Estrogen receptor β induces antiinflammatory and antitumorigenic networks in colon cancer cells

Abstract

Several studies suggest estrogen to be protective against the development of colon cancer. Estrogen receptor β (ERβ) is the predominant estrogen receptor expressed in colorectal epithelium and is the main candidate to mediate the protective effects. We have previously shown that expression of ERβ reduces growth of colorectal cancer in xenografts. Little is known of the actions of ERβ and its effect on gene transcription in colon cancers. To dissect the processes that ERβ mediates and to investigate cell-specific mechanisms, we reexpressed ERβ in three colorectal cancer cell lines (SW480, HT29, and HCT-116) and conducted genome-wide expression studies in combination with gene-pathway analyses and cross-correlation to ERβ-chromatin-binding sites. Although induced gene regulation was cell specific, overrepresentation analysis of functional classes indicated that the same biological themes, including apoptosis, cell differentiation, and regulation of the cell cycle, were affected in all three cell lines. Novel findings include a strong ERβ-mediated down-regulation of IL-6 and downstream networks with significant implications for inflammatory mechanisms involved in colon carcinogenesis. We also discovered cross talk between the suggested nuclear receptor coregulator PROX1 and ERβ, demonstrating that ERβ both regulates and shares target genes with PROX1. The influence of ERβ on apoptosis was further explored using functional studies, which suggested an increased DNA-repair capacity. We conclude that reexpression of ERβ induces transcriptome changes that, through several parallel pathways, converge into antitumorigenic capabilities in all three cell lines. We propose that enhancing ERβ action has potential as a novel therapeutic approach for prevention and/or treatment of colon cancer.

Fig. 1.

Levels of ERβ in the three colon cancer cell lines. A, Real-time PCR showing transcript levels of ERβ in three separately ERβ transfected clone mixes for each cell line. B, Flag-ERβ Western blot. rERβ has a molecular weight of 59 kb, actin has a molecular weight of 42 kb. Not many differentially expressed genes were commonly regulated in the three colon cancer cell lines as illustrated by the Venn diagram (panel C) and Heatmap (panel D) showing hierarchical clustering of the 2000 genes with highest B value from each array experiment. Clustering of rows and columns was made with average linkage of Pearson correlations.

Fig. 2.

Comparison of our microarray data with ERβ-promoter-binding data showed that ERβ binds directly to the three transcription factors MYC, MYB, and RUNX2. Bioinformatics analysis showed that these, in turn, regulate several of the genes observed on the array. A combination of change in primary, secondary, tertiary, and further downstream genes leads to downstream effects such as decreased cell proliferation and apoptosis. Red genes are up-regulated on the microarray; blue genes are down-regulated. The intensity of color is related to fold change of the gene transcript from our array study: stronger color is more significant. Real-time PCR confirmations to the right, y-axis: relative expression (fold change).

Fig. 3.

PROX1 is involved carcinogenesis and cell proliferation in colorectal cancer. PROX1 is down-regulated by ERβ in SW480 at protein (panel A) and mRNA (panel B) levels. C, PROX1 ChIP real-time PCR where samples are normalized to IgG. Primers for pS2 3′-untranslated region (UTR) are used as a negative control (Ctrl). There is a statistically significant reduction (unpaired one-tailed t test) of CITED2 binding in ERβ cells compared with the control cells (PROX1). D, Several differentially expressed genes have previously been defined to be regulated in the same direction when PROX1 is silenced, indicating that regulation of these genes results from the ERβ-driven down-regulation of PROX1 (I–III). In addition, several ERβ-regulated genes have been shown to have binding sites for both ERβ and PROX1 (IV). Proteins in red are transcriptionally up-regulated, as a consequence of ERβ expression described in our study and in agreement with a study by Petrova et al. (24), describing results of PROX1 silencing. Proteins in blue are transcriptionally down-regulated. Blue arrow, PROX1 transcriptional regulation. Dark green arrow with blue filling, PROX1 DNA binding and transcriptional regulation. Green arrow, ERβ DNA binding.

Fig. 4.

Treatment (24 h) with cisplatin in SW480 control cells and ERβ-expressing cells. A higher percentage of attached control cells were dead after cisplatin treatment compared with ERβ-expressing cells, indicating that ERβ is protective against DNA damage in these colon cancer cells. The protective effect against DNA damage in ERβ cells is most likely a combination of lowered cell proliferation and increased DNA repair. DMSO. Dimethylsulfoxide; NS, nonsignificant (P = 0.201).