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Patients with metastatic pancreatic ductal adenocarcinoma survive longer if disease spreads to the lung but not the liver. Here we generated overlapping, multi-omic datasets to identify molecular and cellular features that distinguish patients whose disease develops liver metastasis (liver cohort) from those whose disease develops lung metastasis without liver metastases (lung cohort). Lung cohort patients survived longer than liver cohort patients, despite sharing the same tumor subtype. We developed a primary organotropism (pORG) gene set enriched in liver cohort versus lung cohort primary tumors. We identified ongoing replication stress response pathways in high pORG/liver cohort tumors, whereas low pORG/lung cohort tumors had greater densities of lymphocytes and shared T cell clonal responses. Our study demonstrates that liver-avid pancreatic ductal adenocarcinoma is associated with tolerance to ongoing replication stress, limited tumor immunity and less-favorable outcomes, whereas low replication stress, lung-avid/liver-averse tumors are associated with active tumor immunity that may account for favorable outcomes., Competing Interests: Competing interests: Disclosures relevant to PDAC are as follows. R.C.S. declares Scientific Advisory Board (SAB)/consultancy for Rappta Therapeutics, PanCAN, PRECEDE, MOHCCN & PanCuRx Canada and Precision Panc CRUK; sponsored research for Cardiff Oncology, AstraZeneca. G.B.M. declares SAB/consultancy for Amphista, Astex, AstraZeneca, BlueDot, Chrysallis Biotechnology, Ellipses Pharma, ImmunoMET, Infinity, Ionis, Lilly, Medacorp, Nanostring, Nuvectis, PDX Pharmaceuticals, Roche, Signalchem Lifesciences, Tarveda, Turbine and Zentalis Pharmaceuticals; Stock/options/financial for AstraZeneca, Catena Pharmaceuticals, ImmunoMet, SignalChem, Tarveda and Turbine; licensed technology HRD assay to Myriad Genetics and Digital Spatial Profiler patents with Nanostring. L.M.C. declares consulting services for Cell Signaling Technologies, AbbVie, the Susan G Komen Foundation and Shasqi, received reagents and/or research support from Cell Signaling Technologies, Syndax Pharmaceuticals, ZelBio, Hibercell and Acerta Pharma, and participates in advisory boards for Pharmacyclics, Syndax, Carisma, Verseau, CytomX, Kineta, Hibercell, Cell Signaling Technologies, Alkermes, Zymeworks, Genenta Sciences, Pio Therapeutics, PDX Pharmaceuticals, NextCure, the AstraZeneca Partner of Choice Network, the Lustgarten Foundation and the NIH–NCI-Frederick National Laboratory Advisory Committee. J.R.B. declares SAB for Perthera, Advisory for IDEAYA and is an editor for Springer and Taylor & Francis publishing. The other authors declare no competing interests., (© 2025. The Author(s).)

Pancreatic cancer remains a formidable challenge due to limited treatment options and its aggressive nature. In recent years, the naturally occurring anticancer compound juglone has emerged as a potential therapeutic candidate, showing promising results in inhibiting tumor growth and inducing cancer cell apoptosis. However, concerns over its toxicity have hampered juglone's clinical application. To address this issue, we have explored the use of polymeric micelles as a delivery system for juglone in pancreatic cancer treatment. These micelles, formulated using Poloxamer 407 and D-α-Tocopherol polyethylene glycol 1000 succinate, offer an innovative solution to enhance juglone's therapeutic potential while minimizing toxicity. In-vitro studies have demonstrated that micelle-formulated juglone (JM) effectively decreases proliferation and migration and increases apoptosis in pancreatic cancer cell lines. Importantly, in-vivo, JM exhibited no toxicity, allowing for increased dosing frequency compared to free drug administration. In mice, JM significantly reduced tumor growth in subcutaneous xenograft and orthotopic pancreatic cancer models. Beyond its direct antitumor effects, JM treatment also influenced the tumor microenvironment. In immunocompetent mice, JM increased immune cell infiltration and decreased stromal deposition and activation markers, suggesting an immunomodulatory role. To understand JM's mechanism of action, we conducted RNA sequencing and subsequent differential expression analysis on tumors that were treated with JM. The administration of JM treatment reduced the expression levels of the oncogenic protein MYC, thereby emphasizing its potential as a focused, therapeutic intervention. In conclusion, the polymeric micelles-mediated delivery of juglone holds excellent promise in pancreatic cancer therapy. This approach offers improved drug delivery, reduced toxicity, and enhanced therapeutic efficacy.

