CircRNF13 Promotes the Malignant Progression of Pancreatic Cancer through Targeting miR-139-5p/IGF1R Axis

Background. Mounting evidence has shown circular RNAs (circRNAs) play an important role in the initiation and progression of pancreatic cancer (PC). Meanwhile, circRNAs may serve as the biomarkers for the diagnosis, treatment, and prognosis of PC. Therefore, it is urgent to elucidate the function and underlying mechanism of circRNAs in the development of PC. Methods. The Cancer-Specific CircRNA Database (CSCD), Circular RNA Interactome database (circinteractome database), and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to verify the expression level of circRNF13 in PC cell lines. Fluorescence in situ hybridization (FISH) and RNase protection assay were used to detect the localization and structure of circRNF13. Then, cell functional experiments were employed to estimate the proliferated, migrated, and invasive abilities in PC. Furthermore, bioinformatic tools, luciferase dual reporter assay, and RT-qPCR were used to investigate the interaction among circRNF13, miR-139-5p, and IGF1R. Eventually, the rescue functional experiments were employed to confirm that circRNF13 targeted the miR-139-5p/IGF1R axis to participate in the development of PC. Results. CircRNF13 was overexpressed in PC cell lines compared with the normal pancreatic duct cell line. Additionally, inhibition of circRNF13 impaired the proliferation, migration, and invasion of PC cells. CircRNF13 could serve as the molecular sponge of miR-139-5p to inhibit its association with IGF1R that eventually accelerated the malignant progression of PC. Conclusion. CircRNF13 serves as a competitive endogenous RNA of IGF1R to inhibit the function of miR-139-5p that eventually reinforces the malignant phenotype of PC.

Establishment of Tumor Treating Fields Combined With Mild Hyperthermia as Novel Supporting Therapy for Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant tumor with poor prognosis and limited therapeutic options. Alternating electrical fields with low intensity called “Tumor Treating Fields” (TTFields) are a new, non-invasive approach with almost no side effects and phase 3 trials are ongoing in advanced PDAC. We evaluated TTFields in combination with mild hyperthermia. Three established human PDAC cell lines and an immortalized pancreatic duct cell line were treated with TTFields and hyperthermia at 38.5°C, followed by microscopy, assays for MTT, migration, colony and sphere formation, RT-qPCR, FACS, Western blot, microarray and bioinformatics, and in silico analysis using the online databases GSEA, KEGG, Cytoscape-String, and Kaplan-Meier Plotter. Whereas TTFields and hyperthermia alone had weak effects, their combination strongly inhibited the viability of malignant, but not those of nonmalignant cells. Progression features and the cell cycle were impaired, and autophagy was induced. The identified target genes were key players in autophagy, the cell cycle and DNA repair. The expression profiles of part of these target genes were significantly involved in the survival of PDAC patients. In conclusion, the combination of TTFields with mild hyperthermia results in greater efficacy without increased toxicity and could be easily clinically approved as supporting therapy.

Discovery and 3D imaging of a novel ΔNp63-expressing basal cell type in human pancreatic ducts with implications in disease

ObjectiveThe aggressive basal-like molecular subtype of pancreatic ductal adenocarcinoma (PDAC) harbours a ΔNp63 (p40) gene expression signature reminiscent of a basal cell type. Distinct from other epithelia with basal tumours, ΔNp63+ basal cells reportedly do not exist in the normal pancreas.DesignWe evaluated ΔNp63 expression in human pancreas, chronic pancreatitis (CP) and PDAC. We further studied in depth the non-cancerous tissue and developed a three-dimensional (3D) imaging protocol (FLIP-IT, Fluorescence Light sheet microscopic Imaging of Paraffin-embedded or Intact Tissue) to study formalin-fixed paraffin-embedded samples at single cell resolution. Pertinent mouse models and HPDE cells were analysed.ResultsIn normal human pancreas, rare ΔNp63+ cells exist in ducts while their prevalence increases in CP and in a subset of PDAC. In non-cancer tissue, ΔNp63+ cells are atypical KRT19+ duct cells that overall lack SOX9 expression while they do express canonical basal markers and pertain to a niche of cells expressing gastrointestinal stem cell markers. 3D views show that the basal cells anchor on the basal membrane of normal medium to large ducts while in CP they exist in multilayer dome-like structures. In mice, ΔNp63 is not found in adult pancreas nor in selected models of CP or PDAC, but it is induced in organoids from larger Sox9low ducts. In HPDE, ΔNp63 supports a basal cell phenotype at the expense of a classical duct cell differentiation programme.ConclusionIn larger human pancreatic ducts, basal cells exist. ΔNp63 suppresses duct cell identity. These cells may play an important role in pancreatic disease, including PDAC ontogeny, but are not present in mouse models.

