Distinguishing epigenetic features of preneoplastic testis tissues adjacent to seminomas and nonseminomas

Abstract

PIWI pathway proteins are expressed during spermatogenesis where they play a key role in germ cell development. Epigenetic loss of PIWI proteins expression was previously demonstrated in testicular germ cell tumors (TGCTs), implying their involvement in TGCT development. In this work, apart from studying only normal testis and TGCT samples, we also analyzed an intermediate stage, i.e. preneoplastic testis tissues adjacent to TGCTs. Importantly, in this study, we minimized the contribution of patient-to-patient heterogeneity by using matched preneoplastic/TGCT samples. Surprisingly, expression of germ cell marker DDX4 suggests that spermatogenesis is retained in premalignant testis tissues adjacent to nonseminoma, but not those adjacent to seminoma. Moreover, this pattern is followed by expression of PIWI pathway genes, which impacts one of their functions: DNA methylation level over LINE-1 promoters is higher in preneoplastic testis tissues adjacent to nonseminomas than those adjacent to seminomas. This finding might imply distinct routes for development of the two types of TGCTs and could be used as a novel diagnostic marker, possibly, noninvasively. Finally, we studied the role of CpG island methylation in expression of PIWI genes in patient samples and using in vitro experiments in cell line models: a more complex interrelation between DNA methylation and expression of the corresponding genes was revealed.

Keywords: DDX4; DNA methylation; LINE-1; PIWI; testicular germ cell tumor.

Figure 1. Expression of CIS and germ cell markers and PIWI protein genes in testis and testicular germ cell tumors

(A) Schematic description of the sample types used in the work and the approach to study normal-to-malignant transition. (B) Expression of carcinoma in situ markers POU5F1 (OCT3/4) and NANOG and germ cell marker DDX4. (C) Expression of PIWI protein genes PIWIL1, PIWIL2, and PIWIL4. Average value and SEM (Standard Error of the Mean) are shown for all samples. P value summary for Mann-Whitney non-paired U test and paired Wilcoxon signed-rank test (in brackets) are shown only for significant differences. Due to logariphmic scale several near-zero values are not depicted: DDX4 – 1 TGCT(SE), PIWIL1 – 1 TGCT(SE), PIWIL4 – 3 TGCT(SE), 1 CIS (SE), 1 CIS (NS) and 2 TGCT(NS).

Figure 2. Assessment of sensitivity and specificity of PIWIL2 expression level as a biomarker to distinguish between seminomas and nonseminomas

ROC curve analysis was performed and AUC (Area Under the Curve) was calculated for the cohorts from the present study (A) and The Cancer Genome Atlas project (B). The number of samples in each cohort and P values are indicated.

Figure 3. Integral methylation status of LINE-1 CpG islands

Melting curve analysis was used to obtain methylation level in healthy testis tissues (N), premalignant testis tissues (CIS samples) and testicular germ cell tumors (TGCT). SE – seminoma, NS – nonseminoma.

Figure 4. Integral methylation status of PIWIL1 and PIWIL2 CpG islands

Melting curve analysis was used to obtain methylation level of PIWIL1 (A) and PIWIL2 (B) CpG islands in healthy testis tissues (N samples), premalignant testis tissues (CIS samples) and testicular germ cell tumors (TGCT). SE – seminoma, NS – nonseminoma.

Figure 5. Luciferase reporter assay with methylated or unmethylated PIWIL1 and PIWIL2 promoters

Luciferase reporter constructs pGL4.10 with either methylated or unmethylated PIWIL1 (A) or PIWIL2 (B) promoter were transfected into TERA1 and A549 cells. The basic vector (no promoter control) was used as the reference.

Figure 6. Drug-induced demethylation of genomic promoters of PIWIL1 and PIWIL2

TERA1 and A549 cells were treated with 5-Aza-2′-deoxycytidine (5-aza-dC) and trichostatin A (TSA) and analyzed for promoter CpG methylation (A), chromatin modifications of active transcription start sites (H3K4me3, trimethylated lysine 4 in histone 3) along promoters (B), and transcription level (C) of PIWIL1 and PIWIL2 genes.