A Non-Systemic Phosphodiesterase-5 Inhibitor Suppresses Colon Proliferation in Mice.

Phosphodiesterase-5 inhibitors (PDE5i) are under investigation for repurposing for colon cancer prevention. A drawback to conventional PDE5i are their side-effects and drug-drug interactions. We designed an analog of the prototypical PDE5i sildenafil by replacing the methyl group on the piperazine ring with malonic acid to reduce lipophilicity, and measured its entry into the circulation and effects on colon epithelium. This modification did not affect pharmacology as malonyl-sildenafil had a similar IC50 to sildenafil but exhibited an almost 20-fold reduced EC50 for increasing cellular cGMP. Using an LC-MS/MS approach, malonyl-sildenafil was negligible in mouse plasma after oral administration but was detected at high levels in the feces. No bioactive metabolites of malonyl-sildenafil were detected in the circulation by measuring interactions with isosorbide mononitrate. The treatment of mice with malonyl-sildenafil in the drinking water resulted in a suppression of proliferation in the colon epithelium that is consistent with results previously published for mice treated with PDE5i. A carboxylic-acid-containing analog of sildenafil prohibits the systemic delivery of the compound but maintains sufficient penetration into the colon epithelium to suppress proliferation. This highlights a novel approach to generating a first-in-class drug for colon cancer chemoprevention.

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.

Inhibition of Colon Cancer Cell Growth by Phosphodiesterase Inhibitors Is Independent of cGMP Signaling.

There is growing interest in the potential use of phosphodiesterase (PDE) inhibitors for colorectal cancer (CRC) prevention and treatment. The present study has tested the idea that PDE inhibitors inhibit growth and viability of CRC cell lines by increasing cyclic guanosine monophosphate (cGMP) and activating cGMP-dependent protein kinase (PKG). Colon cancer cell lines and those with ectopic PKG2 expression were treated with membrane-permeable 8Br-cGMP or inhibitors of PDE5, PDE9, and PDE10a. Levels of cGMP capable of activating PKG were measured by immunoblotting for phosphorylation of vasodilator-stimulated phosphoprotein (VASP). The effects of treatment on CRC cell proliferation and death were measured using hemocytometry with trypan blue. Treatment with 8Br-cGMP had no effect on CRC cell proliferation or death. Endogenous PKG activity was undetectable in any of the CRC cells, but expression of ectopic PKG2 conferred modest inhibition of proliferation but did not affect cell death. Extremely high concentrations of all the PDE inhibitors reduced proliferation in CRC cell lines, but none of them increased cGMP levels, and the effect was independent of PKG expression. The inability of the PDE inhibitors to increase cGMP was due to the lack of endogenous cGMP generating machinery. In conclusion, PDE inhibitors that target cGMP only reduce CRC growth at clinically unachievable concentrations, and do so independent of cGMP signaling through PKG. SIGNIFICANCE STATEMENT: A large number of in vitro studies have reported that PDE inhibitors block growth of colon cancer cells by activating cGMP signaling, and that these drugs might be useful for cancer treatment. Our results show that these drugs do not activate cGMP signaling in colon cancer cells due to a lack of endogenous guanylyl cyclase activity, and that growth inhibition is due to toxic effects of clinically unobtainable drug concentrations.

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.

Upregulation of the EGFR/MEK1/MAPK1/2 signaling axis as a mechanism of resistance to antiestrogen­induced BimEL dependent apoptosis in ER+ breast cancer cells.

The epidermal growth factor receptor (EGFR) is commonly upregulated in multiple cancer types, including breast cancer. In the present study, evidence is provided in support of the premise that upregulation of the EGFR/MEK1/MAPK1/2 signaling axis during antiestrogen treatment facilitates the escape of breast cancer cells from BimEL­dependent apoptosis, conferring resistance to therapy. This conclusion is based on the findings that ectopic BimEL cDNA overexpression and confocal imaging studies confirm the pro­apoptotic role of BimEL in ERα expressing breast cancer cells and that upregulated EGFR/MEK1/MAPK1/2 signaling blocks BimEL pro­apoptotic action in an antiestrogen­resistant breast cancer cell model. In addition, the present study identified a pro­survival role for autophagy in antiestrogen resistance while EGFR inhibitor studies demonstrated that a significant percentage of antiestrogen­resistant breast cancer cells survive EGFR targeting by pro­survival autophagy. These pre­clinical studies establish the possibility that targeting both the MEK1/MAPK1/2 signaling axis and pro­survival autophagy may be required to eradicate breast cancer cell survival and prevent the development of antiestrogen resistance following hormone treatments. The present study uniquely identified EGFR upregulation as one of the mechanisms breast cancer cells utilize to evade the cytotoxic effects of antiestrogens mediated through BimEL­dependent apoptosis.

