Implementing cell-free DNA of pancreatic cancer patient-derived organoids for personalized oncology.

One of the major challenges in using pancreatic cancer patient-derived organoids (PDOs) in precision oncology is the time from biopsy to functional characterization. This is particularly true for endoscopic ultrasound-guided fine-needle aspiration biopsies, typically resulting in specimens with limited tumor cell yield. Here, we tested conditioned media of individual PDOs for cell-free DNA to detect driver mutations already early on during the expansion process to accelerate the genetic characterization of PDOs as well as subsequent functional testing. Importantly, genetic alterations detected in the PDO supernatant, collected as early as 72 hours after biopsy, recapitulate the mutational profile of the primary tumor, indicating suitability of this approach to subject PDOs to drug testing in a reduced time frame. In addition, we demonstrated that this workflow was practicable, even in patients for whom the amount of tumor material was not sufficient for molecular characterization by established means. Together, our findings demonstrate that generating PDOs from very limited biopsy material permits molecular profiling and drug testing. With our approach, this can be achieved in a rapid and feasible fashion with broad implications in clinical practice.

Mir34a constrains pancreatic carcinogenesis.

Several studies have shown that over 70 different microRNAs are aberrantly expressed in pancreatic ductal adenocarcinoma (PDAC), affecting proliferation, apoptosis, metabolism, EMT and metastasis. The most important genetic alterations driving PDAC are a constitutive active mutation of the oncogene Kras and loss of function of the tumour suppressor Tp53 gene. Since the MicroRNA 34a (Mir34a) is a direct target of Tp53 it may critically contribute to the suppression of PDAC. Mir34a is epigenetically silenced in numerous cancers, including PDAC, where Mir34a down-regulation has been associated with poor patient prognosis. To determine whether Mir34a represents a suppressor of PDAC formation we generated an in vivo PDAC-mouse model harbouring pancreas-specific loss of Mir34a (KrasG12D; Mir34aΔ/Δ). Histological analysis of KrasG12D; Mir34aΔ/Δ mice revealed an accelerated formation of pre-neoplastic lesions and a faster PDAC development, compared to KrasG12D controls. Here we show that the accelerated phenotype is driven by an early up-regulation of the pro-inflammatory cytokines TNFA and IL6 in normal acinar cells and accompanied by the recruitment of immune cells. Our results imply that Mir34a restrains PDAC development by modulating the immune microenvironment of PDAC, thus defining Mir34a restauration as a potential therapeutic strategy for inhibition of PDAC development.

Loss of Wasl improves pancreatic cancer outcome.

Several studies have suggested an oncogenic role for the neural Wiskott-Aldrich syndrome protein (N-WASP, encoded by the Wasl gene), but thus far, little is known about its function in pancreatic ductal adenocarcinoma (PDAC). In this study, we performed in silico analysis of WASL expression in PDAC patients and found a correlation between low WASL expression and prolonged survival. To clarify the role of Wasl in pancreatic carcinogenesis, we used 2 oncogenic Kras-based PDAC mouse models with pancreas-specific Wasl deletion. In line with human data, both mouse models had an increased survival benefit due to either impaired tumor development in the presence of the tumor suppressor Trp53 or the delayed tumor progression and senescent phenotype upon genetic ablation of Trp53. Mechanistically, loss of Wasl resulted in cell-autonomous senescence through displacement of the N-WASP binding partners WASP-interacting protein (WIP) and p120ctn; vesicular accumulation of GSK3ß, as well as YAP1 and phosphorylated ß-catenin, which are components of the destruction complex; and upregulation of Cdkn1a(p21), a master regulator of senescence. Our findings, thus, indicate that Wasl functions in an oncogenic manner in PDAC by promoting the deregulation of the p120-catenin/ß-catenin/p21 pathway. Therefore, strategies to reduce N-WASP activity might improve the survival outcomes of PDAC patients.

Context-Dependent Epigenetic Regulation of Nuclear Factor of Activated T Cells 1 in Pancreatic Plasticity.

