Abstract
Reduced expression and activity of the proapoptotic, double-stranded RNA-dependent protein kinase, PKR (protein kinase R) is observed in breast, lung and various leukemias, suggesting that loss of PKR potentiates transformation. Now we report that decreased PKR activity inhibits chemotherapy-induced apoptosis of leukemia cells both in vitro and in vivo. Inhibition of PKR expression or activity reduces protein phosphatase 2A (PP2A) activity, a B-cell lymphoma 2 (Bcl-2) phosphatase, resulting in enhanced Bcl-2 phosphorylation. Thus, inhibition of PKR activity leads to hyperphosphorylation of Bcl-2, stabilization of Bcl-2/Bax interaction and decreased Bax insertion into the outer mitochondrial membrane. Treatment with the PP2A activator, FTY720, restores Bcl-2 dephosphorylation and apoptosis in cells with reduced PKR expression following stress. Significantly, xenografts of REH leukemic cells with reduced PKR display significantly increased tumor volume, increased resistance to doxorubicin treatment and shorter survival. Importantly, FTY720 treatment restores sensitivity to chemotherapy and prolongs overall survival of these mice. Collectively, these findings suggest that PP2A activation is a downstream target of PKR and the PKR/PP2A signaling axis is required for rapid and potent stress-induced apoptosis. Importantly, loss of PKR promotes leukemia progression and may serve as a biomarker for predicting chemosensitivity.
Highlights
(PP2A) was reported to be a substrate of PKR, and phosphatase 2A (PP2A) activation has been reported to regulate the intrinsic apoptosis pathway by directly dephosphorylating B-cell lymphoma 2 (Bcl-2).8,24 As studies indicate that PP2A-mediated dephosphorylation of Bcl-2 is critical to initiate apoptosis, and Bcl-2 is commonly overexpressed in hematologic malignancies, we focused our studies on elucidating the role for PKR in regulating apoptosis in human leukemia cells.[25,26,27,28,29]
K562 or REH cells with reduced PKR expression or those treated with PKR inhibitor (PKRI) display decreased substrate phosphorylation of eukaryotic initiation factor 2a (eIF2a), decreased PP2A activity, increased Bcl-2 phosphorylation and reduced apoptosis following treatment with either DOX or H2O2
These results both confirm and extend our previous report that the anti-apoptotic activity of Bcl-2 is enhanced by phosphorylation and that dysregulation of the mitochondria is inhibited by phosphorylation of Bcl[2] that leads to reduced Bax insertion into the outer mitochondria membrane (OMM).[37]
Summary
In hematopoietic stem/progenitor cells, the interferon-inducible, double-stranded RNA-dependent protein kinase, PKR (protein kinase R), is a central mediator for the antiproliferative effects of a wide range of hematopoietic cellular stresses including viral infection, hematopoietic growth factor deprivation, inflammatory cytokines, Toll-like receptor ligands and chemotherapy treatment.[1,2,3,4,5,6] Once activated by cellular stress, PKR inhibits proliferation and initiates apoptosis by phosphorylation of eukaryotic initiation factor 2a (eIF2a) to inhibit new protein synthesis, inhibition of B-cell lymphoma 2 (Bcl-2) function and activation of signaling pathways including p38 mitogen-activated protein kinase, nuclear factor-kB, p53 and signal transducer and activator of transcription 1.2,3,7–10 Loss of PKR expression/activity has been associated with more aggressive human breast carcinoma, non-small-cell lung cancer and various acute and chronic leukemias including B-cell chronic lymphocytic leukemia and T-cell acute lymphoblastic leukemia.[4,5,11,12,13,14,15,16,17,18] In addition, PKR expression has been reported to be increased in some solid tumors such as breast, PKR activity is inhibited either directly or indirectly in these cells, resulting in prosurvival conditions.[17,18,19] Taken together, these findings suggest that loss of PKR is an important driver of tumorigenesis.[14,16,20,21,22]Deregulation of apoptosis is a major contributor to the development of malignant phenotype and chemoresistance in hematopoietic cells.[23]. Protein phosphatase 2A (PP2A) was reported to be a substrate of PKR, and PP2A activation has been reported to regulate the intrinsic apoptosis pathway by directly dephosphorylating Bcl-2.8,24 As studies indicate that PP2A-mediated dephosphorylation of Bcl-2 is critical to initiate apoptosis, and Bcl-2 is commonly overexpressed in hematologic malignancies, we focused our studies on elucidating the role for PKR in regulating apoptosis in human leukemia cells.[25,26,27,28,29] Significantly, our results indicate that expression of enzymatically active PKR is required for PP2A activation and apoptosis in response to cellular stress including hydrogen peroxide (H2O2) or doxorubicin (DOX) treatment. A xenograft model of human leukemia demonstrates that acute leukemia cells with reduced PKR proliferate more rapidly and are resistant to chemotherapy in vivo. These results establish that the PKR/PP2A signaling axis negatively regulates leukemia progression and is required for sensitivity of leukemia cells to chemotherapy in vitro and in vivo
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