RNA demethylase ALKBH5 prevents pancreatic cancer progression by posttranscriptional activation of PER1 in an m6A-YTHDF2-dependent manner.

BACKGROUND: N6-methyladenosine (m6A) is the most abundant reversible methylation modification of eukaryotic mRNA, and it plays vital roles in tumourigenesis. This study aimed to explore the role of the m6A demethylase ALKBH5 in pancreatic cancer (PC). METHODS: The expression of ALKBH5 and its clinicopathological impact were evaluated in PC cohorts. The effects of ALKBH5 on the biological characteristics of PC cells were investigated on the basis of gain-of-function and loss-of-function analyses. Subcutaneous and orthotopic models further uncovered the role of ALKBH5 in tumour growth. mRNA and m6A sequencing and assays of m6A methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR) were performed to identify the targeted effect of ALKBH5 on PER1. P53-binding sites in the ALKBH5 promoter were investigated by ChIP and luciferase assays to reveal the interplay between ALKBH5 and PER1-activated ATM-CHK2-P53/CDC25C signalling. RESULTS: ALKBH5 loss characterized the occurrence and poor clinicopathological manifestations in patients with PC. Overexpression of ALKBH5 reduced tumoural proliferative, migrative, invasive activities in vitro and ameliorated tumour growth in vivo, whereas ALKBH5 knockdown facilitated PC progression. Mechanistically, ALKBH5 posttranscriptionally activated PER1 by m6A demethylation in an m6A-YTHDF2-dependent manner. PER1 upregulation led to the reactivation of ATM-CHK2-P53/CDC25C signalling, which inhibited cell growth. P53-induced activation of ALKBH5 transcription acted as a feedback loop regulating the m6A modifications in PC. CONCLUSION: ALKBH5 serves as a PC suppressor by regulating the posttranscriptional activation of PER1 through m6A abolishment, which may highlight a demethylation-based approach for PC diagnosis and therapy.

The role of iron and ferroptosis in the pathogenesis of acute pancreatitis.

Acute pancreatitis (AP) is an inflammatory disease of the pancreas. Iron is an essential element for life and is involved in many metabolic processes. Ferroptosis is a type of regulated cell death that is triggered by iron and oxidative stress. A well-established mouse AP model was adopted to study the role of iron and ferroptosis in the pathogenesis of pancreatitis. Mice were injected with cerulein to induce AP, and pancreatic tissue samples were analyzed to determine the pathology, cell death, iron deposition, expression of iron transporters, and lipid peroxidation. The role of iron was studied by giving mice extra iron or iron chelator. In vitro studies with acinar cells with ferroptosis activator and inhibitor were also performed to assess the inflammatory response. Iron was found accumulated in the pancreatic tissue of mice who suffered cerulein-induced pancreatitis. Cell death and lipid peroxidation increased in these tissues and could be further modulated by iron dextran or iron chelator. Mice given Hemin through gavage had reduced levels of GSH in pancreatic tissue and increased inflammatory response. Studies with acinar cells showed increased levels of lipid peroxidation and ferroptosis-specific mitochondrial damage when treated with ferroptosis inducer and inflammatory cytokines.

Prediction of Tumor Prognosis of Pancreatic Neuroendocrine Tumors Using Image, Surgical and Pathologic Findings.

OBJECTIVES: To evaluate the magnetic resonance imaging (MRI) and computed tomography (CT) findings along with other surgical and pathologic features as prognosis predictors in pancreatic neuroendocrine tumors (PNETs). METHODS: In this study, we retrospectively analyzed a clinical data pool of patients with pathologically confirmed PNETs. CT and MRI findings were evaluated as potential prediction parameters of tumor-nodes-metastases (TNM) stage and grade, using Fisher's exact test. Univariate and multivariate logistic regression models were used to estimate the risk factors associated with tumor recurrence after surgery. The Kaplan-Meier method and Cox proportional hazards model were used for recurrence-free survival analysis. RESULTS: The predictors of higher TNM stages were tumor diameter, tumor boundary, distant metastases, and lymphadenopathy on CT scan. From MRI images, tumor diameter, T2-weighted image, tumor enhancement, and pancreatic duct dilatation showed statistically significant differences among TNM stages. Univariate analysis showed that American Joint Committee on Cancer (AJCC) TNM stage, World Health Organization (WHO) tumor grade, sex, smoking, and drinking were associated with tumor recurrence and disease-free survival (DFS); while tumor and metastasis also affected DFS. Multivariate survival analysis confirmed that AJCC TNM was an independent predictor after adjusting other covariates. Peripancreatic invasion and lymph node metastases as well as blurred boundary detected by CT or MRI may be independent risk factors for TNM stage and clinical outcome of PNETs. CONCLUSION: TNM stage is a valuable predictor of prognosis in PNETs. Information from CT and MRI imaging can be used to determine the TNM stage, and to estimate the tumor prognosis, guide the follow-up, and avoid ineffective treatments.

