PRL-3 Mediates Survival of Primary Myeloma Cells

Introduction:Phosphatase of regenerating liver-3 (PRL-3) is a marker of aggressive and metastatic disease in a number of solid tumors. PRL-3 is expressed in primary multiple myeloma (MM) cells and MM cell lines, but not in normal plasma cells. We have previously shown that PRL-3 is induced in MM cells by interleukin (IL)-6 and other growth factors, and that PRL-3 promotes MM-cell migration (Fagerli et al, Blood, 2008). A study by Broyl et al (Blood, 2010) identified PRL-3 as a marker gene for a subgroup of patients with MM. In this study we examined the effect of the small molecular inhibitor PRL-3 Inhibitor I on primary MM cells, and we examined possible cellular targets of PRL-3 using a proteomics approach.

Methods: Co-cultures of primary myeloma cells and pooled bone marrow stromal cells from several MM patients were grown for 3 days in the presence or absence of PRL-3 Inhibitor I. Apoptosis was determined by staining cells with YO-PRO-1. To examine cellular targets of PRL-3 we made PRL-3 overexpressing cells from the human MM cell line INA-6 by retroviral transduction (INA-6-PRL-3) and control cells (INA-6-MOCK). Stable isotope labeling by amino acids in cell culture (SILAC) was used for in vivo incorporation of ¹²C-Lysine (“light”) and ¹³C-Lysine (“heavy”) in these transduced cell lines, preparing them for Orbitrap mass spectrometry based quantitative proteomics. The experiments were performed three times. The results were analyzed using Ingenuity Pathway Analysis software (Quiagen).

Results: PRL-3 Inhibitor I significantly reduced viability in all 9 primary MM cell samples tested, ranging from 5-70 % absolute reductions in viability at the top concentration of 40 µM. Proteomics analysis was performed on INA-6-PRL-3 and INA-6-MOCK cell lines at baseline and following incubation in IL-6-free media for 12 hours to deplete INA-6-MOCK cells of PRL-3. At 12 hours, a total of 6747 proteins were identified. 481 proteins were upregulated and 22 proteins downregulated by more than 2-fold in the INA-6-PRL-3 cells compared to INA-6-MOCK cells in 2 out of 3 experiments. Several proteins known to be important for cell survival and migration were upregulated in INA-6-PRL-3 cells, like CD44, Myc assosciated factor X (MAX) and Lyn. Pro-apoptotic proteins Caspase-1 and Caspase-2 were downregulated. Among canonical signaling pathways, there were most proteins belonging to the protein ubiquitination pathway (9,8 % of proteins; p<0,001).

Conclusion: PRL-3 Inhibitor I induced apoptosis in primary MM cells. Quantitative proteomics analyses comparing PRL-3 overexpressing INA-6 cells to normal INA-6 cells identifies several proteins important for MM cell migration and survival. This study indicates that PRL-3 could be important in the pathogenesis of MM and a potential target in the treatment of MM.