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International audience; A library of ultra-small red photoluminescent gold nanoclusters (Au NCs) were synthesized with an increasing amount of positive charges provided by the addition of mono-, di-or trivalent-glutathione modified arginine peptides. We then studied how the arginine content impacted on the interaction of Au NCs with negatively charged artificial lipid bilayers and cell membranes. Results indicated that increasing the arginine content enhanced Au NCs' adsorption on lipid bilayers and on cell membranes followed by an increased cellular uptake in melanoma cells (COLO 829). Surprisingly, the presence of up to 40% serum for highly positively charged Au NCs did not hinder their interaction with lipid bilayers that contain glycolipids, suggesting a reduced opsonization of these Au NCs. In addition, these Au NCs are usually not toxic, except those with the highest arginine contents. Thus, controlled grafting of arginine peptides onto Au NCs is an elegant strategy to improve their binding and internalization by tumor cells while still keeping their anti-fouling properties.

Background Angiogenesis is the process by which new blood vessels arise from pre-existing ones. Fibroblast growth factor-2 (FGF-2), a leading member of the FGF family of heparin-binding growth factors, contributes to normal as well as pathological angiogenesis. Pre-mRNA alternative splicing plays a key role in the regulation of cellular and tissular homeostasis and is highly controlled by splicing factors, including SRSFs. SRSFs belong to the SR protein family and are regulated by serine/threonine kinases such as SRPK1. Up to now, the role of SR proteins and their regulators in the biology of endothelial cells remains elusive, in particular upstream signals that control their expression. Results By combining 2D endothelial cells cultures, 3D collagen sprouting assay, a model of angiogenesis in cellulose sponges in mice and a model of angiogenesis in zebrafish, we collectively show that FGF-2 promotes proliferation, survival, and sprouting of endothelial cells by activating a SRSF1/SRSF3/SRPK1-dependent axis. In vitro, we further demonstrate that this FGF-2-dependent signaling pathway controls VEGFR1 pre-mRNA splicing and leads to the generation of soluble VEGFR1 splice variants, in particular a sVEGFR1-ex12 which retains an alternative last exon, that contribute to FGF-2-mediated angiogenic functions. Finally, we show that sVEGFR1-ex12 mRNA level correlates with that of FGF-2/FGFR1 in squamous lung carcinoma patients and that sVEGFR1-ex12 is a poor prognosis marker in these patients. Conclusions We demonstrate that FGF-2 promotes angiogenesis by activating a SRSF1/SRSF3/SRPK1 network that regulates VEGFR1 alternative splicing in endothelial cells, a process that could also contribute to lung tumor progression. Supplementary Information The online version contains supplementary material available at 10.1186/s12915-021-01103-3.

Additional File 2: Figure S2. Schematic representation of VEGFR1 and VEGFR1 splice variants. sVEGFR1-ex15a results from activation of a cryptic 3’-splice acceptor site upon the use of an alternative polyadenylation site in the latter half of intron 14. sVEGFR1-i13 short and sVEGFR1-i13 long result from alternative polyadenylation at different sites in intron 13 to yield mRNAs encoding the same 867 amino acid sVEGFR1 protein isoform, but with either a 17 or 4146 nt 3’-UTR region. sVEGFR1-ex12 retains an alternative last exon (exon 12). The location of forward and reverse primers used in RT-PCR analyses are indicated as black arrows on each transcript. siRNA sequences target exon 12 for sVEGFR1-ex12 and the junction between exon 13 and retained intron 13 for sVEGFR1-i13. (PPTX 68 kb)

Additional File 1: Figure S1. Effects of FGFR (AZD4547) and SRPK1 (SRPIN340, SPHINX31) inhibitors on VEGF165b protein and total VEGF-A, VEGF121, VEGF165 and VEGF189 mRNA levels in endothelial cells treated or not with FGF-2. (a) Representative VEGF165b immunoblots in HUVEC and HDMEC treated or not (NT) for 72 hours with 3nM FGF-2 in the presence or absence of 10nM AZD4547 (FGFRinh), 10μM SRPIN340 or 5μM SPHINX31 as indicated. GAPDH was used as a loading control. Representative immunoblots of two (HUVEC) and three (HDMEC) independent experiments are presented. (b) HDMEC cells were treated (FGF) or not (NT) with 3nM FGF-2 for 72 hours in the presence or absence of 5μM SPHINX31 (SPH31) or 10μM SRPIN340 as indicated. Graphs represent mean values ± SD of normalized expression of each transcript according to GAPDH mRNA level in 3 independent experiments. For each transcript, the fold change was calculated with value 1 assigned to the normalized expression value obtained in the non treated (control) condition. Unpaired t test, *p

Paclitaxel is a microtubule stabilizing agent and a successful drug for cancer chemotherapy inducing, however, adverse effects. To reduce the effective dose of paclitaxel, we searched for pharmaceutics which could potentiate its therapeutic effect. We screened a chemical library and selected Carba1, a carbazole, which exerts synergistic cytotoxic effects on tumor cells grown in vitro, when co-administrated with a low dose of paclitaxel. Carba1 targets the colchicine binding-site of tubulin and is a microtubule-destabilizing agent. Catastrophe induction by Carba1 promotes paclitaxel binding to microtubule ends, providing a mechanistic explanation of the observed synergy. The synergistic effect of Carba1 with paclitaxel on tumor cell viability was also observed in vivo in xenografted mice. Thus, a new mechanism favoring paclitaxel binding to dynamic microtubules can be transposed to in vivo mouse cancer treatments, paving the way for new therapeutic strategies combining low doses of microtubule targeting agents with opposite mechanisms of action.

We report a method for deriving the absolute value of absorption coefficients at depth in bilayered media. The method was simplified from that of time-resolved diffuse optical tomography (TR-DOT) into one dimension to validate and set up the main parameters with the help of simulations, and to test it in an easy preclinical model. The method was applied to buried flaps as used in reconstructive surgery, and absolute chromophore concentrations in the flap and in the upper (skin and fat) layer were derived. The encouraging results obtained lay a foundation for developing more complex multidimensional models.

International audience; In parallel with the rapidly growing and widespread use of nanomedicine in the clinic, we are also witnessing the development of so-called theranostic agents that combine diagnostic and therapeutic properties. Among them, ultra-small gold nanoclusters (Au NCs) are showing promising potential due to their optical properties and activatable therapeutic activities under irradiation. Furthermore, due to their size smaller than 6 nm and unique biophysical properties, they also present intriguing behaviors in biological and physio-pathological environments. In this review, we aim to present the last researches published on such nanoparticles in animals. We also propose guidelines to identify the main physico-chemical parameters that govern Au NCs behavior after administration in small animals, notably concerning their renal elimination and their ability to accumulate in tumors. Then, we present recent advances for their use as theranostic agents putting them in parallel with others contrast agents.

