DNA Polymerase Theta Plays a Critical Role in Pancreatic Cancer Development and Metastasis.

Pancreatic ductal adenocarcinoma (PDAC), due to its genomic heterogeneity and lack of effective treatment, despite decades of intensive research, will become the second leading cause of cancer-related deaths by 2030. Step-wise acquisition of mutations, due to genomic instability, is considered to drive the development of PDAC; the KRAS mutation occurs in 95 to 100% of human PDAC, and is already detectable in early premalignant lesions designated as pancreatic intraepithelial neoplasia (PanIN). This mutation is possibly the key event leading to genomic instability and PDAC development. Our study aimed to investigate the role of the error-prone DNA double-strand breaks (DSBs) repair pathway, alt-EJ, in the presence of the KRAS G12D mutation in pancreatic cancer development. Our findings show that oncogenic KRAS contributes to increasing the expression of Polθ, Lig3, and Mre11, key components of alt-EJ in both mouse and human PDAC models. We further confirm increased catalytic activity of alt-EJ in a mouse and human model of PDAC bearing the KRAS G12D mutation. Subsequently, we focused on estimating the impact of alt-EJ inactivation by polymerase theta (Polθ) deletion on pancreatic cancer development, and survival in genetically engineered mouse models (GEMMs) and cancer patients. Here, we show that even though Polθ deficiency does not fully prevent the development of pancreatic cancer, it significantly delays the onset of PanIN formation, prolongs the overall survival of experimental mice, and correlates with the overall survival of pancreatic cancer patients in the TCGA database. Our study clearly demonstrates the role of alt-EJ in the development of PDAC, and alt-EJ may be an attractive therapeutic target for pancreatic cancer patients.

Overexpression of Renin-B Induces Warburg-like Effects That Are Associated with Increased AKT/mTOR Signaling.

The classical secretory renin-a is known to be involved in angiotensin generation, thereby regulating not only blood pressure, but also promoting oxidative stress as well as apoptotic and necrotic cell death. In contrast, another cytosolic renin isoform named renin-b has been described, exerting protective effects under ischemia-related conditions in H9c2 cardiomyoblasts. Using microarray-based transcriptome analyses, we aimed to identify the signaling pathways involved in mediating cardioprotection in H9c2 cells overexpressing renin-b. By transcriptome profiling, we identified increased gene expression of several genes encoding glycolytic enzymes and glucose transporters, while the transcript levels of TCA-cycle enzymes were decreased. Complementing data from metabolic analyses revealed enhanced glucose consumption and lactate accumulation due to renin-b overexpression. Renin-b overexpression further stimulated AKT/mTOR signaling, where numerous genes involved in this pathway showed altered transcript levels. For AKT, we also detected enhanced phosphorylation levels by means of Western blotting, suggesting an activation of this kinase. Moreover, analysis of the ROS levels identified an increase in ROS accumulation in renin-b-overexpressing cells. Altogether, our data demonstrate that renin-b overexpression induces the metabolic remodeling of H9c2 cells similar to that seen under oxygen deprivation. This metabolic phenotype exerting so-called aerobic glycolysis is also known as the Warburg effect.

The Thyroid Hormone Transporter Mct8 Restricts Cathepsin-Mediated Thyroglobulin Processing in Male Mice through Thyroid Auto-Regulatory Mechanisms That Encompass Autophagy.

