Exploring the complementarity of pancreatic ductal adenocarcinoma preclinical models

Purpose: Compare pancreatic ductal adenocarcinoma (PDAC), preclinical models, by their transcriptome and drug response landscapes to evaluate their complementarity. Experimental De-sign: Three paired PDAC preclinical models—patient‐derived xenografts (PDX), xenograft‐derived pancreatic organoids (XDPO) and xenograft‐derived primary cell cultures (XDPCC)—were derived from 20 patients and analyzed at the transcriptomic and chemosensitivity level. Transcriptomic characterization was performed using the basal‐like/classical subtyping and the PDAC molecular gradient (PAMG). Chemosensitivity for gemcitabine, irinotecan, 5‐fluorouracil and oxaliplatin was established and the associated biological pathways were determined using independent component analysis (ICA) on the transcriptome of each model. The selection criteria used to identify the different components was the chemosensitivity score (CSS) found for each drug in each model. Results: PDX was the most dispersed model whereas XDPO and XDPCC were mainly classical and basal-like, respectively. Chemosensitivity scoring determines that PDX and XDPO display a positive correlation for three out of four drugs tested, whereas PDX and XDPCC did not correlate. No match was observed for each tumor chemosensitivity in the different models. Finally, pathway analysis shows a significant association between PDX and XDPO for the chemosensitivity‐associated pathways and PDX and XDPCC for the chemoresistance‐associated pathways. Conclusions: Each PDAC preclinical model possesses a unique basal‐like/classical transcriptomic phenotype that strongly in-fluences their global chemosensitivity. Each preclinical model is imperfect but complementary, sug-gesting that a more representative approach of the clinical reality could be obtained by combining them. Translational Relevance: The identification of molecular signatures that underpin drug sensitivity to chemotherapy in PDAC remains clinically challenging. Importantly, the vast majority of studies using preclinical in vivo and in vitro models fail when transferred to patients in a clinical setting despite initially promising results. This study presents for the first time a comparison between three preclinical models directly derived from the same patients. We show that their applica-bility to preclinical studies should be considered with a complementary focus, avoiding tumor-based direct extrapolations, which might generate misleading conclusions and consequently the overlook of clinically relevant features.Fil: Hoare, Owen. Centre National de la Recherche Scientifique; FranciaFil: Fraunhoffer Navarro, Nicolas Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Elkaoutari, Abdessamad. Centre National de la Recherche Scientifique; FranciaFil: Gayet, Odile. Centre National de la Recherche Scientifique; FranciaFil: Bigonnet, Martin. Centre National de la Recherche Scientifique; FranciaFil: Roques, Julie. Centre National de la Recherche Scientifique; FranciaFil: Nicolle, Rémy. No especifíca;Fil: McGuckin, Colin. Cell Therapy Research Institute; FranciaFil: Forraz, Nico. Cell Therapy Research Institute; FranciaFil: Sohier, Emilie. Le Centre Régional de Lutte Contre Le Cancer Léon Bérard; FranciaFil: Tonon, Laurie. Le Centre Régional de Lutte Contre Le Cancer Léon Bérard; FranciaFil: Wajda, Pauline. Le Centre Régional de Lutte Contre Le Cancer Léon Bérard; FranciaFil: Boyault, Sandrine. Le Centre Régional de Lutte Contre Le Cancer Léon Bérard; FranciaFil: Attignon, Valéry. Le Centre Régional de Lutte Contre Le Cancer Léon Bérard; FranciaFil: Tabone, Luciana Belen. Le Centre Régional de Lutte Contre Le Cancer Léon Bérard; FranciaFil: Barbier, Sandrine. No especifíca;Fil: Mignard, Caroline. No especifíca;Fil: Duchamp, Olivier. No especifíca;Fil: Iovanna, Juan. Centre National de la Recherche Scientifique; FranciaFil: Dusetti, Nelson J.. Centre National de la Recherche Scientifique; Franci

