the expression levels of CXCL12, the recoded up- or downregulated expression group as defined above and the genomic loss/gain of CXCL12 are displayed.
Figure 5
Figure 5
Strategies to re-express CXCL12 mRNA in SW480, HCT116 and TC7 cell lines after treatment for 24 h with (A) the natural short-chain fatty acid (SCFA) HDAC inhibitors Butyrate (10 mM) and propionate (15 mM) and (B) pharmacological HDAC inhibitors SAHA (S, 5 and 50 μM) and (C) VPA (1 mM). Results represent experiments run in triplicate with 4 wells per experiment.
Figure 6
Figure 6. Role of histone acetylation in the regulation of CXCL12 gene expression and functional study
(A) Representative experiment of butyrate- and VPA-induced histone H3 acetylation as quantified by western blotting in SW480, HCT116 and TC7 cell lines. (B) ChIP assays were performed on colon cells treated with but (10 mM) or VPA (1 mM) for 24 h, and the levels of the CXCL12 promoter in the immunoprecipitates were measured by RT-qPCR in SW480, HCT116 and TC7 cell lines.
Figure 7
Figure 7. Histone H3 acetylation and PCAF expression
(A) ChIP assays were performed on SW480 and HEK293 cell lines and the levels of the CXCL12 promoter in the immunoprecipitates were measured by RT-QPCR. (B) PCAF mRNA expression distribution. Boxplot of intensity values according to sample types (59 non-tumor tissues, 47 adenomas, 75 MSI carcinomas, 444 MSS carcinomas). (C) Correlation at the mRNA level (RT-qPCR) between CXL12 and PCAF expression in 26 colon tumor samples (black circles), a carcinoma expressing CXCL12 as in normal mucosa (open circle), two colon cancer cell lines (SW480 and HT29, open squares) and the human kidney embryonic cell line expressing CXCL12 (HEK293, black triangle). (D) Effect of PCAF forced-expression on CXCL12 in SW480 cells. PCAF and CXCL12 mRNA expression were evaluated 24 h after transfection with 3 μg of a control plasmid (SW480-EGFP) and a PCAF-expressing plasmid (SW480-PCAF). The mean Ct for CXCL12 and PCAF was normalized with PBGD and SW480 as controls and the results were expressed as 2−ΔΔCt. Experiments have been repeated in triplicate and results are representative of three sets of independent samples per group.
Figure 8
Figure 8. SW480 cell migration in Boyden chamber assays
Cells remaining in the upper chamber were mechanically removed after 24 h of incubation w/o or with 10% fetal calf serum, 50 nM CXCL12 and/or 10 mM but or 1 mM VPA. The cells that had migrated to the lower chamber were counted after staining with the fluorescent dye DAPI (or 4′,6-diamidino-2-phenylindole). Quantification was performed by counting five random fields for each chamber under a microscope (Mann-Whitney parametric test, **p < 0.001; ns: non-significant).
Figure 9
Figure 9. VPA treatment in a subcutaneous human colon tumor xenograft
(A) Tumor growth was measured daily before and throughout the intraperitoneal administration of VPA (500 mg/kg) for 30 days. Data represent the relative tumor volume plotted as a function of time. Five mice, each bearing two individual tumors, were included in each group. Mann-Whitney parametric test, **p < 0.02. (B) Immunohistochemical CXCL12 expression in human colon cancer xenografts treated with 500 mg/kg VPA for 30 days and control (untreated) xenografts. (C) Enlargement of a part of the pictures shown in B. The results represent five mice per group.
Figure 10
Figure 10. Intestinal CXCL12 expression and carcinoma development in control and VPA-treated APCΔ14/+ mice
(A) Quantitative evaluation of the number of carcinomas in the entire intestinal tract of control and VPA-treated APCΔ14/+ mice. The APCΔ14/+ mice were intraperitoneally injected with PBS (n = 6) or VPA (n = 5; 500 mg/kg/day) for 30 days. Mann-Whitney parametric test, **p < 0.001. (BD) Immuno-histochemical CXCL12 expression in the normal colon mucosa of control APCΔ14/+ mice (PBS treated, (B) in a VPA-untreated tumor of an APCΔ14/+ mouse (C) and in the colon of APCΔ14/+ mice treated with 500 mg/kg VPA for 30 days (D).

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