Improving trainee colonoscopy performance by investigating the withdrawal time from individual colonic segments: a single-center observational study.

BACKGROUND: The colonoscopy withdrawal time (WT) and adenoma detection rate (ADR) are widely used quality indicators for colonoscopy. However, no study has investigated the appropriate colonoscopy WTs of individual colonic segments that will allow trainees to achieve a higher ADR. Thus, we analyzed for the first time the relationship between colonoscopy WT and the ADR/polyp detection rate (PDR) in the proximal, left-sided and entire colon among trainees. METHODS: This retrospective study involved 611 consecutive patients who underwent colonoscopy from March 2018 to March 2019 performed by 6 trainees in the Endoscopy Center of Shanghai General Hospital. The WTs for the individual colonic segments and any significant findings of colonoscopies were retrospectively retrieved from the trainees' records and verified in the endoscopy center database. ADR/PDR was defined as the number of colonoscopies detecting at least 1 polyp/adenoma divided by the total number of colonoscopies. Comparisons of PDR and ADR between the 2 groups were conducted using chi-square test. Multilevel analysis was performed to consider individual differences among the 6 trainees. Multilevel binary logistic regression analysis was performed to analyze the factors that influenced the PDR, ADR and advanced adenoma detection rate (AADR) for the entire colon, and trainee status was included as a random effect. RESULTS: The mean WTs were 4.20±1.09, 4.27±1.12, and 8.48±1.87 minutes for the proximal, left-sided, and entire colon, respectively. A longer WT [odds ratio (OR) 1.499, 95% confidence interval (CI): 1.381-1.628, P<0.001; OR 1.409, 95% CI: 1.265-1.569, P<0.001, respectively] was significantly associated with a higher PDR and ADR. The PDR (P<0.001) and ADR (P<0.001) were significantly higher when the WT was >4 minutes than when the WT was ≤4 minutes in both the proximal and left-sided colon, while the PDR (P<0.001) and ADR (P<0.001) were significantly higher when the WT was >8 minutes in the entire colon. CONCLUSIONS: In order to improve trainee colonoscopy performance, trainees were recommended to have WTs of at least 4 minutes in the proximal colon, 4 minutes in the left-sided colon and 8 minutes in the entire colon during negative screening colonoscopies.

Class A1 scavenger receptors mediated macrophages in impaired intestinal barrier of inflammatory bowel disease.

BACKGROUND: This study was to investigate the cytokines and phenotype of macrophages pre-treated with class A1 scavenger receptor (SR-A1) antibody in vitro and the influence on apoptotic pathway of colonic epithelial cells, and to explore the role of SR-A1 mediated macrophages in impaired intestinal barrier of inflammatory bowel diseases (IBDs). METHODS: Mouse macrophage RAW264.7 was pre-treated with SR-A1 antibody in the presence of lipopolysaccharide (LPS). Transwell system was employed for co-culture of RAW264.7 and Caco-2 in the presence of LPS and IFN-γ, with or without SR-A1 antibody pre-treatment. The percentage of F4/80+CD11c+ macrophages, apoptosis rate of Caco-2 cells, and expression of apoptosis and tight junction proteins in Caco-2 cells was determined. RESULTS: Pre-treatment with SR-A1 antibody up-regulated IL-10 expression in RAW264.7, whereas down-regulated the expression of TNF and iNOS. Immunofluorescence staining indicated the upregulation of NF-κB p-p56 after LPS stimulation was significantly inhibited in the presence of SR-A1 antibody. The increase in p-JNK expression was inhibited by SR-A1 antibody. Transwell assay showed the percentage of F4/80+CD11c+ macrophages and apoptotic Caco-2 cells increased after treatment with LPS and IFN-γ, which could be reversed in the presence of SR-A1 antibody. The induction of cleaved caspase-3 and claudin-1 in Caco-2 cells was also suppressed when SR-A1 antibody pre-treatment. CONCLUSIONS: Pre-treatment with SR-A1 antibody can inhibit inflammatory response in LPS-induced macrophages in a NF-κB dependent manner. Pre-treatment with SR-A1 antibody also inhibits M1 phenotype expression of macrophages, and attenuates the pro-apoptotic effect on colonic epithelial cells and disruption of intestinal barrier integrity induced by macrophages.

