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

Stéphanie Buart,Philippe Vielh,Virginie Marty,Luc Legrès,Jane Muret,Philippe Dessen,Yann Gaston-Mathé,Paul Fogel,Guillaume Meurice,Sophie Ferlicot,Stéphane Terry,Caroline Robert,Alexander M.m Eggermont ,Nyam Kamsu‐Kom ,Morad El Bouchtaoui ,C Boutros ,Éric Deutsch ,Émilie Lanoy ,Jean‐Charles Soria ,Muhammad Zaeem Noman ,Émilie Routier ,Séverine Le Roy ,Salem Chouaı̈b

doi:10.18632/oncotarget.22150

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.

Highlights

Cancer immunotherapy has recently emerged as an important treatment modality
The analysis revealed the existence of a functional interaction between 15 genes of the hypomel signature (13 genes overexpressed: BNIP3, AK4, SLC2A1, ADM, PFKFB4, ENO2, VEGFA, DDIT4, PGK1, GBE1, ALDOC, CCL28 and 2 genes underexpressed: EPRS and DDX21) (Figure 1C)
The hypomel signature was validated in tumors fixed-paraffin-embedded www.impactjournals.com/oncotarget (FFPE) from patients who developed primary melanoma, metastatic lymph node, or cutaneous metastasis

Summary

Introduction

Cancer immunotherapy has recently emerged as an important treatment modality. FDA approval of sipuleucel-T (Provenge*), ipilimumab (Yervoy*), nivolumab (Opdivo*) and pembrolizumab (Keytruda*) has started to deliver on the long awaited promise of cancer immunotherapy [1]. The tumor microenvironment harbors a variety of host-derived cells It is a complex system playing an important role in tumor development and progression [5]. It involves soluble factors and metabolic changes. Tumor microenvironmental hypoxia is in this regard a relevant example demonstrating how the tumor microenvironment can paralyze and neutralize T cell functions [11]. It is a negative prognostic and predictive factor owing to its multiple contributions to chemoresistance, radioresistance, angiogenesis, resistance to cell death, altered metabolism and genomic instability [12]

Methods

Results

Conclusion