PB2104 INHIBITION OF NUCLEOLIN REVERSED BORTEZOMIB RESISTANCE VIA THE REGULATION OF NF‐κB PATHWAY IN MULTIPLE MYELOMA CELLS
Authors: Hu J. , Liu W. , Yang T. , Chen Y. 
Published: June 2019

Background:The proteasome inhibitor bortezomib has remarkable efficacy against multiple myeloma (MM). However, intrinsic and acquired resistance to bortezomib could limit its efficacy. The potential resistance mechanisms have yet to be clarified. The nucleo‐cytoplasmic protein nucleolin (NCL) is high expression in several tumors, especially in resistance cells, that may act in the drug resistance. Previously we first observed the higher expression of NCL in KM3 bortezomib resistant cells (KBTZ) than its parental KM3 MM cells, indicating the role of NCL in bortezomib resistance and needing further research to elucidate.Aims:To investigate the potential mechanism(s) by which NCL regulates bortezomib resistance in MM cell line.MethodsKM3 MM cells and its bortezomib resistant cells KBTZ were transfected with lentiviruses carrying the shRNA sequences that target the endogenous NCL gene and control scrambled gene respectively. Post‐transfected cells were named as followed: KM3‐NCL‐Knockdown(KM3‐NCL‐KD), KM3‐NCL‐Scramble(KM3‐NCL‐Scr), KBTZ‐NCL‐Knockdown(KBTZ‐NCL‐KD), KBTZ‐NCL‐Scramble(KBTZ‐NCL‐Scr). Effect of NCL on cell viability and cells drug sensitivity were measured. Cell cycle related proteins and drug resistance related molecules expression level were evaluated. Related signaling pathways were also examined by western blot.Results:NCL silencing significantly inhibited cell proliferation and colony formation ability in KM3 and KBTZ cells. Colony sizes and numbers were both decreased in NCL silenced cells. Colony formation rates in KM3‐NCL‐KD, KM3‐NCL‐Scr, KBTZ‐NCL‐KD, KBTZ‐NCL‐Scr were 9.33% ± 0.47%, 27.22% ± 1.85%, 7.16% ± 0.24% and 28.22% ± 3.57%, respectively (P < 0.05). Moreover, NCL silencing arrested cell cycle transition in G0/G1, resulted in an increasing trend in cell apoptosis. CyclinD, cyclinE, CDK2 proteins were downregulated and P21 protein was upregulated after knockdown of NCL. Notably, NCL silencing increased the sensitivity to bortezomib in KM3 cells and prominently reversed the resistance to bortezomib in KBTZ cells. IC50of bortezomib were 17.29 ± 3.35 ng/mlin KM3‐NCL‐Scr cells and 10.13 ± 1.5 ng/mlin KM3‐NCL‐KD cells (P < 0.05), indicating a 1.7‐fold increase of bortezomib sensitivity after NCL knockdown. IC50of bortezomib were 590.78 ± 147.92 ng/mlin KBTZ‐NCL Scr cells and 63.48 ± 13.16 ng/mlin KBTZ‐NCL KD cells (P < 0.05), meaning a 10.8‐fold reversal of bortezomib resistance after NCL knockdown (Figure 1). Besides, compared with Scr cells, both KM3‐ and KBTZ‐NCL‐KD cells displayed lessrhodamine‐123 effluxion, suggesting that NCL silencing suppressed drug efflux activity. Knockdown of NCL declined ATP‐binding cassette transporter proteins MRP1, LRP, BCRP at mRNA levels and protein levels. Defined mRNA expression level of KM3‐NCL‐Scr and KBTZ‐NCL‐Scr cells equal to 1, relative mRNA expression of MRP1, LRP, BCRPwere 0.56 ± 0.26, 0.13 ± 0.03, 0.13 ± 0.09 in KM3‐NCL‐KD cells and 0.69 ± 0.11, 0.35 ± 0.07, 0.53 ± 0.20 in KBTZ‐NCL‐KD cells, respectively. Furthermore,downregulated expression of P‐NF‐κB, P‐IκBα, IKKα and P‐IKKα protein, decreased intranuclear NF‐κB level and upregulated expression of IκBα protein were observed in both KM3‐ and KBTZ–NCL‐KD cells, indicating that NCL may act by regulating activity of NF‐κB signaling pathway.Summary/Conclusion:In conclusion, our resultssuggested that inhibition of NCL reversed bortezomib resistance via the regulation of NF‐κB pathway in multiple myeloma cells, indicatingthe novel molecular understanding of bortezomib resistance and the role of NCL in MM resistance, and the emerging marker of NCL as a potential target.image