CKS1B Improves Cell Proliferation And Drug-resistance And Disease Progression In Multiple Myeloma Through SKP2/P27-independent Pathways: Activation Of STAT3, MEK/ERK And BCL2 Signal Pathways

Multiple myeloma (MM) is a plasma cell malignancy, which remains largely incurable with current therapeutic strategies. The molecular bases of MM progression and drug-resistance are not completely understood. Gain of chromosome 1q21 is one of the most recurrent chromosomal aberrations observed in MM and is correlated with poor prognosis. We previously identified a 70 high-risk signature pattern in MM, with overrepresentation of over-expressed genes mapping to chromosome 1q, further supporting the hypothesis that 1q21 gain is important in MM progression. High expression of CKS1B, a Cdc28 protein kinase regulatory subunit 1B, mapping to the 1q21 amplicon and one of the 70 high-risk signature gene pattern, was inversely associated with survival in MM. Alternatively, knockdown of CKS1B in MM cells potently induced growth inhibition and apoptosis, suggesting that CKS1B plays a crucial role in MM cell survival. However, the functional role of CKS1B in MM cell survival and MM disease progression remains to be elucidated.CKS1B has a well-documented role in the cell-cycle regulation. Cks1 is a member of the Cks/Suc1 family of proteins, which are essential components of cyclin-dependent kinases (CDKs) that regulate mitosis in all eukaryotes. CKS1B was identified as an essential accessory protein to the SCFSKP2-CKS1 ubiquitin ligase complex. In this complex, Cksl enhances the interaction between SKP2 and p27Kip1 (a CDK inhibitor),, resulting in cell proliferation. The role of Cks1 in regulating p27Kip1 proteasome degradation in the cell cycle is particularly evident in CKS1-/-mice. The key feature of these mice is their small body size, which is due to lack of degradation of the G1-/S-phase CDK inhibitor p27Kip1. Interestingly, besides influencing cell growth and survival through regulation of p27Kip1, silencing of CKSIB also induces cell death and inhibits growth in MM cells in the presence of a bi-allelic deletion of the CDKN1B (p27Kip1) locus, indicating that CKSIB mediates its effects on cell growth and survival also through mechanisms that are independent p27Kip1 and SKP2.In this study, we found that forced expression of CKS1B by lentivirus vector-mediated CKS1B-cDNA transfection in MM cells increased cell proliferation and multidrug-resistance, providing direct evidence of the crucial role of CKSIB in MM progression. Furthermore, we also identified STAT3 and MEK/ERK/BCL2 pathways to be downstream targets of CKS1B activation independent on the complex of SKP2/p27Kip1, responsible for MM cell growth and survival.1. CKS1B expression increased in relapsed MM and conferred a short post-relapse survival.Our previous studies showed that CKS1B was one of the 70 high-risk genes, which was inversely associated with survival in newly diagnosed MM. In this study, we compared CKS1B expression from 51 patients with paired baseline (diagnostic) and relapse samples. The median signals of CKS1B from microarray data at diagnosis and at relapse were 1398 (range:370-4433) and 2174 (range:405-9867), respectively. CKSIB expression increased in 76% relapsed MMs and was in more than 1.5 folds in 51%.As we expected, patients from the 51 paired relapses who had CKS1B expression in quartile 4 (high-risk) at baseline and receiving various salvage therapies had the worst 4-year post-relapse survival compared with those who had CKS1B expression in quartiles 1 to 3 (low-risk) at baseline (P= 0.0012). The quartile 4 reference line is taken from the complete (n=351) sample of arrays at diagnosis. Interestingly, among 38 relapse patients with low CKS1B expression (quartiles 1-3) at baseline, patients increased CKS1B expression at least 1.5 folds at relapse had inferior 4-year postrelapse survival compared with those lacking CKS1B up-regulation at relapse (P=0.032). Furthermore, among 36 relapsed patients with high CKS1B expression at relapse,4-year postrelapse survival in patients with high CKS1B at baseline was significantly poor compared with those with high CKS1B expression only at relapse. These data further confirmed that CKS1B expression is a prognositic marker at the stage of newly diagnosis in MM and also demonstrated that CKSIB expression can be used to predict patient survival in postrelapse