International audience; In parallel with the rapidly growing and widespread use of nanomedicine in the clinic, we are also witnessing the development of so-called theranostic agents that combine diagnostic and therapeutic properties. Among them, ultra-small gold nanoclusters (Au NCs) are showing promising potential due to their optical properties and activatable therapeutic activities under irradiation. Furthermore, due to their size smaller than 6 nm and unique biophysical properties, they also present intriguing behaviors in biological and physio-pathological environments. In this review, we aim to present the last researches published on such nanoparticles in animals. We also propose guidelines to identify the main physico-chemical parameters that govern Au NCs behavior after administration in small animals, notably concerning their renal elimination and their ability to accumulate in tumors. Then, we present recent advances for their use as theranostic agents putting them in parallel with others contrast agents.

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Extracellular matrix (ECM) proteins are major components of most tissues and organs. In addition to their crucial role in tissue cohesion and biomechanics, they chiefly regulate various important biological processes during embryonic development, tissue homeostasis and repair. In essence, ECM proteins were defined as secreted proteins that localized in the extracellular space. The characterization of the human and mouse matrisomes provided the first definition of ECM actors by comprehensively listing ECM proteins and classified them into categories. Because zebrafish is becoming a popular model to study ECM biology, we sought to characterize the zebrafish matrisome using an in-silico gene-orthology-based approach. We report the identification of 1002 genes encoding the in-silico zebrafish matrisome. Using independent validations, we provide evidence for the robustness of the orthology-based approach. Moreover, we evaluated the orthology relationships between human and zebrafish genes at the whole-genome and matrisome levels and showed that the different categories of ECM genes are differentially subjected to evolutionary pressure. Last, we illustrate how the zebrafish matrisome list can be employed to annotate big data using the example of a previously published proteomic study of the skeletal ECM. The establishment of the zebrafish matrisome will undoubtedly facilitate the analysis of ECM components in "-omic" data sets.

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International audience; Like several 50 nm-large nanocarriers, lipid nanoparticles (LNPs) can passively accumulate in tumors through the Enhanced Permeability and Retention effect. In this study, we developed PEGylated LNPs loaded with IR780 iodide as a contrast agent for NIR fluorescence imaging and modified them with cyclic RGD peptides in order to target integrin αvβ3. We demonstrate a specific targeting of the receptor with cRGD-LNPs but not with cRAD-LNP and standard LNP using HEK293(β3), HEK293(β3)-αvRFP, DU145 and PC3 cell lines. We also demonstrate that cRGD-LNPs bind to αvβ3, interfere with cell adhesion to vitronectin and co-internalize with αvβ3 within one hour. We then investigated their biodistribution and tumor targeting in mice bearing DU145 or M21 tumors. We observed no significant differences between cRGD-LNP and the non-targeted ones regarding their biodistribution and accumulation/retention in tumors. This suggested that despite an efficient formulation of the cRGD-LNPs, the cRGD-mediated targeting was not increasing the total amount of LNP that can already accumulate passively in the subcutaneous tumors via the Enhanced Permeability and Retention effect (EPR).

International audience; Heptaheteroaryl compounds comprised of oxazole and pyridine units (TOxaPy) are quadruplex DNA (G4)-interactive compounds. Herein, we report on the synthesis of parent compounds bearing either amino side chains (TOxaPy-1− 5) or featuring an isomeric oxazole-pyridine central connectivity (iso-TOxapy, iso-TOxapy 1−3) or a bipyridine core (iso-TOxabiPy). The new isomeric series showed significant G4-binding activity in vitro, and remarkably, three compounds (iso-TOxaPy, iso-TOxaPy-1, and iso-TOxabiPy) exhibited high antiproliferative activity toward a tumor panel of cancer cell lines. However, these compounds do not behave as typical G-quadruplex (G4) binders, and the kinase profiling assay revealed that the best antiproliferative molecule iso-TOxaPy selectively inhibited Rock-2. The targeting of Rock kinase was confirmed in cells by the dephosphorylation of Rock-2 substrates, the decrease of stress fibers, and peripheral focal adhesions, as well as the induction of long neurite-like extensions. Remarkably, two of these molecules were able to inhibit the growth of cells organized as spheroids.

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International audience; Ovarian cancer is the leading cause of death from gynecological malignancies worldwide, and innate or acquired chemoresistance of ovarian cancer cells is the major cause of therapeutic failure. It has been demonstrated that the concomitant inhibition of Bcl-x L and Mcl-1 anti-apoptotic activities is able to trigger apoptosis in chemoresistant ovarian cancer cells. In this context, siRNA-mediated Bcl-x L and Mcl-1 inhibition constitutes an appealing strategy by which to eliminate chemoresistant cancer cells. However, the safest and most efficient way to vectorize siRNAs in vivo is still under debate. In the present study, using in vivo bioluminescence imaging, we evaluated the interest of atelocollagen to vectorize siRNAs by intraperitoneal (i.p.) or intravenous (i.v.) administration in 2 xenografted ovarian cancer models (peritoneal carcinomatosis and subcutaneous tumors in nude mice). Whereas i.p. administration of atelocollagen-vectorized siRNA in the peritoneal carcinomatosis model did not induce any gene downregulation, a 70% transient downregulation of luciferase expression was achieved after i.v. injection of atelocollagen-vectorized siRNA in the subcutaneous (s.c.) model. However, the use of siRNA targeting Bcl-x L or Mcl-1 did not induce target-specific downregulation in vivo in nude mice. Our results therefore show that atelocollagen complex formulation, the administration route, tumor site and the identity of the siRNA target influence the efficiency of atelocollagen-mediated siRNA delivery.

LIM kinases (LIMK) are emerging targets for cancer therapy, and they function as network hubs to coordinate actin and microtubule dynamics. When LIMKs are inhibited, actin microfilaments are disorganized and microtubules are stabilized. Owing to their stabilizing effect on microtubules, LIMK inhibitors may provide a therapeutic strategy to treat taxane-resistant cancers. In this study, we investigated the effect of LIMK inhibition on breast tumor development and on paclitaxel-resistant tumors, using a novel selective LIMK inhibitor termed Pyr1. Treatment of breast cancer cells, including paclitaxel-resistant cells, blocked their invasion and proliferation in vitro and their growth in vivo in tumor xenograft assays. The tumor-invasive properties of Pyr1 were investigated in vivo by intravital microscopy of tumor xenografts. A striking change of cell morphology was observed with a rounded phenotype arising in a subpopulation of cells, while other cells remained elongated. Notably, although Pyr1 decreased the motility of elongated cells, it increased the motility of rounded cells in the tumor. Pyr1 administration prevented the growth of metastasis but not their spread. Overall, our results provided a preclinical proof of concept concerning how a small-molecule inhibitor of LIMK may offer a strategy to treat taxane-resistant breast tumors and metastases. Cancer Res; 76(12); 3541–52. ©2016 AACR.

