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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; The unfolded protein response (UPR) is an evolutionarily conserved adaptive signaling pathway triggered by a stress of the endoplasmic reticulum (ER) lumen compartment, which is initiated by the accumulation of unfolded proteins. This response, mediated by three sensors-Inositol Requiring Enzyme 1 (IRE1), Activating Transcription Factor 6 (ATF6), and Protein Kinase RNA-Like Endoplasmic Reticulum Kinase (PERK)-allows restoring protein homeostasis and maintaining cell survival. UPR represents a major cytoprotective signaling network for cancer cells, which frequently experience disturbed proteostasis owing to their rapid proliferation in an usually unfavorable microenvironment. Increased basal UPR also participates in the resistance of tumor cells against chemotherapy. UPR activation also occurs during hematopoiesis, and growing evidence supports the critical cytoprotective role played by ER stress in the emergence and proliferation of leukemic cells. In case of severe or prolonged stress, pro-survival UPR may however evolve into a cell death program called terminal UPR. Interestingly, a large number of studies have revealed that the induction of proapoptotic UPR can also strongly contribute to the sensitization of leukemic cells to chemotherapy. Here, we review the current knowledge on the consequences of the deregulation of UPR signaling in leukemias and their implications for the treatment of these diseases.

International audience; The most frequent genetic alteration in acute myeloid leukemia (AML) is the mutation of nucleophosmin 1 (NPM1). Yet, its downstream oncogenic routes are not fully understood. Here, we report the identification of one long noncoding RNA (lncRNA) overexpressed in NPM1-mutated AML patients (named LONA) whose intracellular localization inversely reflects that of NPM1. While NPM1 is nuclear and LONA cytoplasmic in wild-type NPM1 AML cells, LONA becomes nuclear as mutant NPM1 moves toward the cytoplasm. Gain or loss of function combined with a genome-wide RNA-seq search identified a set of LONA mRNA targets encoding proteins involved in myeloid cell differentiation (including THSB1, MAFB, and ASB2) and interaction with its microenvironment. Consistently, LONA overexpression in mutant NPM1 established cell lines and primary AML cells exerts an anti-myeloid differentiation effect, whilst it exerts an opposite promyeloid differentiation effect in a wild type NPM1 setting. In vivo, LONA overexpression acts as an oncogenic lncRNA reducing the survival of mice transplanted with AML cells and rendering AML tumors more resistant to AraC chemotherapy. These data indicate that mutation-dependent nuclear export of NPM1 leads to nuclear retention and consequent oncogenic functions of the overexpressed lncRNA LONA, thus uncovering a novel NPM1 mutation-dependent pathway in AML pathogenesis.

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

International audience; One essential step of museum and clinical specimen preservation is immersion in a fixative fluid to prevent degradation. Formalin is the most largely used fixative, but its benefit is balanced with its toxic and carcinogenic status. Moreover, because formalin-fixation impairs nucleic acids recovery and quality, current museum wet collections and formalin- fixed, paraffin-embedded clinical samples do not represent optimal tanks of molecular information. Our study has been developed to compare formalin to two alternative fixatives (RCL2® and ethanol) in a context of molecular exploitation. Based on a unique protocol, we created mammalian fixed collections, simulated the impact of time on preservationusing an artificial ageing treatment and followed the evolution of specimens’ DNA quality. DNA extraction yield, purity, visual integrity and qualitative and quantitative ability to amplify the Cox1 gene were assessed. Our results show that both RCL2 and ethanol exhibit better performances than formalin. They do not impair DNA extraction yield, and more importantly, DNA alteration is delayed over the preservation step. The use of RCL2 or ethanol as fixative in biological collections may insure a better exploitation of the genetic resources they propose.

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.

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.

The vascular endothelial growth factor VEGF-D promotes metastasis by inducing lymphangiogenesis and dilatation of the lymphatic vasculature, facilitating tumor cell extravasion. Here we report a novel level of control for VEGF-D expression at the level of protein translation. In human tumor cells, VEGF-D colocalized with eIF4GI and 4E-BP1, which can program increased initiation at IRES motifs on mRNA by the translational initiation complex. In murine tumors, the steady-state level of VEGF-D protein was increased despite the overexpression and dephosphorylation of 4E-BP1, which downregulates protein synthesis, suggesting the presence of an internal ribosome entry site (IRES) in the 5′ UTR of VEGF-D mRNA. We found that nucleolin, a nucleolar protein involved in ribosomal maturation, bound directly to the 5′UTR of VEGF-D mRNA, thereby improving its translation following heat shock stress via IRES activation. Nucleolin blockade by RNAi-mediated silencing or pharmacologic inhibition reduced VEGF-D translation along with a subsequent constriction of lymphatic vessels in tumors. Our results identify nucleolin as a key regulator of VEGF-D expression, deepening understanding of lymphangiogenesis control during tumor formation. Cancer Res; 76(15); 4394–405. ©2016 AACR.