Type 2 cGMP-dependent protein kinase regulates proliferation and differentiation in the colonic mucosa.

Signaling through cGMP has emerged as an important regulator of tissue homeostasis in the gastrointestinal tract, but the mechanism is not known. Type 2 cGMP-dependent protein kinase (PKG2) is a major cGMP effector in the gut epithelium, and the present studies have tested its importance in the regulation of proliferation and differentiation in the mouse colon and in colon cancer cell lines. Tissue homeostasis was examined in the proximal colon of Prkg2(-/-) mice using histological markers of proliferation and differentiation. The effect of ectopic PKG2 on proliferation and differentiation was tested in vitro using inducible colon cancer cell lines. PCR and luciferase reporter assays were used to determine the importance of Sox9 downstream of PKG2. The colons of Prkg2(-/-) mice exhibited crypt hyperplasia, increased epithelial apoptosis, and reduced numbers of differentiated goblet and enteroendocrine cells. Ectopic PKG2 was able to inhibit proliferation and induce Muc2 and CDX2 expression in colon cancer cells, but did not significantly affect cell death. PKG2 reduced Sox9 levels and signaling, suggesting possible involvement of this pathway downstream of cGMP in the colon. The work presented here demonstrates a novel antiproliferative and prodifferentiation role for PKG2 in the colon. These homeostatic functions of PKG2 were reproducible in colon cancer cells lines where downregulation of Sox9 is a possible mechanism. The similarities in phenotype between PKG2 and GCC knockout mice positions PKG2 as a likely mediator of the homeostatic effects of cGMP signaling in the colon.

Cyclic 3',5'-guanosine monophosphate-dependent protein kinase inhibits colon cancer cell adaptation to hypoxia.

BACKGROUND: Type 1 cyclic 3',5'-guanosine monophosphate-dependent protein kinase (PKG) has recently been reported to inhibit tumor growth and angiogenesis. These effects suggest that PKG activation may have therapeutic value for colon cancer treatment, but the signaling downstream of this enzyme is poorly understood. The present study examined the mechanism underlying the inhibition of angiogenesis by PKG. METHODS: The effect of ectopically expressed PKG on colon cancer cell adaptation to a 1% O(2) (hypoxic) environment was examined in vitro by measuring hypoxic markers, cell death/viability, and hypoxia inducible factor (HIF) activity. RESULTS: Ectopic PKG inhibited angiogenesis in SW620 xenografts and significantly attenuated hypoxia-induced increases in vascular endothelial growth factor at both the mRNA and protein levels. PKG activation also blocked hypoxia-induced hexokinase 2 expression, which corresponded with reduced cellular adenosine triphosphate levels. Moreover, PKG expression significantly reduced cell viability and promoted necrotic cell death after 2 days in a hypoxic environment. To gain some mechanistic insight, the effect of PKG on HIF activation was determined using luciferase reporter assays. PKG activation inhibited HIF transcriptional activity in several colon cancer cell lines, including SW620, HCT116, and HT29. The mechanism by which PKG can inhibit HIF activity is not known, but it does not affect HIF-1α protein accumulation or nuclear translocation. CONCLUSIONS: These findings demonstrate for the first time that PKG can block the adaptation of colon cancer cells to hypoxia and highlights this enzyme for further evaluation as a potential target for colon cancer treatment.

The Campylobacter jejuni Response Regulator and Cyclic-Di-GMP Binding CbrR Is a Novel Regulator of Flagellar Motility.