International audience; Background. Appropriate animal models are required to test novel therapeutics for head and neck squamous cell carcinoma (HNSCC) such as near-infrared (NIR) imaging-guided surgery. Methods. We developed an optimized animal model of orthotopic HNSCC (in female athymic NMRI (Naval Medical Research Institute) nude mice) with a prolonged survival time. Resection of the orthotopic tumors was performed 30 days after implantation with or without the aid of a minia-turized clinical grade NIR optical imaging device, after systemic administration of a fluorescent RGD-based probe that targets a v b 3 integrin. Results. NIR optical imaging-guided surgery increased the recurrence-free survival rate by 50% through the detection of fluorescent cancer residues as small as 185 mm; these fragments could remain unidentified if resection was performed exclusively under unaided visual guidance. Conclusion. NIR optical imaging-guided surgery showed an improved HNSCC tumor resection quality in our optimized orthotopic animal model.

64Cu-cyclam-RAFT-c(-RGDfK-)4 is a novel multimeric positron emission tomography (PET) probe foraVb3 integrin imaging. Its uptake and aVb3 expression in tumors showed a linear correlation. Since aVb3 integrin is strongly expressed on activated endothelial cells during angiogenesis, we aimed to determine whether 64Cu-cyclam- RAFT-c(-RGDfK-)4 PET can be used to image tumor angiogenesis and monitor the antiangiogenic effect of a novel multi-targeted tyrosine kinase inhibitor, TSU-68. Athymic nude mice bearing human hepatocellular carcinoma HuH-7 xenografts, which expressed negligible aVb3 levels on the tumor cells, received intraperitoneal injections of TSU-68 or the vehicle for 14 days. Antiangiogenic effects were determined at the end of therapy in terms of 64Cu-cyclam-RAFTc(-RGDfK-)4 uptake evaluated using PET, biodistribution assay, and autoradiography, and they were compared with microvessel density (MVD) determined by CD31 immunostaining. 64Cu-cyclam-RAFT-c(-RGDfK-)4 PET enabled clear tumor visualization by targeting the vasculature, and the biodistribution assay indicated high tumor-to-blood andtumor-to-muscle ratios of 31.6 +/- 6.3 and 6.7 +/- 1.1, respectively, 3 h after probe injection. TSU-68 significantly slowed tumor growth and reduced MVD; these findings were consistent with a significant reduction in the tumor 64Cucyclam-RAFT-c(-RGDfK-)4 uptake. Moreover, a linear correlation was observed between tumor MVD and the corresponding standardized uptake value (SUV) (r = 0.829, P = 0.011 for SUVmean; r = 0.776, P = 0.024 for SUVmax)determined by quantitative PET. Autoradiography and immunostaining showed that the distribution of intratumoral radioactivity and tumor vasculature corresponded. We concluded that 64Cu-cyclam-RAFT-c(-RGDfK-)4 PET can be used for in vivo angiogenesis imaging and monitoring oftumor response to antiangiogenic therapy.

INTRODUCTION: \nThe a(V)B(3) integrin is a well-known transmembrane receptor involved in tumor invasion, angiogenesis and metastasis. Our aim was to evaluate a novel positron emission tomography (PET) probe, (64)Cu-cyclam-RAFT-c(-RGDfK-)(4), for noninvasive visualization and quantification of a(V)B(3) integrin expression. \nMETHODS: \nRAFT-c(-RGDfK-)(4), a tetrameric cyclic Arg-Gly-Asp (RGD)-based peptide, was conjugated with a bifunctional chelator, 1,4,8,11-tetraazacyclotetradecane (cyclam), radiolabeled with the positron emitter (64)Cu and evaluated in vitro by cell binding and competitive inhibition assays and in vivo by biodistribution and receptor blocking studies, and PET imaging. The following cell lines, human embryonic kidney HEK293(B(1)) [a(V)B(3)-negative] and HEK293(B(3)) [a(V)B(3)-overexpressing] and human glioblastoma U87MG [naturally expressing a(V)B(3)], together with their subcutaneous xenografts in athymic nude mice, were used for the present study. The expression levels of a(V)B(3) on these cell lines and tumor xenografts were analyzed by flow cytometry and sodium dodecyl sulfate-polyacrylamide gel electrophoresis/autoradiography, respectively. \nRESULTS: \n(64)Cu-cyclam-RAFT-c(-RGDfK-)(4) demonstrated the in vitro and in vivo specificity for the a(V)B(3) integrin and displayed rapid blood clearance, predominantly renal excretion and low uptake in nontumor tissues. Tumor uptake of (64)Cu-cyclam-RAFT-c(-RGDfK-)(4) (3 h postinjection) in HEK293(B(3)) (high levels of a(V)B(3)), U87MG (moderate levels of a(V)B(3)) and HEK293(B(1)) (undetectable levels of a(V)B(3)) tumors was 9.35%+-1.19%, 3.46%+-0.45% and 1.18%+-0.30% injected dose per gram, respectively, with a strong and positive correlation with the tumor a(V)B(3)expression levels (correlation coefficient=0.967; P

International audience; Cell surface glycoconjugates present alterations of their structures in chronic diseases and distinct oligosaccharide epitopes have been associated with cancer. Among them, truncated glycans present terminal non-reducing β-N-acetylglucosamine (GlcNAc) residues that are rare on healthy tissues. Lectins from unconventional sources such as fungi or algi provide novel markers that bind specifically to such epitopes, but their availability may be challenging. A GlcNAc-binding lectin from the fruiting body of the fungus Psathyrella velutina (PVL) has been produced in good yield in bacterial culture. A strong specificity for terminal GlcNAc residues was evidenced by glycan array. Affinity values obtained by microcalorime-try and surface plasmon resonance demonstrated a micromolar affinity for GlcNAcβ1-3Gal epitopes and for biantennary N-glycans with GlcNAcβ1-2Man capped branches. Crystal structure of PVL complexed with GlcNAcβ1-3Gal established the structural basis of the specificity. Labeling of several types of cancer cells and use of inhibitors of glycan metabolism indicated that rPVL binds to terminal GlcNAc but also to sialic acid (Neu5Ac). Analysis of glycosyltransferase expression confirmed the higher amount of GlcNAc present on cancer cells. rPVL binding is specific to cancer tissue and weak or no labeling is observed for healthy ones, except for stomach glands that present unique αGlcNAc-presenting mucins. In lung, breast and colon carcinomas, a clear delineation could be observed between cancer regions and surrounding healthy tissues. PVL is therefore a useful tool for labeling aga-lacto-glycans in cancer or other diseases.

