Intestinal GUCY2C prevents TGF-β secretion coordinating desmoplasia and hyperproliferation in colorectal cancer.

Tumorigenesis is a multistep process that reflects intimate reciprocal interactions between epithelia and underlying stroma. However, tumor-initiating mechanisms coordinating transformation of both epithelial and stromal components are not defined. In humans and mice, initiation of colorectal cancer is universally associated with loss of guanylin and uroguanylin, the endogenous ligands for the tumor suppressor guanylyl cyclase C (GUCY2C), disrupting a network of homeostatic mechanisms along the crypt-surface axis. Here, we reveal that silencing GUCY2C in human colon cancer cells increases Akt-dependent TGF-β secretion, activating fibroblasts through TGF-β type I receptors and Smad3 phosphorylation. In turn, activating TGF-β signaling induces fibroblasts to secrete hepatocyte growth factor (HGF), reciprocally driving colon cancer cell proliferation through cMET-dependent signaling. Elimination of GUCY2C signaling in mice (Gucy2c(-/-)) produces intestinal desmoplasia, with increased reactive myofibroblasts, which is suppressed by anti-TGF-β antibodies or genetic silencing of Akt. Thus, GUCY2C coordinates intestinal epithelial-mesenchymal homeostasis through reciprocal paracrine circuits mediated by TGF-β and HGF. In that context, GUCY2C signaling constitutes a direct link between the initiation of colorectal cancer and the induction of its associated desmoplastic stromal niche. The recent regulatory approval of oral GUCY2C ligands to treat chronic gastrointestinal disorders underscores the potential therapeutic opportunity for oral GUCY2C hormone replacement to prevent remodeling of the microenvironment essential for colorectal tumorigenesis

GUCY2C Opposes Systemic Genotoxic Tumorigenesis by Regulating AKT-Dependent Intestinal Barrier Integrity

The barrier separating mucosal and systemic compartments comprises epithelial cells, annealed by tight junctions, limiting permeability. GUCY2C recently emerged as an intestinal tumor suppressor coordinating AKT1-dependent crypt-villus homeostasis. Here, the contribution of GUCY2C to barrier integrity opposing colitis and systemic tumorigenesis is defined. Mice deficient in GUCY2C (Gucy2c−/−) exhibited barrier hyperpermeability associated with reduced junctional proteins. Conversely, activation of GUCY2C in mice reduced barrier permeability associated with increased junctional proteins. Further, silencing GUCY2C exacerbated, while activation reduced, chemical barrier disruption and colitis. Moreover, eliminating GUCY2C amplified, while activation reduced, systemic oxidative DNA damage. This genotoxicity was associated with increased spontaneous and carcinogen-induced systemic tumorigenesis in Gucy2c−/− mice. GUCY2C regulated barrier integrity by repressing AKT1, associated with increased junction proteins occludin and claudin 4 in mice and Caco2 cells in vitro. Thus, GUCY2C defends the intestinal barrier, opposing colitis and systemic genotoxicity and tumorigenesis. The therapeutic potential of this observation is underscored by the emerging clinical development of oral GUCY2C ligands, which can be used for chemoprophylaxis in inflammatory bowel disease and cancer

GUCY2C Opposes Premalignant Transformation in Intestine by Regulating PTEN-PI3K/AKT Signaling

An evolving paradigm expanding the genetic basis of cancer suggests that tissue-specific developmental programs indelibly imprint restricted repertoires governing homeostasis, forming the substrate for lineage-dependent tumor induction by dysregulation of survival pathways. GUCY2C, the intestinal receptor for the paracrine hormones guanylin and uroguanylin whose early loss characterizes transformation, has emerged as a component of developmental programs organizing spatiotemporal patterning along the crypt-surface axis whose dysregulation promotes hyperproliferation and genetic instability underlying neoplasia. Here we identify GUCY2C as a reversible switch for lineage-dependent tumorigenesis whose disruption amplifies survival circuits essential for regenerative homeostasis and required for transformation. Elimination of GUCY2C in mice expands proliferating crypts, accelerating the cell cycle at the G1/S transition. Proliferative induction is coupled with altered metabolic programming, with an increase in aerobic glycolysis and a reciprocal reduction in mitochondrial biogenesis and oxidative phosphorylation, recapitulating the metabolic phenotype of human tumors. Conversely, GUCY2C signaling restores homeostatic circuits in colon cancer cells, decelerating the cell cycle and switching ATP production from glycolysis to mitochondrial metabolism, recapitulating normal enterocytes. Moreover, oral administration of the downstream mediator of GUCY2C signaling, cyclic GMP, reverses crypt hyperplasia and restores normal proliferative and metabolic programs in mice deficient in GUCY2C. Coordination of survival circuits by GUCY2C is orchestrated through the oncogene AKT, whose inhibition mimics, and activation eliminates, GUCY2C regulation of proliferation and metabolism. Modulation of AKT signaling by GUCY2C is, in part, mediated by the tumor suppressor, PTEN. Silencing PTEN eliminates GUCY2C regulation of AKT, proliferation and metabolism. Thus, disruption of developmentally restricted signaling by GUCY2C, reflecting loss of paracrine hormones, induces maladaptive survival pathways underlying crypt-surface homeostasis whose tissue-specific deregulation contributes to lineage dependency in intestinal tumorigenesis. With the role of guanylin and uroguanylin loss in transformation, the universal compensatory over-expression of GUCY2C by colorectal tumors offers a unique therapeutic opportunity for cancer prevention through oral hormone replacement therapy

Constitutive GUCA2A expression in intestine suppresses DSS-induced colitis.