A leading cause of bacterial gastroenteritis, Campylobacter jejuni is also associated with broad sequelae, including extragastrointestinal conditions such as reactive arthritis and Guillain-Barré Syndrome (GBS). CbrR is a C. jejuni response regulator that is annotated as a diguanylate cyclase (DGC), an enzyme that catalyzes the synthesis of c-di-GMP, a universal bacterial second messenger, from GTP. In C. jejuni DRH212, we constructed an unmarked deletion mutant, cbrR-, and complemented mutant, cbrR+. Motility assays indicated a hyper-motile phenotype associated with cbrR-, whereas motility was deficient in cbrR+. The overexpression of CbrR in cbrR+ was accompanied by a reduction in expression of FlaA, the major flagellin. Biofilm assays and scanning electron microscopy demonstrated similarities between DRH212 and cbrR-; however, cbrR+ was unable to form significant biofilms. Transmission electron microscopy showed similar cell morphology between the three strains; however, cbrR+ cells lacked flagella. Differential radial capillary action of ligand assays (DRaCALA) showed that CbrR binds GTP and c-di-GMP. Liquid chromatography tandem mass spectrometry detected low levels of c-di-GMP in C. jejuni and in E. coli expressing CbrR. CbrR is therefore a negative regulator of FlaA expression and motility, a critical virulence factor in C. jejuni pathogenesis.

p50 suppresses cytotoxic T lymphocyte effector function to regulate tumor immune escape and response to immunotherapy.

BACKGROUND: NF-κB is a key link between inflammation and cancer. Previous studies of NF-κB have largely focused on tumor cells, and the intrinsic function of NF-κB in T cells in tumor development and response to immunotherapy is largely unknown. We aimed at testing the hypothesis that NF-κB1 (p50) activation in T cells underlies human colon cancer immune escape and human cancer non-response to anti-PD-1 immunotherapy. METHODS: We screened NF-κB activation in human colon carcinoma and used mouse models to determine p50 function in tumor cells and immune cells. RNA-Seq was used to identify p50 target genes. p50 binding to target gene promoters were determined by electrophoresis mobility shift assay and chromatin immunoprecipitation. A p50 activation score was generated from gene expression profiling and used to link p50 activation to T-cell activation and function pre-nivolumab and post-nivolumab immunotherapy in human patients with cancer. RESULTS: p50 is the dominant form of NF-κB that is highly activated in immune cells in the human colorectal carcinoma microenvironment and neighboring non-neoplastic colon epithelial cells. Tumor cell intrinsic p50 signaling and T-cell intrinsic p50 signaling exert opposing functions in tumor growth control in vivo. Deleting Nfkb1 in tumor cells increased whereas in T cells decreased tumor growth in preclinical mouse models. Gene expression profiling identified Gzmb as a p50 target in T cells. p50 binds directly to a previously uncharacterized κB sequence at the Gzmb promoter in T cells, resulting in repression of Gzmb expression in tumor-infiltrating cytotoxic T lymphocytes (CTLs) to induce a dysfunctional CTL phenotype to promote tumor immune escape. p50 activation is inversely correlated with both GZMB expression and T-cell tumor infiltration in human colorectal carcinoma. Furthermore, nivolumab immunotherapy decreased p50 activation and increased GZMB expression in human patients with melanoma. CONCLUSIONS: Inflammation activates p50 that binds to the Gzmb promoter to repress granzyme B expression in T cells, resulting in CTL dysfunction to confer tumor immune escape and decreased response to anti-PD-1 immunotherapy.

Mutation of protein kinase C phosphorylation site S1076 on alpha-subunits affects BK(Ca) channel activity in HEK-293 cells.