The efficacy of anti-angiogenic therapies on cancer patients is limited by the emergence of drug resistance, urging the search for second-generation drugs. In this study, we screened an academic chemical library (DCM, University of Grenoble-Alpes) and identified a leader molecule, COB223, that inhibits endothelial cell migration and proliferation. It inhibits also Lewis lung carcinoma (LLC/2) cell proliferation whereas it does not affect fibroblast proliferation. The anti-angiogenic activity of COB223 was confirmed using several in vitro and in vivo assays. In a mouse LLC/2 tumor model, ip administration of doses as low as 4 mg/kg COB223 efficiently reduced the tumor growth rate. We observed that COB223 inhibits endothelial cell ERK1/2 phosphorylation induced by VEGF, FGF-2 or serum and that it acts downstream of PKC and upstream of Ras. This molecule represents a novel anti-angiogenic and anti-tumorigenic agent with an original mechanism of action that deserves further development as an anti-cancer drug.

International audience; One of the main reasons for the dismal prognosis of lung cancer is related to the late diagnosis of this pathology. In this study, we evaluated the potential of optimized lung MRI techniques as a completely non-invasive approach for non-small-cell lung cancer (NSCLC) MRI in vivo detection and follow-up in a mouse model of lung adenocarcinoma expressing the luciferase gene.Bioluminescent lung tumour cells were orthotopically implanted in immuno-deficient mice. Ultra-short echo-time (UTE) MRI free-breathing acquisitions were compared with standard gradient-echo lung MRI (FLASH) using both respiratory-gated and free-breathing protocols. The MRI findings were validated against bioluminescence imaging (BLI) and gold-standard histopathology analysis.Adenocarcinoma-like pathological tissue was successfully identified in all the mice with gated-FLASH and non-gated UTE MRI, and good tumour co-localization was found between MRI, BLI and histological analyses. An excellent or good correlation was found between the measured bioluminescent signal and the total tumour volumes quantified with UTE MRI or gated-FLASH MRI, respectively. No significant correlation was found when the tumours were segmented on non-gated MR FLASH images.MRI was shown to be a powerful imaging tool able to detect, quantify and longitudinally monitor the development of sub-millimetric NSCLCs. To our knowledge, this is the first study which proves the feasibility of a completely non-invasive MRI quantitative detection of lung adenocarcinoma in freely breathing mice. The absence of ionizing radiation and the high-resolution of MRI, along with the complete non-invasiveness and good reproducibility of the proposed non-gated protocol, make this imaging tool ideal for direct translational applications. Copyright © 2014 John Wiley & Sons, Ltd.

International audience; To select the appropriate patients for treatment with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), it is important to gain a better understanding of the intracellular pathways leading to EGFR-TKI resistance, which is a common problem in patients with lung cancer. We recently reported that mutant KRAS adenocarcinoma is resistant to gefitinib as a result of amphi-regulin and insulin-like growth factor-1 receptor overexpression. This resistance leads to inhibition of Ku70 acetylation, thus enhancing the BAX/Ku70 interaction and preventing apoptosis. Here, we determined the intracellular pathways involved in gefitinib resistance in lung cancers and explored the impact of their inhibition. We analyzed the activation of the phosphatidyl inositol-3-kinase (PI3K)/AKT pathway and the mitogen-activated protein kinase/extracellular-signal regulated kinase (MAPK/ERK) pathway in lung tumors. The activation of AKT was associated with disease progression in tumors with wild-type EGFR from patients treated with gefitinib (phase II clinical trial IFCT0401). The administration of IGF1R-TKI or amphiregulin-directed shRNA decreased AKT signaling and restored gefitinib sensitivity in mutant KRAS cells. The combination of PI3K/AKT inhibition with gefitinib restored apoptosis via Ku70 downregulation and BAX release from Ku70. Deacetylase inhibitors, which decreased the BAX/Ku70 interaction, inhibited AKT signaling and induced gefitinib-dependent apoptosis. The PI3K/AKT pathway is thus a major pathway contributing to gefitinib resistance in lung tumors with KRAS mutation, through the regulation of the BAX/Ku70 interaction. This finding suggests that combined treatments could improve the outcomes for this subset of lung cancer patients, who have a poor prognosis.

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Objective: 64Cu-cyclam-RAFT-c(-RGDfK-)4 (64Cu-RAFTRGD) is a novel tetrameric cyclic RGD peptide probe for PET imaging of tumor angiogenesis via targeting the V3 integrin. Because 64Cu also emits -, it can be used for internal radiotherapy. We studied the effects of a succinylated gelatin-containing plasma expander Gelofusine and L-lysine on the renal uptake and retention of 64Cu-RAFTRGD in tumor-bearing mice, aiming to find measures to lower the risk of nephrotoxicity in internal radiotherapy using 64Cu-RAFTRGD. Methods: Normal or tumor-bearing mice received injection of 64Cu-RAFTRGD with or without co-injection of Gelofusine, L-lysine, or the both. Biodistribution studies were performed at 3 and 24 h post-injection (p.i.). Dynamic PET scans (5 min/frame) were performed at 0-60 min p.i., followed by static PET scans at 3.5 and 24 h p.i. Radio-TLC was done to analyze the radioactive metabolites in blood, urine, liver, and kidney samples at 1 and 24 h p.i. Results: Co-injection of Gelofusine significantly reduced the renal uptake and retention of 64Cu-RAFTRGD. Although L-lysine alone had no influence on the renal radioactivity accumulation, it showed the tendency to enhance the inhibitory effect of Gelofusine. The uptake of 64Cu-RAFTRGD in the tumors was not influenced by co-injection of Gelofusine or together with L-lysine. Dynamic PET imaging of mice and metabolic analysis of tissue samples clearly showed that Gelofusine did block the renal reabsorption of 64Cu-RAFTRGD, and did not interfere with the metabolism and excretion pathway of the probe. Conclusion: Administration of Gelofusine or together with L-lysine is a promising measure for kidney protection in internal radiotherapy using 64Cu-RAFTRGD., SNMMI 2013 Annual Meeting

