Pharmacological targeting of the protein synthesis mTOR /4E‐ BP 1 pathway in cancer‐associated fibroblasts abrogates pancreatic tumour chemoresistance

• DOI: 10.15252/emmm.201404346
• Publication Date: 2015-04-02T14:57:00Z
• Authors: Camille Duluc; Siham Moatassim‐B...; Mounira Chalabi‐D...; Aurélie Perraud; Rémi Samain; Florence Breibach; Marion Gayral; Pierre Cordelier; Marie‐Bernadette ...; Marie‐Pierre Bous...; Richard Tomasini; Herbert Schmid; Muriel Mathonnet; Stéphane Pyronnet; Yvan Martineau; Corinne Bousquet
• Keywords: 0301 basic medicine; STELLATE CELLS; Medicine (General); Pancreatic Neuroendocrine Tumors; Epidemiology; [SDV]Life Sciences [q-bio]; pancreatic cancer; Drug Resistance; MICROENVIRONMENT; Cell Cycle Proteins; QH426-470; Signal transduction; THERAPY; Deoxycytidine; Biochemistry; pharmacotherapy; MOUSE MODELS; Tumor Microenvironment; TUMOR PROGRESSION; Internal medicine; Research Articles; Cells, Cultured; STARRING ROLE; Cancer; PI3K PATHWAY; TOR Serine-Threonine Kinases; chemoresistance; Life Sciences; 3. Good health; Chemistry; Treatment Outcome; Oncology; Hedgehog Signaling in Development and Cancer; PI3K/AKT/mTOR pathway; Heterografts; Medicine; Somatostatin; cancer-associated fibroblasts; Carcinoma, Pancreatic Ductal; Pancreatic Cancer Research and Treatment; STROMA; 610; Mice, Nude; DUCTAL ADENOCARCINOMA; [SDV.CAN]Life Sciences [q-bio]/Cancer; Antineoplastic Agents; Cancer research; Adenocarcinoma; Tumor Classification; 03 medical and health sciences; R5-920; [SDV.CAN] Life Sciences [q-bio]/Cancer; Biochemistry, Genetics and Molecular Biology; Health Sciences; Genetics; Animals; Humans; Molecular Biology; Biology; Adaptor Proteins, Signal Transducing; cancer‐associated fibroblasts; Epidemiology and Management of Neuroendocrine Tumors; Pancreatic cancer; Fibroblasts; Phosphoproteins; Gemcitabine; SST2 SOMATOSTATIN RECEPTOR; Disease Models, Animal; protein synthesis and secretion

Research Article1 April 2015Open Access Source Data Pharmacological targeting of the protein synthesis mTOR/4E-BP1 pathway in cancer-associated fibroblasts abrogates pancreatic tumour chemoresistance Camille Duluc INSERM UMR-1037, Cancer Center Toulouse (CRCT), Equipe labellisée Ligue Contre le and Laboratoire d'Excellence (TOUCAN), Université de Toulouse, France Search for more papers by this author Siham Moatassim-Billah Biochemistry-Immunology Laboratory, Faculty Sciences Rabat, University Mohammed V – Agdal, Morocco Mounira Chalabi-Dchar Aurélie Perraud EA 3842 Medicine Pharmacy Faculties, Limoges University, Limoges, Rémi Samain Florence Breibach Pathology Department, Hôpitaux Marion Gayral Pierre Cordelier Marie-Bernadette Delisle Marie-Pierre Bousquet-Dubouch CNRS UMR-5089, Institut Pharmacologie et Biologie structurale (IPBS), Richard Tomasini CRCM, INSERM, U1068; Paoli-Calmettes Institute; Aix-Marseille UM105; CNRS, UMR7258, Marseille, Herbert Schmid Novartis Pharmaceuticals, Bales, Switzerland Muriel Mathonnet Stéphane Pyronnet Yvan Martineau Corinne Bousquet Corresponding Author Information Duluc1, Moatassim-Billah1,2, Chalabi-Dchar1, Perraud3, Samain1, Breibach4, Gayral1, Cordelier1, Delisle4, Bousquet-Dubouch5, Tomasini6, Schmid7, Mathonnet3, Pyronnet1, Martineau1 1 1INSERM 2Biochemistry-Immunology 3EA 4Pathology 5CNRS 6CRCM, 7Novartis *Corresponding author. Tel: [phone]; E-mail: [email protected] EMBO Mol Med (2015)7:735-753https://doi.org/10.15252/emmm.201404346 PDFDownload PDF article text main figures. Peer ReviewDownload a summary editorial decision process including letters, reviewer comments responses to feedback. ToolsAdd favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Pancreatic ductal adenocarcinoma (PDAC) is extremely stroma-rich. Cancer-associated (CAFs) secrete proteins that activate survival promote cancer cells. Our results demonstrate CAF secretome-triggered abolished upon inhibition regulatory which we found highly activated primary cultures α-SMA-positive CAFs, isolated from human PDAC resections. CAFs selectively express sst1 somatostatin receptor. The SOM230 analogue (Pasireotide) activates receptor inhibits resultant secreted IL-6. Consequently, growth nude mice xenografted with cells or pieces resected PDACs, are reduced when chemotherapy (gemcitabine) combined