Stéphane Pyronnet

Irresistible IRES. Attracting the translation machinery to internal ribosome entry sites.

Studies on the control of eukaryotic translation initiation by a cap‐independent recruitment of the 40S ribosomal subunit to internal messenger RNA sequences called internal ribosome entry sites (IRESs) have shown that these sequence elements are present in a growing list of viral and cellular RNAs. Here we discuss their prevalence, mechanisms whereby they may function and their uses in regulating gene expression.

Molecular Signaling of Somatostatin Receptors

Abstract: 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.

Antitumor effects of somatostatin

Since its discovery three decades ago as an inhibitor of GH release from the pituitary gland, somatostatin has attracted much attention because of its functional role in the regulation of a wide variety of physiological functions in the brain, pituitary, pancreas, gastrointestinal tract, adrenals, thyroid, kidney and immune system. In addition to its negative role in the control of endocrine and exocrine secretions, somatostatin and analogs also exert inhibitory effects on the proliferation and survival of normal and tumor cells. Over the past 15 years, studies have begun to reveal some of the molecular mechanisms underlying the antitumor activity of somatostatin. This review covers the present knowledge in the antitumor effect of somatostatin and analogs and discusses the perspectives of novel clinical strategies based on somatostatin receptor sst2 gene transfer therapy.

Netrin-1 Mediates Early Events in Pancreatic Adenocarcinoma Progression, Acting on Tumor and Endothelial Cells

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

Current Scientific Rationale for the Use of Somatostatin Analogs and mTOR Inhibitors in Neuroendocrine Tumor Therapy

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 preferentially target subtype 2) and thus has a broader spectrum of activity. Everolimus is a rapamycin analog that inhibits mTOR, but it is more soluble than rapamycin and can be administered orally. mTOR is a protein kinase involved in many signaling pathways, primarily those initiated by tyrosine kinase receptors. Sustained mTOR activity leads to the induction of cell growth, proliferation, and cell survival. Everolimus therefore has obvious potential in cancer therapy. CONCLUSIONS The combination of somatostatin analogs and everolimus in therapeutic trials offers a promising treatment option for neuroendocrine tumors.

Restoration of Functional Gap Junctions through Internal Ribosome Entry Site-Dependent Synthesis of Endogenous Connexins in Density-Inhibited Cancer Cells

ABSTRACT Gap junctions are composed of connexins and are critical for the maintenance of the differentiated state. Consistently, connexin expression is impaired in most cancer cells, and forced expression of connexins following cDNA transfection reverses the tumor phenotype. We have found that the restoration of density inhibition of human pancreatic cancer cells by the antiproliferative somatostatin receptor 2 (sst2) is due to overexpression of endogenous connexins Cx26 and Cx43 and consequent formation of functional gap junctions. Immunoblotting along with protein metabolic labeling and mRNA monitoring revealed that connexin expression is enhanced at the level of translation but is not sensitive to the inhibition of cap-dependent translation initiation. Furthermore, we identified a new internal ribosome entry site (IRES) in the Cx26 mRNA. The activity of Cx26 IRES and that of the previously described Cx43 IRES are enhanced in density-inhibited cells. These data indicate that the restoration of functional gap junctions is likely a critical event in the antiproliferative action of the sst2 receptor. We further suggest that the existence of IRESes in connexin mRNAs permits connexin expression in density-inhibited or differentiated cells, where cap-dependent translation is generally reduced.

Pharmacological targeting of the protein synthesis mTOR/4E‐BP1 pathway in cancer‐associated fibroblasts abrogates pancreatic tumour chemoresistance

Pancreatic ductal adenocarcinoma (PDAC) is extremely stroma‐rich. Cancer‐associated fibroblasts (CAFs) secrete proteins that activate survival and promote chemoresistance of cancer cells. Our results demonstrate that CAF secretome‐triggered chemoresistance is abolished upon inhibition of the protein synthesis mTOR/4E‐BP1 regulatory pathway which we found highly activated in primary cultures of α‐SMA‐positive CAFs, isolated from human PDAC resections. CAFs selectively express the sst1 somatostatin receptor. The SOM230 analogue (Pasireotide) activates the sst1 receptor and inhibits the mTOR/4E‐BP1 pathway and the resultant synthesis of secreted proteins including IL‐6. Consequently, tumour growth and chemoresistance in nude mice xenografted with pancreatic cancer cells and CAFs, or with pieces of resected human PDACs, are reduced when chemotherapy (gemcitabine) is combined with SOM230 treatment. While gemcitabine alone has marginal effects, SOM230 is permissive to gemcitabine‐induced cancer cell apoptosis and acts as an antifibrotic agent. We propose that selective inhibition of CAF protein synthesis with sst1‐directed pharmacological compounds represents an anti‐stromal‐targeted therapy with promising chemosensitization potential.

Pancreatic cell plasticity and cancer initiation induced by oncogenic Kras is completely dependent on wild-type PI 3-kinase p110α

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.

4E‐BP1 is a target of Smad4 essential for TGFβ‐mediated inhibition of cell proliferation

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 TGFβ‐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 TGFβ 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 TGFβ. Thus, 4E‐BP1 gene appears critical for TGFβ/Smad4‐mediated inhibition of cell proliferation.

Pancreatic tumours escape from translational control through 4E-BP1 loss

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.