International audience; Surgery is the only therapy that offers the possibility of cure for patients with hepatic metastatic diseases from colorectal or neuroendocrine carcinoma. Five-year survival rates after resection of all detectable liver metastases can reach 50%. In contrast, the role of hepatectomy in patients with liver metastases from noncolorectal and nonneuroendocrine carcinoma is not well defined. Scientific literature showed that in highly selected patients, resection of noncolorectal and nonneuroendocrine liver metastases can be done safely with survival similar to colorectal metastases. Therefore, preoperative and intraoperative staging are critical parameters in order to anticipate an optimized resectability and a favorable outcome.Indocyanine green (ICG) is a fluorescent agent approved in the clinic for liver function testing, as well as measurement of cardiac output and retinal angiography. It is a water-soluble, anionic, amphilic tricarbocyanine probe with a hydrodynamic diameter of 1.2 nm; the excitation and emission wavelengths in the presence of serum are 778 and 830 nm, respectively. Fluorescent navigation systems with ICG injection are used to detect sentinel lymph nodes. It has only recently shown real interest for optical-guided surgical oncology. Several studies report now that this technique can be useful for assisted intraoperative resection of otherwise undetectable liver cancers (such as hepatocellular and metastatic carcinomas) in patients who received and intravenous injection of ICG 4 to 7 days before surgery for a routine preoperative liver function test.We developed a new, miniaturized version of the Fluobeam (see www.fluoptics.com), which can be used for intra-operative surgery of cancer. We used this system and ICG in a clinical trial on 2 patients with liver metastasis, in order to demonstrate that ICG can efficiently label the rim of these tumors, when it is injected intravenously or infused ex-vivo on the surgical liver specimen. Furthermore, we discuss the quality of the ICG labeling, and the possibility to anticipate it by performing conventional diffusion-weighted magnetic resonance imaging, X-ray imaging and 18-FDG metabolic imaging of these patients before optical guided surgery.

International audience; INTRODUCTION: Understanding the multiple biological functions played by human mesenchymal stem cells (hMSCs) as well as their development as therapeutics in regenerative medicine or in cancer treatment are major fields of research. Indeed, it has been established that hMSCs play a central role in the pathogenesis and progression of tumours, but their impact on tumour growth remains controversial. METHODS: In this study, we investigated the influence of hMSCs on the growth of pre-established tumours. We engrafted nude mice with luciferase-positive mouse adenocarcinoma cells (TSA-Luc+) to obtain subcutaneous or lung tumours. When tumour presence was confirmed by non-invasive bioluminescence imaging, hMSCs were injected into the periphery of the SC tumours or delivered by systemic intravenous injection in mice bearing either SC tumours or lung metastasis. RESULTS: Regardless of the tumour model and mode of hMSC injection, hMSC administration was always associated with decreased tumour growth due to an inhibition of tumour cell proliferation, likely resulting from deep modifications of the tumour angiogenesis. Indeed, we established that although hMSCs can induce the formation of new blood vessels in a non-tumoural cellulose sponge model in mice, they do not modify the overall amount of haemoglobin delivered into the SC tumours or lung metastasis. We observed that these tumour vessels were reduced in number but were longer. CONCLUSIONS: Our results suggest that hMSCs injection decreased solid tumour growth in mice and modified tumour vasculature, which confirms hMSCs could be interesting to use for the treatment of pre-established tumours.

Objective: Pancreatic cancer has the worst prognosis among solid tumors. Early diagnosis and efficient therapeutic measure remain as the major challenges. A suitable animal model that mimics the clinical situation as closely as possible is essential for developing and comparing new diagnostic and therapeutic strategies. A new PET imaging probe, 64Cu-labeled cyclam-RAFT-c(-RGDfK-)4 (64Cu-RAFT-RGD) peptide has been examined for visualization and quantification of avB3 integrin expression in animal tumor models by our group. The purpose of this study is to develop a clinically relevant orthotopic xenotransplantation model of pancreatic cancer that reflects the clinical situation and to perform preclinical evaluation of 64Cu-RAFT-RGD probe, allowed in this model. Methods: Expression of avB3 integrin in several human pancreatic cancer cell lines was examined by flow cytometry and Western blotting. The representative cell line BxPC-3 which was stably transfected with red fluorescence reporter protein (RFP) that could be detected with optical imaging system, was used for surgical orthotopic implantation. In vivo probe biodistribution and receptor blocking study, preclinical PET imaging co-registered with contrast enhanced computed tomography (CECT) comparing 64Cu-RAFT-RGD and 18F-FDG accumulation in tumor, post imaging ex vivo autoradiography and histological examination were done. Results: Most of pancreatic cancer cell lines showed integrin avB3 expression. Orthotopic xenograft was established in pancreas of recipient nude mice by implantation of a piece of subcutaneous tumor developed in donor mice. In vivo biodistribution with blocking study confirmed that the efficient binding of probe to tumor is highly avB3 integrin specific. 64Cu-RAFT-RGD PET imaging combined with CECT that provided anatomical delineation of tumor, encouraged for precise and easy detection of pancreatic cancer lesion. In comparative PET study, 64Cu-RAFT-RGD accumulation provided better tumor contrast to background than 18F-FDG. Post imaging autoradiography and histological examination validated the accumulation of 64Cu-RAFT-RGD probe in tumor versus non-tumor tissues. Conclusion: Our results suggest that 64Cu-RAFT-RGD PET imaging can be potentially applicable for the diagnosis of avB3-expressing pancreatic cancer., 2012 World Molecular Imaging Congress