Triple-negative breast cancer (TNBC) patients have a poor prognosis and few treatment options. Mouse models of TNBC are important for development of new therapies, however, few mouse models represent the complexity of TNBC. Here, we develop a female TNBC murine model by mimicking two common TNBC mutations with high co-occurrence: amplification of the oncogene MYC and deletion of the tumor suppressor PTEN. This Myc;Ptenfl model develops heterogeneous triple-negative mammary tumors that display histological and molecular features commonly found in human TNBC. Our research involves deep molecular and spatial analyses on Myc;Ptenfl tumors including bulk and single-cell RNA-sequencing, and multiplex tissue-imaging. Through comparison with human TNBC, we demonstrate that this genetic mouse model develops mammary tumors with differential survival and therapeutic responses that closely resemble the inter- and intra-tumoral and microenvironmental heterogeneity of human TNBC, providing a pre-clinical tool for assessing the spectrum of patient TNBC biology and drug response., (© 2023. Springer Nature Limited.)

Nuclear receptors (NRs) are implicated in the regulation of tumors and immune cells. We identify a tumor-intrinsic function of the orphan NR, NR2F6, regulating antitumor immunity. NR2F6 was selected from 48 candidate NRs based on an expression pattern in melanoma patient specimens (i.e., IFN-γ signature) associated with positive responses to immunotherapy and favorable patient outcomes. Correspondingly, genetic ablation of NR2F6 in a mouse melanoma model conferred a more effective response to PD-1 therapy. NR2F6 loss in B16F10 and YUMM1.7 melanoma cells attenuated tumor development in immune-competent but not -incompetent mice via the increased abundance of effector and progenitor-exhausted CD8 + T cells. Inhibition of NACC1 and FKBP10, identified as NR2F6 effectors, phenocopied NR2F6 loss. Inoculation of NR2F6 KO mice with NR2F6 KD melanoma cells further decreased tumor growth compared with NR2F6 WT mice. Tumor-intrinsic NR2F6 function complements its tumor-extrinsic role and justifies the development of effective anticancer therapies.

Introduction: The response of the brain to space radiation is an important concern for astronauts during space missions. Therefore, we assessed the response of the brain to 28 Si ion irradiation (600 MeV/n), a heavy ion present in the space environment, on cognitive performance and whether the response is associated with altered DNA methylation in the hippocampus, a brain area important for cognitive performance. Methods: We determined the effects of 28 Si ion irradiation on object recognition, 6-month-old mice irradiated with 28 Si ions (600 MeV/n, 0.3, 0.6, and 0.9 Gy) and cognitively tested two weeks later. In addition, we determined if those effects were associated with alterations in hippocampal networks and/or hippocampal DNA methylation. Results: At 0.3 Gy, but not at 0.6 Gy or 0.9 Gy, 28 Si ion irradiation impaired cognition that correlated with altered gene expression and 5 hmC profiles that mapped to specific gene ontology pathways. Comparing hippocampal DNA hydroxymethylation following proton, 56 Fe ion, and 28 Si ion irradiation revealed a general space radiation synaptic signature with 45 genes that are associated with profound phenotypes. The most significant categories were glutamatergic synapse and postsynaptic density. Discussion: The brain's response to space irradiation involves novel excitatory synapse and postsynaptic remodeling., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Impey, Pelz, Riparip, Tafessu, Fareh, Zuloaga, Marzulla, Stewart, Rosi, Turker and Raber.)

Objective: The purpose of this study was to assess the reliability and educational quality of content available on Google and YouTube regarding subacromial impingement syndrome (SAIS)., Methods: Google and YouTube were queried for English and German results on SAIS using the search terms "shoulder impingement" and the German equivalent "Schulter Impingement". The analysis was restricted to the first 30 results of each query performed. Number of views and likes as well as upload source and length of content were recorded. Each result was evaluated by two independent reviewers using the Journal of the American Medical Association (JAMA) benchmark criteria (score range, 0-5) to assess reliability and the DISCERN score (score range, 16-80) and a SAIS-specific score (SAISS, score range, 0-100) to evaluate educational content., Results: The 58 websites found on Google and 48 videos found on YouTube were included in the analysis. The average number of views per video was 220,180 ± 415,966. The average text length was 1375 ± 997 words and the average video duration 456 ± 318 s. The upload sources were mostly non-physician based (74.1% of Google results and 79.2% of YouTube videos). Overall, there were poor results in reliability and educational quality, with sources from doctors having a significantly higher mean reliability measured in the JAMA score (p < 0.001) and educational quality in DISCERN (p < 0.001) and SAISS (p = 0.021). There was no significant difference between German and English results but texts performed significantly better than videos in terms of reliability (p = 0.002) and educational quality (p < 0.001)., Conclusion: Information on SAIS found on Google and YouTube is of low reliability and quality. Therefore, orthopedic health practitioners and healthcare providers should inform patients that this source of information may be unreliable and make efforts to provide patients with higher quality alternatives., Level of Evidence: IV, case series., (© 2022. The Author(s).)

