Adar3 Is Involved in Learning and Memory in Mice.

The amount of regulatory RNA encoded in the genome and the extent of RNA editing by the post-transcriptional deamination of adenosine to inosine (A-I) have increased with developmental complexity and may be an important factor in the cognitive evolution of animals. The newest member of the A-I editing family of ADAR proteins, the vertebrate-specific ADAR3, is highly expressed in the brain, but its functional significance is unknown. In vitro studies have suggested that ADAR3 acts as a negative regulator of A-I RNA editing but the scope and underlying mechanisms are also unknown. Meta-analysis of published data indicates that mouse Adar3 expression is highest in the hippocampus, thalamus, amygdala, and olfactory region. Consistent with this, we show that mice lacking exon 3 of Adar3 (which encodes two double stranded RNA binding domains) have increased levels of anxiety and deficits in hippocampus-dependent short- and long-term memory formation. RNA sequencing revealed a dysregulation of genes involved in synaptic function in the hippocampi of Adar3-deficient mice. We also show that ADAR3 transiently translocates from the cytoplasm to the nucleus upon KCl-mediated activation in SH-SY5Y cells. These results indicate that ADAR3 contributes to cognitive processes in mammals.

Microsatellite Instability in Mouse Models of Colorectal Cancer.

Microsatellite instability (MSI) is caused by DNA mismatch repair deficiency and is an important prognostic and predictive biomarker in colorectal cancer but relatively few studies have exploited mouse models in the study of its clinical utility. Furthermore, most previous studies have looked at MSI in the small intestine rather than the colon of mismatch repair deficient Msh2-knockout (KO) mice. Here we compared Msh2-KO, p53-KO, and wild type (WT) mice that were treated with the carcinogen azoxymethane (AOM) and the nonsteroidal anti-inflammatory drug sulindac or received no treatment. The induced tumors and normal tissue specimens from the colon were analysed with a panel of five mononucleotide repeat markers. MSI was detected throughout the normal colon in untreated Msh2-KO mice and this involved contraction of the repeat sequences compared to WT. The markers with longer mononucleotide repeats (37-59) were the most sensitive for MSI while the markers with shorter repeats (24) showed only minor change. AOM exposure caused further contraction of the Bat37 and Bat59 repeats in the distal colon of Msh2-KO mice which was reversed by sulindac. Thus AOM-induced carcinogenesis is associated with increased instability of mononucleotide repeats in the colon of Msh2-KO mice but not in WT or p53-KO mice. Chemoprevention of these tumors by sulindac treatment reversed or prevented the increased MSI.

HIF1α deficiency reduces inflammation in a mouse model of proximal colon cancer.

Hypoxia-inducible factor 1α (HIF1α) is a transcription factor that regulates the adaptation of cells to hypoxic microenvironments, for example inside solid tumours. Stabilisation of HIF1α can also occur in normoxic conditions in inflamed tissue or as a result of inactivating mutations in negative regulators of HIF1α. Aberrant overexpression of HIF1α in many different cancers has led to intensive efforts to develop HIF1α-targeted therapies. However, the role of HIF1α is still poorly understood in chronic inflammation that predisposes the colon to carcinogenesis. We have previously reported that the transcription of HIF1α is upregulated and that the protein is stabilised in inflammatory lesions that are caused by the non-steroidal anti-inflammatory drug (NSAID) sulindac in the mouse proximal colon. Here, we exploited this side effect of long-term sulindac administration to analyse the role of HIF1α in colon inflammation using mice with a Villin-Cre-induced deletion of Hif1α exon 2 in the intestinal epithelium (Hif1α(ΔIEC)). We also analysed the effect of sulindac sulfide on the aryl hydrocarbon receptor (AHR) pathway in vitro in colon cancer cells. Most sulindac-treated mice developed visible lesions, resembling the appearance of flat adenomas in the human colon, surrounded by macroscopically normal mucosa. Hif1α(ΔIEC) mice still developed lesions but they were smaller than in the Hif1α-floxed siblings (Hif1α(F/F)). Microscopically, Hif1α(ΔIEC) mice had significantly less severe colon inflammation than Hif1α(F/F) mice. Molecular analysis showed reduced MIF expression and increased E-cadherin mRNA expression in the colon of sulindac-treated Hif1α(ΔIEC) mice. However, immunohistochemistry analysis revealed a defect of E-cadherin protein expression in sulindac-treated Hif1α(ΔIEC) mice. Sulindac sulfide treatment in vitro upregulated Hif1α, c-JUN and IL8 expression through the AHR pathway. Taken together, HIF1α expression augments inflammation in the proximal colon of sulindac-treated mice, and AHR activation by sulindac might lead to the reduction of E-cadherin protein levels through the mitogen-activated protein kinase (MAPK) pathway.