Angiogenesis plays important roles in tumor growth and metastasis, and has been increasingly recognized as an essential target for tumor imaging and therapy. Positron emission tomography (PET) is one of the most promising tools for noninvasive detection and quantification of molecular activities. The cell adhesion protein, alfaVbeta-3-integrin, is highly expressed on the activated vascular endothelial cells during angiogenesis. We have developed a novel multivalent PET probe, 64Cu-cyclam-RAFT-c(-RGDfK-)4, for alfaVbeta3-targeted imaging, and found a strong and positive correlation between the tumor uptake of this probe and the corresponding tumor alfaVbeta 3 expression levels. Our present study is to subsequently investigate the potential of this PET probe for visualization and quantification of tumor angiogenesis and furthermore for monitoring of the therapeutic efficacy of a tyrosine kinase inhibitor (TKI) as an antiangiogenic drug. Methods: A hypervascular subcutaneous xenograft of a alfaVbeta3-negative human hepatocellular carcinoma cell line (HCC) in athymic nude mice was used as a tumor angiogenesis model for the present study. PET imaging and biodistribution studies were performed at 1 or 3 h after probe administration, and autoradiography and immunostaining of serial frozen tumor sections were carried out to compare the intratumoral distribution of the probe and that of microvasculature. Another cohort of HCC tumor-bearing mice were divided into 2 groups: vehicle-treated control group and TKI-treated antiangiogenic therapy group. Mouse body weight and tumor size were measured throughout the study. After 2 weeks of treatment, tumor microvessel density (MVD), tumor uptake of the probe measured by biodistribution study and PET imaging were compared between the control and treatment group. Results: Biodistribution and PET studies revealed the favorable tumor uptake of 64Cu-cyclam-RAFT-c(-RGDfK-)4 with high tumor-to-background contrast, and autoradiography and immunostaining demonstrated the approximate colocalization of the probe and microvasculature. Antiangiogenic therapy did not show obvious side effects, and could delay the tumor growth although the significant difference in tumor volumes was not obtained between the treatment and control groups. Antiangiogenic therapy resulted in obvious reduction of tumor MVD. The corresponding decrease in tumor uptake of 64Cu-cyclam-RAFT-c(-RGDfK-)4 was expected from biodistribution and PET studies. Conclusion: 64Cu-cyclam-RAFT-c(-RGDfK-)4-based PET is able to visualize tumor angiogenesis by targeting the alfaVbeta3-integrin, and would be useful for monitoring the relatively early response to antiangiogenic therapy., 2011 World Molecular Imaging Congress (WMIC)

International audience; RGD peptides recognize the α(v)β(3) integrin, a receptor that is overexpressed on the surface of both tumor blood vessels and cancerous cells. These peptides are powerful tools that act as single antiangiogenic molecules, but recently also have been used for tumor imaging and drug targeting. We designed the molecule RAFT-(c[-RGDfK-])(4), a constrained and chemically defined entity that can be produced at clinical-grade quality. This scaffold was covalently coupled via a labile bridge to the proapoptotic peptide (KLAKLAK)(2) (RAFT-RGD-KLA). A fluorescent, activatable probe was also introduced, allowing intracellular localization. At 2.5 µM, this molecule induced the intracellular release of an active KLA peptide, which in turn caused mitochondrial depolarization and cell death in vitro in tumor cells. In a mouse model, the RAFT-RGD-KLA peptide was found to prevent the growth of remote subcutaneous tumors. This study demonstrated that the antitumor peptide is capable of killing tumor cells in an RGD-dependent manner, thus lowering the nonspecific cytotoxic effects expected to occur when using cationic cytotoxic peptides. Thus, this chemistry is suitable for the design of complex, multifunctional molecules that can be used for both imaging and therapeutics, representing the next generation of perfectly controlled, targeted drug-delivery systems.

International audience; Although many cancer cells are primed for apoptosis, they usually develop resistance to cell death at several levels. Permeabilization of the outer mitochondrial membrane, which is mediated by proapoptotic Bcl-2 family members such as Bax, is considered as a point of no return for initiating apoptotic cell death. This crucial role has placed Bcl-2 family proteins as recurrent targets for anticancer drug development. Here, we propose and demonstrate a new concept based on minimal active versions of Bax to induce cell death independently of endogenous Bcl-2 proteins. We show that membrane-active segments of Bax can directly induce the release of mitochondria-residing apoptogenic factors and commit tumor cells promptly and irreversibly to caspase-dependent apoptosis. On this basis, we designed a peptide encompassing part of the Bax pore-forming domain, which can target mitochondria, induce cytochrome c release and trigger caspase-dependent apoptosis. Moreover, this Bax-derived 'poropeptide' produced effective tumor regression after peritumoral injection in a nude mouse xenograft model. Thus, peptides derived from proteins that form pores in the mitochondrial outer membrane represent novel templates for anticancer agents.

International audience; The appropriate selection of patients is a major challenge in the treatment of non-small cell lung cancer (NSCLC) with epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs). Prospective trials in adenocarcinoma demonstrated that the mucinous subtype presents a poorer outcome under EGFR-TKI treatment than the non-mucinous subtype. Our aim was to determine the molecular characteristics associated with resistance to EGFR-TKIs in mucinous and non-mucinous adenocarcinoma. Eighty adenocarcinoma samples, including 34 tumours from patients treated with gefitinib in a phase II clinical trial (IFCT0401), were classified as mucinous (n = 32) or non-mucinous (n = 48) adenocarcinoma. We demonstrated that four biological markers were differentially expressed between the two subtypes: mucinous tumours that overexpressed IGF1R (p < 0.0001) and amphiregulin (p = 0.004) with a tendency for more frequent KRAS mutations, in contrast to non-mucinous tumours that overexpressed EGFR (p < 0.0001) and TTF-1 (p < 0.0001) with more frequent EGFR mutations (p = 0.037). Higher IGF1R (p = 0.02) and lower TTF-1 (p = 0.02) expression was associated with disease progression under gefitinib treatment. We observed in vitro cross-talk between EGFR and IGF1R signalling pathways in gefitinib-resistant H358 mucinous cells. Anti-amphiregulin siRNAs and anti-IGF1R treatments sensitized the H358 cells to gefitinib-induced apoptosis with additive effects, suggesting that these treatments could overcome the resistance of mucinous tumours to EGFR-TKIs, including those with KRAS mutation. Our results highlighted that mucinous and non-mucinous adenocarcinoma subtypes are different entities with different therapeutic responses to EGFR-TKIs. These data will foster the development of therapeutic strategies for treating adenocarcinoma with mucinous component.

International audience; Molecular resistance mechanisms affecting the efficiency of receptor tyrosine kinase inhibitors such as gefitinib in non-small-cell lung cancer (NSCLC) cells are not fully understood. Amphiregulin (Areg) overexpression has been proposed to predict NSCLC resistance to gefitinib and we have established that Areg-overexpressing H358 NSCLC cells resist apoptosis. Here, we demonstrate that Areg prevents gefitinib-induced apoptosis in NSCLC cells. We show that H358 cells are resistant to gefitinib in contrast to H322 cells, which do not overexpress Areg. Inhibition of Areg expression by small-interfering RNAs (siRNAs) restores gefitinib sensitivity in H358 cells, whereas addition of recombinant Areg confers resistance in H322 cells. Areg knockdown overcomes resistance to gefitinib and induced apoptosis in NSCLC H358 cells in vitro and in vivo. Under gefitinib treatment, Areg decreases the expression of the proapoptotic protein BAX, inhibits its conformational change and its mitochondrial translocation. Thus, in the presence of Areg, gefitinib-mediated apoptosis is reduced because BAX is sequestered in the cytoplasm. This suggests that treatments using epidermal growth factor receptor (EGFR) inhibitors may be poorly efficient in patients with elevated levels of Areg. These findings indicate the need for inhibition of Areg to enhance the efficiency of the EGFR inhibitors in patients suffering NSCLC.

