Transcriptional response to hypoxic stress in melanoma and prognostic potential of GBE1 and BNIP3

2017 
// Stephanie Buart 1 , Stephane Terry 1, * , Muhammad Z. Noman 1, * , Emilie Lanoy 2 , Celine Boutros 3 , Paul Fogel 4 , Philippe Dessen 5 , Guillaume Meurice 5 , Yann Gaston-Mathe 6 , Philippe Vielh 7 , Severine Roy 3 , Emilie Routier 3 , Virginie Marty 7 , Sophie Ferlicot 8 , Luc Legres 9 , Morad El. Bouchtaoui 9 , Nyam Kamsu-Kom 10 , Jane Muret 1 , Eric Deutsch 11, 12, 13, 14 , Alexander Eggermont 3, 14 , Jean-Charles Soria 10, 12 , Caroline Robert 3, 10, 14 and Salem Chouaib 1 1 INSERM UMR1186, Integrative Tumor Immunology and Genetic Oncology, Gustave Roussy, Equipe Labellisee par La Ligue Contre Le Cancer, EPHE, Faculte de Medecine, Universite Paris-Sud, Universite Paris-Saclay, Villejuif, France 2 INSERM UMR 1018, Gustave Roussy, Universite Paris-Sud, Universite Paris-Saclay, Villejuif, France 3 Department of Medical Oncology, Gustave Roussy, Villejuif, France 4 Independent Consultant, Paris, France 5 Plateforme de Bioinformatique, UMS AMMICA, Gustave Roussy, Villejuif, France 6 YGM Consult, CEO, Paris, France 7 Departement de Biologie et Pathologie Medicales, Gustave Roussy, Villejuif, France 8 Service d’Anatomie Pathologique, Hopitaux Universitaires Paris Sud, AP-HP, Le Kremlin Bicetre, France 9 Laboratoire de Pathologie, INSERM UMR_S-1165/Universite Paris-Diderot, Sorbonne Paris Cite, Paris, France 10 INSERM UMR 981, Gustave Roussy, Universite Paris-Sud, Universite Paris-Saclay, Villejuif, France 11 Department of Radiation Oncology, Gustave Roussy, Villejuif, France 12 Drug Development Department (DITEP), Gustave Roussy, Villejuif, France 13 INSERM U1030, Molecular Radiotherapy, Gustave Roussy, Universite Paris-Sud, Universite Paris-Saclay, Villejuif, France 14 Faculty of Medicine, Universite Paris-Sud, Universite Paris-Saclay, Le Kremlin-Bicetre, France * These authors have contributed equally to this work Correspondence to: Stephanie Buart, email: stephanie.buart@gustaveroussy.fr Salem Chouaib, email: salem.chouaib@gustaveroussy.fr Keywords: hypoxia; melanoma; GBE1; glucose transporter 1; BNIP3 Received: April 19, 2017     Accepted: June 27, 2017     Published: October 30, 2017 ABSTRACT Gradients of hypoxia occur in most solid tumors and cells found in hypoxic regions are associated with the most aggressive and therapy-resistant fractions of the tumor. Despite the ubiquity and importance of hypoxia responses, little is known about the variation in the global transcriptional response to hypoxia in melanoma. Using microarray technology, whole genome gene expression profiling was first performed on established melanoma cell lines. From gene set enrichment analyses, we derived a robust 35 probes signature (hypomel for HYPOxia MELanoma) associated with hypoxia-response pathways, including 26 genes up regulated, and 9 genes down regulated. The microarray data were validated by RT-qPCR for the 35 transcripts. We then validated the signature in hypoxic zones from 8 patient specimens using laser microdissection or macrodissection of Formalin fixed-paraffin-embedded (FFPE) material, followed with RT-qPCR. Moreover, a similar hypoxia-associated gene expression profile was observed using NanoString technology to analyze RNAs from FFPE melanoma tissues of a cohort of 19 patients treated with anti-PD1. Analysis of NanoString data from validation sets using Non-Negative Matrix Factorization (NMF) analysis (26 genes up regulated in hypoxia) and dual clustering (samples and genes) further revealed that the increased level of BNIP3 (Bcl-2 adenovirus E1B 19 kDa-interacting protein 3)/GBE1 (glycogen branching enzyme1) differential pair correlates with the lack of response of melanoma patients to anti-PD1 (pembrolizumab) immunotherapy. These studies suggest that through elevated glycogenic flux and induction of autophagy, hypoxia is a critical molecular program that could be considered as a prognostic factor for melanoma.
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