MM.2. CKS1B over-expression promotes MM cell proliferation and drug-resistance.Increased expression of CKS1B is a progression event suggesting that CKS1B may be heterogeneously expressed in a small population of myeloma cells at diagnosis, and current treatments ineffectively eliminate the small populations of CKS1B high-expression myeloma cells, which results in myeloma relapse and treatment failure.To further investigate the functional role of CKS1B played in MM cells, CKS1B was over-expressed in OCI-MY5 and XG1 MM cells by lentivirus vector-mediated CKS1B-cDNA transfection. CKS1B-transfected MM cells cultured respectively in medium with 1%,5%,10%FBS showed higher proliferation ratio than MM cells transfected by empty vector(EV).To test the hypothesis that MM cells with high expression of CKSIB are more drug-resistant and responsible for MM poor prognosis, CKS1B-transfected OCI-MY5 and XG-1 cells were treated with bortezomib (Vel) at a dose of 5 nM for 48 hours. Cell growth and cell survival were examined. Untreated and EV-transfected cells with or without bortezomib were used as controls. Bortezomib treatment induced significantly less growth inhibition and cell death in CKS1B-transfected cells compared with EV-transfected controls (P<0.05). Similarly, treatment of doxorubicin (Dox) 100nM and etoposide (Epo) 100nM for 48 hours, induced significantly less growth inhibition and cell death in CKS1B-transfected cells compared with EV-transfected controls (P< 0.05). Clearly, increased CKS1B expression induced chemotherapeutic resistance in myeloma.3. CKS1B over-expression activates STAT3 and MEK/ERK through SKP2 and p27Kip1-independent pathways.To identify these CKSIB-mediated SKP2/p27Kip1-independent signaling pathways, western blots were applied to screen for activation of the key signaling pathways which relate to cell survival and apoptosis, including MAPK, NF-κB, TP53, PI3K/AKT and STAT3 using OCI-MY5 cells after CKS1B-silencing by specific CKS1B-shRNA transfection. Decreased phosphorylated (p)-MEK1/2, p-ERK1/2, p-STAT3 and p-BCL2 were detected in CKS1B silenced OCI-MY5 cells compared with wild-type cells and cells transfected with a non-targeting scramble sequence (SCR). Similar results were also observed in CKS1B silenced KMS28PE and XG-1 cells.We further examined the alteration of STAT3, MEK/ERK and BCL2 in CKS1B-transfected OCI-MY5 and XG1 cells. Increased levels of p-MEK1/2, p-ERK1/2, p-STAT3 and p-BCL2 were observed in CKS1B-transfected OCI-MY5 and XG-1 cells compared with the EV-transfected controls. These results strongly suggest STAT3, MEK/ERK, and BCL2 are the downstream signaling pathways of CKS1B, which may mediate MM cell growth, survival, and disease progression.To examine the functional relationship between SKP2 and these CKS1B activated STAT3, MEK/ERK and BCL2 signaling pathways, SKP2-shRNA was used to silence SKP2 in KMS28PE,OCI-MY5, and XG-1 cells and protein levels of p-MEK1/2, p-ERK1/2, p-STAT3, and p-BCL2 in these cells were examined. Wild-type (WT) and SCR-transfected cells were used as controls. SKP2-knockdown resulted in an increase rather than a decrease of p-MEK1/2, p-ERK1/2 and p-STAT3 levels in KMS28PE,OCI-MY5 and XG-1 cells, and with no remarkable change in p-BCL2 level. We then examined the functional relationship between p27Kip1 and STAT3, MEK/ERK and BCL2 by over-expressing p27Kipl in KMS28PE,OCI-MY5 and XG-1 cells using a lentivirus system. Western blots detected increased levels of p-MEK1/2 and p-ERK1/2 in p27Kip1-transfected myeloma cells with no remarkable effect on p-STAT3,p-BCL2 compared with WT and EV controls.4. [Activation of STAT3 is involved in CKS1B-mediated myeloma cell growth and survival]A specific STAT3 inhibitor Nifuroxazide at the dose of 10 nM for 48 hours was used to treat CKS1B-transfected OCI-MY5 and XG-1 cells. Nifuroxazide treatment decreased p-STAT3 in both EV-and CKS1B-transfected myeloma cells compared with untreated controls by western blot, confirming its specificity. Nifuroxazide-induced cell growth and cell death were also evaluated in CKS1B-transfected OCI-MY5 and XG-1 cells. Cells were treated with 10 nM Nifuroxazide for 48 hours, Nifuroxazide treatment induced more inhibition of cell growth and more increase in cell death in CKS1B-transfected cells compared with EV-transfected cells, indicating that myeloma cells with higher CKS1B-expression levels are more sensitive to STAT3 