Nucleolin Targeting by N6L Inhibits Wnt/Β-Catenin Pathway Activation in Pancreatic Ductal Adenocarcinoma
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cancers Article Nucleolin Targeting by N6L Inhibits Wnt/β-Catenin Pathway Activation in Pancreatic Ductal Adenocarcinoma Fabio Raineri 1, Sandrine Bourgoin-Voillard 1,2,3,4 ,Mélissande Cossutta 1,5 , Damien Habert 1, Matteo Ponzo 1, Claire Houppe 1, Benoît Vallée 1, Michele Boniotto 1, Mounira Chalabi-Dchar 6, Philippe Bouvet 6,7 , Anne Couvelard 8, Jerome Cros 8 , Anais Debesset 1, José L. Cohen 1,5 , José Courty 1,5 and Ilaria Cascone 1,5,* 1 University Paris Est Créteil, INSERM, IMRB, 94010 Créteil, France; [email protected] (F.R.); [email protected] (S.B.-V.); [email protected] (M.C.); [email protected] (D.H.); [email protected] (M.P.); [email protected] (C.H.); [email protected] (B.V.); [email protected] (M.B.); [email protected] (A.D.); [email protected] (J.L.C.); [email protected] (J.C.) 2 University of Grenoble Alpes, CNRS, Grenoble INP, Inserm U1055, LBFA and BEeSy, PROMETHEE Proteomic Platform, 38400 Saint-Martin d’Heres, France 3 University of Grenoble Alpes, CNRS, Grenoble INP, TIMC, PROMETHEE Proteomic Platform, 38000 Grenoble, France 4 CHU Grenoble Alpes, Institut de Biologie et de Pathologie, 38043 Grenoble, France 5 AP-HP, Groupe Hospitalo-Universitaire Chenevier Mondor, Centre d’Investigation Clinique Biotherapie, 94010 Créteil, France 6 Centre de Recherche en Cancérologie de Lyon, Cancer Cell Plasticity Department, University of Lyon, UMR INSERM 1052 CNRS 5286, Centre Léon Bérard, 69008 Lyon, France; Citation: Raineri, F.; [email protected] (M.C.-D.); [email protected] (P.B.) 7 University of Lyon, Ecole Normale Supérieure de Lyon, 69342 Lyon, France Bourgoin-Voillard, S.; Cossutta, M.; 8 Département de Pathologie, Hôpital Bichat APHP DHU UNITY, 75018 Paris, France; Habert, D.; Ponzo, M.; Houppe, C.; [email protected] (A.C.); [email protected] (J.C.) Vallée, B.; Boniotto, M.; Chalabi-Dchar, * Correspondence: [email protected]; Tel.: +33-149-813-765 M.; Bouvet, P.; et al. Nucleolin Targeting by N6L Inhibits Simple Summary: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer that has no Wnt/β-Catenin Pathway Activation effective treatment. Nucleolin targeting by the pseudopeptide N6L inhibits the tumor growth and in Pancreatic Ductal Adenocarcinoma. Cancers 2021, 13, 2986. https:// metastasis of pancreatic ductal adenocarcinoma (PDAC). Here, we explored the pathways regulated doi.org/10.3390/cancers13122986 by nucleolin and N6L in PDAC. We demonstrated that both interact with β-catenin, and that the Wnt/β-catenin pathway is activated in PDAC mouse models. Nucleolin inhibition decreases Wnt/β- Academic Editors: Louis BUSCAIL catenin pathway activation in PDAC cells and tumors, and represents a new druggable pathway and Niccola Funel regulated by nucleolin. Received: 30 March 2021 Abstract: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive and resistant cancer with Accepted: 10 June 2021 no available effective therapy. We have previously demonstrated that nucleolin targeting by N6L Published: 15 June 2021 impairs tumor growth and normalizes tumor vessels in PDAC mouse models. Here, we investigated new pathways that are regulated by nucleolin in PDAC. We found that N6L and nucleolin interact Publisher’s Note: MDPI stays neutral with β-catenin. We found that the Wnt/β-catenin pathway is activated in PDAC and is necessary with regard to jurisdictional claims in for tumor-derived 3D growth. N6L and nucleolin loss of function induced by siRNA inhibited published maps and institutional affil- Wnt pathway activation by preventing β-catenin stabilization in PDAC cells. N6L also inhibited iations. the growth and the activation of the Wnt/β-catenin pathway in vivo in mice and in 3D cultures derived from MIA PaCa2 tumors. On the other hand, nucleolin overexpression increased β-catenin stabilization. In conclusion, in this study, we identified β-catenin as a new nucleolin interactor and suggest that the Wnt/β-catenin pathway could be a new target of the nucleolin antagonist N6L Copyright: © 2021 by the authors. in PDAC. Licensee MDPI, Basel, Switzerland. This article is an open access article Keywords: pancreatic ductal adenocarcinoma; nucleolin; N6L; Wnt/β-catenin pathway; tumor distributed under the terms and conditions of the Creative Commons microenvironment; cancer therapy Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Cancers 2021, 13, 2986. https://doi.org/10.3390/cancers13122986 https://www.mdpi.com/journal/cancers Cancers 2021, 13, 2986 2 of 19 1. Introduction Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and lethal cancers. The incidence rate continues to increase for this type of cancer, and only 9% of PDAC patients survive beyond 5 years [1], often due to late symptom onset and diagnosis. Nucleolin is overexpressed in several tumors and in pancreatic ductal adenocarcinoma (PDAC); 74.5% of patients have a high level of this protein’s