Expression of guide RNAs in the CRISPR/Cas9 system typically requires the use of RNA polymerase III promoters, which are not cell-type specific. Flanking the gRNA with self-cleaving ribozyme motifs to create a self-cleaving gRNA overcomes this limitation. Here, we use self-cleaving gRNAs to create drug-selectable gene editing events in specific hepatocyte loci. A recombinant Adeno Associated Virus vector targeting the Albumin locus with a promoterless self-cleaving gRNA to create drug resistance is linked in cis with the therapeutic transgene. Gene expression of both are dependent on homologous recombination into the target locus. In vivo drug selection for the precisely edited hepatocytes allows >30-fold expansion of gene-edited cells and results in therapeutic levels of a human Factor 9 transgene. Importantly, self-cleaving gRNA expression is also achieved after targeting weak hepatocyte genes. We conclude that self-cleaving gRNAs are a powerful system to enable cell-type specific in vivo drug resistance for therapeutic gene editing applications., (© 2022. The Author(s).)

Yes-associated protein 1 (YAP1) is indispensable for the development of mutant KRAS -driven pancreatic ductal adenocarcinoma (PDAC). High YAP1 mRNA is a prognostic marker for worse overall survival in patient samples; however, the regulatory mechanisms that mediate its overexpression are not well understood. YAP1 genetic alterations are rare in PDAC, suggesting that its dysregulation is likely not due to genetic events. HuR is an RNA-binding protein whose inhibition impacts many cancer-associated pathways, including the "conserved YAP1 signature" as demonstrated by gene set enrichment analysis. Screening publicly available and internal ribonucleoprotein immunoprecipitation (RNP-IP) RNA sequencing (RNA-Seq) data sets, we discovered that YAP1 is a high-confidence target, which was validated in vitro with independent RNP-IPs and 3' untranslated region (UTR) binding assays. In accordance with our RNA sequencing analysis, transient inhibition (e.g., small interfering RNA [siRNA] and small-molecular inhibition) and CRISPR knockout of HuR significantly reduced expression of YAP1 and its transcriptional targets. We used these data to develop a HuR activity signature (HAS), in which high expression predicts significantly worse overall and disease-free survival in patient samples. Importantly, the signature strongly correlates with YAP1 mRNA expression. These findings highlight a novel mechanism of YAP1 regulation, which may explain how tumor cells maintain YAP1 mRNA expression at dynamic times during pancreatic tumorigenesis.

As a transcription factor that promotes cell growth, proliferation, and apoptosis, c-MYC (MYC) expression in the cell is tightly controlled. Disruption of oncogenic signaling pathways in human cancers can increase MYC protein stability, due to altered phosphorylation ratios at two highly conserved sites, Threonine 58 (T58) and Serine 62 (S62). The T58 to Alanine mutant (T58A) of MYC mimics the stabilized, S62 phosphorylated, and highly oncogenic form of MYC. The S62A mutant is also stabilized, lacks phosphorylation at both Serine 62 and Threonine 58, and has been shown to be nontransforming in vitro. However, several regulatory proteins are reported to associate with MYC lacking phosphorylation at S62 and T58, and the role this form of MYC plays in MYC transcriptional output and in vivo oncogenic function is understudied. We generated conditional c-Myc knock-in mice in which the expression of wild-type MYC (MYCWT), the T58A mutant (MYCT58A), or the S62A mutant (MYCS62A) with or without expression of endogenous Myc is controlled by the T-cell-specific Lck-Cre recombinase. MYCT58A expressing mice developed clonal T-cell lymphomas with 100% penetrance and conditional knock-out of endogenous Myc accelerated this lymphomagenesis. In contrast, MYCS62A mice developed clonal T-cell lymphomas at a much lower penetrance, and the loss of endogenous MYC reduced the penetrance while increasing the appearance of a non-transgene driven B-cell lymphoma with splenomegaly. Together, our study highlights the importance of regulated phosphorylation of MYC at T58 and S62 for T-cell transformation., Implications: Dysregulation of phosphorylation at conserved T58 and S62 residues of MYC differentially affects T-cell development and lymphomagenesis., (©2022 The Authors; Published by the American Association for Cancer Research.)