inhibition than cells with lower CKS1B-expression.Insulin-like growth factorⅠ(IGF-1) is a well-recognized myeloma cell survival factor activating the STAT3 signaling pathway. To further confirm the functional role of STAT3 signaling in CKS1B-mediated myeloma cell growth and survival, CKS1B-silenced KMS28PE, OCI-MY5 and XG-1 cells were treated with IGF-1 (100ng/ml) for 72 hours. Western-blots detected increased p-STAT3 in these CKS1B-silenced myeloma cells compared with untreated, SCR- or CKS1B-shRNA-transfected controls, confirming activation of STAT3 in IGF-1-treated cells. The effects of IGF-1 treatment on CKS1B-shRNA-induced myeloma cell death and growth inhibition were evaluated. IGF-1 treatment partially abrogated growth inhibition and cell death induced by CKS1B-knockdown (P<0.05).STAT3 regulates the expression of many target genes involved in cell survival and growth. To investigate whether CKSIB activates STAT3 signaling through regulation of STAT3 target genes, the protein of cell growth and apoptosis related gene MCL1, was measured by western blot. The level of MCL1, a gene that enhances cell survival by inhibiting apoptosis, was decreased in CKSIB silencing MM cells and increased in CKS1B over-expressing MM cells; whereas MCL1 expression did not show alteration in MM cells manipulated by inhibition of SKP2 and over-expression of p27Kip1. Furthermore, MCL1, following STAT3 expression pattern, was significantly increased in IGF1 treated MM cells inhibited CKS1B expression by CKS1B-shRNA.These data provide evidence that CKSIB regulates STAT3 signaling dependent on the STAT3 transcription of MCL1.5. Activation of the MEK/ERK signaling pathway is also involved in CKS1B-mediated myeloma cell growth and survivalA specific MEK inhibitor U0126 was used to treat CKS1B-transfected OCI-MY5 and XG-1 cells for 48 hours at the dose of 10μM. Cell lysates were prepared and subjected to western analysis using antibodies recognizing p-MEK1/2 and p-ERK1/2 proteins. U0126 decreased p- MEK1/2 and p-ERK1/2 in both EV-and CKS1B-transfected myeloma cells, confirming its specificity. Interestingly, U0126 treatment down-regulated BCL2 in myeloma cells, suggesting that BCL2 represents a downstream target of the MEK/ERK signaling pathway. We also evaluated U0126-induced cell growth and cell viability in CKS1B-transfected OCI-MY5 and XG-1 cells. Cells were treated with 10μM U0126 for 48 hours, U0126-treatment induced more cell growth inhibition and cell death in CKS1B-transfected cells than those in EV-transfected cells (P< 0.05).Since inhibitors of MEK/ERK signaling pathway may induce some non-specific effects on myeloma cell growth and survival, we constitutively activated the MEK/ERK signaling pathway by lentivirus-vector-mediated MEK1-cDNA transfection in CKS1B-silenced KMS28PE,OCI-MY5 and XG1 cells. Western blots showed a larger increase of p-MEK1/2 and p-ERK1/2 levels in MEK1-cDNA and CKS1B-shRNA double-transfected cells compared with SCR-and CKS1B-transfected controls. MEK1-transfection also increased protein levels of p-BCL2 in CKS1B-silenced cells, indicating that MEK1-transfection could overcome CKS1B-knockdown-mediated inhibition of BCL2 signaling pathway, further demonstrating that BCL2 is the downstream target of CKS1B and MEK/ERK signaling. Although MEK1-cDNA and CKS1B-shRNA doubly-transfected cells clearly showed increased cell death and growth inhibition compared with SCR-transfected control cells (P< 0.05), these double-transfected cells exhibited significantly less cell death and growth inhibition than CKS1B-silenced myeloma cells (P<0.05), indicating that MEK1-transfection partially abrogated cell death and growth inhibition induced by CKS1B-knockdown. Our results indicate that BCL2 is a downstream target of the CKS1B and MEK/ERK signaling pathway. Therefore, a specific BCL2 inhibitor (2,9-Dimethoxy-11,12-dihydrodibenzo[c,g][1,2]-diazocine 5,6-dioxide and 5,5'-Dimethoxy-2,2'-dinitrosobenzyl) at the dose of 10 nM for 48 hours was used to treat CKS1B-transfected OCI-MY5 and XG-1 cells. Treatment with this BCL2-inhibitor resulted in significantly more cell growth inhibition and cell death in CKS1B-transfected cells compared with EV-transfected cells, indicating that myeloma cells with higher CKS1B-expression are more sensitive to BCL2 inhibition than cells with lower CKS1B-expression. We subsequently over-expressed BCL2 by lentivirus-mediated BCL2 