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Cancer-associated fibroblasts (CAFs) are orchestrators of the pancreatic ductal adenocarcinoma (PDAC) microenvironment. Previously we described four CAF subtypes with specific molecular and functional features. Here, we have refined our CAF subtype signatures using RNAseq and immunostaining with the goal of defining bioinformatically the phenotypic stromal and tumor epithelial states associated with CAF diversity. We used primary CAF cultures grown from patient PDAC tumors, human data sets (in-house and public, including single-cell analyses), genetically engineered mouse PDAC tissues, and patient-derived xenografts (PDX) grown in mice. We found that CAF subtype RNAseq signatures correlated with immunostaining. Tumors rich in periostin-positive CAFs were significantly associated with shorter overall survival of patients. Periostin-positive CAFs were characterized by high proliferation and protein synthesis rates and low α-smooth muscle actin expression and were found in peri-/pre-tumoral areas. They were associated with highly cellular tumors and with macrophage infiltrates. Podoplanin-positive CAFs were associated with immune-related signatures and recruitment of dendritic cells. Importantly, we showed that the combination of periostin-positive CAFs and podoplanin-positive CAFs was associated with specific tumor microenvironment features in terms of stromal abundance and immune cell infiltrates. Podoplanin-positive CAFs identified an inflammatory CAF (iCAF)-like subset, whereas periostin-positive CAFs were not correlated with the published myofibroblastic CAF (myCAF)/iCAF classification. Taken together, these results suggest that a periostin-positive CAF is an early, activated CAF, associated with aggressive tumors, whereas a podoplanin-positive CAF is associated with an immune-related phenotype. These two subpopulations cooperate to define specific tumor microenvironment and patient prognosis and are of putative interest for future therapeutic stratification of patients. © 2022 The Authors. The Journal of Pathology published by John WileySons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

mRNA translation is a key mechanism for cancer cell proliferation and stress adaptation. Regulation of this machinery implicates upstream pathways such as PI3K/AKT/mTOR, RAS/MEK/ERK and the integrated stress response (ISR), principally coordinating the translation initiation step. During the last decade, dysregulation of the mRNA translation process in pancreatic cancer has been widely reported, and shown to critically impact on cancer initiation, development and survival. This includes translation dysregulation of mRNAs encoding oncogenes and tumor suppressors. Hence, cancer cells survive a stressful microenvironment through a flexible regulation of translation initiation for rapid adaptation. The ISR pathway has an important role in chemoresistance and shows high potential therapeutic interest. Despite the numerous translational alterations reported in pancreatic cancer, their consequences are greatly underestimated. In this review, we summarize the different translation dysregulations described in pancreatic cancer, which make it invulnerable, as well as the latest drug discoveries bringing a glimmer of hope.

International audience; Background: The pancreatic ductal adenocarcinoma (PDAC) microenvironment is highly fibrotic and hypoxic, with poor immune cell infiltration. Recently, we showed that nucleolin (NCL) inhibition normalizes tumour vessels and impairs PDAC growth.Methods: Immunocompetent mouse models of PDAC were treated by the pseudopeptide N6L,which selectively inhibits NCL. Tumour-infiltrating immune cells and changes in the tumour microenvironment were analysed.Results: N6L reduced the proportion of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) and increased tumour-infiltrated T lymphocytes (TILs) with an activated phenotype. Low-dose anti-VEGFR2 treatment normalized PDAC vessels but did not modulate the immune suppressive microenvironment. RNAseq analysis of N6L-treated PDAC tumours revealed a reduction of cancer-associated fibroblast (CAF) expansion in vivo and in vitro. Notably, N6L treatment decreased IL-6 levels both in tumour tissues and in serum. Treating mPDAC by an antibody blocking IL-6 reduced the proportion of Tregs and MDSCs and increased the amount of TILs, thus mimicking the effects of N6L.Conclusions: These results demonstrate that NCL inhibition blocks the amplification of lymphoid and myeloid immunosuppressive cells and promotes T cell activation in PDAC through a new mechanism of action dependent on the direct inhibition of the tumoral stroma.

The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.15538. G protein-coupled receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

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Pancreatic ductal adenocarcinoma (PDAC) aggressiveness results from its high metastatic and chemoresistance potential. Pancreatic cancer cells are immersed in an exuberant tumor microenvironment (or stroma), which represents up to 80% of PDAC tumor volume, and is hijacked by cancer cells for their own survival and metastasis. Additionally, PDAC tumors are heterogeneous, e.g. transcriptomic (RNAseq) analyses of human PDAC tumors have allowed patients to be stratified into two tumor subgroups (basal and classical) with different prognoses. Therefore, we aimed to understand which tumor-stroma crosstalks are critical for PDAC aggressiveness in the context of its heterogeneity, and how they can be pharmacologically targeted, to then propose more effective personalized therapeutic strategies. By performing bioinformatical analysis on RNAseq data from PDX (Patient Derived Xenograft), a hybrid tumor model from which human-derived tumor cell sequences are distinguishable from murine-derived stromal cell sequences, we searched for different stromal behaviors linked to the tumor aggressiveness, to be then validated in published PDAC patient databases. According to NMF (Non-negative matrix factorization) and GSEA (Gene Set Enrichment Analyses), we identified two components, which classify the samples according to a stromal gradient. Interestingly, these components are characterized by distinct functional signatures of aggressiveness and they are highly prognostic (correlated with survival). In addition, these components stratify patients independently on the described molecular tumor (basal or classical) classification. Importantly, we validated those results on other PDAC published databases. Moreover, ligand-receptor bioinformatics analyses identified novel players of the TGFβ superfamily in the most aggressive stromal component. Indeed, we have highlighted the involvement of the ligand activin a member of this large superfamily. Using in situ hybridization coupled with immunofluorescence, we were able to demonstrate that activin is only expressed by stromal cells, and more specifically by cancer associated fibroblasts (CAF), with however a strong heterogeneity (inter and intra-tumor). In vitro, activin strongly increases the migratory capacity of fibroblasts but also of specific cancer cells, this effect being effectively blocked pharmacologically with an activin receptor-targeting drug that is now tested in a pre-clinical PDAC murine model. Therapeutic targeting of the stroma must take into account its functional heterogeneity. We have identified in specific aggressive PDAC tumors novel stromal players involved in the tumor-stroma symbiosis, and which presumably play a critical role in CAFs and cancer cells migration and invasion. Our work should enable to delineate which stroma-targeting drug to co-deliver, in addition to chemotherapies, in stratified patients, to improve PDAC prognosis. Citation Format: Alexia Brunel, Jérôme Raffenne, Remy Nicolle, Ilse Rooman, Elisa Espinet, Marjorie Fanjul, Aurélie Perraud, Muriel Mathonnet, Sophie Vasseur, Nelson Dusetti, Yvan Martineau, Christine Jean, Corinne Bousquet. Bioinformatical identification of cancer-associated-fibroblasts with tumor cells pro-migratory crosstalks depending on activin [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C034.

