mTORC1 and mTORC2 Converge on the Arp2/3 Complex to Promote KrasG12D-Induced Acinar-to-Ductal Metaplasia and Early Pancreatic Carcinogenesis.

BACKGROUND & AIMS: Oncogenic KrasG12D induces neoplastic transformation of pancreatic acinar cells through acinar-to-ductal metaplasia (ADM), an actin-based morphogenetic process, and drives pancreatic ductal adenocarcinoma (PDAC). mTOR (mechanistic target of rapamycin kinase) complex 1 (mTORC1) and 2 (mTORC2) contain Rptor and Rictor, respectively, and are activated downstream of KrasG12D, thereby contributing to PDAC. Yet, whether and how mTORC1 and mTORC2 impact on ADM and the identity of the actin nucleator(s) mediating such actin rearrangements remain unknown. METHODS: A mouse model of inflammation-accelerated KrasG12D-driven early pancreatic carcinogenesis was used. Rptor, Rictor, and Arpc4 (actin-related protein 2/3 complex subunit 4) were conditionally ablated in acinar cells to deactivate the function of mTORC1, mTORC2 and the actin-related protein (Arp) 2/3 complex, respectively. RESULTS: We found that mTORC1 and mTORC2 are markedly activated in human and mouse ADM lesions, and cooperate to promote KrasG12D-driven ADM in mice and in vitro. They use the Arp2/3 complex as a common downstream effector to induce the remodeling the actin cytoskeleton leading to ADM. In particular, mTORC1 regulates the translation of Rac1 (Rac family small GTPase 1) and the Arp2/3-complex subunit Arp3, whereas mTORC2 activates the Arp2/3 complex by promoting Akt/Rac1 signaling. Consistently, genetic ablation of the Arp2/3 complex prevents KrasG12D-driven ADM in vivo. In acinar cells, the Arp2/3 complex and its actin-nucleation activity mediated the formation of a basolateral actin cortex, which is indispensable for ADM and pre-neoplastic transformation. CONCLUSIONS: Here, we show that mTORC1 and mTORC2 attain a dual, yet nonredundant regulatory role in ADM and early pancreatic carcinogenesis by promoting Arp2/3 complex function. The role of Arp2/3 complex as a common effector of mTORC1 and mTORC2 fills the gap between oncogenic signals and actin dynamics underlying PDAC initiation.

AGR2-Dependent Nuclear Import of RNA Polymerase II Constitutes a Specific Target of Pancreatic Ductal Adenocarcinoma in the Context of Wild-Type p53.

BACKGROUND & AIMS: Promoted by pancreatitis, oncogenic KrasG12D triggers acinar cells' neoplastic transformation through acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia. Anterior gradient 2 (Agr2), a known inhibitor of p53, is detected at early stage of pancreatic ductal adenocarcinoma (PDAC) development. RNA polymerase II (RNAPII) is a key nuclear enzyme; regulation of its nuclear localization in mammalian cells represents a potential therapeutic target. METHODS: A mouse model of inflammation-accelerated KrasG12D-driven ADM and pancreatic intraepithelial neoplasia development was used. Pancreas-specific Agr2 ablation was performed to access its role in pancreatic carcinogenesis. Hydrophobic hexapeptides loaded in liposomes were developed to disrupt Agr2-RNAPII complex. RESULTS: We found that Agr2 is up-regulated in ADM-to-pancreatic intraepithelial neoplasia transition in inflammation and KrasG12D-driven early pancreatic carcinogenesis. Genetic ablation of Agr2 specifically blocks this metaplastic-to-neoplastic process. Mechanistically, Agr2 directs the nuclear import of RNAPII via its C-terminal nuclear localization signal, undermining the ATR-dependent p53 activation in ADM lesions. Because Agr2 binds to the largest subunit of RNAPII in a peptide motif-dependent manner, we developed a hexapeptide to interfere with the nuclear import of RNAPII by competitively disrupting the Agr2-RNAPII complex. This novel hexapeptide leads to dysfunction of RNAPII with concomitant activation of DNA damage response in early neoplastic lesions; hence, it dramatically compromises PDAC initiation in vivo. Moreover, the hexapeptide sensitizes PDAC cells and patient-derived organoids harboring wild-type p53 to RNAPII inhibitors and first-line chemotherapeutic agents in vivo. Of note, this therapeutic effect is efficient across various cancer types. CONCLUSIONS: Agr2 is identified as a novel adaptor protein for nuclear import of RNAPII in mammalian cells. Also, we provide genetic evidence defining Agr2-dependent nuclear import of RNAPII as a pharmaceutically accessible target for prevention and treatment in PDAC in the context of wild-type p53.