<p>Constitutive expression of GUCA2A was initiated by Cre recombinase expressed in intestinal epithelial cells after 5 days of IP tamoxifen administration in <i>ROSA-Guca2a</i>×<i>Vil-Cre/ER^T2</i> male mice. Two days later, 3% DSS was administrated for 7 d to induce colitis. (<b>A</b>) Intestinal macromolecular permeability was assessed on day 10 (3 d post-DSS exposure) by serum fluorescence 90 min after FITC-dextran gavage (n≥5). Severity of colitis was quantified by (<b>B</b>) body weight and (<b>C</b>) survival (n≥15). Data represent one of three independent experiments. Inflammation was quantified by (<b>D</b>) colon length and (<b>E</b>) histological score on day 10 (n≥5). Each point represents one mouse. (<b>F</b>) Representative H&E colon sections (20×). Data are mean ± SEM. *, p<0.05.</p

GUCY2C ligand supplementation decreases intestinal permeability and susceptibility to DSS-induced colitis.

<p>C57B6 female mice were administered control peptide (CP) or ST to examine the effect of GUCY2C activation on intestinal permeability after 14 d of oral ligand supplementation. (<b>A</b>) Serum fluorescence was analyzed 90 min after FITC-dextran gavage (n = 21) following 6 d of ligand supplementation. (<b>B, C</b>) Following 14 d of ST pre-conditioning, 3.5% DSS was administrated for 7 d to induce colitis which was quantified by (<b>B</b>) body weight and (<b>C</b>) survival. Data represent one of two independent experiments. Severity of colitis was quantified by (<b>D</b>) colon length, measured on day 10 (3 d post-DSS exposure), and gross anatomic analysis of colons from ST-, compared to CP-, preconditioned mice demonstrated normal stool formation (n = 21) and (<b>E</b>) histological score on day 10. Each point represents one mouse. (<b>F</b>) Representative H&E colon sections (20×). *, p<0.05, **, p<0.01.</p

GUCY2C-deficiency increases susceptibility to DSS-induced colitis.

<p><i>Gucy2c^+/+</i> and <i>Gucy2c^−/−</i> male mice were administered 3% DSS in drinking water for 7 d to disrupt the epithelial barrier and induce colitis. Severity of colitis was analyzed by (<b>A</b>) body weight (n = 11) and (<b>B</b>) survival (n≥11). (<b>C</b>) Colon length was measured from the colocecal junction to the anal verge on day 12 (5 d post-DSS exposure) to quantify chronic inflammation. (<b>D</b>) Histological sections obtained from jejunum and distal colon on day 12 were stained with H&E and scored for epithelial and mesenchymal inflammation. Each point represents one mouse. (<b>E</b>) Representative H&E colon sections (20×). Data are mean ± SD (in C) and SEM (in D) obtained from one of three independent experiments. **, p<0.01, ***, p<0.001.</p

GUCY2C modulates systemic genotoxicity and tumorigenesis.

<p>(<b>A</b>) Eliminating GUCY2C increases basal DNA oxidation in leukocytes. Each point represents one mouse. (<b>B</b>) Oral ST supplementation decreases DSS-induced hepatic genotoxicity. <i>Gucy2c^+/+</i> mice were preconditioned with oral ST for 6 d, and then treated with 3.5% DSS for 7 d, followed by quantification of DNA oxidation in liver on days 10 (n≥5) and 13 (n≥2; 3 and 6 d post-DSS exposure). Data represent mean ± SD. (<b>C</b>) <i>Gucy2c^−/−</i> mice exhibited a higher incidence of spontaneous tumors comparing with age-matched 2-year-old <i>Gucy2c^+/+</i> mice (n≥14, p = 0.02). (<b>D</b>) The carcinogen AOM induced hepatoma in <i>Gucy2c^−/−</i>, but not <i>Gucy2c^+/+</i>, mice (n≥10, p = 0.04). C and D are analyzed by two-sided Fisher's exact test. *, p<0.05.</p

GUCY2C regulation of barrier integrity is AKT1-dependent.

<p>(<b>A</b>) Immunoblot analysis of tight junction proteins from jejuna of 12 week-old mice (n≥7). Data represent means ± SEM. Blue asterisks compare results to <i>Gucy2c^+/+</i>, black asterisks compare results to <i>Gucy2c^−/</i>. (<b>B</b>) Intestinal permeability was examined by serum fluorescence 90 min after FITC-dextran gavage. Each point represents one mouse. (<b>C</b>) Regulation of AKT phosphorylation by GUCY2C signaling in Caco2 human colon cancer cells was examined by immunoblot analysis. Data represent means ± SEM of 3 experiments done in duplicate. (<b>D</b>) AKT1 signaling in Caco2 cells was manipulated by adenovirus-delivered AKT1 (WTAKT1), constitutive active AKT1 mutant (MyrAKT1), or siRNA against AKT1 (siAKT1). Two days after infection, cells were treated with ST for 6 d. Barrier permeability was examined by FITC-dextran diffusion. Data represent mean ± SEM obtained from one of five experiments done in triplicate. (<b>E</b>) Tight junction protein expression was examined by immunoblot analysis in Caco2 cells stably expressing empty vector (MSCV) or shRNA against AKT1 (shAKT1) after 6 d of ST treatment. Data represent means ± SEM of three experiments done in duplicate. Blue asterisks compare results to MSCV-PBS, black asterisks compare results to MSCV-ST. *, p<0.05, **, p<0.01, ***, p<0.001. In C–E, statistical analyses were compared to PBS control.</p