Simple Summary Stroma modifications observed in solid cancer are now recognized as critical events for cancer progression and as potential therapeutic or diagnostic targets. The recent appreciation of multiple but complex roles of the extracellular matrix (ECM) in cancer, and of the cancer-associated fibroblast (CAF) diversity, has revolutionized the field and raised innovative but challenging questions. In this review, we summarize the latest knowledge regarding the role of the ECM in cancer progression, discuss the potential use of such stromal pro-tumoral modifications as therapeutic or diagnostic targets, and, finally, discuss benefits, disappointments, or even failures, of recently reported stroma-targeting strategies. Abstract Solid cancer progression is dictated by neoplastic cell features and pro-tumoral crosstalks with their microenvironment. Stroma modifications, such as fibroblast activation into cancer-associated fibroblasts (CAFs) and extracellular matrix (ECM) remodeling, are now recognized as critical events for cancer progression and as potential therapeutic or diagnostic targets. The recent appreciation of the key, complex and multiple roles of the ECM in cancer and of the CAF diversity, has revolutionized the field and raised innovative but challenging questions. Here, we rapidly present CAF heterogeneity in link with their specific ECM remodeling features observed in cancer, before developing each of the impacts of such ECM modifications on tumor progression (survival, angiogenesis, pre-metastatic niche, chemoresistance, etc.), and on patient prognosis. Finally, based on preclinical studies and recent results obtained from clinical trials, we highlight key mechanisms or proteins that are, or may be, used as potential therapeutic or diagnostic targets, and we report and discuss benefits, disappointments, or even failures, of recently reported stroma-targeting strategies.

Pancreatic ductal adenocarcinoma remains a major challenge in cancer medicine. Given the increase in incidence and mortality, interdisciplinary research is necessary to translate basic knowledge into therapeutic strategies improving the outcome of patients. On the 4th and 5th of February 2021, three German pancreatic cancer research centers, the Clinical Research Unit 5002 from Gottingen, the Collaborative Research Center 1321 from Munich, and Clinical Research Unit 325 from Marburg organized the 1st Virtual Gottingen-Munich-Marburg Pancreatic Cancer Meeting in order to foster scientific exchange. This report summarizes current research and proceedings presented during that meeting.

Cancer‐associated fibroblasts (CAFs) are considered the most abundant type of stromal cells in pancreatic ductal adenocarcinoma (PDAC), playing a critical role in tumour progression and chemoresistance; however, a druggable target on CAFs has not yet been identified. Here we report that focal adhesion kinase (FAK) activity (evaluated based on 397 tyrosine phosphorylation level) in CAFs is highly increased compared to its activity in fibroblasts from healthy pancreas. Fibroblastic FAK activity is an independent prognostic marker for disease‐free and overall survival of PDAC patients (cohort of 120 PDAC samples). Genetic inactivation of FAK within fibroblasts (FAK kinase‐dead, KD) reduces fibrosis and immunosuppressive cell number within primary tumours and dramatically decreases tumour spread. FAK pharmacologic or genetic inactivation reduces fibroblast migration/invasion, decreases extracellular matrix (ECM) expression and deposition by CAFs, modifies ECM track generation and negatively impacts M2 macrophage polarization and migration. Thus, FAK activity within CAFs appears as an independent PDAC prognostic marker and a druggable driver of tumour cell invasion., Understanding how cancer‐associated fibroblasts (CAFs) promote PDAC progression is of major interest given the poor prognosis of patients. This study identifies a druggable key regulator of CAF‐induced tumour cell metastasis and a prognostic factor: the protein Focal Adhesion Kinase (FAK).

Background & Aims Cancer-associated fibroblasts (CAFs) from pancreatic adenocarcinoma (PDA) present high protein synthesis rates. CAFs express the G-protein–coupled somatostatin receptor sst1. The sst1 agonist SOM230 blocks CAF protumoral features in vitro and in immunocompromised mice. We have explored here the therapeutic potential of SOM230, and underlying mechanisms, in immunocompetent models of murine PDA mimicking the heavy fibrotic and immunosuppressive stroma observed in patient tumors. Methods Large-scale mass spectrometry analyses were performed on media conditioned from 9 patient PDA-derived CAF primary cultures. Spontaneous transgenic and experimental (orthotopic co-graft of tumor cells plus CAFs) PDA-bearing mice were longitudinally ultrasound-monitored for tumor and metastatic progression. Histopathology and flow cytometry analyses were performed on primary tumors and metastases. Stromal signatures were functionally validated through bioinformatics using several published, and 1 original, PDA database. Results Proteomics on the CAF secretome showed that SOM230 controls stromal activities including inflammatory responses. Among the identified secreted proteins, we validated that colony-stimulating factor 1 (CSF-1) (a macrophage growth factor) was reduced by SOM230 in the tumor and plasma of PDA-harboring mice, alongside intratumor stromal normalization (reduced CAF and macrophage activities), and dramatic metastasis reduction. In transgenic mice, these SOM230 benefits alleviate the chemotherapy-induced (gemcitabine) immunosuppressive stroma reshaping. Mechanistically, SOM230 acts in vivo on CAFs through sst1 to disrupt prometastatic CAF production of CSF-1 and cross-talk with macrophages. We found that in patients, stromal CSF-1 was associated with aggressive PDA forms. Conclusions We propose SOM230 as an antimetastatic therapy in PDA for its capacity to remodel the fibrotic and immunosuppressive myeloid stroma. This pharmacotherapy should benefit PDA patients treated with chemotherapies., Graphical abstract

Simple Summary A growing number of studies suggest that cancer-associated fibroblasts (CAFs) modulate both myeloid and lymphoid cells through secretion of molecules (i.e., chemical function) and production of the extracellular matrix (ECM), i.e., physical function. Even though targeting functions CAFs is a relevant strategy, published clinical trials solely aimed at targeting the stroma showed disappointing results, despite being based on solid preclinical evidence. Our review dissects the interactions between CAFs and immune cells and explains how a deeper understanding of CAF subpopulations is the cornerstone to propose relevant therapies that will ultimately improve survival of patients with cancer. Abstract Cancer-associated fibroblasts (CAFs) are prominent cells within the tumor microenvironment, by communicating with other cells within the tumor and by secreting the extracellular matrix components. The discovery of the immunogenic role of CAFs has made their study particularly attractive due to the potential applications in the field of cancer immunotherapy. Indeed, CAFs are highly involved in tumor immune evasion by physically impeding the immune system and interacting with both myeloid and lymphoid cells. However, CAFs do not represent a single cell entity but are divided into several subtypes with different functions that may be antagonistic. Considering that CAFs are orchestrators of the tumor microenvironment and modulate immune cells, targeting their functions may be a promising strategy. In this review, we provide an overview of (i) the mechanisms involved in immune regulation by CAFs and (ii) the therapeutic applications of CAFs modulation to improve the antitumor immune response and the efficacy of immunotherapy.

