ABSTRACT
Inositide signaling pathways can have a role in the Myelodysplastic Syndromes (MDS) progression to acute myeloid leukemia. Erythropoietin (EPO) is currently used in low-risk MDS, where it successfully corrects anemia in 50-70% of patients. However, some MDS patients are refractory to this treatment and little is known about the exact molecular mechanisms underlying the effect of EPO in these subjects. Here, we investigated the role of inositide pathways in low-risk MDS treated with EPO, mainly focusing on the Akt/PI-PLC (Phosphoinositide-Phospholipase C) gamma1 axis, which is activated by the EPO receptor, and PI-PLCbeta1/Cyclin D3 signaling, as Cyclin D3 is associated with hematopoietic proliferation and differentiation. Interestingly, EPO responder patients showed a specific activation of both the Akt/PI-PLCgamma1 pathway and beta-Globin gene expression, while nonresponders displayed an increase in PI-PLCbeta1 signaling. Moreover, in normal CD34+ cells induced to erythroid differentiation, PI-PLCbeta1 overexpression abrogated both EPO-induced Akt phosphorylation and beta-Globin expression. Overall, these findings suggest that PI-PLCbeta1 can act as a negative regulator of erythroid differentiation and confirm the involvement of Akt/PI-PLCgamma1 pathway in EPO signaling, therefore contributing to the comprehension of the effect of EPO in low-risk MDS and possibly paving the way to the identification of MDS patients at higher risk of refractoriness to EPO treatment.
Subject(s)
Cell Nucleus/metabolism , Erythropoietin/therapeutic use , Myelodysplastic Syndromes/metabolism , Signal Transduction/drug effects , Aged , Aged, 80 and over , Blotting, Western , Case-Control Studies , Cell Differentiation , Cell Nucleus/genetics , Cyclin D3 , Follow-Up Studies , Humans , Immunoenzyme Techniques , Middle Aged , Myelodysplastic Syndromes/drug therapy , Myelodysplastic Syndromes/pathology , Phosphatidylinositols/metabolism , Phospholipase C beta/genetics , Phospholipase C beta/metabolism , Phospholipase C gamma/genetics , Phospholipase C gamma/metabolism , Phosphorylation , Prognosis , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Receptors, Erythropoietin/genetics , Receptors, Erythropoietin/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Risk Factors , beta-Globins/genetics , beta-Globins/metabolismABSTRACT
Phosphoinositide-phospholipase C (PI-PLC) beta1 can be considered a specific target for demethylating therapy in high-risk myelodysplastic syndrome (MDS) patients, as azacitidine treatment has been associated with a PI-PLCbeta1-specific promoter demethylation, and induction of PI-PLCbeta1 gene and protein expression. However, little is known about the molecular effect of azacitidine in low-risk MDS or the functional mechanisms linked with azacitidine effect on PI-PLCbeta1 promoter. In the present study, we further investigated the role of epigenetic regulation of PI-PLCbeta1, mainly focusing on the structure of the PI-PLCbeta1 promoter. We first examined the effect of azacitidine on PI-PLCbeta1 promoter methylation and gene expression in low-risk MDS. Moreover, we studied the expression of key molecules associated with the nuclear inositide signaling pathways, such as cyclin D3. By applying a chromatin immunoprecipitation method, we also studied the correlation between the demethylating effect of azacitidine and the degree of recruitment to PI-PLCbeta1 promoter of some transcription factors implicated in hematopoietic stem cell proliferation and differentiation, as well as of the methyl-CpG-binding domain proteins, which specifically interact with methylated DNA. Taken together, our results hint at a specific involvement of PI-PLCbeta1 in epigenetic mechanisms, and are particularly consistent with the hypothesis of a role for PI-PLCbeta1 in azacitidine-induced myeloid differentiation.
Subject(s)
Antimetabolites, Antineoplastic/therapeutic use , Azacitidine/therapeutic use , Epigenesis, Genetic , Myelodysplastic Syndromes/drug therapy , Phosphatidylinositols/metabolism , Phospholipase C beta/metabolism , Signal Transduction , Aged , Aged, 80 and over , Base Sequence , DNA Methylation , DNA Primers , Female , Humans , Male , Middle Aged , Molecular Sequence Data , Myelodysplastic Syndromes/enzymology , Myelodysplastic Syndromes/pathology , Phospholipase C beta/genetics , Promoter Regions, GeneticABSTRACT
The association between azacitidine (AZA) and valproic acid (VPA) has shown high response rates in high-risk myelodysplastic syndromes (MDS) cases with unfavorable prognosis. However, little is known about the molecular mechanisms underlying this therapy, and molecular markers useful to monitor the disease and the effect of the treatment are needed. Phosphoinositide-phospholipase C (PI-PLC) ß1 is involved in both genetic and epigenetic mechanisms of MDS progression to acute myeloid leukemia. Indeed, AZA as a single agent was able to induce PI-PLCß1 expression, therefore providing a promising new tool in the evaluation of response to demethylating therapies. In this study, we assessed the efficacy of the combination of AZA and VPA on inducing PI-PLCß1 expression in high-risk MDS patients. Furthermore, we observed an increase in Cyclin D3 expression, a downstream target of PI-PLCß1 signaling, therefore suggesting a potential combined activity of AZA and VPA in high-risk MDS in activating PI-PLCß1 signaling, thus affecting cell proliferation and differentiation. Taken together, our findings might open up new lines of investigations aiming at evaluating the role of the activation of PI-PLCß1 signaling in the epigenetic therapy, which may also lead to the identification of innovative targets for the epigenetic therapy of high-risk MDS.
