Identification of Gene Expression Profiles Associated with an Increased Risk of Post-Operative Recurrence in Crohn's Disease.

BACKGROUND AND AIMS: Ileocolonic resection is frequently needed in the course of Crohn's disease [CD] treatment and post-operative recurrence is extremely common. Our main objective was to analyse gene expression in the mucosa of CD patients at the time of surgery and at post-operative endoscopy, in order to identify predictors and mechanisms of early endoscopic recurrence. METHODS: We conducted transcriptome analyses on ileal mucosa samples collected from inflamed sections of the surgical specimens [n = 200], from ileal resection margins [n = 149] and in the neo-terminal ileum 6 months after surgery [n = 122]; these were compared with non-inflammatory bowel disease controls [n = 25]. The primary endpoint was post-operative endoscopic recurrence at 6 months. We applied regression models to identify gene signatures predicting endoscopic recurrence. RESULTS: Chronic inflammation was associated with strong expression of inflammatory genes [IL-6, IL-8, IL-1B] and decreased expression of genes involved in metabolic processes, but with a high inter-individual heterogeneity. Gene signatures associated with early endoscopic recurrence were mainly characterized by upregulation of TNFα, IFNγ, IL23A and IL17A. Pathway analyses showed that upregulation of mitochondrial dysfunction within the inflamed sections and JAK/STAT at the ileal margin were predictive of post-operative recurrence. A combined model integrating these top pathway signatures improved the prediction of endoscopic recurrence [area under the curve of 0.79]. STAT3 phosphorylation at the surgical ileal margin was associated with severe recurrence at 6 months. CONCLUSION: We identified several biological pathways in surgical ileal mucosa specimens associated with an increased risk of disease recurrence. Integration of the JAK/STAT and mitochondrial dysfunction pathways in the clinical model improved the prediction of post-operative recurrence.

Meta-Analysis of IBD Gut Samples Gene Expression Identifies Specific Markers of Ileal and Colonic Diseases.

BACKGROUND: Inflammatory bowel diseases (IBDs) are characterized by chronic inflammation and tissue damages in limited segments of the digestive tract. Pathogenesis in the tissue and mucosal inflammation probably differs according to disease location. Our aim was to further analyze transcriptomic profiles in different locations of IBD, differentiating ulcerative colitis (UC), colonic Crohn's disease (CD), ileal CD, and pouchitis, with respect to normal colonic and ileal mucosa. We thus performed a meta-analysis focusing on specific transcriptomic signatures of ileal and colonic diseases. METHODS: We identified 5 cohorts with available transcriptomic data in ileal or colonic samples from active IBD and non-IBD control samples. The meta-analysis was performed on 1047 samples. In each cohort separately, we compared gene expression in CD ileitis and normal ileum; in CD colitis, UC, and normal colon; and finally in pouchitis and normal ileum. RESULTS: We identified specific markers of ileal (FOLH1, CA2) and colonic (REG3A) inflammation and showed that, with disease, some cells from the ileum start to express colonic markers. We confirmed by immunohistochemistry that these markers were specifically present in ileal or colonic diseases. We highlighted that, overall, colonic CD resembles UC and is distinct from ileal CD, which is in turn closer to pouchitis. CONCLUSIONS: We demonstrated that ileal and colonic diseases exhibit specific signatures, independent of their initial clinical classification. This supports molecular, rather than clinical, disease stratification, and may be used to design drugs specifically targeting ileal or colonic diseases.

We perform a meta-analysis of publicly available inflammatory bowel disease transcriptomes and identify FOLH1, REG3A, and CA2 as specific markers of ileal and colonic diseases. We demonstrate that Crohn's colitis resembles ulcerative colitis, while Crohn's ileitis resembles pouchitis.

KLRG1 and CD103 Expressions Define Distinct Intestinal Tissue-Resident Memory CD8 T Cell Subsets Modulated in Crohn's Disease.

