Aging and cancer-related loss of insulin-like growth factor 2 imprinting in the mouse and human prostate

Abstract

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, is important in cancer progression. Epigenetic modifications may be altered by environmental factors. However, is not known whether changes in imprinting occur with aging in prostate and other tissues susceptible to cancer development. We found a LOI for Igf2 occurs specifically in the mouse prostate associated with increased Igf2 expression during aging. In older animals, expression of the chromatin insulator protein CTCF and its binding to the Igf2-H19 imprint control region was reduced. Forced down-regulation of CTCF leads to Igf2 LOI. We further show that Igf2 LOI occurs with aging in histologically normal human prostate tissues and that this epigenetic alteration was more extensive in men with associated cancer. This finding may contribute to a postulated field of cancer susceptibility that occurs with aging. Moreover, Igf2 LOI may serve as a marker for the presence of prostate cancer.

Figure 1

Re-expression of inactive Igf2 allele in aging mouse tissues. A, DLP tissues were microdissected from aging mice (3, 11, 19, and 24 mo) and the maternal and paternal allelic expression was measured using FluPE. The ratio of the inactive allele (Aⁱ) to active allele (A^a) was calculated for each aging cohort (n = 6). All DLP samples in the 11-, 19-, and 24-month cohorts have significantly higher Aⁱ/A^a ratios when compared with the 3-mo-old cohort (**, P < 0.01; *, P < 0.05). B, Igf2 imprinting is maintained with aging in the VP and other mouse tissues. Allelic expression from the liver, kidney, and VP was determined using FluPE. Tissues from 3- and 19-mo-old cohorts (n = 6) were compared and no age-associated relaxation of Igf2 imprinting was seen.

Figure 2

Igf2 expression increases in aging mouse DLP. A, QPCR was used to measure Igf2 expression levels in the mouse DLP of the 3-, 11-, 19-, and 24-mo-old cohorts (n = 6; **, P < 0.01; *, P < 0.05). B and C, mouse DLP sections were analyzed using immunofluorescence for Igf2 and α-tubulin (control). Images from five different random fields were acquired per section (n = 3) and the integrated density of each whole single-color image was measured with NIH ImageJ as described. Igf2 measurements were then normalized to that of α-tubulin. The older 24-mo cohort expresses significantly higher levels of Igf2 protein (P < 0.01) when compared with the 3-mo group.

Figure 3

Decrease of CTCF binding in aging mouse DLP. A, schematic of the Igf2-H19 genomic region showing the ICR containing four CTCF binding sites. Methylation analysis using Methylation-sensitive PCR and bisulfite cloning of CTCF binding sites 3 (63% ± 6% versus 52% ± 3%; P = 0.16) and 4 (58% ± 5% versus 47% ± 5%; P = 0.26) did not show any differences when young and old animals were compared (n = 5). CTCF 3 and 4 represent the major target sites for CTCF binding in this region, whereas CTCF 2 does not display nuclease hypersensitivity. B, ChIP assay was performed to examine CTCF protein binding alterations at CTCF binding sites 2, 3, and 4 within the H19 ICR in aging mouse DLP. A significant decrease in binding is noted in older (24 mo) versus younger (3 mo) cohorts at both CTCF 4 and CTCF 3 (**, P < 0.01; n = 6). No alteration in CTCF 2 binding (a site that does not regulate Igf2 promoter activity) is seen. C, no significant change in CTCF binding is seen when the older cohort is compared with younger in the VP, a tissue found to retain imprinting. Binding to IgG was used as a nonspecific control for the experiments.

Figure 4

Relaxation of Igf2 imprinting in aging histologically normal human prostate tissues with and without associated cancer. A, prostate tissues from donors of various ages were analyzed using FluPE. The imprint status from younger patients ages <40 y (mean, 27 y) was significantly (P = 0.02) more imprinted when compared with a cohort of tissues from patients older than 55 y (mean, 64 y). Histologically normal peripheral prostate tissues from men with associated prostate cancer (mean, 63 y) showed a greater associated LOI (60%) than an age-matched noncancer associated cohorts (**, P < 0.01). Interestingly, variations in Aⁱ/A^a ratios within groups of younger men even without associated cancer are seen. These were not associated with the presence of inflammation on histology. B, model of epigenetic changes in the prostate with aging. The imprinting of Igf2 (expression of single allele depicted in cell) is maintained in prostate tissues from young mammals. In rodents, who poorly maintain epigenetic patterns, and in humans with associated cancer, a marked loss of Igf2 imprinting (LOI) occurs. This LOI generates a “field effect” throughout the peripheral zone of the prostate that is associated with the development of prostate cancer in multiple areas. Other genetic and epigenetic factors are required for the development of malignancy. Igf2 imprinting patterns are generally maintained and cancer risk is lower in individuals who maintain Igf2 imprinting.