Tumor suppressive function of protein tyrosine phosphatase receptor-type T and mechanism of its loss in NK-cell malignancies

Abstract

Nature killer (NK) cell malignancies are a group of rare but highly aggressive hematolymphoid malignancies. Extranodal NK-/T-cell lymphoma, nasal type and aggressive NK-cell leukemia are the most common and aggressive types of NK-cell malignancies. They occur more frequently in southern Chinese compared to western populations. The outcomes of current therapies for NK-cell malignancies including radiotherapies and chemotherapies are unsatisfactory. Therefore, it is necessary to study the molecular mechanism of NK-cell malignancies and identify new molecular targets for treatment. Recently, protein tyrosine phosphatase, receptor-type kappa (PTPRK) was identified as a tumor suppressor and directly target signal transducer and activator of transcription 3 (STAT3) in NK-cell malignancies. It leads us to investigate the role of protein tyrosine phosphatase, receptor-type T (PTPRT), which is a potential upstream protein of STAT3 in a same family (R2B family) of PTPRK, in NK-cell malignancies. In the present study, PTPRT expression was assessed in normal NK cells, NK malignant cell lines with or without demethylation treatment by semi-quantitative reverse transcriptase polymerase chain reaction and in specimens of primary NK-cell malignancies cases by immunohistochemistry. Promoter methylation of PTPRT in cell lines and the cases were investigated by bisulphite genomic sequencing and methylation-specific polymerase chain reaction respectively. Correlation between PTPRT promoter methylation and protein expression in tumor cells was estimated in patients. In addition, PTPRT was overexpressed in a PTPRT-deficient NK malignant cell line. Cell growth, apoptosis and cell cycle distribution after PTPRT restoration were analyzed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay and flow cytometry. Expression level of phospho-STAT3 (tyrosine 705) after PTPRT restoration was evaluated by western blot. Correlations between PTPRT and clinical features/ prognosis were analyzed. In contrast to normal NK cells, PTPRT was lost in all the NK malignant cell lines. It was also lost or downregulated in 75% of the patients in our cohort. Promoter hypermethylation was sufficient to silence PTPRT in 3 of the NK malignant cell lines (SNK6, KHYG-1 and YT) but not in NKYS and NK92 cells. Although promoter methylation of PTPRT was detected in most of the cases in our cohort, correlation between PTPRT promoter methylation and protein expression in tumor cells was not identified. In the functional assays, PTPRT exhibits tumor suppressive properties including inhibiting tumor cell growth, increasing cell apoptosis and keeping cell arrested in G0/G1 phase. In addition, phospho-STAT3 (tyrosine 705) expression level was decreased by PTPRT restoration in NK malignant cells. Lastly, no significant correlation between PTPRT promoter methylation/ protein expression and patients' clinical features/ overall survival/ disease free survival was identified in the patients in our cohort. In conclusion, PTPRT is a candidate tumor suppressor being downregulated in NK-cell malignancies. Promoter methylation may be one of the mechanism of its loss in the disease and STAT3 is a putative downstream target of PTPRT in NK malignant cells. However, the clinical significance of PTPRT in NK-cell malignancies still need to be further investigated in a larger cohort.