The thyroid gland is both a thyroid hormone (TH) generating as well as a TH responsive organ. It is hence crucial that cathepsin-mediated proteolytic cleavage of the precursor thyroglobulin is regulated and integrated with the subsequent export of TH into the blood circulation, which is enabled by TH transporters such as monocarboxylate transporters Mct8 and Mct10. Previously, we showed that cathepsin K-deficient mice exhibit the phenomenon of functional compensation through cathepsin L upregulation, which is independent of the canonical hypothalamus-pituitary-thyroid axis, thus, due to auto-regulation. Since these animals also feature enhanced Mct8 expression, we aimed to understand if TH transporters are part of the thyroid auto-regulatory mechanisms. Therefore, we analyzed phenotypic differences in thyroid function arising from combined cathepsin K and TH transporter deficiencies, i.e., in Ctsk-/-/Mct10-/-, Ctsk-/-/Mct8-/y, and Ctsk-/-/Mct8-/y/Mct10-/-. Despite the impaired TH export, thyroglobulin degradation was enhanced in the mice lacking Mct8, particularly in the triple-deficient genotype, due to increased cathepsin amounts and enhanced cysteine peptidase activities, leading to ongoing thyroglobulin proteolysis for TH liberation, eventually causing self-thyrotoxic thyroid states. The increased cathepsin amounts were a consequence of autophagy-mediated lysosomal biogenesis that is possibly triggered due to the stress accompanying intrathyroidal TH accumulation, in particular in the Ctsk-/-/Mct8-/y/Mct10-/- animals. Collectively, our data points to the notion that the absence of cathepsin K and Mct8 leads to excessive thyroglobulin degradation and TH liberation in a non-classical pathway of thyroid auto-regulation.

Angiotensin dependent and angiotensin independent protective effects of renin-b in H9c2 cells after anoxia.

The renin-angiotensin system is known to regulate blood pressure as well as water- and electrolyte balance. An activated RAS is involved in the development of hypertension and hypertension-related organ damage. Thus, inhibitors of the RAS are protective and markedly increasing the life span of patients. In contrast, renin transcripts have been discovered encoding a cytoplasmatic renin isoform, termed renin-b, which is not harmful but may be even protective. Here we demonstrate that depletion of renin-b encoding transcripts by small interference RNA decreased ATP levels and increased basal necrosis as well as apoptosis rates. Furthermore, renin-b depletion potentiated the anoxia-induced increase of necrosis rates. Vice versa, overexpression of renin-b prevented the anoxia-induced increase of caspase-mediated apoptosis rates. Besides, cells overexpressing renin-b exhibited even reduced mitochondrial mediated apoptosis rates under anoxia, when compared with normoxic conditions, as indicated by Annexin V labeling. However, whereas the protective effect of renin-b on caspase-mediated apoptosis was completely blocked by the renin inhibitor CH732, the effect on mitochondrial-mediated apoptosis was not affected by CH732 at all. From these data we conclude that renin-b overexpression mediates cardioprotective effects under anoxia with respect to mitochondrial induced apoptosis angiotensin-independently, but with respect to caspase induced apoptosis likely in an angiotensin-dependent manner.

NLRP3 Inflammasome Regulates Development of Systemic Inflammatory Response and Compensatory Anti-Inflammatory Response Syndromes in Mice With Acute Pancreatitis.

