Swapna Mahurkar-Joshi

A long noncoding RNA signature for ulcerative colitis identifies IFNG-AS1 as an enhancer of inflammation

High-throughput technologies revealed new categories of genes, including the long noncoding RNAs (lncRNAs), involved in the pathogenesis of human disease; however, the role of lncRNAs in the ulcerative colitis (UC) has not been evaluated. Gene expression profiling was used to develop lncRNA signatures in UC samples. Jurkat T cells were activated by PMA/ionomycin subsequently interferon-γ (IFNG) and tumor necrosis factor (TNF)-α protein levels were assessed by ELISA. Anti-sense molecules were designed to block IFNG-AS1 expression. A unique set of lncRNAs was differentially expressed between UC and control samples. Of these, IFNG-AS1 was among the highest statistically significant lncRNAs (fold change: 5.27, P value: 7.07E-06). Bioinformatic analysis showed that IFNG-AS1 was associated with the IBD susceptibility loci SNP rs7134599 and its genomic location is adjacent to the inflammatory cytokine IFNG. In mouse models of colitis, active colitis samples had increased colonic expression of this lncRNA. Utilizing the Jurkat T cell model, we found IFNG-AS1 to positively regulate IFNG expression. Novel lncRNA signatures differentiate UC patients with active disease, patients in remission, and control subjects. A subset of these lncRNAs was found to be associated with the clinically validated IBD susceptibility loci. IFNG-AS1 was one of these differentially expressed lncRNAs in UC patients and found to regulate the key inflammatory cytokine, IFNG, in CD4 T cells. Taking these findings together, our study revealed novel lncRNA signatures deregulated in UC and identified IFNG-AS1 as a novel regulator of IFNG inflammatory responses, suggesting the potential importance of noncoding RNA mechanisms on regulation of inflammatory bowel disease-related inflammatory responses.

The effect of sex and irritable bowel syndrome on HPA axis response and peripheral glucocorticoid receptor expression

BACKGROUND AND AIMS Dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis has been reported in irritable bowel syndrome (IBS). Enhanced HPA axis response has been associated with reduced glucocorticoid receptor (GR) mediated negative feedback inhibition. We aimed to study the effects of IBS status, sex, or presence of early adverse life events (EAL) on the cortisol response to corticotropin-releasing factor (CRF) and adrenocorticotropic hormone (ACTH), and on GR mRNA expression in peripheral blood mononuclear cells (PBMCs). METHODS Rome III+ IBS patients and healthy controls underwent CRF (1μg/kg ovine) and ACTH (250μg) stimulation tests with serial plasma ACTH and cortisol levels measured (n=116). GR mRNA levels were measured using quantitative PCR (n=143). Area under the curve (AUC) and linear mixed effects models were used to compare ACTH and cortisol response measured across time between groups. RESULTS There were divergent effects of IBS on the cortisol response to ACTH by sex. In men, IBS was associated with an increased AUC (p=0.009), but in women AUC was blunted in IBS (p=0.006). Men also had reduced GR mRNA expression (p=0.007). Cumulative exposure to EALs was associated with an increased HPA response. Lower GR mRNA was associated with increased pituitary HPA response and increased severity of overall symptoms and abdominal pain in IBS. CONCLUSION This study highlights the importance of considering sex in studies of IBS and the stress response in general. Our findings also provide support for PBMC GR mRNA expression as a peripheral marker of central HPA response.

Sigmoid colon mucosal gene expression supports alterations of neuronal signaling in irritable bowel syndrome with constipation

Peripheral factors likely play a role in at least a subset of irritable bowel syndrome (IBS) patients. Few studies have investigated mucosal gene expression using an unbiased approach. Here, we performed mucosal gene profiling in a sex-balanced sample to identify relevant signaling pathways and gene networks and compare with publicly available profiling data from additional cohorts. Twenty Rome III+ IBS patients [10 IBS with constipation (IBS-C), 10 IBS with diarrhea (IBS-D), 5 men/women each), and 10 age-/sex-matched healthy controls (HCs)] underwent sigmoidoscopy with biopsy for gene microarray analysis, including differential expression, weighted gene coexpression network analysis (WGCNA), gene set enrichment analysis, and comparison with publicly available data. Expression levels of 67 genes were validated in an expanded cohort, including the above samples and 18 additional participants (6 each of IBS-C, IBS-D, HCs) using NanoString nCounter technology. There were 1,270 differentially expressed genes (FDR < 0.05) in IBS-C vs. HCs but none in IBS or IBS-D vs. HCs. WGNCA analysis identified activation of the cAMP/protein kinase A signaling pathway. Nine of 67 genes were validated by the NanoString nCounter technology (FDR < 0.05) in the expanded sample. Comparison with publicly available microarray data from the Mayo Clinic and University of Nottingham supports the reproducibility of 17 genes from the microarray analysis and three of nine genes validated by nCounter in IBS-C vs. HCs. This study supports the involvement of peripheral mechanisms in IBS-C, particularly pathways mediating neuronal signaling. NEW & NOTEWORTHY Peripheral factors play a role in the pathophysiology of irritable bowel syndrome (IBS), which, to date, has been mostly evident in IBS with diarrhea. Here, we show that sigmoid colon mucosal gene expression profiles differentiate IBS with constipation from healthy controls. These profiling data and analysis of additional cohorts also support the concept that peripheral neuronal pathways contribute to IBS pathophysiology.

