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In this study, our aim is to assess the role played by autophagy and its inhibition in the different PDAC cellular compartments, and its involvement in chemo-resistance using primary human pancreatic cancer-derived cells (PCC) and Cancer Associated Fibroblasts (CAF). Autophagy flux, as measured by LC3-I and -II in the presence of Chloroquine, showed a variable level in PCC and CAFs. We found no correlation between autophagy level and degree of tumor differentiation. Association of Chloroquine with gemcitabine, 5FU, oxaliplatin, irinotecan and docetaxel revealed that its effect on survival is cell- and drug-dependent in vitro and in vivo. In addition, we demonstrated that autophagy in CAFs can play an important role in sensitizing PDAC to anticancer treatments since its inhibition increased the resistance of PCCs to gemcitabine. In conclusion, this work clearly shows a heterogeneity in the effect of Chloroquine and highlights a role of CAFs autophagy in sensitizing tumors to treatments. It also reveals that the role of autophagy is more complex than expected in PDAC as well as its sensitivity to treatments. Fil: Molejon, Maria Ines. Institut National de la Santé et de la Recherche Médicale; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Ciencias de la Tierra y Ambientales de La Pampa. Universidad Nacional de La Pampa. Facultad de Ciencias Exactas y Naturales. Instituto de Ciencias de la Tierra y Ambientales de La Pampa; Argentina Fil: Swayden, Mirna. Institut National de la Santé et de la Recherche Médicale; Francia Fil: Fanale, Daniele. 3 Department of Surgical, Oncological and Oral Sciences, Section of Medical Oncology, University of Palermo, 90127 Palermo, Italy.; Argentina. University of Palermo. Department of Surgical, Oncological and Oral Sciences; Italia Fil: Bintz, Jennifer. Institut National de la Santé et de la Recherche Médicale; Francia Fil: Gayet, Odile. Institut National de la Santé et de la Recherche Médicale; Francia Fil: Soubeyran, Philippe. Institut National de la Santé et de la Recherche Médicale; Francia Fil: Iovanna, Juan Lucio. Institut National de la Santé et de la Recherche Médicale; Francia

Pancreatic ductal adenocarcinoma (PDAC) offers an optimal model for discovering >druggable> molecular pathways that participate in inflammation-associated cancer development. Chronic pancreatitis, a common prolonged inflammatory disease, behaves as a well-known premalignant condition that contributes to PDAC development. Although the mechanisms underlying the pancreatitis-to-cancer transition remain to be fully elucidated, emerging evidence supports the hypothesis that the actions of proinflammatory mediators on cells harboring Kras mutations promote neoplastic transformation. Recent elegant studies demonstrated that the IL17 pathway mediates this phenomenon and can be targeted with antibodies, but the downstream mechanisms by which IL17 functions during this transition are currently unclear. In this study, we demonstrate that IL17 induces the expression of REG3β, a wellknown mediator of pancreatitis, during acinar-to-ductal metaplasia and in early pancreatic intraepithelial neoplasia (PanIN) lesions. Furthermore, we found that REG3β promotes cell growth and decreases sensitivity to cell death through activation of the gp130-JAK2-STAT3-dependent pathway. Genetic inactivation of REG3β in the context of oncogenic Kras-driven PDAC resulted in reduced PanIN formation, an effect that could be rescued by administration of exogenous REG3β. Taken together, our findings provide mechanistic insight into the pathways underlying inflammation-associated pancreatic cancer, revealing a dual and contextual pathophysiologic role for REG3β during pancreatitis and PDAC initiation., This work was supported by La Ligue Contre le Cancer, INCa, Canceropole PACA, DGOS (labellisation SIRIC), and INSERM to J.L. Iovanna; NIH grants DK52913, the Mayo Clinic Center for Cell Signaling in Gastroenterology (P30DK084567), and the Mayo Foundation to R. Urrutia; by Fraternal Order of Eagles Cancer Award to G. Lomberk; and the FIS grant from Instituto de Salud Carlos III (PI13/01224) to E. Folch-Puy