Selective and reversible suppression of intestinal stem cell differentiation by pharmacological inhibition of BET bromodomains.

Absorptive and secretory cells of the small intestine are derived from a single population of Lgr5-expressing stem cells. While key genetic pathways required for differentiation into specific lineages have been defined, epigenetic programs contributing to this process remain poorly characterized. Members of the BET family of chromatin adaptors contain tandem bromodomains that mediate binding to acetylated lysines on target proteins to regulate gene expression. In this study, we demonstrate that mice treated with a small molecule inhibitor of BET bromodomains, CPI203, exhibit greater than 90% decrease in tuft and enteroendocrine cells in both crypts and villi of the small intestine, with no changes observed in goblet or Paneth cells. BET bromodomain inhibition did not alter the abundance of Lgr5-expressing stem cells in crypts, but rather exerted its effects on intermediate progenitors, in part through regulation of Ngn3 expression. When BET bromodomain inhibition was combined with the chemotherapeutic gemcitabine, pervasive apoptosis was observed in intestinal crypts, revealing an important role for BET bromodomain activity in intestinal homeostasis. Pharmacological targeting of BET bromodomains defines a novel pathway required for tuft and enteroendocrine differentiation and provides an important tool to further dissect the progression from stem cell to terminally differentiated secretory cell.

Regulation of GLI Underlies a Role for BET Bromodomains in Pancreatic Cancer Growth and the Tumor Microenvironment.

PURPOSE: The initiation, progression, and maintenance of pancreatic ductal adenocarcinoma (PDAC) results from the interplay of genetic and epigenetic events. While the genetic alterations of PDAC have been well characterized, epigenetic pathways regulating PDAC remain, for the most part, elusive. The goal of this study was to identify novel epigenetic regulators contributing to the biology of PDAC. EXPERIMENTAL DESIGN: In vivo pooled shRNA screens targeting 118 epigenetic proteins were performed in two orthotopic PDAC xenograft models. Candidate genes were characterized in 19 human PDAC cell lines, heterotopic xenograft tumor models, and a genetically engineered mouse (GEM) model of PDAC. Gene expression, IHC, and immunoprecipitation experiments were performed to analyze the pathways by which candidate genes contribute to PDAC. RESULTS: In vivo shRNA screens identified BRD2 and BRD3, members of the BET family of chromatin adaptors, as key regulators of PDAC tumor growth. Pharmacologic inhibition of BET bromodomains enhanced survival in a PDAC GEM model and inhibited growth of human-derived xenograft tumors. BET proteins contribute to PDAC cell growth through direct interaction with members of the GLI family of transcription factors and modulating their activity. Within cancer cells, BET bromodomain inhibition results in downregulation of SHH, a key mediator of the tumor microenvironment and canonical activator of GLI. Consistent with this, inhibition of BET bromodomains decreases cancer-associated fibroblast content of tumors in both GEM and xenograft tumor models. CONCLUSIONS: Therapeutic inhibition of BET proteins offers a novel mechanism to target both the neoplastic and stromal components of PDAC. Clin Cancer Res; 22(16); 4259-70. ©2016 AACR.

Different Clinical Presentations of Hyperlipidemic Acute Pancreatitis: A Retrospective Study.