treatment. While gemcitabine alone has marginal effects, permissive gemcitabine-induced cell apoptosis acts as an antifibrotic agent. We propose selective sst1-directed pharmacological compounds represents anti-stromal-targeted therapy promising chemosensitization potential. Synopsis A novel drug association combining current chemotherapies Cancer-Associated Fibroblast secretome (Pasireotide), effectively reverses cancer. critically contributes stroma-triggered chemoresistance. Elevated PI3K/mTOR activity high synthesis/secretion rates. Inhibition fibroblasts, targets subtype not expressed non-activated α-SMA-negative combination fibroblast provides synergistic breakdown. Introduction one most intractable solid malignancies humans. rate at years < 5%. Due silent evolution several lack biomarkers, patients usually have late-stage cancer, metastasis time diagnosis. Surgery, only available strategy may increase rate, feasible very few cases (< 15%), patient rarely extends beyond years. Standard using targeted therapies directed molecular alterations provided almost no benefit clinical trials, despite cytostatic vitro vivo preclinical models (Hidalgo Von Hoff, 2012). Therapeutic inadequacy be attributed, part, under-estimation influences exerted microenvironment on cells, use do mimic critical feature (Singh al, 2010; Feig 2012; Perez-Mancera stroma-rich cancers, stroma forming than 80% mass. abundant present α-SMA (alpha-smooth muscle actin)-expressing (CAFs), pancreas also called stellate contains immune, inflammatory nerve blood vessels, surrounded acellular components form extracellular matrix (ECM) (Erkan 2012b; These features been observed other advanced stage carcinomas (e.g. breast cancer) (De Palma Hanahan, In pancreas, involved deposition ECM secretion soluble factors factors), regulate normal epithelial differentiation homeostasis (Apte Upon 'activation' during inflammation, principal source constituents considered mediators scar formation tissue fibrosis large amounts factors. Once wound repaired, resting phenotype thought restored. Conversely, organ fibrosis, site remain perpetually activated. fibrotic produced constitutes mechanical scaffold physical barrier against effective delivery therapeutic agents (Olive 2009). Antifibrotic therefore appears treatment PDAC, although it 'symptomatic' non-selective (Erkan, 2013). Besides secreting fibrillar components, growth, angiogenic factors, engage stromal metastatic signalling promotes invasion (Hwang 2008; Vonlaufen 2008). Importantly, signals stimulated redundant those therapies, conferring innate resistance 2013; Erkan, Since master 'secretors' insoluble these specific features, hypothesized would represent option PDAC. Further understanding mechanisms governing assist development overcome CAF-triggered resistance. manuscript, role activation promoting elucidated. Additionally, stop through proposed strategy, used Results exocrine essentially composed acinar whose network supported discrete matrix, mostly interlobular spaces around tubular structures (Supplementary Fig S1A, H&E Masson's trichrome stainings). contrast, rich α-SMA-expressing reside both inside boundaries between invasive host tissue. inactive state S1A). It shown confer chemoprotective 2008), though underlying and, consequently, chemoprotection unclear. To explore this, were established outgrowth method (Fig 1A) 2012a) fifteen surgically obtained tumours different disease stages Table S1). After days, migrated tissues exhibited fibroblast-like confirmed expression vimentin 'activated' since nearly 100% 1B). This was maintained throughout 10 passages before senescence occurred (not shown). By vimentin-positive (PaSCs), samples, did doubling-time period longer (6 days) PaSCs (2-days) S1B), Ki67 negative, whereas significant number positive S1C). Interestingly, two-fold PaSCs, measured their respective secretomes (hereafter referred conditioned media, CM) S1D). into correlated increased mediate novo (Meads Figure 1. but providing inhibited pre-treatment A. isolation resections (left panel) primo-culture (right panel). B. Isolated PaSC characterization immunofluorescence anti-vimentin anti-α-SMA antibody (one representative field n = 3). C. Experimental schematic representation. treated (10−7 M) 48 h. Conditioned media (CM) collected. incubated h indicated CM, presence (100 μg/ml). D, E. Panc-1 viability assessed MTT. (mean ± SD) presented each (CM PaSC, CAF, SOM230, SOM230) percentage gemcitabine-untreated (= 100%) (n 4; left right: **P 0.007, ##P 0.006 D; *P 0.032, #P 0.041, 0.047 E). F. Apoptosis induced evaluated Western blot anti-cleaved caspase-3 anti-PARP (representative G. analysed flow cytometry. Percentages annexin V-positive 3; 0.005, 0.004, 0.006). information: * effect (gemcitabine versus untreated cells; #CM-incubated non-incubated gemcitabine-treated data online figure. [emmm201404346-sup-0002-SourceDataFig1.pdf] Download figure PowerPoint verify possibility, chemosensitivity lines tested CM had grown without foetal calf serum (passages 3 8) 1C). An MTT assay demonstrated cytotoxic action 1D), 5-fluorouracil (5FU) oxaliplatin S2A–B) completely reversed co-treatment provide any chemoprotection. addition, heating 95°C significantly diminished capacity, suggesting S2C). derived paracrine peptides could drugs currently medical treatments inhibit excessive hormone/peptide secretions neuroendocrine tumours, analogues. test hypothesis, response gemcitabine, 5FU after previously multi-receptor (Pasireotide® Novartis) directly proliferation survival, nor reduce marker property dose-dependently once first 1E Supplementary S2D–F). When applied direct addition chemotherapeutic drugs, CAF-CM 1E). Various assays (caspase-3 PARP cleavage, 1F S2G–H; apoptotic 1G; executioner caspase activity, S3A) restored used. two additional lines, Capan-1 BxPC-3 S3B–D). calculated IC50 values cytotoxicity S3E–G. potential treating enhance sensitivity drugs. High mTORC1 responsible factors—phenotypic reversion relies elevated cytokines and/or chemokines. rates monitored SUnSET (a non-radioactive equivalent 35S-Met based puromycin incorporation nascent polypeptides) (Schmidt 2009) serum-starved incorporated much polypeptides 2A, compare lanes totally suppressed treatment, while detected 2A). inhibitory further polysomal fractionation assay, fewer polysomes (containing translated mRNAs) formed 2B). might due pathway, strong regulator mRNA translation. our previous showing analogues PI3K (Bousquet 2006). Consistently, Akt S6 appeared intrinsically phosphorylated (i.e. activated) fully sensitive constitutively hence 2C). Similarly, 4E-BP1, major target regulating translation (Martineau 2013), severely hypophosphorylated active inhibiting translation) 2D). global concentrations extracts dramatically decreased 2E) affect total RNA concentration S4A), indicating SOM230-triggered mTORC1/4E-BP1 axis. Silencing 4E-BP1 siRNA (si4E-BP1) 2F) circumvented effects 2G) CAF-CM-dependent protection gemcitabine-triggered 2H) 2I). Furthermore, least potent (RAD001) mTOR (PP242) inhibitors suppressing re-sensitizing S4B–C). 2. mediates cells—reversion Immunoblotting (+) h, anti-puromycin Polysomes profiles anti-P-Akt, anti-P-S6 anti-β-actin (loading control) 30 min times, anti-4E-BP1 Protein SOM230-treated normalized per × 106 0.008, 0.007). anti-GAPDH siCTR- si4E-BP1-transfected equal transfected siCTR si4E-BP-1. 0.003, §§P 0.002). Caspase-3 cleavage antibodies Arrow indicates cleaved forms PARP. # SOM230-untreated § siCTR-transfected 2 [emmm201404346-sup-0003-SourceDataFig2.pdf] constitutive neutralization translational repressor intrinsic permanently higher PaSCs. reveal abrogation its ability efficiently phosphorylation CAFs. Somatostatin five G protein-coupled receptors named sst1–sst5. However, low affinity sst4 (Schmid, 2008) compatible suspected involvement four receptors. qRT–PCR experiments performed revealed BON known contain levels (Xiao 2012) 3A). analyses showed neither 3B) 3C) sst1. Immunofluorescence localized 3D). Knock-down interference (siRNA sst1, sisst1) specificity signal anti-sst1 (Western immunofluorescence, S5A–B). confocal microscopy serial sections 42 PDACs cytokeratin-19 staining co-localize, demonstrating 3E). 69% 19 (quantified PDACs) 3F, S2A). stroma, all sst1-positive 3F). proportion (40% 16) (39% 17) 15 yielded (inhibited) (r 0.96), sst1-expressing 3G, S5C, Tables S2B–C). (sisst1) importance (GPCR) (dephosphorylation Akt, 4E-BP1) 3H–I), restoration 3J). alter 3H, top). affects via Accordingly,