International audience; Molecular resistance mechanisms affecting the efficiency of receptor tyrosine kinase inhibitors such as gefitinib in non-small-cell lung cancer (NSCLC) cells are not fully understood. Amphiregulin (Areg) overexpression has been proposed to predict NSCLC resistance to gefitinib and we have established that Areg-overexpressing H358 NSCLC cells resist apoptosis. Here, we demonstrate that Areg prevents gefitinib-induced apoptosis in NSCLC cells. We show that H358 cells are resistant to gefitinib in contrast to H322 cells, which do not overexpress Areg. Inhibition of Areg expression by small-interfering RNAs (siRNAs) restores gefitinib sensitivity in H358 cells, whereas addition of recombinant Areg confers resistance in H322 cells. Areg knockdown overcomes resistance to gefitinib and induced apoptosis in NSCLC H358 cells in vitro and in vivo. Under gefitinib treatment, Areg decreases the expression of the proapoptotic protein BAX, inhibits its conformational change and its mitochondrial translocation. Thus, in the presence of Areg, gefitinib-mediated apoptosis is reduced because BAX is sequestered in the cytoplasm. This suggests that treatments using epidermal growth factor receptor (EGFR) inhibitors may be poorly efficient in patients with elevated levels of Areg. These findings indicate the need for inhibition of Areg to enhance the efficiency of the EGFR inhibitors in patients suffering NSCLC.

International audience; The transcription factor E2F1 has a crucial role in the control of cell growth and has been shown to regulate neoangiogenesis in a p53-dependent manner through inhibition of activity of the VEGF-A (vascular endothelial growth factor) promoter. Besides being regulated by transcription, VEGF-A is also highly regulated by pre-mRNA alternative splicing, resulting in the expression of several VEGF isoforms with either pro-(VEGF(xxx)) or anti-(VEGF(xxx)b) angiogenic properties. Recently, we identified the SR (Ser-Rich/Arg) protein SC35, a splicing factor, as a new transcriptional target of E2F1. Here, we show that E2F1 downregulates the activity of the VEGF-A promoter in tumour cells independently of p53, leading to a strong decrease in VEGF(xxx) mRNA levels. We further show that, strikingly, E2F1 alters the ratio of pro-VEGF(xxx) versus anti-VEGF(xxx)b angiogenic isoforms, favouring the antiangiogenic isoforms, by a mechanism involving the induction of SC35 expression. Finally, using lung tumour xenografts in nude mice, we provide evidence that E2F1 and SC35 proteins increase the VEGF(165)b/VEGF ratio and decrease tumour neovascularization in vivo. Overall, these findings highlight E2F1 and SC35 as two regulators of the VEGF(xxx)/VEGF(xxx)b angiogenic switch in human cancer cells, a role that could be crucial during tumour progression, as well as in tumour response to antiangiogenic therapies.

National audience

Multivalent interactions are frequently used to enhance ligand-receptor binding affinity. In this study a mono-, di- and trimeric AVTGRGDSY peptide derived from fibronectin were compared concerning the integrin receptor binding affinity and/or specificity. Methods: AVTGRGDSY monomer, dimer and trimer were synthesized, labeled with 125I or Cy5.5. Using human embryonic kidney cells HEK293 (naturally alphaV-positive and beta3-negative), HEK293(beta1) (beta1-transfected and alphaVbeta3-negative), HEK293(beta3)( beta3-transfected and strongly alphaVbeta3 -positive), and human glioblastoma cells U87MG (naturally alphaVbeta3-positive), cell-binding assay, competitive inhibition assay, cell adhesion assay and confocal laser scanning microscopic study were performed to determine the bioactivities of these peptides. Results: The monomeric AVTGRGDSY showed specific binding to both HEK293(beta1) and HEK293(beta3) cells. Multimerization resulted in no change with HEK293 cells, diminished binding for HEK293(beta1) cells, but substantially enhanced binding for alphaVbeta3-positive HEK293(beta3) and U87MG cells in the presence of Mn2+. Moreover, the multimeric AVTGRGDSY peptides were found to be nearly comparable with th alphaVbeta3-specific cyclo(RGDfV) peptide in specificity and affinity for targeting alphaVbeta3 integrin. Conclusion: The multimeric AVTGRGDSY peptide might be an efficient alphaVbeta3-targeting molecule. The present study would be useful for better understanding of the molecular basis of the interaction between RGD ligands and integrin receptors., World Molecular Imaging Congress

International audience; Integrin alpha(v)beta(3) is overexpressed on neoendothelial cells and frequently on tumor cells. We have developed a peptide-like scaffold (regioselectively addressable functionalized template, RAFT), which holds four cyclo(-RGDfK-) (cRGD) motifs and proved that this molecule (called regioselectively addressable functionalized template-arginine-glycine-aspartic acid, RAFT-RGD) targets integrin alpha(v)beta(3) in vitro and in vivo. Using fluorescence correlation spectroscopy (FCS), we measured the constant of affinity (K(D)) of the RAFT-RGD for purified integrins. K(D) values rose from 3.87 nmol/l for RAFT-RGD to 41.70 nmol/l for cyclo(-RGDfK-). In addition, RAFT-RGD inhibited alpha(v)beta(3) lateral mobility in the cell membrane, probably due to the formation of integrin clusters as demonstrated by fluorescence recovery after photobleaching (FRAP). This was confirmed by electronic microscopy data, which established the formation of molecular complexes containing two integrins in the presence of RAFT-RGD but not cRGD or regioselectively addressable functionalized template-arginine-alanine- aspartic acid (RAFT-RAD). Using an enzyme-linked immunosorbent assay (ELISA), we proved that 1 micromol/l RAFT-RGD increased by 79% alpha(v)beta(3) internalization via clathrin-coated vesicles. Conversely, cRGD was internalized without modifying alpha(v)beta(3) internalization. Although RGD has been known for >20 years, this is the first study to formerly establish the relationships among multimeric presentation, increased affinity, and subsequent integrin-mediated cointernalization. These results strongly support the rationale for using multimeric RGD-peptides as targeting vectors for imaging, diagnosis, or therapy of cancers.

