Methylation of the PTPRO gene in human hepatocellular carcinoma and identification of VCP as its substrate

Abstract

We have previously reported that the gene encoding protein tyrosine phosphatase receptor type-O (PTPRO) is suppressed by promoter methylation in a rat model of hepatocellular carcinoma (HCC) and it functions as tumor suppressor in leukemia and lung cancer. Here, we explored the methylation and expression of PTPRO as well as its function in human HCC. MassARRAY analysis of primary human HCC and matching liver samples (n = 24) revealed significantly higher (P = 0.004) methylation density at the promoter CGI in tumors. Combined bisulfite restriction analysis (COBRA) of another set of human HCC samples (n = 17) demonstrated that the CGI was methylated in 29% of tumors where expression of PTPRO was lower than that in corresponding matching livers. A substrate-trapping mutant of PTPRO that stabilizes the bound substrates was used to identify its novel substrate(s). VCP/p97 was found to be a PTPRO substrate by mass spectrometry of the peptides pulled down by the substrate-trapping mutant of PTPRO. Tyrosyl dephosphorylation of VCP following ectopic expression of wild-type PTPRO in H293T and HepG2 cells confirmed that it is a bona fide substrate of PTPRO. Treatment of PTPRO overexpressing HepG2 cells with Doxorubicin, a DNA damaging drug commonly used in therapy of primary HCC, sensitized these cells to this potent anticancer drug that correlated with dephosphorylation of VCP. Taken together, these results demonstrate methylation and downregulation of PTPRO in a subset of primary human HCC and establish VCP as a novel functionally important substrate of this tyrosine phosphatase that could be a potential molecular target for HCC therapy.

Fig. 1

Methylation and expression of PTPRO in primary human HCC. A: Schematic representation of the amplicon where CpG methylation was measured by Mass Array. B: Heatmap displaying quantitative DNA methylation level of single CpG units (columns). Rows represent samples (red = tumor samples; blue = normal controls, green = cell lines). Dark blue encodes high methylation values (>50%), bright green stands for low methylation levels and gray for missing data. Unsupervised clustering was performed using Eucledian distance. C: Scatter plot displaying the average DNA methylation values for the CpG island at the PTPRO transcriptional start site (TSS) in matching benign livers (N), HCC specimens (HCC) and cell lines. Red bars represent the median. Significance was assessed by non-parametric two-sided Mann–Whitney test. D: Real-time RT-PCR analysis of PTPRO in 14 pairs of primary HCCs and matching liver tissues. The data is represented as fold change in tumor over normal. The samples labeled with asterisks were used for COBRA assay. E: COBRA assay for methylation in a few representative pairs where PTPRO expression was lower in tumors (top panel) and where PTPRO expression was higher in tumors (bottom panel).

Fig. 2

Identification of PTPRO substrate(s) in the HCC cell line by mass spectrometry. A: Schematic view of the substrate trapping method. B: Total lysate prepared from pervanadate treated HepG2 cells was incubated with GSH-beads conjugated to GST-tagged WT form or CS/DA mutant form of PTPRO. After washing, the associated proteins and beads were boiled in SDS–PAGE sample buffer, resolved on a SDS–PAGE gel followed by Western blot analysis with anti-pY Ab (left panel) or Coomassie blue staining (right panel). Arrows indicate the position of protein bands excised from gel for LC/MS-MS analysis. C: Tabular list of proteins identified from the excised band by mass spectrometry. Hit scores were generated by MassMatrix database [Xu and Freitas, 2007]. D: Twenty peptides matched the VCP protein sequence (grayed), spanning 31% of the full-length protein.

Fig. 3

Identification of PTPRO substrate(s) in the HCC cell lines by Western blot analysis. A: (i) Enlarged middle section at p97 position of Figure 2B, right panel. (ii) Immunoblot analysis with anti-VCP, anti-Villin-1, anti-α-Spectrin, anti-GST antibodies was performed on a duplicate blot. B: Substrate-trapping followed by Western blot analysis was performed using whole cell extracts from pervanadate treated Hep3B cells. Since Hep3B cells express Syk, a previously identified substrate of PTPRO, immunoblotting with anti-Syk was used as a positive control.

Fig. 4

Ectopic expression of PTPROt sensitizes HCC cells to Doxorubicin treatment. A: Growth of vector-transfected and PTPROt expressing HepG2 cells seeded in a 96-well plate was followed by MTT assay over a period of 72 h. B: Vector-transfected or PTPROt expressing HepG2 cells were treated with 2.5 µM Doxorubicin for 72 h and cell survival was measured by MTT assay. Survival of untreated cells was taken as 1. C: Phosphotyrosine-containing proteins were immunoprecipitated from cell lysates of pervanadate-treated HepG2 cells (vector control and PTPROt stable transfectants) or, D, PTPRO-FL expressing H293T cells using anti-phosphotyrosine antibody 4G10 + Y20 followed by pull down with protein G agarose beads. The bound proteins were then analyzed by immunoblotting with antibodies as indicated in the figure. P-value: * P <0.05; ** P <0.01.

Fig. 5

PTPRO targets Y805 of VCP. HA-tagged VCP (WT and mutants) were transfected into H293T cells and the cells were treated with 100 µM pervanadate to enhance tyrosine phosphorylation. Equal amount of cell extracts were incubated with bacterially expressed and purified 6X-His-PTPROt-CS/DA. Extracts (E) and proteins bound to PTPROt-CS/DA (I) were separated on SDS–PAGE and immunoblotted with anti-HA (for VCP) and anti-His (for PTPROt).