Human Antigen R (HuR/ELAVL1) is known to regulate stability of mRNAs involved in pancreatic ductal adenocarcinoma (PDAC) cell survival. Although several HuR targets are established, it is likely that many remain currently unknown. Here, we identified BARD1 mRNA as a novel target of HuR. Silencing HuR caused a >70% decrease in homologous recombination repair (HRR) efficiency as measured by the double-strand break repair (pDR-GFP reporter) assay. HuR-bound mRNAs extracted from RNP-immunoprecipitation and probed on a microarray, revealed a subset of HRR genes as putative HuR targets, including the BRCA1-Associated-Ring-Domain-1 (BARD1) (p < 0.005). BARD1 genetic alterations are infrequent in PDAC, and its context-dependent upregulation is poorly understood. Genetic silencing (siRNA and CRISPR knock-out) and pharmacological targeting of HuR inhibited both full length (FL) BARD1 and its functional isoforms (α, δ, Φ). Silencing BARD1 sensitized cells to olaparib and oxaliplatin; caused G2-M cell cycle arrest; and increased DNA-damage while decreasing HRR efficiency in cells. Exogenous overexpression of BARD1 in HuR-deficient cells partially rescued the HRR dysfunction, independent of an HuR pro-oncogenic function. Collectively, our findings demonstrate for the first time that BARD1 is a bona fide HuR target, which serves as an important regulatory point of the transient DNA-repair response in PDAC cells.

Background & Aims: Mature hepatocytes have limited expansion capability in culture and rapidly loose key functions. Recently however, tissue culture conditions have been developed that permit rodent hepatocytes to proliferate and transform into progenitor-like cells with ductal characteristics in vitro. Analogous cells expressing both hepatic and duct markers can be found in human cirrhotic liver in vivo and may represent an expandable population., Methods: An in vitro culture system to expand epithelial cells from human end stage liver disease organs was developed by inhibiting the canonical TGF-β, Hedgehog and BMP pathways., Results: Human cirrhotic liver epithelial cells became highly proliferative in vitro. Both gene expression and DNA methylation site analyses revealed that cirrhosis derived epithelial liver cells were intermediate between normal hepatocytes and cholangiocytes. Mouse hepatocytes could be expanded under the same conditions and retained the ability to re-differentiate into hepatocytes upon transplantation. In contrast, human cirrhotic liver derived cells had only low re-differentiation capacity., Conclusions: Epithelial cells of intermediate ductal-hepatocytic phenotype can be isolated from human cirrhotic livers and expanded in vitro. Unlike their murine counterparts they have limited liver repopulation potential., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: One key approach for anticancer therapy is drug combination. Drug combinations can help reduce doses and thereby decrease side effects. Furthermore, the likelihood of drug resistance is reduced. Distinct alterations in tumor metabolism have been described in past decades, but metabolism has yet to be targeted in clinical cancer therapy. Recently, we found evidence for synergism between dichloroacetate (DCA), a pyruvate dehydrogenase kinase inhibitor, and the HIF-1α inhibitor PX-478. In this study, we aimed to analyse this synergism in cell lines of different cancer types and to identify the underlying biochemical mechanisms., Methods: The dose-dependent antiproliferative effects of the single drugs and their combination were assessed using SRB assays. FACS, Western blot and HPLC analyses were performed to investigate changes in reactive oxygen species levels, apoptosis and the cell cycle. Additionally, real-time metabolic analyses (Seahorse) were performed with DCA-treated MCF-7 cells., Results: The combination of DCA and PX-478 produced synergistic effects in all eight cancer cell lines tested, including colorectal, lung, breast, cervical, liver and brain cancer. Reactive oxygen species generation and apoptosis played important roles in this synergism. Furthermore, cell proliferation was inhibited by the combination treatment., Conclusions: Here, we found that these tumor metabolism-targeting compounds exhibited a potent synergism across all tested cancer cell lines. Thus, we highly recommend the combination of these two compounds for progression to in vivo translational and clinical trials.

Periodontitis is one of the most common oral diseases worldwide and is caused by a variety of interactions between oral bacteria and the host. Here, pathogens induce inflammatory host responses that cause the secretion of proinflammatory cytokines such as IL-1β, IL-6, and IL-8 by oral epithelial cells. In various systems, it has been shown that inflammation compromises physical barriers, which enables bacteria to invade the tissue. In this study, we investigated the barrier properties of the oral mucosa under physiological and inflamed conditions. For this purpose, we assessed the influence of IL-1β on the transepithelial electrical resistance and in particular on tight junctions in vitro in human stratified squamous epithelium models. Indirect immunofluorescence and western blot analyses were performed to investigate localization and expression of tight junction proteins in primary gingival cells, immortalized gingival cells and native gingiva. Furthermore, the TEER of gingival keratinocytes was assessed. The results showed that IL-1β led to strengthening of the gingival keratinocyte barrier. This was demonstrated by an increase in TEER, the upregulation of TJ proteins, and an increase in the formation of TJ strands. The IL-1β-mediated upregulation of occludin was prevented by the NF-κB inhibitor BAY 11-7085. These observations provide insights into host responses in the early stages of periodontal disease and offer information about TJ formation in human gingival epithelial cells under physiological and inflammatory conditions. Comprehensive knowledge of the physical barrier during inflammation may help in developing strategies to effectively target the inflammatory barrier to improve the bioavailability of drugs for the treatment of periodontitis.

