GTPase regulator associated with the focal adhesion kinase (GRAF) transcript was down-regulated in patients with myeloid malignancies

Background GTPase regulator associated with the focal adhesion kinase (GRAF), a putative tumor suppressor gene, is found inactivated in hematopoietic malignancies by either genetic or epigenetic abnormalities. However, the expression level of GRAF gene has not yet been studied in leukemia. The aim of this study was to investigate the expression level of GRAF gene in those patients with myeloid malignancies including acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) and chronic myeloid leukemia (CML). Methods The expression levels of GRAF transcript were determined in 94 patients using real-time quantitative PCR (RQ-PCR). Clinical and laboratory data of these patients were collected and analyzed. Results The significantly decreased level of GRAF transcript was observed in three myeloid malignancies compared to controls. Within AML, there was no difference in the level of GRAF transcript among different FAB subtypes (P > 0.05). Difference was not observed in the amount of GRAF mRNA between CML at chronic phase and controls. As CML progressed, GRAF transcript significantly decreased. In MDS, three cases with 5q deletion had lower GRAF transcript than four without 5q deletion (median 0.76 vs 2.99) (P > 0.05). Conclusion our results demonstrate that the GRAF transcript is decreased in myeloid malignancies.


Background
Focal adhesion kinase (FAK), a non-receptor tyrosine kinase that resides at the sites of integrin clustering [1], plays an important role in the modulation of cell growth, proliferation, survival and migration [2]. Recently, FAK has been found to be overexpressed and/ or constitutively activated and correlated with increased motility, invasiveness, and proliferation of neoplastic cells of various tissue types [2]. Two published articles revealed that aberrant expression of FAK was observed in CD34+ leukemic cells and associated with enhanced blast migration, increased cellularity and poor prognosis [3,4]. Le et al showed that FAK silencing inhibited leukemogenesis in BCR/ABL-transformed hematopoietic cells [5]. Tyner et al also identified FAK as one of therapeutic molecular targets in acute myeloid leukemia (AML) [6].
FAK protein is composed of an N-terminal FERM domain, a central kinase domain, and a C-terminal domain that includes the focal adhesion targeting (FAT) sequence responsible for FAK's localization to focal adhesions. Both the N-terminal and C-terminal domains have been shown to mediate FAK interaction with a variety of other proteins critical for activation of FAK by integrins or other cell surface receptors as well as FAK regulation of different cellular functions [2].
GTPase regulator associated with focal adhesion kinase (GRAF) is a newly identified protein specifically binding to the proline-rich region in the COOH terminus of FAK and negatively regulates the small GTPbinding protein RhoA, which is well known for its growth-promoting effect in RAS-mediated malignant transformation [7,8]. GRAF gene is located at chromosome 5q31 and its protein is ubiquitously expressed in various tissues [9]. Mutations and deletions of GRAF gene were found in some cases with AML or myelodysplastic syndrome (MDS) with a deletion 5q [9]. Furthermore, Bojesen et al [10] found that GRAF gene promoter was methylated in AML and MDS. The suppressed GRAF expression could be restored in leukemic cell lines by treatment with a demethyating agent and an inhibitor of histone deacytylases. However, the expression level of GRAF gene has not yet been studied in leukemia. We established the real-time quantitative polymerase chain reaction (RQ-PCR) assay with Eva-Green dye and examined the expression level of GRAF mRNA in myeloid malignancies.
The clinical characteristics of patients were listed in Table 1. Karyotypes were analyzed using conventional R-banding method. Karyotype risk in AML and MDS was classified according to the reported studies [15,17]. t (15;17) was also included in the group of low risk. BMNCs, collected from 3 donors of bone marrow transplantation, 5 patients with immune thrombocytopenia (ITP), and 13 with iron deficiency anemia (IDA), were used as controls.

Preparation of RNA and cDNA synthesis
BMNCs were separated using Lymphoprep and lysed with Trizol (In Vitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Two micrograms of total RNA was reverse transcribed to cDNA in a total reaction volume of 40 μl containing 5× buffer, dNTPs 10 mM each, random hexamers 10 μM, RNAsin 80 units and 200 units of MMLV reverse transcriptase (MBI Fermentas, USA). Samples were incubated for 10 min at 25°C, 60 min at 42°C, and then stored at -20°C.

