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

Time: Fri 2021-05-28 09.30

Location:, Stockholm (English)

Subject area: Medical Technology

Doctoral student: Rajitha Indukuri , Cellulär och klinisk proteomik, Science for Life Laboratory, SciLifeLab, Karolinska Institute

Opponent: Professor Jason Carroll, University of Cambridge, UK

Supervisor: Professor Cecilia Williams, Science for Life Laboratory, SciLifeLab, Cellulär och klinisk proteomik, Karolinska Institute

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Colorectal cancer (CRC) is the third leading cause of death from cancer in both men andwomen in the Western world. Improved screening efforts, surveillance, and treatment havereduced 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 increasingobesity, changed dietary patterns, and lifestyle factors. The progression of CRC is a multistepprocedure that takes 10-15 years, thus offering a time to implement preventative measures andearly detection. There is a critical need to develop more effective preventive therapies due tothe risks posed by current prevention therapies. The best CRC prophylactic agent should beboth safe and suitable to use for a long time (1).

In preclinical studies, estrogen has been shown to protect from CRC, and substantial evidencesuggests it is through estrogen receptor beta (ERβ). Natural ERβ selective agonists have beentested 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 inpreventing adenomatous polyps progression into CRC. However, the mechanism of thesebeneficial ERβ effects is not well understood, and there is a significant knowledge gap in thisarea.

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

Another aim was to explore transcriptional regulation by ERβ through mapping of chromatinbinding sites and interaction with NFκB, which is studied in Paper II and IV. Commonly usedERβ antibodies have been shown to be unspecific towards ERβ; this study used a validatedERβ antibody to map genome-wide ERβ binding sites in colon cancer cells. We observed thatthe presence of ERβ also modulated the regulatory chromatin mark H3K27AC in potentialenhancers of transcriptional regulation, Wnt signaling, and cell proliferation. Further, motifanalysis indicated a novel ERβ colon-specific cross-talk with TCF, and KLF motifs supporteda interaction between β-catenin/TCF and ERβ. We found that ERβ binds and regulates severalimportant tumor suppressors and oncogenes in CRC cells, such as CST5 and LRP6, consistentwith 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. Wefound that ERβ chromatin binding sites were enriched among circadian clock genes and alsothat 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 13weeks and treated them with estrogenic ligands for the last three weeks prior to sacrifice. Thecolon transcriptome showed predominant sex differences, and selective activation of ERβreduced macrophage infiltration and epithelial cell proliferation induced by HFD. Wedemonstrated that ERβ opposes HFD-induced dysregulation of core circadian clock genes invivo, further strengthening the role of ERβ in circadian rhythm.

Taken together, these results highlight the chemopreventive potential of ERβ in CRC in bothsexes. The identified cross-talk with TNFα/NFκB pathway, Wnt signaling, regulating genesinvolved circadian clock, and tumorigenesis reflected ERβ protection/antitumor activityagainst CRC progression and development (as illustrated in Figure 1).