Intestinal tissue-resident memory CD8 T cells (Trm) are non-recirculating effector cells ideally positioned to detect and react to microbial infections in the gut mucosa. There is an emerging understanding of Trm cell differentiation and functions, but their implication in inflammatory bowel diseases, such as Crohn's disease (CD), is still unknown. Here, we describe CD8 cells in the human intestine expressing KLRG1 or CD103, two receptors of E-cadherin. While CD103 CD8 T cells are present in high numbers in the mucosa of CD patients and controls, KLRG1 CD8 T cells are increased in inflammatory conditions. Mucosal CD103 CD8 T cells are more responsive to TCR restimulation, but KLRG1 CD8 T cells show increased cytotoxic and proliferative potential. CD103 CD8 T cells originate mostly from KLRG1 negative cells after TCR triggering and TGFß stimulation. Interestingly, mucosal CD103 CD8 T cells from CD patients display major changes in their transcriptomic landscape compared to controls. They express Th17 related genes including CCL20, IL22, and IL26, which could contribute to the pathogenesis of CD. Overall, these findings suggest that CD103 CD8 T cells in CD induce a tissue-wide alert increasing innate immune responses and recruitment of effector cells such as KLRG1 CD8 T cells.

Association Between Microscopic Lesions at Ileal Resection Margin and Recurrence After Surgery in Patients With Crohn's Disease.

BACKGROUND AND AIMS: Different types of histologic lesions at the ileal margin, detected by histology, have been associated with increased rates of recurrence after ileocaecal surgery in patients with Crohn's disease (CD). We aimed to characterize histologic features of the ileal margin and to evaluate their association with disease recurrence. METHODS: We collected histologic data from 211 patients with ileal or ileocolonic CD who underwent ileocolonic resections at hospitals in France from September 2010 through December 2016. Ileal margins were analyzed. Early endoscopic recurrence was defined by a Rutgeerts score of i2 or more, 6 months after surgery. We also collected data from 10 adults with healthy ileum who underwent ileocecal resection for colonic tumors (controls). Clinical relapse was defined by CD-related symptoms confirmed by imaging, endoscopy, therapy intensification, CD-related complication, or subsequent surgery. RESULTS: Six months after surgery, 49% of patients had endoscopic recurrence; 5 years after surgery, 57% of patients had clinical relapse. Ileal margins were macroscopically affected in 20.9% of patients. CD transmural lesions at the margin (defined by mucosal ulceration or cryptitis, submucosal fibrosis and lymphoplasmacytic infiltrate of the subserosa) were observed in 13.6% of patients. Endoscopic recurrence was observed in 75% of patients with CD transmural lesions vs 46% of patients without (P =.005). In multivariate analysis, CD transmural lesions at the margin were independently associated with early endoscopic recurrence (OR, 3.83; 95% CI, 1.47-11.05; P =.008) and clinical recurrence (OR 2.04; 95% CI, 1.09-3.99; P =.026). CONCLUSION: In patients with CD, transmural lesions at the ileal margin were associated with an increased risk of post-operative recurrence. Histologic features of the ileal margin should be included in making decisions about post-operative therapy.

T cell clonal expansions in ileal Crohn's disease are associated with smoking behaviour and postoperative recurrence.

T cell clonal expansions are present in the inflamed mucosa of patients with Crohn's disease (CD) and may be implicated in postoperative recurrence after ileocolonic resection. METHODS: T cell receptor (TCR) analysis was performed in 57 patients included in a prospective multicentre cohort. Endoscopic recurrence was defined by a Rutgeerts score >i0. DNA and mRNA were extracted from biopsies collected from the surgical specimen and endoscopy, and analysed by high throughput sequencing and microarray, respectively. RESULTS: TCR repertoire in the mucosa of patients with CD displayed diverse clonal expansions. Active smokers at time of surgery had a significantly increased proportion of clonal expansions as compared with non-smokers (25.9%vs17.9%, p=0.02). The percentage of high frequency clones in the surgical specimen was significantly higher in patients with recurrence and correlated with postoperative endoscopic recurrence (area under the curve (AUC) 0.69, 95% CI 0.54 to 0.83). All patients with clonality above 26.8% (18/57) had an endoscopic recurrence. These patients with a high clonality were more frequently smokers than patients with a low clonality (61% vs 23%, p=0.005). The persistence of a similar TCR repertoire at postoperative endoscopy was associated with smoking and disease recurrence. Patients with high clonality showed increased expression of genes associated with CD8 T cells and reduced expression of inflammation-related genes. Expanded clones were found predominantly in the CD8 T cell compartment. CONCLUSION: Clonal T cell expansions are implicated in postoperative endoscopic recurrence. CD patients with increased proportion of clonal T cell expansions in the ileal mucosa represent a subgroup associated with smoking and where pathogenesis appears as T cell driven. TRIAL REGISTRATION NUMBER: NCT03458195.

