Comparative Evolutionary and Epigenetic Analysis of the Insulin-Like Growth Factor 2 P1 and P4 Promoters in Hepatocellular Carcinoma

Insulin-like growth factor 2 (IGF2) is transcribed from five promoters with distinct developmental and tissue-specific activities. P1 is the dominant promoter in healthy adult liver, whereas P4 is predominantly active during fetal development and is largely silent in adult liver. Because hepatocellular carcinoma (HCC) can reactivate fetal IGF2 transcriptional programs, promoter-specific regulation may be obscured by gene-level measurements. This study tested whether the adult-liver P1 and fetal P4 promoters represent distinct regulatory states by integrating comparative sequence analysis, CpG-island architecture, predicted transcription-factor binding, regulatory-locus annotation, and methylation-expression relationships in TCGA-LIHC. Overall sequence conservation was similar for P1 and P4, but their regulatory architecture and methylation-expression behavior differed. P4 retained CpG-island architecture across the examined primates and showed an inverse association between methylation and IGF2 expression, consistent with oncofetal reactivation. In contrast, P1 lacked a CpG island despite 68% GC content and showed a positive association between methylation and both total IGF2 expression and expression of the P1-specific first exon; this association persisted within HCC. These findings indicate that P1 and P4 exhibit promoter-specific epigenetic behavior in HCC that is not captured by a uniform promoter-methylation model. Independent validation and multivariable analysis are needed to determine the mechanisms underlying the unexpected P1 relationship.