Regulative loop between beta-catenin and protein tyrosine phosphatase receptor type gamma (PTPRG) in chronic myeloid leukemia

Introduction. Chronic Myeloid Leukemia (CML) is a myeloproliferative disease characterized by the presence of the oncogene BCR-ABL1, which acts as tyrosine kinase. PTPRG (Protein Tyrosine Phoshatase Receptor type γ) is a tumor suppressor gene down- regulated by hypermethylation of its promoter region in CML. Previous studies demonstrated that a re-expression of PTPRG is correlated with a decreased clonogenic capability of CML cells, as shown by the down-regulation of Ki67, and with an increased cellular differentiation related to a PTPRG-mediated overexpression of GATA-1 and Cyclin D1. In addition, its restored expression was observed in patients with a good response to TKI therapy. In order to understand the regulation between this phosphatase and BCR- ABL1, we searched for PTPRG putative interactors among proteins downstream BCR-ABL1 driven pathways and we focused on b-Catenin, that is at the same time a PTPRG substrate and its transcriptional regulator. Methods. Cells: PTPRG negative cell line K562, with a stable transfection of exogenous PTPRG, and PTPRG positive cell line LAMA-84, treated with a specific siRNA and with a new PTPRG small drug inhibitor. Pull-down assay with purified, recombinant intracellular domain of PTPRG demonstrated a direct interaction between PTPRG and b-Catenin, while Western Blotting or Immunofluorescence were applied to detect a specific dephospho- rylation pattern in presence of PTPRG. Chromatin Immunoprecipitation showed us the binding between DNMT1(b-Catenin transcriptional target) and PTPRG promoter region. Results. We demonstrated that PTPRG binds and dephosphorylates b-Catenin, phosphorylated by BCR-ABL1, causing its cytoplasmic destabilization and the resulting degradation in CML cell lines with an exogenous or endogenous expression of PTPRG (K562 and LAMA-84 cell lines). Consequently, this regulation leads to MYC down-expression and p21/WAF1 increased expression, explaining the slow-down of proliferation in presence of PTPRG. On the contrary, we demonstrated that an increased expression of b-Catenin in PTPRG negative CML cell lines is correlated with an over-expression of the DNA (cytosine-5)-methyltransferase 1 (DNMT1) that is responsible of PTPRG promoter hypermethylation and that an inhibition after a treatment with 5-Azacydine or a down-regulation of this enzyme is closely related to PTPRG re-expression both at mRNA and protein levels. Conclusions. We show for the first time a mechanism that involves b-Catenin degradation control and the consequent down-regulation of genes regulated by the TCF/b-Catenin transcription complex. In return, b-Catenin up-regulation is correlated with an over-expression of DNMT1 that contributes to an hypermethylation of PTPRG promoter region. We hypothesized a regulative loop between PTPRG and b-Catenin and that an imbalance of the system in favor of one or the other could determine a different proliferation fate of CML cells and their clinical aggressiveness.