Sca-1 is a marker for cell plasticity in murine pancreatic epithelial cells and induced by IFN-ß in vitro.

BACKGROUND & AIMS: Sca-1 is a surface marker for murine hematopoietic stem cells (HSCs) and type-I interferon is a key regulator for Lin-Sca-1+ HSCs expansion through Ifnar/Stat-1/Sca-1-signaling. In this study we aimed to characterize the role and regulation of Sca-1+ cells in pancreatic regeneration. METHODS: To characterize Sca-1 in vivo, immunohistochemistry and immunofluorescence staining of Sca-1 was conducted in normal pancreas, in cerulein-mediated acute pancreatitis, and in Kras-triggered cancerous lesions. Ifnar/Stat-1/Sca-1-signaling was studied in type-I IFN-treated epithelial explants of adult wildtype, Ifnar-/-, and Stat-1-/- mice. Sca-1 induction was analyzed by gene expression and FACS analysis. After isolation of pancreatic epithelial Lin-Sca-1+cells, pancreatosphere-formation and immunofluorescence-assays were carried out to investigate self-renewal and differentiation capabilities. RESULTS: Sca-1+ cells were located in periacinar and periductal spaces and showed an enrichment during cerulein-induced acute pancreatitis (23.2/100 µm2 ± 4.9 SEM) and in early inflammation-mediated carcinogenic lesions of the pancreas of KrasG12D mice (35.8/100 µm2 ± SEM 1.9) compared to controls (3.6/100 µm2 ± 1.3 SEM). Pancreatic Lin-Sca-1+ cells displayed a small population of 1.46% ± 0.12 SEM in FACS. In IFN-ß treated pancreatic epithelial explants, Sca-1 expression was increased, and Lin-Sca-1+ cells were enriched in vitro (from 1.49% ± 0.36 SEM to 3.85% ± 0.78 SEM). Lin-Sca-1+ cells showed a 12 to 51-fold higher capacity for clonal self-renewal compared to Lin-Sca-1- cells and generated cells express markers of the acinar and ductal compartment. CONCLUSIONS: Pancreatic Sca-1+ cells enriched during parenchymal damage showed a significant capacity for cell renewal and in vitro plasticity, suggesting that corresponding to the type I interferon-dependent regulation of Lin-Sca-1+ hematopoietic stem cells, pancreatic Sca-1+ cells also employ type-I-interferon for regulating progenitor-cell-homeostasis.

Expression of the EWSR1-FLI1 fusion oncogene in pancreas cells drives pancreatic atrophy and lipomatosis.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) harbors mutant KRAS as the most common driver mutation. Studies on mouse models have uncovered the tumorigenic characteristics of the Kras oncogene driving pancreatic carcinogenesis. Similarly, Ewing sarcoma predominantly depends on the occurrence of the EWSR1-FLI1 fusion oncogene. The expression of EWSR1-FLI1 affects pro-tumorigenic pathways and induces cell transformation. In this study, we investigated whether mutant Kras could be exchanged by another potent oncogene, such as EWSR1-FLI1, to initiate pancreatic cancer development. METHODS: We generated two conditional mouse models expressing mutant KrasG12D (KC) or the EWSR1-FLI1 oncogene (E/F) in pancreas cells. Pancreatic tissue was collected from the mice at 4-6 weeks and 11-13 weeks of age as well as from survival cohorts to determine the development of spontaneous acinar-to-ductal metaplasia (ADM) and neoplastic lesions. Immunohistochemistry and immunofluorescence staining were performed to characterize and quantify changes in tissue morphology. RESULTS: The expression of the EWSR1-FLI1 fusion protein in pancreas cells was confirmed by positive FLI1 immunohistochemistry staining. Notably, the EWSR1-FLI1 expression in pancreas cells resulted in a strong depletion of the acinar cell mass and an extensive lipomatosis. Although the E/F mice exhibited spontaneous ADM formation and a shorter overall survival rate compared to KC mice, no development of neoplastic lesion was observed in aging E/F mice. CONCLUSIONS: The expression of the EWSR1-FLI1 oncogene leads to a strong pancreatic atrophy and lipomatosis. ADM formation indicates that pancreatic acinar cells are susceptible for EWSR1-FLI1-mediated oncogenic transformation to a limited extent. However, the EWSR1-FLI1 oncogene is insufficient to induce pancreatic cancer development.

