Insulin-like growth factor-2 (IGF2) loss of imprinting marks a field defect within human prostates containing cancer

Abstract

Background: Loss of imprinting (LOI) is an epigenetic alteration involving loss of parental origin-specific expression at normally imprinted genes. A LOI for IGF2, a paracrine growth factor, has been implicated in the development of prostate and other cancers. In the current study, we define IGF2 LOI in histologically normal prostate tissues in relationship to tumor foci and gene expression.

Methods: Microdissected tumor associated (TA) adjacent (2 mm) and distant (10 mm) tissues surrounding tumor foci were generated. IGF2 imprinting in informative prostate tissue sets was quantitated using a fluorescent primer extension assay and expression analyzed utilizing quantitative PCR. DNA methylation analyses were performed using quantitative pyrosequencing.

Results: A marked IGF2 LOI was found in adjacent TA tissues (39 ± 3.1%) and did not significantly decrease in tissues distant (38 ± 5.3%) from tumor foci (45 ± 2.9%; P = 0.21). IGF2 imprinting correlated with IGF2 expression in TA tissues, but not within the tumor foci. Hypomethylation of the IGF2 DMR0 region correlated with decreased IGF2 expression in tumors (P < 0.01). The expression of IGF2 and its adjacent imprinted gene H19 were increased in adjacent and distant tissues compared to tumors (P < 0.05) indicating the importance of factors other than LOI in driving IGF2 expression.

Conclusions: LOI of IGF2 occurs not only adjacent to prostate tumor foci, but is widely prevalent even in distant areas within the peripheral zone. These data provide evidence for a widespread epigenetic field defect in histologically normal tissues that might be employed to identify prostate cancer in patients.

Fig. 1

(A) Schematic showing prostate tumor, adjacent and distant normal tissue microdissection. Radical prostatectomy samples containing tumor foci were sectioned and samples microdissected for subsequent analyses. Adjacent and distant tumor-associated (TA) tissues did not contain histologic cancer. Samples were assessed in a three-dimensional pattern. (B) Fluorescent primer extension (FluPE) assay for IGF2 loss ofimprinting (LOI). Analysis of 9 informative sample sets using FluPE was performed and image analysis done using ImageJ software. LOI in prostate tumor foci ranged from 32 to 68% (mean 45%). Mean LOI in adjacent and distant TA regions was 39 and 38%, respectively. Analyses were performed in duplicate.

Fig. 2. IGF2 and H19 expression in tumor and TA tissues

Gene expression was measured using quantitative PCR(qPCR) in adjacent and distant TA tissues relative to the tumor for 18 microdissected sample sets (A), IGF2 mRNA expression (B), H19 mRNA expression (C). In contrast to predicted results, a positive correlation between IGF2 and H19 expression in distant TA tissues was seen (r = 0.82, P = 0.0001). No correlation was found between IGF2 and H19 gene expressionin tumors (data not shown) (* = P < 0.05).

Fig. 3. Correlation between IGF2 LOI and IGF2 expression

Gene expression was analyzed using qPCR and FLuPE utilized for imprinting analyses. Correlation analyses were performed using Spearman’s correlation. (A) Tumor samples (r = −0.49, P = 0.09), (B) distant TA tissues (r = 0.58, P = 0.017).

Fig. 4. Analysis of methylation status in paired prostate tumors and TA tissues

(A) Alterations in IGF2 expression have been linked to changes in methylation within DMR0 of the IGF2 promoter, as well as within CTCF#6 in the imprint control region (ICR) [24,54]. Methylation was analyzed using pyrosequencing specific primers. Tissues analyzed included prostate tumors with low or high IGF2 expression and distant TA tissues. An additional set of tissues from bladder, kidney, and prostates without cancer (NTA) that demonstrated maintained IGF2 imprinting were also analyzed for methylation. (B) At DMR0, tumors with low IGF2 expression demonstrate significantly decreased methylation compared to distant TA tissues. (C) CpG methylation analysis of CTCF6 in NTA tissues. TA prostate tissues from men with associated cancer (n = 6) demonstrating LOI is compared to imprinted NTA tissues (n = 7). A trend towards increased methylation in distant TA tissues is noted with significance seen at CpG 10 (P = 0.02).