Abstract
High expression of NEK2 mediated by p53 contributes to progression and relapse of multiple myeloma
Xiangling Feng1,2, Jiaojiao Guo1, Bowen Ouyang2, Yinghong Zhu1,Gang An3, Hao Zhen1, Jiliang Xia1, Yongjun Guan1, Xinying Zhao2, Lugui Qiu3, Jiaxi Zhou3, Fenghuang Zhan4,Wen Zhou1
1, Cancer Research Institute,Central South University, Changsha 410078, China. 2Xiang Ya School of Public Health, Central South University, Changsha, Hunan, China. USA. 3State Key Laboratory of Experimental Hematology, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Science & Peking Union Medical College, Tianjin, China. 4Department of Internal Medicine, Division of Hematology, Oncology, and Blood & Marrow Transplantation, University of Iowa, Iowa City, USA. E-mail: [email protected].
Background: Loss of p53 is an independent prognostic factor in patients with multiple myeloma (MM). Our previous studies found abnormal high expression of NEK2 was closely related to the poor prognosis and drug resistance of myeloma patients. However, it's unclear how NEK2 was up-regulated in MM. Through bioinformatics analysis, the binding site of p53 protein is found in NEK2 promoter, but the relationship and function of p53 and NEK2 in MM are poorly understood.
Materials and Methods: In this study, p53-/- MM cell lines (ARP1 and KMS11) and p53 p53+/+ MM cell lines (MM1S and H929) were used for investigating the role of NEK2 in p53-/- MM cell. FISH was performed on interphase nuclei of MM primary cells to detect p53 and NEK2 copy numbers. Chromatin immunoprecipitation and fluorescence reporter system were applied for examining the binding site of p53 protein in the distal NEK2 promoter. CGH and RNA-seq were performed to validate copy number changes and variations in the expression of several transcripts.
Results: The top 10% of MM patients with the highest NEK2 expression and lowest p53 had a significantly inferior OS (P<0.001) in TT2 and TT3 patients (GSE2658) and the expression of NEK2 increased significantly in myeloma cells during chemotherapy(GSE19554), while p53 decreased with the disease progression, suggesting a strong relationship with drug resistance. Single cell PCR showed increased NEK2 expression correlated with decreased p53 expression in single CD138+ plasma cell. FISH confirmed the loss of p53 in CD138+ plasma cells with amplification of NEK2 copies. Furthermore, NEK2 was also high expressed in p53 low expressed MM cells by Immunofluorescence (IF) (P<0.01). In addition, NEK2 was upregulated in p53-/- MM cell lines and HEK293 cells by deleted p53 gene with CRISPR technique both on mRNA and protein level (P<0.01), suggesting a negative correlation between the p53 and the expression level of NEK2. Meanwhile, when p53 deletion and NEK2 overexpression occur simultaneously, the phenomena of asymmetric mitosis and multipolar division are more obvious (P<0.001), suggested that the double hit of p53 deletion and NEK2 overexpression increases the chromosomal instability. Further in vivo study indicated the subcutaneous tumorigenesis in p53 deletion and NEK2 overexpression group was significantly greater than that of the single overexpression of NEK2 and deletion of p53 group (P<0.001), suggested that NEK2 overexpression and p53 deletion enhances the tumorigenic ability in vivo. While down-regulation of NEK2 by shRNA in p53 deletion cells, cell growth was inhibited in vitro and in vivo.To explore the relationship between p53 and NEK2, chromatin immunoprecipitation and fluorescence reporter system showed that p53 could bind to the promoter region of NEK2 and regulate its transcriptional expression. Further CGH analysis of the deletion of p53 expression in HEK293 cells can cause 1q21.4-44 amplification of the chromosome region of the NEK2 directly, which further confirmed by FISH. Finally, RNA-seq revealed several chromosome instability genes were abnormal expressed in NEK2 overexpression and p53 deletion double-hit group.
Conclusion: In summary, p53 deletion and NEK2 overexpression induced cancer cell drug resistance, proliferation and chromosomal instability. p53 could bind to the promoter region of NEK2 and cause NEK2 amplification. Down-regulation of NEK2 by shRNA in p53 deletion cells inhibited cell growth in vitro and in vivo. Thus,The significance of this study will provide the pre-clinical application of the NEK2 inhibitor to overcome the drug resistance induced by p53 in MM.
Disclosures
No relevant conflicts of interest to declare.
Immunologically distinct p53 molecules generated by alternative splicing.