OBJECTIVES: The goal of this study was to summarize the clinical features of hyperlipidemic acute pancreatitis (HLAP), and help clinicians understand the characteristic presentations of HLAP. METHODS: From July 2009 to June 2013, 1073 cases of acute pancreatitis were retrospectively assessed. The clinical characteristics of HLAP and non-HLAP were statistically analyzed. RESULTS: The etiologic ratio of HLAP in acute pancreatitis rose from 13% in 2009 to 25.6% in 2013. Diabetes mellitus, fatty liver, and acute pancreatitis recurrence were positively correlated with HLAP, and female sex, age (>60 years), and alkaline phosphatase level were negatively correlated with HLAP. The diagnostic accuracy of amylase in HLAP was only 40.38%, compared with lipase (91.83%). Different cutoff points of serum triglyceride on day 1 (5.33 mmol/L), day 2 (2.77 mmol/L), and day 3 (2.18 mmol/L) could be used to obtain an accurate diagnosis of HLAP. Higher incidences of acute peripancreatic fluid collection, renal failure, and severe acute pancreatitis were also observed in patients with HLAP. CONCLUSIONS: Different clinical presentations of HLAP should be applied to be distinguished from non-HLAP in the clinic.

C-reactive protein: rethinking its role in evaluating the severity of hyperlipidemic acute pancreatitis.

OBJECTIVES: The goal of this study is to evaluate the role of C-reactive protein (CRP) in predicting the severity of hyperlipidemic acute pancreatitis (HLAP) compared with non-HLAP (NHLAP). METHODS: A total of 1073 episodes of acute pancreatitis between July 2009 and June 2013 were retrospectively studied. The clinical characteristics and laboratory data of HLAP and NHLAP were statistically analyzed on days 1, 2, 3, 4, and 6, especially the CRP level. RESULTS: There was a significant difference in CRP levels between HLAP and NHLAP (P < 0.01) on days 1, 2, 3, 4, and 6. The cutoff value for CRP in HLAP should be greater than NHLAP to obtain an accurate prediction of severity. Higher serum CRP levels in HLAP cases were correlated with higher incidences of diabetes and fatty liver and lower incidences in women, elevated very-low-density lipoprotein levels, and lower high-density lipoprotein levels. CONCLUSIONS: The significant difference in CRP cutoff values in predicting severity between patients with HLAP and NHLAP should be noted in the clinic.

Identification of RegIV as a novel GLI1 target gene in human pancreatic cancer.

BACKGROUND AND AIMS: GLI1 is the key transcriptional factor in the Hedgehog signaling pathway in pancreatic cancer. RegIV is associated with regeneration, and cell growth, survival, adhesion and resistance to apoptosis. We aimed to study RegIV expression in pancreatic cancer and its relationship to GLI1. METHODS: GLI1 and RegIV expression were evaluated in tumor tissue and adjacent normal tissues of pancreatic cancer patients and 5 pancreatic cancer cell lines by qRT-PCR, Western blot, and immunohistochemistry (IHC), and the correlation between them. The GLI1-shRNA lentiviral vector was constructed and transfected into PANC-1, and lentiviral vector containing the GLI1 expression sequence was constructed and transfected into BxPC-3. GLI1 and RegIV expression were evaluated by qRT-PCR and Western blot. Finally we demonstrated RegIV to be the target of GLI1 by chromatin immunoprecipitation (CHIP) and electrophoretic mobility shift assays (EMSA). RESULTS: The results of IHC and qRT-PCR showed that RegIV and GLI1 expression was higher in pancreatic cancer tissues versus adjacent normal tissues (p<0.001). RegIV expression correlated with GLI1 expression in these tissues (R = 0.795, p<0.0001). These results were verified for protein (R = 0.939, p = 0.018) and mRNA expression (R = 0.959, p = 0.011) in 5 pancreatic cancer cell lines. RegIV mRNA and protein expression was decreased (94.7±0.3%, 84.1±0.5%; respectively) when GLI1 was knocked down (82.1±3.2%, 76.7±2.2%; respectively) by the RNAi technique. GLI1 overexpression in mRNA and protein level (924.5±5.3%, 362.1±3.5%; respectively) induced RegIV overexpression (729.1±4.3%, 339.0±3.7%; respectively). Moreover, CHIP and EMSA assays showed GLI1 protein bound to RegIV promotor regions (GATCATCCA) in pancreatic cancer cells. CONCLUSION: GLI1 promotes RegIV transcription by binding to the RegIV gene promoter in pancreatic cancer.