CKS1B Mediates SKP2/p27Kip1-Independent Myeloma Cell Survival and Disease Progression through Activation of MEK/ERK and JAK/STAT3 Signaling Pathways.

Abstract 126

We previously reported that CKS1B may influence myeloma (MM) cell growth and survival through SKP2/p27^Kip1-dependent and -independent mechanisms. However, there is still no direct evidence to prove that CKS1B has a role in MM cell proliferation and disease progression. The present study was performed to establish its functional role and define CKS1B-mediated SKP2/p27^Kip1-independent down-stream signaling pathways. CKS1B was over-expressed in OCI-MY5 and XG1 MM cell lines by lentivirus. Western blots confirmed CKS1B over-expression. Cells were cultured in medium containing 1% fetal bovine serum for 7 days. CKS1B-transfection resulted in increased cell proliferation compared to empty-vector (EV)-transfected controls. We also examined the role of CKS1B in myeloma resistance to the general used chemotherapeutic drugs, such as bortezomib (5nM), doxorubicin (100nM) and etoposide (100nM). Untreated cells and empty-vector (EV)-transfected cells with or without drug treatments served as controls. Significant less inhibition of cell growth and cell death was observed after drug treatment in CKS1B-transfected cells compared with controls (P < .05). To screen down-stream signaling pathways associated with cell growth and survival in OCI-MY5, MS28PE and XG-1 cells were transfected with specific CKS1B-shRNA, which resulted in decreased phosphorylation of MEK1/2, ERK1/2, STAT3, MCL1 and BCL2 compared to wild-type and control cells, transfected with scrambled CKS1B-shRNA. To confirm these results, we examined the alteration of STAT3, MEK/ERK and BCL2 signaling pathways in OCI-MY5 and XG1 cells after forced over-expression of CKS1B. Increased levels of p-MEK1/2, p-ERK1/2, p-STAT3, MCL1 and p-BCL2 were observed compared to the EV-transfected controls, confirming that CKS1B activates STAT3, MEK/ERK and BCL2 signaling pathways. In Contrast, SKP2 over-expression or p27^Kip1 inhibition resulted in inhibition of STAT3 and MEK/ERK pathways with no remarkable changes inBCL2. Further investigation showed that BCL2 is a downstream target of MEK/ERK signaling. Stimulation of STAT3, MEK/ERK and BCL2 signaling pathways only partially abrogated MM cell death and growth inhibition induced by CKS1B knockdown. Targeting either the STAT3, MEK/ERK or BCL2 signaling pathway with specific inhibitors induced significant MM cell death and growth inhibition in CKS1B-over-expressing MM cells; their combination had a synergistic effect on cell death and growth inhibition. Our findings provide a rationale for targeting STAT3 and MEK/ERK/BCL2 signaling in the therapy of aggressive CKS1B-overexpressing MM, which shows increased proliferation and drug-resistance