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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.

ObjectiveIntercellular communication within pancreatic ductal adenocarcinoma (PDAC) dramatically contributes to metastatic processes. The underlying mechanisms are poorly understood, resulting in a lack of targeted therapy to counteract stromal-induced cancer cell aggressiveness. Here, we investigated whether ion channels, which remain understudied in cancer biology, contribute to intercellular communication in PDAC.DesignWe evaluated the effects of conditioned media from patient-derived cancer-associated fibroblasts (CAFs) on electrical features of pancreatic cancer cells (PCC). The molecular mechanisms were deciphered using a combination of electrophysiology, bioinformatics, molecular and biochemistry techniques in cell lines and human samples. An orthotropic mouse model where CAF and PCC were co-injected was used to evaluate tumour growth and metastasis dissemination. Pharmacological studies were carried out in the Pdx1-Cre, Ink4afl/flLSL-KrasG12D(KICpdx1) mouse model.ResultsWe report that the K+channel SK2 expressed in PCC is stimulated by CAF-secreted cues (8.84 vs 2.49 pA/pF) promoting the phosphorylation of the channel through an integrin–epidermal growth factor receptor (EGFR)–AKT (Protein kinase B) axis. SK2 stimulation sets a positive feedback on the signalling pathway, increasing invasiveness in vitro (threefold) and metastasis formation in vivo. The CAF-dependent formation of the signalling hub associating SK2 and AKT requires the sigma-1 receptor chaperone. The pharmacological targeting of Sig-1R abolished CAF-induced activation of SK2, reduced tumour progression and extended the overall survival in mice (11.7 weeks vs 9.5 weeks).ConclusionWe establish a new paradigm in which an ion channel shifts the activation level of a signalling pathway in response to stromal cues, opening a new therapeutic window targeting the formation of ion channel-dependent signalling hubs.

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).

The MNK1 protein kinase is directly activated by the MAPK pathway and is specifically expressed in pancreatic acinar cells. Both the MNK1 kinase and the MAPK pathway are required for response to pancreatitis, suggesting that their pharmacological targeting would be of therapeutic interest. Because the mRNA cap-binding protein and translation initiation factor eIF4E is the major known MNK1 substrate, one could anticipate that the protective function of MNK1 in pancreatitis is mediated by eIF4E phosphorylation.Acute pancreatitis was induced by the intraperitoneal administration of cerulein in wild-type mice and in transgenic mice carrying two non-phosphorylatable Eif4e alleles. The expression and phosphorylation of proteins of the MNK1-eIF4E pathway was visualized by western-blotting. The severity of pancreatitis was monitored by the measure of serum amylase levels and by histopathology and immunohistochemistry using apoptosis and immune infiltrate markers.Despite a strong induction in MNK1 kinase activity in both wild-type and transgenic mice, precluding eIF4E phosphorylation has no impact on the severity of acute pancreatitis. Serum amylase is equally induced in both mouse genotypes and neither acinar cell apoptosis nor immune infiltrate is exacerbated.eIF4E phosphorylation is not required for response to pancreatitis indicating that the acinar-cell-specific MNK1 kinase acts in acute pancreatitis via another substrate.

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.

