TET1-expressing cells and cell clusters are frequently encountered in PCa and seldom present in benign prostate tissues
Using conventional tissue slides, we examined TET1 expression by IHC in NOR and PCa specimens obtained from 50 PCa patients (Table 1, Fig. 1A). Particular regard was given to the cell specificity of TET1 expression. The basal cell marker CK903 and PCa cell marker AMACR were examined in parallel to TET1. In NOR, TET1 was exclusively expressed in CK903-positive basal cells, and TET1-expressing cells were scattered in the basal epithelium (Fig. 1A.1 and Additional file 1: Fig. S1A). In contrast, in PCa, TET1 was abundantly expressed exclusively in AMACR-positive cancer cells, whereby not all AMACR-positive cells expressed TET1 (Fig. 1A.3 and Additional file 1: Fig. S1C).
For a comprehensive overview, we performed a large-scale IHC study of TET1 expression using TMAs representing NOR and PCa specimens from 371 PCa patients (Table 2). In total, we could analyze 1371 single PCa-TMA spots and 669 single NOR-TMA spots. Observations in TMAs confirmed the results obtained from IHC analyses using conventional slides. In NOR-TMA, TET1 showed a scattered expression pattern in basal epithelial cells (Fig. 1A.2 and Additional file 1: Fig. S1B). In PCa-TMA, TET1-expressing cells appeared much more frequently and formed clusters (Fig. 1B, C.1, and Additional file 1: Fig. S1D). In both NOR and PCa, TET1 expression was detectable in the cytoplasm (Fig. 1B.1) as well as in the nucleus (Fig. 1B.2). Single PCa- and NOR-TMA spots could be categorized with respect to numbers of TET1-expressing cells as “TET1-high” (presence of numerous TET1-expressing cell clusters) (Fig. 1C.1), “TET1-moderate” (presence of a few scattered TET1-expressing cells) (Fig. 1C.2), or “TET1-negative” (absence of TET1-expressing cells) (Fig. 1C.3). From 371 PCa patients, 65 (17.5%) exhibited at least one PCa-TMA spot with high TET1 expression, 160 (43.1%) exhibited at least one PCa-TMA spot with moderate TET1 expression (Fig. 1C.1, C.2), and 146 (39.4%) exhibited only TET1-negative PCa-TMA spots (Fig. 1C.3). Among 366 PCa patients for which NOR-TMA were analyzed, ten (3%) exhibited at least one NOR-TMA spot with high TET1 expression, 181 (50%) at least one NOR-TMA spot with moderate TET1 expression, and 175 (48%) exhibited only TET1-negative NOR-TMA spots (Fig. 1C.4). Comparison of PCa patients considering the tumor stages (pT2 to pT4) and Gleason scores (GS ≤ 6 to ≥ 9) revealed that “TET1-high” foci were detectable in PCa at any tumor stage and Gleason grade (Fig. 1C.4). The frequencies of occurrence of TET1-high, moderate, and negative TMA spots were similar in PCa specimens from tumors of different stages and Gleason grades (Fig. 1C.4).
Upregulation of TET1 in PCa is caused by aberrant DNA methylation in the TET1 promoter, 5′-UTR, and gene body
Cancer-associated up- and downregulation of proteins is often caused by epigenetic dysregulation of the genes. To understand the epigenetic reasons for TET1 upregulation in PCa, we used TCGA, in particular the methylome and transcriptome data generated on 341 PCa and 35 NOR specimens (Additional file 2: Table S1).