Molecular profiling of Pancreatic Ductal Adenocarcinoma (PDA), based on transcriptomic analyses, identifies two main prognostic subtypes (basal-like and classical), but does not allow personalized first-line treatment. To date, tumors have not been profiled based on protein synthesis rates, yet the step of mRNA translation is highly dysregulated in both PDA cancer cells and their microenvironment. We aim at assessing whether quantification of mRNA translation could provide a distinct perspective on PDA and identify novel tumor subtypes. Using a collection of twenty-seven pancreatic Patient-Derived Xenografts (PDX), we performed transcriptome-wide analysis of translated mRNA (translatome). Unsupervised bioinformatics analysis allowed PDA tumors classification according to mRNA translation rate. PDX-derived cancer cells as well as common PDA cell lines were used to functionally characterize newly identified subtype. Independent component analysis revealed a new tumor subtype harboring a low protein synthesis rate, but associated with a robust translation of mRNAs encoding effectors of the integrated stress response (ISR), including the transcription factor ATF4. Functional characterization of the “ISR-activated” human cancer cells revealed a high resistance to drugs, low autophagic capacities, and importantly, metabolic impairments in the serine synthesis and transsulfuration pathways. Therefore, the drug-resistant cancer cell phenotype showing auxotrophy to both serine and cysteine may be amenable to targeted therapy. Overall, our study highlights profiling of mRNA translation in cancer as an underexplored avenue for identification of previously unrecognized subtypes together with potential treatments. Citation Format: Sauyeun Shin, Remy Nicolle, Mehdi Liauzun, Jacobo Solorzano, Alexia Brunel, Christine Jean, Remi Samain, Jerôme Raffenne, Cindy Neuzillet, Carine Joffre, Stephane Rocci, Juan Iovanna, Nelson Dusetti, Ola Larsson, Stephane Pyronnet, Corinne Bousquet, Yvan Martineau. Classification based on efficiency of mRNA translation reveals a metabolically-dependent subtype of pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-088.

Molecular profiling of Pancreatic Ductal Adenocarcinoma (PDA), based on transcriptomic analyses, identifies two main prognostic subtypes (basal-like and classical), but does not allow personalized first-line treatment. To date, tumors have not been profiled based on protein synthesis rates, yet the step of mRNA translation is highly deregulated in both PDA cancer cells and their microenvironment. Using a collection of twenty-seven pancreatic Patient-Derived Xenografts (PDX), we performed genome-wide analysis of translated mRNA (translatome). Unsupervised bioinformatics analysis revealed a new tumor subtype harboring a low protein synthesis rate, but associated with a robust translation of mRNAs encoding effectors of the integrated stress response (ISR), including the transcription factor ATF4. Functional characterization of the “ISR-activated” human cancer cells revealed a high resistance to drugs, low autophagic capacities, and importantly, metabolic impairments in the serine synthesis and transsulfuration pathways. Overall, our study highlights the strength of translatomic profiling on PDA, which here revealed an unforeseen drug-resistant cancer cell phenotype, whose auxotrophy to both serine and cysteine may be amenable to targeted therapy.

Pancreatic ductal adenocarcinoma (PDAC) relies on hyperactivated protein synthesis. Consistently, human and mouse PDAC lose expression of the translational repressor and mTOR target 4E-BP1. Using genome-wide polysome profiling, we here explore mRNAs whose translational efficiencies depend on the mTOR/4E-BP1 axis in pancreatic cancer cells. We identified a functional enrichment for mRNAs encoding DNA replication and repair proteins, including RRM2 and CDC6. Consequently, 4E-BP1 depletion favors DNA repair and renders DNA replication insensitive to mTOR inhibitors, in correlation with a sustained protein expression of CDC6 and RRM2, which is inversely correlated with 4E-BP1 expression in PDAC patient samples. DNA damage and pancreatic lesions induced by an experimental pancreatitis model uncover that 4E-BP1/2-deleted mice display an increased acinar cell proliferation and a better recovery than WT animals. Targeting translation, independently of 4E-BP1 status, using eIF4A RNA helicase inhibitors (silvestrol derivatives) selectively modulates translation and limits CDC6 expression and DNA replication, leading to reduced PDAC tumor growth. In summary, 4E-BP1 expression loss during PDAC development induces selective changes in translation of mRNA encoding DNA replication and repair protein. Importantly, targeting protein synthesis by eIF4A inhibitors circumvents PDAC resistance to mTOR inhibition.

WOS:000462822100001; International audience; The 4G family of eukaryotic mRNA translation initiation factors is composed of three members (eIF4GI, eIF4GII, and DAP5). Their specific roles in translation initiation are under intense investigations, but how their respective intracellular amounts are controlled remains poorly understood. Here we show that eIF4GI and eIF4GII exhibit much shorter half-lives than that of DAP5. Both eIF4GI and eIF4GII proteins, but not DAP5, contain computer-predicted PEST motifs in their N-termini conserved across the animal kingdom. They are both sensitive to degradation by the proteasome. Under normal conditions, eIF4GI and eIF4GII are protected from proteasomal destruction through binding to the detoxifying enzyme NQO1 [NAD(P)H:quinone oxidoreductase]. However, when cells are exposed to oxidative stress both eIF4GI and eIF4GII, but not DAP5, are degraded by the proteasome in an N-terminal-dependent manner, and cell viability is more compromised upon silencing of DAP5. These findings indicate that the three eIF4G proteins are differentially regulated by the proteasome and that persistent DAP5 plays a role in cell survival upon oxidative stress.

International audience; Neuregulin 1 (NRG1), a ligand for HER3 and HER4 receptors, is secreted by both pancreatic tumor cells (PC) and cancer-associated fibroblasts (CAFs), the latter representing the most abundant compound of pancreatic stroma. This desmoplastic stroma contributes to Pancreatic Ductal Adenocarcinoma (PDAC) aggressiveness and therapeutic failure by promoting tumor progression, invasion and resistance to chemotherapies. In the present work, we aimed at disrupting the complex crosstalk between PC and CAF in order to prevent tumor cell proliferation. To do so, we demonstrated the promising tumor growth inhibitory effect of the 7E3, an original antibody directed to NRG1. This antibody promotes antibody dependent cellular cytotoxicity in NRG1-positive PC and CAFs and inhibits NRG1-associated signaling pathway induction, by blocking NRG1-mediated HER3 activation. Moreover, 7E3 inhibits migration and growth of pancreatic cancer cells co-cultured with CAFs, both in vitro and in vivo using orthotopic pancreatic tumor xenografts. Our preclinical results demonstrate that the anti-NRG1 antibody 7E3 could represent a promising approach to target pancreatic stroma and cancer cells, thereby providing novel therapeutic options for PDAC.

