Abstract 196: The oncogenic role of PRL-3 in multiple myeloma through regulation of Src kinase family members

Introduction: Phosphatase of regenerating liver 3 (PRL-3) is a dual specificity phosphatase and its up-regulation in cancer cells is related to poor prognosis. We have previously described PRL-3 as a downstream target of IL-6 in multiple myeloma (MM) by demonstrating that it was upregulated in response to this cytokine. The Src kinase family (SFK) is composed of 8 members and regulates proto-oncogenic cellular pathways. As it has been reported that the SFK members Lyn, Fyn and Hck are important in signal transduction of IL-6 signals in MM cells and that Src is reported to be the major kinase regulated by PRL-3 in colon cancer, we evaluated effects of PRL-3 on activation of Fyn, Lyn, Hck and Src in MM. Methods: By retroviral transduction in the IL-6-dependent MM cell line INA-6,we generated functional PRL-3(INA-PRL-3) overexpressing cells, and control cells expressing catalytically inactive mutant PRL-3 (C104S) or empty vector (Mock). We measured global tyrosine (Y) phosphorylation (P) by immunoblotting using (P-Y1000) antibody. We measured the expression of 4 SFK members by qRT PCR and immunoblotting. Activity of SFK members was evaluated using MILLIPLEX MAP8-plex Human SFK kit. We confirmed MILLIPLEX result for Src by immunoblotting with antibody against P-Y416 Src (activating phosphorylation site). We also measured Src activity after inhibiting PRL-3 by PRL-3 inhibitor I or shRNA. Finally, we showed the influence of PRL-3 on cell growth and cell sensitivity to two Src inhibitors (PP2 and Su6656) by using CellTiter-Glo Luminescent Cell Viability Assay. Results: INA-PRL-3 had more global P-Y than C104S and Mock cells in the absence of IL-6 and showed a pattern of P-Y more similar to that of the parental INA-6 cells grown in the presence of IL-6. C104S and INA-PRL-3 cells showed lowest and highest activity, respectively, of Lyn and Src. Stimulation of cells with IL-6 increased active Lyn and Src but still C104S had lowest activity. We did not observe significant difference in activity of Fyn and Hck. Measuring total amount of 4 SFK members showed up-regulation of total Hck and Fyn in C104S cells in mRNA and protein level. This indicated that inactive PRL-3 in C104S cells leads to less SFK activity compared to both INA-PRL-3 and Mock cells. Increased total amount of Hck and Fyn in C104S could be the result of diminished negative feedback regulation of expression of SFK members by their active forms. INA-PRL-3 showed more P-Y416 Src also by immunoblotting and its inhibition by PRL-3 inhibitor or shRNA decreased Src activity. INA-PRL-3 had higher growth rate than both C104S and Mock cells. This difference was more prominent in the absence of IL-6 and high SFK activity in INA-PRL-3 could be one of the possible reasons for this. INA-PRL-3 also showed high sensitivity to both PP2 and Su6656. Conclusion: Inhibition of PRL-3 and SFK members could be considered as a possible treatment for MM. Citation Format: Pegah Abdollahi, Esten Nymoen Vandsemb, Magnus Aassved Hjort, Kristine Misund, Toril Holien, Torstein Baade Ro, Tobias Schmidt Slordahl, Magne Borset. The oncogenic role of PRL-3 in multiple myeloma through regulation of Src kinase family members. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 196.