Ring1b-dependent epigenetic remodelling is an essential prerequisite for pancreatic carcinogenesis.

BACKGROUND AND AIMS: Besides well-defined genetic alterations, the dedifferentiation of mature acinar cells is an important prerequisite for pancreatic carcinogenesis. Acinar-specific genes controlling cell homeostasis are extensively downregulated during cancer development; however, the underlying mechanisms are poorly understood. Now, we devised a novel in vitro strategy to determine genome-wide dynamics in the epigenetic landscape in pancreatic carcinogenesis. DESIGN: With our in vitro carcinogenic sequence, we performed global gene expression analysis and ChIP sequencing for the histone modifications H3K4me3, H3K27me3 and H2AK119ub. Followed by a comprehensive bioinformatic approach, we captured gene clusters with extensive epigenetic and transcriptional remodelling. Relevance of Ring1b-catalysed H2AK119ub in acinar cell reprogramming was studied in an inducible Ring1b knockout mouse model. CRISPR/Cas9-mediated Ring1b ablation as well as drug-induced Ring1b inhibition were functionally characterised in pancreatic cancer cells. RESULTS: The epigenome is vigorously modified during pancreatic carcinogenesis, defining cellular identity. Particularly, regulatory acinar cell transcription factors are epigenetically silenced by the Ring1b-catalysed histone modification H2AK119ub in acinar-to-ductal metaplasia and pancreatic cancer cells. Ring1b knockout mice showed greatly impaired acinar cell dedifferentiation and pancreatic tumour formation due to a retained expression of acinar differentiation genes. Depletion or drug-induced inhibition of Ring1b promoted tumour cell reprogramming towards a less aggressive phenotype. CONCLUSIONS: Our data provide substantial evidence that the epigenetic silencing of acinar cell fate genes is a mandatory event in the development and progression of pancreatic cancer. Targeting the epigenetic repressor Ring1b could offer new therapeutic options.

Ductal obstruction promotes formation of preneoplastic lesions from the pancreatic ductal compartment.

Pancreatitis is a significant risk factor for pancreatic ductal adenocarcinoma (PDAC). Previous studies in mice have demonstrated that pancreatitis contributes to oncogenic Kras-driven carcinogenesis, probably initiated in acinar cells; however, oncogenic Kras alone or in combination with caerulein-induced pancreatitis is not sufficient in initiating PDAC from the ductal compartment. We thus introduced ductal obstruction - which induces a more severe form of pancreatitis - by pancreatic ductal ligation in mice harbouring oncogenic Kras. This induced a particular phenotype with highly proliferative nonmucinous cells with nuclear atypia. Around these lesions, there was a significant proliferation of activated fibroblasts and infiltration of immune cells, corroborating the pathological features of preneoplastic lesions. Lineage-tracing experiments revealed that these preneoplastic cells derived from two distinctive cellular sources: acinar and ductal cells. Phenotypic characterisation revealed that the duct-derived preneoplastic lesions show a high proliferative potential with persistent activation of tumour-promoting inflammatory pathways while the acinar-derived ones were less proliferative with persistent p53 activation. Furthermore, the duct-derived preneoplastic cells have a particularly high nuclear-to-cytoplasmic ratio. These data demonstrate that ductal obstruction promotes preneoplastic lesion formation from the pancreatic ductal compartment.