Somatostatin, also known as somatotropin-release inhibitory factor, is a cyclopeptide that exerts potent inhibitory actions on hormone secretion and neuronal excitability. Its physiologic functions are mediated by five G protein–coupled receptors (GPCRs) called somatostatin receptor (SST)1–5. These five receptors share common structural features and signaling mechanisms but differ in their cellular and subcellular localization and mode of regulation. SST2 and SST5 receptors have evolved as primary targets for pharmacological treatment of pituitary adenomas and neuroendocrine tumors. In addition, SST2 is a prototypical GPCR for the development of peptide-based radiopharmaceuticals for diagnostic and therapeutic interventions. This review article summarizes findings published in the last 25 years on the physiology, pharmacology, and clinical applications related to SSTs. We also discuss potential future developments and propose a new nomenclature.

Increased PI 3-kinase (PI3K) signaling in pancreatic ductal adenocarcinoma (PDAC) correlates with poor prognosis, but the role of class I PI3K isoforms during its induction remains unclear. Using genetically engineered mice and pharmacological isoform-selective inhibitors, we found that the p110α PI3K isoform is a major signaling enzyme for PDAC development induced by a combination of genetic and nongenetic factors. Inactivation of this single isoform blocked the irreversible transition of exocrine acinar cells into pancreatic preneoplastic ductal lesions by oncogenic Kras and/or pancreatic injury. Hitting the other ubiquitous isoform, p110β, did not prevent preneoplastic lesion initiation. p110α signaling through small GTPase Rho and actin cytoskeleton controls the reprogramming of acinar cells and regulates cell morphology in vivo and in vitro. Finally, p110α was necessary for pancreatic ductal cancers to arise from Kras-induced preneoplastic lesions by increasing epithelial cell proliferation in the context of mutated p53. Here we identify an in vivo context in which p110α cellular output differs depending on the epithelial transformation stage and demonstrate that the PI3K p110α is required for PDAC induced by oncogenic Kras, the key driver mutation of PDAC. These data are critical for a better understanding of the development of this lethal disease that is currently without efficient treatment.

Given the failure of chemo- and biotherapies to fight advanced pancreatic cancer, one major challenge is to identify critical events that initiate invasion. One priming step in epithelia carcinogenesis is the disruption of epithelial cell anchorage to the basement membrane which can be provided by hemidesmosomes (HDs). However, the existence of HDs in pancreatic ductal epithelium and their role in carcinogenesis remain unexplored. HDs have been explored in normal and cancer pancreatic cells, and patient samples. Unique cancer cell models where HD assembly can be pharmacologically manipulated by somatostatin/sst2 signaling have been then used to investigate the role and molecular mechanisms of dynamic HD during pancreatic carcinogenesis. We surprisingly report the presence of mature type-1 HDs comprising the integrin α6β4 and bullous pemphigoid antigen BP180 in the human pancreatic ductal epithelium. Importantly, HDs are shown to disassemble during pancreatic carcinogenesis. HD breakdown requires phosphoinositide 3-kinase (PI3K)-dependent induction of the matrix-metalloprotease MMP-9, which cleaves BP180. Consequently, integrin α6β4 delocalizes to the cell-leading edges where it paradoxically promotes cell migration and invasion through S100A4 activation. As S100A4 in turn stimulates MMP-9 expression, a vicious cycle maintains BP180 cleavage. Inactivation of this PI3K-MMP-9-S100A4 signaling loop conversely blocks BP180 cleavage, induces HD reassembly and inhibits cell invasion. We conclude that mature type-1 HDs are critical anchoring structures for the pancreatic ductal epithelium whose disruption, upon PI3K activation during carcinogenesis, provokes pancreatic cancer cell migration and invasion.

The mRNA cap-binding protein eIF4E (eukaryotic translation initiation factor 4E) permits ribosome recruitment to capped mRNAs, and its phosphorylated form has an important role in cell transformation. The oncogenic function of eIF4E is, however, antagonised by the hypophosphorylated forms of the inhibitory eIF4E-binding proteins 1 and 2. eIF4E-binding protein 1 and 2 (4E-BP1 and 2) are two major targets of the protein kinase mTOR, and are essential for the antiproliferative effects of mTOR inhibitors. Herein, we report that pancreas expresses specifically and massively 4E-BP1 (4E-BP2 is nearly undetectable). However, 4E-BP1 expression is extinguished in more than half of the human pancreatic ductal adenocarcinomas (PDAC). 4E-BP1 shutoff is recapitulated in a mouse genetic model of PDAC, which is based on a pancreas-specific mutation of Kras, the more frequently mutated oncogene in human pancreatic tumours. 4E-BP1 downregulation enhances eIF4E phosphorylation and facilitates pancreatic cancer cell proliferation in vitro and tumour development in vivo. Furthermore, 4E-BP1 loss combined with the absence of 4E-BP2 renders eIF4E phosphorylation, protein synthesis and cell proliferation resistant to mTOR inhibition. However, proliferation can be better limited by a recently developed compound that mimics the function of 4E-BP1 and 2 independently of mTOR inhibition.

The eukaryotic translation initiation factor 4E (eIF4E) is necessary for the translation of capped mRNAs into proteins. Cap-dependent mRNA translation can be however inhibited by the eIF4E-binding protein 1 (4E-BP1). The hypophosphorylated forms of 4E-BP1 indeed sequester eIF4E and thus block translation initiation and consequent protein synthesis. Different reports indicate that, in addition to hypophosphorylation, 4E-BP1 function can be also regulated at the level of protein expression. This is the case in contact-inhibited cells or in cells exposed to hypoxia. The molecular mechanisms responsible for 4E-BP1 protein accumulation in these conditions remain however unknown. In the present study, we found that 4E-BP1 gene promoter contains a hypoxia-responsive element (HRE) that mediates 4E-BP1 gene upregulation via the hypoxia-inducible factor-1 alpha (HIF-1α) transcription factor. Gene reporter assays then revealed that the presence of such HRE in the promoter of 4E-BP1 gene is involved in 4E-BP1 accumulation in contact-inhibited cells and in cells exposed to hypoxia. We also reveal that the TGF-β–dependent transcription factor SMAD4 cooperates with HIF-1α to fully activate 4E-BP1 gene transcription under hypoxia. These data therefore suggest that HIF-1α contributes to 4E-BP1 gene expression under different conditions. Mol Cancer Res; 11(1); 54–61. ©2012 AACR.

