Obesity promotes colonic stem cell expansion during cancer initiation

Abstract

There is an urgent need to elucidate the mechanistic links between obesity and colon cancer. Convincing evidence for the role of Lgr5(+) stem cells in colon tumorigenesis has been established; however, the influence of obesity on stem cell maintenance is unknown. We assessed the effects of high fat (HF) feeding on colonic stem cell maintenance during cancer initiation (AOM induced) and the responsiveness of stem cells to adipokine signaling pathways. The number of colonic GFP(+) stem cells was significantly higher in the AOM-injected HF group compared to the LF group. The Lgr5(+) stem cells of the HF fed mice exhibited statistically significant increases in cell proliferation and decreases in apoptosis in response to AOM injection compared to the LF group. Colonic organoid cultures from lean mice treated with an adiponectin receptor agonist exhibited a reduction in Lgr5-GPF(+) stem cell number and an increase in apoptosis; however, this response was diminished in the organoid cultures from obese mice. These results suggest that the responsiveness of colonic stem cells to adiponectin in diet-induced obesity is impaired and may contribute to the stem cell accumulation observed in obesity.

Keywords: Adiponectin; Colon cancer; Lgr5 stem cells; Obesity.

Figure 1

Effect of high fat (HF) feeding and AOM treatment on colonic stem cells. (A) Total cells per crypt, (B) GFP-negative cells per crypt, (C) Lgr5-GFP⁺cells per crypt, and (D) the correlation between body weight and Lgr5-GFP⁺ stem cells. Mice were fed a low fat (LF) control diet or a HF diet for 12 wks then injected with AOM or saline. Data represent mean ± SEM (n=3-7 per group). Groups with a p-value <0.05 were considered statistically significant by Bonferroni’s multiple comparisons test following a two-way ANOVA.

Figure 2

Proliferation of Lgr5-GFP⁺ and GFP negative cells from obese (HF) mice 12 h after AOM-injection. Colon samples were fixed in 4% PFA and paraffin embedded. Levels of cell proliferation were measured by the EdU Click-It assay. Data are expressed as percentage of EdU-labeled cells relative to the total number of (A) Lgr5-GFP+ stem cells and (B) non-GFP cells. (C) Representative micrographs for EdU—Alexa-647-stained (pink) proliferating cells, Lgr5-GFP⁺-Alexa-488-stained (green) stem cells and nuclei (blue). Data represent mean ± SEM (n=3-7 per group). Groups with a p-value <0.05 were considered statistically significant by Bonferroni’s multiple comparisons test following a two-way ANOVA.

Figure 3

AOM-induced apoptosis is increased in Lgr5-GFP⁺ colonic stem cells. Colon samples were fixed in 4% PFA and paraffin embedded. Cell apoptosis was measured by the Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Data are expressed as the percentage of apoptotic cells relative to the total number of (A) Lgr5-GFP⁺ stem cells and (B) non-GFP cells. (C) Representative micrographs for TUNEL—Alexa-647-stained (pink) apoptotic cells, Lgr5-GFP⁺ -Alexa-488-stained (green) stem cells and nuclei (blue). Data represent mean ± SEM (n=3-7 per group). Groups with a p-value <0.05 were considered statistically significant by Bonferroni’s multiple comparisons test following a two-way ANOVA.

Figure 4

(A) Gene expression of adipokine receptors and several downstream targets identified in stem cells by RNA-sequencing. Data represent mean RPKM values ± SEM (n=20 mice). Statistical difference between stem cell and non-stem cell populations was determined by the Student t-test. (B)Recombinant adiponectin and AdipoRon treatment reduces Lgr5-GFP+ stem cell number in colonic organoid cultures from healthy mice. Colonic crypts were isolated and pooled from regular Lgr5-EGFP-IRES-creER mice (n=2). Organoids were treated with varying doses recombinant leptin, adiponectin or AdipoRon for 48 hours and subsequently analyzed for the percentage of Lgr5-GFP⁺ stem cells by flow cytometry. Data represent mean ± SEM (treatments were performed in triplicate). Groups with a p-value <0.05 were considered statistically significant by Bonferroni’s multiple comparisons test following a one-way ANOVA.

Figure 5

AdipoRon-induced colonic cell apoptosis and reduction of Lgr5-GFP⁺ stem cell numbers is blunted in organoids cultured from obese (HF) mice. Colonic crypts were isolated from LF (n=3) and HF (n=3) fed Lgr5-EGFP-IRES-creER mice. Organoid cultures from each mouse were treated in triplicate with 10 uM AdipoRon for 48 h and subsequently analyzed for (A) apoptosis and (B) the percentage of Lgr5-GFP⁺ stem cells by flow cytometry. Data represent mean ± SEM. Groups with a p-value <0.05 were considered statistically significant by Bonferroni’s multiple comparisons test following a two-way ANOVA.