BACKGROUND & AIMS: The ability of exocrine pancreatic cells to change the cellular phenotype is required for tissue regeneration upon injury, but also contributes to their malignant transformation and tumor progression. We investigated context-dependent signaling and transcription mechanisms that determine pancreatic cell fate decisions toward regeneration and malignancy. In particular, we studied the function and regulation of the inflammatory transcription factor nuclear factor of activated T cells 1 (NFATC1) in pancreatic cell plasticity and tissue adaptation. METHODS: We analyzed cell plasticity during pancreatic regeneration and transformation in mice with pancreas-specific expression of a constitutively active form of NFATC1, or depletion of enhancer of zeste 2 homologue 2 (EZH2), in the context of wild-type or constitutively activate Kras, respectively. Acute and chronic pancreatitis were induced by intraperitoneal injection of caerulein. EZH2-dependent regulation of NFATC1 expression was studied in mouse in human pancreatic tissue and cells by immunohistochemistry, immunoblotting, and quantitative reverse transcription polymerase chain reaction. We used genetic and pharmacologic approaches of EZH2 and NFATC1 inhibition to study the consequences of pathway disruption on pancreatic morphology and function. Epigenetic modifications on the NFATC1 gene were investigated by chromatin immunoprecipitation assays. RESULTS: NFATC1 was rapidly and transiently induced in early adaptation to acinar cell injury in human samples and in mice, where it promoted acinar cell transdifferentiation and blocked proliferation of metaplastic pancreatic cells. However, in late stages of regeneration, Nfatc1 was epigenetically silenced by EZH2-dependent histone methylation, to enable acinar cell redifferentiation and prevent organ atrophy and exocrine insufficiency. In contrast, oncogenic activation of KRAS signaling in pancreatic ductal adenocarcinoma cells reversed the EZH2-dependent effects on the NFATC1 gene and was required for EZH2-mediated transcriptional activation of NFATC1. CONCLUSIONS: In studies of human and mouse pancreatic cells and tissue, we identified context-specific epigenetic regulation of NFATc1 activity as an important mechanism of pancreatic cell plasticity. Inhibitors of EZH2 might therefore interfere with oncogenic activity of NFATC1 and be used in treatment of pancreatic ductal adenocarcinoma.

Oncogenic KRas-induced Increase in Fluid-phase Endocytosis is Dependent on N-WASP and is Required for the Formation of Pancreatic Preneoplastic Lesions.

Fluid-phase endocytosis is a homeostatic process with an unknown role in tumor initiation. The driver mutation in pancreatic ductal adenocarcinoma (PDAC) is constitutively active KRasG12D, which induces neoplastic transformation of acinar cells through acinar-to-ductal metaplasia (ADM). We have previously shown that KRasG12D-induced ADM is dependent on RAC1 and EGF receptor (EGFR) by a not fully clarified mechanism. Using three-dimensional mouse and human acinar tissue cultures and genetically engineered mouse models, we provide evidence that (i) KRasG12D leads to EGFR-dependent sustained fluid-phase endocytosis (FPE) during acinar metaplasia; (ii) variations in plasma membrane tension increase FPE and lead to ADM in vitro independently of EGFR; and (iii) that RAC1 regulates ADM formation partially through actin-dependent regulation of FPE. In addition, mice with a pancreas-specific deletion of the Neural-Wiskott-Aldrich syndrome protein (N-WASP), a regulator of F-actin, have reduced FPE and impaired ADM emphasizing the in vivo relevance of our findings. This work defines a new role of FPE as a tumor initiating mechanism.

Hes1 Controls Exocrine Cell Plasticity and Restricts Development of Pancreatic Ductal Adenocarcinoma in a Mouse Model.

Perturbation of pancreatic acinar cell state can lead to acinar-to-ductal metaplasia (ADM), a precursor lesion to the development of pancreatic ductal adenocarcinoma (PDAC). In the pancreas, Notch signaling is active both during development and in adult cellular differentiation processes. Hes1, a key downstream target of the Notch signaling pathway, is expressed in the centroacinar compartment of the adult pancreas as well as in both preneoplastic and malignant lesions. In this study, we used a murine genetic in vivo approach to ablate Hes1 in pancreatic progenitor cells (Ptf1a+/Cre; Hes1fl/fl). Using this model, we studied the role of Hes1 in both acinar cell plasticity and pancreatic regeneration after caerulein-induced pancreatitis and in KrasG12D-driven PDAC development. We show that, although pancreatic development is not perturbed on the deletion of Hes1, terminal acinar differentiation in the adult pancreas is compromised. Moreover, the loss of Hes1 leads to the impaired regeneration of the exocrine compartment, accelerated fatty metaplasia, and persistent ADM after acute caerulein-induced pancreatitis. In KrasG12D-driven carcinogenesis, Hes1 ablation resulted in increased ADM, decreased formation of high-grade pancreatic intraepithelial neoplasias, and accelerated development of PDAC with shortened survival time. In conclusion, Hes1 plays a key role in acinar cell integrity and plasticity on cellular insults. Furthermore, Hes1 is an essential component of the pancreatic intraepithelial neoplasias-to-PDAC route in KrasG12D-driven mouse pancreatic carcinogenesis.