cDNA transfection in CKS1B-silenced KMS28PE, OCI-MY5 and XG-1 cells. Cells were cultured for 4 days and western blots confirmed increased p-BCL2 levels in BCL2 cDNA and CKS1B-shRNA doubly-transfected cells. The effects of BCL2-transfection on CKS1B-shRNA induced myeloma cell growth inhibition and death were also evaluated. Although BCL2-cDNA and CKS1B-shRNA doubly-transfected cells clearly showed cell growth inhibition and death compared with SCR-transfected control cells (P<0.05), BCL2 transfection partially abrogated myeloma cell growth inhibition and death induced by CKS1B-silencing (P< 0.05).6. Combination of STAT3 and MEK1 inhibitors induced synergistic cytotoxicity on myeloma cells with high CKS1B expressionSince the STAT3 and MEK/ERK signaling pathways were both involved in CKS1B-induced myeloma cell growth and survival, a combined targeting of these signaling pathways might induce more cytotoxicity in myeloma cells. CKS1B-transfected OCI-MY5 cells were treated with combination of STAT3 and MEK1 inhibitors (i.e. U0126 [10μM] plus Nifuroxazide [10 nM]), or combination of STAT3 and BCL2 inhibitors (i.e. U0126 [10μM] plus BCL2 inhibitor [10 nM]) for 48 hours. EV-transfected cells treated with the same combinations were used as controls. Untreated CKS1B-and EV-transfected cells also served as controls. The combinations of the STAT3 with MEK1 and the STAT3 with BCL2 inhibitors induced significant less cell growth and more cell death in CKS1B-transfected OCI-MY5 compared with EV-transfected control cells (P<0.05). CKS1B-transfected XG-1 cells were also treated with the same combinations for 48 hours. Similar to the CKS1B-transfected OCI-MY5 cells, the combinations of the STAT3 with MEK1 and STAT3 with BCL2 inhibitors induced significant less cell growth and more cell death in CKS1B-transfected XG-1 cells compared with EV-transfected control cells (P< 0.05). These results further demonstrate that myeloma cells with higher CKS1B-expression are more sensitive to combinations therapy with STAT3 and MEK/ERK inhibitors than cells with lower CKS1B-expression.7. FOXM1 inhibited expression of CKS1B and was up-regulated by p27kip1Kaplan-meior analysis showed that high expression of Foxm1 was associated with shorter survival in MM patients. To investigate the functional relation between FOXM1 and CKS1B, we transfected FOXM1 cDNA into MM cells. WB was used to examine protein expression of FOXM1, CKS1B and p27. Expression of CKS1B was increased, while expression of p27 was decreased in FOXM1-high expressed MM cells. Furthermore, FOXM1 was up-regulated in p27-cDNA transfected MM cells but was not significantly regulated in CKS1B-knockdown MM cells. Our datas confirmed that FOXM1 up-regulated expression of gene CKS1B and down-regulated expression of p27, while over-expression of p27 positively regulated the expression of FOXM1. [Conclusion]The role of the present study is to further clarify the functional role of CKS1B in myeloma cell survival and drug-resistance and investigate CKS1B induced SKP2-and p27Kip1-independent signaling pathways. Our results show that forced-expression of CKSIB in MM cells induced multidrug-resistance, providing direct evidence of the crucial role of CKS1B in myeloma progression. Using western blots, we found that over-expression of CKS1B stimulated STAT3 and MEK/ERK, whereas SKP2 knockdown or p27Kip1 over-expression activated rather than suppressed STAT3 and MEK/ERK pathways, suggesting that SKP2 over-expression or p27Kip1 inhibition exerted the opposite effect of CKS1B over-expression on STAT3 and MEK/ERK. Further investigation showed that BCL2 is a downstream target of CKS1B-induced MEK/ERK signaling. Moreover, stimulation of STAT3 and MEK/ERK/BCL2 signaling pathways partially abrogated MM cell death and growth inhibition induced by CKS1B-knockdown. Targeting STAT3 and MEK/ERK/BCL2 activity by specific inhibitors resulted in significant MM cell death and growth inhibition and their combinations had a synergistic effect on cytotoxicity of myeloma cells. Furthermore, feedback of inter-regulation of FOXM1-CKS1B-P27 was involved in mechanisms of MM cell growth and survival induced by CKS1B.Thus, our findings clarify the SKP2/p27Kip1-independent mechanisms of CKS1B activity in maintaining myeloma cell growth and survival, and also provide a rationale for specifically targeting STAT3 and MEK/ERK/BCL2 in aggressive CKS1B-overexpressing MM.