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.

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.

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Pancreatic cancer cells show varying sensitivity to the anticancer effects of gemcitabine. However, as a chemotherapeutic agent, gemcitabine can cause intolerably high levels of toxicity and patients often develop resistance to the beneficial effects of this drug. Combination studies show that use of gemcitabine with the pro-apoptotic cytokine TRAIL can enhance the inhibition of survival and induction of apoptosis of pancreatic cancer cells. Additionally, following combination treatment there is a dramatic increase in the level of the hypophosphorylated form of the tumour suppressor protein 4E-BP1. This is associated with inhibition of mTOR activity, resulting from caspase-mediated cleavage of the Raptor and Rictor components of mTOR. Use of the pan-caspase inhibitor Z-VAD-FMK indicates that the increase in level of 4E-BP1 is also caspase-mediated. ShRNA-silencing of 4E-BP1 expression renders cells more resistant to cell death induced by the combination treatment. Since the levels of 4E-BP1 are relatively low in untreated pancreatic cancer cells these results suggest that combined therapy with gemcitabine and TRAIL could improve the responsiveness of tumours to treatment by elevating the expression of 4E-BP1.

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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.

Somatostatin is an endogenous inhibitor of secretion and cell proliferation. These features render somatostatin a logical candidate for the management of neuroendocrine tumors that express somatostatin receptors. Synthetic somatostatin analogs (SSAs) have longer half-lives than somatostatin, but have similar activities, and are used for the treatment of these types of disorders. Interest has focused on novel multireceptor analogs with broader affinity to several of the five somatostatin receptors, thereby presenting putatively higher antitumor activities. Recent evidence indicates that SSAs cannot be considered mimics of native somatostatin in regulating signaling pathways downstream of receptors. Here we review this knowledge, discuss the concept of biased agonism, and highlight what considerations need to be taken into account for the optimal clinical use of SSAs. Refereed/Peer-reviewed

The simultaneous interaction of poly(A)-binding protein (PABP) with eukaryotic translation initiation factor 4G (eIF4G) and the mRNA 3' poly(A) tail promotes translation initiation. We previously showed that the interaction of PABP-interacting protein 1 (Paip1) with PABP and eukaryotic translation initiation factor 3 (eIF3; via the eIF3g subunit) further stimulates translation. Here, we demonstrate that the interaction of eIF3 with Paip1 is regulated by amino acids through the mTORC1 signaling pathway. The Paip1-eIF3 interaction is impaired by the mTORC1 inhibitors, rapamycin and PP242. We show that ribosomal protein S6 kinases 1 and 2 (S6K1/2) promote the interaction of eIF3 with Paip1. The enhancement of Paip1-eIF3 interaction by amino acids is abrogated by an S6K inhibitor or shRNA against S6K1/2. S6K1 interacts with eIF3f and, in vitro, phosphorylates eIF3. Finally, we show that S6K inhibition leads to a reduction in translation by Paip1. We propose that S6K1/2 phosphorylate eIF3 to stimulate Paip1-eIF3 interaction and consequent translation initiation. Taken together, these data demonstrate that eIF3 is a new translation target of the mTOR/S6K pathway.

Anti-angiogenic and anti-lymphangiogenic drugs slow tumor progression and dissemination. However, an important difficulty is that a tumor reacts and compensates to obtain the blood supply needed for tumor growth and lymphatic vessels to escape to distant loci. Therefore, there is a growing consensus on the requirement of multiple anti-(lymph)angiogenic molecules to stop cell invasion efficiently. Here we studied the cooperation between endogenous anti-angiogenic molecules, endostatin and fibstatin, and a chemokine, the Platelet Factor-4 variant 1, CXCL4L1. Anti-angiogenic factors were co-expressed by IRES-based bicistronic vectors and their cooperation was analyzed either by local delivery following transduction of pancreatic adenocarcinoma cells with lentivectors, or by distant delivery resulting from intramuscular administration in vivo of adeno-associated virus derived vectors followed by tumor subcutaneous injection. In this study, fibstatin and CXCL4L1 cooperate to inhibit endothelial cell proliferation, migration and tubulogenesis in vitro. No synergistic effect was found for fibstatin–endostatin combination. Importantly, we demonstrated for the first time that fibstatin and CXCL4L1 not only inhibit in vivo angiogenesis, but also lymphangiogenesis and tumor spread to the lymph nodes, whereas no beneficial effect was found on tumor growth inhibition using molecule combinations compared to molecules alone. These data reveal the synergy of CXCL4L1 and fibstatin in inhibition of tumor angiogenesis, lymphangiogenesis and metastasis and highlight the potential of IRES-based vectors to develop anti-metastasis combined gene therapies.

