Role And Mechanism Of WAVE1 In Multidrug Resistance In Children With Acute Myeloblastic Leukemia

Leukemia, a malignant disease of the bone marrow and blood, is the most common form of cancer in children. Chemotherapy is the main treatment for nearly all type of childhood leukemia. Multidrug resistance (MDR) is one of the primary reasons causing suboptimal chemotherapy in children with acute myeloblastic leukemia (AML). The occurrence of MDR restricts the long-term event-free survival (EFS). It is important to reveal the role of WAVE1 in MDR of AML and to find a new target for therapeutics. WASP-family verprolin-homologous protein 1 (WAVE1) is a member of the actin regulatory protein family and plays an important role in the regulation with actin's polymerization and cytoskeleton's reorganization. In our previous study, we demonstrated that WAVE1 was a novel regulator of apoptosis, and was involved in the MDR in K562/A02 leukemia cells.At first, Real-time fluorescence quantitative PCR(RQ-PCR) and Western blotting analysis the mRNA and protein expression levels of WAVE1, P-glycoprotein, MRP 1 (Multiple resistance-associated protein-1), LRP(lung-resistance related protein) and BCRP(breast cancer resistance protein) in bone marrow mononuclear cells (BMMCs) in a cohort of 52 children with AML. We found the expression levels of these genes or proteins in refractory/relapsing group were much higher than complete continuous remission (CCR) group. Moreover, the mRNA and protein expression levels of WAVE1 in BMMCs of AML patients were higher in the phase of newly diagnosed or relapsed than complete continuous remission, suggesting that WAVE1 is correlated to the development of AML. Furthermore, overexpression of WAVE1 in K562 and HL60 cell lines increased the expression levels of P-gp, MRP and BCRP respectively; whereas suppression of WAVE1 expression in K562/A02 and HL60/ADR cells by RNA interference decreased the expression levels of P-gp, MRP1 and BCRP respectively.At second, in order to evaluate the mechanisms of WAVE1-mediated MDR, WAVE1 binding proteins were separated and identified in AML BMMCs and leukemia cell lines respectively. WAVE1 binding proteins were captured by anti-WAVE1 antibody using immunop-recipitation with the total proteins from BMMCs and leukemia cell lines. The captured protein complexes were subjected to SDS-PAGE. Protein bands were cut off from gel and in-gel digested analyzed by ESI-Q-TOF. Ezrin protein were identified by peptide sequence tags (PST) and database searching, and it was confirmed by co-immunoprecipitation and Western blotting analysis. Furthermore,43 proteins were identified in the WAVE1 immunoprecipitates complex by preteomic analysis. These proteins were divided into seven main groups based on their functions: tumor-associated proteins, cytoskeletal proteins, receptors, metabolism-related enzymes, immune-related proteins, membrane and ion channel proteins and signaling pathway proteins.At third, to find the interaction of WAVE1-Ezrin-P-gp in vitro, we use immunofluoresence to observe the co-localization of WAVE1 and Ezrin in cell membrane. Indeed, overexpression of WAVE1 in K562 cell lines increased the expression levels of P-gp and ezrin, whereas suppression of WAVE1 or Ezrin expression by RNA interference decreased the expression levels of P-gp in K562/A02 cell lines and further restored leukemia cells'sensitivity to ADM. In contrast, overexpression of WAVE1 and suppression of ezrin didn't increase the expression levels of P-gp. Thus, Ezrin may be a downstream acting factor of WAVE1. These results suggested that WAVE1 regulated P-gp via ezrin in leukemia cells.Finally, we use magnetic activated cell sorting (MACS) to isolate CD34+/- cells from AML BMMCs and leukemia cell lines. We found that the expression of WAVE1 in CD34+ cells was significantly higher than CD34- cells by Western blotting. WAVE1 may be involved in the formation and maturation of hematopoietic stem cell (HSC) and leukemia stem cell (LSC).In conclusion, WAVE1 was involved in the regulation of multidrug resistance (MDR) and pathogenesis of acute myeloblastic leukemia. WAVE1 regulated MDR partly via a WAVE1-Ezrin-P-gp dependent way in AML. WAVE1 may be associated with the formation of malignant clones of LSC. Our experimental data revealed a noval function of WAVE1 in leukemia and provided clues to reverse MDR in AML.