MCC inhibits beta-catenin transcriptional activity by sequestering DBC1 in the cytoplasm.

The mutated in colorectal cancer (MCC) is a multifunctional gene showing loss of expression in colorectal and liver cancers. MCC mutations can drive colon carcinogenesis in the mouse and in vitro experiments suggest that loss of MCC function promotes cancer through several important cellular pathways. In particular, the MCC protein is known to regulate beta-catenin (ß-cat) signaling, but the mechanism is poorly understood. Here we show that the ß-cat repressor function of MCC is strongly impaired by the presence of a disease-associated mutation. We also identify deleted in breast cancer 1 (DBC1) as a new MCC interacting partner and regulator of ß-cat signaling. RNA interference experiments show that DBC1 promotes ß-cat transcriptional activity and that the presence of DBC1 is required for MCC-mediated ß-cat repression. In contrast to all other DBC1 interacting partners, MCC does not interact through the DBC1 Leucine Zipper domain but with a glutamic-acid rich region located between the Nudix and EF-hand domains. Furthermore, MCC overexpression relocalizes DBC1 from the nucleus to the cytoplasm and reduces ß-cat K49 acetylation. Treatment of cells with the SIRT1 inhibitor Nicotinamide reverses MCC-induced deacetylation of ß-cat K49. These data suggest that the cytoplasmic MCC-DBC1 interaction sequesters DBC1 away from the nucleus, thereby removing a brake on DBC1 nuclear targets, such as SIRT1. This study provides new mechanistic insights into the DBC1-MCC axis as a new APC independent ß-cat inhibitory pathway.

Sulindac activates NF-κB signaling in colon cancer cells.

BACKGROUND: The non-steroidal anti-inflammatory drug (NSAID) sulindac has shown efficacy in preventing colorectal cancer. This potent anti-tumorigenic effect is mediated through multiple cellular pathways but is also accompanied by gastrointestinal side effects, such as colon inflammation. We have recently shown that sulindac can cause up-regulation of pro-inflammatory factors in the mouse colon mucosa. The aim of this study was to determine the signaling pathways that mediate the transcriptional activation of pro-inflammatory cytokines in colon cancer epithelial cells treated with sulindac sulfide. RESULTS: We found that sulindac sulfide increased NF-κB signaling in HCT-15, HCT116, SW480 and SW620 cells, although the level of induction varied between cell lines. The drug caused a decrease in IκBα levels and an increase of p65(RelA) binding to the NF-κB DNA response element. It induced expression of IL-8, ICAM1 and A20, which was inhibited by the NF-κB inhibitor PDTC. Sulindac sulfide also induced activation of the AP-1 transcription factor, which co-operated with NF-κB in up-regulating IL-8. Up-regulation of NF-κB genes was most prominent in conditions where only a subset of cells was undergoing apoptosis. In TNFα stimulated conditions the drug treatment inhibited phosphorylation on IκBα (Ser 32) which is consistent with previous studies and indicates that sulindac sulfide can inhibit TNFα-induced NF-κB activation. Sulindac-induced upregulation of NF-κB target genes occurred early in the proximal colon of mice given a diet containing sulindac for one week. CONCLUSIONS: This study shows for the first time that sulindac sulfide can induce pro-inflammatory NF-κB and AP-1 signaling as well as apoptosis in the same experimental conditions. Therefore, these results provide insights into the effect of sulindac on pro-inflammatory signaling pathways, as well as contribute to a better understanding of the mechanism of sulindac-induced gastrointestinal side effects.

Review: Mouse models of inflammatory bowel disease--insights into the mechanisms of inflammation-associated colorectal cancer.

The association between chronic inflammation and cancer has been noted for at least a century but the exact molecular mechanisms of cancer initiation and promotion by such inflammation are still poorly understood. The gastrointestinal tract is a unique organ where maintaining a balance between the colonic epithelial cells, the immune system and a fine-tuned response to the resident microflora is crucial for preserving the gut homeostasis. A breakdown of the tight interdependent regulation of the epithelium-immunity-microbiota triangle leads to inflammatory bowel disorders and may promote cancer. This review focuses on inflammation-associated colorectal cancer in mouse models of the disease and highlights emerging research trends.