An initial comparison of TET1 expression in PCa and NOR without any categorization of tissue samples showed no significant differences (Additional file 1: Fig. S2A). A comparison of PCa samples with regard to TET1 expression in consideration of Gleason grade and tumor stage also showed no significant differences (Additional file 1: Fig. S2B). As methylation changes leading to aberrant gene expression may happen at specific sites throughout a gene, we aimed to systematically analyze all available TET1 CpG probes in TCGA and to identify those critically important for TET1 expression. Altogether, 30 CpG probes, including 8 in the promoter (TSS200 and TSS1500), 16 in the 5′-UTR, and 6 in the gene body of TET1 were available in TCGA (Additional file 2: Table S2). Spearman’s analysis of possible correlation between TET1 methylation at 30 CpG probes and TET1 expression revealed the methylation at 4 CpG sites in NOR (one in the promoter and 3 in the 5′-UTR) and 10 CpG sites in PCa (4 in the promoter, 3 in the 5′-UTR, and 3 in the gene body) to be significantly correlated with TET1 expression (Fig. 2A.1 and Additional file 2: Table S3). In NOR, methylation of all four CpG sites was negatively correlated with TET1 expression (i.e., the more they were methylated, the less the gene was expressed). In contrast, in PCa, methylation of 6 out of 10 detected CpG sites was negatively correlated with TET1 expression, and methylation of 4 was positively correlated (i.e., the more methylation, the more TET1 was expressed) (Fig. 2A.1 and Additional file 2: Table S3). These data show the complexity of TET1 dysregulation in PCa. Further differential methylation analyses of 30 TET1 CpG probes in PCa versus NOR revealed 18 significantly hypermethylated CpG probes in PCa (4 in the promoter, 12 in the 5′-UTR, and 2 in the gene body) and 4 significantly hypomethylated CpG sites in PCa (one in the 5′-UTR and 3 in the gene body) (Fig. 2A.1, Additional file 2: Table S3 and Additional file 1: Fig. S3). Comparison of PCa specimens with regard to Gleason scores revealed 7 CpG sites in TET1 (2 in the promoter and 5 in the gene body) to be significantly hypermethylated or hypomethylated in the course of prostate carcinogenesis (Additional file 1: Fig. S3).
Next, we selected the TET1 CpG probe showing the most significant correlation with TET1 expression in PCa and NOR, and analyzed it in PCa cell lines with regard to TET1 mRNA and TET1 protein expression. In both PCa and NOR, TET1_cg02952701 located in the TET1 promoter showed the strongest correlation with TET1 expression (Fig. 2A.2, A.3, Additional file 2: Table S3). Analyses in PCa cell lines showed that hypermethylation of TET1_cg02952701 in LNCaP cells (Fig. 2B.1) was associated with downregulation of both mRNA and protein expression (Fig. 2B.2, B.3). In comparison, in DU145 and PC3 cells, TET1_cg02952701 was hypomethylated (Fig. 2B.1) and TET1 mRNA and protein were expressed (Fig. 2B.2, B.3). Thus, using PCa cell lines, direct causality between TET1 promoter hypomethylation and upregulation of TET1 mRNA and TET1 protein expression was confirmed.
Next, we categorized the TCGA-PCa specimens into three groups according to their TET1 expression levels: TET1-high (expression level above the 85th percentile, n = 51), TET1-low (expression below the 40th percentile, n = 136), and TET1-moderate (expression between the 40th and 85th percentiles, n = 154) (Fig. 2C.1), and analyzed the methylation of TET1_cg02952701. As expected, hypomethylation of TET1_cg02952701 was observed in the TET1-high group, and hypermethylation in the TET1-low group (Fig. 2C.2). We then performed differential methylation analyses considering all 30 TET1 CpG sites in TET1-high versus TET1-low PCa. In TET1-high PCa, four significantly hypomethylated CpG-sites in the TET1-promoter and five significantly hypermethylated CpG-sites in the TET1 5′-UTR and gene body were detected (Fig. 2C.3, Additional file 2: Table S4 and Additional file 1: Fig. S4). Importantly, the four hypomethylated CpG-sites were significantly negatively correlated with TET1 expression, that is, they contributed to a gain of TET1 expression, and the three hypermethylated CpG sites were significantly positively correlated with TET1 expression, that is, they also contributed to a gain of TET1 expression (Fig. 2C.3 and Additional file 2: Table S3). Thus, seven differentially methylated CpG-sites in TET1 caused an overexpression of TET1 in PCa. Further analyses showed that TET1-high PCa specimens were more frequently detected among PCa with higher Gleason scores (Gleason 6: 8.4%, Gleason 7: 13.3%, Gleason 8: 20.5%, Gleason ≥ 9: 17.6%) and higher tumor stages (T2a–c: 11.5%, T3a–b: 16.6%, T4: 40%) (Fig. 2C.4).
