NEDD4-1 Modulates the Resistance of Multiple Myeloma to Bortezomib Via PTEN/PI3K/Akt Signaling Pathway

Background: Multiple myeloma (MM) is the second most common hematologic malignancy. Proteasome inhibitor bortezomib (Bor) is one of the most effective drugs for treatment of MM. However, during long-term Bor treatment, MM cells may eventually develop acquired-resistance to Bor which results in recurrence and a poor prognosis. Several researches show that E3 ubiquitin ligases (E3s) primarily determine the substrate specificity of ubiquitin proteasome system and play an essential role in Bor resistance of MM. NEDD4-1 E3s, a founding member of the Neural precursor cell-Expressed Developmentally Downregulated gene 4 (NEDD4) family, was proved to involve in the proliferation, migration, invasion of cancer cells and the sensitivity of anticancer therapies. Our current study aims to explore the role and underlying mechanism of NEDD4-1 in acquired resistance of Bor in MM.

Methods: The level of NEDD4-1 and its substrates in MM cell lines (NCI-H929, ARP-1, RPMI8226, OPM2, CAG, MM.1S) and MM cells (CD138+ plasma cells of the bone marrow) from patients were determined by semiquantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and Western Blotting. Lentiviral plasmids containing shRNA against NEDD4-1 were transfected into MM cells. NEDD4-1 was over-expressed in MM cells by lentivirus vector-mediated NEDD4-1-cDNA transfection. Cell viability, proliferation and apoptosis of MM cells were measured by Cell Counting kit8(CCK8) and flow cytometry. To immunoprecipitate endogenous proteins, whole cell extracts were immunoprecipitated with antibodies against NEDD4-1, PTEN, PI3K or Akt, along with IgG control. Clinical significance of NEDD4-1 expression in MM was assessed with the publicly available gene expression profiling (Oncomine) datasets.

Results: The Oncomine showed that expression of NEDD4-1 was higher in MM cells from patients with Monoclonal Gammopathy of Undetermined Significance (MGUS) and MM, in comparison to those from patients with Plasma Cell Leukemia. Patients in recurrence showed a lower NEDD4-1 expression than in primary occurrence. Also, MM cell lines NCI-H929, ARP-1 and RPMI8226 highly expressed NEDD4-1 at mRNA and protein levels. CAG and OPM2 showed relatively low expression. Cell growth assay displayed no significant difference in proliferation between the NEDD4-1 knockdown (KD) and the control group (P>0.05). After suppression of NEDD4-1 using shRNA, the killing effect of Bor in MM was significantly weaker than the control group (P<0.05), MM cells cycle arrested in G2/M phase (P<0.05) and NEDD4-1 over-expression sensitized MM cells to Bor (P<0.05). In the add back rescue experiment, NEDD4-1 overexpressed in NEDD4-1 KD cells re-sensitized the cells to Bor (P<0.05). In KD cells, Bcl-2, CDK4 and CDK6 expressions were markedly activated, while cleaved capase-3, PARP-1, P21 and P27 were repressed after Bor treatment; the results were opposite in NEDD4-1 over-expressed cells. NEDD4-1 KD promoted the activation of PI3K, pAkt (Ser473), but markedly diminished the expression of PTEN compared with the control group. On the other hand, NEDD4-1 overexpression inactivated the PTEN/PI3K/Akt pathway. Furthermore, we detected PTEN, PI3K and Akt in NEDD4-1 immunoprecipitates, but not in the IgG control. Reciprocally, NEDD4-1, PI3K and PTEN were detected in Akt immunoprecipitates.

Conclusion: Our study reveals that NEDD4-1 can promote MM sensitivity to Bor via inhibiting PTEN/PI3K/Akt signaling pathway and NEDD4-1 can interact with PTEN/PI3K/Akt to form a putative complex. These results suggest that NEDD4-1 may serve as a novel drug target in the treatment of MM.