Oncotarget Roscovitine enhances all-trans retinoic acid (ATRA)-induced nuclear enrichment

Oncotarget Volume 11, Issue 12 reported that using the HL-60 human non-APL AML model where ATRA causes nuclear enrichment of c-Raf that drives differentiation/G0-arrest, the research team now observe that roscovitine enhanced nuclear enrichment of certain traditionally cytoplasmic signaling molecules and enhanced differentiation and cell cycle arrest.

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.

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.

Dr. Mengsu Yang from the Department of Biomedical Sciences, City University of Hong Kong, Kowloon, Hong Kong SAR, People's Republic of China and Dr. Andrew Yen from the Department of Biomedical Sciences, Cornell University, Ithaca, NY, USA said, "All-trans retinoic acid (ATRA), a retinoid metabolite of vitamin A, regulates gene expression in a number of physiological processes, including morphogenesis, vision, growth, metabolism, differentiation and cellular homeostasis."

"All-trans retinoic acid (ATRA), a retinoid metabolite of vitamin A, regulates gene expression in a number of physiological processes, including morphogenesis, vision, growth, metabolism, differentiation and cellular homeostasis."

- Dr. Mengsu Yang, Department of Biomedical Sciences, City University of Hong Kong & Dr. Andrew Yen, Department of Biomedical Sciences, Cornell University

ATRA induces these cells to undergo myeloid differentiation and G0 cell cycle arrest that depends on a sustained MAPK pathway signal with up-regulation and unanticipated translocation of c-Raf to the nucleus.

In ATRA-induced differentiation of leukemia cells, Vav1 also interacts with PU.1, recruiting it to the promoter to transcriptionally activate expression of the CD11b differentiation marker.

This enhances its interest in chemotherapy and, as the authors now report, particularly for ATRA-based differentiation induction therapy, which is an entirely novel mechanism for this drug that reveals novel therapeutic vulnerabilities as well as basic molecular mechanistic features of ATRA-induced differentiation of leukemic cells.

Figure 1: Roscovitine enhances the amount of ATRA-induced phosphorylated c-Raf and phosphorylated c-Raf in the nucleus modulates the RB protein functions. (A–C) Western blot of c-Raf and its phospho-regulatory residues in HL-60 cells cultured with ATRA for 72 h showed that ATRA upregulated nuclear c-Raf, pS259 and pS289/296/301 c-Raf expression and co-treatment with ATRA plus roscovitine further increased of c-Raf and its active phosphorylation sites, pS259 and pS289/296/301, compared to ATRA alone. (D) TATA binding protein (TBP) is the loading control. (E) c-Raf immunoprecipitates probed for RB or S608 RB show that roscovitine enhances ATRA-induced downregulation of the amount of nuclear c-Raf complexed with RB and specifically with its serine 608 phosphorylated form (pS608 RB). An equal amount of pre-cleared nuclear lysate was collected 72 h post treatment and incubated overnight with 2.5 μg of the precipitating antibody with magnetic beads and resolved on 12 % polyacrylamide gels. All blots shown are representative of three replicates.

Lyn knockdown augmented certain roscovitine enhancements of ATRA effects on nuclear signaling and cell cycle regulatory molecules.

The novel activation of the signaling molecules and translocation to the nucleus during ATRA-induced differentiation is enhanced by roscovitine with concomitant enhancement of induced differentiation.

The Yang/Yen Research Team concluded in their Oncotarget Research Paper, "the current study showed that roscovitine exhibits effects beyond its original presentation as a CDK inhibitor (Figure 9A and 9B). Roscovitine may modulate nuclear molecules and enhance the therapeutic effects of ATRA in HL-60 cells. To the best of our knowledge, this study is the first to report that roscovitine potentiates ATRA in inducing myeloid leukemia cell differentiation, the mechanistic insights of which suggests new therapeutic targets to improve the clinical efficiency of ATRA to treat myeloid leukemia."

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DOI - https://doi.org/10.18632/oncotarget.27511

Full text - https://www.oncotarget.com/article/27508/text/

Correspondence to - Mengsu Yang - bhmyang@cityu.edu.hk and Andrew Yen - ay13@cornell.edu

Keywords - ATRA, roscovitine, APL, HL-60, Lyn

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