Intestinal-specific Hdac3 deletion increases susceptibility to colitis and small intestinal tumor development in mice fed a high-fat diet.

High-fat (HF) diets (HFDs) and inflammation are risk factors for colon cancer; however, the underlying mechanisms remain to be fully elucidated. The transcriptional corepressor HDAC3 has recently emerged as a key regulator of intestinal epithelial responses to diet and inflammation with intestinal-specific Hdac3 deletion (Hdac3IKO) in mice increasing fatty acid oxidation genes and the rate of fatty acid oxidation in enterocytes. Hdac3IKO mice are also predisposed to experimentally induced colitis; however, whether this is driven by the intestinal metabolic reprogramming and whether this predisposes these mice to intestinal tumorigenesis is unknown. Herein, we examined the effects of intestinal-specific Hdac3 deletion on colitis-associated intestinal tumorigenesis in mice fed a standard (STD) or HFD. Hdac3IKO mice were highly prone to experimentally induced colitis, which was further enhanced by an HFD. Hdac3 deletion also accelerated intestinal tumor development, specifically when fed an HFD and most notably in the small intestine where lipid absorption is maximal. Expression of proteins involved in fatty acid metabolism and oxidation (SCD1, EHHADH) were elevated in the small intestine of Hdac3IKO mice fed an HFD, and these mice displayed increased levels of lipid peroxidation, DNA damage, and apoptosis in their villi, as well as extensive expansion of the stem cell and progenitor cell compartment. These findings reveal a novel role for Hdac3 in suppressing colitis and intestinal tumorigenesis, particularly in the context of consumption of an HFD, and reveal a potential mechanism by which HFDs may increase intestinal tumorigenesis by increasing fatty acid oxidation, DNA damage, and intestinal epithelial cell turnover.NEW & NOTEWORTHY We reveal a novel role for the transcriptional corepressor Hdac3 in suppressing colitis and intestinal tumorigenesis, particularly in the context of consumption of an HFD, and reveal a potential mechanism by which HFDs may increase intestinal tumorigenesis by increasing fatty acid oxidation, DNA damage, and intestinal epithelial cell turnover. We also identify a unique mouse model for investigating the complex interplay between diet, metabolic reprogramming, and tumor predisposition in the intestinal epithelium.

An acetate-yielding diet imprints an immune and anti-microbial programme against enteric infection.

OBJECTIVES: During gastrointestinal infection, dysbiosis can result in decreased production of microbially derived short-chain fatty acids (SCFAs). In response to the presence of intestinal pathogens, we examined whether an engineered acetate- or butyrate-releasing diet can rectify the deficiency of SCFAs and lead to the resolution of enteric infection. METHODS: We tested whether a high acetate- or butyrate-producing diet (HAMSA or HAMSB, respectively) condition Citrobacter rodentium infection in mice and assess its impact on host-microbiota interactions. We analysed the adaptive and innate immune responses, changes in gut microbiome function, epithelial barrier function and the molecular mechanism via metabolite sensing G protein-coupled receptor 43 (GPR43) and IL-22 expression. RESULTS: HAMSA diet rectified the deficiency in acetate production and protected against enteric infection. Increased SCFAs affect the expression of pathogen virulence genes. HAMSA diet promoted compositional and functional changes in the gut microbiota during infection similar to healthy microbiota from non-infected mice. Bacterial changes were evidenced by the production of proteins involved in acetate utilisation, starch and sugar degradation, amino acid biosynthesis, carbohydrate transport and metabolism. HAMSA diet also induced changes in host proteins critical in glycolysis, wound healing such as GPX1 and epithelial architecture such as EZR1 and PFN1. Dietary acetate assisted in rapid epithelial repair, as shown by increased colonic Muc-2, Il-22, and anti-microbial peptides. We found that acetate increased numbers of colonic IL-22 producing TCRαß+CD8αß+ and TCRγδ+CD8αα+ intraepithelial lymphocytes expressing GPR43. CONCLUSION: HAMSA diet may be an effective therapeutic approach for fighting inflammation and enteric infections and offer a safe alternative that may impact on human health.

Molecular regulators of lipid metabolism in the intestine - Underestimated therapeutic targets for obesity?