Loss of TLR3 and its downstream signaling accelerates acinar cell damage in the acute phase of pancreatitis.

BACKGROUND: Acute pancreatitis is accompanied by acinar cell damage releasing potential toll-like receptor 3 (TLR3) ligands. So far, TLR3 is known as a pattern recognition receptor in the immune signaling cascade triggering a type I interferon response. In addition, TLR3 signaling contributes to programmed cell death through the activation of caspase 8. However, the functional role of TLR3 and its downstream toll-like receptor adaptor molecule 1 (TICAM1) in the inflamed pancreas is unknown. METHODS: To uncover the role of TLR3 signaling in acute pancreatitis, we induced a cerulein-mediated pancreatitis in Tlr3 and Ticam1 knockout (KO) mice and in wildtype animals. The exocrine damage was determined by blood serum analysis and histological examination. Immunohistochemistry, gene expression and immunoblot analysis were conducted to study TLR3 function. RESULTS: After the induction of an acute pancreatitis, wildtype mice showed a high endosomal TLR3 expression in acinar cells. In comparison to wildtype and Ticam1 KO mice, Tlr3 KO mice exhibited the highest severity of pancreatitis with an increased NF-κB activation and elevated expression of the pro-inflammatory cytokines Il6 and Tnf, although the amount of infiltrating immune cells was unaffected. Additionally, we detected a strong elevation of acinar cell necrosis and reduced levels of cleaved caspase 8 in Tlr3 and Ticam1 KO mice. CONCLUSIONS: TLR3 and its downstream adaptor TICAM1 are important mediators of acinar cell damage in acute pancreatitis. They possess a critical role in programmed cell death and our data suggest that TLR3 signaling controls the onset and severity of acute pancreatitis.

Dynamic landscape of pancreatic carcinogenesis reveals early molecular networks of malignancy.

OBJECTIVE: The initial steps of pancreatic regeneration versus carcinogenesis are insufficiently understood. Although a combination of oncogenic Kras and inflammation has been shown to induce malignancy, molecular networks of early carcinogenesis remain poorly defined. DESIGN: We compared early events during inflammation, regeneration and carcinogenesis on histological and transcriptional levels with a high temporal resolution using a well-established mouse model of pancreatitis and of inflammation-accelerated KrasG12D-driven pancreatic ductal adenocarcinoma. Quantitative expression data were analysed and extensively modelled in silico. RESULTS: We defined three distinctive phases-termed inflammation, regeneration and refinement-following induction of moderate acute pancreatitis in wild-type mice. These corresponded to different waves of proliferation of mesenchymal, progenitor-like and acinar cells. Pancreas regeneration required a coordinated transition of proliferation between progenitor-like and acinar cells. In mice harbouring an oncogenic Kras mutation and challenged with pancreatitis, there was an extended inflammatory phase and a parallel, continuous proliferation of mesenchymal, progenitor-like and acinar cells. Analysis of high-resolution transcriptional data from wild-type animals revealed that organ regeneration relied on a complex interaction of a gene network that normally governs acinar cell homeostasis, exocrine specification and intercellular signalling. In mice with oncogenic Kras, a specific carcinogenic signature was found, which was preserved in full-blown mouse pancreas cancer. CONCLUSIONS: These data define a transcriptional signature of early pancreatic carcinogenesis and a molecular network driving formation of preneoplastic lesions, which allows for more targeted biomarker development in order to detect cancer earlier in patients with pancreatitis.

A subset of metastatic pancreatic ductal adenocarcinomas depends quantitatively on oncogenic Kras/Mek/Erk-induced hyperactive mTOR signalling.

