Glutamate has been implicated in tumorigenesis through activation of alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptors (AMPAR). However, the function of a glutamate‐to‐AMPAR ...signal in pancreatic ductal adenocarcinoma (PDAC) has remained elusive. We now show that glutamate‐mediated AMPA receptor activation increases invasion and migration of pancreatic cancer cells via activation of the classical MAPK pathway. Glutamate levels were increased in pancreatic cancer accompanied by downregulation of GluR subunits 1, 2, and 4. In pancreatic cancer precursor lesions, pancreatic intraepithelial neoplasia (PanIN), GluR1 subunit levels were strikingly and step‐wise increased but its expression was rare in PDAC. Pharmacological inhibition or RNAi‐mediated suppression of GluR1 or GluR2 did not affect cancer cell growth but significantly decreased invasion. In a K‐ras wildtype cell line, AMPA receptor activation enhanced K‐ras activity and—further downstream—phosphorylation of p38 and of p44/42. Preemptive blockade of AMPA receptors in a mouse model of pancreatic cancer inhibited tumor cell settling. AMPA receptor activation thus not only activates MAPK signalling but also directly increases activity of K‐ras. Glutamate might serve as a molecular switch that decreases the threshold of K‐ras‐induced oncogenic signalling and increases the chance of malignant transformation of pancreatic cancer precursor lesions.
The aim of this study was to investigate the expression of Krüppel-like factor 9 (KLF9) and its clinicopathological significance and prognostic value in pancreatic ductal adenocarcinoma.
A total of ...149 patients diagnosed with pancreatic ductal adenocarcinoma who underwent curative surgery were enrolled in this study. The expression of KLF9 was examined immunohistochemically. The correlation of KLF9 with clinicopathological parameters and overall survival rate of patients were analyzed.
Low expression of KLF9 was observed in 62.82% of tumors, which was related to poor differentiation (p=0.036) and vascular invasion (p=0.017). Furthermore, the overall survival of patients with low KLF9 expression was significantly shorter than that of those with high KLF9 expression (p=0.001). Multivariate analysis confirmed KLF9 as an independent prognostic factor (p=0.000).
KLF9 expression was found to be a valuable prognostic factor for patients with pancreatic ductal adenocarcinoma.
We have identified syndecan-2 as a protein potentially involved in perineural invasion of pancreatic adenocarcinoma (PDAC) cells.
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.
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.
SDC-2 is a novel (perineural) invasion-associated gene in PDAC which cooperates with K-ras to induce a more invasive phenotype.
Peroxisome Proliferator-Activated Receptor gamma (PPARγ) is a nuclear receptor demonstrated to play an important role in various biological processes. The aim of this study was to determine the ...effect of PPARγ on liver regeneration upon partial hepatectomy (PH) in mice. Mice were subjected to two-thirds PH. Before surgery, mice were either treated with the PPARγ agonist rosiglitazone, the PPARγ antagonist GW9662 alone, or with the c-met inhibitor SGX523. Liver-to-body-weight ratio, lab values, and proliferation markers were assessed. Components of the PPARγ-specific signaling pathway were identified by western blot and qRT-PCR. Our results show that liver regeneration is being inhibited by rosiglitazone and accelerated by GW9662. Inhibition of c-Met by SGX523 treatment abrogates GW9662-induced liver regeneration and hepatocyte proliferation. Hepatocyte growth factor (HGF) protein levels were significantly downregulated after rosiglitazone treatment. Activation of HGF/c-Met pathways by phosphorylation of c-Met and ERK1/2 were inhibited in rosiglitazone-treated mice. In turn, blocking phosphorylation of c-Met significantly abrogated the augmented effect of GW9662 on liver regeneration. Our data support the concept that PPARγ abrogates liver growth and hepatocellular proliferation by inhibition of the HGF/c-Met/ERK1/2 pathways. These pathways may represent potential targets in response to liver disease and could impact on the development of molecular therapies.
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.
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.
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-β 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.
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.
Type I interferon constitutes an essential component of the combinational therapy against viral disease. Acute pancreatitis is one side effect of type I interferon-based therapy, implying that ...activation of type I interferon signaling affects the homeostasis and integrity of pancreatic acinar cells. Here, we investigated the role of type I interferon signaling in pancreatic acinar cells using a caerulein-induced murine model of acute pancreatitis. Pancreas-specific ablation of interferon (alpha and beta) receptor 1 (Ifnar1) partially protected animals from caerulein-induced pancreatitis, as demonstrated by reduced tissue damage. Profiling of infiltrating immune cells revealed that this dampened tissue damage response correlated with the number of macrophages in the pancreas. Pharmacologic depletion of macrophages reversed the protective effect of Ifnar1 deficiency. Furthermore, expression of chemokine (C-C motif) ligand 2 (Ccl2), a potent factor for macrophage recruitment, was significantly increased in the Ifnar1-deficient pancreas. Thus, type I interferon signaling in pancreatic acinar cells controls pancreatic homeostasis by affecting the macrophage-mediated inflammatory response in the pancreas.