Global analysis of H3K27me3 as an epigenetic marker in prostate cancer progression.

BACKGROUND: H3K27me3 histone marks shape the inhibition of gene transcription. In prostate cancer, the deregulation of H3K27me3 marks might play a role in prostate tumor progression. METHODS: We investigated genome-wide H3K27me3 histone methylation profile using chromatin immunoprecipitation (ChIP) and 2X400K promoter microarrays to identify differentially-enriched regions in biopsy samples from prostate cancer patients. H3K27me3 marks were assessed in 34 prostate tumors: 11 with Gleason score > 7 (GS > 7), 10 with Gleason score ≤ 7 (GS ≤ 7), and 13 morphologically normal prostate samples. RESULTS: Here, H3K27me3 profiling identified an average of 386 enriched-genes on promoter regions in healthy control group versus 545 genes in GS ≤ 7 and 748 genes in GS > 7 group. We then ran a factorial discriminant analysis (FDA) and compared the enriched genes in prostate-tumor biopsies and normal biopsies using ANOVA to identify significantly differentially-enriched genes. The analysis identified ALG5, EXOSC8, CBX1, GRID2, GRIN3B, ING3, MYO1D, NPHP3-AS1, MSH6, FBXO11, SND1, SPATS2, TENM4 and TRA2A genes. These genes are possibly associated with prostate cancer. Notably, the H3K27me3 histone mark emerged as a novel regulatory mechanism in poor-prognosis prostate cancer. CONCLUSIONS: Our findings point to epigenetic mark H3K27me3 as an important event in prostate carcinogenesis and progression. The results reported here provide new molecular insights into the pathogenesis of prostate cancer.

H3K4 acetylation, H3K9 acetylation and H3K27 methylation in breast tumor molecular subtypes.

AIM: Here, we investigated how the St Gallen breast molecular subtypes displayed distinct histone H3 profiles. PATIENTS & METHODS: 192 breast tumors divided into five St Gallen molecular subtypes (luminal A, luminal B HER2-, luminal B HER2+, HER2+ and basal-like) were evaluated for their histone H3 modifications on gene promoters. RESULTS: ANOVA analysis allowed to identify specific H3 signatures according to three groups of genes: hormonal receptor genes (ERS1, ERS2, PGR), genes modifying histones (EZH2, P300, SRC3) and tumor suppressor gene (BRCA1). A similar profile inside high-risk cancers (luminal B [HER2+], HER2+ and basal-like) compared with low-risk cancers including luminal A and luminal B (HER2-) were demonstrated. CONCLUSION: The H3 modifications might contribute to clarify the differences between breast cancer subtypes.

Epigenetic Modifications with DZNep, NaBu and SAHA in Luminal and Mesenchymal-like Breast Cancer Subtype Cells.

BACKGROUND/AIM: Numerous studies have shown that breast cancer and epigenetic mechanisms have a very powerful interactive relation. The MCF7 cell line, representative of luminal subtype and the MDA-MB 231 cell line representative of mesenchymal-like subtype were treated respectively with a Histone Methyl Transferase Inhibitors (HMTi), 3-Deazaneplanocin hydrochloride (DZNep), two histone deacetylase inhibitors (HDACi), sodium butyrate (NaBu), and suberoylanilide hydroxamic acid (SAHA) for 48 h. MATERIALS AND METHODS: Chromatin immunoprecipitation (ChIP) was used to observe HDACis (SAHA and NaBu) and HMTi (DZNep) impact on histones and more specifically on H3K27me3, H3K9ac and H3K4ac marks with Q-PCR analysis of BRCA1, SRC3 and P300 genes. Furthermore, the HDACi and HMTi effects on mRNA and protein expression of BRCA1, SRC3 and P300 genes were checked. In addition, statistical analyses were used. RESULTS: In the MCF7 luminal subtype with positive ER, H3k4ac was significantly increased on BRCA1 with SAHA. On the contrary, in the MDA-MB 231 breast cancer cell line, representative of mesenchymal-like subtype with negative estrogen receptor, HDACis had no effect. Also, DZNEP decreased significantly H3K27me3 on BRCA1 in MDA-MB 231. Besides, on SRC3, a significant increase for H3K4ac was obtained in MCF7 treated with SAHA. And DZNEP had no effect in MCF7. Also, in MDA-MB 231 treated with DZNEP, H3K27me3 significantly decreased on SRC3 while H3K4ac was significantly increased in MDA-MB-231 treated with SAHA or NaBu for P300. CONCLUSION: Luminal and mesenchymal-like breast cancer subtype cell lines seemed to act differently to HDACis (SAHA and NaBu) or HMTi (DZNEP) treatments.

