M6A RNA modification and its writer/reader VIRMA/YTHDF3 in testicular germ cell tumors: a role in seminoma phenotype maintenance

João Lobo,Luís Antunes,Jorge Oliveira,Ana Laura Costa,Mattia Pelizzola,Isaac Braga,Rui Henrique,Carmen Jerónimo,Mariana Cantante,Rita Guimarães,Paula Lopes

doi:10.1186/s12967-019-1837-z

Abstract

BackgroundCovalent RNA modifications, such as N-6-methyladenosine (m6A), have been associated with various biological processes, but their role in cancer remains largely unexplored. m6A dynamics depends on specific enzymes whose deregulation may also impact in tumorigenesis. Herein, we assessed the differential abundance of m6A, its writer VIRMA and its reader YTHDF3, in testicular germ cell tumors (TGCTs), looking for clinicopathological correlates.MethodsIn silico analysis of TCGA data disclosed altered expression of VIRMA (52%) and YTHDF3 (48%), prompting subsequent validation. Formalin-fixed paraffin-embedded tissues from 122 TGCTs (2005–2016) were selected. RNA extraction, cDNA synthesis and real-time qPCR (Taqman assays) for VIRMA and YTHDF3 were performed, as well as immunohistochemistry for VIRMA, YTHDF3 and m6A, for staining intensity assessment. Associations between categorical variables were assessed using Chi square and Fisher’s exact test. Distribution of continuous variables between groups was compared using the nonparametric Mann–Whitney and Kruskal–Wallis tests. Biomarker performance was assessed through receiver operating characteristics (ROC) curve construction and a cut-off was established by Youden’s index method. Statistical significance was set at p < 0.05.ResultsIn our cohort, VIRMA and YTHDF3 mRNA expression levels differed among TGCT subtypes, with Seminomas (SEs) depicting higher levels than Non-Seminomatous tumors (NSTs) (p < 0.01 for both). A positive correlation was found between VIRMA and YTHDF3 expression levels. VIRMA discriminated SEs from NSTs with AUC = 0.85 (Sensitivity 77.3%, Specificity 81.1%, PPV 71.6%, NPV 85.3%, Accuracy 79.7%). Immunohistochemistry paralleled transcript findings, as patients with strong m6A immunostaining intensity depicted significantly higher VIRMA mRNA expression levels and stronger VIRMA immunoexpression intensity (p < 0.001 and p < 0.01, respectively).ConclusionAbundance of m6A and expression of VIRMA/YTHDF3 were different among TGCT subtypes, with higher levels in SEs, suggesting a contribution to SE phenotype maintenance. VIRMA and YTHDF3 might cooperate in m6A establishment in TGCTs, and their transcript levels accurately discriminate between SEs and NSTs, constituting novel candidate biomarkers for patient management.

Highlights

Covalent ribonucleic acid (RNA) modifications, such as N-6-methyladenosine (m6A), have been associated with various biological processes, but their role in cancer remains largely unexplored. m6A dynamics depends on specific enzymes whose deregulation may impact in tumorigenesis
The Cancer Genome Atlas (TCGA) dataset analysis revealed that KIAA1429/VIRMA and YTHDF3 (m6A reader) were the two most commonly altered m6A-related genes in testicular germ cell tumor (TGCT) (52% and 48% of the samples, respectively)
Most alterations consisted of transcript upregulation, with no mutations found for YTHDF3 and only one depicted for VIRMA (Fig. 1)

Summary

Introduction

Covalent RNA modifications, such as N-6-methyladenosine (m6A), have been associated with various biological processes, but their role in cancer remains largely unexplored. m6A dynamics depends on specific enzymes whose deregulation may impact in tumorigenesis. More than 95% of cases are derived from germ cells—testicular germ cell tumors (TGCTs)—and the vast majority of these correspond to germ-cell neoplasia in situ (GCNIS)-related tumors, according to the most recent World Health Organization (WHO) classification [4]. This category comprises two major subtypes—seminomas (SEs) and non-seminomatous tumors (NSTs)—and discrimination between them is of paramount clinical importance, entailing different prognosis and treatment algorithms [5, 6]. Heterogeneity is the hallmark of GCNIS-related TGCTs, reflecting this complex tumor model, they share a common cytogenetic background, i.e., isochromosome 12p [4, 8]. There is an increasing need for reliable and clinically validated TGCT biomarkers that might improve diagnosis, subtype discrimination, prognostication and patient monitoring, overcoming the limitations of classical serum markers currently employed in the clinical setting [9,10,11,12,13,14]

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Discussion

Conclusion