OBJECTIVE: Oncogenic Kras-activated robust Mek/Erk signals phosphorylate to the tuberous sclerosis complex (Tsc) and deactivates mammalian target of rapamycin (mTOR) suppression in pancreatic ductal adenocarcinoma (PDAC); however, Mek and mTOR inhibitors alone have demonstrated minimal clinical antitumor activity. DESIGN: We generated transgenic mouse models in which mTOR was hyperactivated either through the Kras/Mek/Erk cascade, by loss of Pten or through Tsc1 haploinsufficiency. Primary cancer cells were isolated from mouse tumours. Oncogenic signalling was assessed in vitro and in vivo, with and without single or multiple targeted molecule inhibition. Transcriptional profiling was used to identify biomarkers predictive of the underlying pathway alterations and of therapeutic response. Results from the preclinical models were confirmed on human material. RESULTS: Reduction of Tsc1 function facilitated activation of Kras/Mek/Erk-mediated mTOR signalling, which promoted the development of metastatic PDACs. Single inhibition of mTOR or Mek elicited strong feedback activation of Erk or Akt, respectively. Only dual inhibition of Mek and PI3K reduced mTOR activity and effectively induced cancer cell apoptosis. Analysis of downstream targets demonstrated that oncogenic activity of the Mek/Erk/Tsc/mTOR axis relied on Aldh1a3 function. Moreover, in clinical PDAC samples, ALDH1A3 specifically labelled an aggressive subtype. CONCLUSIONS: These results advance our understanding of Mek/Erk-driven mTOR activation and its downstream targets in PDAC, and provide a mechanistic rationale for effective therapeutic matching for Aldh1a3-positive PDACs.

Pancreas-specific activation of mTOR and loss of p53 induce tumors reminiscent of acinar cell carcinoma.

BACKGROUND: Pancreatic acinar cell carcinoma (ACC) is a rare tumor entity with an unfavorable prognosis. Recent whole-exome sequencing identified p53 mutations in a subset of human ACC. Activation of the mammalian target of rapamycin (mTOR) pathway is associated with various pancreatic neoplasms. We thus aimed at analyzing whether activation of mTOR with a concomitant loss of p53 may initiate ACC. METHODS: We generated transgenic mouse models in which mTOR was hyperactivated through pancreas-specific, homozygous tuberous sclerosis 1 (Tsc1) deficiency, with or without deletion of p53 (Tsc1 (-/-) and Tsc1 (-/-) ; p53 (-/-) ). Activity of mTOR signaling was investigated using mouse tissues and isolated murine cell lines. Human ACC specimens were used to corroborate the findings from the transgenic mouse models. RESULTS: Hyperactive mTOR signaling in Tsc1 (-/-) mice was not oncogenic but rather induced a near-complete loss of the pancreatic acinar compartment. Acinar cells were lost as a result of apoptosis which was associated with p53 activation. Concomitantly, ductal cells were enriched. Ablation of p53 in Tsc1-deficient mice prevented acinar cell death but promoted formation of acinar cells with severe nuclear abnormalities. One out of seven Tsc1 (-/-) ; p53 (-/-) animals developed pancreatic tumors showing a distinctive tumor morphology, reminiscent of human ACC. Hyperactive mTOR signaling was also detected in a subset of human ACC. CONCLUSION: Hyperactive mTOR signaling combined with loss of p53 in mice induces tumors similar to human ACC.

Next-generation sequencing reveals novel differentially regulated mRNAs, lncRNAs, miRNAs, sdRNAs and a piRNA in pancreatic cancer.

BACKGROUND: Previous studies identified microRNAs (miRNAs) and messenger RNAs with significantly different expression between normal pancreas and pancreatic cancer (PDAC) tissues. Due to technological limitations of microarrays and real-time PCR systems these studies focused on a fixed set of targets. Expression of other RNA classes such as long intergenic non-coding RNAs or sno-derived RNAs has rarely been examined in pancreatic cancer. Here, we analysed the coding and non-coding transcriptome of six PDAC and five control tissues using next-generation sequencing. RESULTS: Besides the confirmation of several deregulated mRNAs and miRNAs, miRNAs without previous implication in PDAC were detected: miR-802, miR-2114 or miR-561. SnoRNA-derived RNAs (e.g. sno-HBII-296B) and piR-017061, a piwi-interacting RNA, were found to be differentially expressed between PDAC and control tissues. In silico target analysis of miR-802 revealed potential binding sites in the 3' UTR of TCF4, encoding a transcription factor that controls Wnt signalling genes. Overexpression of miR-802 in MiaPaCa pancreatic cancer cells reduced TCF4 protein levels. Using Massive Analysis of cDNA Ends (MACE) we identified differential expression of 43 lincRNAs, long intergenic non-coding RNAs, e.g. LINC00261 and LINC00152 as well as several natural antisense transcripts like HNF1A-AS1 and AFAP1-AS1. Differential expression was confirmed by qPCR on the mRNA/miRNA/lincRNA level and by immunohistochemistry on the protein level. CONCLUSIONS: Here, we report a novel lncRNA, sncRNA and mRNA signature of PDAC. In silico prediction of ncRNA targets allowed for assigning potential functions to differentially regulated RNAs.