The intratumoral microenvironment, or stroma, is of major importance in the pathobiology of pancreatic ductal adenocarcinoma (PDA), and specific conditions in the stroma may promote increased cancer aggressiveness. We hypothesized that this heterogeneous and evolving compartment drastically influences tumor cell abilities, which in turn influences PDA aggressiveness through crosstalk that is mediated by extracellular vesicles (EVs). Here, we have analyzed the PDA proteomic stromal signature and identified a contribution of the annexin A6/LDL receptor-related protein 1/thrombospondin 1 (ANXA6/LRP1/TSP1) complex in tumor cell crosstalk. Formation of the ANXA6/LRP1/TSP1 complex was restricted to cancer-associated fibroblasts (CAFs) and required physiopathologic culture conditions that improved tumor cell survival and migration. Increased PDA aggressiveness was dependent on tumor cell-mediated uptake of CAF-derived ANXA6+ EVs carrying the ANXA6/LRP1/TSP1 complex. Depletion of ANXA6 in CAFs impaired complex formation and subsequently impaired PDA and metastasis occurrence, while injection of CAF-derived ANXA6+ EVs enhanced tumorigenesis. We found that the presence of ANXA6+ EVs in serum was restricted to PDA patients and represents a potential biomarker for PDA grade. These findings suggest that CAF-tumor cell crosstalk supported by ANXA6+ EVs is predictive of PDA aggressiveness, highlighting a therapeutic target and potential biomarker for PDA.

Single-agent immunotherapy has achieved limited clinical benefit to date in patients suffering from pancreatic ductal adenocarcinoma (PDAC). This may be due to the presence of a uniquely immunosuppressive tumor microenvironment (TME). Critical obstacles to immunotherapy in PDAC tumors include a high number of tumor-associated immunosuppressive cells and a uniquely desmoplastic stroma that acts as a barrier to T-cell infiltration. We have identified hyperactivated focal adhesion kinase (FAK) activity in neoplastic PDAC cells as a significant regulator of the fibrotic and immunosuppressive TME. We found that FAK activity was elevated in human PDAC tissues and correlates with high levels of fibrosis and poor CD8+ cytotoxic T-cell infiltration. Single-agent FAK inhibition using the selective FAK inhibitor VS-4718 significantly limited tumor progression, resulting in a doubling of survival in the p48-Cre/LSL-KrasG12D/p53Flox/+ (KPC) mouse model of human PDAC. This delay in tumor progression was associated with dramatically reduced tumor fibrosis, and decreased numbers of tumor-infiltrating immunosuppressive cells. We also found that FAK inhibition rendered the previously unresponsive KPC mouse model responsive to T cell immunotherapy and PD-1 antagonists. These data suggest that FAK inhibition increases immune surveillance by overcoming the fibrotic and immunosuppressive PDAC TME and renders tumors responsive to immunotherapy.

In stressed cells, a general decrease in the rate of protein synthesis occurs due to modifications in the activity of translation initiation factors. Compelling data now indicate that these changes also permit a selective post-transcriptional expression of proteins necessary for either cell survival or completion of apoptosis when cells are exposed to severe or prolonged stress. In this review, we summarize the modifications that inhibit the activity of the main canonical translation initiation factors, and the data explaining how certain mRNAs encoding proteins involved in either cell survival or apoptosis can be selectively translated.

The eIF4E-binding proteins (4E-BPs) are inhibitors of protein synthesis that sequester the mRNA cap-binding protein eIF4E and consequently block cell growth and proliferation. In most tumors however, their inhibitory function is compromised by major oncogenic signaling pathways. Recently, thanks to the generation of mouse genetic models, considerable progress has been made in elucidating the involvement of 4E-BPs and their unique target, eIF4E, in the process of carcinogenesis. Increasing evidence indicates that an 'addiction' to protein synthesis emerges in cancer cells, highlighting the potential that 4E-BPs have as targets for therapeutics. In this review, we summarize the biochemical function, regulation and anti-oncogenic activity of the 4E-BPs.

Context: Among the innovative molecules used to manage neuroendocrine tumors, there is growing interest in combining the somatostatin analogs octreotide or pasireotide (SOM230) and everolimus (RAD001), an inhibitor that targets the protein kinase mammalian target of rapamycin (mTOR). Evidence Acquisition: The aims of this review were to describe the signaling pathways targeted independently by somatostatin analogs and everolimus and to summarize the scientific rationale for the potential additive or synergistic antitumor effects of combined therapy. Evidence Synthesis: The somatostatin analogs (octreotide and lanreotide) have potent inhibitory effects on hypersecretion, thereby alleviating the symptoms associated with neuroendocrine tumors. Furthermore, the antitumor potential of octreotide is now well documented. Pasireotide, a somatostatin analog, has the advantage of targeting a wider range of somatostatin receptors (subtypes 1, 2, 3, and 5) than the analogs previously used in clinical practice (which ...

International audience; Assembly of the multi-subunit eukaryotic translation initiation factor-4F (eIF4F) is critical for protein synthesis and cell growth and proliferation. eIF4F formation is regulated by the translation-inhibitory protein 4E-BP1. While proliferation factors and intracellular pathways that impinge upon 4E-BP1 phosphorylation have been extensively studied, how they control 4E-BP1 expression remains unknown. Here, we show that Smad4, a transcription factor normally required for TGFbeta-mediated inhibition of normal cell proliferation, enhances 4E-BP1 gene-promoter activity through binding to a conserved element. 4E-BP1 expression is specifically modulated by treatment with TGFbeta and by manipulations of the natural Smad4 regulators (co-Smads) in cells isolated from Smad4(+/+) human tumours, whereas no response is observed in cells isolated from Smad4(-/-) human tumours or in cells where Smad4 has been knocked down by specific siRNAs. In addition, cells where 4E-BP1 has been knocked down (inducible shRNAs in human pancreatic cancer cells or siRNAs in non-malignant human keratinocytes) or has been knocked out (mouse embryonic fibroblasts isolated from 4E-BP1(-/-) mice) proliferate faster and are resistant to the antiproliferative effect of TGFbeta. Thus, 4E-BP1 gene appears critical for TGFbeta/Smad4-mediated inhibition of cell proliferation.

Comment inSomatostatin receptor subtype 2 as pancreatic tumorigenesis suppressor: identification of a new targetable signaling node. [Gastroenterology. 2015]; International audience; BACKGROUND & AIMS:The KRAS gene is mutated in most pancreatic ductal adenocarcinomas (PDAC). Expression of this KRAS oncoprotein in mice is sufficient to initiate carcinogenesis but not progression to cancer. Activation of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) is required for KRAS for induction and maintenance of PDAC in mice. The somatostatin receptor subtype 2 (sst2) inhibits PI3K, but sst2 expression is lost during the development of human PDAC. We investigated the effects of sst2 loss during KRAS-induced PDAC development in mice.METHODS:We analyzed tumor growth in mice that expressed the oncogenic form of KRAS (KRAS(G12D)) in pancreatic precursor cells, as well as sst2+/- and sst2-/-, and in crossed KRAS(G12D);sst2+/- and KRAS(G12D);sst2-/- mice. Pancreatic tissues and acini were collected and assessed by histologic, immunoblot, immunohistochemical, and reverse-transcription polymerase chain reaction analyses. We also compared protein levels in paraffin-embedded PDAC samples from patients vs heathy pancreatic tissues from individuals without pancreatic cancer.RESULTS:In sst2+/- mice, PI3K was activated and signaled via AKT (PKB; protein kinase B); when these mice were crossed with KRAS(G12D) mice, premalignant lesions, tumors, and lymph node metastases developed more rapidly than in KRAS(G12D) mice. In crossed KRAS(G12D);sst2+/- mice, activation of PI3K signaling via AKT resulted in activation of nuclear factor-κB (NF-κB), which increased KRAS activity and its downstream pathways, promoting initiation and progression of neoplastic lesions. We found this activation loop to be mediated by PI3K-induced production of the chemokine CXCL16. Administration of a CXCL16-neutralizing antibody to KRAS(G12D) mice reduced activation of PI3K signaling to AKT and NF-κB, blocking carcinogenesis. Levels of CXCL16 and its receptor CXCR6 were significantly higher in PDAC tissues and surrounding acini than in healthy pancreatic tissues from mice or human beings. In addition, expression of sst2 was progressively lost, involving increased PI3K activity, in mouse lesions that expressed KRAS(G12D) and progressed to PDAC.CONCLUSIONS:Based on analyses of mice, loss of sst2 from pancreatic tissues activates PI3K signaling via AKT, leading to activation of NF-κB, amplification of oncogenic KRAS signaling, increased expression of CXCL16, and pancreatic tumor formation. CXCL16 might be a therapeutic target for PDAC.

