PRL-3 Regulates Myeloma Cell Survival, Proliferation, Adhesion and Migration

Multiple myeloma (MM) is a neoplastic monoclonal proliferation of bone marrow plasma cells. Despite advances in treatment in recent years, MM is still a fatal disease. Phosphatase of regenerating liver-3 (PRL-3) is a protein expressed in primary MM cells and MM cell lines, but not in normal plasma cells. A recent study showed that siRNA against PRL-3 suppresses MM-cell migration (Fagerli et al, 2008) and another study has identified PRL-3 as a marker gene for a subgroup of patients with MM (Broyl et al, 2010).

The human myeloma cell lines INA-6, ANBL-6, IH-1, OH-2 and RPMI-8226 were used in this study. Apoptosis was measured by Annexin V-FITC binding by flow cytometry, proliferation was measured by methyl-³H-thymidine incorporation, migration against a stromal cell derived factor-1α (SDF-1α) gradient was studied in a Transwell two-chamber assay and adhesion was measured as percentage adhered cells to fibronectin after stimulation with the pro-adhesive cytokines hepatocyte growth factor (HGF), insulin like growth factor-1 (IGF-1) and SDF-1α. RT-PCR and Western blotting were used to measure expression of pro- and anti-apoptotic proteins. Western blotting was used to map intracellular signaling pathways. INA-6 cells stably expressing exogenous PRL-3 were generated using retroviral transduction. The small molecular inhibitor PRL-3 Inhibitor I was used for PRL-3 inhibition.

PRL-3 inhibitor I reduced survival of the myeloma cell lines INA-6, ANBL-6, IH-1, OH-2 and RPMI-8226. IC₅₀ was between 15 and 50 μM depending on cell line. Treatment with the inhibitor down-regulated the anti-apoptotic protein Mcl-1 and led to activation of the intrinsic apoptotic pathway. Inhibition of PRL-3 reduced interleukin (IL)-6-induced phosphorylation of STAT-3. Treatment with 10 μM of PRL-3 inhibitor I also significantly reduced migration against a SDF-1α gradient and HGF-, IGF-1- and SDF-1α -mediated adhesion of the cell line INA-6. By retroviral transduction we made PRL-3-overexpressing INA-6 cells. INA-6 cells are dependent on IL-6 to grow, but overexpression of PRL-3 led to a major increase in cell proliferation even in the absence of IL-6 as well as increased survival at suboptimal concentrations of IL-6.

PRL-3 is expressed in MM cells but not in normal plasma cells and can represent a cancer-specific target. Overexpression of PRL-3 in the IL-6-dependent human myeloma cell line INA-6 renders the cells less dependent of IL-6 and increases survival and proliferation. On the other hand, the use of an inhibitor against PRL-3 significantly decreases survival of five MM-cell lines and reduces adhesion and migration of the cell line INA-6. IL-6-STAT3 signaling is important in MM cell survival and proliferation and we here show that PRL-3 possibly influences cell survival as a positive regulator of this signaling pathway. This study indicates that PRL-3 could be important in the pathogenesis of MM and a potential target in the treatment of MM.

No relevant conflicts of interest to declare.