Sarah K. Sharman

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

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.

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 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.

Abstract 1918: Type 2 cGMP-dependent protein kinase activates antineoplastic signaling in the colon

Signaling through cGMP has emerged as a potentially important suppressor of tumorigenesis in the colon but the mechanisms are poorly defined. Studies of guanylyl-cyclase (GCC) knockout mice indicate that cGMP signaling inhibits proliferation in the colon epithelium and that AKT may have an important role. Type-2 cGMP-dependent protein kinase (PKG2) is a central effector of cGMP in the colon epithelium and likely mediates the suppressive signaling. An important growth-inhibitory effect of AKT is the inactivation of forkhead transcription factors (FoxO). FoxO proteins are tumor suppressors that regulate growth-inhibitory and pro-apoptotic target gene expression but these proteins have not previously been examined in the intestinal tract. The present study sought to determine whether PKG2 can activate FoxO in colon cancer cells and in the colon epithelium. Activation of PKG2 in LS174T colon cancer cells inhibited cell proliferation but did not significantly affect apoptosis. Suggestive of a mechanism, it was found that PKG2 inhibited the activity of AKT but not ERK in these cells. Activation of PKG2 also significantly increased luciferase reporter activity driven by FoxO-responsive elements in LS174T cells. In support of FoxO activation, PKG2 also increased the expression of several FoxO target genes, including catalase, GADD45, and p27kip. Treatment of colon explants with 8Br-cGMP also activated FoxO target gene expression at both RNA and protein levels, and this was associated with reduced redox stress. The regulation of FoxO by cGMP in vivo most likely requires PKG2, because target gene expression was reduced in the colon mucosa of Prkg2-/- mice compared to wild type siblings. Since FoxO has not previously been studied in the colon, we first examined expression using IHC. These data showed that FoxO3a is the most prominent isoform, and was concentrated almost exclusively at the lumenal border. While FoxO1 showed negligible staining, FoxO4 was observed deeper in the crypt. In order to determine whether cGMP could activate FoxO in vivo, the PDE-5 inhibitor vardenafil (Levitra™) was used. Mice treated with vardenafil showed a dramatic mobilization of FoxO3a to the nucleus of lumenal epithelial cells. Analysis of the colon mucosa from treated animals showed increased levels of FoxO target genes and reduced redox stress. This was likely to be mediated by FoxO3a since vardenafil did not significantly affect the localization of FoxO1 or FoxO4. Taken together these data define a novel signaling pathway in the colon epithelium, where PKG2 leads to activation of FoxO. The established tumor suppressor role of FoxO proteins highlights the potential therapeutic role for cGMP signaling in colon cancer prevention. Citation Format: Allison Bridges, Bianca Islam, Sarah Sharman, Rui Wang, Subbaramiah Sridhar, Darren Douglas Browning. Type 2 cGMP-dependent protein kinase activates antineoplastic signaling in the colon. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1918. doi:10.1158/1538-7445.AM2015-1918

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.

Abstract B86: The NF-κB p65 and p50 homodimer cooperate with pSTAT1 to synergistically activate iNOS transcription

Inducible nitric oxide synthase (iNOS) metabolizes L-arginine to produce NO that was originally identified in myeloid cells as a host defense mechanism against pathogens. Recent studies, however, have revealed that iNOS is often induced in both tumor and myeloid cells in the tumor microenvironment. More intriguingly, compelling experimental data have shown that iNOS promotes tumor development in certain cellular context and suppress tumor development under other cellular conditions. The molecular mechanisms underlying this contrasting function of iNOS is unknown. Because iNOS is often induced by inflammatory signals, it is therefore likely that the opposite functions of iNOS might be controlled by the inflammatory signaling pathways. We show here that proinflammatory IFN-γ and NF-κB signals synergistically induce iNOS expression in human colon cancer cells. We further demonstrated that NF-κB directly binds to the NOS2 promoter to regulate iNOS expression. Although the IFN-γ signaling pathway or the NF-κB signaling pathway alone is insufficient to induce iNOS expression in myeloid cells, IFN-γ and NF-κB synergistically induce iNOS expression in myeloid cells. Furthermore, we determined that IFN-γ 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. Our results thus determine that the IFN-γ-induced IRF8 acts in concert with the NF-κB p65/p65 and p50/p50 homodimers to regulate iNOS expression. Citation Format: Priscilla Simon, Sarah Sharman, Chunwan Lu, Dafeng Yang, Amy Paschall, Kebin Liu. The NF-κB p65 and p50 homodimer cooperate with pSTAT1 to synergistically activate iNOS transcription. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B86.