Loss of special AT-rich binding protein 1 expression is a marker of poor survival in lung cancer.

INTRODUCTION: Lung cancer is the leading cause of cancer-related mortality and requires more effective molecular markers of prognosis and therapeutic responsiveness. Special AT-rich binding protein 1 (SATB1) is a global genome organizer that recruits chromatin remodeling proteins to epigenetically regulate hundreds of genes in a tissue-specific manner. Initial studies suggest that SATB1 overexpression is a predictor of poor prognosis in breast cancer, but the prognostic significance of SATB1 expression has not been evaluated in lung cancer. METHODS: A cohort of 257 lung cancers was evaluated by immunohistochemistry. Epigenetic silencing of SATB1 was examined in cell lines by 5-Aza 2-deoxycytidine and trichostatin A treatment, and chromatin immunoprecipitation. RESULTS: Significant loss of SATB1 expression was found in squamous preinvasive lesions (p < 0.04) and in non-small cell lung cancers (p < 0.001) compared with matched normal bronchial epithelium. Loss of SATB1 independently predicted poor cancer-specific survival in squamous cell carcinomas (SCCs; hazard ratio: 2.06, 95% confidence interval: 1.2-3.7, p = 0.016). Treatment of lung cancer cell lines with the histone deacetylase inhibitor trichostatin A resulted in up-regulation of SATB1. SATB1 was associated with a decrease in the active chromatin mark acetylated histone H3K9 and an increase in the repressive polycomb mark trimethylated H3K27 in a SCC cell line relative to a normal bronchial epithelial cell line. CONCLUSIONS: This is the first study showing that SATB1 expression is lost in early preinvasive squamous lesions and that loss of SATB1 is associated with poor prognosis in lung SCC. We hypothesize that the SATB1 gene is epigenetically silenced through histone modifications.

The NSAID sulindac is chemopreventive in the mouse distal colon but carcinogenic in the proximal colon.

BACKGROUND AND AIMS: The non-steroidal anti-inflammatory drug sulindac is an effective chemopreventive agent in sporadic colorectal cancer but its potential benefit in mismatch repair deficient cancers remains to be defined. We wanted to determine whether genetic defects that are relevant for colorectal cancer, such as Msh2 or p53 deficiency, would influence the efficiency of sulindac chemoprevention or increase the side effects. METHODS: Msh2 or p53 deficient and wild-type mice received feed containing 160-320 ppm sulindac for up to 25 weeks with or without a concurrent treatment with the carcinogen azoxymethane. Colon tissue was analysed by histopathology and molecular biology methods. RESULTS: We show that sulindac prevented azoxymethane-induced distal colon tumours in all mice. In the proximal colon, however, sulindac induced new inflammatory lesions on the mucosal folds, which further developed into adenocarcinoma in up to 18-25% of the p53 or Msh2 deficient mice but rarely in wild-type mice. This region in the proximal colon was characterised by a distinct profile of pro- and anti-inflammatory factors, which were modulated by the sulindac diet, including upregulation of hypoxia inducible factor 1α and macrophage inflammatory protein 2. CONCLUSIONS: These data show that the sulindac diet promotes carcinogenesis in the mouse proximal colon possibly through chronic inflammation. Sulindac has both beneficial and harmful effects in vivo, which are associated with different microenvironments within the colon of experimental mice. Deficiency for the Msh2 or p53 tumour suppressor genes increases the harmful side effects of long-term sulindac treatment in the mouse colon.

The "Mutated in Colorectal Cancer" Protein Is a Novel Target of the UV-Induced DNA Damage Checkpoint.

MCC is a potential tumor suppressor gene, which is silenced by promoter hypermethylation in a subset of colorectal cancers. However, its functions have remained poorly understood. In the present study, we describe a novel function of MCC in the DNA damage response. Several novel phosphorylation sites were identified by mass spectrometry, including 2 highly conserved ATM/ATR consensus sites at serine 118 and serine 120. In addition, exposure to ultraviolet radiation (UV), but not phleomycin, caused PI3K-dependent phosphorylation of MCC and its nuclear localization. Re-expression of MCC in HCT15 colorectal cancer cells led to a G2/M arrest, and MCC knockdown impaired the induction of a G2/M arrest following UV radiation. Finally, mutation of S118/120 to alanine did not affect MCC nuclear shuttling following UV but did impair MCC G2/M checkpoint activity. Thus, these results suggest that MCC is a novel target of the DNA damage checkpoint and that MCC is required for the complete cell cycle arrest in the G2/M phase in response to UV.