MKAD-21 suppresses the oncogenic activity of the miR-21/PPP2R2A/ERK molecular network in bladder cancer

Bladder cancer represents a disease associated with significant morbidity and mortality. MiR-21 has been found to have oncogenic activity in multiple cancers, including bladder cancer, whereas inhibition of its expression suppresses tumor growth. Here, we examine the molecular network regulated by miR-21 in bladder cancer and evaluate the effects of i.v. and i.p. administration of a novel miR-21 chemical inhibitor in vivo. LNA miR-21 reduced the oncogenic potential of bladder cancer cells, whereas the MKAD-21 chemically modified antisense oligo against miR-21 dose-dependently blocked xenograft growth. I.v. administration of LNA miR-21 was more effective in suppressing tumor growth than was i.p. administration. Integration of computational and transcriptomic analyses in a panel of 28 bladder cancer lines revealed a 15-gene signature that correlates with miR-21 levels. Protein Phosphatase 2 Regulatory Subunit Balpha (PPP2R2A) was one of these 15 genes and was experimentally validated as a novel miR-21 direct target gene. Gene network and molecular analyses showed that PPP2R2A is a potent negative regulator of the ERK pathway activation and bladder cancer cell proliferation. Importantly, we show that PPP2R2A acts as a mediator of miR-21–induced oncogenic effects in bladder cancer. Integrative analysis of human bladder cancer tumors and a large panel of human bladder cancer cell lines revealed a novel 15-gene signature that correlates with miR-21 levels. Importantly, we provide evidence that PPP2R2A represents a new miR-21 direct target and regulator of the ERK pathway and bladder cancer cell growth. Furthermore, i.v. administration of the MKAD-21 inhibitor effectively suppressed tumor growth through regulation of the PPP2R2A–ERK network in mice. Mol Cancer Ther; 17(7); 1430–40. ©2018 AACR.

Lysine methyltransferase 2D regulates pancreatic carcinogenesis through metabolic reprogramming

Objective Despite advances in the identification of epigenetic alterations in pancreatic cancer, their biological roles in the pathobiology of this dismal neoplasm remain elusive. Here, we aimed to characterise the functional significance of histone lysine methyltransferases (KMTs) and demethylases (KDMs) in pancreatic tumourigenesis. Design DNA methylation sequencing and gene expression microarrays were employed to investigate CpG methylation and expression patterns of KMTs and KDMs in pancreatic cancer tissues versus normal tissues. Gene expression was assessed in five cohorts of patients by reverse transcription quantitative-PCR. Molecular analysis and functional assays were conducted in genetically modified cell lines. Cellular metabolic rates were measured using an XF24-3 Analyzer, while quantitative evaluation of lipids was performed by liquid chromatography-mass spectrometry (LC-MS) analysis. Subcutaneous xenograft mouse models were used to evaluate pancreatic tumour growth in vivo. Results We define a new antitumorous function of the histone lysine (K)-specific methyltransferase 2D (KMT2D) in pancreatic cancer. KMT2D is transcriptionally repressed in human pancreatic tumours through DNA methylation. Clinically, lower levels of this methyltransferase associate with poor prognosis and significant weight alterations. RNAi-based genetic inactivation of KMT2D promotes tumour growth and results in loss of H3K4me3 mark. In addition, KMT2D inhibition increases aerobic glycolysis and alters the lipidomic profiles of pancreatic cancer cells. Further analysis of this phenomenon identified the glucose transporter SLC2A3 as a mediator of KMT2D-induced changes in cellular, metabolic and proliferative rates. Conclusion Together our findings define a new tumour suppressor function of KMT2D through the regulation of glucose/fatty acid metabolism in pancreatic cancer.

