Clinic of Urology, Pediatric Urology and Andrology, Justus-Liebig-University Giessen, Rudolf-Buchheim-Str. 7, 35392, Giessen, Germany.
Epigenetics of Urogenital System, Justus-Liebig-University Giessen, Schubertstr. 81, 35392, Giessen, Germany.
Cell Commun Signal. 2017 Oct 10;15(1):40. doi: 10.1186/s12964-017-0197-7.
Human cancer cells often exhibit impaired IGF2 expression and the underlying mechanisms are multifaceted and complex. Besides the well-known imprinting control region IGF2/H19-ICR, the involvement of a differentially methylated region in the promoter P0 of IGF2 gene (IGF2-DMR0) has been suggested. Here, we evaluate several mechanisms potentially leading to up- and/or down-regulation of IGF2 expression in prostate cancer and present a novel role of Kruppel-like factor 4 (KLF4) as a transcriptional regulator of IGF2 binding in IGF2-DMR0.
Putative binding sites for transcription factors were identified in IGF2-DMR0 using JASPAR CORE database. Gene expressions were analyzed by RT-qPCR in prostate carcinoma and adjacent benign prostate hyperplasia samples obtained by radical prostatectomy (86 RP-PCa and 47 RP-BPH) and BPH obtained by transurethral prostate resection (13 TUR-BPH). Pyrosequencing and qMSP were used for DNA methylation studies in IGF2-DMR0, IGF2/H19-ICR and Glutathione-S-transferase-P1 (GSTP1) promoter. Loss of imprinting (LOI) was analyzed by RFLP. Copy number variation (CNV) test was performed using qBiomarker CNV PCR Assay. KLF4-binding and histone-modifications were analyzed by ChIP-qPCR in prostate cancer cell lines exhibiting differentially methylated IGF2-DMR0 (LNCaP hypomethylated and DU145 hypermethylated). KLF4 protein was analyzed by western blot. Statistical associations of gene expression to methylation, IGF2 LOI and CNV were calculated by Mann-Whitney-U-test. Correlations between gene expression and methylation levels were evaluated by Spearman's-Rank-Correlation-test.
We found a significant reduction of IGF2 expression in the majority of RP-PCa and RP-BPH in comparison to TUR-BPH. Analyzing potential molecular reasons, we found in RP-PCa and RP-BPH in comparison to TUR-BPH a significant hypomethylation of IGF2-DMR0, which coincided with hypermethylation of GSTP1-promoter, a prominent marker of prostate tumors. In contrast, IGF2 LOI and CNV did not associate significantly with up- and/or down-regulation of IGF2 expression in prostate tumors. By analyzing IGF2-DMR0, we detected a consensus sequence for KLF4 with a z-score of 7.6. Interestingly, we found that KLF4 binds to hypomethylated (17%) IGF2-DMR0 enriched with H3K9me3 and H3K27me3 (LNCaP), but does not bind under hypermethylated (85%) and H3K4me3-enriched conditions (DU145). KLF4 expression was detected in TUR-BPH as well as in RP-BPH and RP-PCa and showed a highly significant correlation to IGF2 expression.
Our study demonstrated that in human prostate cancer the impairment of IGF2 expression is accompanied by hypomethylation of IGF2-DMR0. We revealed that KLF4 is a putative transcriptional regulator of IGF2, which binds in IGF2-DMR0 in dependence of the prevailing epigenetic state in this region. Herewith we provide complementary new insights into IGF2 dysregulation mechanisms as a critical process in prostate tumorigenesis.
人类癌细胞常表现出 IGF2 表达受损，其潜在机制复杂多样。除了众所周知的 IGF2/H19-ICR 印迹控制区外，IGF2 基因启动子 P0 中的差异甲基化区域 (IGF2-DMR0) 的参与也已被提出。在这里，我们评估了几种可能导致前列腺癌中 IGF2 表达上调和/或下调的机制，并提出了 Kruppel 样因子 4 (KLF4) 作为 IGF2 结合在 IGF2-DMR0 中转录调节因子的新作用。
使用 JASPAR CORE 数据库在 IGF2-DMR0 中识别潜在的转录因子结合位点。通过 RT-qPCR 分析前列腺癌和根治性前列腺切除术 (86 例 RP-PCa 和 47 例 RP-BPH) 中获得的相邻良性前列腺增生样本和经尿道前列腺切除术 (TUR) 中获得的 BPH (13 例 TUR-BPH) 中的基因表达。使用焦磷酸测序和 qMSP 进行 IGF2-DMR0、IGF2/H19-ICR 和谷胱甘肽 S-转移酶 P1 (GSTP1) 启动子的 DNA 甲基化研究。通过 RFLP 分析印迹缺失 (LOI)。使用 qBiomarker CNV PCR assay 进行拷贝数变异 (CNV) 检测。在表现出 IGF2-DMR0 差异甲基化的前列腺癌细胞系 (LNCaP 低甲基化和 DU145 高甲基化) 中，通过 ChIP-qPCR 分析 KLF4 结合和组蛋白修饰。通过 Western blot 分析 KLF4 蛋白。通过 Mann-Whitney-U 检验计算基因表达与甲基化、IGF2 LOI 和 CNV 之间的统计学关联。通过 Spearman's-Rank-Correlation-test 评估基因表达与甲基化水平之间的相关性。
我们发现与 TUR-BPH 相比，大多数 RP-PCa 和 RP-BPH 中 IGF2 的表达显著降低。分析潜在的分子原因，我们发现与 TUR-BPH 相比，RP-PCa 和 RP-BPH 中 IGF2-DMR0 甲基化程度显著降低，这与 GSTP1 启动子的高甲基化相一致，GSTP1 启动子是前列腺肿瘤的一个显著标志物。相比之下，IGF2 LOI 和 CNV 与前列腺肿瘤中 IGF2 表达的上调和/或下调没有显著关联。通过分析 IGF2-DMR0，我们检测到 KLF4 的一个共识序列，其 z 值为 7.6。有趣的是，我们发现 KLF4 结合低甲基化 (17%) IGF2-DMR0，富含 H3K9me3 和 H3K27me3 (LNCaP)，但在高甲基化 (85%) 和富含 H3K4me3 的条件下不结合 (DU145)。KLF4 在 TUR-BPH 以及 RP-BPH 和 RP-PCa 中均有检测到，与 IGF2 表达呈高度显著相关。
我们的研究表明，在人类前列腺癌中，IGF2 表达的受损伴随着 IGF2-DMR0 的低甲基化。我们揭示了 KLF4 是 IGF2 的一个潜在转录调节因子，它在该区域的表观遗传状态的影响下在 IGF2-DMR0 中结合。在此，我们提供了关于 IGF2 失调机制的互补新见解，这是前列腺肿瘤发生的关键过程。
