Upstream open reading frames regulate translation of cancer-associated transcripts and encode HLA-presented immunogenic tumor antigens

Annika Nelde,Juliane S Walz,Christoph Schliemann,Georg Lenz,Torsten Kessler,Lea Flötotto,Jens Bauer,Hans-Georg Rammensee,Elvira Hubert,Klaus Wethmar,Laura Szymik,Lara Jürgens

doi:10.1007/s00018-022-04145-0

Abstract

BackgroundUpstream open reading frames (uORFs) represent translational control elements within eukaryotic transcript leader sequences. Recent data showed that uORFs can encode for biologically active proteins and human leukocyte antigen (HLA)-presented peptides in malignant and benign cells suggesting their potential role in cancer cell development and survival. However, the role of uORFs in translational regulation of cancer-associated transcripts as well as in cancer immune surveillance is still incompletely understood.MethodsWe examined the translational regulatory effect of 29 uORFs in 13 cancer-associated genes by dual-luciferase assays. Cellular expression and localization of uORF-encoded peptides (uPeptides) were investigated by immunoblotting and immunofluorescence-based microscopy. Furthermore, we utilized mass spectrometry-based immunopeptidome analyses in an extensive dataset of primary malignant and benign tissue samples for the identification of naturally presented uORF-derived HLA-presented peptides screening for more than 2000 uORFs.ResultsWe provide experimental evidence for similarly effective translational regulation of cancer-associated transcripts through uORFs initiated by either canonical AUG codons or by alternative translation initiation sites (aTISs). We further demonstrate frequent cellular expression and reveal occasional specific cellular localization of uORF-derived peptides, suggesting uPeptide-specific biological implications. Immunopeptidome analyses delineated a set of 125 naturally presented uORF-derived HLA-presented peptides. Comparative immunopeptidome profiling of malignant and benign tissue-derived immunopeptidomes identified several tumor-associated uORF-derived HLA ligands capable to induce multifunctional T cell responses.ConclusionOur data provide direct evidence for the frequent expression of uPeptides in benign and malignant human tissues, suggesting a potentially widespread function of uPeptides in cancer biology. These findings may inspire novel approaches in direct molecular as well as immunotherapeutic targeting of cancer-associated uORFs and uPeptides.

Highlights

Upstream open reading frames represent translational control elements within eukaryotic transcript leader sequences
Aiming to characterize the functional impact of Upstream open reading frames (uORFs)-mediated translational regulation and the prevalence of uPeptide expression in cancer-related genes, we first selected a set of 29 upstream AUG (uAUG) and alternative translation initiation sites (aTISs) uORFs from 13 cancer-associated genes (Fig. 1a, Supplementary Table 3)
Upstream ORFs were selected based on literature research [4, 38, 39] and categorized according to the type of initiation codon and the computationally defined uORF and uPeptide scores [3] predicting functional relevance

Summary

Introduction

Upstream open reading frames (uORFs) represent translational control elements within eukaryotic transcript leader sequences. Recent data showed that uORFs can encode for biologically active proteins and human leukocyte antigen (HLA)-presented peptides in malignant and benign cells suggesting their potential role in cancer cell development and survival. We utilized mass spectrometry-based immunopeptidome analyses in an extensive dataset of primary malignant and benign tissue samples for the identification of naturally presented uORFderived HLA-presented peptides screening for more than 2000 uORFs. Results We provide experimental evidence for effective translational regulation of cancer-associated transcripts through uORFs initiated by either canonical AUG codons or by alternative translation initiation sites (aTISs). Conclusion Our data provide direct evidence for the frequent expression of uPeptides in benign and malignant human tissues, suggesting a potentially widespread function of uPeptides in cancer biology These findings may inspire novel approaches in direct molecular as well as immunotherapeutic targeting of cancer-associated uORFs and uPeptides. Only a few reports provided individual experimental evidence for the regulatory impact of uAUG and aTIS uORFs in human proto-oncogenes [11–13, 15, 26, 27]

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Discussion

Conclusion