The incidence of obesity and type 2 diabetes continues to rise across the globe necessitating the need to identify new therapeutic approaches to manage these diseases. In this review, we explore the potential for therapeutic interventions focussed on the intestinal epithelium, by targeting the role of this tissue in lipid uptake, lipid-mediated cross talk and lipid oxidation. We focus initially on ongoing strategies to manage obesity by targeting the essential role of the intestinal epithelium in lipid uptake, and in mediating tissue cross talk to regulate food intake. Subsequently, we explore a previously underestimated capacity of intestinal epithelial cells to oxidize fatty acids. In this context, we describe recent findings which have unveiled a key role for the peroxisome proliferator-activated receptor (PPAR) family of nuclear receptors and histone deacetylases (HDACs) in the regulation of lipid oxidation genes in enterocytes and how targeted genetic manipulation of these factors in enterocytes reduces weight gain, identifying intestinal PPARs and HDACs as potential therapeutic targets in the management of obesity.

Deletion of intestinal Hdac3 remodels the lipidome of enterocytes and protects mice from diet-induced obesity.

Histone deacetylase 3 (Hdac3) regulates the expression of lipid metabolism genes in multiple tissues, however its role in regulating lipid metabolism in the intestinal epithelium is unknown. Here we demonstrate that intestine-specific deletion of Hdac3 (Hdac3IKO) protects mice from diet induced obesity. Intestinal epithelial cells (IECs) from Hdac3IKO mice display co-ordinate induction of genes and proteins involved in mitochondrial and peroxisomal ß-oxidation, have an increased rate of fatty acid oxidation, and undergo marked remodelling of their lipidome, particularly a reduction in long chain triglycerides. Many HDAC3-regulated fatty oxidation genes are transcriptional targets of the PPAR family of nuclear receptors, Hdac3 deletion enhances their induction by PPAR-agonists, and pharmacological HDAC3 inhibition induces their expression in enterocytes. These findings establish a central role for HDAC3 in co-ordinating PPAR-regulated lipid oxidation in the intestinal epithelium, and identify intestinal HDAC3 as a potential therapeutic target for preventing obesity and related diseases.

Enhanced Solubility, Permeability and Anticancer Activity of Vorinostat Using Tailored Mesoporous Silica Nanoparticles.

Suberoylanilide hydroxamic acid (SAHA) or vorinostat (VOR) is a potent inhibitor of class I histone deacetylases (HDACs) that is approved for the treatment of cutaneous T-cell lymphoma. However, it has the intrinsic limitations of low water solubility and low permeability which reduces its clinical potential especially when given orally. Packaging of drugs within ordered mesoporous silica nanoparticles (MSNs) is an emerging strategy for increasing drug solubility and permeability of BCS (Biopharmaceutical Classification System) class II and IV drugs. In this study, we encapsulated vorinostat within MSNs modified with different functional groups, and assessed its solubility, permeability and anti-cancer efficacy in vitro. Compared to free drug, the solubility of vorinostat was enhanced 2.6-fold upon encapsulation in pristine MSNs (MCM-41-VOR). Solubility was further enhanced when MSNs were modified with silanes having amino (3.9 fold) or phosphonate (4.3 fold) terminal functional groups. Moreover, permeability of vorinostat into Caco-2 human colon cancer cells was significantly enhanced for MSN-based formulations, particularly MSNs modified with amino functional group (MCM-41-NH2-VOR) where it was enhanced ~4 fold. Compared to free drug, vorinostat encapsulated within amino-modified MSNs robustly induced histone hyperacetylation and expression of established histone deacetylase inhibitor (HDACi)-target genes, and induced extensive apoptosis in HCT116 colon cancer cells. Similar effects were observed on apoptosis induction in HH cutaneous T-cell lymphoma cells. Thus, encapsulation of the BCS class IV molecule vorinostat within MSNs represents an effective strategy for improving its solubility, permeability and anti-tumour activity.

Interleukin 33 Signaling Restrains Sporadic Colon Cancer in an Interferon-γ-Dependent Manner.

