Bortezomib-Resistance Is Characterized By Upregulation of E3 Ligase Pirh2 Which Protects Bortezomib Induced Apoptosis in Multiple Myeloma Cells

Background Proteasome inhibitor bortezomib is an effective approach to the treatment of multiple myeloma (MM), but drug resistance often emerges and limits its retreatment. However, the action mechanisms are not fully understood. Several lines of evidence link E3 ligase to myeloma and its drug resistance. Pirh2 (p53-induced RING-H2) E3 ubiquitin ligase with intrinsic ubiquitin-protein ligase activity for polyubiquitination and subsequently proteasomal degradation was predisposed for plasma cell hyperplasia and tumorigenesis as reported. In this study, we examined Pirh2 expression in plasma cells of the bone marrow (CD138+ cells) from MM patients and investigate the effect of Pirh2 on the viability, proliferation, survival and drug resistance of MM cells.

Methods Bortezomib-sensitive malignant haematopoetic cells can acquire secondary resistance to Bortezomib in vitro. To identify some of the mechanisms involved, we developed myeloma cell lines OPM-2 and RPMI8226 bortezomib resistant (OPM-2-BR, RPMI8226-BR) derivative lines by continuous culture in sub-lethal concentrations of bortezomib. Clones of OPM-2-BR and RPMI8226-BR were obtained after several months, and acquired resistance to bortezomib remained stable over months with extended stimulus duration in the presence of bortezomib. Pirh2 mRNA and protein expressions were detected by real time quantitative PCR(Q-RT PCR) and Western blotting. We also generated stable Pirh2 knock down cell lines and stable Pirh2 overexpression cell lines.

Results We found differential expression of Pirh2 in bortezomib-resistant (BR) cells, compared to wild type (WT) controls by Q-RT PCR and Western blotting. We confirmed generally Pirh2 mRNA and protein expressions in MM cell lines. Besides, Pirh2 mRNA expression was also determined from bone marrow samples of MM patients. In summary, we showed that Pirh2 is more highly expressed in MM cell lines and patient MM cells (from newly diagnosed or refractory/recurrence patients with MM) than in normal bone marrow mononuclear cells from normal donors (P<.0001). Interestingly, We found that Pirh2 overexpression cell lines showed negatively effect on the response to bortezomib treatment. Consistent with this, Pirh2 knock down cell lines can overcome bortezomib resistance.

Conclusions Collectively, Our findings demonstrate a novel role for Pirh2, which suggests that Pirh2 may act by an unknown mechanism involved in bortezomib resistance of MM. An improved understanding of it can lead to better therapeutics for bortezomib retreatment which will be a rational strategy for clinical translation.