Proteomic analysis identifies deregulated metabolic and oxidative-associated proteins in Italian intrahepatic cholangiocarcinoma patients

Background Cholangiocarcinoma (CCA) is an aggressive disease with poor prognosis. A molecular classification based on mutational, methylation and transcriptomic features could allow identifying tailored therapies to improve CCA patient outcome. Proteomic remains partially unexplored; here, we analyzed the proteomic profile of five intrahepatic cholangiocarcinoma (ICC) derived from Italian patients undergone surgery and one normal bile duct cell line. Methods Proteome profile was investigated by using 2D electrophoresis followed by Mass Spectrometry (MS). To validate proteomic data, the expression of four overexpressed proteins (CAT, SOD, PRDX6, DBI/ACBP) was evaluated by immunohistochemistry in an independent cohort of formalin fixed, paraffin-embedded (FFPE) ICC tissues. We also compared proteomic data with those obtained by transcriptomic profile evaluated by microarray analysis of the same tissues. Results We identified 19 differentially expressed protein spots, which were further characterized by MS; 13 of them were up- and 6 were down-regulated in ICC. These proteins are mainly involved in redox processes (CAT, SODM, PRDX2, PRDX6), in metabolism (ACBP, ACY1, UCRI, FTCD, HCMS2), and cell structure and organization (TUB2, ACTB). CAT is overexpressed in 86% of patients, PRDX6 in 73%, SODM in 100%, and DBI/ACBP in 81% compared to normal adjacent tissues. A concordance of 50% between proteomic and transcriptomic data was observed. Conclusions This study pointed out that the impairment of the metabolic and antioxidant systems, with a subsequent accumulation of free radicals, might be a key step in CCA development and progression.

Correlation of the expression of estrogen receptor, progesterone receptor and HER2/neu with the clinical features and the tumour histopathology in patients of breast carcinoma at mahavir cancer sansthan

Background: Breast carcinoma is the most common malignant tumor and the leading cause of cancer death in women, with more than 10,00,000 cases occurring worldwide annually. Its incidence varies from one geographical area to the other.Methods: Cases of all breast conservative surgeries and modified radical mastectomy specimen received for routine histopathological evaluation from the department of onco Surgery, Mahavir Cancer Sansthan, Patna, Bihar, form the source of data for the study.Results: Infiltrating duct cell carcinoma (NOS) type was the commonest type of carcinoma breast in our institute with significant group occurring in more than 45 years of age. ER and PR positive expression was seen in grade 2 tumors and negative expression was seen with tumor size more than 2cm, positive lymph nodes and higher stage of disease. HER2/neu negative expression was seen in the post-menopausal age group, tumor size more than 2 cm, positive lymph nodes and higher stage of disease indicating bad prognosis. HER2/neu expression was inversely related to ER and PR expression. HER2/neu expression was seen in 50% of medullary carcinoma which is rare. Triple negative cases were seen in 22.08% cases of infiltrating duct cell carcinoma indicating bad prognosis.Conclusions: A beforehand knowledge of these immunohistochemical markers can help oncotherapists to initiate a correct form of treatment and improve the survival rate in breast carcinoma patients.

Role of hepcidin in oxidative stress and cell death of cultured mouse renal collecting duct cells: protection against iron and sensitization to cadmium