International audience; Integrin alpha(v)beta(3) is overexpressed on neoendothelial cells and frequently on tumor cells. We have developed a peptide-like scaffold (regioselectively addressable functionalized template, RAFT), which holds four cyclo(-RGDfK-) (cRGD) motifs and proved that this molecule (called regioselectively addressable functionalized template-arginine-glycine-aspartic acid, RAFT-RGD) targets integrin alpha(v)beta(3) in vitro and in vivo. Using fluorescence correlation spectroscopy (FCS), we measured the constant of affinity (K(D)) of the RAFT-RGD for purified integrins. K(D) values rose from 3.87 nmol/l for RAFT-RGD to 41.70 nmol/l for cyclo(-RGDfK-). In addition, RAFT-RGD inhibited alpha(v)beta(3) lateral mobility in the cell membrane, probably due to the formation of integrin clusters as demonstrated by fluorescence recovery after photobleaching (FRAP). This was confirmed by electronic microscopy data, which established the formation of molecular complexes containing two integrins in the presence of RAFT-RGD but not cRGD or regioselectively addressable functionalized template-arginine-alanine- aspartic acid (RAFT-RAD). Using an enzyme-linked immunosorbent assay (ELISA), we proved that 1 micromol/l RAFT-RGD increased by 79% alpha(v)beta(3) internalization via clathrin-coated vesicles. Conversely, cRGD was internalized without modifying alpha(v)beta(3) internalization. Although RGD has been known for >20 years, this is the first study to formerly establish the relationships among multimeric presentation, increased affinity, and subsequent integrin-mediated cointernalization. These results strongly support the rationale for using multimeric RGD-peptides as targeting vectors for imaging, diagnosis, or therapy of cancers.

International audience

International audience; Amphiregulin (AR) and insulin-like growth factor-1 (IGF1) are growth factors known to promote non-small cell lung cancer (NSCLC) survival. We have previously published that 1) AR and IGF1, secreted by H358 NSCLC cells, cooperate to protect those cells and H322 NSCLC cells from serum-starved apoptosis; 2) H358 cells resist Bax-induced apoptosis through an inhibition of Bax conformational change. We show here that the antiapoptotic activity of the AR/IGF1 combination is specifically abolished by the PKC inhibitors calphostin C and staurosporine, but not by the MAPK and phosphatidylinositol 3-kinase inhibitors PD98059 and wortmannin, suggesting the involvement of a PKC-dependent and MAPK- and phosphatidylinositol 3-kinase-independent survival pathway. The PKCdelta inhibitor rottlerin restores apoptosis induced by serum deprivation. In addition, phosphorylation of PKCdelta and PKCzeta/lambda, but not of PKCalpha/beta(II), increases in serum-starved H358 cells and in H322 cells treated with an AR/IGF1 combination and is blocked by calphostin C. The combination of AR and IGF1 increases p90(rsk) and Bad phosphorylation as well as inhibiting the conformational change of Bax by a PKC-dependent mechanism. Finally, PKCdelta, PKCzeta, or p90(rsk) small interfering RNAs block the antiapoptotic activity of AR/IGF1 combination but have no effect on partial apoptosis inhibition observed with each factor used alone. Constitutively active PKC expression inhibits serum deprivation-induced apoptosis, whereas a catalytically inactive form of p90(rsk) restores it. Thus, AR and IGF1 cooperate to prevent apoptosis by activating a specific PKC-p90(rsk)-dependent pathway, which leads to Bad and Bax inactivation. This signaling pathway is different to that used by single factor.

International audience; Amphiregulin (AR) and insulin-like growth factor-1 (IGF1) are growth factors known to promote non-small cell lung cancer (NSCLC) survival. We have previously published that 1) AR and IGF1, secreted by H358 NSCLC cells, cooperate to protect those cells and H322 NSCLC cells from serum-starved apoptosis; 2) H358 cells resist Bax-induced apoptosis through an inhibition of Bax conformational change. We show here that the antiapoptotic activity of the AR/IGF1 combination is specifically abolished by the PKC inhibitors calphostin C and staurosporine, but not by the MAPK and phosphatidylinositol 3-kinase inhibitors PD98059 and wortmannin, suggesting the involvement of a PKC-dependent and MAPK- and phosphatidylinositol 3-kinase-independent survival pathway. The PKCdelta inhibitor rottlerin restores apoptosis induced by serum deprivation. In addition, phosphorylation of PKCdelta and PKCzeta/lambda, but not of PKCalpha/beta(II), increases in serum-starved H358 cells and in H322 cells treated with an AR/IGF1 combination and is blocked by calphostin C. The combination of AR and IGF1 increases p90(rsk) and Bad phosphorylation as well as inhibiting the conformational change of Bax by a PKC-dependent mechanism. Finally, PKCdelta, PKCzeta, or p90(rsk) small interfering RNAs block the antiapoptotic activity of AR/IGF1 combination but have no effect on partial apoptosis inhibition observed with each factor used alone. Constitutively active PKC expression inhibits serum deprivation-induced apoptosis, whereas a catalytically inactive form of p90(rsk) restores it. Thus, AR and IGF1 cooperate to prevent apoptosis by activating a specific PKC-p90(rsk)-dependent pathway, which leads to Bad and Bax inactivation. This signaling pathway is different to that used by single factor.

International audience; Until recently, the ability of ARF (human p14(ARF), murine p19(ARF)) tumour-suppressor protein, encoded by the INK4A/ARF locus, to inhibit cell growth in response to various stimuli was related to its ability to stabilize p53 through the so-called ARF/MDM2/p53 pathway. However, recent data have demonstrated that ARF is not implicated in this unique p53-dependent pathway. By use of transient and stable expression, we show here that human p14(ARF) inhibits the growth of human tumoral cells lacking functional p53 by inducing a transient G(2) arrest and subsequently apoptosis. This p14(ARF)-induced G(2) arrest was correlated with inhibition of CDC2 activity, inactivation of CDC25C phosphatase and induction of the CDK inhibitor p21(WAFI). Apoptosis was demonstrated using Hoechst 33352 staining, proteolytic activation of caspase-3 and PARP cleavage. Similar results were obtained in experiments with cells synchronized by hydroxyurea block. Importantly, we were able to reproduce these effects 'in vivo' by showing that p14(ARF) inhibits the growth of p53 nullizygous human tumours in nude mice and induces the regression of p53 -/- established tumours. In these experiments, tumoral regression was associated with inhibition of cell proliferation as well as induction of apoptosis confirming the data obtained in cell lines.