Estrogen signaling in colon inflammation and colorectal cancer

Colorectal cancer (CRC) is the third most deadly form of cancer in the Western world. Although screening efforts have reduced the overall mortality, the incidence is increasing among young adults. The frequency of inflammatory bowel disease (IBD) and obesity are increasing in parallel, which suggest a common underlying environmental link between the conditions. This increase is thought to correlate to an increased intake of high fat diets, and obesity is a major risk factor for CRC. Chronic inflammation, which is a hallmark for CRC promotion, is a well-known underlying factor in both obesity and IBD. The gut microbiota is another hallmark, and an impaired relationship between the host and gut microbes can contribute to obesity, IBD and CRC. The risk-benefit balance of current CRC-preventative treatments is poor, and there is a need for safer and better preventatives in order to reduce the CRC mortality. Both obesity and IBD place men at a significant higher risk of CRC compared to women. This indicates a protective role for estrogen. The use of full Estrogen receptor (ER) β knockout mice has demonstrated ERβ protective effects against experimentally induced CRC. However, it is unknown through which cells these protective effects are mediated. There are only low mRNA levels of ERβ in the colon, unclear if adequate for a functional role, and ERβ may also be expressed in intestinal immune cells. Understanding the CRC-preventative effects of intestinal epithelial ERβ in both sexes is important and may provide the background for a novel CRC chemo-preventive approach.

The overall aim of the thesis is the functional characterization of intestinal epithelial ERβ during colon inflammation and colitis-induced CRC and identification of potential sex differences, which can ultimately provide novel opportunities for chemo-preventive exploitation (Figure 1). In paper I we utilized intestinal epithelial ERβ knockout mice (ERβKOVil) of both sexes and induced colitis and colitis associated CRC (CA-CRC). We found that intestinal epithelial ERβ is protective against colitis and CA-CRC in both sexes, but in a sex-dependent manner. The underlying mechanism includes an intricate crosstalk with TNFα- induced NFκB signaling. In paper II we identify that both sex and intestinal epithelial ERβ impact the microbiota composition. This may contribute to the exacerbated colitis and colitis-induced tumor formation observed in ERβKOVil mice. In paper III we induced colon inflammation by feeding the mice a high-fat diet (HFD, 60%) for 13 weeks and explored treatment with estrogen receptor-selective ligands. We identified that estrogen signaling, via ERβ, modulated the HFD-induced changes in the colon microenvironment. This included sex-dependent effects on epithelial cell proliferation, macrophage infiltration, and regulation of core circadian clock gene expression. In paper IV we utilized paired-normal and CRC clinical samples and identified sex differences in the transcriptome of both normal colon and CRC. By applying data-driven feature selection and machine learning on sex-separated TCGA data, we proposed sex-specific diagnostic biomarkers and prognostic biomarkers using survival analysis.

In summary, this thesis characterizes intestinal epithelial ERβ as a novel chemo-preventative target for CA-CRC in both sexes, and identifies related biological pathways, including crosstalk with nuclear factor κB (NFκB) signaling and modulation of circadian clock genes. ERβ activity in intestinal epithelial cells is manifested by altered microbiota composition, cell proliferation and immune cell infiltration. The identification of several significant sex differences provides evidence for the need to take sex into account in colitis and CRC research to improve health interventions.

List of scientific papers

I. Hases L, Indukuri R, Birgersson M, Nguyen-Vu T, Lozano R, Saxena A, Hartman, J, Frasor J, Gustafsson JÅ, Katajisto P, Archer A, Williams C. Intestinal estrogen receptor beta suppresses colon inflammation and tumorigenesis in both sexes. (2020) Cancer Letters. 492:54-62.
https://doi.org/10.1016/j.canlet.2020.06.021

II. Ibrahim A, Hugerth LW, Hases L, Saxena A, Seifert M, Thomas Q, Gustafsson, JÅ, Engstrand L, Williams C. Colitis-induced colorectal cancer and intestinal epithelial estrogen receptor beta impact gut microbiota diversity. (2019) Int J Cancer. 144:3086-3098.
https://doi.org/10.1002/ijc.32037

III. Hases L*, Archer A*, Indukuri R, Birgersson M, Savva C, Korach-André M, Williams C. High-fat diet and estrogen impact the colon and its transcriptome in a sex-dependent manner. (2020) Scientific Reports. *Equal contribution.
https://doi.org/10.1038/s41598-020-73166-1

IV. Hases L, Ibrahim A, Birgersson M, Liu Y, Hartman J, Williams C. The importance of sex in colorectal cancer biomarker discovery. [Manuscript]