Introduction L’octreotide a une activite anti-secretoire sur les insulinomes, en ciblant notamment le sous type 2 du recepteur a la somatostatine (SSTR2). Recemment, une mutation somatique T372R du facteur de transcription YY1 a ete identifiee dans 30 % des insulinomes. Des analyses de transcriptomes suggerent qu’elle augmenterait leur niveau d’expression de SSTR2. L’objectif de ce travail etait de rechercher une correlation entre la mutation d’YY1, l’expression de SSTR2 et la reponse anti-secretoire a l’octreotide dans les insulinomes. Materiel et methode Nous avons analyse 49 echantillons d’insulinomes de patients operes dans quatre CHU francais, pour lesquels la reponse a l’octreotide avait ete evaluee au prealable. La mutation YY1T372R a ete detectee par la technique de ddPCR, l’expression de SSTR2 par immuno-histochimie (IHC). Resultats Huit des 49 insulinomes (16 %) presentaient une mutation YY1T372R. Cette mutation etait associee a une expression de SSTR2 en IHC plus frequente dans les insulinomes (88 % vs 37 % ; p = 0,02), ainsi qu’a une reponse anti-secretoire a l’octreotide plus frequente (88 % vs 34 % ; p = 0,02). Les patients presentant un insulinome mute YY1T372R etaient plus âges que ceux avec un insulinome non mute (64,5 ans vs 50,0 ans ; p = 0,04). Il n’y avait pas de difference significative entre les deux groupes sur le reste des parametres etudies (sexe, NEM1, taille de l’insulinome, grade OMS). Discussion En conclusion, la mutation YY1T372R semble associee a la reponse a l’octreotide dans les insulinomes, probablement via une augmentation de la transcription du gene SSTR2.

International audience

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.

International audience

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.

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.

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.

Increasingly, evidence suggests that phosphorylation of the mRNA translation initiation factor eIF4E plays an important role in carcinogenesis, downstream of Ras. The eIF4E factor is phosphorylated by MAPK-interacting protein kinases 1 and 2 (MNK1 and MNK2). Due to alternative splicing, two MNK2 proteins exist (MNK2a and MNK2b). While MNK2a possesses a binding site for the stress-induced p38-MAPK, MNK2b does not. Recently, Maimon et al. revealed that a splicing shift towards the MNK2b isoform, in Ras-activated cells, sustains transformation, due to a defect in p38-induced cell death, while the MNK2b-dependent phosphorylation of eIF4E is maintained.

L’octreotide, un analogue de la somatostatine, peut etre une option therapeutique pour controler les hypoglycemies des patients atteints d’un insulinome, lorsque la chirurgie (seul traitement curatif) n’est pas realisable. L’octreotide agit via le sous-type 2 des recepteurs de somatostatine (sst2) et le 5 (sst5) dans une moindre mesure. De nombreux cas cliniques ont demontre que malgre l’expression de sst2, la reponse therapeutique (normalisation de la glycemie) n’etait pas systematique apres traitement a l’octreotide. Nous avons donc evalue la relation entre l’efficacite therapeutique de l’octreotide et la presence de sst2. Une analyse proteique (immunohistochimie (IHC) et western blot (WB)) et de l’ARNm (RT-qPCR) de sst2 a ete realisee pour 32 patients traites par octreotide dans le cadre d’un insulinome. La presence de la proteine sst2 (IHC et/ou WB) a ete retrouvee chez 17 patients dont 13 repondent au traitement (taux de reponse de 76 % en presence de sst2). La reponse a l’octreotide est independante de la localisation membranaire et/ou cytosolique de sst2. A l’inverse, moins de 14 % des patients repondent en l’absence d’expression proteique de sst2 (2/15 patients). Enfin, en dessous d’un certain seuil d’amplification de l’ARN sst2 (DeltaCt > a 7,1), il n’y a plus de reponse therapeutique. Le prolongement de cette etude permettra de caracteriser les autres sous-types de recepteur de somatostatine (sst5, 3 et 1) presentant une forte affinite pour les analogues de somatostatine de deuxieme generation (pasireotide). Ces derniers pourraient alors etre proposes comme traitement aux patients n’exprimant pas sst2.