Phosphatidylinositol 3-kinase (PI3K) regulates many cellular functions including growth and survival, and its excessive activation is a hallmark of cancer. Somatostatin, acting through its G protein-coupled receptor (GPCR) sst2, has potent proapoptotic and anti-invasive activities on normal and cancer cells. Here, we report a novel mechanism for inhibiting PI3K activity. Somatostatin, acting through sst2, inhibits PI3K activity by disrupting a pre-existing complex comprising the sst2 receptor and the p85 PI3K regulatory subunit. Surface plasmon resonance and molecular modeling identified the phosphorylated-Y71 residue of a p85-binding pYXXM motif in the first sst2 intracellular loop, and p85 COOH-terminal SH2 as direct interacting domains. Somatostatin-mediated dissociation of this complex as well as p85 tyrosine dephosphorylation correlates with sst2 tyrosine dephosphorylation on the Y71 residue. Mutating sst2-Y71 disabled sst2 to interact with p85 and somatostatin to inhibit PI3K, consequently abrogating sst2's ability to suppress cell survival and tumor growth. These results provide the first demonstration of a physical interaction between a GPCR and p85, revealing a novel mechanism for negative regulation by ligand-activated GPCR of PI3K-dependent survival pathways, which may be an important molecular target for antineoplastic therapy.

Somatostatin receptor subtype 2 (sst2) gene expression is lost in 90% of human pancreatic adenocarcinomas. We previously demonstrated that stable sst2 transfection of human pancreatic BxPC-3 cells, which do not endogenously express sst2, inhibits cell proliferation, tumorigenicity, and metastasis. These sst2 effects occur as a consequence of an autocrine sst2-dependent loop, whereby sst2 induces expression of its own ligand, somatostatin. Here we investigated whether sst2 induces apoptosis in sst2-transfected BxPC-3 cells. Expression of sst2 induced a 4.4- +/- 0.05-fold stimulation of apoptosis in BxPC-3 through the activation of tyrosine phosphatase SHP-1. sst2 also sensitized these cells to apoptosis induced by tumor necrosis factor alpha (TNFalpha), enhancing it 4.1- +/- 1.5-fold. Apoptosis in BxPC-3 cells mediated by TNF-related apoptosis-inducing ligand (TRAIL) and CD95L was likewise increased 2.3- +/- 0.5-fold and 7.4- +/- 2.5-fold, respectively. sst2-dependent activation and cell sensitization to death ligand-induced apoptosis involved activation of the executioner caspases, key factors in both death ligand- or mitochondria-mediated apoptosis. sst2 affected both pathways: first, by up-regulating expression of TRAIL and TNFalpha receptors, DR4 and TNFRI, respectively, and sensitizing the cells to death ligand-induced initiator capase-8 activation, and, second, by down-regulating expression of the antiapoptotic mitochondrial Bcl-2 protein. These results are of interest for the clinical management of chemoresistant pancreatic adenocarcinoma by using a combined gene therapy based on the cotransfer of genes for both the sst2 and a nontoxic death ligand.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone derived from the proglucagon gene, capable of regulating the transcription of the three major genes that determine the pancreatic beta-cell-specific phenotype: insulin, GLUT-2, and glucokinase. The aim of this study was to investigate the potential role of GLP-1 for the gene therapy of glucose-insensitive pancreatic beta-cells. We transfected mouse insulinoma cells with a DNA fragment of the human proglucagon gene containing the nucleotide sequence encoding for human GLP-1 but lacking the coding region for glucagon. Two constructs were generated: In one, the expression of GLP-1 was under the control of the cytomegalovirus (CMV) promoter (CMV/GLP-1), and the second was regulated by the rat insulin II promoter (RIP)/GLP-1). Northern blot, HPLC, and RIA analyses confirmed that the minigene was transcribed and the protein appropriately translated, processed, and secreted in the extracellular environment. Gene expression studies revealed that although CMV/GLP-1 cells did not gain a greater glucose sensitivity as a result of the transfection with GLP-1, compared with cells transfected with the plasmid alone, RIP/GLP-1 was capable of regulating the gene expression of insulin and GLP-1 based on the concentration of glucose in the culture medium. Detection of the counterpart proteins (insulin and GLP-1) in the culture medium paralleled the observation derived from the Northern blot analysis. GLP-1 action was mediated by an IDX-1 (islet/duodenum homeobox-1) dependent transactivation of the endogenous insulin promoter, as demonstrated by gel shift analysis. This was further suggested by a significant increase of the glucose-dependent binding of IDX-1 to the insulin promoter in RIP/GLP-1 cells but not in CMV/GLP-1 cells or control cells. Finally, we observed that although the GLP-1-dependent secretion of insulin was mediated by an increase in cAMP levels, the transcription of the insulin gene, in response to GLP-1, was in large part cAMP independent. The present study lays the research foundation to investigate the potential use of GLP-1 for the gene or cell therapy of diabetes.