Kif20a inhibition reduces migration and invasion of pancreatic cancer cells.

BACKGROUND: The Translational Genome Research Network in Pancreatic Cancer performed a meta-analysis of publicly available various high-throughput gene analysis panels to identify drugable targets. There, the most differentially expressed gene between normal and cancerous pancreas was Kif20a. The aim of the study was to verify this expression pattern and further characterize Kif20a in pancreatic cancer. MATERIALS AND METHODS: Detailed expression analyses were carried out in pancreatic tissues and in a wide panel of pancreatic cells including ductal adenocarcinoma (PDAC) and neuroendocrine-cancer cell lines as well as immortalized human pancreatic ductal epithelial and primary stellate cells using quantitative real-time polymerase chain reaction, immunohistochemistry, immunofluorescence, and immunoblot analyses. Effects on proliferation, apoptosis, and cell cycle were assessed by MTT assays, caspase-cleavage assays, and fluorescence-activated cell sorting analysis after Kif20a silencing. Cell motility was assessed by migration and invasion assays as well as time-lapse microscopy. RESULTS: Mean Kif20a messenger RNA expression was 18.4-fold upregulated in PDAC tissues compared with that in the normal pancreas. In line, neuroendocrine-cancer cell lines display a 1.6-fold increase and ductal adenocarcinoma cell lines a 11-fold increase of Kif20a messenger RNA (P = 0.009) in comparison with primary stellate cells. A 7.3-fold overexpression was also found in immortalized pancreatic ductal epithelial cells. Kif20a silencing with small interfering RNA molecules resulted in an inhibition of proliferation, motility, and invasion of pancreatic cancer cell lines. CONCLUSIONS: Targeting Kif20a reduces proliferation, migration, and invasion of pancreatic cancer cells. Together with its significant overexpression in PDAC, this makes it a potential target for diagnostic and interventional purposes.

In Vivo Microbeam Radiation Therapy at a Conventional Small Animal Irradiator.

Microbeam radiation therapy (MRT) is a still pre-clinical form of spatially fractionated radiotherapy, which uses an array of micrometer-wide, planar beams of X-ray radiation. The dose modulation in MRT has proven effective in the treatment of tumors while being well tolerated by normal tissue. Research on understanding the underlying biological mechanisms mostly requires large third-generation synchrotrons. In this study, we aimed to develop a preclinical treatment environment that would allow MRT independent of synchrotrons. We built a compact microbeam setup for pre-clinical experiments within a small animal irradiator and present in vivo MRT application, including treatment planning, dosimetry, and animal positioning. The brain of an immobilized mouse was treated with MRT, excised, and immunohistochemically stained against γH2AX for DNA double-strand breaks. We developed a comprehensive treatment planning system by adjusting an existing dose calculation algorithm to our setup and attaching it to the open-source software 3D-Slicer. Predicted doses in treatment planning agreed within 10% with film dosimetry readings. We demonstrated the feasibility of MRT exposures in vivo at a compact source and showed that the microbeam pattern is observable in histological sections of a mouse brain. The platform developed in this study will be used for pre-clinical research of MRT.

Syndecan-2 promotes perineural invasion and cooperates with K-ras to induce an invasive pancreatic cancer cell phenotype.