expression, and it correlates with a poor prognosis [2,3]. Nucleolin is a nucleocytoplasmic protein involved in ribosomal assembly and mRNA metabolism, promoting cell proliferation and survival of tumor cells [4]. Our group patented the nucleolin antagonist N6L, which shows antitumor activi- ties in breast cancer [5], prostate cancer [6], leukemia [7], non-small cell lung carcinoma [8], brain tumors [9,10], and pancreatic ductal adenocarcinoma [2,11], and furthermore can be coupled to nanoparticles [11–13] or toxins [10]. The Wnt/β-catenin signaling pathway has a central role in embryogenesis and is dys- regulated in several types of cancers [14,15]. It enhances cell proliferation, migration, and survival. When the Wnt/β-catenin signaling pathway is not active, β-catenin is phospho- rylated by casein kinase 1 (CK1) and glycogen synthase kinase 3β (GSK3β), ubiquitinated by β-transducin repeat-containing protein (β-TrCP) and degraded by the proteasome [16]. The activation of the pathway by Wnt ligands to the receptor Frizzled [17,18] induces the accumulation of β-catenin, and the transcription of target genes [19,20]. The Wnt/β-catenin pathway seems to be involved in carcinogenesis and progression of PDAC [21–24]. We are investigating molecular pathways in order to find new diagnostic markers and therapeutic targets in PDAC, in order to develop more effective therapies. N6L localizes to tumor tissue and reduces tumor growth by inducing apoptosis and inhibiting cell proliferation [2,5]. However, the mechanism of its effects on tumor cell growth was still unclear. The aim of this work was to investigate new molecular interactors of nucleolin, and we found that the Wnt/β-catenin pathway is a new pathway that is inhibited by N6L in PDAC. 2. Results 2.1. N6L Interacts with b-Catenin The nucleolin antagonist N6L inhibits PDAC tumor growth by impairing tumor cell proliferation and survival [2]. We sought to investigate the mechanism of action of N6L by studying the N6L interactome in PDAC cells. Since N6L binds to extranuclear nucleolin, an approach of N6L pull-down of the extranuclear cellular fraction of PDAC cells and identification of proteins by mass-spectrometry was applied. Increasing concentrations of biotinylated-N6L were incubated on murine PDAC (mPDAC) cells. Cells were lysed with a specific protocol to harvest only the extranuclear fraction of cells [25], and proteins bound to biotinylated-N6L were isolated by a pull-down assay. Protein bands showed an increasing intensity from 5 µM to 10 µM in biotinylated-N6L pull-down samples, supporting a dose-dependent efficacy of the interaction with N6L (Figure S1). Bands of proteins from the 10 µM biotinylated-N6L pull-down sample or from the pull-down of biotin alone were processed for mass-spectrometry analysis (Table S1). Non- specific proteins found in the negative control were subtracted from the list of the spe- cific proteins interacting with N6L. Forty-five proteins were identified as N6L partners (Table S1). Nucleolin and 18 known interactors of nucleolin, such as integrin αvβ3 [26], belong to the list of 45 N6L partners (Table S1). The gene ontology (GO) of the 45 proteins pulled down by N6L is built in a ClueGO network by using Cytoscape software and larger hexagons have lower p-value (Figure1A). The more highly represented biological processes were the ones in which nucleolin was involved, such as “regulation of rRNA processing”, “ribosomal large and small subunit biogenesis”, “mRNA stabilization”, and “regulation of translational initiation” (Figure1A). The same statistically overrepresented biological processes were obtained by GO enrichment analysis (Table S2). This evidence supported the idea that this approach isolated specific N6L targets and could be used to find new N6L targets. We were interested in the biological process of “response to antineoplastic Cancers 2021, 13, x 3 of 19 Cancers 2021, 13, 2986 the idea that this approach isolated specific N6L targets and could be used3 ofto 19 find new N6L targets. We were interested in the biological process of “response to antineoplastic agents”, in which we identified β-catenin as a new interesting potential target of N6L. The interactionagents”, between in which β we-catenin identified andβ-catenin N6L aswas a new validated interesting by potential a pull-down target of N6L.assay The in murine (mPDAC)interaction and two between humanβ-catenin (MIA and PaCa-2 N6L wasand validated Panc-1) byPDAC a pull-down cells. β assay-catenin in murine and nucleolin were present(mPDAC) in and N6L two humanpull-down (MIA PaCa-2of mPDAC and Panc-1) (Figure PDAC 1B), cells. MIAβ-catenin PaCa-2 and (Figure nucleolin 1C), and Panc-1were (Figure present S3A) in N6Lcells. pull-down of mPDAC (Figure1B), MIA PaCa-2 (Figure1C), and Panc-1 (Figure S3A) cells. A L L 6 6 N - -N B d C d e t te la a l y l o n l y t tr i o n u t t r ti p n o u t o i n io in c B p o in c B -catenin -catenin nucleolin nucleolin mPDAC MIA PaCa-2 Figure 1.