Proteome changes induced by c-myb silencing in human chronic myeloid leukemia cells suggest molecular mechanisms and putative biomarkers of hematopoietic malignancies

Abstract

To shed light on the molecular mechanisms associated with aberrant accumulation of c-Myb in chronic myeloid leukemia, comparative proteomic analysis was performed on c-myb RNAi-specifically silenced K562 cells, sampled on a time-course basis. 2D-DIGE technology highlighted 37 differentially-represented proteins that were further characterized by nLC-ESI-LIT-MS/MS and validated by western blotting and qRT-PCR analysis. Most of the deregulated proteins were related to protein folding, energy/primary metabolism, transcription/translation regulation and oxidative stress response. Protein network analysis suggested that glycolysis, gluconeogenesis and protein ubiquitination biosynthesis pathways were highly represented, confirming also the pivotal role of c-Myc. A specific reduced representation was observed for glyceraldehyde-3-phosphate-dehydrogenase and α-enolase, suggesting a possible role of c-Myb in the activation of aerobic glycolysis. A reduced amount was also observed for stress responsive heat shock 70kDa protein and 78kDa glucose-regulated protein, previously identified as direct targets of c-Myb. Among over-represented proteins, worth mentioning is the chromatin modifier chromobox protein homolog 3 that contributes to silencing of E2F- and Myc-responsive genes in quiescent G0 cells. Data here presented, while providing novel insights onto the molecular mechanisms underlying c-Myb activity, indicate potential protein biomarkers for monitoring the progression of chronic myeloid leukemia.

Biological significance: Myeloid leukemia is a malignant disease of the hematopoietic system in which cells of myeloid lineages accumulate to an undifferentiated state. In particular, it was shown that an aberrant accumulation of the c-Myb transcriptional factor is associated with the suppression of normal differentiation processes promoting the development of the hematopoietic malignancies. Many efforts have been recently made to identify novel genes directly targeted by c-Myb at a transcriptome level. In this work, we originally describe a differential proteomic approach to facilitate the comprehension of the regulation of the protein networks exerted by c-Myb. Our study reveals a complex network of proteins regulated by c-Myb. The functional heterogeneity of these proteins emphasizes the pleiotropic role of c-Myb as a regulator of genes that are crucial for energy production and stress response in leukemia. In fact, variations in glyceraldehyde-3-phosphate-dehydrogenase and α-enolase suggest a possible role of c-Myb in the activation of aerobic glycolysis. Moreover, significant differences were found for heat shock 70kDa protein and 78kDa glucose-regulated protein known as direct c-Myb targets. This work highlights potential protein biomarkers to look into disease progression and to develop translational medicine approaches in myeloid leukemia.

Keywords: 2D-DIGE; Cell biology; Chronic myeloid leukemia; c-Myb.