Interleukin 33 (IL33) is an inflammatory cytokine released during necrotic cell death. The epithelium and stroma of the intestine express large amounts of IL33 and its receptor St2. IL33 is therefore continuously released during homeostatic turnover of the intestinal mucosa. Although IL33 can prevent colon cancer associated with inflammatory colitis, the contribution of IL33 signaling to sporadic colon cancer remains unknown. Here, we utilized a mouse model of sporadic colon cancer to investigate the contribution of IL33 signaling to tumorigenesis in the absence of preexisting inflammation. We demonstrated that genetic ablation of St2 enhanced colon tumor development. Conversely, administration of recombinant IL33 reduced growth of colon cancer cell allografts. In reciprocal bone marrow chimeras, the concurrent loss of IL33 signaling within radioresistant nonhematopoietic, and the radiosensitive hematopoietic, compartments was associated with increased tumor burden. We detected St2 expression within the radioresistant mesenchymal cell compartment of the colon whose stimulation with IL33 induced expression of bona fide NF-κB target genes. Mechanistically, we discovered that St2 deficiency within the nonhematopoietic compartment coincided with increased abundance of regulatory T cells and suppression of an IFNγ gene expression signature, whereas IL33 administration triggered IFNγ expression by tumor allograft-infiltrating T cells. The decrease of this IFNγ gene expression signature was associated with more aggressive disease in human colon cancer patients, suggesting that lack of IL33 signaling impaired the generation of a potent IFNγ-mediated antitumor immune response. Collectively, our data reveal that IL33 functions as a tumor suppressor in sporadic colon cancer. Cancer Immunol Res; 6(4); 409-21. ©2018 AACR.

DUSP5 is methylated in CIMP-high colorectal cancer but is not a major regulator of intestinal cell proliferation and tumorigenesis.

The ERK signalling pathway regulates key cell fate decisions in the intestinal epithelium and is frequently dysregulated in colorectal cancers (CRCs). Variations in the dynamics of ERK activation can induce different biological outcomes and are regulated by multiple mechanisms, including activation of negative feedback loops involving transcriptional induction of dual-specificity phosphatases (DUSPs). We have found that the nuclear ERK-selective phosphatase DUSP5 is downregulated in colorectal tumours and cell lines, as previously observed in gastric and prostate cancer. The DUSP5 promoter is methylated in a subset of CRC cell lines and primary tumours, particularly those with a CpG island methylator phenotype (CIMP). However, this epigenetic change alone could not account for reduced DUSP5 expression in CRC cells. Functionally, DUSP5 depletion failed to alter ERK signalling or proliferation in CRC cell lines, and its transgenic overexpression in the mouse intestine had minimal impact on normal intestinal homeostasis or tumour development. Our results suggest that DUSP5 plays a limited role in regulating ERK signalling associated with the growth of colorectal tumours, but that methylation the DUSP5 gene promoter can serve as an additional means of identifying CIMP-high colorectal cancers.

ATF3 Repression of BCL-XL Determines Apoptotic Sensitivity to HDAC Inhibitors across Tumor Types.

Purpose: Histone deacetylase inhibitors (HDACi) are epigenome-targeting small molecules approved for the treatment of cutaneous T-cell lymphoma and multiple myeloma. They have also demonstrated clinical activity in acute myelogenous leukemia, non-small cell lung cancer, and estrogen receptor-positive breast cancer, and trials are underway assessing their activity in combination regimens including immunotherapy. However, there is currently no clear strategy to reliably predict HDACi sensitivity. In colon cancer cells, apoptotic sensitivity to HDACi is associated with transcriptional induction of multiple immediate-early (IE) genes. Here, we examined whether this transcriptional response predicts HDACi sensitivity across tumor type and investigated the mechanism by which it triggers apoptosis.Experimental Design: Fifty cancer cell lines from diverse tumor types were screened to establish the correlation between apoptotic sensitivity, induction of IE genes, and components of the intrinsic apoptotic pathway.Results: We show that sensitivity to HDACi across tumor types is predicted by induction of the IE genes FOS, JUN, and ATF3, but that only ATF3 is required for HDACi-induced apoptosis. We further demonstrate that the proapoptotic function of ATF3 is mediated through direct transcriptional repression of the prosurvival factor BCL-XL (BCL2L1) These findings provided the rationale for dual inhibition of HDAC and BCL-XL, which we show strongly cooperate to overcome inherent resistance to HDACi across diverse tumor cell types.Conclusions: These findings explain the heterogeneous responses of tumor cells to HDACi-induced apoptosis and suggest a framework for predicting response and expanding their therapeutic use in multiple cancer types. Clin Cancer Res; 23(18); 5573-84. ©2017 AACR.