Pancreatic ductal adenocarcinoma (PDAC) is extremely rich in stroma. By secreting tremendous amounts of soluble and un-soluble proteins, stromal cancer-associated fibroblasts (CAFs) can induce tumor chemoresistance by activating survival signals in cancer cells and preventing drug delivery through increased fibrosis. Our results demonstrate that chemoresistance triggered by the CAF secretome is strongly reduced upon inhibition of CAF protein synthesis. This is achieved by impinging upon the PI3K/mTOR/4E-BP1 pathway that we found highly activated in primary cultures of alpha-SMA-positive CAFs isolated from human PDAC. CAFs, but not normal pancreatic fibroblasts, express primarily the sstr1 somatostatin receptor subtype whose activation by the somatostatin analogue SOM230 (Pasireotide) strongly inhibits the PI3K/mTOR/4E-BP1 pathway and the resulting synthesis of secreted proteins including IL-6 and collagen-I. As a consequence, the growth and chemoresistance of pancreatic tumors provided by co-injection of CAFs in nude mice are overcome when chemotherapy (such as gemcitabine) is combined to pasireotide treatment. While gemcitabine or pasireotide alone have marginal effects, the combined treatment markedly decreases fibrosis and strongly enhances cancer cell apoptosis due to increased cell sensitivity to gemcitabine. We propose that selective inhibition of CAF protein synthesis and secretion with sstr1-directed pharmacological compounds represents an anti-stromal targeted therapy with a promising potential in chemosensitization. Citation Format: Camille Duluc, Siham Moatassim, Mounira Chalabi, Aurélie Perraud, Yvan Martineau, Florence Breibach, Marie-Bernadette Delisle, Muriel Mathonnet, Herbert A. Schmid, Stéphane Pyronnet, Corinne Bousquet. Pasireotide reduces chemoresistance in pancreatic tumor cells by inhibiting the synthesis and secretion of growth factors from tumor associated fibroblasts. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 402. doi:10.1158/1538-7445.AM2015-402

Colorectal cancer (CRC) represents the second leading cause of cancer-related death worldwide. One of the main reasons for this high mortality rate is because CRC develops into invasive lesions with high metastasis potential. The cancer-associated fibroblasts (CAFs) present in the tumour reactive stroma promote epithelial cancer cell motility and aggressiveness. However, whereas the role of tumour-stroma crosstalks in cancer progression is increasingly documented, how “normal” resident fibroblasts are converted into pro-invasive CAFs and how CAFs participate to the epithelial-stroma dialogue remains an open question. Precursor of the hormone gastrin, progastrin, now established as a growth factor for colon cells, is detected in colorectal polyps and tumours while it is absent in healthy colon. Using immunohistochemical and biochemical approaches we found that progastrin induces the expression of fibroblast activation markers (alphaSMA, FAPalpha) in the normal colon mucosa of progastrin-overexpressing mice as well as in the human colon fibroblast cell line CCD18Co. To investigate the role of progastrin-induced fibroblast activation, we compared the migration of colon epithelial cancer cells expressing or not the hormone in presence or absence of fibroblasts in Boyden chamber. Progastrin-expressing epithelial cells have increased migration capacities in presence of fibroblasts while not difference between the two epithelial cell lines was observed in absence of fibroblasts. Moreover, we identified the implication of different chemokines in this process. Thus, here we demonstrate that progastrin, secreted by tumour epithelial cells, contributes to the activation of the fibroblasts present in the cancer-associated stroma and participates to the epithelial-stroma dialogue. Citation Format: Nicolas Fénié, Claudine Bertrand, Aurélien Lacombe, Serge Roche, Corinne Bousquet, Valérie Gouazé-Andersson, Christine Toulas, Elizabeth Moyal, Audrey Ferrand. Progastrin activates colon fibroblasts and participates to the dialogue between tumor epithelial cells and stromal fibroblasts in colorectal cancer. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B16. doi:10.1158/1538-7445.CHTME14-B16