We have previously demonstrated a crucial role of nuclear protein 1 (NUPR1) in tumor development and progression. In this work, we report the functional characterization of a novel Nupr1-like isoform (NUPR1L) and its functional interaction with the protumoral factor NUPR1. Through the use of primary sequence analysis, threading, and homology-based molecular modeling, as well as expression and immunolocalization, studies reveal that NUPR1L displays properties, which are similar to member of the HMG-like family of chromatin regulators, including its ability to translocate to the cell nucleus and bind to DNA. Analysis of the NUPR1L promoter showed the presence of two p53-response elements at positions −37 and −7, respectively. Experiments using reporter assays combined with site-directed mutagenesis and using cells with controllable p53 expression demonstrate that both of these sequences are responsible for the regulation of NUPR1L expression by p53. Congruently, NUPR1L gene expression is activated in response to DNA damage induced by oxaliplatin treatment or cell cycle arrest induced by serum starvation, two well-validated methods to achieve p53 activation. Interestingly, expression of NUPR1L downregulates the expression of NUPR1, its closely related protumoral isoform, by a mechanism that involves the inhibition of its promoter activity. At the cellular level, overexpression of NUPR1L induces G1 cell cycle arrest and a decrease in their cell viability, an effect that is mediated, at least in part, by downregulating NUPR1 expression. Combined, these experiments constitute the first functional characterization of NUPR1L as a new p53-induced gene, which negatively regulates the protumoral factor NUPR1. J. Cell. Physiol. 230: 2936–2950, 2015. © 2015 Wiley Periodicals, Inc.

Pancreatic ductal adenocarcinoma (PDAC) has the lowest survival rate of all cancers and shows remarkable resistance to cell stress. Nuclear protein 1 (Nupr1), which mediates stress response in the pancreas, is frequently upregulated in pancreatic cancer. Here, we report that Nupr1 plays an essential role in pancreatic tumorigenesis. In a mouse model of pancreatic cancer with constitutively expressed oncogenic Kras(G12D), we found that loss of Nupr1 protected from the development of pancreatic intraepithelial neoplasias (PanINs). Further, in cultured pancreatic cells, nutrient deprivation activated Nupr1 expression, which we found to be required for cell survival. We found that Nupr1 protected cells from stress-induced death by inhibiting apoptosis through a pathway dependent on transcription factor RelB and immediate early response 3 (IER3). NUPR1, RELB, and IER3 proteins were coexpressed in mouse PanINs from Kras(G12D)-expressing pancreas. Moreover, pancreas-specific deletion of Relb in a Kras(G12D) background resulted in delayed in PanIN development associated with a lack of IER3 expression. Thus, efficient PanIN formation was dependent on the expression of Nupr1 and Relb, with likely involvement of IER3. Finally, in patients with PDAC, expression of NUPR1, RELB, and IER3 was significantly correlated with a poor prognosis. Cumulatively, these results reveal a NUPR1/RELB/IER3 stress-related pathway that is required for oncogenic Kras(G12D)-dependent transformation of the pancreas.

Pancreatic ductal adenocarcinoma (PDAC) has the highest mortality rate of all cancers and the lowest overall survival rate of less than 5% at 5 years. The incidence of PDAC almost equals its mortality rate and is increasing every year, with more than 10,000 predicted new cases in France, 38,000 in the United States and 65,000 in Europe [1]. PDAC is characterized by a stepwise progression of ductal-like cells into malignant adenocarcinoma. This starts with the acinar-ductal metaplasia (ADM), a reprogramming event that induces transdifferentiation to a duct-like phenotype, and results in the pancreatic intraepithelial neoplasia (PanIN) 1A, 1B, 2 and 3 precancerous lesions [2], the latter corresponding to the in situ PDAC. The earliest activating mutation occurring in human PDAC is on the GTPase Kras oncogene. Previously, we described the importance of the pancreatitis-activated protein NUPR1, which activates RELB during the abovementioned process of transformation [3]. We went on to report that this NUPR1- RELB pathway affects the expression of genes encoding proteins that can serve as effectors of their function in this process, such as the immediate early response 3 (IER3) gene defined as essential in PanIN and PDAC development [4]. IER3 is a stress-inducible gene involved in cellular response to stress. The protein it encodes has multiple actions in signaling pathways associated with the activation of ERK, itself implicating a cascade of phosphorylation events initiated by the stimulation of RAS and leading to phosphorylation of ERK1/2. The ERK-dependent pathway plays a major role in diverse cellular processes, including proliferation, differentiation and survival, and its activity is critical for Kras-dependent PDAC development [5]. Considering that ERK1/2 is mostly inactivated by PP2A, targeting this phosphatase appears an obvious mechanism for regulating Kras oncogenic function. PP2A is a multifarious complex composed of catalytic, scaffold and regulatory subunits which have been divided into three families named PR55, B56 and PR72. In addition, each subunit consists of several members [6]. Using the pancreas as a model, we showed that genetic inactivation of IER3 in Kras oncogene-driven PanIN to PDAC formation leads to a strong delay in PanIN and consequently also in PDAC development. This finding demonstrated, as expected, that IER3 is an essential contributor to the ADM, PanIN, and PDAC development induced by oncogenic Kras. Mechanistically, we demonstrated that all processes resulting in Nupr1- dependent IER3 overexpression led to a sustained activation of ERK1/2. Indeed, we showed that IER3 interacts directly with the regulatory subunit of PP2A - B56 - resulting in a sustained phosphorylation of ERK1/2, consequently facilitating the Kras-mediated transformation [4]. Interestingly, IER3 expression levels, easily detectable during the early transformation processes such as ADM and early PanINs, appeared to decrease or even disappear in late PanIN and PDAC. This would suggest a necessity for IER3 during early steps of tumor transformation rather than later steps of pancreatic carcinogenesis. This corroborates the Kras-addiction observed during the early but not the later PDAC stages [7]. In this way we demonstrated that IER3 expression can promote ERK1/2 phosphorylation in PDAC cells but has no influence over tumor growth in xenografts. This explains the inefficiency of anticancer strategies that target ERK1/2 phosphorylation in PDAC treatment. In conclusion our data indicate that NUPR1 regulates PDAC development through the activation of ERK1/2 phosphorylation in an IER3-dependent manner. IER3 is therefore an important molecule that, by stabilizing ERK1/2 activation, enhances Kras-oncogenic effects and thus contributes towards driving PDAC formation. Since Kras is found to be mutated in several cancers we consider the possibility that this original molecular mechanism may not be pancreas-specific.

