-catenin is constitutively active and increases STAT3 expression/activation in anaplastic lymphoma kinase-positive anaplastic large cell lymphoma

Abstract

The role of β-catenin in cancer has been most studied in tumors of epithelial cell origin. The functional status and biological significance of this protein in anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma is unknown. ALK-positive anaplastic large cell lymphoma cell lines and patients' tumor samples were examined for status of β-catenin expression and signaling. The subcellular localization of β-catenin was assessed using immunohistochemistry, sub-cellular fractionation and confocal microscopy, while its transcriptional activity was studied using the TOPFlash/FOPFlash luciferase reporter assay. To examine the biological significance of β-catenin, short interfering RNA was used to knock-down its expression; the resulting biological effects were studied using trypan-blue exclusion and MTS assay, and the impact on its various downstream targets was assessed using quantitative real-time polymerase chain reaction and western blots. β-catenin was transcriptionally active in three of three ALK-positive anaplastic large cell lymphoma cell lines, and this finding correlates with the nuclear localization of β-catenin in these cells and the neoplastic cells identified in most of the patients' tumor samples. β-catenin is biologically significant in ALK-positive anaplastic large cell lymphoma, since down-regulation of β-catenin resulted in a significant reduction in their cell growth. Down-regulation of β-catenin led to a marked reduction in both the total protein level and the activated/phosphorylated form of STAT3, another signaling protein previously shown to be important in the pathogenesis of ALK-positive anaplastic large cell lymphoma. In contrast to some of the oncogenic tyrosine kinases, modulation of nucleophosmin-anaplastic lymphoma kinase expression did not result in any detectable change in the protein level, nuclear localization or tyrosine phosphorylation of β-catenin; however, inhibition of nucleophosmin-anaplastic lymphoma kinase expression significantly down-regulated the transcriptional activity of β-catenin. β-catenin signaling is constitutively active in ALK-positive anaplastic large cell lymphoma and represents a previously unknown mechanism by which the high levels of STAT3 expression and activation in these tumors are sustained. Our results suggest that the interaction between oncogenic tyrosine kinases and various cell signaling proteins may be more complex than previously believed.