A bivalent role of TIP60 histone acetyl transferase in human cancer.

Acetylation is a major modification that is required for gene regulation, genome maintenance and metabolism. A dysfunctional acetylation plays an important role in several diseases, including cancer. A group of enzymes-lysine acetyltransferases are responsible for this modification and act in regulation of transcription as cofactors and by acetylation of histones and other proteins. Tip60, a member of MYST family, is expressed ubiquitously and is the acetyltransferase catalytic subunit of human NuA4 complex. This HAT has a well-characterized involvement in many processes, such as cellular signaling, DNA damage repair, transcriptional and cellular cycle. Aberrant lysine acetyltransferase functions promote or suppress tumorigenesis in different cancers such as colon, breast and prostate tumors. Therefore, Tip60 might be a potential and important therapeutic target in the cancer treatment; new histone acetyl transferase inhibitors were identified and are more selective inhibitors of Tip60.

The Role of Soy Phytoestrogens on Genetic and Epigenetic Mechanisms of Prostate Cancer.

Soy phytoestrogens are dietary components with considerable effects on reducing the incidence of prostate cancer. Epidemiological studies demonstrated that occurrence of prostate cancer is relatively low in Asia and Southern Europe, a status associated with consuming of soy isoflavones, such as genistein, daidzein, and glycitein. Soy phytoestrogens exert their activity on molecular mechanisms, including cell-cycle control, induction of apoptosis, inhibition of angiogenesis, and metastasis. In addition, they have antioxidant activity and show regulatory effect on the expression of genes involved in DNA damage and repair. Furthermore, the epigenetic regulation of gene expression can be modified by soy phytoestrogens. They show regulatory effects on gene activity by altering DNA methylation and/or histone modification patterns. In this chapter, we discuss the role of soy phytoestrogens on the genetic and epigenetic mechanisms of prostate cancer. We attempt to provide further insight in order to understand the underlying mechanisms of protective effects of soy phytoestrogens in preventing prostate cancer.

Genome-wide DNA methylation modified by soy phytoestrogens: role for epigenetic therapeutics in prostate cancer?

In prostate cancer, DNA methylation is significantly associated with tumor initiation, progression, and metastasis. Previous studies have suggested that soy phytoestrogens might regulate DNA methylation at individual candidate gene loci and that they play a crucial role as potential therapeutic agents for prostate cancer. The purpose of our study was to examine the modulation effects of phytoestrogens on a genome-wide scale in regards to DNA methylation in prostate cancer. Prostate cancer cell lines DU-145 and LNCaP were treated with 40 µM of genistein and 110 µM of daidzein. DNMT inhibitor 5-azacytidine (2 µM) and the methylating agent budesonide (2 µM) were used to compare their demethylation/methylation effects with phytoestrogens. The regulatory effects of phytoestrogens on DNA methylation were analyzed by using a methyl-DNA immunoprecipitation method coupled with Human DNA Methylation Microarrays (MeDIP-chip). We observed that the methylation profiles of 58 genes were altered by genistein and daidzein treatments in DU-145 and LNCaP prostate cancer cells. In addition, the methylation frequencies of the MAD1L1, TRAF7, KDM4B, and hTERT genes were remarkably modified by genistein treatment. Our results suggest that the modulation effects of phytoestrogens on DNA methylation essentially lead to inhibition of cell growth and induction of apoptosis. Genome-wide methylation profiling reported here suggests that epigenetic regulation mechanisms and, by extension, epigenetics-driven novel therapeutic candidates warrant further consideration in future "omics" studies of prostate cancer.

