Type-2 cGMP-dependent protein kinase suppresses proliferation and carcinogenesis in the colon epithelium.

A large body of evidence has demonstrated that cyclic-guanosine monophosphate (cGMP), signaling has anti-tumor effects that might be used for colon cancer prevention. The tumor-suppressive mechanism and the signaling components downstream of cGMP remain largely unknown. The present study has characterized the expression of cGMP-dependent protein kinases (PKG1, PKG2) in normal and cancerous tissue from human colon. PKG1 was detected in both normal and tumor tissue, where it localized exclusively to the lamina propria and stroma (respectively). In contrast, PKG2 localized specifically to the epithelium where its expression decreased markedly in tumors compared to matched normal tissue. Neither PKG isoform was detected at the RNA or protein level in established colon cancer cell lines. To test for a potential tumor-suppressor role of PKG2 in the colon epithelium, Prkg2 knockout (KO) mice were subjected to azoxymethane/dextran sulfate-sodium (AOM/DSS) treatment. PKG2 deficiency was associated with crypt hyperplasia (Ki67) and almost twice the number of polyps per mouse as wild-type (WT) siblings. In vitro culture of mouse colon epithelium as organoids confirmed that PKG2 was the only isoform expressed, and it was detected in both proliferating and differentiating epithelial compartments. Colon organoids derived from Prkg2 KO mice proliferated more rapidly and exhibited a reduced ability to differentiate compared to WT controls. Taken together our results highlight PKG2 as the central target of cGMP in the colon, where it suppresses carcinogenesis by controlling proliferation in an epithelial-cell intrinsic manner.

cGMP Signaling Increases Antioxidant Gene Expression by Activating Forkhead Box O3A in the Colon Epithelium.

Signaling through cGMP has therapeutic potential in the colon, where it has been implicated in the suppression of colitis and colon cancer. In this study, we tested the ability of cGMP and type 2 cGMP-dependent protein kinase (PKG2) to activate forkhead box O (FoxO) in colon cancer cells and in the colon epithelium of mice. We show that activation of PKG2 in colon cancer cells inhibited cell proliferation, inhibited AKT, and activated FoxO. Treatment of colon explants with 8Br-cGMP also activated FoxO target gene expression at both RNA and protein levels, and reduced epithelial reduction-oxidation (redox) stress. FoxO3a was the most prominent isoform in the distal colon epithelium, with prominent luminal staining. FoxO3a levels were reduced in Prkg2-/- animals, and FoxO target genes were unaffected by 8Br-cGMP challenge in vitro. Treatment of mice with the phosphodiesterase-5 inhibitor vardenafil (Levitra) mobilized FoxO3a to the nucleus of luminal epithelial cells, which corresponded to increased FoxO target gene expression, reduced redox stress, and increased epithelial barrier integrity. Treatment of human colonic biopsy specimens with 8Br-cGMP also activated catalase and manganese superoxide dismutase expression, indicating that this pathway is conserved in humans. Taken together, these results identify a novel signaling pathway in the colon epithelium, where FoxO tumor suppressors could provide protection from redox stress. Moreover, this pathway is regulated by endogenous cGMP/PKG2 signaling, and can be targeted using phosphodiesterase-5 inhibitors.

The NF-κB p65 and p50 homodimer cooperate with IRF8 to activate iNOS transcription.

BACKGROUND: Inducible 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. METHODS: iNOS 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. RESULTS: We 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. CONCLUSIONS: IFNγ-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.

Clinical utility of plecanatide in the treatment of chronic idiopathic constipation.

