| Aim:Multiple myeloma(MM)is a malignancy characterized by the monoclonal expansion of plasma cells and has become the second most frequent haematological malignancy.Proteasome inhibitor bortezomib(Bor)is one of the most effective drugs for treatment of MM which may eventually develop acquired-resistance to Bor during long-term treatment.Several researches show that E3 ubiquitin ligases primarily determine the substrate specificity of ubiquitin proteasome system and play an essential role in Bor resistance of MM.Neural precursor cell-expressed developmentally downregulated gene 4-1(NEDD4-1,also known as NEDD4)is a founding member of the HECT family of E3 ligases and is involved in the proliferation,migration,invasion,and drug sensitivity of cancer cells.Our current study aims to explore the role and underlying mechanism of NEDD4-1 in acquired resistance of Bor in MM.Methods:1. The level and localization of NEDD4-1 in MM cell lines,MM cells(CD138~+ plasma cells of the bone marrow)from patients,and PBMCs were determined by semi-quantitative Real Time-Polymerase Chain Reaction(q RT-PCR),Western blot and immunofluorescence staining;NEDD4-1 expression in bone marrow tissue of healthy donors and MM patients was detected by immunohistochemical staining;Clinical significance of NEDD4-1 expression in MM was assessed with the publicly available gene expression profiling(Oncomine)datasets.2. MM cells were treated with different concentrations of Bor or 2 n M Bor for different durations.The m RNA and protein levels of NEDD4-1 were evaluated by q RT-PCR and Western blot;A variety of apoptosis inhibitors were used to explore whether the changes in NEDD4-1 caused by Bor can be reversed.3. Constructed of NEDD4-1 knockdown and overexpression MM cell lines.Cell viability,proliferation,apoptosis and related classic pathways of MM cells were measured by cell counting kit-8(CCK-8),flow cytometry,and Western blot;Constructed of enzyme-dead NEDD4-1-C867S mutant overexpression MM cell lines,the sites of NEDD4-1 modulating MM Bor resistance were explored by flow cytometry.4.The substrate of NEDD4-1 was identified by Western blot,co-IP,GST-pull down assay,and immunofluorescence staining.In vivo ubiquitination experiments were used to detect the ubiquitination level and ubiquitination sites of the substrate modulated by NEDD4-1.5.The substrate was activated or inhibited by drugs or gene transfection technology.The role of substrate in regulating Bor resistance of MM Cells by NEDD4-1was explored by Western blot and flow cytometry.6.Constructed of a MM xenograft mouse model by subcutaneously injecting NEDD4-1 knockdown or overexpression MM cells in NOD-SCID mice.Bor treated the mice.The tumor burden of mice was observed,and the expression of related proteins in tumor tissue was detected by immunohistochemical staining.Results:1.Various amounts of NEDD4-1 were found in MM cell lines.ARK and LP-1had relatively high expression levels of NEDD4-1 m RNA and protein,but JJN3 and CAG had relatively low expression levels,all the NEDD4-1 expression levels of MM cells were lower than PBMCs.NEDD4-1 was localized to both the cytoplasm and nucleus but was primarily found in the cytoplasm of MM cells.PBMCs from healthy donors expressed higher NEDD4-1 levels than CD138~+cells from MM patients.Low expression of NEDD4-1 was associated with poor response to Bor,progression,and relapse in patients with MM.2.Bor treatment was associated with a reduction in NEDD4-1 in a dose/time-dependent manner.The level of NEDD4-1 did not decrease when MM cells were incubated with Bor in the presence of pan-caspase inhibitors,such as Q-VD-Oph and Z-VAD-FMK,but the level of NEDD4-1 was still reduced in the presence of other apoptosis inhibitors,such as NQDI-1 and Baxi.3.NEDD4-1 knockdown MM cells showed increased cell proliferation,less apoptosis and increased proportion of cells in the G2/M phase than the control group when treated with Bor.NEDD4-1 overexpression MM cells showed the opposite results.Cell viability in the presence of Bor gradually decreased as the level of HA-NEDD4-1increased in MM cells.The above results were also confirmed by further examining the changes in apoptosis and cell cycle-related proteins through Western blot.4. NEDD4-1 overexpressed in the NEDD4-1 knockdown MM cells induced more cell death than that observed in NEDD4-1 knockdown cells when treated with Bor.The Bor sensitivity caused by NEDD4-1 appeared to be a direct effect of NEDD4-1 E3ligase activity because the enzyme-dead HECT domain mutant,NEDD4-1-C867S,failed to promote Bor sensitivity.The NEDD4-1 knockdown MM cells showed no obvious increases in viability compared with the control when treated with another classic drug for the treatment of myeloma,melphalan.5. Co-IP and GST-pull down assay indicated that NEDD4-1 directly bound to Akt,and these two proteins were colocalized.In vivo ubiquitination experiments showed that NEDD4-1 ubiquitinated Akt through K63 conjugation and not K48 conjugation but catalysed both the K63-linked and K48-linked ubiquitination of p Akt-Ser473.6. p Akt-Ser473 is a NEDD4-1-interacting protein in MM and that its expression is required for NEDD4-1-mediated Bor resistance.NEDD4-1 overexpressed MM cells showed an inactivation of the PTEN/PI3K/Akt signalling pathway.7. The tumour size was significantly increased and the median level of soluble lambda light chains was higher in the NEDD4-1 knockdown group compared to the control group in the absence or presence of Bor treatment.Immunohistochemical analysis showed that NEDD4-1 repressed tumors exhibited more positive staining for p Akt-Ser473 and Ki67,while the NEDD4-1 overexpression group showed the opposite results.Conclusion:Overexpression of NEDD4-1 resulted in less bortezomib resistance in MM cells in vitro and in vivo,and inactivated the PTEN/PI3K/Akt signalling pathway.E3 NEDD4-1ubiquitinated Akt and targeted p Akt-Ser473 for proteasomal degradation and and thereby exerting anti-MM effects.The data warrant further investigation of NEDD4-1reactivation as a novel therapeutic strategy for MM. |
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