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. 2016 Dec;94(12):1385-1395.
doi: 10.1007/s00109-016-1447-7. Epub 2016 Aug 13.

C/EBPβ Regulates Homeostatic and Oncogenic Gastric Cell Proliferation

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Free PMC article

C/EBPβ Regulates Homeostatic and Oncogenic Gastric Cell Proliferation

Goncalo Regalo et al. J Mol Med (Berl). .
Free PMC article

Abstract

Cancer of the stomach is among the leading causes of death from cancer worldwide. The transcription factor C/EBPβ is frequently overexpressed in gastric cancer and associated with the suppression of the differentiation marker TFF1. We show that the murine C/EBPβ knockout stomach displays unbalanced homeostasis and reduced cell proliferation and that tumorigenesis of human gastric cancer xenograft is inhibited by knockdown of C/EBPβ. Cross-species comparison of gene expression profiles between C/EBPβ-deficient murine stomach and human gastric cancer revealed a subset of tumors with a C/EBPβ signature. Within this signature, the RUNX1t1 tumor suppressor transcript was down-regulated in 38 % of gastric tumor samples. The RUNX1t1 promoter was frequently hypermethylated and ectopic expression of RUNX1t1 in gastric cancer cells inhibited proliferation and enhanced TFF1 expression. These data suggest that the tumor suppressor activity of both RUNX1t1 and TFF1 are mechanistically connected to C/EBPβ and that cross-regulation between C/EBPβ-RUNX1t1-TFF1 plays an important role in gastric carcinogenesis.

Key message: C/EBPβ controls proliferation and differentiation balance in the stomach. Homeostatic differentiation/proliferation balance is altered in gastric cancer. RUNX1t1 is a C/EBPβ-associated tumor suppressor. RUNX1t1 negatively regulates C/EBPβ pro-oncogenic functions.

Keywords: C/EBPβ; Gastric cancer; Homeostasis; Proliferation; RUNX1t1; TFF1.

Conflict of interest statement

The authors declare no conflict of interests related to this study.

Figures

Fig. 1
Fig. 1
C/EBPβ controls gastric cancer cell proliferation. a Stable knockdown (KO) of C/EBPβ in MKN74 cell line evaluated by protein blotting. b Cell proliferation was determined by BrdU analysis. Cells were labeled with BrdU and incorporation was determined by flow cytometry (FACS) and plotted against 7-AAD-positive cells, as a measure of DNA content. c Expression of the gastric differentiation marker TFF1 was assessed on the stably transfected cells. Expression was increased in MKN74. d Equal amounts of control and stable C/EBPβ KO MKN74 cells were injected into nude mice and tumor volume and weight was assessed at different time points. Tumors originated from C/EBPβ KO cells were smaller than tumors in the controls. e Ki67 staining revealed reduction of proliferation in the KO-derived tumors. All bar graphs represent the result of at least three independent measurements; asterisk indicates p < 0.05
Fig. 2
Fig. 2
Analysis of the gastric phenotype of the C/EBPβ knockout (KO) mouse. a Quantification of the C/EBPβ KO mice and WT antral gastric mucosa thickness (in arbitrary units). Adjacent immunohistochemical panel depicts the reduction of Ki67-positive cells in the C/EBPβ KO mucosa. Lower panels show qPCR evaluation of Ki67, PCNA, Cyclin A1, D3, E1, and p15 in the gastric mucosa of WT and C/EBPβ KO mouse stomach (five animals/group, 3 months old). Expression values were first normalized to GAPDH expression and values are presented as fold of WT expression. b Mutually exclusive expression of TFF1 and C/EBPβ in the normal human (upper panel) and mouse (lower panel) stomach epithelium; C/EBPβ is expressed in proliferative cells of the neck zone and TFF1 in differentiated mucous epithelium. c Increased expression of mRNA of differentiation protein TFF1, in the C/EBPβ KO mouse mucosa as measured by qPCR. Lower panel show ChIP assay on disaggregated wt mouse stomach cells, showing in vivo binding of C/EBPβ to the TFF1 promoter both on an agarose gel (left) and by qPCR quantification. Results are presented as ratio to anti-IgG control binding. Binding to the Muc5ac promoter was used as a negative control. All bar graphs represent the result of at least three independent measurements; asterisk indicates p < 0.005
Fig. 3
Fig. 3
a Cross-species comparison of gene expression. Two-way hierarchical clustering was performed on the human gastric cancer samples using a strongly regulated gene cluster (shown in Supplementary Fig. 2) from microarray-derived genes that differed between murine C/EBPβ KO and WT stomach (p ≤ 0.01, FC ≥ 1.5). Depicted are the resultant gene and sample dendrograms and the corresponding expression intensity heatmap. The black box indicates a tumor cluster in which most of the genes show down-regulation (bluish spots). This tumor group consisted of 16 of the original 59 (≈27 %) samples and contained primarily cancers of the intestinal histological type. b Transfection of C/EBPβ isoforms LAP*, LAP, and LIP into gastric cell lines MKN28 and MKN45 repressed RUNX1t1 expression as measured by quantitative PCR. All bar graphs represent the result of at least three independent measurements; asterisk indicates p < 0.001
Fig. 4
Fig. 4
RUNX1t1 and gastric cancer. a RUNX1t1 expression was evaluated by immunohistochemistry in 64 human gastric cancer samples, and staining was classified by comparison to the expression in the normal mucosa (left panel). Thirty-eight percent of the cases showed reduced expression of nuclear RUNX1t1 (Tumor 1–3) in comparison to staining in the normal epithelium. b In 10 gastric tumors with reduced RUNX1t1, RNA levels were examined for C/EBPβ expression by qPCR. Only 3 out of 10 cases showed higher C/EBPβ expression as compared to WT. c The methylation status of the RUNX1t1 promoter was evaluated by methylation-specific PCR. Bisulfite treatment of tumor DNA converts unmethylated but not methylated cytosines to uracil, and subsequent methylation-specific PCR detects either methylated (M) or unmethylated (U) DNA. Fifty percent of the analyzed human gastric cancer cases (rows a-b, columns 1–5) present RUNX1t1 promoter hypermethylation. An increase in the methylation status is considered when the PCR product with methylation-specific primers is more intense than the one produced by non-methylated specific primers. d Ectopic expression of RUNX1t1 in MKN28 and MKN45 gastric cancer cell lines reduces gastric cancer cell proliferation as measured by BrdU incorporation assay. S-phase percentages are indicated in the FACS plots
Fig. 5
Fig. 5
RUNX1t1 modulates C/EBPβ activity. a Immunostaining shows C/EBPβ and RUNX1t1 colocalization in the neck zone of the normal human gastric mucosa. b Flag-Immunoprecipitation after transfection of gastric cell lines with a flag-tagged RUNX1t1 pulls down C/EBPβ. Visible in the input Western blot is also that RUNX1t1 does not affect C/EBPβ expression. c EMSA using a radiolabeled C/EBPβ consensus probe shows that transfection of RUNX1t1 to gastric cancer cells reduces the binding of C/EBPβ to DNA in MKN28 and MKN45 cell lines. Arrow indicated the super-shift. Also visible in the control Western blots, RUNX1t1 has no effect on basal C/EBPβ expression. d A RUNX1t1-dependent increase in the expression of TFF1 was visible by real-time PCR in the MKN74 cell line. e Luciferase assay in MKN45 cells transfected with the TFF1-luciferase fused promoter shows that co-transfection with RUNX1t1 reverts the repressive potential of C/EBPβ on the TFF1 promoter

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