Suppression of the protein tyrosine phosphatase receptor type O gene (PTPRO) by methylation in hepatocellular carcinomas

Abstract

A diet lacking folic acid and choline and low in methionine (folate/methyl deficient diet, FMD diet) fed to rats is known to produce preneoplastic nodules (PNNs) after 36 weeks and hepatocellular carcinomas (tumors) after 54 weeks. FMD diet-induced tumors exhibit global hypomethylation and regional hypermethylation. Restriction landmark genome scanning analysis with methylationsensitive enzyme NotI (RLGS-M) of genomic DNA isolated from control livers, PNNs and tumor tissues was performed to identify the genes that are differentially methylated or amplified during multistage hepatocarcinogenesis. Out of the 1250 genes analysed, 2 to 5 genes were methylated in the PNNs, whereas 5 to 45 genes were partially or completely methylated in the tumors. This analysis also showed amplification of 3 to 12 genes in the primary tumors. As a first step towards identifying the genes methylated in the PNNs and primary hepatomas, we generated a rat NotI-EcoRV genomic library in the pBluescriptKS vector. Here, we describe identification of one methylated and downregulated gene as the rat protein tyrosine phosphatase receptor type O (PTPRO) and one amplified gene as rat C-MYC. Methylation of PTPRO at the NotI site located immediate upstream of the trancription start site in the PNNs and tumors, and amplification of C-MYC gene in the tumors were confirmed by Southern blot analyses. Bisulfite genomic sequencing of the CpG island encompassing exon 1 of the PTPRO gene revealed dense methylation in the PNNs and tumors, whereas it was methylation free in the livers of animals on normal diet. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed significant decrease in the expression of PTPRO in the tumors and in a transplanted rat hepatoma. The expression of PTPRO mRNA in the transplanted hepatoma after demethylation with 5-azacytidine, a potent inhibitor of DNA methyltransferases, further confirmed the role of methylation in PTPRO gene expression. These results demonstrate alteration in methylation profile and expression of specific genes during tumor progression in the livers of rats in response to folate/methyl deficiency, and further implicate the potential role of PTPRO as a novel growth regulatory gene at least in the hepatocellular carcinomas.

Clinicopathologic and prognostic values of apolipoprotein D alterations in hepatocellular carcinoma

“…17,18 In human HCC, a number of methylation-induced inactivation of genes, such as E-cadherin, p16INK4a, 14-3-3 sigma, SOCS-1 and DLC-1, have already been documented. [19][20][21][22][23][24][25][26][27] However, there is no information on the methylation-mediated silencing of Apo D in HCC. Therefore, we first analyzed the pharmacological induction of Apo D by 5-Azacitidine in human liver cancer cell lines.…”

Epigenetic regulation of protein tyrosine phosphatases: potential molecular targets for cancer therapy

“…17,68 In this context, it is noteworthy that methyl deficient diet can lead to both global DNA hypomethylation and localized or regional hypermethylation of genes such as tumor suppressor genes. 33,69 The most accepted concept is to envisage early changes in the hypomethylation of genes such as oncogenes followed by hypermethylation of genes such as tumor suppressor genes during cell transformation or in tumorigenesis, both processes resulting in gene deregulation. It remains to be determined as to what fraction of tumor incidence is caused by alteration in genomic methylation.…”

“…This observation set the stage for many studies in the ensuing decades that explored reactivation of suppressed genes in different cell types, particularly cancer cells (for reviews, see Baylin, 46 Karpf and Jones, 47 and Jain 48 ). Reactivation of the suppressed genes can also occur in animals treated with 5-AzaC, 33,49,50 which rules out the possibility of any artifacts produced in cell culture. Epigenetic modification alters the expression of a gene without affecting DNA sequence.…”

“…This gene was identified in a genome-wide scan for methylation changes during tumor progression. 8,33 It is also heavily methylated and silenced in a transplanted rat hepatoma. 33 Further, when the animals bearing the tumor were treated with the DNA hypomethylating agent 5-azacytidine (5-AzaC), demethylation of the PTPRO promoter resulted in gene re-expression and reduction in tumor size.…”

“…All these observations pointed towards the potential role of PTPRO as a tumor suppressor. The observation that PTPTO is methylated in preneoplastic liver of rats fed methyl-deficient diet 33 suggests that this modification could emerge as an early tumor marker in hepatocellular and probably other tumors. We have since extended these studies to human tumors and have identified tumor-specific methylation of the PTPRO CpG island, located within the promoter region, in primary human hepatocellular carcinoma relative to the matching normal liver tissue.…”