The Combination of 9-cis Retinoic Acid Plus MEK Inhibitor Induces Apoptosis Via Dephosphorylation of Retinoid X Receptor α in HL-60R Cells.

Resistance against retinoic acid (RA) is a serious problem of differentiation-induction therapy for acute promyelocytic leukemia (APL) in clinical practice. RA exerts its biological activities primarily through the nuclear receptor dimmer, consisting of retinoic acid receptor (RAR) and retinoid X receptor (RXR). Both receptors consist of three subtypes (α, β and γ) and form heterodimeric RAR/RXR and homodimeric RXR/RXR complexes. 9-cis RA, which is a high-affinity ligand for RXR but also binds to RAR, induces cell differentiation in wild type HL-60 human myeloid leukemia cells. However, in HL-60R cells, the RA-resistant subclone of HL-60, 9-cis RA can not induce cell differentiation and apoptosis. We recently reported that malfunction of RXRα due to posttranslational modification by phosphorylation to be associated with carcinogenesis of hepatocellular carcinoma (HCC). Phosphorylated form of RXRα (p-RXRα) at serine 260 by Ras/MAPK is resistant to ubiquitin/proteasome-mediated degradation and the accumulation of p-RXRα interferes with the function of remaining normal RXRα in a dominant negative manner, thereby promoting growth of HCC cells. We also found that in the presence of MEK inhibitor PD98059, 9-cis RA can induce the degradation of p-RXRα and thus restoring the function of this receptor in RXRα-phosphorylated human HCC cells. Based on the results as described above, we initiated this study to examine whether 9-cis RA can exert growth inhibitory effects on RA-resistant HL-60R cells when combined with MEK inhibitor, with focusing on the inhibition of expression of p-RXRα protein. We found that RXRα protein was originally expressed in both HL-60 and HL-60R cells, and that the expression level of RXRα protein was inhibited by about 60% and 20%, respectively, when those cells were treated with 0.5 μM 9-cis RA. Not only total RXRα protein, but also the level of p-RXRα protein was constitutively expressed in both HL-60 and HL-60R cells, and was significantly decreased in HL-60 cells by treatment with 9-cis RA alone. On the other hand, in HL-60R cells, 9-cis RA alone nor 20 μM PD98059 alone did not cause a down regulation of these proteins. However, when HL-60R cells were treated with the combination of 9-cis RA plus PD98059, the expression level of p-RXRα protein was markedly decreased. Moreover, the combination of these agents induced apoptosis in HL-60R cells, whereas similar effect was not obtained when the cells were treated with either agent alone. The combined treatment of these agents also cooperatively inhibited the growth of HL-60 R cells. The present findings suggest that the accumulation of p-RXRα might impair the function of normal RXRα as a master regulator of nuclear receptors and, thus, contributing to the resistance to RA in HL-60R cells. Combination of 9-cis RA plus MEK inhibitor might be an effective regimen for patients with RA resistant APL.