Large conductance, calcium- and voltage-activated potassium (BK(Ca)) channels are important modulators of pulmonary vascular smooth muscle membrane potential, and phosphorylation of BK(Ca) channels by protein kinases regulates pulmonary arterial smooth muscle function. However, little is known about the effect of phosphorylating specific channel subunits on BK(Ca) channel activity. The present study was done to determine the effect of mutating protein kinase C (PKC) phosphorylation site serine 1076 (S1076) on transfected human BK(Ca) channel alpha-subunits in human embryonic kidney (HEK-293) cells, a heterologous expression system devoid of endogenous BK(Ca) channels. Results showed that mutating S1076 altered the effect of PKC activation on BK(Ca) channels in HEK-293 cells. Specifically, the phospho-deficient mutation BK(Ca)-alpha(S1076A)/beta(1) attenuated the excitatory effect of the PKC activator phorbol myristate acetate (PMA) on BK(Ca) channels, whereas the phospho-mimetic mutation BK(Ca)-alpha(S1076E)/beta(1) increased the excitatory effect of PMA on BK(Ca) channels. In addition, the phospho-null mutation S1076A blocked the activating effect of cGMP-dependent protein kinase G (PKG) on BK(Ca) channels. Collectively, these results suggest that specific putative PKC phosphorylation site(s) on human BK(Ca) channel alpha-subunits influences BK(Ca) channel activity, which may subsequently alter pulmonary vascular smooth muscle function and tone.

Nitric oxide inactivates the retinoblastoma pathway in chronic inflammation.

Patients with chronic inflammatory bowel disease have a high risk of colon cancer. The molecules that initiate and promote colon cancer and the cancer pathways altered remain undefined. Here, using in vitro models and a mouse model of colitis, we show that nitric oxide (NO) species induce retinoblastoma protein (pRb) hyperphosphorylation and inactivation, resulting in increased proliferation through the pRb-E2F1 pathway. NO-driven pRb hyperphosphorylation occurs through soluble guanylyl cyclase/guanosine 3',5'-cyclic monophosphate signaling and is dependent on the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase MEK/ERK and phosphatidylinositol 3-kinase/AKT pathways. Our results reveal a link between NO and pRb inactivation and provide insight into molecules that can be targeted in the prevention of the inflammation-to-cancer sequence.

Repression of IFN regulatory factor 8 by DNA methylation is a molecular determinant of apoptotic resistance and metastatic phenotype in metastatic tumor cells.

Apoptotic resistance is often associated with metastatic phenotype in tumor cells and is considered a hallmark of tumor progression. In this study, IFN regulatory factor 8 (IRF8) expression was found to be inversely correlated with an apoptotic-resistant and metastatic phenotype in human colon carcinoma cell lines in vitro. This inverse correlation was further extended to spontaneously arising primary mammary carcinoma and lung metastases in a mouse tumor model in vivo. Exogenous expression of IRF8 in the metastatic tumor cell line restored, at least partially, the sensitivity of the tumor cells to Fas-mediated apoptosis, and disruption of IRF8 function conferred the poorly metastatic tumors with enhanced apoptotic resistance and metastatic capability. DNA demethylation restored IRF8 expression and sensitized the metastatic tumor cells to Fas-mediated apoptosis. Analysis of genomic DNA isolated from both primary and metastatic tumor cells with methylation-sensitive PCR revealed hypermethylation of the IRF8 promoter in metastatic tumor cells but not in primary tumor cells. Taken together, our data suggest that IRF8 is both an essential regulator in Fas-mediated apoptosis pathway and a metastasis suppressor in solid tumors and that metastatic tumor cells use DNA hypermethylation to repress IRF8 expression to evade apoptotic cell death and to acquire a metastatic phenotype.

A role for macroautophagy in protection against 4-hydroxytamoxifen-induced cell death and the development of antiestrogen resistance.

This study identifies macroautophagy as a key mechanism of cell survival in estrogen receptor-positive (ER+) breast cancer cells undergoing treatment with 4-hydroxytamoxifen (4-OHT). This selective ER modifier is an active metabolite of tamoxifen commonly used for the treatment of breast cancer. Our study provides the following key findings: (a) only 20% to 25% of breast cancer cells treated with 4-OHT in vitro die via caspase-dependent cell death; more typically, the antiestrogen-treated ER+ breast cancer cells express increased levels of macroautophagy and are viable; (b) 4-OHT-induced cell death, but not 4-OHT-induced macroautophagy, can be blocked by the pan-caspase inhibitor z-VAD-fmk, providing strong evidence that these two outcomes of antiestrogen treatment are not linked in an obligatory manner; (c) 4-OHT-resistant cells selected from ER+ breast cancer cells show an increased ability to undergo antiestrogen-induced macroautophagy without induction of caspase-dependent cell death; and (d) 4-OHT, when used in combination with inhibitors of autophagosome function, induces robust, caspase-dependent apoptosis of ER+, 4-OHT-resistant breast cancer cells. To our knowledge, these studies provide the first evidence that macroautophagy plays a critical role in the development of antiestrogen resistance. We propose that targeting autophagosome function will improve the efficacy of hormonal treatment of ER+ breast cancer.

