Insulin-like growth factor 2 expression in prostate cancer is regulated by promoter-specific methylation

Abstract

Deregulation of the insulin-like growth factor (IGF) axis and dysbalance of components of the IGF system as potential therapeutic targets have been described in different tumor types. IGF2 is a major embryonic growth factor and an important activator of IGF signaling. It is regulated by imprinting in a development- and tissue-dependent manner and has been implicated in a broad range of malignancies including prostate cancer (PCa). Loss of imprinting (LOI) usually results in bi-allelic gene expression and increased levels of IGF2. However, the regulatory mechanisms and the pathophysiological impact of altered IGF2 expression in PCa remain elusive. Here, we show that in contrast to many other tumors, IGF2 mRNA and protein levels were decreased in 80% of PCa in comparison with non-neoplastic adjacent prostate and were independent of LOI status. Instead, IGF2 expression in both tumors and adjacent prostate depended on preferential usage of the IGF2 promoters P3 and P4. Decreased IGF2 expression in tumors was strongly related to hypermethylation of these two promoters. Methylation of the A region in promoter P4 correlated specifically with IGF2 expression in the 20% of PCa where IGF2 was higher in tumors than in adjacent prostate. We conclude that IGF2 is downregulated in most PCa and may be particularly relevant during early stages of tumor development or during chemotherapy and androgen deprivation. PCa differs from other tumors in that IGF2 expression is mainly regulated through methylation of promoter-specific and not by imprinting. Targeting of promoter-specific regions may have relevance for the adjuvant treatment of PCa.

Keywords: IGF2; imprinting; promoter methylation; prostate cancer; targeted therapy.

Figure 1

Schematic overview of the IGF2/H19 gene locus with protein coding exons (dark gray) (NG_008849.1) and the four promoter regions P1–P4. The P4 region can be further subdivided into CpG regions P4A, P4B1, and P4B2 (not shown) (Qian et al., 2011). The ApaI SNP rs680 is located at the 5′‐UTR. The ICR is located 90 kb downstream of the IGF gene and closer to the H19 locus.

Figure 2

Distribution of imprinting status and IGF2 expression in T and N. (A) Frequency of LOI and ROI in T (n = 32) and N (n = 32). (B) Concordance of LOI vs. ROI in 22 paired T and N samples. (C) IGF2 mRNA ratio in 60 paired T and N samples. Twelve cases (20%) showed a higher level of IGF2 in T than in N (IGF2high). (D) ELISA measurement of 13 paired samples showed significantly lower IGF2 protein concentration in T than in N (P < 0.0012).

Figure 3

Promoter‐specific IGF2 transcripts (P1–P4) in T and N. (A) Expression of promoter‐specific transcripts P1–P4 in T and N. P3 and P4 were dominant in both T and N. (B) P3‐ and P4‐specific transcripts were significantly higher expressed in N than in T (P < 0.0002 and P < 0.004, respectively). (C+E) Significant correlation between total IGF2 mRNA and promoter transcripts P3 in T (r = 0.42, P < 0.009) and N (r = 0.45, P < 0.002). (D+F) The correlation between total IGF2 mRNA and promoter transcripts P4 was statistically significant only in N (r = 0.71, P < 0.0001), but not in T (r = 0.05295, P > 0.05). **P > 0.005, ***P > 0.0005.

Figure 4

Methylation of promoter P3 and P4 correlates with IGF2 mRNA expression in PCa and normal prostate. (A) Overall higher methylation of P3 (P < 0.05) and (B) P4 (P < 0.05) in T (n = 21) compared to N (n = 22). (C+D) However, when cases were separated into IGF2_low and IGF2_high tumors, this difference was found only in IGF2low tumors for P3 (P < 0.05) and P4 (P < 0.05). (E) In IGF2_high tumors, there was a significant correlation between IGF2 expression and methylation of P4 (r = 0.64, P < 0.05). (F) The methylation of the CpG in P4A correlated significantly with the P4 promoter‐specific transcript (r = 0.80, P < 0.05). *P > 0.05.