Constipation is an important health burden that reduces the quality of life for countless millions of people. Symptom-centric therapeutics are often used to treat constipation due to unknown etiology, but in many cases, these drugs are either inadequate or have significant side effects. More recently, synthetic peptide agonists for epithelial guanylyl cyclase C (GC-C) have been developed which are effective at treating constipation in a sub-population of adult constipation patients. The first to market was linaclotide that is structurally related to the diarrheagenic enterotoxin, but this was followed by plecanatide, which more closely resembles endogenous uroguanylin. Both the drugs exhibit almost identical clinical efficacy in about 20% of patients, with diarrhea being a common side effect. Despite the potential for reduced side effects with plecanatide, detailed analysis suggests that clinically, they are very similar. Ongoing clinical and preclinical studies with these drugs suggest that treating constipation might be the tip of the iceberg in terms of clinical utility. The expression of cGMP signaling components could be diagnostic for functional bowel disorders, and increasing cGMP using GC-C agonists or phosphodiesterase inhibitors has huge potential for treating enteric pain, ulcerative colitis, and for the chemoprevention of colorectal cancer.

Cyclic-GMP-Elevating Agents Suppress Polyposis in ApcMin mice by Targeting the Preneoplastic Epithelium.

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 ApcMin/+ mouse model of intestinal cancer. Treatment of ApcMin/+ 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, ApcMin/+ 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. Cancer Prev Res; 11(2); 81-92. ©2018 AACR.

Sildenafil normalizes bowel transit in preclinical models of constipation.

Guanylyl cyclase-C (GC-C) agonists increase cGMP levels in the intestinal epithelium to promote secretion. This process underlies the utility of exogenous GC-C agonists such as linaclotide for the treatment of chronic idiopathic constipation (CIC) and irritable bowel syndrome with constipation (IBS-C). Because GC-C agonists have limited use in pediatric patients, there is a need for alternative cGMP-elevating agents that are effective in the intestine. The present study aimed to determine whether the PDE-5 inhibitor sildenafil has similar effects as linaclotide on preclinical models of constipation. Oral administration of sildenafil caused increased cGMP levels in mouse intestinal epithelium demonstrating that blocking cGMP-breakdown is an alternative approach to increase cGMP in the gut. Both linaclotide and sildenafil reduced proliferation and increased differentiation in colon mucosa, indicating common target pathways. The homeostatic effects of cGMP required gut turnover since maximal effects were observed after 3 days of treatment. Neither linaclotide nor sildenafil treatment affected intestinal transit or water content of fecal pellets in healthy mice. To test the effectiveness of cGMP elevation in a functional motility disorder model, mice were treated with dextran sulfate sodium (DSS) to induce colitis and were allowed to recover for several weeks. The recovered animals exhibited slower transit, but increased fecal water content. An acute dose of sildenafil was able to normalize transit and fecal water content in the DSS-recovery animal model, and also in loperamide-induced constipation. The higher fecal water content in the recovered animals was due to a compromised epithelial barrier, which was normalized by sildenafil treatment. Taken together our results show that sildenafil can have similar effects as linaclotide on the intestine, and may have therapeutic benefit to patients with CIC, IBS-C, and post-infectious IBS.

SETD1B Activates iNOS Expression in Myeloid-Derived Suppressor Cells.

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 antitumor 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 MDSCs, iNOS expression was significantly elevated in tumor-induced MDSCs, suggesting that the expression of iNOS is regulated by an IRF8-independent mechanism under pathologic conditions. Furthermore, tumor-induced MDSCs exhibited diminished STAT1 and NF-κB Rel protein levels, the essential inducers of iNOS in myeloid cells. Instead, tumor-induced MDSCs showed increased SETD1B expression as compared with 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 MDSCs, and inhibition or silencing of SETD1B diminished iNOS expression in tumor-induced MDSCs. 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 MDSCs when they are generated under pathologic conditions. Cancer Res; 77(11); 2834-43. ©2017 AACR.

Sildenafil Suppresses Inflammation-Driven Colorectal Cancer in Mice.

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 dextran-sulfate sodium (DSS)-induced barrier dysfunction. In mice treated with AOM/DSS, oral administration of sildenafil throughout the disease course reduced polyp multiplicity by 50% compared with untreated controls. Polyps that did form in sildenafil treated mice were less proliferative and more differentiated compared with 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 IL6 compared with 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. Cancer Prev Res; 10(7); 377-88. ©2017 AACRSee related editorial by Piazza, p. 373.