We have elucidated a novel mechanism through which the autophagy-specific class III phosphatidylinositol 3-kinase (PtdIns3K) complex can be recruited to the PAS in mammalian cells, through the interaction between BECN1 and the vacuole membrane protein 1 (VMP1), an integral autophagosomal membrane protein. This interaction involves the binding between the C-terminal 20 amino acids of the VMP1 hydrophilic domain, which we have named the VMP1 autophagy-related domain (VMP1-AtgD), and the BH3 domain of BECN1. The association between these two proteins allows the formation of the autophagy-specific PtdIns3K complex, which activity favors the generation of phosphatidylinositol-3-phosphate (PtdIns3P) and the subsequent association of the autophagy-related (ATG) proteins, including ATG16L1, with the phagophore membranes. Therefore, VMP1 regulates the PtdIns3K activity on the phagophore membrane through its interaction with BECN1. Our data provide a novel model describing one of the key steps in phagophore assembly site (PAS) formation and autophagy regulation, and positions VMP1 as a new interactor of the autophagy-specific PtdIns3K complex in mammalian cells. Fil: Molejon, Maria Ines. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Fisiopatología; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular; Argentina; Fil: Ropolo, Alejandro Javier. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Fisiopatología; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular; Argentina; Fil: Vaccaro, Maria Ines. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Fisiopatología; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular; Argentina

The Vacuole Membrane Protein 1 -VMP1- is a pancreatitis-associated transmembrane protein whose expression triggers autophagy in several human diseases. In the current study, we unveil the mechanism through which this protein induces autophagosome formation in mammalian cells. We show that VMP1 autophagy-related function requires its 20-aminoacid C-terminus hydrophilic domain (VMP1-AtgD). This is achieved through its direct binding to the BH3 motif of Beclin 1 leading to the formation of a complex with the Class III phosphatidylinositol-3 kinase (PI3K) hVps34, a key positive regulator of autophagy, at the site where autophagosomes are generated. This interaction also concomitantly promotes the dissociation of Bcl-2, an autophagy inhibitor, from Beclin 1. Moreover, we show that the VMP1-Beclin 1-hVps34 complex favors the association of Atg16L1 and LC3 with the autophagosomal membranes. Collectively, these findings reveal that VMP1 expression recruits and activates the Class III PI3K complex at the site of autophagosome formation during mammalian autophagy.

Fil: Loncle, Celine. Aix-Marseille Universite; Francia. Centre National de la Recherche Scientifique; Francia

Autophagy is an evolutionarily conserved degradation process of cytoplasmic cellular constituents. It has been suggested that autophagy plays a role in tumor promotion and progression downstream oncogenic pathways; however, the molecular mechanisms underlying this phenomenon have not been elucidated. Here, we provide both in vitro and in vivo evidence of a novel signaling pathway whereby the oncogene KRAS induces the expression of VMP1, a molecule needed for the formation of the authophagosome and capable of inducing autophagy, even under nutrient-replete conditions. RNAi experiments demonstrated that KRAS requires VMP1 to induce autophagy. Analysis of the mechanisms identified GLI3, a transcription factor regulated by the Hedgehog pathway, as an effector of KRAS signaling. GLI3 regulates autophagy as well as the expression and promoter activity of VMP1 in a Hedgehog-independent manner. Chromatin immunoprecipitation assays demonstrated that GLI3 binds to the VMP1 promoter and complexes with the histone acetyltransferase p300 to regulate promoter activity. Knockdown of p300 impaired KRAS- and GLI3-induced activation of this promoter. Finally, we identified the PI3K-AKT1 pathway as the signaling pathway mediating the expression and promoter activity of VMP1 upstream of the GLI3-p300 complex. Together, these data provide evidence of a new regulatory mechanism involved in autophagy that integrates this cellular process into the molecular network of events regulating oncogene-induced autophagy.