Identification of 626 TET1-coactivated genes in PCa
To investigate the impact of cells and cell clusters in PCa expressing TET1 and TET1 at high levels, we performed genome-wide correlation and gene regulatory network analyses using transcriptome and methylome data of PCa patients listed in TCGA (Fig. 3). Based on transcriptome data, we calculated the Pearson’s correlation coefficients between TET1 expression and the expression values of all 19,252 protein-coding genes. Of 7533 genes exhibiting a statistically significant positive correlation to TET1 expression (p < 0.05), 626 showed particularly strong correlation (Pearson’s r > 0.5, p < 0.0001). These 626 genes were referred to as TET1-coactivated genes and were subjected to further detailed bioinformatics studies (Fig. 3). TET1 expression was not correlated to AMACR expression (Pearson’s r = 0.01, p = 0.8). As TET1 is a 5-methylcytosine dioxygenase, and demethylation of gene promoters leads to increased expression of the respective mRNA, we also calculated the Spearman’s correlation coefficients for the promoter methylation of 626 TET1-coactivated genes and TET1 expression. Of 618 genes represented in the Illumina 450 K array, 594 possessed CpG sites in their promoters (sequences within 1500 and 200 bp of the transcriptional start site—TSS1500 and TSS200, respectively—were considered). In 279 out of 594 genes, the promoter was hypomethylated when TET1 expression was increased, and in TET1-high PCa these gene promoters were significantly less methylated than in TET1-low PCa. In 235 of those 279 genes, promoter methylation was significantly negatively correlated with the gene’s expression, that is, the hypomethylation of the promoter led to increased gene expression.
Upregulation of TET1 in PCa strongly correlates with promoter demethylation and enhanced expression of genes encoding zinc-finger transcription factors
According to the Human Transcription Factor (TF) databank [30], 161 out of 626 TET1-coactivated genes in PCa are identified TFs. Remarkably, 133 out of 161 TET1-coactivated TFs (82.6%) belong to the group of zinc-finger TFs (Fig. 4) and were significantly enriched for the GO “Molecular function” terms “DNA-binding transcription activator activity” (23/133, p = 5.0e−08) and “DNA-binding transcription repressor activity” (15/133, p = 1.0e−07). Among the top 30 TET1-coactivated TFs, we found primarily stem cell- and cancer-associated TFs, for example, RFX7 (an X-box-recognizing TF involved in cellular specialization and differentiation), REST (a Kruppel-type TF involved in the regulation of stem cell pluripotency), ZNF292 (a growth hormone-dependent TF with tumor suppressor activity), ARID2 (an AT-rich interactive domain-containing TF involved in embryonic patterning and cell lineage gene regulation), ZXDB (an X-linked TF promoting MHC gene expression), NR2C2 (a nuclear hormone receptor involved in aging-associated diseases), and SP1 (a Kruppel-like TF involved in cell growth and differentiation) (Fig. 4A). Importantly, the genes encoding the top 30 TET1-coactivated TFs all possess at least one promoter CpG site that was significantly demethylated when TET1 was significantly upregulated, and all these genes possessed significantly hypomethylated promoters in TET1-high PCa versus TET1-low PCa (Fig. 4B and Additional file 2: Table S6). In total, we identified 68 TFs in PCa whose gene promoters were significantly hypomethylated when TET1 was significantly upregulated (Additional file 2: Table S6).