The c-MYC (MYC) oncoprotein is often overexpressed in human breast cancer; however, its role in driving disease phenotypes is poorly understood. Here, we investigate the role of MYC in HER2+ disease, examining the relationship between HER2 expression and MYC phosphorylation in HER2+ patient tumors and characterizing the functional effects of deregulating MYC expression in the murine NeuNT model of amplified-HER2 breast cancer. Deregulated MYC alone was not tumorigenic, but coexpression with NeuNT resulted in increased MYC Ser62 phosphorylation and accelerated tumorigenesis. The resulting tumors were metastatic and associated with decreased survival compared with NeuNT alone. MYC;NeuNT tumors had increased intertumoral heterogeneity including a subtype of tumors not observed in NeuNT tumors, which showed distinct metaplastic histology and worse survival. The distinct subtypes of MYC;NeuNT tumors match existing subtypes of amplified-HER2, estrogen receptor-negative human tumors by molecular expression, identifying the preclinical utility of this murine model to interrogate subtype-specific differences in amplified-HER2 breast cancer. We show that these subtypes have differential sensitivity to clinical HER2/EGFR-targeted therapeutics, but small-molecule activators of PP2A, the phosphatase that regulates MYC Ser62 phosphorylation, circumvents these subtype-specific differences and ubiquitously suppresses tumor growth, demonstrating the therapeutic utility of this approach in targeting deregulated MYC breast cancers.

In this study, an untargeted metabolomics approach was used to assess the effects of proton irradiation (1 Gy of 150 MeV) on the metabolome and DNA methylation pattern in the murine hippocampus and left ventricle of the heart 22 weeks following exposure using an integrated metabolomics-DNA methylation analysis. The integrated metabolomics-DNA methylation analysis in both tissues revealed significant alterations in aminoacyl-tRNA biosynthesis, but the direction of change was tissue-dependent. Individual and total amino acid synthesis were downregulated in the left ventricle of proton-irradiated mice but were upregulated in the hippocampus of proton-irradiated mice. Amino acid tRNA synthetase methylation was mostly downregulated in the hippocampus of proton-irradiated mice, whereas no consistent methylation pattern was observed for amino acid tRNA synthetases in the left ventricle of proton-irradiated mice. Thus, proton irradiation causes long-term changes in the left ventricle and hippocampus in part through methylation-based epigenetic modifications. Integrated analysis of metabolomics and DNA methylation is a powerful approach to obtain converging evidence of pathways significantly affected. This in turn might identify biomarkers of the radiation response, help identify therapeutic targets, and assess the efficacy of mitigators directed at those targets to minimize, or even prevent detrimental long-term effects of proton irradiation on the heart and the brain.

The transcription factor MYC (also c-Myc) induces histone modification, chromatin remodeling, and the release of paused RNA polymerase to broadly regulate transcription. MYC is subject to a series of post-translational modifications that affect its stability and oncogenic activity, but how these control MYC's function on the genome is largely unknown. Recent work demonstrates an intimate connection between nuclear compartmentalization and gene regulation. Here, we report that Ser62 phosphorylation and PIN1-mediated isomerization of MYC dynamically regulate the spatial distribution of MYC in the nucleus, promoting its association with the inner basket of the nuclear pore in response to proliferative signals, where it recruits the histone acetyltransferase GCN5 to bind and regulate local gene acetylation and expression. We demonstrate that PIN1-mediated localization of MYC to the nuclear pore regulates MYC target genes responsive to mitogen stimulation that are involved in proliferation and migration pathways. These changes are also present at the chromatin level, with an increase in open regulatory elements in response to stimulation that is PIN1-dependent and associated with MYC chromatin binding. Taken together, our study indicates that post-translational modification of MYC controls its spatial activity to optimally regulate gene expression in response to extrinsic signals in normal and diseased states., (© 2018 Su et al.; Published by Cold Spring Harbor Laboratory Press.)

