Abstract 3734: Progressive spreading of CpG methylation in the CpG island of Glutathione S-Transferase pi 1 (GSTP1) alleles across transitions from precursor to invasive prostate cancer
Authors: Gupta Harshath , Inoue Hitoshi , Nakai Yasutomo , Nakayama Masashi , Nelson William G. , De Marzo Angelo M. , Yegnasubramanian Srinivasan 
Published: June 2022

Abstract
Glutathione S-transferase pi 1 (GSTP1) is expressed at low levels in normal prostate luminal cells and becomes induced in most proliferative inflammatory atrophy lesions (PIA). GSTP1 becomes silenced in prostatic intraepithelial neoplasia (PIN) and prostate adenocarcinoma (CaP) via CpG island promoter hypermethylation. However, the specific methylation patterns in CaP and its precursors have not been investigated. We used bisulfite genomic sequencing to examine methylation of 39 CpG sites in the GSTP1 promoter. Radical prostatectomy specimens were selected from 32 subjects that underwent treatment for CaP. Serial sections were subjected to laser capture microdissection for enrichment of epithelial cells from benign, PIA, PIN, and CaP regions. Isolated DNA was bisulfite converted. Sequences corresponding to the GSTP1 promoter CpG island were amplified with nested PCR, and PCR products were cloned. 10 independent clones were sequenced and analyzed with a Java program, DNAMethylMap, to determine the methylation pattern of 39 CpG sites of the GSTP1 promoter in each lesion. The extent of methylation on an individual allele was classified as negative (&lt;10% CpGs), mild (10-25%), moderate (25-50%), or high (&gt;50%). 212 clones from 24 normal epithelium regions, 327 clones from 37 PIA regions, 167 clones from 18 PIN regions, and 202 clones from 23 CaP regions were sequenced, totaling 34863 CpG sites. Normal and PIA lesions were mostly unmethylated with 0.52% and 1.3% of total CpG sites methylated, respectively. Methylated PIA lesions were adjacent to CaP or PIN regions significantly more often than unmethylated PIA lesions (χ2, p = 0.012). PIN and CaP lesions had greater methylation with 24% and 51% of total CpG sites methylated, respectively. PIN lesions generally showed partial methylation with 28.7% of alleles in PIN having mild/moderate methylation density compared to 5.5% in PIA and 11% in CaP. PIA and PIN lesions were enriched for methylation changes at 6 CpG sites that aligned with AP1 and SP1 binding sites. Among 16 PIN and CaP lesions with an overall intermediate level of methylation, 5 of the lesions had clones that clustered into either fully methylated or unmethylated. One patient with 3 CaP lesions displayed significant heterogeneity in methylation patterns: full, moderate, or no methylation across all clones in a given lesion. The results demonstrate that methylation density in the GSTP1 CpG island from PIN was intermediate between normal prostate epithelium/PIA and CaP lesions. The observed methylation in PIA and PIN was enriched at binding sites of key transcription factors, AP1 and SP1. The results are consistent with gradual spreading of DNA methylation centered at the transcription factor binding sites in the putative precursor lesions, with subsequent spreading of methylation across the entire CpG island in transition to CaP.
Citation Format: Harshath Gupta, Hitoshi Inoue, Yasutomo Nakai, Masashi Nakayama, William G. Nelson, Angelo M. De Marzo, Srinivasan Yegnasubramanian. Progressive spreading of CpG methylation in the CpG island of Glutathione S-Transferase pi 1 (GSTP1) alleles across transitions from precursor to invasive prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3734.