Membranous CD24 drives the epithelial phenotype of pancreatic cancer.

Surface CD24 has previously been described, together with CD44 and ESA, for the characterization of putative cancer stem cells in pancreatic ductal adenocarcinoma (PDAC), the most fatal of all solid tumors. CD24 has a variety of biological functions including the regulation of invasiveness and cell proliferation, depending on the tumor entity and subcellular localization. Genetically engineered mouse models (GEMM) expressing oncogenic KrasG12D recapitulate the human disease and develop PDAC. In this study we investigate the function of CD24 using GEMM of endogenous PDAC and a model of cerulein-induced acute pancreatitis. We found that (i) CD24 expression was upregulated in murine and human PDAC and during acute pancreatitis (ii) CD24 was expressed exclusively in differentiated PDAC, whereas CD24 absence was associated with undifferentiated tumors and (iii) membranous CD24 expression determines tumor subpopulations with an epithelial phenotype in grafted models. In addition, we show that CD24 protein is stabilized in response to WNT activation and that overexpression of CD24 in pancreatic cancer cells upregulated ß-catenin expression augmenting an epithelial, non-metastatic signature. Our results support a positive feedback model according to which (i) WNT activation and subsequent ß-catenin dephosphorylation stabilize CD24 protein expression, and (ii) sustained CD24 expression upregulates ß-catenin expression. Eventually, membranous CD24 augments the epithelial phenotype of pancreatic tumors. Thus we link the WNT/ß-catenin pathway with the regulation of CD24 in the context of PDAC differentiation.

EGF receptor is required for KRAS-induced pancreatic tumorigenesis.

Initiation of pancreatic ductal adenocarcinoma (PDA) is definitively linked to activating mutations in the KRAS oncogene. However, PDA mouse models show that mutant Kras expression early in development gives rise to a normal pancreas, with tumors forming only after a long latency or pancreatitis induction. Here, we show that oncogenic KRAS upregulates endogenous EGFR expression and activation, the latter being dependent on the EGFR ligand sheddase, ADAM17. Genetic ablation or pharmacological inhibition of EGFR or ADAM17 effectively eliminates KRAS-driven tumorigenesis in vivo. Without EGFR activity, active RAS levels are not sufficient to induce robust MEK/ERK activity, a requirement for epithelial transformation.

Early requirement of Rac1 in a mouse model of pancreatic cancer.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease without effective chemopreventive or therapeutic approaches. Although the role of oncogenic Kras in initiating development of PDAC is well established, downstream targets of aberrant Ras signaling are poorly understood. Acinar-ductal metaplasia (ADM) appears to be an important prerequisite for development of pancreatic intraepithelial neoplasia (PanIN), a common precursor to PDAC. RAS-related C3 botulinum substrate 1 (Rac1), which controls actin reorganization, can be activated by Ras, is up-regulated in several human cancers, and is required for cerulein-induced morphologic changes in acini. We investigated effects of loss of Rac1 in Kras-induced pancreatic carcinogenesis in mice. METHODS: Using a Cre/lox approach, we deleted Rac1 from pancreatic progenitor cells in different mouse models of PDAC and in mice with cerulein-induced acute pancreatitis. Acinar epithelial explants of mutant mice were used to investigate the role of Rac1 in vitro. RESULTS: Rac1 expression increased in mouse and human pancreatic tumors, particularly in the stroma. Deletion of Rac1 in Kras(G12D)-induced PDAC in mice reduced formation of ADM, PanIN, and tumors and significantly prolonged survival. Pancreatic epithelial metaplasia was accompanied by apical-basolateral redistribution of F-actin, along with basal expression of Rac1. Acinar epithelial explants that lacked Rac1 or that were incubated with inhibitors of actin polymerization had a reduced ability to undergo ADM in 3-dimensional cultures. CONCLUSIONS: In mice, Rac1 is required for early metaplastic changes and neoplasia-associated actin rearrangements in development of pancreatic cancer. Rac1 might be developed as a diagnostic marker or therapeutic target for PDAC.