Erratum to: "Monoallelic germline methylation and sequence variant in the promoter of the RB1 gene: a possible constitutive epimutation in hereditary retinoblastoma".

[This corrects the article DOI: 10.1186/s13148-015-0167-0.].

Transketolase-like 1 ectopic expression is associated with DNA hypomethylation and induces the Warburg effect in melanoma cells.

BACKGROUND: The metabolism of cancer cells is often reprogrammed by dysregulation of metabolic enzymes. Transketolase-like 1 (TKTL1) is a homodimeric transketolase linking the pentose-phosphate pathway with the glycolytic pathway. It is generally silenced at a transcriptional level in somatic tissues. However, in human cancers its expression is associated with the acquisition of a glycolytic phenotype (the Warburg effect) by cancer cells that contributes to the progression of malignant tumors. In melanoma, defective promoter methylation results in the expression of genes and their products that can affect the tumor cell's phenotype including the modification of immune and functional characteristics. The present study evaluates the role of TKTL1 as a mediator of disease progression in melanoma associated with a defective methylation phenotype. METHODS: The expression of TKTL1 in metastatic melanoma tumors and cell lines was analysed by qRT-PCR and immunohistochemistry. The promoter methylation status of TKTL1 in melanoma cells was evaluated by quantitative methylation specific PCR. Using qRT-PCR, the effect of a DNA demethylating agent 5-aza-2'-deoxycytidine (5aza) on the expression of TKTL1 was examined. Biochemical and molecular analyses such as glucose consumption, lactate production, invasion, proliferation and cell cycle progression together with ectopic expression and siRNA mediated knockdown were used to investigate the role of TKTL1 in melanoma cells. RESULTS: Expression of TKTL1 was highly restricted in normal adult tissues and was overexpressed in a subset of metastatic melanoma tumors and derived cell lines. The TKTL1 promoter was activated by hypomethylation and treatment with 5aza induced TKTL1 expression in melanoma cells. Augmented expression of TKTL1 in melanoma cells was associated with a glycolytic phenotype. Loss and gain of function studies revealed that TKTL1 contributed to enhanced invasion of melanoma cells. CONCLUSIONS: Our data provide evidence for an important role of TKTL1 in aerobic glycolysis and tumor promotion in melanoma that may result from defective promoter methylation. This epigenetic change may enable the natural selection of tumor cells with a metabolic phenotype and thereby provide a potential therapeutic target for a subset of melanoma tumors with elevated TKTL1 expression.

"Monoallelic germline methylation and sequence variant in the promoter of the RB1 gene: a possible constitutive epimutation in hereditary retinoblastoma".

BACKGROUND: Retinoblastoma is a malignant tumor of the retina in children <5 years of age and occurs after two mutations in the RB1 gene. The first mutation (M1) is germinal and confers predisposition to the hereditary type, which is transmitted as an autosomal dominant highly penetrant trait, so 90 % of carriers develop retinoblastoma; however, 10 % of carriers either do not develop the tumor or develop it unilaterally. Most mutations are point mutations. Inactivation of the RB1 gene is usually caused by mutations affecting the coding region. Silencing by methylation of the RB1 promoter has been observed in retinoblastoma tumors as a second mutation (M2) and is classified as somatic epimutation. Germline methylation of the RB1 gene promoter was studied in a particular pedigree of six generations from the paternal side, with incomplete penetrance and bias towards healthy male carriers and those affected with unilateral retinoblastoma. RESULTS: The methylation status of the 27 CpGs dinucleotides that constitute the core of the RB1 gene promoter, analyzed by cloning and genomic sequencing after DNA sodium bisulfite conversion, demonstrated a monoallelic methylation pattern which coincides with a c. [-187T > G; -188T > G] sequence variant that is found in peripheral blood lymphocytes and tumor DNA. Unexpectedly, it was the mother who transmitted this variant to two more generations. Microsatellite markers of D chromosome showed a biparental contribution of both D13 chromosomes to the retinoblastoma phenotype, conferring double heterozygosity in the affected cases. CONCLUSIONS: The monoallelic genetic-epigenetic finding, the sequence variant, and methylation suggest a constitutive epimutation and probably a genetic-epigenetic hereditary predisposition for retinoblastoma in this family.