CDK2 and PKA Mediated-Sequential Phosphorylation Is Critical for p19INK4d Function in the DNA Damage Response

DNA damage triggers a phosphorylation-based signaling cascade known as the DNA damage response. p19INK4d, a member of the INK4 family of CDK4/6 inhibitors, has been reported to participate in the DNA damage response promoting DNA repair and cell survival. Here, we provide mechanistic insight into the activation mechanism of p19INK4d linked to the response to DNA damage. Results showed that p19INK4d becomes phosphorylated following UV radiation, β-amyloid peptide and cisplatin treatments. ATM-Chk2/ATR-Chk1 signaling pathways were found to be differentially involved in p19INK4d phosphorylation depending on the type of DNA damage. Two sequential phosphorylation events at serine 76 and threonine 141 were identified using p19INK4d single-point mutants in metabolic labeling assays with 32P-orthophosphate. CDK2 and PKA were found to participate in p19INK4d phosphorylation process and that they would mediate serine 76 and threonine 141 modifications respectively. Nuclear translocation of p19INK4d induced by DNA damage was shown to be dependent on serine 76 phosphorylation. Most importantly, both phosphorylation sites were found to be crucial for p19INK4d function in DNA repair and cell survival. In contrast, serine 76 and threonine 141 were dispensable for CDK4/6 inhibition highlighting the independence of p19INK4d functions, in agreement with our previous findings. These results constitute the first description of the activation mechanism of p19INK4d in response to genotoxic stress and demonstrate the functional relevance of this activation following DNA damage

The functional landscape of Hsp27 reveals new cellular processes such as DNA repair and alternative splicing and proposes novel anticancer targets.

International audiencePreviously, we identified the stress-induced chaperone, Hsp27, as highly overexpressed in castration-resistant prostate cancer and developed an Hsp27 inhibitor (OGX-427) currently tested in phase I/II clinical trials as a chemosensitizing agent in different cancers. To better understand the Hsp27 poorly-defined cytoprotective functions in cancers and increase the OGX-427 pharmacological safety, we established the Hsp27-protein interaction network using a yeast two-hybrid approach and identified 226 interaction partners. As an example, we showed that targeting Hsp27 interaction with TCTP, a partner protein identified in our screen increases therapy sensitivity, opening a new promising field of research for therapeutic approaches that could decrease or abolish toxicity for normal cells. Results of an in-depth bioinformatics network analysis allying the Hsp27 interaction map into the human interactome underlined the multifunctional character of this protein. We identified interactions of Hsp27 with proteins involved in eight well known functions previously related to Hsp27 and uncovered 17 potential new ones, such as DNA repair and RNA splicing. Validation of Hsp27 involvement in both processes in human prostate cancer cells supports our system biology-predicted functions and provides new insights into Hsp27 roles in cancer cells

Absence of Tumor Suppressor Tumor Protein 53-Induced Nuclear Protein 1 (TP53INP1) Sensitizes Mouse Thymocytes and Embryonic Fibroblasts to Redox-Driven Apoptosis

International audienceThe p53-transcriptional target TP53INP1 is a potent stress-response protein promoting p53 activity. We previously showed that ectopic overexpression of TP53INP1 facilitates cell cycle arrest as well as cell death. Here we report a study investigating cell death in mice deficient for TP53INP1. Surprisingly, we found enhanced stress-induced apoptosis in TP53INP1-deficient cells. This observation is underpinned in different cell types in vivo (thymocytes) and in vitro (thymocytes and MEFs), following different types of injury inducing either p53-dependent or-independent cell death. Nevertheless, absence of TP53INP1 is unable to overcome impaired cell death of p53-deficient thymocytes. Stress-induced ROS production is enhanced in the absence of TP53INP1, and antioxidant NAC complementation abolishes increased sensitivity to apoptosis of TP53INP1-deficient cells. Furthermore, antioxidant defenses are defective in TP53INP1-deficient mice in correlation with ROS dysregu-lation. Finally, we show that autophagy is reduced in TP53INP1-deficient cells both at the basal level and upon stress. Altogether, these data show that impaired ROS regulation in TP53INP1-deficient cells is responsible for their sensitivity to induced apoptosis. In addition, they suggest that this sensitivity could rely on a defect of autophagy. Therefore, these data emphasize the role of TP53INP1 in protection against cell injury. Antioxid. Redox Signal. 15, 1639–1653