Pancreatic ductal adenocarcinoma (PDAC) correlate with a poor prognosis, because of its late diagnosis, its aggressiveness, and its propensity to metastasize quickly. New therapeutic strategies are urgently needed to improve this prognosis. One of these strategies is to target PDAC microenvironment, which is very dense and active and represents a large part of the tumor mass. It is composed of an extracellular matrix, endothelial cells, immune cells and cancer-associated fibroblasts (CAFs). Through physical and chemical relations, the microenvironment, and particularly CAFs and their secretions, promotes tumor progression at in the primary tumor and at metastatic sites. Our team has demonstrated an antitumor effect of a somatostatin analog, pasireotide or SOM230. Somatostatin is a hormone that inhibits many biological functions, including endocrine and exocrine secretions and cell proliferation, and with an anti-tumor effect demonstrated in different types of cancer. Via the receptor sst1 expressed by CAFs, pasireotide is able to block CAFs secretions, and thus restore pancreatic cancer cells sensitivity to chemotherapy and slow down tumor growth. The aim of my PhD consisted in demonstrating the efficacy of the combination of gemcitabine, the reference chemotherapy in PDAC, and the somatostatin analog SOM230 in mouse models faithfully mimicking the physiopathology of human PDAC. My results show a major effect of the combination of treatments on the development of metastases, both on orthotopic co-graft models and on the KPC genetic model. This effect is associated with a reduction in the recruitment of pro-tumor immune populations, particularly tumor-associated macrophages and MDSCs. We identified the macrophage growth factor CSF-1 as a key protein implicated in the immunomodulatory effect of CAFs, and its secretion is strongly reduced by the SOM230 treatment. Considering recent failures with treatment that aimed to suppress CAFs, target their secretions seems to be a more promising strategy in the treatment of PDAC.; L'adénocarcinome canalaire pancréatique (PDAC) est un cancer au pronostic sombre, en raison de son diagnostic souvent trop tardif, de son agressivité, et de sa propension à métastaser rapidement. De nouvelles stratégies thérapeutiques sont nécessaires rapidement pour améliorer ce pronostic. Une de ces stratégies consiste à cibler le microenvironnement de PDAC, très dense et actif, représentant une part importante de la masse tumorale. Il est composé d'une matrice extracellulaire, de cellules endothéliales, de cellules immunitaires et de fibroblastes associés au cancer (CAFs). Par l'intermédiaire de relations physiques et chimiques, le microenvironnement, et particulièrement les CAFs et leurs secrétions, favorise la progression tumorale au niveau de la tumeur primaire et sur les sites métastatiques. Notre équipe a mis en évidence un effet antitumoral d'un analogue de somatostatine, le pasiréotide ou SOM230. La somatostatine est une hormone qui entraine une inhibition de nombreuses fonctions biologiques, notamment les sécrétions endocrines et exocrines et la prolifération cellulaire, et dont l'effet anti-tumoral est démontré dans différentes types de cancer. Via le récepteur sst1 exprimé par les CAFs, le pasiréotide est capable de bloquer les secrétions des CAFs, et ainsi de restaurer la sensibilité des cellules cancéreuses pancréatiques à la chimiothérapie et de ralentir la croissance tumorale. Mes travaux de thèse consistaient à démontrer l'efficacité de l'association de gemcitabine, la chimiothérapie de référence dans le PDAC, et de l'analogue de somatostatine SOM230 dans des modèles murins mimant fidèlement la physiopathologie du PDAC humain. Mes résultats montrent un effet majeur de la combinaison des traitements sur le développement de métastases, à la fois sur les modèles de co-greffes orthotopique et sur le modèle génétique KPC. Cet effet est associé à une réduction du recrutement de populations immunitaires pro-tumorales, particulièrement les macrophages associés aux tumeurs et les MDSCs. Nous avons identifié le facteur de croissance des macrophages CSF-1 comme une protéine clé dans l'effet immunomodulateur des CAFs, et dont la sécrétion est réduite par le traitement au SOM230. En considérant les récents échecs de traitements visant à supprimer les CAFs, il semble que le ciblage de leurs secrétions soit une stratégie plus prometteuse dans le traitement du PDAC.

