Elevated Expression Of CKS1B Inhibits Senescence Thorough Enhanced Degradation Of p21 In Multiple Myeloma
Authors: Gu Zhimin , Zhou Yi , Wang He , Yang Ye , Xu Hongwei , Huang Junwei , He Jie , Tao Yi , Tricot Guido J. , Zhan Fenghuang 
Published: October 2019

Abstract

Background
High expression of CKS1B gene as a result of amplification of region 1q21 identifies a subset of patients with poor prognosis in multiple myeloma (MM). We have previously shown that over-expression of CKS1B enhances degradation of p27 and activates both MEK/ERK and JAK/STAT3 signaling pathways, promotes MM cell drug-resistance. However, other mechanisms of drug resistance induced by high expression of CKS1B remain to be identified.

Materials and methods
CKS1B was over-expressed or knocked-down by shRNA in OCI-MY5 and KMS28PE myeloma cell lines. Cell viability was detected by PrestoBlue assay; Annexin-V assay was employed to detect cell apoptosis. Colony formation was assessed in soft agar. The direct dye efﬂux to assess multidrug resistance was performed using eFluxx-ID Multidrug resistance assay. Senescence was detected by SA-β-galactosidase (β-Gal) staining. Expression of SCF-Skp2 substrates was evaluated by western blots.

Results
We demonstrated that overexpression of CKS1B significant decreased sensitivity to bortezomib and increased the clonogenic capacity compared to cells transfected with empty vector in the presence of bortezomib (p < 0.05). A prominent mechanism of drug resistance involved upregulation of multidrug resistance associated proteins (MRPs). We investigated whether the level of MDR1, BCRP, and MRP1, mitotic checkpoint protein MAD2 and ABC transporter family members was increased in cells with elevated CKS1B. However, none of drug pump proteins was up-regulated or stabilized by over-expression of CKS1B in OCI-MY5 and KMS28PE. Accordingly, over-expression of CKS1B did not promote efflux of the hydrophilic eFluxx-ID™ gold fluorescent dye from MM cells any greater than control cells, suggesting alternative mechanisms. We therefore tested whether CKS1B can inhibit drug-induced senescence in MM cells after treatment with bortezomib by quantifying the percentage of cells expressing senescence-associated β-Gal. Interestingly, MM cells with elevated CKS1B were significantly less β-Gal positive following treatment with bortezomib compared to control cells (p < 0.05). To explore molecular mechanism underlying CKS1B induced anti-senescence, expression of SCF-Skp2 substrates involved in senescence pathway was examined as CKS1B is an essential cofactor for SCF-Skp2 complex. Over-expression of CKS1B significantly reduced expression of p21, p27, and p57 while knock-down of CKS1B stabilized these proteins. Notably, cells with high expression of CKS1B failed to accumulate p21 protein in response to bortezomib.

Conclusion
Our findings suggest that anti-senescence is a novel mechanism in CKS1B-induced drug resistance in MM and the turnover of p21 regulated by CKS1B could have important implications not only for bortezomib therapy but also to develop therapies aimed at enhancing p21 function independent of p53.

Disclosures:
No relevant conflicts of interest to declare.