MicroRNA-1301-Mediated RanGAP1 Downregulation Induces BCR-ABL Nuclear Entrapment to Enhance Imatinib Efficacy in Chronic Myeloid Leukemia Cells.

Tsung-Yao Lin,Hsing-Jin Eugene Liu,Ann-Jeng Liu,Ku-Chung Chen,Chwen-Ming Shih,Kun-Li Wang,Persio Dello Sbarba

doi:10.1371/journal.pone.0156260

Abstract

Chronic myeloid leukemia (CML) is a myeloproliferative disease. Imatinib (IM), the first line treatment for CML, is excessively expensive and induces various side effects in CML patients. Therefore, it is essential to investigate a new strategy for improving CML therapy. Our immunoblot data revealed that RanGTPase activating protein 1 (RanGAP1) protein levels increased by approximately 30-fold in K562 cells compared with those in normal cells. RanGAP1 is one of the important components of RanGTPase system, which regulates the export of nuclear protein. However, whether RanGAP1 level variation influences BCR-ABL nuclear export is still unknown. In this report, using shRNA to downregulate RanGAP1 expression level augmented K562 cell apoptosis by approximately 40% after treatment with 250 nM IM. Immunofluorescence assay also indicated that three-fold of nuclear BCR-ABL was detected. These data suggest that BCR-ABL nuclear entrapment induced by RanGAP1 downregulation can be used to improve IM efficacy. Moreover, our qRT-PCR data indicated a trend of inverse correlation between the RanGAP1 and microRNA (miR)-1301 levels in CML patients. MiR-1301, targeting the RanGAP1 3′ untranslated region, decreased by approximately 100-fold in K562 cells compared with that in normal cells. RanGAP1 downregulation by miR-1301 transfection impairs BCR-ABL nuclear export to increase approximately 60% of cell death after treatment of 250 nM IM. This result was almost the same as treatment with 1000 nM IM alone. Furthermore, immunofluorescence assay demonstrated that Tyr-99 of nuclear P73 was phosphorylated accompanied with nuclear entrapment of BCR-ABL after transfection with RanGAP1 shRNA or miR-1301 in IM-treated K562 cells. Altogether, we demonstrated that RanGAP1 downregulation can mediate BCR-ABL nuclear entrapment to activate P73-dependent apoptosis pathway which is a novel strategy for improving current IM treatment for CML.

Highlights

Imatinib (IM) is used as a first line drug for chronic myeloid leukemia (CML) therapy
The mouse anti-RanGTPase activating protein 1 (RanGAP1), rabbit anti-c-ABL, mouse anti-Bax, rabbit anti-Lamin B, and rabbit anti-phospho-P73 (Tyr99) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); the rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), rabbit anti-poly (ADP-ribose) polymerase (PARP), and rabbit anti-phospho-Crk-like protein (CRKL) (Tyr-207) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA)
These data demonstrated that the protein levels of RanGAP1 was increased in CML cells, suggesting that RanGAP1 may be crucial in CML

Summary

Introduction

Imatinib (IM) is used as a first line drug for chronic myeloid leukemia (CML) therapy. CML drugs including IM and second generation drugs are very expensive, and this expense may reduce the opportunity for CML patients to receive appropriate therapy [1]. Nuclear BCR-ABL can be re-activated either by the removal of IM or through the metabolic decay of IM, and subsequently phosphorylated the Tyr-99 of P73 to trigger apoptosis [9,10,11]. These results suggest that impairment of BCR-ABL nuclear export can induce P73-dependent apoptosis which would be used as a strategy for improving IM efficacy

Methods

Results

Conclusion