Indispensable role of the Ubiquitin-fold modifier 1-specific E3 ligase in maintaining intestinal homeostasis and controlling gut inflammation.

Intestinal exocrine secretory cells, including Paneth and goblet cells, have a pivotal role in intestinal barrier function and mucosal immunity. Dysfunction of these cells may lead to the pathogenesis of human diseases such as inflammatory bowel disease (IBD). Therefore, identification and elucidation of key molecular mechanisms that regulate the development and function of these exocrine cells would be crucial for understanding of disease pathogenesis and discovery of new therapeutic targets. The Ufm1 conjugation system is a novel ubiquitin-like modification system that consists of Ufm1 (Ubiquitin modifier 1), Uba5 (Ufm1-activating enzyme, E1), Ufc1 (Ufm1-conjugating enzyme, E2) and poorly characterized Ufm1 E3 ligase(s). Recent mouse genetic studies have demonstrated its indispensable role in embryonic development and hematopoiesis. Yet its role in other tissues and organs remains poorly defined. In this study, we found that both Ufl1 and Ufbp1, two key components of the Ufm1 E3 ligase, were highly expressed in the intestinal exocrine cells. Ablation of either Ufl1 and Ufbp1 led to significant loss of both Paneth and goblet cells, which in turn resulted in dysbiotic microbiota and increased susceptibility to experimentally induced colitis. At the cellular and molecular levels, Ufbp1 deficiency caused elevation of endoplasmic reticulum stress and activation of the Unfolded Protein Response (UPR) and cell death program. Administration of small molecular chaperone partially prevented loss of Paneth cells caused by acute Ufbp1 deletion. Taken together, our results have provided unambiguous evidence for the crucial role of the Ufm1 E3 ligase in maintenance of intestinal homeostasis and protection from inflammatory diseases.

Targeting lymphotoxin beta receptor with tumor-specific T lymphocytes for tumor regression.

PURPOSE: One of the impediments of immunotherapy against cancer is the suppression of tumor-specific CTLs in the tumor microenvironment, partly due to the selective inhibition of the perforin pathway and the emergence of Fas-resistant tumors. Therefore, we sought to identify perforin- and Fas-independent cytotoxic pathways and explored the potential of targeting LTbetaR with tumor-specific CTLs to induce tumor rejection in vivo. EXPERIMENTAL DESIGN: Fas-resistant tumors were examined for their susceptibility to perforin-deficient (pfp) CTLs via CTL adoptive transfer in mouse models of experimental lung metastasis. The specificity of LTbetaR, a cell surface death receptor, in causing tumor rejection by CTLs was analyzed by LTbetaR-specific neutralizing monoclonal antibody in vitro. The specificity and efficacy of LTbetaR in the suppression of established tumors was further investigated by silencing LTbetaR in tumor cells in vivo. RESULTS: pfp CTLs exhibited significant cytotoxicity against Fas-resistant tumors in vivo. The perforin- and Fas-independent cytotoxicity was directly mediated, at least in part, by the adoptively transferred CTLs. It was observed that LTbetaR was expressed on the tumor cell surface, and LTalpha, LTbeta, and LIGHT, all of which are ligands for LTbetaR, were either constitutively expressed or activated in the tumor-specific CTLs and primary CD8(+) T cells. Blocking LTbetaR with LTbetaR-specific neutralizing monoclonal antibody decreased CTL cytotoxicity in vitro. Silencing LTbetaR using LTbetaR-specific short hairpin RNA reduced the ability of pfp CTLs to induce tumor rejection in vivo. CONCLUSION: LTbetaR directly mediates CTL-directed tumor rejection in vivo. Targeting LTbetaR with tumor-specific CTLs is a potential therapeutic approach.

