PTEN/PI3K/AKT PATHWAY DYSREGULATION IN MYELODYSPLASTIC GRANULOCYTES

Background. Patients with Myelodysplastic Syndromes are at increased risk of infections not only because of the reduced number of granulocytes, but also for their functional abnormalities. Myelodysplastic neutrophils were shown to present impaired fungicidal and bactericidal activities and reduced chemotaxis. PTEN/PI3K/AKT pathway plays a crucial role in mediating cellular response to chemotactic stimuli. Aims. To study gene expression of PTEN/PI3K/AKT signaling pathways in myelodysplastic granulocytes compared to the normal counterpart. Methods. We evaluated PTEN mRNA expression in peripheral blood granulocytes isolated by Ficoll gradient centrifugation and osmotic erythrolysis from 30 patients with MDS and 12 normal controls by Sybr Green-based realtime RT-PCR, using Abl as reference gene. Data were expressed as 2-ΔCt. DNA methylation of two regions of the PTEN promoter (from -1223 bp to -1123 bp and from -300 bp to -128 bp) was studied by Methylation-specific PCR on bisulfite-treated DNA from 19 MDS and 6 controls. The gene expression profile of 84 genes belonging to the PTEN/PI3K/AKT signaling pathway was studied on granulocytes from 10 MDS patients and 10 matched normal control using the Human PI3K-AKT Signaling Pathway RT² Profiler® PCR Arrays (SABiosciences, Qiagen). Patients’ cohort used for PCR arrays included the following MDS subtypes: 2 RCMD, 2 RAEB1, 4 RAEB2, 1 CMML and 1 t-MDS. Selected housekeeping genes were GAPDH, RPL13A and HPRT1. Data analysis was performed using the RT² Profiler® PCR Array Analysis Template v3.3. Results. PTEN mRNA was significantly down-regulated in myelodysplastic versus normal granulocytes (median 157,59 and 401,22, respectively, p=0.029). No significant differences in PTEN mRNA levels were observed in different MDS subtypes. PTEN promoter was not methylated in MDS and control granulocytes. Ten genes belonging to the PTEN/PI3K/AKT signaling pathway were found to be significantly down-regulated in MDS versus controls, including NFKBIA, FOS, AKT1, MAPK3, RBL2, RPS6KA1, GRB2, PIK3CG, RAF1 and ADAR (fold change less than -2 and p<0.05). Among studied genes, the most significantly suppressed was NFKBIA (fold change: -5,63, p=0.004) which encodes for the NFKB inhibitor-α, whose reduction might contribute to deregulated signaling across the PI3KT-AKT pathway in myelodysplastic granulocytes.Summary/ Conclusions. Deregulation of PTEN/PI3K/AKT signaling pathway can contribute to granulocytic dysfunction in MDS.