Prostate cancer: The main risk and protective factors-Epigenetic modifications.

With 13 million new cases worldwide every year, prostate cancer is as a very real public health concern. Prostate cancer is common in over-50s men and the sixth-leading cause of cancer-related death in men worldwide. Like all cancers, prostate cancer is multifactorial - there are non-modifiable risk factors like heredity, ethnicity and geographic location, but also modifiable risk factors such as diet. Diet-cancer linkages have risen to prominence in the last few years, with accruing epidemiological data pointing to between-population incidence differentials in numerous cancers. Indeed, there are correlations between fat-rich diet and risk of hormone-dependent cancers like prostate cancer and breast cancer. Diet is a risk factor for prostate cancer, but certain micronutrients in specific diets are considered protective factors against prostate cancer. Examples include tomato lycopene, green tea epigallocatechin gallate, and soy phytoestrogens. These micronutrients are thought to exert cancer-protective effects via anti-oxidant pathways and inhibition of cell proliferation. Here, we focus in on the effects of phytoestrogens, and chiefly genistein and daidzein, which are the best-researched to date. Soy phytoestrogens are nonsteroid molecules whose structural similarity lends them the ability to mimic the effects of 17ß-estradiol. On top of anti-oxidant effects, there is evidence that soy phytoestrogens can modulate the epigenetic modifications found in prostate cancer. We also studied the impact of phytoestrogens on epigenetic modifications in prostate cancer, with special focus on DNA methylation, miRNA-mediated regulation and histone modifications.

The association between histone 3 lysine 27 trimethylation (H3K27me3) and prostate cancer: relationship with clinicopathological parameters.

BACKGROUND: It is well established that genetic and epigenetic alterations are common events in prostate cancer, which may lead to aberrant expression of critical genes. The importance of epigenetic mechanisms in prostate cancer carcinogenesis is increasingly evident. In this study, the focus will be on histone modifications and the primary objectives are to map H3K27me3 marks and quantify RAR beta 2, ER alpha, SRC3, RGMA, PGR, and EZH2 gene expressions in prostate cancer tissues compared to normal tissues. In addition, a data analysis was made in connection with the clinicopathological parameters. METHODS: 71 normal specimens and 66 cancer prostate tissues were randomly selected in order to assess the proportion of the repressive H3K27me3 mark and gene expression. H3K27me3 level was evaluated by ChIP-qPCR and mRNA expression using RT-qPCR between prostate cancer and normal tissues. Subsequently, western-blotting was performed for protein detection. The analysis of variance (ANOVA) was performed, and Tukey's test was used to correct for multiple comparisons (p-value threshold of 0.05). The principal component analysis (PCA) and discriminant factorial analysis (DFA) were used to explore the association between H3K27me3 level and clinicopathological parameters. RESULTS: The study demonstrated that H3K27me3 level was significantly enriched at the RAR beta 2, ER alpha, PGR, and RGMA promoter regions in prostate cancer tissues compared to normal tissues. After stratification by clinicopathological parameters, the H3K27me3 level was positively correlated with Gleason score, PSA levels and clinical stages for RAR beta 2, ER alpha, PGR, and RGMA. High H3K27me3 mark was significantly associated with decreased RAR beta 2, ER alpha, PGR and RGMA gene expressions in prostate cancer sample compared to the normal one. Moreover, the results showed that mRNA level of EZH2, AR and SRC3 are upregulated in prostate cancer compared to normal prostate tissues and this correlates positively with Gleason score, PSA levels and clinical stages. Obviously, these observations were confirmed by protein level using western-blot. CONCLUSIONS: This data clearly demonstrated that H3K27me3 level correlated with aggressive tumor features. Also this study revealed that reverse correlation of RAR beta 2, ER alpha, PGR, and RGMA expressions with EZH2, SRC3, and AR expressions in prostate cancer tissues suggests that these genes are the target of EZH2. Therefore, all therapeutic strategies leading to histone demethylation with epigenetic drugs such as histone methyltransferase inhibitor may be relevant treatments against prostate cancer.

