The Regulatory Roles Of Spred2 In Chronic Myelocytic Leukemia Cells And Its Mechanisms

Chronic myelogenous leukemia (CML) arises mostly from the reciprocal t(9;22)(q34;q11) chromosomal translocation in pluripotent hematopoietic stem cells. The chromosomal translocation-generated Bcr-Abl fusion gene encodes a p210 protein with wild tyrosine kinase activity that stimulating multiple downstream signaling pathways, thereby conferring growth factor-independent overproduction and expansion of myeloid cell pool. CML is clinically characterized by three phases: an initial chronic phase displaying almost normal myeloid differentiation, followed by an accelerated phase and then the final blast crisis, in which myeloid and lymphoid blasts fail to differentiate and lead to abnormal accumulation of immature leukemic blast cells in blood and bone marrow. The application of imatinib that specifically targets the causative BCR-ABL oncoprotein represents a major therapeutic advance in CML. However, leuekmic cells have still evolved clinical imatinib resistance leading to therapeutic failure. Thus, extensive research in pathogenesis of CML and development of novel tyrosine kinase inhibitors targeting BCR-ABL downstream signaling have become more important in CML therapy.Recently discovered endogenous ERK pathway inhibitors are reported to be implicated in a series of cancers'development, involving leukemogenesis. Spred proteins are newly identified as members of a new membrane-associated negative regulator family which strongly inhibit Ras-ERK activation. Although Spreds have been implicated in some tumors such as hepatocellular carcinoma (HCC) and Neurofibromatosis 1 like syndrome (NFLS), their regulatory roles in CML development still remain unclear. Our hypothesis is whether Spred2 have effects on proliferation, aopoptosis, and differentiation of CML cells. To reveal this assumption, we have conducted a series of experiments as follow.Firstly, we developed recombinant adenovirus vectors harboring the full length spred2 CDS and packaged virus using HEK293 cells contransfected with Ad5f11p plasmid. In order to silence the SPRED2 expression by CML cells, we also constructed plasmid vectors with short hairpin RNAs (shRNAs) specific targeting spred2. The next, we infected the K562 cells, a widely used CML cell line with BCR-ABL positive character, with Ad5f11p-Spred2 and confirmed the spred2 overexpression. The K562 cells were also transfected with the SPRED2 shRNA using electroporation methods with NeonTM system. Based on the spred2 overespression and silence strategies, we investigated the regulatory roles of spred2 in CML cells as below.1. The effects of Spred2 overexpresion on K562 cell proliferation. The proliferation of Spred2 overxpressing K562 cells as well as of control cells were monitored using CCK-8 kit. The results indicate that Spred2 overexpression suppresses K562 cell proliferation. 2. The effects of Spred2 on ERK activity and SPHK1, MCL-1 expression. Using Western blot assays, we observed that Spred2 inhibits constitutive activity of ERK and expression of SPHK1 and Mcl-1, as well as SCF-induced upregulation of SPHK1 and MCL-1. It is indicated that Spred2 possesses of potential roles in inducing apoptosis of CML cells.3. The impact of Spred2 on CML cells apoptosis. Using Annexin-V-PE/7-AAD or Annexin-V-FITC/PI lableling followed by FACS analysis, we found that Spred2 overexpression induced CML cell apoptosis and sensitized cells to imatinib-induced cytotoxicity. In contrary, Spred2 silencing protected K562 cells from imatinib-triggered apoptosis. These effects were confirmed by active Caspase3 and cleaved PARP assay using Western blot and FACS.4. Increase expression of Spred2 in imatinib-induced CML cell death. We detected the expression of SPRED2 by K562 cells and primary CML cells exposed to imatinib for different periods.The endogenous Spred2 expression of both K562 and primary CML cells was increased after exposure to imatinib. Similar results were obtained for Spred1 in the K562 cells.5. Spred2 is involved in megakaryocytic differentiation of K562 cells. K562 cells were transfected with shRNA-Spred2 vector and the transient Spred2 silencing were confirmed by Western blot. Spred2 silencing enlarges cell size and upregulats CD41a expression which were well established as a cell surface marker of megakaryocytes. We also investigated the PMA-induced differentiation of K562 cells with Spred2 stable silencing. The data indicated that Spred2 stable silencing upregulated GATA-2, Egr-1 mRNA levels, increased PMA-induced CD41a expression, and downregulated GPA mRNA. These results indicated tha Spred2 silencing promotes K562 cells differention toward megakaryocytic lineage.6. Spred2 promotes K562 cell differentiation toward erythroid lineage. GPA is the pecific surface marker of erythroid lineage. GPA mRNA levels of K562 cells overexpressing Spred2 and control cells were determined by real time quantitative RT-PCR and their expression was confirmed on protein level by using anti-GPA-PE lableling and FACS analysis, The results showed that Spred2 overexpression promoted GPA expression at both mRNA and protein levels. Furthermore, the Spred2 sensitized K562 cells to imatinib-induced GPA mRNA promotion. These data showed that Spred2 ovexpression promoted erythroid differentiation of K562 cells.Taken together, we conclude that Spred2 regulates proliferation, apoptosis and differentiation of CML cells, most probably by negatively regulating ERK signaling pathway. Spred2 is also involved in imatinib-induced CML cytotoxicity and erythroid differentiation. Spreds may play important roles in CML pathogenesis and act as potential targets in CML therapy.