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.

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.

International audience; Fibroblast growth factor 1 (FGF1) is involved in muscle development and regeneration. The FGF1 gene contains four tissue-specific promoters allowing synthesis of four transcripts with distinct leader regions. Two of these transcripts contain internal ribosome entry sites (IRESs), which are RNA elements allowing mRNA translation to occur in conditions of blockade of the classical cap-dependent mechanism. Here, we investigated the function and the regulation of FGF1 during muscle differentiation and regeneration. Our data show that FGF1 protein expression is induced in differentiating myoblasts and regenerating mouse muscle, whereas siRNA knock-down demonstrated FGF1 requirement for myoblast differentiation. FGF1 induction occurred at both transcriptional and translational levels, involving specific activation of both promoter A and IRES A, whereas global cap-dependent translation was inhibited. Furthermore, we identified, in the FGF1 promoter A distal region, a cis-acting element able to activate the IRES A-driven translation. These data revealed a mechanism of molecular coupling of mRNA transcription and translation, involving a unique process of IRES activation by a promoter element. The crucial role of FGF1 in myoblast differentiation provides physiological relevance to this novel mechanism. This finding also provides a new insight into the molecular mechanisms linking different levels of gene expression regulation.

SummaryLoss of skeletal muscle mass and force aggravates age-related sarcopenia and numerous pathologies, such as cancer and diabetes. The AKT-mTORC1 pathway plays a major role in stimulating adult muscle growth; however, the functional role of its downstream mediators in vivo is unknown. Here, we show that simultaneous inhibition of mTOR signaling to both S6K1 and 4E-BP1 is sufficient to reduce AKT-induced muscle growth and render it insensitive to the mTORC1-inhibitor rapamycin. Surprisingly, lack of mTOR signaling to 4E-BP1 only, or deletion of S6K1 alone, is not sufficient to reduce muscle hypertrophy or alter its sensitivity to rapamycin. However, we report that, while not required for muscle growth, S6K1 is essential for maintaining muscle structure and force production. Hypertrophy in the absence of S6K1 is characterized by compromised ribosome biogenesis and the formation of p62-positive protein aggregates. These findings identify S6K1 as a crucial player for maintaining muscle function during hypertrophy.

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.

Hypoxia has recently been shown to activate the endoplasmic reticulum kinase PERK, leading to phosphorylation of eIF2alpha and inhibition of mRNA translation initiation. Using a quantitative assay, we show that this inhibition exhibits a biphasic response mediated through two distinct pathways. The first occurs rapidly, reaching a maximum at 1-2 h and is due to phosphorylation of eIF2alpha. Continued hypoxic exposure activates a second, eIF2alpha-independent pathway that maintains repression of translation. This phase is characterized by disruption of eIF4F and sequestration of eIF4E by its inhibitor 4E-BP1 and transporter 4E-T. Quantitative RT-PCR analysis of polysomal RNA indicates that the translation efficiency of individual genes varies widely during hypoxia. Furthermore, the translation efficiency of individual genes is dynamic, changing dramatically during hypoxic exposure due to the initial phosphorylation and subsequent dephosphorylation of eIF2alpha. Together, our data indicate that acute and prolonged hypoxia regulates mRNA translation through distinct mechanisms, each with important contributions to hypoxic gene expression.

Somatostatin is a neuropeptide that acts as an endogenous inhibitor of various cellular functions including endocrine and exocrine secretions and the proliferation of normal and tumour cells. Its action is mediated by a family of G-protein-coupled receptors (sst1–sst5) that are widely distributed in normal and tumour cells. Gastroenteropancreatic endocrine tumours express multiple somatostatin receptors, sst2 being clearly predominant. These receptors represent the molecular basis for the clinical use of somatostatin analogues in the treatment of endocrine tumours and their in vivo localisation. This review covers current knowledge in somatostatin receptor biology and signalling.