Phosphorylation of serine 76 and threonine 141 is required for p19 function linked to the response to DNA damage

<p>(<b>A</b>) DNA repair ability of cells overexpressing p19wt or p19 phosphorylation deficient mutants. WI-38 fibroblasts were transfected with p19wt or the indicated p19 mutants. Cells were maintained in an arginine-free medium containing 1% fetal bovine serum during 48 h, damage with 4 mJ/cm² UV and incubated with [³H]-thymidine. Following 10 h, cell lysates were tested for Unscheduled DNA Synthesis assay (UDS). Bars represent the mean ± s.e.m of three independent experiments performed in triplicate. Student's <i>t</i>-test was used to compare UV-treated control sample (none) with UV-treated p19wt or p19 mutant samples. (*<i>p</i><0,005). Protein expression was analyzed by immunoblot. (<b>B</b>) Similarly as in (<b>A</b>) but overexpressing the phosphomimetic p19 mutants. (<b>C</b>) UV-dependent apoptotic response of cells overexpressing p19wt or phosphorylation deficient mutants of p19. WI-38 fibroblasts were transfected with p19wt or the indicated p19 mutants. Twelve hours following UV irradiation, cell lysates were tested for caspase-3 activity. Results are expressed as percentage of caspase-3 activity with respect to basal activity of cell lysates nontransfected and without UV-treatment, which was set to 100. Bars represent the mean ± s.e.m. of three independent experiments performed in triplicate. Student <i>t</i>-test was used to compare, UV-treated control sample (none) with UV-treated p19wt or p19 mutant samples (^*<i>p</i><0.005). (<b>D</b>) Similarly as in (<b>C)</b> but overexpressing the phosphomimetic p19 mutants.</p

p19 phosphorylation is induced in response to DNA damage.

<p>(<b>A, B</b>) WI-38 fibroblasts were labeled with [³²P]-orthophosphate and treated with β-amyloid peptide (20 µM), cisplatin (10 µM) or UV light (4 mJ/cm²) for the indicated times. Equal amounts of whole cell extracts were subjected to immunoprecipitation with anti-p19 antibody and the immune complexes were analyzed by SDS-PAGE and autoradiography (upper panels; P-p19, phosphorylated p19) or immunoblotting (lower panels; p19). (C; Control, untreated cells).</p

ATM/ATR signaling pathways are differentially involved in p19 phosphorylation.

<p>(<b>A</b>) Inhibition of p19 phosphorylation by caffeine treatment. WI-38 fibroblasts were incubated with caffeine (5 mM) for 1 hour, then treated with cisplatin (10 µM) or β-amyloid peptide (20 µM) for the indicated times and endogenous p19 phosphorylation analyzed by autoradiography. (<b>B</b>) Evaluation of ATM/ATR involvement in p19 phosphorylation by wortmannin treatment. WI-38 fibroblasts were incubated with the indicated doses of wortmannin for 1 hour, followed by treatment with cisplatin (10 µM) or β-amyloid peptide (20 µM) for 2 hours. (<b>C</b>) Effect of Chk1 and Chk2 inhibitors on p19 phosphorylation. WI-38 fibroblasts were incubated with SB-218078 (SB, 15 nM) or dopamine ß-hidroxylase inhibitor (DBH, 3 µM), both Chk1 inhibitors, or with Chk2 Inhibitor Calbiochem (ICHK2, 20 nM) for 1 hour before treatment with UV light (4 mJ/cm²), cisplatin (10 µM) or β-amyloid peptide (20 µM). After 2 hours, cell extracts were analyzed as in A.</p