Intestinal cyclic guanosine monophosphate (cGMP) signaling regulates epithelial homeostasis and has been implicated in the suppression of colitis and colon cancer. In this study, we investigated the cGMP-elevating ability of the phosphodiesterase-5 (PDE5) inhibitor sildenafil to prevent disease in the azoxymethane/dextran sulfate sodium (AOM/DSS) inflammation-driven colorectal cancer model. Treatment of mice with sildenafil activated cGMP signaling in the colon mucosa and protected against DSS-induced barrier dysfunction. In mice treated with AOM/DSS, oral administration of sildenafil throughout the disease course reduced polyp multiplicity by 50% compared to untreated controls. Polyps that did form in sildenafil treated mice were less proliferative and more differentiated compared to polyps from untreated mice, but apoptosis was unaffected. Polyps in sildenafil treated mice were also less inflamed; they exhibited reduced myeloid-cell infiltration, and reduced expression of iNOS, IFNγ, and IL-6 compared to untreated controls. Most of the protection conferred by sildenafil was during the initiation stage of carcinogenesis (38% reduction in multiplicity). Administration of sildenafil during the later promotion stages did not affect multiplicity but had a similar effect on the polyp phenotype, including increased mucus production, and reduced proliferation and inflammation. In summary, the results demonstrate that oral administration of sildenafil suppresses polyp formation and inflammation in mice treated with AOM/DSS. This validation of PDE5 as a target highlights the potential therapeutic value of PDE5 inhibitors for the prevention of colitis-driven colon cancer in humans.

BackgroundInducible nitric oxide synthase (iNOS) metabolizes L-arginine to produce nitric oxide (NO) which was originally identified in myeloid cells as a host defense mechanism against pathogens. Recent studies, however, have revealed that iNOS is often induced in tumor cells and myeloid cells in the tumor microenvironment. Compelling experimental data have shown that iNOS promotes tumor development in certain cellular context and suppresses tumor development in other cellular conditions. The molecular mechanisms underlying these contrasting functions of iNOS is unknown. Because iNOS is often induced by inflammatory signals, it is therefore likely that these contrasting functions of iNOS could be controlled by the inflammatory signaling pathways, which remains to be determined.MethodsiNOS is expressed in colon carcinoma and myeloid cells in the tumor microenvironment. Colon carcinoma and myeloid cell lines were used to elucidate the molecular mechanisms underlying iNOS expression. Chromatin immunoprecipitation and electrophoretic mobility shift assay were used to determine the IFNγ-activated pSTAT1 and NF-κB association with the chromatin DNA of the nos2 promoter.ResultsWe show here that iNOS is dramatically up-regulated in inflammed human colon tissues and in human colon carcinoma as compared to normal colon tissue. iNOS is expressed in either the colon carcinoma cells or immune cells within the tumor microenvironment. On the molecular level, the proinflammatory IFNγ and NF-κB signals induce iNOS expression in human colon cancer cells. We further demonstrate that NF-κB directly binds to the NOS2 promoter to regulate iNOS expression. Although neither the IFNγ signaling pathway nor the NF-κB signaling pathway alone is sufficient to induce iNOS expression in myeloid cells, IFNγ and NF-κB synergistically induce iNOS expression in myeloid cells. Furthermore, we determine that IFNγ up-regulates IRF8 expression to augment NF-κB induction of iNOS expression. More interestingly, we observed that the p65/p65 and p50/p50 homodimers, not the canonical p65/p50 heterodimer, directly binds to the nos2 promoter to regulate iNOS expression in myeloid cells.ConclusionsIFNγ-induced IRF8 acts in concert with NF-κB to regulate iNOS expression in both colon carcinoma and myeloid cells. In myeloid cells, the NF-κB complexes that bind to the nos2 promoter are p65/p65 and p50/p50 homodimers.

Inducible nitric oxide synthase (iNOS) generates nitric oxide (NO) in myeloid cells that acts as a defense mechanism to suppress invading microorganisms or neoplastic cells. In tumor-bearing mice, elevated iNOS expression is a hallmark of myeloid-derived suppressor cells (MDSC). MDSCs use NO to nitrate both the T cell receptor and STAT1, thus inhibiting T cell activation and the anti-tumor immune response. The molecular mechanisms underlying iNOS expression and regulation in tumor-induced MDSCs are unknown. We report here that deficiency in IRF8 results in diminished iNOS expression in both mature CD11b+Gr1− and immature CD11b+Gr1+ myeloid cells in vivo. Strikingly, although IRF8 was silenced in tumor-induced MDSC, iNOS expression was significantly elevated in tumor-induced MDSC, suggesting that the expression of iNOS is regulated by an IRF8-independent mechanism under pathological conditions. Furthermore, tumor-induced MDSC exhibited diminished STAT1 and NF-κB Rel protein levels, the essential inducers of iNOS in myeloid cells. Instead, tumor-induced MDSC showed increased SETD1B expression as compared to their cellular equivalents in tumor-free mice. Chromatin immunoprecipitation revealed that H3K4me3, the target of SETD1B, was enriched at the nos2 promoter in tumor-induced MDSC, and inhibition or silencing of SETD1B diminished iNOS expression in tumor-induced MDSC. Our results show how tumor cells use the SETD1B-H3K4me3 epigenetic axis to bypass a normal role for IRF8 expression in activating iNOS expression in MDSC, when they are generated under pathological conditions.

The cGMP signaling axis has been implicated in the suppression of intestinal cancers, but the inhibitory mechanism and the extent to which this pathway can be targeted remains poorly understood. This study has tested the effect of cGMP-elevating agents on tumorigenesis in the mouse model of intestinal cancer. Treatment of mice with the receptor guanylyl-cyclase C (GCC) agonist linaclotide, or the phosphodiesterase-5 (PDE5) inhibitor sildenafil, significantly reduced the number of polyps per mouse (67% and 50%, respectively). Neither of the drugs affected mean polyp size, or the rates of apoptosis and proliferation. This was possibly due to increased PDE10 expression, as endogenous GCC ligands were not deficient in established polyps. These results indicated that the ability of these drugs to reduce polyp multiplicity was primarily due to an effect on nonneoplastic tissues. In support of this idea, mice exhibited reduced levels of endogenous GCC agonists in the nonneoplastic intestinal mucosa compared with wild-type animals, and this was associated with crypt hyperplasia and a loss of goblet cells. Administration of either sildenafil or linaclotide suppressed proliferation, and increased both goblet cell numbers and luminal apoptosis in the intestinal mucosa. Taken together, the results demonstrate that targeting cGMP with either PDE5 inhibitors or GCC agonists alters epithelial homeostasis in a manner that reduces neoplasia, and suggests that this could be a viable chemoprevention strategy for patients at high risk of developing colorectal cancer..