BACKGROUND: We have identified syndecan-2 as a protein potentially involved in perineural invasion of pancreatic adenocarcinoma (PDAC) cells. METHODS: Syndecan-2 (SDC-2) expression was analyzed in human normal pancreas, chronic pancreatitis and PDAC tissues. Functional in vitro assays were carried out to determine its role in invasion, migration and signaling. RESULTS: SDC-2 was expressed in the majority of the tested pancreatic cancer cell lines while it was upregulated in nerve-invasive PDAC cell clones. There were 2 distinct expression patterns of SDC-2 in PDAC tissue samples: SDC-2 positivity in the cancer cell cytoplasm and a peritumoral expression. Though SDC-2 silencing (using specific siRNA oligonucleotides) did not affect anchorage-dependent growth, it significantly reduced cell motility and invasiveness in the pancreatic cancer cell lines T3M4 and Su8686. On the transcriptional level, migration-and invasion-associated genes were down-regulated following SDC-2 RNAi. Furthermore, SDC-2 silencing reduced K-ras activity, phosphorylation of Src and--further downstream--phosphorylation of ERK2 while levels of the putative SDC-2 signal transducer p120GAP remained unaltered. CONCLUSION: SDC-2 is a novel (perineural) invasion-associated gene in PDAC which cooperates with K-ras to induce a more invasive phenotype.

Energy metabolism and proliferation in pancreatic carcinogenesis.

INTRODUCTION: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer entity with a high proliferative potential. Uncontrolled cell proliferation is mediated by a number of core signaling pathways. Recently, novel data of PDAC biology suggest that these core signal pathways affect cell proliferation and metabolism simultaneously. METHODS: Here, we reviewed the literature on core metabolic signaling pathways in pancreatic carcinogenesis. RESULTS: Results obtained from mouse genetics and in vitro experiments have demonstrated the significance of the Kras, p53, c-Myc, and Lkb1 networks in the proliferation of pancreatic epithelial and cancer cells. At the same time, these major pathways also affect energy metabolism by influencing glucose and glutamine utilization. In particular, Kras-mediated metabolic changes seem to be directly involved in carcinogenesis. However, there is a lack of solid evidence on how metabolism and proliferation are connected in pancreatic carcinogenesis. CONCLUSION: Understanding early and subtle changes in cellular metabolism of pancreatic epithelial-and specifically of acinar-cells, which accompany or directly influence malignant transformation and uncontrolled proliferation, will be paramount to define novel imaging and other modalities for earlier detection of PDAC.

The Emerging Role of miRNAs for the Radiation Treatment of Pancreatic Cancer.

Today, pancreatic cancer is the seventh leading cause of cancer-related deaths worldwide with a five-year overall survival rate of less than 7%. Only 15-20% of patients are eligible for curative intent surgery at the time of diagnosis. Therefore, neoadjuvant treatment regimens have been introduced in order to downsize the tumor by chemotherapy and radiotherapy. To further increase the efficacy of radiotherapy, novel molecular biomarkers are urgently needed to define the subgroup of pancreatic cancer patients who would benefit most from radiotherapy. MicroRNAs (miRNAs) could have the potential to serve as novel predictive and prognostic biomarkers in patients with pancreatic cancer. In the present article, the role of miRNAs as blood biomarkers, which are associated with either radioresistance or radiation-induced changes of miRNAs in pancreatic cancer, is discussed. Furthermore, the manuscript provides own data of miRNAs identified in a pancreatic cancer mouse model as well as radiation-induced miRNA changes in the plasma of tumor-bearing mice.

Glycemic Variability Promotes Both Local Invasion and Metastatic Colonization by Pancreatic Ductal Adenocarcinoma.

