ST-Producing E. coli Oppose Carcinogen-Induced Colorectal Tumorigenesis in Mice

Abstract

There is a geographic inequality in the incidence of colorectal cancer, lowest in developing countries, and greatest in developed countries. This disparity suggests an environmental contribution to cancer resistance in endemic populations. Enterotoxigenic bacteria associated with diarrheal disease are prevalent in developing countries, including enterotoxigenic E. coli (ETEC) producing heat-stable enterotoxins (STs). STs are peptides that are structurally homologous to paracrine hormones that regulate the intestinal guanylyl cyclase C (GUCY2C) receptor. Beyond secretion, GUCY2C is a tumor suppressor universally silenced by loss of expression of its paracrine hormone during carcinogenesis. Thus, the geographic imbalance in colorectal cancer, in part, may reflect chronic exposure to ST-producing organisms that restore GUCY2C signaling silenced by hormone loss during transformation. Here, mice colonized for 18 weeks with control E. coli or those engineered to secrete ST exhibited normal growth, with comparable weight gain and normal stool water content, without evidence of secretory diarrhea. Enterotoxin-producing, but not control, E. coli, generated ST that activated colonic GUCY2C signaling, cyclic guanosine monophosphate (cGMP) production, and cGMP-dependent protein phosphorylation in colonized mice. Moreover, mice colonized with ST-producing E. coli exhibited a 50% reduction in carcinogen-induced colorectal tumor burden. Thus, chronic colonization with ETEC producing ST could contribute to endemic cancer resistance in developing countries, reinforcing a novel paradigm of colorectal cancer chemoprevention with oral GUCY2C-targeted agents.

Keywords: GUCY2C-cGMP axis; azoxymethane; chemoprevention; colorectal cancer; enterotoxigenic E. coli; heat-stable enterotoxins.

Figure 1

Colonization of mice with recombinant bacteria. (A,B) BL21(ST+) [ST(+)] produced heat-stable enterotoxin (ST) in vitro compared to BL21(ST−) [ST(−)] (n ≥ 3 cultures, each in triplicate). (C) BL21(ST+) [ST(+)] and BL21(ST−) [ST(−)] were recovered in stool from colonized mice for up to 18 weeks of age, compared to control mice [no recombinant bacteria (CTRL)]. CFU, colony forming units; F, female; M, male. n = 5 mice per group; error bars represent 95% CI; *** p < 0.001.

Figure 2

BL21(ST+) produce ST that activates GUCY2C downstream signaling in mouse intestine. (A) ST was quantified in intestinal content recovered from mice colonized for 3 weeks by BL21(ST+) [ST(+)], but not from BL21(ST−) [ST(−)]. (B) Cyclic GMP accumulated in epithelial cells recovered from intestinal segments of from mice colonized for 3 weeks by recombinant BL21(ST+) [ST(+)], but not from BL21(ST−) [ST(−)]. (C,D) VASP was phosphorylated in epithelial cells recovered from intestinal segments from mice colonized for 3 weeks by BL21(ST+) [ST(+)], but not by BL21(ST−) [ST(−)]. Je, jejunum, Ile, ileum, PC, proximal colon, DC, distal colon. n = 5 mice per group; assays were performed in triplicate; error bars represent 95% CI; * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 3

Synthetic ST mimicked the effects of ST-producing enterotoxigenic E. coli (ETEC) in mouse intestine. (A) Bioactivity of synthetic ST was verified using the suckling mouse assay. (B) Synthetic ST (10 μg) activated guanylyl cyclase C (GUCY2C) and cGMP production in epithelial cells recovered from the small intestine of adult mice. (C) Synthetic ST induced the phosphorylation of VASP in intestinal epithelial cells in a dose-dependent fashion. n = 5 adult mice per group; assays were performed in triplicate; error bars represent 95% CI; * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 4

Mice chronically colonized by recombinant bacteria maintained normal growth. Mice chronically colonized with BL21(ST+) [ST(+)] or BL21(ST−) [ST(−)]. (A) experienced growth, quantified by weight gain, and (B) exhibited stool water content, that was comparable to control mice [no recombinant bacteria (CTRL, Control)]. n = 5 mice per group; assays were performed in triplicate; error bars represent 95% CI. F, female; M, male.

Figure 5

Chronic colonization with BL21(ST+), but not BL21(ST−), opposed azoxymethane (AOM)-induced tumorigenesis. (A,B) AOM specifically produced colorectal tumors (arrows) in mice. (C–E) Colonization of mice with BL21(ST+) [ST(+); n = 22] diminished the number (C) and size (D) of colorectal tumors, (E) reducing the tumor burden compared to mice colonized with BL21(ST−) [ST(−); n = 23]. Horizontal bars in (C–E) represent medians; * p < 0.05; *** p < 0.001.