Estrogen receptor beta transcriptional regulation : a potential mechanism for colon cancer prevention

Abstract

Colorectal cancer (CRC) is the third leading cause of death from cancer in both men and women in the Western world. Improved screening efforts, surveillance, and treatment have reduced CRC mortality in older patients. However, the incidence is increasing in young adults, even in the absence of CRC family history. This may indicate an influence of increasing obesity, changed dietary patterns, and lifestyle factors. The progression of CRC is a multistep procedure that takes 10-15 years, thus offering a time to implement preventative measures and early detection. There is a critical need to develop more effective preventive therapies due to the risks posed by current prevention therapies. The best CRC prophylactic agent should be both safe and suitable to use for a long time (1). In preclinical studies, estrogen has been shown to protect from CRC, and substantial evidence suggests it is through estrogen receptor beta (ERβ). Natural ERβ selective agonists have been tested in phase II clinical trials to treat menopause symptoms and proven to be safe and welltolerated with no side effects (2, 3). Thus, selective activation of ERβ with selective agonists, which do not activate estrogen receptor alpha (ERα), is a potential clinical approach in preventing adenomatous polyps progression into CRC. However, the mechanism of these beneficial ERβ effects is not well understood, and there is a significant knowledge gap in this area.

The overall aim of this thesis was to identify the mechanistic background of the intestinal ERβ- mediated antitumorigenic effects in the colon and further to explore ERβ as a preventative approach in CRC. One specific aim was to determine whether ERβ present specifically in colon epithelium is responsible for protecting from CRC, which is addressed in Paper I. To understand the impact of ERβ in protecting from colitis-associated CRC (CA-CRC), we have induced colitis in intestinal-specific ERβ knockout mice of both sexes. The loss of intestinal ERβ aggravated CA-CRC in a sex-dependent manner. The incidence of tumors increased in males, while in females, the size of the tumors was enhanced. We identified that ERβ attenuates tumor necrosis factor alpha (TNFα) induced epithelial cell damage and modulates the regulation of key nuclear factor-κB (NFκB) members. As a direct consequence, ERβ was found to reduce inflammation and to control intestinal crypt cell proliferation.

Another aim was to explore transcriptional regulation by ERβ through mapping of chromatin binding sites and interaction with NFκB, which is studied in Paper II and IV. Commonly used ERβ antibodies have been shown to be unspecific towards ERβ; this study used a validated ERβ antibody to map genome-wide ERβ binding sites in colon cancer cells. We observed that the presence of ERβ also modulated the regulatory chromatin mark H3K27AC in potential enhancers of transcriptional regulation, Wnt signaling, and cell proliferation. Further, motif analysis indicated a novel ERβ colon-specific cross-talk with TCF, and KLF motifs supported a interaction between β-catenin/TCF and ERβ. We found that ERβ binds and regulates several important tumor suppressors and oncogenes in CRC cells, such as CST5 and LRP6, consistent with its proposed antitumorigenic activity. We also revealed the p65 cistrome in CRC cell lines and showed that ERβ alters the p65 chromatin binding in a cell-type-dependent manner. We found that ERβ chromatin binding sites were enriched among circadian clock genes and also that ERβ modulates p65 binding to core clock genes in CRC cells, indicating potential crosstalk between ERβ and circadian clock gene regulation.

The final aim was to investigate the impact of ERβ, and estrogen signaling in high-fat diet (HFD) induced inflammation in colon, explored in paper III. We fed mice with an HFD for 13 weeks and treated them with estrogenic ligands for the last three weeks prior to sacrifice. The colon transcriptome showed predominant sex differences, and selective activation of ERβ reduced macrophage infiltration and epithelial cell proliferation induced by HFD. We demonstrated that ERβ opposes HFD-induced dysregulation of core circadian clock genes in vivo, further strengthening the role of ERβ in circadian rhythm. Taken together, these results highlight the chemopreventive potential of ERβ in CRC in both sexes. The identified cross-talk with TNFα/NFκB pathway, Wnt signaling, regulating genes involved circadian clock, and tumorigenesis reflected ERβ protection/antitumor activity against CRC progression and development (as illustrated in Figure 1 in thesis).