The liver hormone hepcidin regulates systemic iron homeostasis. Hepcidin is also expressed by the kidney, but exclusively in distal nephron segments. Several studies suggest hepcidin protects against kidney damage involving Fe2+ overload. The nephrotoxic non-essential metal ion Cd2+ can displace Fe2+ from cellular biomolecules, causing oxidative stress and cell death. The role of hepcidin in Fe2+ and Cd2+ toxicity was assessed in mouse renal cortical [mCCD(cl.1)] and inner medullary [mIMCD3] collecting duct cell lines. Cells were exposed to equipotent Cd2+ (0.5–5 μmol/l) and/or Fe2+ (50–100 μmol/l) for 4–24 h. Hepcidin (Hamp1) was transiently silenced by RNAi or overexpressed by plasmid transfection. Hepcidin or catalase expression were evaluated by RT-PCR, qPCR, immunoblotting or immunofluorescence microscopy, and cell fate by MTT, apoptosis and necrosis assays. Reactive oxygen species (ROS) were detected using CellROX™ Green and catalase activity by fluorometry. Hepcidin upregulation protected against Fe2+-induced mIMCD3 cell death by increasing catalase activity and reducing ROS, but exacerbated Cd2+-induced catalase dysfunction, increasing ROS and cell death. Opposite effects were observed with Hamp1 siRNA. Similar to Hamp1 silencing, increased intracellular Fe2+ prevented Cd2+ damage, ROS formation and catalase disruption whereas chelation of intracellular Fe2+ with desferrioxamine augmented Cd2+ damage, corresponding to hepcidin upregulation. Comparable effects were observed in mCCD(cl.1) cells, indicating equivalent functions of renal hepcidin in different collecting duct segments. In conclusion, hepcidin likely binds Fe2+, but not Cd2+. Because Fe2+ and Cd2+ compete for functional binding sites in proteins, hepcidin affects their free metal ion pools and differentially impacts downstream processes and cell fate.

Hnf1b-CreER, a model used for fate mapping pancreatic lineages, achieves efficient Cre-mediated recombination in duct and islet δ cells

The Hnf1b-CreERT2 BAC transgenic (Tg(Hnf1b-cre/ERT2)1Jfer) has been used extensively to trace the progeny of pancreatic ducts in development, regeneration, or cancer. This model originally showed that duct-like plexus cells of the embryonic pancreas are bipotent duct-endocrine progenitors, whereas adult mouse duct cells are not a common source of δ cells in various regenerative settings. We have now examined Hnf1b-CreERT2 mice with a Rosa26-RFP reporter transgene. This showed inducible recombination of up to 96% adult duct cells, a much higher efficiency than the previously used β-galactosidase reporter. Despite this high duct-cell excision, recombination in α and β cells remained very low, similar to the previously used reporter transgene (Rosa26-βgalactosidase). However, nearly half of somatostatin-expressing δ cells showed reporter activation, which was due to Cre expression in δ cells rather than an indication of duct to δ cell conversions. The high recombination efficiency in duct cells indicates that the Hnf1b-CreERT2 model can be useful for both ductal fate mapping and genetic inactivation studies. The recombination in δ cells does not modify the interpretation of studies that failed to show duct conversions to other cell types, but needs to be considered in studies that use this model to modify the plasticity of pancreatic duct cells.

Single cell transcriptome analysis defines heterogeneity of the murine pancreatic ductal tree

To study disease development, an inventory of an organ's cell types and understanding of physiologic function is paramount. Here, we performed single-cell RNA sequencing to examine heterogeneity of murine pancreatic duct cells, pancreatobiliary cells, and intrapancreatic bile duct cells. We describe an epithelial-mesenchymal transitory axis in our three pancreatic duct subpopulations and identify osteopontin as a regulator of this fate decision as well as human duct cell dedifferentiation. Our results further identify functional heterogeneity within pancreatic duct subpopulations by elucidating a role for geminin in accumulation of DNA damage in the setting of chronic pancreatitis. Our findings implicate diverse functional roles for subpopulations of pancreatic duct cells in maintenance of duct cell identity and disease progression and establish a comprehensive road map of murine pancreatic duct cell, pancreatobiliary cell, and intrapancreatic bile duct cell homeostasis.

Accuracy of brush cytology in biliopancreatic strictures: a single-center cohort study

Objective False positive and negative results are associated with biliary tract cell brushing cytology during endoscopic retrograde cholangiopancreatography (ERCP). The causes are uncertain. The purpose of this study was to evaluate the accuracy of diagnoses made via cell brushing in our center, and to explore the factors influencing diagnosis. Methods The clinical data of patients who underwent cell brushing at our center from January 2016 to August 2019 were retrospectively analyzed. These included age, gender, stricture location, thickness of the bile duct wall in the narrow segment, maximum diameter of the biliary duct above the stricture, number of cell brush smears, carbohydrate antigen 19-9, and carcinoembryonic antigen. Positive brush cytology results were compared with results of surgical histology or tumor biopsy as well as with the patient’s clinical course. Results Of the 48 patients who underwent cell brushing cytology, 27 (56.3%) had positive results. The sensitivity and specificity of biliary duct cell brushing was 79.4%, and 85.7%, respectively. None of the above-mentioned factors were associated with positive cytology brushing results. Conclusions Cell brushing cytology remains a reliable method for diagnosis of pancreaticobiliary malignancies.