Autophagy is an evolutionarily preserved degradation process of cytoplasmic cellular constituents and plays important physiological roles in human health and disease. It has been proposed that autophagy plays an important role both in tumor progression and in promotion of cancer cell death, although the molecular mechanisms responsible for this dual action of autophagy in cancer have not been elucidated. Pancreatic ductal adenocarcinoma is one of the most aggressive human malignancies with 2–3% five-year survival rate. Its poor prognosis has been attributed to the lack of specific symptoms and early detection tools, and its relatively refractory to traditional cytotoxic agents and radiotherapy. Experimental evidence pointed at autophagy as a pancreatic cancer cell mechanism to survive under adverse environmental conditions, or as a defective programmed cell death mechanism that favors pancreatic cancer cell resistance to treatment. Here, we consider several phenotypical alterations that have been related to increase or decrease the autophagic process in pancreatic tumor cells. We specially review autophagy as a cell death mechanism in response to chemotherapeutic drugs. Fil: Ropolo, Alejandro Javier. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Fisiopatología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina Fil: Bagnes, Claudia I.. Universidad de Buenos Aires. Facultad de Medicina; Argentina Fil: Molejon, Maria Ines. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Fisiopatología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina Fil: Lo Ré, Andrea Emilia. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Fisiopatología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina Fil: Boggio, Veronica. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Fisiopatología; Argentina Fil: Gonzalez, Claudio D.. Universidad de Buenos Aires. Facultad de Medicina; Argentina Fil: Vaccaro, Maria Ines. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Fisiopatología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina

Autophagy has recently elicited significant attention as a mechanism that either protects or promotes cell death, although different autophagy pathways, and the cellular context in which they occur, remain to be elucidated. We report a thorough cellular and biochemical characterization of a novel selective autophagy that works as a protective cell response. This new selective autophagy is activated in pancreatic acinar cells during pancreatitis-induced vesicular transport alteration to sequester and degrade potentially deleterious activated zymogen granules. We have coined the term “zymophagy” to refer to this process. The autophagy-related protein VMP1, the ubiquitin-protease USP9x, and the ubiquitin-binding protein p62 mediate zymophagy. Moreover, VMP1 interacts with USP9x, indicating that there is a close cooperation between the autophagy pathway and the ubiquitin recognition machinery required for selective autophagosome formation. Zymophagy is activated by experimental pancreatitis in genetically engineered mice and cultured pancreatic acinar cells and by acute pancreatitis in humans. Furthermore, zymophagy has pathophysiological relevance by controlling pancreatitis-induced intracellular zymogen activation and helping to prevent cell death. Together, these data reveal a novel selective form of autophagy mediated by the VMP1-USP9x-p62 pathway, as a cellular protective response. Fil: Grasso, Daniel Hector. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Fisiopatología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Ropolo, Alejandro Javier. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Fisiopatología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Lo Ré, Andrea Emilia. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Fisiopatología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Boggio, Verónica. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Fisiopatología; Argentina Fil: Molejon, Maria Ines. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Fisiopatología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Iovanna, Juan L.. Inserm; Francia Fil: Gonzalez, Claudio D.. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Fisiopatología; Argentina Fil: Urrutia, Raúl. Mayo Clinic; Estados Unidos Fil: Vaccaro, Maria Ines. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Fisiopatología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

s / Pancreatology 14 (2014) S1eS129 S21 remodelling and ADM formation was evaluated in vitro using a 3D culture system of pancreatic explants from mice expressing the activating Kras (G12D) mutation. Results: Lack of 5-HT in vivo or inhibition of its uptake 5-HT in vitro resulted in increased cell-cell adhesion. In both cerulein regimens, WT mice developed typical ADM lesions four days after the induction of pancreatitis. Conversely, TPH-1-/mice were characterized by stronger cell-cell adhesion and showed smaller ADM foci with incomplete acinar cell trans-differentiation. In vitro, inhibition of intracellular 5-HT up-take completely prevented the formation of ADMs in Kras explants. Conclusion: These data indicate that cytoskeletal remodeling mediated by intracellular 5-HT is critical for the formation of ADM lesions. Thus, inhibition of 5-HT uptake may constitute a novel pharmacological intervention to interfere with ADM formation and prevent the spreading of pancreatic pre-malignant lesions.