Next, using JASPAR 2020 we analyzed the binding motifs and binding scores of TET1-coactivated TFs in the TET1 promoter and in the promoters of 626 TET1-coactivated genes (TSS1500 was considered). Of 161 TFs, 21 were listed in JASPAR 2020 and could be analyzed. In the TET1 promoter, TF-BSs were found for all 21 analyzed TFs (Additional file 2: Table S5). Segments in the TET1 promoter that were found to be significantly hypomethylated in TET1-high PCa and hence decisive for TET1-upregulation (Fig. 2C.3 and Additional file 2: Table S4) exhibited TF-BSs for RFX7, NR2C2, SP1, CREB1, MGA, SMAD5, ZBTB6, ZSCAN9, and ZNF354C (Additional file 2: Table S4). The vast majority of 626 TET1-coactivated genes also exhibited TF-BSs for 19 out of 21 TFs (Additional file 2: Table S5). The mean number of TF-BSs per gene promoter varied from a few (1 to 10), for example, in ZBTB26, ZKSCAN1, ZKSCAN29, SMAD5, POU2F1, ZBTB6, SP4, ZNF148, EHF, GABPA, CREB1, RREB1, and REST, to a great many (> 10), for example, in ZNF384, NR2C2, ZNF354C, RFX7, MGA, CLOCK, SP3, and SP1 (Additional file 2: Table S5). Nine out of 21 TET1-coactivated TFs, namely RFX7, SP1, SP3, SP4, POU2F1, ZBTB6, REST, CLOCK, and ZSCAN29, possessed CGs in their binding motifs and hence were sensitive to CpG-methylation and demethylation (Additional file 1: Fig. S5).
Many TFs of different families do not work alone and form complex homotypic or heterotypic interactions through dimerization [38]. Therefore, we analyzed the protein–protein interactions (PPI) and PPI networks (interactomes) of 161 TET1-coactivated TFs. Interactome analyses centered on 21 TFs that were available in JASPAR 2020 and showed true binding sites in the TET1 promoter and promoters of TET1-coactivated genes. Two interactomes, one SP1-centered and one CREB1-centered, were revealed (Additional file 1: Fig. S6). SP1, a zinc-finger transcription activator binding to GC-rich motifs, showed a direct interaction with six TFs (POU2F1, GABPA, ARNT, SP3, REST, and PURA) (Additional file 1: Fig. S6A.1) and an indirect interaction (i.e., through a bridge TF) with another 33 TFs (Additional file 1: Fig. S6A.2). CREB1, a transcription activator that binds to the cAMP response elements in many mammalian and viral promoters, showed a direct interaction with three TFs (ZHX1, POU2F1, and ZNF92) (Additional file 1: Fig. S6B.1) and an indirect interaction with a further 31 TFs (Additional file 1: Fig. S6B.2).
TET1-coactivated genes in PCa point to a cumulative gain of chromatin remodeling and mitotic activities
To characterize the functional features shared by 626 TET1-coactivated genes in PCa, we performed functional genomics studies (Fig. 3). GO analyses revealed a significant enrichment of biological processes primarily responsible for chromatin remodeling, such as “covalent chromatin modification,” “histone modification,” and “peptidyl-lysine modification” (Fig. 5A and Additional file 2: Table S7). In total, 78 genes encoding epigenetic modifiers were activated in PCa together with TET1 (Additional file 2: Table S7 and Additional file 1: Fig. S7). Moreover, significant enrichments of biological processes involved in regulating DNA metabolism and chromosome organization were also detected (Fig. 5A). Among TET1-coactivated genes, we found TET2, TET3, and DNMT3A. In both NOR and PCa, strong positive correlations between the expression of TET1, TET2, TET3, and DNMT1 were found (Additional file 1: Fig. S8). However, only in PCa, strong positive correlations were also found between TET1 and DNMT3A and DNMT3B expression (Additional file 1: Fig. S8). Further, GSEA of “Hallmark gene sets” on 626 TET1-coactivated genes showed a significant enrichment of mitotic and DNA replication hallmarks, in particular “mitotic spindle,” “G2M checkpoint,” and “E2F targets” (Fig. 5B and Additional file 2: Table S7). In total, 41 genes encoding mitotic factors were activated in PCa, together with TET1 (Additional file 2: Table S7). In order to examine, whether the P53-pathway is affected alongside with the E2F targets, we performed a GSEA of “Curated gene sets,” in particular gene sets representing canonical pathways. Among 626 TET1-coactivated genes, we found significant enrichments for pathways related to regulation of P53 activity by phosphorylation and methylation, and to P53-hypoxia (Additional file 2: Table S7). Moreover, GSEA of “Oncogenic signature gene sets” representing cellular pathways often dysregulated in cancer [35] on 626 TET1-coactivated genes showed highly significantly enrichments for gene sets related to Kirsten rat sarcoma viral oncogene homolog (KRAS) and TANK Binding Kinase 1 (TBK1), Placental Growth Factor (PGF), Janus Kinase 2 (JAK2), Catenin Beta 1 (CTNNB1) and Vascular Endothelial Growth Factor A (VEGFA) (Additional file 1: Fig. S9 and Additional file 2: Table S8).
