Bortezomib Downregulates The Expression Of HCDC14A In Human Myeloma Cell Line By Inhibiting NF/κB Signaling Pathway

Background and objective:chromosome 1p21 deletion could be identified in around 20%to 30%patients diagnosed with multiple myeloma (MM). Patients with 1p21 deletions had significant shorter progression-free survival and overall survival than those without shuch abnormality. High-dose chemotherapy could not prolong the survival of patients with 1p21 deletions.1p21 deletion was an independent risk factor. We searched the genome database and identified a gene named cyclin-dependent kinase phosphatase CDC 14A. CDC14A had been shown to play an important role in regulating mitotic exit and the centrosome duplication cycle. Our study group found that hCDC14A mRNA expression was lower than controls in around 40%patient with MM, based on this observation, hCDC14A dysregulation might be involved in the development of MM. The aim of the present study was to identify the role of hCDC14A in MM and to explore whether bortezomib could modulate the expression of hCDC14A.Methods:Part I:Bone marrow aspirates were obtained from 29 patients with MM. Mononuclear cells were enriched by Ficoll-gradient centrifugation and myeloma cells were positively sorted using anti-CD138 magnetic microbeads. The hCDC14A and p53 mRNA levels in CD 138 positive cells from MM patients were detected by RQ-PCR. The correlationship between hCDC14A and p53 were analyzied by Spearman test. PartⅡ:1) hCDC14A protein expression in human myeloma cell lines (HMCLs, including RPMI-8226, U266, MM1.RL, MM1.S, CZ-1, OPM-2, and LP-1) was determined by Western-Blot; 2) the localization of hCDC14A in U266 and MM1.S were determined by confocal microscope.3) U266, OPM-2 and MM1.S cells were co-cultured with different concentrations of bortezomib (PS-341) (OnM,2.6nM,5.2nM, and 10.4nM) for 12h to 24h. The mRNA transcripts and protein levels of hCDC14A were detected by RQ-PCR and Western-Blot before and after the co-culture with bortezomib; 4) OPM-2 cells were co-cultured with 2.6nM bortezomib for 0h-12h, or OPM-2 cells were treated by different concentrations of bortezomib (OnM, 1.3nM,2.6nM,3.9nM, and 5.2nM) for 6h, then the protein levels of hCDC14A, P53 and phospho-P53 (S315) were indetified by Western-Blot; 5) to explore the role of NF/κB signal pathway in the process of bortezomib modulating hCDC14A expression, we treated OPM-2 cells with bortezomib (OnM,2.6nM, and 10.4nM) for 6h or with 2.6nM bortezomib for 6h and 12h. The activities of NF/kB were determined by EMSA. U266, OPM-2, and MM1.S cells were treated by bortezomib (2.6nM), or MG-132 (500nM), or BAY 11-7082 (5μM) last for 12h, the change of hCDC14A was determined by Western-Blot; 6) the constructed lentivirus named pLKO.1-shRNA-hCDC14A were produced in 293T cells and infected to U266。Results:PartⅠ:the transcripts expression level of hCDC14A had no correlationship with p53 in all the 29 patients (Correlation Coefficient= 0.351, P=0.062) or in 24 patients with untreated MM (Correlation Coefficient=0.291, P=0.167). PartⅠ:1) RPMI-8226, U266, OPM-2, and LP-1 were highly expressed hCDC14A protein while hCDC14A levels were lower in CZ-1, MM1.RL, and MM1.S cells. Based on this result, U266, OPM-2, and MM1.S cells were the main subjects in the following study; 2) hCDC14A localized in nucleus and plasma in U266 and MM1.S cells. In some cells, it also localized in centrosome; 3) bortezomib down-regulated hCDC14A expression in time-and dose-dependent manners; 4) bortezomib down-regulated hCDC14A while up-regulated the phospho-P53 (S315) in OPM-2 cell; 5) all the three drugs (bortezomib, MG-132, and BAY 11-7082 could inhibit hCDC14A expression, EMSA test determined that the activity of NF/κB in OPM-2 cell was inhibited by bortezomib. It suggested that bortezomib inhibited hCDC14A expression in time-and dose-dependent manner in HMCLs via the inhibition of NF/κB signal pathway; 6) the constructed lentivirus named pLKO.1-shRNA-hCDC14A were successfully produced in 293T cells and infected to U266.Conclusion:there was no efficient evidence to confirm that hCDC14A was the right molecular located in 1p21 invovled in the pathogenesis of MM until now. But indeed we found that bortezomib can down-regulate the expression of hCDC14A via the inhibition of NF/κB pathway. It suggested that further studies are requied to understand the molecular pathogenesis of hCDC14A in MM.