Purpose: Previous studies have identified alkyl-phospholipids as promising compounds for cancer therapy by targeting constituents of the cell membrane and different signaling pathways. We previously showed that the alkylphospholipid Inositol-C2-PAF inhibits the proliferation and migration of immortalized keratinocytes and the squamous carcinoma-derived cell line SCC-25. Here, we investigated the effect of this compound on growth and motility as well as its mode of action in mammary carcinoma-derived cell lines., Methods: Using BrdU incorporation and haptotactic cell migration assays, we assessed the effects of Inositol-C2-PAF on MCF-7 and MBA-MB-231 cell proliferation and migration. The phosphorylation status of signaling molecules was investigated by Western blotting as well as indirect immunofluorescence analysis and capillary isoelectric focusing., Results: We found that Inositol-C2-PAF inhibited the growth as well as the migration in MCF-7 and MBA-MB-231 cells. Furthermore, we found that this compound inhibited phosphorylation of the protein kinase Akt at serine residue 473, but had no impact on phosphorylation at threonine 308. Phosphorylation of other kinases, such as Erk1/2, FAK and Src, which are targeted by Inositol-C2-PAF in other cells, remained unaffected by the compound in the mammary carcinoma-derived cell lines tested. In MCF-7 cells, we found that IGF-1-induced growth, as well as phosphorylation of Akt S473 , mTOR and the tumor suppressor pRB, was inhibited in the presence of Inositol-C2-PAF. Moreover, we found that in these cells IGF-1 had no impact on migration and did not seem to be linked to full Akt activity. Therefore, MCF-7 cell migration appears to be inhibited by Ino-C2-PAF in an Akt-independent manner., Conclusion: The antagonistic effects of Inositol-C2-PAF on cell migration and proliferation are indicative for its potential for breast cancer therapy, alone or in combination with other cytostatic drugs.

Intratumoral phenotypic heterogeneity has been described in many tumor types, where it can contribute to drug resistance and disease recurrence. We analyzed ductal and neuroendocrine markers in pancreatic ductal adenocarcinoma, revealing heterogeneous expression of the neuroendocrine marker Synaptophysin within ductal lesions. Higher percentages of Cytokeratin-Synaptophysin dual positive tumor cells correlate with shortened disease-free survival. We observe similar lineage marker heterogeneity in mouse models of pancreatic ductal adenocarcinoma, where lineage tracing indicates that Cytokeratin-Synaptophysin dual positive cells arise from the exocrine compartment. Mechanistically, MYC binding is enriched at neuroendocrine genes in mouse tumor cells and loss of MYC reduces ductal-neuroendocrine lineage heterogeneity, while deregulated MYC expression in KRAS mutant mice increases this phenotype. Neuroendocrine marker expression is associated with chemoresistance and reducing MYC levels decreases gemcitabine-induced neuroendocrine marker expression and increases chemosensitivity. Altogether, we demonstrate that MYC facilitates ductal-neuroendocrine lineage plasticity in pancreatic ductal adenocarcinoma, contributing to poor survival and chemoresistance.

The brain's response to radiation exposure is an important concern for patients undergoing cancer therapy and astronauts on long missions in deep space. We assessed whether this response is specific and prolonged and is linked to epigenetic mechanisms. We focused on the response of the hippocampus at early (2-weeks) and late (20-week) time points following whole body proton irradiation. We examined two forms of DNA methylation, cytosine methylation (5mC) and hydroxymethylation (5hmC). Impairments in object recognition, spatial memory retention, and network stability following proton irradiation were observed at the two-week time point and correlated with altered gene expression and 5hmC profiles that mapped to specific gene ontology pathways. Significant overlap was observed between DNA methylation changes at the 2 and 20-week time points demonstrating specificity and retention of changes in response to radiation. Moreover, a novel class of DNA methylation change was observed following an environmental challenge (i.e. space irradiation), characterized by both increased and decreased 5hmC levels along the entire gene body. These changes were mapped to genes encoding neuronal functions including postsynaptic gene ontology categories. Thus, the brain's response to proton irradiation is both specific and prolonged and involves novel remodeling of non-random regions of the epigenome.

The gallbladder and cystic duct (GBCs) are parts of the extrahepatic biliary tree and share a common developmental origin with the ventral pancreas. Here, we report on the very first genetic reprogramming of patient-derived human GBCs to β-like cells for potential autologous cell replacement therapy for type 1 diabetes. We developed a robust method for large-scale expansion of human GBCs ex vivo. GBCs were reprogrammed into insulin-producing pancreatic β-like cells by a combined adenoviral-mediated expression of hallmark pancreatic endocrine transcription factors PDX1, MAFA, NEUROG3, and PAX6 and differentiation culture in vitro. The reprogrammed GBCs (rGBCs) strongly induced the production of insulin and pancreatic endocrine genes and these responded to glucose stimulation in vitro. rGBCs also expressed an islet-specific surface marker, which was used to enrich for the most highly reprogrammed cells. More importantly, global mRNA and microRNA expression profiles and protein immunostaining indicated that rGBCs adopted an overall β-like state and these rGBCs engrafted in immunodeficient mice. Furthermore, comparative global expression analyses identified putative regulators of human biliary to β cell fate conversion. In summary, we have developed, for the first time, a reliable and robust genetic reprogramming and culture expansion of primary human GBCs-derived from multiple unrelated donors-into pancreatic β-like cells ex vivo, thus showing that human gallbladder is a potentially rich source of reprogrammable cells for autologous cell therapy in diabetes.