AODTH-010 A mirna-epigenetic network in pancreatic cancer

Introduction The dismal prognosis of pancreatic cancer due to the delayed diagnosis, rapid metastasis and resistance to current therapeutics, signifies the importance of identifying novel therapeutic approaches. Although the past two decades of research have focused on identifying genetic alterations, in the recent years it has become apparent that pancreatic cancer is as much a disease of DNA mutations as it is a disease of misregulated epigenetics. Method We analysed alterations at the epigenomic (genome-wide DNA methylation), transcriptomic (gene expression) and miRNomic (miRNA expression) level, in 20 pancreatic cancer and 14 normal tissues. We then performed data integration through the Starburst analysis and explored for overlapping changes in DNA methylation and gene expression. To validate our data, gene expression was further studied through real time RT-PCR and immunohistochemistry. To elucidate the clinical significance of our findings, we conducted immunohistochemistry in a tissue microarray containing 154 tissue specimens from pancreatic cancer and matched non-neoplastic tissue samples. To address the functional importance and characterise the pathways involved, we performed a series of cell-based pancreatic cancer assays. Results This analysis revealed hepatocyte nuclear factor 4A (HNF4A), a key transcription factor in the development and proper function of the pancreas, as a novel target for aberrant DNA methylation. Hypermethylation is identified in several loci of the HNF4A gene promoter and there is a positive correlation between HNF4A hypermethylation and down-regulation in pancreatic cancer tissues. Our preliminary data indicate the activity of an epigenetic feedback circuit that involves HNF4A, microRNAs and DNA methyltransferases (DNMTs). Clinicopathological analysis revealed that low HNF4A expression correlates with poor survival. Our functional studies indicate that the HNF4A-circuit acts as a tumour suppressor, regulating pancreatic cancer growth, invasiveness and chemoresistance. Conclusion Our data suggest that the HNF4A-miRNA-DNMTs molecular network plays a central role in pancreatic cancer progression and its perturbation holds promise as a novel therapeutic approach. Disclosure of Interest None Declared

Abstract 4528: Chromatin regulation by ING3 leads to tumor suppressive effects in pancreatic cancer through distinct signaling pathways

Background: Despite our increased knowledge of the molecular events underlying the multi-step development of pancreatic cancer (PanCa), clinically meaningful improvement in survival rates has not yet been achieved. A growing body of evidence supports that pancreatic tumorigenesis is not only led by genetic alterations but also aberrant epigenetic regulation. Identification of epigenetic drivers of the clinical disease is of major importance, as it could potentially offer a means to interfere with novel targets in PanCa therapy. Methods: Gene expression microarrays were employed in PanCa tissues and adjacent uninvolved tissues. mRNA and protein levels were assessed by qRT-PCR and immunohistochemical analysis of tissue microarrays in extended cohorts of patients. RNAi interference assays or lentiviral expression vector systems were applied for knockdown/overexpression experiments, as evidenced by qRT-PCR and Western Blot Analysis. Molecular analysis, cell proliferation, invasion and colony formation assays were conducted in genetically modified cells. Subcutaneous xenograft mouse models were used to evaluate tumor growth in vivo. Immunoprecipitation followed by mass-spectroscopy analysis was used to evaluate protein-protein interactions. Histone H4 at lysine 16 acetylation (H4K16ac) levels were assessed by Western Blot analysis, while chromatin immunoprecipitation followed by DNA sequencing (ChIP-Seq) was performed for mapping protein-DNA interactions. Results: Differential expression analysis of chromatin regulators in PanCa versus normal tissues shows that 27 epigenetic molecules are significantly deregulated (>1.5 fold, P Conclusions: Conclusively, we provide evidence that PanCa is characterized by loss of the chromatin regulator ING3 and we elucidated the tumor suppressive role of the latter in PanCa cell growth and invasion in vitro and in vivo. Citation Format: Marina Koutsioumpa, Maria Hatziapostolou, Christos Polytarchou, Angelos Oikonomopoulos, Swapna Mahurkar-Joshi, Sara Huerta-Yepez, Belen Tirado-Rodriguez, Dimitrios Karavias, Helen Kourea, George A. Poultsides, David W. Dawson, Timothy R. Donahue, Dimitrios Iliopoulos. Chromatin regulation by ING3 leads to tumor suppressive effects in pancreatic cancer through distinct signaling pathways. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4528.