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.

Downregulation of miR-221/222 enhances sensitivity of breast cancer cells to tamoxifen through upregulation of TIMP3

“…Studies using the luciferase reporter assay and expression analysis have shown that TIMP 3 is a common target of miR-221/222. 41,42 In the present study, we found that TIMP 3 expression was significantly increased following inhibition of miR-221/222. This increased expression of TIMP 3 was associated with decreased viability of ER-positive MCF-7 cells treated with tamoxifen.…”

PTPROt-mediated regulation of p53/Foxm1 suppresses leukemic phenotype in a CLL mouse model

“…A truncated isoform (PTPROt) identified in B-lymphoid cells was found to promote cell cycle arrest 4 . A series of studies by our group and others have demonstrated its methylation and suppression in different types of cancers 5 - 10 and its in vitro and ex vivo growth suppressive characteristics 5 , 7 , 8 , 11 . In addition to understanding its functions, several studies including ours have identified its substrates in different cell types e.g.…”

“…In addition to understanding its functions, several studies including ours have identified its substrates in different cell types e.g. eph receptors in axons 12 , SYK, Lyn and ZAP70 in lymphocytes 13 , 14 , BCR/ABL in myelogenous leukemia 11 and VCP in HCC 5 . Recent studies using large number of human samples have demonstrated a prognostic function of PTPRO in breast cancer 15 and a biomarker function in esophageal squamous cell carcinoma 16 .…”

Defining the Protein-Protein Interaction Network of the Human Protein Tyrosine Phosphatase Family

“…In addition, PTPN13 was identified as a suppressor of HER2-positive breast cancers through antagonism of HER2 activation (13). Loss of PTPRO (14) and PTPN13 (15) has been observed in hepatocellular carcinoma. DUSP6, a DSP that targets the RAS-MAPK pathway, is lost in pancreatic cancers (16).…”