The hypothalamo-pituitary-adrenal (HPA) axis maintains a homeostatic response to stress, infection, or neoplasia. Inflammatory cytokines, including leukemia inhibitory factor (LIF), stimulate the HPA axis either directly at the pituitary corticotroph, or indirectly through induction of CRH or sympathetic noradrenergic neurons, and mediate the immuno-neuroendocrine interface. Unrestrained HPA axis activation leads, however, to immunosuppression. Because suppressor of cytokine signaling-3 (SOCS-3) is a potent inhibitor of LIF-activated HPA axis, and dynamic interactions between hypothalamus-derived cAMP-inducing neuropeptides and proinflammatory cytokines occur at the corticotroph level, we investigated SOCS-3 expression in response to peptides that stimulate cAMP including CRH, pituitary adenylate cyclase-activating polypeptide, and epinephrine. (Bu)2cAMP mediates induction of SOCS-3 promoter activity (6.7-fold +/- 0.5, P < 0.001) and SOCS-3 gene expression (4-fold +/- 0.8, P < 0.005) in a PKA-dependent manner. LIF and cAMP-inducing agents are additive on SOCS-3 promoter activity (22-fold +/- 2.6, LIF + (Bu)2cAMP vs. 7.3-fold +/- 0.6, LIF alone, P < 0.05) and on SOCS-3 transcription (11.3-fold +/- 2.1, LIF + (Bu)2cAMP vs. 9.3-fold +/- 1, LIF alone, P < 0.05), suggesting alternate pathways for LIF and cAMP-mediated corticotroph signaling. Similarly, LIF and CRH or pituitary adenylate cyclase-activating polypeptide are additive for SOCS-3 promoter activity and transcription (P < 0.05). Whereas signal transducer and activator of transcription 3 binding to the SOCS-3 promoter mediates LIF action, several SOCS-3 promoter regions containing cAMP-responsive elements are required for cAMP-PKA effect. Thus, both classes of POMC-inducing agents, cytokines as well as cAMP-inducing central peptides, regulate SOCS-3, providing a further level of negative HPA axis control during inflammation. These results indicate a sensitive intracellular autoregulation of corticotroph function.

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine that stimulates the hypothalamo-pituitary-adrenal (HPA) axis through JAK-STAT activation. We show here that LIF-induced JAK2 and STAT3 tyrosine phosphorylation is transient, disappearing within 20 and 40 minutes, respectively. LIF activates the SH2 domain-containing tyrosine phosphatase, SHP-1, with maximal stimulation observed at 30 minutes. SHP-1 is constitutively associated with JAK2, and LIF induces recruitment of phosphorylated STAT3 to this complex. Overexpression of wild-type or dominant negative forms of SHP-1 shows decreased or increased LIF-induced proopiomelanocortin (POMC) promoter activity, respectively. LIF-induced JAK2 and STAT3 dephosphorylation is delayed until after 60 minutes in cells that overexpress the mutant SHP-1. In addition, SOCS-3, a negative regulator of LIF signaling, binds to JAK2 after 60 minutes of LIF stimulation, after which the complex is degraded by the proteasome. SOCS-3 overexpression blocks LIF-induced JAK2 tyrosine phosphorylation, confirming a role for SOCS-3 in deactivating JAK2 by direct association. Using SOCS-3 fusion proteins, we also define regions of the SOCS-3 protein that are critical for inhibition of LIF-induced POMC promoter activity. Corticotrophic signaling by LIF is thus subject to 2 forms of negative autoregulation: dephosphorylation of JAK2 and STAT3 by the SHP-1 tyrosine phosphatase, and SOCS-3-dependent inactivation of JAK2.

As pituitary leukemia-inhibitory factor (LIF) mediates neuroimmune signals to the hypothalamo-pituitary-adrenal axis, we tested the role of intracellular SOCS-3 in corticotroph function. SOCS-3, a cytokine-inducible protein of the suppressor of cytokine signaling (SOCS) family, is expressed in the murine pituitary in vivo. After i.p. injection of LIF (5.0 micrograms/mouse) or interleukin-1 beta (0.1 microgram/mouse) pituitary SOCS-3 mRNA was stimulated 9-fold and 6-fold, respectively. Also, in corticotroph AtT-20 cells LIF and interleukin-1 beta both potently stimulated SOCS-3 mRNA expression. In AtT-20 cells, stable overexpression of SOCS-3 inhibits basal and LIF-stimulated ACTH secretion in comparison to mock-transfected AtT-20 cells (basal: 4426 +/- 118 vs. 4973 +/- 138 pg/ml, P0.05; LIF-induced: 5511 +/- 172 vs. 9308 +/- 465 pg/ml, P0.001). Stable overexpression of SOCS-3 cDNA in AtT-20 cells also resulted in a significant 50% decrease of LIF-induced POMC mRNA levels (P0.05) and POMC promoter activity (P0.001), respectively. Western blot analysis revealed an inhibition of LIF-stimulated gp130 and STAT-3 phosphorylation in SOCS-3 overexpressing AtT-20 cells. Thus, SOCS-3 inhibits the Janus kinase (JAK) and signal transducers and activators of transcription (STAT) pathway, which is known to mediate LIF-stimulated ACTH secretion and POMC gene expression. In conclusion, SOCS-3 functions as an intracellular regulator of POMC gene expression and ACTH secretion, acting as a negative feedback mediator of the cytokine-mediated neuro-immuno-endocrine interface.

We have previously reported in Chinese hamster ovary (CHO) cells expressing sst2 that activation of the sst2 somatostatin receptor inhibits insulin-induced cell proliferation by a mechanism involving stimulation of a tyrosine phosphatase activity. Here we show that the tyrosine phosphatase SHP-1 was associated with the insulin receptor (IR) at the basal level. Activation of IR by insulin resulted in a rapid and transient increase of tyrosine phosphorylation of IR, its substrates IRS-1 and Shc, and also of SHP-1. This was then followed by a rapid dephosphorylation of these molecules, which was related to the insulin-induced increase of SHP-1 association to IR and of SHP-1 activity. On the other hand, addition to insulin of the somatostatin analogue, RC160, resulted in a higher and faster increase of SHP-1 association to IR directly correlated with an inhibition of phosphorylation of IR and its substrates, IRS-1 and Shc. RC160 also induced a higher and more sustained increase in SHP-1 activity. Furthermore, RC160 completely suppressed the effect of insulin on SHP-1 phosphorylation. Finally, in CHO cells coexpressing sst2 and a catalytically inactive mutant SHP-1, insulin as well as RC160 could no longer stimulate SHP-1 activity. Overexpression of the SHP-1 mutant prevented the insulin-induced signaling to be terminated by dephosphorylation of IR, suppressed the inhibitory effect of RC160 on insulin-induced IR phosphorylation, and abolished the cell proliferation modulation by insulin and RC160. Our results suggest that SHP-1 plays a role in negatively modulating insulin signaling by association with IR. Furthermore, somatostatin inhibits the insulin-induced mitogenic signal by accelerating and amplifying the effect of SHP-1 on the termination of the insulin signaling pathway.

In eukaryotes, mRNA translation is dependent on the cap-binding protein eIF4E. Through its simultaneous interaction with the mRNA cap structure and with the ribosome-associated eIF4G adaptor protein, eIF4E physically posits the ribosome at the 5' extremity of capped mRNA. eIF4E activity is regulated by phosphorylation on a unique site by the eIF4G-associated kinase MNK. eIF4E assembly with the eIF4G-MNK sub-complex can be however antagonized by the hypophosphorylated forms of eIF4E-binding protein (4E-BP). We show here that eIF4E phosphorylation is dramatically affected by disruption of eIF4E-eIF4G interaction, independently of changes in MNK expression. eIF4E phosphorylation is actually strongly downregulated upon eIF4G shutdown or upon sequestration by hypophosphorylated 4E-BP, consequent to mTOR inhibition. Downregulation of 4E-BP renders eIF4E phosphorylation insensitive to mTOR inhibition. These data highlight the important role of 4E-BP in regulating eIF4E phosphorylation independently of changes in MNK expression.