The biliary system plays an important role in several acquired and genetic disorders of the liver. We have previously shown that biliary duct epithelium contains cells giving rise to proliferative Lgr5 + organoids in vitro. However, it remained unknown whether all biliary cells or only a specific subset had this clonogenic activity. The cell surface protease ST14 was identified as a positive marker for the clonogenic subset of cholangiocytes and was used to separate clonogenic and non-clonogenic duct cells by fluorescence-activated cell sorting. Only ST14 hi duct cells had the ability to generate organoids that could be serially passaged. The gene expression profiles of clonogenic and non-clonogenic duct cells were similar, but several hundred genes were differentially expressed. RNA fluorescence in situ hybridization showed that clonogenic duct cells are interspersed among regular biliary epithelium at a ∼1:3 ratio. We conclude that adult murine cholangiocytes can be subdivided into two populations differing in their proliferative capacity., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Direct reprogramming is a promising approach for the replacement of β cells in diabetes. Reprogramming of cells originating from the endodermal lineage, such as acinar cells in the pancreas, liver cells and gallbladder cells has been of particular interest because of their developmental proximity to β cells. Our previous work showed that mouse gallbladder epithelium can be partially reprogrammed in vitro to generate islet-like cells (rGBC1). Here, the reprogramming protocol was substantially improved, yielding cells (rGBC2) closer to functional β cells than the 1st generation method with higher conversion efficiency and insulin expression. In addition to insulin synthesis and processing, rGBC2 presented many hallmark features of β cells, including insulin secretion in response to high glucose stimulation. Gene expression analysis indicated that rGBC2 clustered closer with β cells and had a metabolic gene expression profile resembling neonatal β cells. When transplanted into immune-deficient animals, rGBC2 were stable for at least 5months and further matured in vivo. Taken together, this approach provides further understanding of endodermal lineage conversion and potential for development of cell replacement therapy for type 1 diabetes patients., (Copyright © 2016 Michael Boutros, German Cancer Research Center, Heidelberg, Germany. Published by Elsevier B.V. All rights reserved.)

Background: Astronauts are exposed to 56 Fe ions that may pose a significant health hazard during and following prolonged missions in deep space. We showed previously that object recognition requiring the hippocampus, a structure critical for cognitive function, is affected in 2-month-old mice irradiated with 56 Fe ions. Here we examined object recognition in 6-month-old mice irradiated with 56 Fe ions, a biological age more relevant to the typical ages of astronauts. Moreover, because the mechanisms mediating the detrimental effects of 56 Fe ions on hippocampal function are unclear, we examined changes in hippocampal networks involved in synaptic plasticity and memory, gene expression, and epigenetic changes in cytosine methylation (5mC) and hydroxymethylation (5hmC) that could accompany changes in gene expression. We assessed the effects of whole body 56 Fe ion irradiation at early (2 weeks) and late (20 weeks) time points on hippocampus-dependent memory and hippocampal network stability, and whether these effects are associated with epigenetic changes in hippocampal DNA methylation (both 5mC and 5hmC) and gene expression., Results: At the two-week time point, object recognition and network stability were impaired following irradiation at the 0.1 and 0.4 Gy dose, but not following irradiation at the 0.2 Gy dose. No impairments in object recognition or network stability were seen at the 20-week time point at any irradiation dose used. Consistent with this pattern, the significance of pathways for gene categories for 5hmC was lower, though not eliminated, at the 20-week time point compared to the 2-week time point. Similarly, significant changes were observed for 5mC gene pathways at the 2-week time point, but no significant gene categories were observed at the 20-week time point. Only the 5hmC changes tracked with gene expression changes., Conclusions: Dose- and time-dependent epigenomic remodeling in the hippocampus following 56 Fe ion exposure correlates with behavioral changes.

