Down-Regulation of Ribosomal S6 kinase RPS6KA6
in Acute Myeloid Leukemia Patients
Signaling pathways such as extracellular regulated kinase/mitogen activated protein kinase (ERK/MAPK) have increased activity in leukemia. Ribosomal 6 kinase
Materials and Methods
This cross-sectional study was undertaken in 2013-2014 at Ghaem Hospital in Mashhad, Iran, on 40 AML patients and 10 non-AML patients as the control group. The expression rate was measured by real-time polymerase change reaction (PCR) and employing the ΔΔCT method. Data were analyzed using Mann-Whitney and Spearman tests using SPSS (version 11.5).
Expression rate of
We detected a down-regulation of
A proportion of acute myeloid leukemia (AML) cases exhibit irregularities in the expression of genes including transcription factors, oncogenes, tumor suppressors, and abnormal activities in tyrosine kinase receptors which regulate blood production (1). Tyrosine kinase receptors recognize a specific sequence of enzymes (kinases) to apply the effect of their ligand on transcription of the genes (2). Extracellular regulated kinase/mitogen activated protein kinase (ERK/MAPK) is one of the wellknown pathways in blood cells which become activated by growth factors and cytokines (such as IL-3), through tyrosine kinase receptors (3). After the ligand attaches to the receptor, the RAS oncogene is activated which in turn activates the ERK (4,5). The activation of this pathway increases the chance of survival, growth and proliferation of hematopoietic cells (6,7) and also inhibits many of the apoptosis-related proteins such as caspase 9 (8). Irregularity in ERK/MPAK adjustment is an important factor in leukemogenesis of AML cells (9,10). In one study, increased activity of this pathway was related to weak/faint prognosis (11). Activation of ERK was also observed in more than 50% of primary AML progenitors, and this activation is an independent prognostic factor for survival in AML(12). Resistance to drugs is the major problem in effectively treating AML(13). It has been observed that inhibition of the ERK/MPAK signaling pathway by lovastatin and PD98059 (ERK inhibitory drug), resulted in apoptosis and treatments were better (14). Therefore the activity of this pathway and its related mediators may be relevant therapeutic targets in AML progression prevention (15).
An important mediator in this pathway is ribosomal 6 kinase (RSK) which is also the most important substrate of ERK (4). The RSK family has four members (RSK1-4) and are activated by ERK (16). In spite of other members increasing growth and proliferation in cells,
Given that previous studies have shown
Materials and Methods
This cross-sectional study was performed in the Cancer Molecular Pathology Research Center, Ghaem Hospital, Mashhad University of Medical Sciences, Mashhad, Iran, in 2013-2014. The local Ethical Committee approved the research protocol and written informed consents were taken from all participants. Peripheral blood and bone marrow samples of diagnosed AML patients [according to World Health Organization (WHO) criteria] and those of non-AML individuals (as the control group) were collected in anticoagulant-containing blood sampling tubes Ethylene Diamine Tetra Acetic acid-K2 (EDTA-K2). For all patients, complete blood count (CBC) was analyzed and peripheral blood and bone marrow smears were prepared and observed after staining. AML was diagnosed as existence of at least 20% blasts in peripheral blood or bone marrow smears that were positive for myeloperoxidase or Sudan Black B staining and myeloid markers CD33, CD13, CD117, CD64 and CD14. According to morphologic features of the smears, other staining including periodic acid schiff and none-specific esterase were also performed. In the next step, for AML patients only, among the recurrent cytogenetic abnormalities determined by the WHO classification, three translocations including t(15;17), t(8;21) and inv(16) were analyzed by PCR (ABI thermo cycler, Applied Biosystems, USA). AML Patients without these genetic abnormalities were considered as a different group defined as "other". In addition, morphologic subtypes of all AML patients were determined according to the FAB classification (M0 to M7). AML patients undergoing treatment or with recurrent leukemia, uncertain diagnosis and improper samples were excluded from the study. The control group included individuals that had no neoplastic disorders in their history or their peripheral blood and bone marrow samples. To determine
PCR; Polymerase chain reaction.
Data were analyzed using SPSS (Version 11.5). Initially, descriptive and then comparative analyses of parameters were performed. Due to the non-normal distribution of expression values, sample means were compared using Mann-Whitney test and correlation analysis was undertaken by Spearman’s test. P≤0.05 was considered to be significant.
After exclusion of five samples, 40 patients diagnosed with AML and 10 individuals as the control group were analyzed. In the patient group, 24 (60%) were male and 16 (40%) were female, while in the control group, 5 (50%) were male and 5 (50%) were female. The average age ± SD was 31 ± 17 and 29 ± 5.9 years in the patient and the con- trol groups respectively. The mean age difference between two groups was not significant (P=0.544). Differences between AML patients and the control group in red blood cell (RBC) and Platelet (PLT) count, Haematocrit (HCT) and Hemoglobin (Hb) were significant (P<0.05), however, white blood cell (WBC) count was not significantly different. Table 2, shows results of CBC in two groups.
Among the AML patients, the most common subtype (according to the FAB classification) was the
AML-M3 (,Table 3,)
and also the most common cytogenetic abnormality was t(15;17) (Table 4,). The
average expression level fold-change of
|Parameters||AML (Mean ± SD)||Control (Mean ± SD)||P value|
|RBC×106/μl||2.94 ± 0.94||4.9 ± 0.59||<0.001|
|WBC×103/μl||26.79 ± 33.3||7.19 ± 1.48||0.174|
|PLT×103/μl||76.45 ± 74.72||204.9 ± 54.5||<0.001|
|HCT (%)||26.4 ± 8.35||42.1 ± 4.3||<0.001|
|Hb (g/dl)||8.46 ± 3.05||15.1 ± 1.6||<0.001|
AML; Acute myeloid leukemia, RBC; Red blood cell, WBC; White blood cell, PLT; Platelet, HCT; Haematocrit and Hb; Hemoglobin.
|Morphologic subtype||n (%)||Fold change (Mean expression rate)|
AML; Acute myeloid leukemia.
|Chromosomal disorder||n (%)||Fold change (Mean expression rate)|
AML; Acute myeloid leukemia and ; Patients negative for the three chromosomal disorders mentioned.
Due to the high activity of ERK in some AML
progenitors and ability of RSK to inhibit ERK,
we hypothesized that
Other members of the RSK family also act
as substrates for ERK and activate some transcription factors which play important roles in
some malignancies and may be effective factors
in leukemias (31). It was suggested that only
We observed down-regulation of
This study was based on M.Sc. student thesis supported financially by the Vice President of Research at Mashhad University of Medical Sciences, Mashhad, Iran. We would like to convey our thanks to him. We also thank Dr. Afzalaghaii for advice on statistical analysis. There is no conflict of interest in this study.