Frequent oncogenic alterations occur in the phosphoinositide 3-kinase (PI3K) pathway, urging identification of novel negative controls. We previously reported an original mechanism for restraining PI3K activity, controlled by the somatostatin G protein-coupled receptor (GPCR) sst2 and involving a ligand-regulated interaction between sst2 with the PI3K regulatory p85 subunit. We here identify the scaffolding protein filamin A (FLNA) as a critical player regulating the dynamic of this complex. A preexisting sst2-p85 complex, which was shown to account for a significant basal PI3K activity in the absence of ligand, is disrupted upon sst2 activation. FLNA was here identified as a competitor of p85 for direct binding to two juxtaposed sites on sst2. Switching of GPCR binding preference from p85 toward FLNA is determined by changes in the tyrosine phosphorylation of p85- and FLNA-binding sites on sst2 upon activation. It results in the disruption of the sst2-p85 complex and the subsequent inhibition of PI3K. Knocking down FLNA expression, or abrogating FLNA recruitment to sst2, reversed the inhibition of PI3K and of tumor growth induced by sst2. Importantly, we report that this FLNA inhibitory control on PI3K can be generalized to another GPCR, the mu opioid receptor, thereby providing an unprecedented mechanism underlying GPCR-negative control on PI3K.

International audience; BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers. It is characterized by substantial tumor cell invasion and early-stage metastasis. We developed an in vivo model to analyze interactions between cancer and stromal cells during early stages of PDAC. METHODS: Human pancreatic adenocarcinoma cells were grafted onto the chick chorioallantoic membrane (CAM). Human and chicken GeneChips were used simultaneously to study gene regulation during PDAC cell invasion. Bioinformatic analysis was used to identify human orthologs and cell specificity of gene expression. The effects of netrin-1 encoded by NTN1 were investigated in adhesion, invasion, and apoptosis assays. The effects of NTN1 silencing with small interfering RNAs were investigated in PDAC cells in vivo. NTN1 expression was measured in human PDAC samples. RESULTS: PDAC cells rapidly invade the CAM stroma and remodel the CAM vasculature. Around 800 stromal genes were up-regulated by >2-fold; the angiogenesis regulators vascular endothelial growth factor D, thrombospondin 1, and CD151 were among the most highly regulated genes. Silencing of tumor cell NTN1, which is up-regulated 4-fold in the PDAC model, inhibited tumor cell invasion in vivo. Netrin-1 conferred apoptosis resistance to tumor and endothelial cells in vitro, induced their invasion, and provided an adhesive substrate for tumor cells. NTN1 and its gene product are strongly overexpressed in human PDAC samples. CONCLUSIONS: We developed a useful tool to study the invasive mechanisms of early-stage PDAC. Netrin-1 might be an important regulator of pancreatic tumor growth that functions in tumor and endothelial cells.

International audience; The eukaryotic translation initiation factor 4GI (eIF4GI) serves as a central adapter in cap-binding complex assembly. Although eIF4GI has been shown to be sensitive to proteasomal degradation, how the eIF4GI steady-state level is controlled remains unknown. Here, we show that eIF4GI exists in a complex with NAD(P)H quinone-oxydoreductase 1 (NQO1) in cell extracts. Treatment of cells with dicumarol (dicoumarol), a pharmacological inhibitor of NQO1 known to preclude NQO1 binding to its protein partners, provokes eIF4GI degradation by the proteasome. Consistently, the eIF4GI steady-state level also diminishes upon the silencing of NQO1 (by transfection with small interfering RNA), while eIF4GI accumulates upon the overexpression of NQO1 (by transfection with cDNA). We further reveal that treatment of cells with dicumarol frees eIF4GI from mRNA translation initiation complexes due to strong activation of its natural competitor, the translational repressor 4E-BP1. As a consequence of cap-binding complex dissociation and eIF4GI degradation, protein synthesis is dramatically inhibited. Finally, we show that the regulation of eIF4GI stability by the proteasome may be prominent under oxidative stress. Our findings assign NQO1 an original role in the regulation of mRNA translation via the control of eIF4GI stability by the proteasome.

International audience; Although contact inhibition is a fundamental process for multicellular organisms, how proliferation is inhibited at high cellular densities remains poorly characterized. Here we show that 4E-BP1, one major repressor of cap-dependent translation, plays a critical role in density-mediated cell cycle arrest. 4E-BP1 promoter is activated and 4E-BP1 protein amount increases as cells reach confluence. Conversely, a much less marked density-dependent inhibition of cell proliferation is observed upon 4E-BP1 silencing. We further show that at high density, progression through the G(1) phase of the cell cycle is faster and Cyclin D1 protein is induced in different cell types where 4E-BP1 has been either downregulated (stable shRNA expression or transient siRNA transfection) or removed (knockout). Thus 4E-BP1 appears as an important mediator of contact inhibition.

International audience; The somatostatin receptor subtype 2 (sst2) behaves as a tumor suppressor when expressed and stimulated by its ligand somatostatin in pancreatic cancer. We reveal a mechanism underlying oncosuppressive action of sst2, whereby this inhibitory receptor upregulates the expression of the secreted angioinhibitory factor thrombospondin-1 (TSP-1), as demonstrated in exocrine BxPC-3 and endocrine BON pancreatic cancer cells. The sst2-dependent upregulation of TSP-1 occurs through the inhibition of the PI3K pathway. It depends on transcriptional and translational events, involving a previously undescribed IRES in the 5'-UTR of TSP-1 mRNA. Chick chorioallantoic membrane was used as an in vivo model to demonstrate that TSP-1 is a critical effector of the inhibitory role of sst2 on the neoangiogenesis and oncogenesis induced by pancreatic cancer cells. TSP-1 reduced in vitro tubulogenesis of endothelial cells when grown in conditioned medium from pancreatic cancer cells expressing sst2, as compared to those expressing the control vector. TSP-1 inhibited tumor cell-induced neoangiogenesis by directly sequestering the proangiogenic factor VEGF, and inactivating the angiogenesis initiated by VEGFR2 phosphorylation in endothelial cells. Using human pancreatic tissue-microarrays, the expression of both sst2 and TSP-1 was shown to be correlated during the pancreatic neoplastic program. Both proteins are nearly undetectable in normal exocrine pancreas and in most invasive cancer lesions, but their expression is strikingly upregulated in most preinvasive cancer-adjacent lesions. The upregulation of both sst2 and TSP-1 tumor suppressors may function as an early negative feedback to restrain pancreatic carcinogenesis.