Epigenetic mechanisms of breast cancer: an update of the current knowledge.

Epigenetic alterations are heritable changes in gene expression that occur without causing any change in DNA sequence. They are important key factors for cancer development and prognosis. Breast cancer is induced by the accumulation of altered gene regulation. Besides genetic mutations, epigenetics mechanisms have an important role in breast cancer tumorigenesis. Investigations related with aberrant epigenetic regulations in breast cancer focus on initiating molecular mechanisms in cancer development, identification of new biomarkers to predict breast cancer aggressiveness and the potential of epigenetic therapy. In this review, we will summarize the recent knowledge about the role of epigenetic alterations related with DNA methylation and histone modification in breast cancer. In addition, altered regulation of breast cancer specific genes and the potential of epigenetic therapy will be discussed according to epigenetic mechanisms.

Epigenetic modifications in prostate cancer.

Prostate cancer is the most common cancer in men and the second leading cause of cancer deaths in men in France. Apart from the genetic alterations in prostate cancer, epigenetics modifications are involved in the development and progression of this disease. Epigenetic events are the main cause in gene regulation and the three most epigenetic mechanisms studied include DNA methylation, histone modifications and microRNA expression. In this review, we summarized epigenetic mechanisms in prostate cancer. Epigenetic drugs that inhibit DNA methylation, histone methylation and histone acetylation might be able to reactivate silenced gene expression in prostate cancer. However, further understanding of interactions of these enzymes and their effects on transcription regulation in prostate cancer is needed and has become a priority in biomedical research. In this study, we summed up epigenetic changes with emphasis on pharmacologic epigenetic target agents.

Comparative effects of soy phytoestrogens and 17ß-estradiol on DNA methylation of a panel of 24 genes in prostate cancer cell lines.

Major phytoestrogens genistein and daidzein have been reported to have the ability to reverse DNA methylation in cancer cell lines. The mechanism by which genistein and daidzein have an inhibiting action on DNA methylation is not well understood. The aim of this study was to investigate the effects of soy phytoestrogens and the natural estrogen 17ß-estradiol (E2) to determine whether one of the estrogen receptors is mobilized for the action of these compounds on DNA methylation. We also made a comparative study with a DNA methylation inhibitor (5-azacytidine) and a DNA methylation activator (budesonide). Three prostate cell lines, PC-3, DU-145, and LNCaP, were treated with 40 µM genistein, 110 µM daidzein, 2 µM budesonide, 2 µM 5-azacytidine, and 10 µM E2. In these 3 human prostate cancer cell lines, we performed methylation quantification using methyl-profiler-DNA-methylation analysis. Soy phytoestrogens and E2 induced a demethylation of all the promoter regions studied except for those that were unmethylated in control cells. Our results showed that E2 induces, like soy phytoestrogen, a decrease in DNA methylation in prostate cancer cell lines. This action may be mediated through ERß.

Gene panel model predictive of outcome in patients with prostate cancer.

In men at high risk for prostate cancer, established clinical and pathological parameters provide only limited prognostic information. Here we analyzed a French cohort of 103 prostate cancer patients and developed a gene panel model predictive of outcome in this group of patients. The model comprised of a 15-gene TaqMan Low-Density Array (TLDA) card, with gene expressions compared to a standardized reference. The RQ value for each gene was calculated, and a scoring system was developed. Summing all the binary scores (0 or 1) corresponding to the 15 genes, a global score is obtained between 0 and 15. This global score can be compared to Gleason score (0 to 10) by recalculating it into a 0-10 scaled score. A scaled score ≥2 suggested that the patient is suffering from a prostate cancer, and a scaled score ≥7 flagged aggressive cancer. Statistical analyses demonstrated a strongly significant linear correlation (p=3.50E-08) between scaled score and Gleason score for this prostate cancer cohort (N=103). These results support the capacity of this designed 15 target gene TLDA card approach to predict outcome in prostate cancer, opening up a new avenue for personalized medicine through future independent replication and applications for rapid identification of aggressive prostate cancer phenotypes for early intervention.