YTHDF3 in testicular germ cell tumors: a role in seminoma phenotype maintenance - PubMed (original) (raw)

m6A RNA modification and its writer/reader VIRMA/YTHDF3 in testicular germ cell tumors: a role in seminoma phenotype maintenance

João Lobo et al. J Transl Med. 2019.

Abstract

Background: 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 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.

Methods: In 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.

Results: In 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).

Conclusion: Abundance 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.

Keywords: Epitranscriptomics; M6A; RNA; Testicular germ-cell tumors; VIRMA; YTHDF3.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1

In silico analysis: frequency of alterations in queried genes in TCGA database. Notice the frequency of alterations in VIRMA and YTHDF3 compared to other queried genes. Also, note the frequent co-existence of alterations in this pair of genes, and the high proportion of seminomas showing deregulation of such genes

Fig. 2

Transcript levels of VIRMA and YTHDF3 among seminoma and non-seminomatous tumor samples. a VIRMA mRNA expression in seminomas vs. non-seminomatous tumors; b YTHDF3 mRNA expression in seminomas vs. Non-seminomatous tumors; c ROC curve for discrimination among seminomas and non-seminomatous tumors based on VIRMA mRNA expression levels. SE Seminoma, NST non-seminomatous tumor, AUC area under the curve, Ref reference genes GUSB and 18S, CI confidence interval

Fig. 3

VIRMA (a) and YTHDF3 (b) mRNA expression levels among different tumor subtypes. SE Seminoma, EC embryonal carcinoma, YST pospubertal-type Yolk sac tumor, CH choriocarcinoma, TE postpubertal-type Teratoma, Ref reference genes GUSB and 18S

Fig. 4

YTHDF3 transcript levels among Stage (a), IGCCCG Prognostic Group (b) and presence of metastases at diagnosis (c). IGCCCG international Germ Cell Cancer Collaborative Group; Ref reference genes GUSB and 18S

Fig. 5

Immunostaining for YTHDF3 (a, b), VIRMA (c, d); and m6A (e, f) in testicular germ cell tumors. a Strong YTHDF3 cytoplasmic immunoexpression in embryonal carcinoma; b Weak/moderate YTHDF3 cytoplasmic immunoexpression in postpubertal-type teratoma; c strong VIRMA nuclear immunoexpression in seminoma; d Weak/moderate VIRMA nuclear immunoexpression in Seminoma; e Strong m6A nuclear immunostaining in seminoma; f Weak/moderate m6A nuclear immunostaining in postpubertal-type yolk sac tumor. Note the granularity of staining, particularly in c, d and e

Fig. 6

Immunostaining intensity of VIRMA, YTHDF3 and m6A. a m6A vs. VIRMA immunostaining intensity; b VIRMA immunostaining intensity among seminomas vs. non-seminomatous Tumors; c–e VIRMA, YTHDF3 and m6A immunostaining intensity among different tumor subtypes. SE Seminoma; EC embryonal carcinoma, YST pospubertal-type Yolk sac tumor, CH choriocarcinoma, TE postpubertal-type teratoma, NST non-seminomatous tumors

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m6A RNA modification and its writer/reader VIRMA/YTHDF3 in testicular germ cell tumors: a role in seminoma phenotype maintenance - PubMed (original) (raw)