Next, we performed TCGAanalyze_SurvivalKM, a univariate Kaplan–Meier (KM) survival analysis on 626 TET1-coactivated genes in PCa (Fig. 6, Additional file 2: Table S9). In total, expression of 35 genes showed a significant impact on survival (Additional file 2: Table S9). Survival curves of top 9 candidates (CCNT2, ZNF197, ORC2, TOPBP1, U2SURP, PWWP2A, ZNF550, SMC3 and ZNF782) are shown in Fig. 6 (p value < 0.02).
Analyzing publicly available TET1- and Tet1-ChIP-seq data, we discovered a strong similarity of MSigDB-hallmark- and GO-enrichment profiles in PCa and mouse trophoblast stem cells (mTSCs) [9, 10]. In mTSCs, 2691 gene promoters (defined as TSS1500) exhibited Tet1 binding sites (Fig. 7). A parallel analysis of TET1-coactivated genes in PCa and genes exhibiting Tet1 binding sites in mTSCs revealed an enrichment of nearly the same GO terms (primarily associated with chromatin modification) and same gene sets (primarily associated with mitosis) (Fig. 7).
TET1-correlated activation of ZNF antiviral genes in PCa
To explore the pathways activated in TET1-high PCa, we performed KEGG pathway analyses on 626 TET1-coactivated genes (Additional file 1: Fig. S10). We found that the “herpes simplex virus 1 infection” (HSV-1 infection) pathway was the most significantly enriched one (Additional file 1: Fig. S10A and S10B). In total, 62 genes encoding ZNF antiviral proteins (ZAPs) showed a strong positive correlation to TET1 expression and were significantly upregulated in TET1-high PCa (Additional file 1: Fig. S10C and Additional file 2: Table S10). Besides the genes encoding ZAPs, we also detected five genes encoding signaling molecules involved in the immune response to cancer, namely TRAF6, EIF2AK2, PIK3CA, APAF1, and POU2F2 (Additional file 1: Fig. S10C). Therefore, to assess the impact of viral infections in PCa, we analyzed 34 PCa and 16 benign prostate hyperplasia (BPH) samples with regard to different viral infections, including the herpesviruses HCMV, EBV and HSV-1/-2, and the polyomaviruses BK and JC (Additional file 2: Table S11). We detected infections with HCMV in 11/34 (32.4%) PCa and 3/16 (18.8%) BPH, and EBV in 5/34 (14.7%) PCa and 1/16 (6.3%) BPH (Additional file 2: Table S11). No infections with HSV-1/-2 or JC-/BK-viruses were found in PCa and BPH.
Furthermore, KEGG pathway analyses also revealed a significant enrichment of genes involved in “signaling pathway regulating pluripotency of stem cells” (Additional file 1: Fig. S10D and Additional file 2: Table S10). The expression of several genes encoding regulators of stem cell pluripotency, including PI3K, ACVR1/2, BMPR1/2, GSK3B, p38, SMARCAD1, SETDB1, JARID2, KAT6A, REST, and RIF1 showed a strong positive correlation with TET1 expression in PCa and were coactivated in TET1-high PCa (Additional file 1: Fig. S10D).