NEK2 Suppresses Cellular Senescence through Regulation of the β-Catenin/CCND2/RB Axis in Multiple Myeloma

Background: Multiple myeloma (MM) is a B-cell malignancy characterized by complex cytogenetic abnormalities and drug-resistance. We have previously shown that increased NEK2 enhances MM cell growth, survival and drug-resistance in vitro and in vivo. In this study, we explore whether high NEK2 is involved in overcoming cellular senescence, a state of irreversible proliferation arrest.

Materials and Methods: MM cell lines (ARP1, OPM2, OCI-MY5, and KMS28PE) with either over-expressed or knocked-down (shRNA) NEK2 (NEK2 OE or KD) by a lentiviral delivery system were used in this study. Cell senescence status was evaluated by staining SA β-galactosidase (SA β-gal). Flow cytometry was performed to examine cell cycle state in NEK2-KD or NEK2-OE MM cells treated with doxorubicin (30nM) for 48 hrs. Mouse embronic fibroblasts (MEFs) derived from NEK2-transgenic mice and wild type control mice were also used to verify the role of NEK2 in suppressing senescence and to elucidate signaling pathways related to NEK2 anti-senescence. Real-time PCR, immunofluorescence, co-immunoprecipitation (Co-IP) assay and western blot were employed for NEK2 mechanistic studies.

Results: NEK2-KD induced MM cellular senescence and cell cycle arrest, while NEK2 OE decreased doxorubicin-induced senescence and cell cycle arrest. Also, MEFs derived from NEK2 transgenic mice showed suppressed cell senescence. Mechanistic studies showed that NEK2 stabilized β-catenin and prevented degradation by the ubiquitin/proteasome pathway in MM cells. Intriguingly, immunofluorescence revealed that NEK2 co-located with β-catenin in the cell nucleus in NEK2 transgenic MEFs. Importantly, increased nuclear β-catenin up-regulated CCND2 expression, (but not CCND1 and CCND3) and phosphorylated RB in NEK2 over-expressed MM cells or NEK2 transgenic MEFs. Furthermore, LiCl, an inhibitor of GSK3β, was used to restore the activity of β-catenin in NEK2-KD cells. LiCl significantly increased CCND2 expression and blocked NEK2-shRNA induced cellular senescence in MM cells.

Conclusion: NEK2 prevented MM cell senescence. This may play an important role in NEK2 induced MM cell proliferation, survival and drug resistance. Prevention of senescence by NEK2 depended on its interaction with β-catenin. Increased β-catenin activity, caused by high NEK2 resulted in up-regulation of CCND2 and hyper-phosphorylation of RB.