Abstract A placebo-controlled phase 3 trial demonstrated that the epidermal growth factor receptor (EGFR) inhibitor erlotinib in combination with gemcitabine was especially efficient in a pancreatic ...ductal adenocarcinoma (PDAC) subgroup of patients developing skin toxicity. However, EGFR expression was not predictive for response, and markers to characterize an erlotinib-responding PDAC group are currently missing. In this work, we observed high erlotinib IC50 values in a panel of human and murine PDAC cell lines. Using EGFR small interfering RNA, we detected that the erlotinib response was marginally influenced by EGFR. To find novel EGFR targets, we used an unbiased chemical proteomics approach for target identification and quality-controlled target affinity determination combined with quantitative mass spectrometry based on stable isotope labeling by amino acids in cell culture. In contrast to gefitinib, we observed a broad target profile of erlotinib in PDAC cells by quantitative proteomics. Six protein kinases bind to erlotinib with similar or higher affinity ( Kd = 0.09-0.358 μ M) than the EGFR ( Kd 0.434 μ M). We provide evidence that one of the novel erlotinib targets, ARG, contributes in part to the erlotinib response in a PDAC cell line. Our data show that erlotinib is a multikinase inhibitor, which can act independent of EGFR in PDAC. These findings may help to monitor future erlotinib trials in the clinic.
IntroductionPancreatic cancer is a devastating disease with an exceptionally poor prognosis. Complete resection of the primary tumour followed by adjuvant chemotherapy is the current standard ...treatment for patients with resectable disease and the only curative treatment option. However, long-term survival remains rare. Tumour cell dissemination due to manipulation during surgery may increase the rate of future metastases and local recurrence, and perioperative chemotherapy might diminish local, distant and circulating minimal residual disease. Yet, safety and feasibility of systemic chemotherapeutic treatments during pancreatic cancer resection have to be evaluated in a first instance.Methods and analysisThis is a prospective, single-centre phase I/II feasibility study to investigate the safety and tolerability of a combination of intraoperative chemotherapy and surgical resection in pancreatic cancer. Forty patients with locally confined or borderline resectable pancreatic cancer, meeting all proposed criteria will be included. Participants will receive 400 mg/m2 calcium folinate over 2 hours and 2000 mg/m2 5-fluorouracil over 48 hours, started on the day before pancreatic surgery and thus continuing during surgery. Participants will be followed until 60 days after surgery. The primary endpoint is the 30-day overall complication rate according to the Clavien-Dindo classification. Secondary endpoints comprise toxicity and treatment associated complications. Patients receiving perioperative chemotherapy will be compared with a propensity score matched contemporary control group of 70 patients with pancreatic cancer receiving the standard treatment. This trial also contains an ancillary translational study to analyse disseminated tumour cells and effects of pharmacological interventions in pancreatic cancer.Ethics and disseminationCombiCaRe has been approved by the German Federal Institute for Drugs and Medical Devices (reference number 4042787) and the Medical Ethics Committee of Heidelberg University (reference number AFmo-269/2018). The results of this trial will be presented at national and international conferences and published in peer-reviewed journals.Trial registration numberGerman Clinical Trials Register (DRKS00015766).
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.
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.
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.
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.
Display omitted
Mammalian Target of Rapamycin complex 2 (mTORC2) and its regulatory component Rapamycin-insensitive companion of mTOR (RICTOR) are increasingly recognized as important players in human cancer ...development and progression. However, the role of RICTOR in human pancreatic ductal adenocarcinoma (PDAC) is unclear so far. Here, we sought to analyze the effects of RICTOR inhibition in human pancreatic cancer cell lines in vitro and in vivo. Furthermore, RICTOR expression was determined in human PDAC samples. Results demonstrate that depletion of RICTOR with siRNA (transient knock-down) or shRNA (stable knock-down) has an inhibitory effect on tumor growth in vitro. Moreover, RICTOR inhibition led to impaired phosphorylation/activity of AGC kinases (AKT, SGK1). Interestingly, hypoxia-induced expression of hypoxia-induced factor-1α (HIF-1α) was diminished and secretion of vascular-endothelial growth factor-A (VEGF-A) was impaired upon targeting RICTOR. Stable RICTOR knock-down led to significant inhibition of tumor growth in subcutaneous and orthotopic tumor models which was accompanied by significant reduction of tumor cell proliferation. Finally, immunohistochemical analyses of 85 human PDAC samples revealed significantly poorer survival in patients with higher RICTOR expression. In conclusion, these findings provide first evidence for mTORC2/RICTOR as an attractive novel target for treatment of human PDAC.
PDF