Background & Aims: Although nearly half of pancreatic ductal adenocarcinoma (PDAC) patients have diabetes mellitus with episodes of hyperglycemia, its tumor microenvironment is hypoglycemic. Thus, it is crucial for PDAC cells to develop adaptive mechanisms dealing with oscillating glucose levels. So far, the biological impact of such glycemic variability on PDAC biology remains unknown. Methods: Murine PDAC cells were cultured in low- and high-glucose medium to investigate the molecular, biochemical, and metabolic influence of glycemic variability on tumor behavior. A set of in vivo functional assays including orthotopic implantation and portal and tail vein injection were used. Results were further confirmed on tissues from PDAC patients. Results: Glycemic variability has no significant effect on PDAC cell proliferation. Hypoglycemia is associated with local invasion and angiogenesis, whereas hyperglycemia promotes metastatic colonization. Increased metastatic colonization under hyperglycemia is due to increased expression of runt related transcription factor 3 (Runx3), which further activates expression of collagen, type VI, alpha 1 (Col6a1), forming a glycemic pro-metastatic pathway. Through epigenetic machinery, retinoic acid receptor beta (Rarb) expression fluctuates according to glycemic variability, acting as a critical sensor relaying the glycemic signal to Runx3/Col6a1. Moreover, the signal axis of Rarb/Runx3/Col6a1 is pharmaceutically accessible to a widely used antidiabetic substance, metformin, and Rar modulator. Finally, PDAC tissues from patients with diabetes show an increased expression of COL6A1. Conclusions: Glycemic variability promotes both local invasion and metastatic colonization of PDAC. A pro-metastatic signal axis Rarb/Runx3/Col6a1 whose activity is controlled by glycemic variability is identified. The therapeutic relevance of this pathway needs to be explored in PDAC patients, especially in those with diabetes.

Polycomb repressor complex 1 promotes gene silencing through H2AK119 mono-ubiquitination in acinar-to-ductal metaplasia and pancreatic cancer cells.

Acinar-to-ductal metaplasia (ADM) occurring in cerulein-mediated pancreatitis or in oncogenic Kras-driven pancreatic cancer development is accompanied by extensive changes in the transcriptional program. In this process, acinar cells shut down the expression of acinar specific differentiation genes and re-express genes usually found in embryonic pancreatic progenitor cells. Previous studies have demonstrated that a loss of acinar-specific transcription factors sensitizes the cells towards oncogenic transformation, ultimately resulting in cancer development. However, the mechanism behind the transcriptional silencing of acinar cell fate genes in ADM and pancreatic cancer is largely unknown. Here, we analyzed whether elevated levels of the polycomb repressor complex 1 (PRC1) components Bmi1 and Ring1b and their catalyzed histone modification H2AK119ub in ADMs and tumor cells, are responsible for the mediation of acinar gene silencing. Therefore, we performed chromatin-immunoprecipitation in in vitro generated ADMs and isolated murine tumor cells against the repressive histone modifications H3K27me3 and H2AK119ub. We established that the acinar transcription factor complex Ptf1-L is epigenetically silenced in ADMs as well as in pancreatic tumor cells. For the first time, this work presents a possible mechanism of acinar gene silencing, which is an important prerequisite in the initiation and maintenance of a dedifferentiated cell state in ADMs and tumor cells.

Periostin and tumor-stroma interactions in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-associated mortality globally. Interactions of the cancer cells with the tumor microenvironment are essential carcinogenic features for the majority of solid tumors, such as pancreatic cancer. The present study investigated the role of stromal activation in NSCLC and analyzed the surgical specimens of 93 patients by immunohistochemistry with regard to periostin (an extracellular matrix protein), α-smooth muscle actin (α-SMA; a marker of myofibroblasts) and cluster of differentiation 31 (CD31; a marker of endothelial cells), and the activated stroma index. There was a trend towards reduced overall survival for patients with high periostin expression (hazard ratio, 1.80; 95% confidence interval, 0.99-3.27; P=0.050). No significant correlations with overall survival were identified for α-SMA (P=0.930), CD31 (P=0.923), collagen (P=0.441) or the activated stroma index (P=0.706). In a multivariable analysis, the histological tumor subtype, tumor stage, lymph node involvement and resection status were independent prognostic factors in NSCLC, but none of the investigated immunohistochemical markers were prognostic factors. Thus, the tumor microenvironment and stroma activation did not prove to be of prognostic relevance for lung cancer, as it has been previously described for pancreatic cancer. Other markers of the microenvironment of NSCLC may be of higher prognostic value, pointing towards tumor-type specific effects.