Pancreatic ductal adenocarcinoma (PDAC) correlate with a poor prognosis, because of its late diagnosis, its aggressiveness, and its propensity to metastasize quickly. New therapeutic strategies are urgently needed to improve this prognosis. One of these strategies is to target PDAC microenvironment, which is very dense and active and represents a large part of the tumor mass. It is composed of an extracellular matrix, endothelial cells, immune cells and cancer-associated fibroblasts (CAFs). Through physical and chemical relations, the microenvironment, and particularly CAFs and their secretions, promotes tumor progression at in the primary tumor and at metastatic sites. Our team has demonstrated an antitumor effect of a somatostatin analog, pasireotide or SOM230. Somatostatin is a hormone that inhibits many biological functions, including endocrine and exocrine secretions and cell proliferation, and with an anti-tumor effect demonstrated in different types of cancer. Via the receptor sst1 expressed by CAFs, pasireotide is able to block CAFs secretions, and thus restore pancreatic cancer cells sensitivity to chemotherapy and slow down tumor growth. The aim of my PhD consisted in demonstrating the efficacy of the combination of gemcitabine, the reference chemotherapy in PDAC, and the somatostatin analog SOM230 in mouse models faithfully mimicking the physiopathology of human PDAC. My results show a major effect of the combination of treatments on the development of metastases, both on orthotopic co-graft models and on the KPC genetic model. This effect is associated with a reduction in the recruitment of pro-tumor immune populations, particularly tumor-associated macrophages and MDSCs. We identified the macrophage growth factor CSF-1 as a key protein implicated in the immunomodulatory effect of CAFs, and its secretion is strongly reduced by the SOM230 treatment. Considering recent failures with treatment that aimed to suppress CAFs, target their secretions seems to be a more promising strategy in the treatment of PDAC.; L'adénocarcinome canalaire pancréatique (PDAC) est un cancer au pronostic sombre, en raison de son diagnostic souvent trop tardif, de son agressivité, et de sa propension à métastaser rapidement. De nouvelles stratégies thérapeutiques sont nécessaires rapidement pour améliorer ce pronostic. Une de ces stratégies consiste à cibler le microenvironnement de PDAC, très dense et actif, représentant une part importante de la masse tumorale. Il est composé d'une matrice extracellulaire, de cellules endothéliales, de cellules immunitaires et de fibroblastes associés au cancer (CAFs). Par l'intermédiaire de relations physiques et chimiques, le microenvironnement, et particulièrement les CAFs et leurs secrétions, favorise la progression tumorale au niveau de la tumeur primaire et sur les sites métastatiques. Notre équipe a mis en évidence un effet antitumoral d'un analogue de somatostatine, le pasiréotide ou SOM230. La somatostatine est une hormone qui entraine une inhibition de nombreuses fonctions biologiques, notamment les sécrétions endocrines et exocrines et la prolifération cellulaire, et dont l'effet anti-tumoral est démontré dans différentes types de cancer. Via le récepteur sst1 exprimé par les CAFs, le pasiréotide est capable de bloquer les secrétions des CAFs, et ainsi de restaurer la sensibilité des cellules cancéreuses pancréatiques à la chimiothérapie et de ralentir la croissance tumorale. Mes travaux de thèse consistaient à démontrer l'efficacité de l'association de gemcitabine, la chimiothérapie de référence dans le PDAC, et de l'analogue de somatostatine SOM230 dans des modèles murins mimant fidèlement la physiopathologie du PDAC humain. Mes résultats montrent un effet majeur de la combinaison des traitements sur le développement de métastases, à la fois sur les modèles de co-greffes orthotopique et sur le modèle génétique KPC. Cet effet est associé à une réduction du recrutement de populations immunitaires pro-tumorales, particulièrement les macrophages associés aux tumeurs et les MDSCs. Nous avons identifié le facteur de croissance des macrophages CSF-1 comme une protéine clé dans l'effet immunomodulateur des CAFs, et dont la sécrétion est réduite par le traitement au SOM230. En considérant les récents échecs de traitements visant à supprimer les CAFs, il semble que le ciblage de leurs secrétions soit une stratégie plus prometteuse dans le traitement du PDAC.