Background and Aims: EUS-guided FNA (EUS-FNA) is the primary method used to obtain pancreatic tissue for preoperative diagnosis. Accumulating evidence suggests diagnostic and prognostic information may be obtained by gene-expression profiling of these biopsy specimens. RNA sequencing (RNAseq) is a newer method of gene-expression profiling, but published data are scant on the use of this method on pancreas tissue obtained via EUS-FNA. The aim of this study was to determine whether RNAseq of EUS-FNA biopsy samples of undiagnosed pancreatic masses can reliably discriminate between benign and malignant tissue., Methods: In this prospective study, consenting adults presented to 2 tertiary care hospitals for EUS of suspected pancreatic mass. Tissue was submitted for RNAseq. The results were compared with cytologic diagnosis, surgical pathology diagnosis, or benign clinical follow-up of at least 1 year., Results: Forty-eight patients with solid pancreatic mass lesions were enrolled. Nine samples were excluded because of inadequate RNA and 3 because of final pathologic diagnosis of neuroendocrine tumor. Data from the first 13 patients were used to construct a linear classifier, and this was tested on the final 23 patients (15 malignant and 8 benign lesions). RNAseq of EUS-FNA biopsy samples distinguishes ductal adenocarcinoma from benign pancreatic solid masses with a sensitivity of .87 (range, .58-.98) and specificity of .75 (range, .35-.96)., Conclusions: This proof-of-principle study suggests RNAseq of EUS-FNA samples can reliably detect adenocarcinoma and may provide a new method to evaluate more diagnostically challenging pancreatic lesions., (Copyright © 2016 American Society for Gastrointestinal Endoscopy. Published by Elsevier Inc. All rights reserved.)

Background: Proton irradiation poses a potential hazard to astronauts during and following a mission, with post-mitotic cells at most risk because they cannot dilute resultant epigenetic changes via cell division. Persistent epigenetic changes that result from environmental exposures include gains or losses of DNA methylation of cytosine, which can impact gene expression. In the present study, we compared the long-term epigenetic effects of whole body proton irradiation in the mouse hippocampus and left ventricle. We used an unbiased genome-wide DNA methylation study, involving ChIP-seq with antibodies to 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) to identify DNA regions in which methylation levels have changed 22 weeks after a single exposure to proton irradiation. We used DIP-Seq to profile changes in genome-wide DNA methylation and hydroxymethylation following proton irradiation. In addition, we used published RNAseq data to assess whether differentially methylated regions were linked to changes in gene expression., Results: The DNA methylation data showed tissue-dependent effects of proton irradiation and revealed significant major pathway changes in response to irradiation that are related to known pathophysiologic processes. Many regions affected in the ventricle mapped to genes involved in cardiovascular function pathways, whereas many regions affected in the hippocampus mapped to genes involved in neuronal functions. In the ventricle, increases in 5hmC were associated with decreases in 5mC. We also observed spatial overlap for regions where both epigenetic marks decreased in the ventricle. In hippocampus, increases in 5hmC were most significantly correlated (spatially) with regions that had increased 5mC, suggesting that deposition of hippocampal 5mC and 5hmC may be mechanistically coupled., Conclusions: The results demonstrate long-term changes in DNA methylation patterns following a single proton irradiation, that these changes are tissue specific, and that they map to pathways consistent with tissue specific responses to proton irradiation. Further, the results suggest novel relationships between changes in 5mC and 5hmC.

miR-132 and miR-212 are structurally related microRNAs that have been found to exert powerful modulatory effects within the central nervous system (CNS). Notably, these microRNAs are tandomly processed from the same noncoding transcript, and share a common seed sequence: thus it has been difficult to assess the distinct contribution of each microRNA to gene expression within the CNS. Here, we employed a combination of conditional knockout and transgenic mouse models to examine the contribution of the miR-132/-212 gene locus to learning and memory, and then to assess the distinct effects that each microRNA has on hippocampal gene expression. Using a conditional deletion approach, we show that miR-132/-212 double-knockout mice exhibit significant cognitive deficits in spatial memory, recognition memory, and in tests of novel object recognition. Next, we utilized transgenic miR-132 and miR-212 overexpression mouse lines and the miR-132/-212 double-knockout line to explore the distinct effects of these two miRNAs on the transcriptional profile of the hippocampus. Illumina sequencing revealed that miR-132/-212 deletion increased the expression of 1138 genes; Venn analysis showed that 96 of these genes were also downregulated in mice overexpressing miR-132. Of the 58 genes that were decreased in animals overexpressing miR-212, only four of them were also increased in the knockout line. Functional gene ontology analysis of downregulated genes revealed significant enrichment of genes related to synaptic transmission, neuronal proliferation, and morphogenesis, processes known for their roles in learning, and memory formation. These data, coupled with previous studies, firmly establish a role for the miR-132/-212 gene locus as a key regulator of cognitive capacity. Further, although miR-132 and miR-212 share a seed sequence, these data indicate that these miRNAs do not exhibit strongly overlapping mRNA targeting profiles, thus indicating that these two genes may function in a complex, nonredundant manner to shape the transcriptional profile of the CNS. The dysregulation of miR-132/-212 expression could contribute to signaling mechanisms that are involved in an array of cognitive disorders., (© 2016 Hansen et al.; Published by Cold Spring Harbor Laboratory Press.)