Roscovitine enhances all-trans retinoic acid (ATRA)-induced nuclear enrichment of an ensemble of activated signaling molecules and augments ATRA-induced myeloid cell differentiation.

Asif Rashid,Feng Gao,Mengsu Yang,Xin Duan,Andrew Yen

doi:10.18632/oncotarget.27508

Asif Rashid, Feng Gao + Show 3 more

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https://doi.org/10.18632/oncotarget.27508

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Abstract

Although ATRA represents a successful differentiation therapy for APL, it is largely ineffective for non-APL AMLs. Hence combination therapies using an agent targeting ATRA-regulated molecules that drive cell differentiation/arrest are of interest. Using the HL-60 human non-APL AML model where ATRA causes nuclear enrichment of c-Raf that drives differentiation/G0-arrest, we now observe that roscovitine enhanced nuclear enrichment of certain traditionally cytoplasmic signaling molecules and enhanced differentiation and cell cycle arrest. Roscovitine upregulated ATRA-induced nuclear c-Raf phosphorylation at S259 and S289/296/301. Nuclear c-Raf interacted with RB protein and specifically with pS608RB, the hinge region phosphorylation controlling E2F binding and cell cycle progression. ATRA-induced loss of pS608RB with cell cycle arrest was associated with loss of RB-sequestered c-Raf, thereby coupling cell cycle arrest and increased availability of c-Raf to promote differentiation. Part of this mechanism reflects promoting cell cycle arrest via ATRA-induced upregulation of the p27 Kip1 CDKI. Roscovitine also enhanced the ATRA-induced nuclear enrichment of other signaling molecules traditionally perceived as cytoplasmic promoters of proliferation, but now known to promote differentiation; in particular: SFKs, Lyn, Fgr; adaptor proteins, c-Cbl, SLP-76; a guanine exchange factor, Vav1; and a transcription factor, IRF-1. Akin to c-Raf, Lyn bound to RB, specifically to pS608RB. Lyn-pS608RB association was greatly diminished by ATRA and essentially lost in ATRA plus roscovitine treated cells. Interestingly Lyn-KD enhanced such ATRA-induced nuclear signaling and differentiation and made roscovitine more effective. ATRA thus mobilized traditionally cytoplasmic signaling molecules to the nucleus where they drove differentiation which were further enhanced by roscovitine.

Highlights

All-trans retinoic acid (ATRA), a retinoid metabolite of vitamin A, regulates gene expression [1] in a number of physiological processes, including morphogenesis, vision, growth, metabolism, differentiation and cellular homeostasis [2]
ATRA reduced the amount of RB and pS608 RB bound with c-Raf, and roscovitine enhanced the reduction in the amount of c-Raf bound with RB and pS608 RB
We found that ATRA treated Lyn knockdown (shLyn) stable transfectant (Lyn KD) cells showed progressively more G1/G0 enrichment than wild type cells, consistent with retardation of growth, and adding roscovitine to ATRA further enhanced the accumulation of these cells in G1/G0 compared to ATRA alone (Figure 7N)

Summary

Introduction

All-trans retinoic acid (ATRA), a retinoid metabolite of vitamin A, regulates gene expression [1] in a number of physiological processes, including morphogenesis, vision, growth, metabolism, differentiation and cellular homeostasis [2]. ATRA is prominent as a differentiation-inducing therapeutic for acute promyelocytic leukemia (APL) [3, 4], which is a FAB (French American British classification) M3 subtype of acute myeloid leukemia (AML). The classical paradigm of ATRA-induced differentiation in leukemia cells focuses on RARα and retinoid X receptors, which are transcription factors www.oncotarget.com activated by binding to their ligands. Other signaling pathways, mitogen-activated protein kinase (MAPK), have been found to be necessary for RAR and RXR to transcriptionally activate and induce differentiation and G1/G0 cell cycle arrest [6,7,8]. The Raf/Mek/Erk axis is imbedded in the ATRA-induced signalsome which includes Src family kinases Fgr and Lyn, PI3K, c-Cbl, SLP-76, Vav and KSR1, plus transcription factors AhR and IRF1 [9,10,11,12]

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