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.

Myeloid-Derived Suppressor Cells Produce IL-10 to Elicit DNMT3b-Dependent IRF8 Silencing to Promote Colitis-Associated Colon Tumorigenesis.

IL-10 functions as a suppressor of colitis and colitis-associated colon cancer, but it is also a risk locus associated with ulcerative colitis. The mechanism underlying the contrasting roles of IL-10 in inflammation and colon cancer is unknown. We report here that inflammation induces the accumulation of CD11b+Gr1+ myeloid-derived suppressor cells (MDSCs) that express high levels of IL-10 in colon tissue. IL-10 induces the activation of STAT3 that directly binds to the Dnmt1 and Dnmt3b promoters to activate their expression, resulting in DNA hypermethylation at the Irf8 promoter to silence IRF8 expression in colon epithelial cells. Mice with Irf8 deleted in colonic epithelial cells exhibit significantly higher inflammation-induced tumor incidence. Human colorectal carcinomas have significantly higher DNMT1 and DNMT3b and lower IRF8 expression, and they exhibit significantly higher IRF8 promoter DNA methylation than normal colon. Our data identify the MDSC-IL-10-STAT3-DNMT3b-IRF8 pathway as a link between chronic inflammation and colon cancer initiation.

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.

A role for cyclic-GMP dependent protein kinase in anoikis.

Anoikis is an essential process in which a loss of adhesion to the substratum alters intracellular signaling pathways that lead to apoptosis. Using phosphorylation of vasodilator stimulated phosphoprotein (VASP) as an indicator of cGMP-dependent protein kinase (PKG) activity in vivo, it was found that suspension of the colon epithelial cell line (CCD841) leads to rapid and transient activation of PKG that lasted several hours. The colon carcinoma lines SW480 and SW620 do not express endogenous PKG, but exogenously expressed PKG was similarly activated upon cell suspension. To determine whether PKG has a role in apoptosis following cell suspension, poly-ADP ribose polymerase (PARP) cleavage and propidium iodide staining were measured. After 24 h in suspension it was found that approximately 50% of CCD841 cells exhibited apoptosis, whereas apoptosis was not detected in either of the colon carcinoma cell lines. Inhibition of type 1 PKG by expression of a dominant negative PKG construct (G1alphaR-GFP), or by incubation with the PKG inhibitor peptide DT-2, blocked apoptosis in suspended CCD841 cells by approximately 50%. Furthermore, expression of exogenous PKG in SW620 and SW480 cells conferred partial sensitivity anoikis. Taken together these findings indicate that PKG has an important role in the induction of apoptosis following suspension of normal colon epithelial cells, and loss of PKG expression in colon tumor cells may contribute to resistance to anoikis.

Identification of tetratricopeptide repeat 1 as an adaptor protein that interacts with heterotrimeric G proteins and the small GTPase Ras.

The biological functions of heterotrimeric G proteins and small GTPases are modulated by both extracellular stimuli and intracellular regulatory proteins. Using Saccharomyces cerevisiae two-hybrid screening, we identified tetratricopeptide repeat 1 (TPR1), a 292-amino-acid protein with three TPR motifs, as a Galpha16-binding protein. The interaction was confirmed both in vitro and in transfected mammalian cells, where TPR1 also binds to several other Galpha proteins. TPR1 was found to interact with Ha-Ras preferentially in its active form. Overexpression of TPR1 promotes accumulation of active Ras. TPR1 was found to compete with the Ras-binding domain (RBD) of Raf-1 for binding to the active Ras, suggesting that it may also compete with Ras GTPase-activating protein, thus contributing to the accumulation of GTP-bound Ras. Expression of Galpha16 strongly enhances the interaction between TPR1 and Ras. Removal of the TPR1 N-terminal 112 residues abolishes potentiation by Galpha16 while maintaining the interaction with Galpha16 and the ability to discriminate active Ras from wild-type Ras. We have also observed that LGN, a Galphai-interacting protein with seven TPR motifs, binds Ha-Ras. Thus, TPR1 is a novel adaptor protein for Ras and selected Galpha proteins that may be involved in protein-protein interaction relating to G-protein signaling.