The protein tyrosine phosphatase receptor type R gene is an early and frequent target of silencing in human colorectal tumorigenesis

Abstract

Background: Tumor development in the human colon is commonly accompanied by epigenetic changes, such as DNA methylation and chromatin modifications. These alterations result in significant, inheritable changes in gene expression that contribute to the selection of tumor cells with enhanced survival potential.

Results: A recent high-throughput gene expression analysis conducted by our group identified numerous genes whose transcription was markedly diminished in colorectal tumors. One of these, the protein-tyrosine phosphatase receptor type R (PTPRR) gene, was dramatically downregulated from the earliest stages of cellular transformation. Here, we show that levels of both major PTPRR transcript variants are markedly decreased (compared with normal mucosal levels) in precancerous and cancerous colorectal tumors, as well in colorectal cancer cell lines. The expression of the PTPRR-1 isoform was inactivated in colorectal cancer cells as a result of de novo CpG island methylation and enrichment of transcription-repressive histone-tail marks, mainly H3K27me3. De novo methylation of the PTPRR-1 transcription start site was demonstrated in 29/36 (80%) colorectal adenomas, 42/44 (95%) colorectal adenocarcinomas, and 8/8 (100%) liver metastases associated with the latter tumors.

Conclusions: Epigenetic downregulation of PTPRR seems to be an early alteration in colorectal cell transformation, which is maintained during the clonal selection associated with tumor progression. It may represent a preliminary step in the constitutive activation of the RAS/RAF/MAPK/ERK signalling, an effect that will later be consolidated by mutations in genes encoding key components of this pathway.

Figure 1

Expression of PTPRR mRNA in normal colorectal mucosa, polypoid precancerous colorectal lesions, colorectal cancers, and colorectal cancer cell lines. (A): mRNA levels based on normalized raw signal values (y-axis) were detected with the Affymetrix U133 Plus 2.0 arrays in the tissue sample series reported in Sabates-Bellver et al., 2007. (B): The two main isoforms of PTPRR are shown aligned from 5' to 3'. Introns have been reduced to minimal lengths, whereas exon sizes are proportional to their actual lengths. Horizontal bars represent the portions of cDNA that were amplified by qRT-PCR to discriminate between the two isoforms (see Methods). (C) and (D): PTPRR-1 and PTPRR-2 expression was investigated with real time qRT-PCR in a subset of the samples collected for the present study (see Methods). Fold changes were calculated as reported in Methods: For polypoid lesions and colorectal cancers, reference expression was the mean observed in the corresponding normal mucosa samples (indicated as 1); for cell lines, the reference consisted in the mean observed for 9 samples of normal mucosa from 9 patients with adenomas (indicated as 1). The differences between normal mucosa and the other groups of samples were highly significant in panels A, C, and D (all p values < 0.0001; bars: mean SE). (E) and (F): Two colorectal cancer cell lines (HCT116 and HT29) that do not express PTPRR-1 were used for the experiments shown in these panels. Re-activation of PTPRR-1 expression was investigated in cells treated with agents that act, through different mechanisms, on DNA and chromatin (see Methods). 5-Aza-dC, 5-Aza-2'-deoxycytidine; TSA, Trichostatin A; DZNep, 3-Deazaneplanocin A. Results are expressed relative to reference expression (mean observed in 9 different samples of normal mucosa, indicated as 1).

Figure 2

Methylation analysis of PTPRR-1. (A): Schematic depiction of the PTPRR-1 CpG island encompassing the transcription start site (black arrow). CpG dinucleotides are represented as short vertical lines. Regions explored with combined bisulfite restriction analysis (COBRA) and with bisulfite genomic sequencing (BGS) on DNA extracted from fresh or formalin-fixed, paraffin-embedded (FFPE) samples are also shown. (B) and (C): Examples of COBRA analysis and BGS in cell lines (B) and clinical samples (C). COBRA: Arrows indicate BstUI-digested DNA fragments representing methylated alleles; slower-migrating fragments correspond to undigested, unmethylated DNA. BGS: Each row shows the methylation status of a cloned target sequence, with circles representing unmethylated (open) and methylated (filled) CpG dinucleotides.

Figure 3

Western blotting and chromatin immunoprecipitation (ChIP) studies. (A): PTPRR-1 protein expression in total cell extracts from the cell lines used for ChIP studies (Colo205 and SK-N-SH) are shown. Proteins were separated on a 10% polyacrylamide gel. Reactivation of PTPRR protein expression was also investigated in Colo205 cells after treatment with 5-Aza-2'-deoxycytidine (5-Aza-dC) or 3-Deazaneplanocin A (DZNep) plus Trichostatin A (TSA). Extracts of 293T cells transiently transfected with PTPRR-1 cDNA were used as positive controls. PTPRR expression was also documented in assays of protein extracts from epithelial cells of normal colonic crypts, as expected. (The slightly higher molecular weight might be due to a post-translational modification occurring in vivo in this tissue.) MSH6 antibodies were used as proliferation-rate controls: In cells treated with drugs, the expression of the DNA mismatch repair protein MSH6 was decreased --- as expected --- due to reduced cell viability. (B): Immunoprecipitated DNA was quantified by real-time PCR. Enrichment was calculated as percentage of input DNA control (details in Methods). Upper 3 panels: Chromatin prepared from Colo205 cells was immunoprecipitated with antibodies against H3K9ac (acetylated-histone H3Lys9, white bars), H3K27me3 (trimethyl-histone H3Lys27, light gray bars), and H3K9me3 (trimethyl-histone H3Lys9, dark gray bars). The specifity of immunoprecipitations was verified by the selective enrichment, in the PCR amplifications, of the actively transcribed RPL30 gene (H3K9ac antibody), the polycomb target KCNA1 promoter (H3K27me3 antibody), and the heterochromatic Sat 2 locus (H3K9me3 antibody). Lower panel: PTPRR-1 promoter histone code differences between the colorectal cancer cell line Colo205 and the neuroblastoma cell line SK-N-SH. (PTPRR-1 transcript and PTPRR-1 protein expression profiles for the 2 lines are shown in Figures 2B and 3A, respectively.)