Notch signaling is required for exocrine regeneration after acute pancreatitis.

BACKGROUND & AIMS: The mechanisms for tissue regeneration and renewal after acute pancreatitis are not well understood but may involve activation of Notch signaling. To study the effect of Notch signaling ablation during acute experimental pancreatitis, we used a chemical and genetic approach to ablate Notch signaling in cerulein-induced pancreatitis in mice. METHODS: Acute pancreatitis was induced by cerulein treatment in mice treated with the gamma-secretase inhibitor dibenzazepine or in conditional Notch1 knockout mice. Mice were characterized using immunohistologic, biochemical, and molecular methods. To investigate Notch and beta-catenin interaction, acinar 266-6 cells were analyzed using transfection and biochemical assays. RESULTS: Loss of Notch signaling results in impaired regeneration after acute pancreatitis with fewer mature acinar cells in dibenzazepine-treated and Notch1-deficient mice in the regenerative phase 3 days after induction. beta-catenin expression was increased and prolonged during exocrine regeneration. Crosstalk between Notch and beta-catenin-mediated signaling was identified, with Notch1-IC inhibiting beta-catenin-mediated transcriptional activity. This inhibition was dependent on a functional RAM domain. CONCLUSIONS: Inhibition of Notch signaling in vivo leads to impaired regeneration of the exocrine pancreas after acute pancreatitis. Our results suggest an interaction of Notch and Wnt signaling in pancreatic acinar cells, providing evidence for a role of these pathways in the regulation of the maturation process of acinar cells.

Concomitant pancreatic activation of Kras(G12D) and Tgfa results in cystic papillary neoplasms reminiscent of human IPMN.

Growth factors have been implicated in pancreatic carcinogenesis. In this study we analyzed the effect of Tgfa overexpression in addition to mutant Kras(G12D) by crossing Elastase-Tgfa mice with p48(+/Cre);Kras(+/LSL-G12D) mice. We show that concomitant expression of TGFalpha and Kras(G12D) accelerates the progression of mPanIN lesions to metastatic pancreatic cancer and leads to the development of cystic papillary lesions resembling human intraductal papillary mucinous neoplasms (IPMN). Microarray data in mice revealed an IPMN signature and IPMNs expressed MUC1 and MUC5AC but not MUC2, similar to human pancreatobiliary IPMNs. Invasive ductal adenocarcinoma developed from PanINs and IPMNs, suggesting precursor lines for both lesion types in this model. In conclusion, Egfr signaling in synergy with oncogenic Kras may be a prerequisite for IPMN development and progression to pancreatic cancer.

Human guanylate binding protein-1 is a secreted GTPase present in increased concentrations in the cerebrospinal fluid of patients with bacterial meningitis.

Interferon-gamma-induced GTPases are key to the protective immunity against microbial and viral pathogens. As yet, the cell interior has been regarded as the exclusive residence of these proteins. Here we show that a member of this group, human guanylate binding protein-1 (hGBP-1), is secreted from cells. Secretion occurred in the absence of a leader peptide via a nonclassical, likely ABC transporter-dependent, pathway, was independent of hGBP-1 GTPase activity and isoprenylation, and did not require additional interferon-gamma-induced factors. Interestingly, hGBP-1 was only secreted from endothelial cells but not from any of the nine different cell types tested. Clinically most important was the detection of significantly (P<0.001, Mann-Whitney U-test) increased hGBP-1 concentrations in the cerebrospinal fluid of patients with bacterial meningitis (n=32) as compared to control patients (n=74). In this first report of a secreted GTPase, we demonstrate that secreted hGBP-1 may be a useful surrogate marker for diagnosis of bacterial meningitis.

Nuclear factor-kappaB motif and interferon-alpha-stimulated response element co-operate in the activation of guanylate-binding protein-1 expression by inflammatory cytokines in endothelial cells.