The intestinal epithelial cell differentiation marker intestinal alkaline phosphatase (ALPi) is selectively induced by histone deacetylase inhibitors (HDACi) in colon cancer cells in a Kruppel-like factor 5 (KLF5)-dependent manner.

The histone deacetylase inhibitor (HDACi) sodium butyrate promotes differentiation of colon cancer cells as evidenced by induced expression and enzyme activity of the differentiation marker intestinal alkaline phosphatase (ALPi). Screening of a panel of 33 colon cancer cell lines identified cell lines sensitive (42%) and resistant (58%) to butyrate induction of ALP activity. This differential sensitivity was similarly evident following treatment with the structurally distinct HDACi, MS-275. Resistant cell lines were significantly enriched for those harboring the CpG island methylator phenotype (p = 0.036, Chi square test), and resistant cell lines harbored methylation of the ALPi promoter, particularly of a CpG site within a critical KLF/Sp regulatory element required for butyrate induction of ALPi promoter activity. However, butyrate induction of an exogenous ALPi promoter-reporter paralleled up-regulation of endogenous ALPi expression across the cell lines, suggesting the presence or absence of a key transcriptional regulator is the major determinant of ALPi induction. Through microarray profiling of sensitive and resistant cell lines, we identified KLF5 to be both basally more highly expressed as well as preferentially induced by butyrate in sensitive cell lines. KLF5 overexpression induced ALPi promoter-reporter activity in resistant cell lines, KLF5 knockdown attenuated butyrate induction of ALPi expression in sensitive lines, and butyrate selectively enhanced KLF5 binding to the ALPi promoter in sensitive cells. These findings demonstrate that butyrate induction of the cell differentiation marker ALPi is mediated through KLF5 and identifies subsets of colon cancer cell lines responsive and refractory to this effect.

Gain of DNA methylation is enhanced in the absence of CTCF at the human retinoblastoma gene promoter.

BACKGROUND: Long-term gene silencing throughout cell division is generally achieved by DNA methylation and other epigenetic processes. Aberrant DNA methylation is now widely recognized to be associated with cancer and other human diseases. Here we addressed the contribution of the multifunctional nuclear factor CTCF to the epigenetic regulation of the human retinoblastoma (Rb) gene promoter in different tumoral cell lines. METHODS: To assess the DNA methylation status of the Rb promoter, genomic DNA from stably transfected human erythroleukemic K562 cells expressing a GFP reporter transgene was transformed with sodium bisulfite, and then PCR-amplified with modified primers and sequenced. Single- and multi-copy integrants with the CTCF binding site mutated were isolated and characterized by Southern blotting. Silenced transgenes were reactivated using 5-aza-2'-deoxycytidine and Trichostatin-A, and their expression was monitored by fluorescent cytometry. Rb gene expression and protein abundance were assessed by RT-PCR and Western blotting in three different glioma cell lines, and DNA methylation of the promoter region was determined by sodium bisulfite sequencing, together with CTCF dissociation and methyl-CpG-binding protein incorporation by chromatin immunoprecipitation assays. RESULTS: We found that the inability of CTCF to bind to the Rb promoter causes a dramatic loss of gene expression and a progressive gain of DNA methylation. CONCLUSIONS: This study indicates that CTCF plays an important role in maintaining the Rb promoter in an optimal chromatin configuration. The absence of CTCF induces a rapid epigenetic silencing through a progressive gain of DNA methylation. Consequently, CTCF can now be seen as one of the epigenetic components that allows the proper configuration of tumor suppressor gene promoters. Its aberrant dissociation can then predispose key genes in cancer cells to acquire DNA methylation and epigenetic silencing.