BACKGROUND & AIMS: Pancreatitis starts with primarily sterile local inflammation that induces systemic inflammatory response syndrome, followed by compensatory anti-inflammatory response syndrome (CARS). We investigated the mechanisms of these processes in mice and human serum. METHODS: We induced severe acute pancreatitis by partial duct ligation with caerulein stimulation or intraperitoneal injection of l-arginine in mice with deletion of interleukin (IL)12B, NLRP3, or IL18 and in mice given MCC950, a small molecule inhibitor of the NLRP3-inflammasome. Pancreata were collected from mice and analyzed by histology, and cytokine levels were measured in serum samples. We measured activation of adaptive immune responses in mice with pancreatitis by flow cytometry analysis of T cells (CD25 and CD69) isolated from the spleen. Differentiation of T-helper (Th1) cells, Th2 cells, and T-regulatory cells was determined by nuclear staining for TBET, GATA3, and FOXP3. We performed transcriptome analysis of mouse lymph nodes and bone marrow-derived macrophages after incubation with acini. We measured levels of cytokines in serum samples from patients with mild and severe acute pancreatitis. RESULTS: Activation of the adaptive immune response in mice was initiated by macrophage-derived, caspase 1-processed cytokines and required activation of NLRP3 (confirmed in serum samples from patients with pancreatitis). Spleen cells from mice with pancreatitis had increases in Th2 cells but not in Th1 cells. Bone marrow-derived macrophages secreted IL1B and IL18, but not IL12, after co-incubation with pancreatic acini. T-cell activation and severity of acute pancreatitis did not differ significantly between IL12B-deficient and control mice. In contrast, NLRP3- or IL18-deficient mice had reduced activation of T cells and no increase in Th2 cell-mediated responses compared with control mice. The systemic type 2 immune response was mediated by macrophage-derived cytokines of the IL1 family. Specifically, IL18 induced a Th2 cell-mediated response in the absence of IL12. MCC950 significantly reduced neutrophil infiltration, T-cell activation, and disease severity in mice. CONCLUSIONS: In mice with severe pancreatitis, we found systemic inflammatory response syndrome and compensatory anti-inflammatory response syndrome developed in parallel. Infiltrating macrophages promote inflammation and simultaneously induce a Th2 cell-mediated response via IL18. Inhibition of NLRP3 reduces systemic inflammatory response syndrome and compensatory anti-inflammatory response syndrome and might be used to treat patients with severe pancreatitis.

Cathepsin B-Mediated Activation of Trypsinogen in Endocytosing Macrophages Increases Severity of Pancreatitis in Mice.

BACKGROUND & AIMS: Acute pancreatitis is characterized by premature intracellular activation of digestive proteases within pancreatic acini and a consecutive systemic inflammatory response. We investigated how these processes interact during severe pancreatitis in mice. METHODS: Pancreatitis was induced in C57Bl/6 wild-type (control), cathepsin B (CTSB)-knockout, and cathepsin L-knockout mice by partial pancreatic duct ligation with supramaximal caerulein injection, or by repetitive supramaximal caerulein injections alone. Immune cells that infiltrated the pancreas were characterized by immunofluorescence detection of Ly6g, CD206, and CD68. Macrophages were isolated from bone marrow and incubated with bovine trypsinogen or isolated acinar cells; the macrophages were then transferred into pancreatitis control or cathepsin-knockout mice. Activities of proteases and nuclear factor (NF)-κB were determined using fluorogenic substrates and trypsin activity was blocked by nafamostat. Cytokine levels were measured using a cytometric bead array. We performed immunohistochemical analyses to detect trypsinogen, CD206, and CD68 in human chronic pancreatitis (n = 13) and acute necrotizing pancreatitis (n = 15) specimens. RESULTS: Macrophages were the predominant immune cell population that migrated into the pancreas during induction of pancreatitis in control mice. CD68-positive macrophages were found to phagocytose acinar cell components, including zymogen-containing vesicles, in pancreata from mice with pancreatitis, as well as human necrotic pancreatic tissues. Trypsinogen became activated in macrophages cultured with purified trypsinogen or co-cultured with pancreatic acini and in pancreata of mice with pancreatitis; trypsinogen activation required macrophage endocytosis and expression and activity of CTSB, and was sensitive to pH. Activation of trypsinogen in macrophages resulted in translocation of NF-kB and production of inflammatory cytokines; mice without trypsinogen activation (CTSB-knockout mice) in macrophages developed less severe pancreatitis compared with control mice. Transfer of macrophage from control mice to CTSB-knockout mice increased the severity of pancreatitis. Inhibition of trypsin activity in macrophages prevented translocation of NF-κB and production of inflammatory cytokines. CONCLUSIONS: Studying pancreatitis in mice, we found activation of digestive proteases to occur not only in acinar cells but also in macrophages that infiltrate pancreatic tissue. Activation of the proteases in macrophage occurs during endocytosis of zymogen-containing vesicles, and depends on pH and CTSB. This process involves macrophage activation via NF-κB-translocation, and contributes to systemic inflammation and severity of pancreatitis.