The large GTPase GBP-1 (guanylate-binding protein-1) is a major IFN-gamma (interferon-gamma)-induced protein with potent anti-angiogenic activity in endothelial cells. An ISRE (IFN-alpha-stimulated response element) is necessary and sufficient for the induction of GBP-1 expression by IFN-gamma. Recently, we have shown that in vivo GBP-1 expression is strongly endothelial-cell-associated and is, in addition to IFN-gamma, also activated by interleukin-1beta and tumour necrosis factor-alpha, both in vitro and in vivo [Lubeseder-Martellato, Guenzi, Jörg, Töpolt, Naschberger, Kremmer, Zietz, Tschachler, Hutzler, Schwemmle et al. (2002) Am. J. Pathol. 161, 1749-1759; Guenzi, Töpolt, Cornali, Lubeseder-Martellato, Jörg, Matzen, Zietz, Kremmer, Nappi, Schwemmle et al. (2001) EMBO J. 20, 5568-5577]. In the present study, we identified a NF-kappaB (nuclear factor kappaB)-binding motif that, together with ISRE, is required for the induction of GBP-1 expression by interleukin-1beta and tumour necrosis factor-alpha. Deactivation of the NF-kappaB motif reduced the additive effects of combinations of these cytokines with IFN-gamma by more than 50%. Importantly, NF-kappaB p50 rather than p65 activated the GBP-1 promoter. The NF-kappaB motif and ISRE were detected in an almost identical spatial organization, as in the GBP-1 promoter, in the promoter regions of various inflammation-associated genes. Therefore both motifs may constitute a cooperative inflammatory cytokine response module that regulates GBP-1 expression. Our findings may open new perspectives for the use of NF-kappaB inhibitors to support angiogenesis in inflammatory diseases including ischaemia.

The guanylate binding protein-1 GTPase controls the invasive and angiogenic capability of endothelial cells through inhibition of MMP-1 expression.

Expression of the large GTPase guanylate binding protein-1 (GBP-1) is induced by inflammatory cytokines (ICs) in endothelial cells (ECs), and the helical domain of the molecule mediates the repression of EC proliferation by ICs. Here we show that the expression of GBP-1 and of the matrix metalloproteinase-1 (MMP-1) are inversely related in vitro and in vivo, and that GBP-1 selectively inhibits the expression of MMP-1 in ECs, but not the expression of other proteases. The GTPase activity of GBP-1 was necessary for this effect, which inhibited invasiveness and tube-forming capability of ECs in three-dimensional collagen-I matrices. A GTPase-deficient mutant (D184N-GBP-1) operated as a transdominant inhibitor of wild-type GBP-1 and rescued MMP-1 expression in the presence of ICs. Expression of D184N-GBP-1, as well as paracrine supplementation of MMP-1, restored the tube-forming capability of ECs in the presence of wild-type GBP-1. The latter finding indicated that the inhibition of capillary formation is specifically due to the repression of MMP-1 expression by GBP-1, and is not affected by the anti-proliferative activity of the helical domain of GBP-1. These findings substantiate the role of GBP-1 as a major regulator of the anti-angiogenic response of ECs to ICs.

Guanylate-binding protein-1 expression is selectively induced by inflammatory cytokines and is an activation marker of endothelial cells during inflammatory diseases.

During angiogenesis and inflammatory processes, endothelial cells acquire different activation phenotypes, whose identification may help in understanding the complex network of angiogenic and inflammatory interactions in vivo. To this goal we investigated the expression of the human guanylate-binding protein (GBP)-1 that is highly induced by inflammatory cytokines (ICs) and, therefore, may characterize IC-activated cells. Using a new rat monoclonal antibody raised against GBP-1, we show that GBP-1 is a cytoplasmic protein and that its expression in endothelial cells is selectively induced by interferon-gamma, interleukin-1alpha, interleukin-1beta, or tumor necrosis factor-alpha, but not by other cytokines, chemokines, or growth factors. Moreover, we found that GBP-1 expression is highly associated with vascular endothelial cells as confirmed by the simultaneous detection of GBP-1 and the endothelial cell-associated marker CD31 in a broad range of human tissues. Notably, GBP-1 expression was undetectable in the skin, but it was highly induced in vessels of skin diseases with a high-inflammatory component including psoriasis, adverse drug reactions, and Kaposi's sarcoma. These results indicate that GBP-1 is a novel cellular activation marker that characterizes the IC-activated phenotype of endothelial cells.