Somatostatin is a neuropeptide family that is produced by neuroendocrine, inflammatory, and immune cells in response to different stimuli. Somatostatin acts as an endogenous inhibitory regulator of various cellular functions including secretions, motility, and proliferation. Its action is mediated by a family of G-protein-coupled receptors (called sst1-sst5) that are widely distributed in the brain and periphery. The five receptors bind the natural peptides with high affinity, but only sst2, sst5, and sst3 bind the short synthetic analogs used to treat acromegaly and neuroendocrine tumors. This review covers the current knowledge in somatostatin receptor biology and signaling.

The G protein-coupled sst2 somatostatin receptor is a critical negative regulator of cell proliferation. sstII prevents growth factor-induced cell proliferation through activation of the tyrosine phosphatase SHP-1 leading to induction of the cyclin-dependent kinase inhibitor p27Kip1. Here, we investigate the signaling molecules linking sst2 to p27Kip1. In Chinese hamster ovary-DG-44 cells stably expressing sst2 (CHO/sst2), the somatostatin analogue RC-160 transiently stimulates ERK2 activity and potentiates insulin-stimulated ERK2 activity. RC-160 also stimulates ERK2 activity in pancreatic acini isolated from normal mice, which endogenously express sst2, but has no effect in pancreatic acini derived from sst2 knock-out mice. RC-160-induced p27Kip1 up-regulation and inhibition of insulin-dependent cell proliferation are both prevented by pretreatment of CHO/sst2 cells with the MEK1/2 inhibitor PD98059. In addition, using dominant negative mutants, we show that sst2-mediated ERK2 stimulation is dependent on the pertussis toxin-sensitive Gi/o protein, the tyrosine kinase Src, both small G proteins Ras and Rap1, and the MEK kinase B-Raf but is independent of Raf-1. Phosphatidylinositol 3-kinase (PI3K) and both tyrosine phosphatases, SHP-1 and SHP-2, are required upstream of Ras and Rap1. Taken together, our results identify a novel mechanism whereby a Gi/o protein-coupled receptor inhibits cell proliferation by stimulating ERK signaling via a SHP-1-SHP-2-PI3K/Ras-Rap1/B-Raf/MEK pathway.

Through its interaction with the 5′ translation initiation factor eIF4G, poly(A) binding protein (PABP) facilitates the translation of 5′-capped and 3′-poly(A)-tailed mRNAs. Rotavirus mRNAs are capped but not polyadenylated, instead terminating in a 3′ GACC motif that is recognized by the viral protein NSP3, which competes with PABP for eIF4G binding. Upon rotavirus infection, viral, GACC-tailed mRNAs are efficiently translated, while host poly(A)-tailed mRNA translation is, in contrast, severely impaired. To explore the roles of NSP3 in these two opposing events, the translational capabilities of three capped mRNAs, distinguished by either a GACC, a poly(A), or a non-GACC and nonpoly(A) 3′ end, have been monitored after electroporation of cells expressing all rotavirus proteins (infected cells) or only NSP3 (stably or transiently transfected cells). In infected cells, we found that the magnitudes of translation induction (GACC-tailed mRNA) and translation reduction [poly(A)-tailed mRNA] both depended on the rotavirus strain used but that translation reduction not genetically linked to NSP3. In transfected cells, even a small amount of NSP3 was sufficient to dramatically enhance GACC-tailed mRNA translation and, surprisingly, to slightly favor the translation of both poly(A)- and nonpoly(A)-tailed mRNAs, likely by stabilizing the eIF4E-eIF4G interaction. These data suggest that NSP3 is a translational surrogate of the PABP-poly(A) complex; therefore, it cannot by itself be responsible for inhibiting the translation of host poly(A)-tailed mRNAs upon rotavirus infection. IMPORTANCE To control host cell physiology and to circumvent innate immunity, many viruses have evolved powerful mechanisms aimed at inhibiting host mRNA translation while stimulating translation of their own mRNAs. How rotavirus tackles this challenge is still a matter of debate. Using rotavirus-infected cells, we show that the magnitude of cellular poly(A) mRNA translation differs with respect to rotavirus strains but is not genetically linked to NSP3. Using cells expressing rotavirus NSP3, we show that NSP3 alone not only dramatically enhances rotavirus-like mRNA translation but also enhances poly(A) mRNA translation rather than inhibiting it, likely by stabilizing the eIF4E-eIF4G complex. Thus, the inhibition of cellular polyadenylated mRNA translation during rotavirus infection cannot be attributed solely to NSP3 and is more likely the result of global competition between viral and host mRNAs for the cellular translation machinery.