RQ-PCR
RQ-PCR was performed using EvaGreen dye (BIO-TIUM, Hayward, CA, USA) on a 7300 Thermo cycler (Applied Biosystems, Foster City, CA, USA). Real-time fluorescent data were collected and analyzed with SDS 1.3 software (Applied Biosystems, Foster City, CA, USA). The baseline fluorescence intensities were fixed at cycles 6-15 by default and 0.01 was set as the threshold to determine the cycle threshold (C T ) value. The The relative amount of GRAF was normalized using the following formula: N GRAF = (copies of GRAF/copies of ABL) × 100. Amplified RQ-PCR products from three samples were sequenced (Shanghai GeneCore Bio-Technologies Co., Ltd., China).

Statistical analyses
Statistics was performed using the SPSS 13.0 software package (SPSS, Chicago, IL). The Kruskal-Wallis test (multiple groups) and Mann-Whitney U-test (two groups) were employed to compare the difference between patient groups and controls. The correlation between the level of GRAF transcript and the sex, age, hematologic parameters, FAB subtypes and karyotypic groups was calculated by Spearman's rho correlation analyses. A P-value < 0.05 was considered significant.

GRAF expression in controls and AML patients
The level of GRAF transcript in controls was 14.49-126.85 (median 56.04). The significantly decreased level of GRAF transcript was observed in different myeloid malignancies (Table 1, Figure 1). There was no correlation between GRAF mRNA amount and the sex, age, peripheral white blood cell count, hemoglobin level, and platelet count (P > 0.05). The association of GRAF levels with cytogenetic abnormalities or CD34 antigen expression was also not observed in AML patients (P > 0.05). Within AML, there was no difference in the level of GRAF transcript among different FAB subtypes (P > 0.05).

GRAF expression in CML patients
The median levels of GRAF transcript in CML patients at CP and BC were 46.82 (1.08-157.42) and 10.69 (0.01-23.51), respectively ( Figure 2). There was no difference in GRAF transcript amount between CML patients at CP and controls (P > 0.05). However, the amount of GRAF mRNA in CML at BC was significantly lower than that in cases at CP and that in controls (P = 0.028 and <0.001, respectively).

GRAF expression in MDS patients
Among MDS patients, three cases were identified with deletions of 5q (5q-) ( Table 2). The level of GRAF

Discussion
In this study, we demonstrated that the expression level of GRAF transcript was decreased in primary leukemic cells of all types of myeloid malignancies. Bojesen et al [10] found that GRAF promoter was hypermethylated in 38% cases with AML and MDS but not in healthy individuals, however, they did not detect the GRAF transcript in primary leukemic cells of AML and MDS. GRAF contains a centrally located GTPaseactivating protein (GAP) domain, followed by a serine/ proline rich domain and a carboxy-terminal Srchomology 3 (SH3) domain. GRAF acts as a negative regulator of RhoA because the GRAF GAP domain enhances GTP hydrolysis of both Cdc42 and RhoA in vitro [7]. Rho family GTPases play a role in the growth control besides regulating the organization of the actin cytoskeleton [18,19]. RhoA inhibits p21Cip1, p27Kip and p16Ink4 activities, permitting cell cycle progression [20][21][22][23][24]. Furthermore, RhoA has been shown involved in the regulation of apoptosis, migration, proliferation, differentiation [18,19]: for example, in vitro, constitutively active RhoA can stimulate transformation. In normal epithelia, RhoA contributes to the generation of epithelial polarity and junction assembly and function but also affects epithelial disruption during tumor progression [25]. Recently, clinical studies have revealed the correlation of increased expression of RhoA and invasion, metastasis and progression of several solid tumors including liver, bladder, esophageal, head and neck, ovary, gastric, testicular, lung and breast carcinomas [18]. As an upstream regulator, the loss of function of GRAF might prevent the physiologic down-regulation of RhoA and lead to the repression of p21. Then, the GRAF-defective cell will be driven into the S phase [9]. Several mechanisms, including translocations, allelic loss, insertions and promoter methylation observed in AML and MDS, can lead to the inactivation of GRAF [9,10].
The mechanisms responsible for the disease progression of CML remained poorly understood. Recent studies have suggested that several alterations promote this progress, including differentiation arrest caused by the suppression of translation of the transcription factor CEBPα induced by the BCR-ABL oncoprotein in CML cell, increasing genomic instability in CML cell resulting from the reduced capability of genome surveillance system, telomere shortening and loss of tumor suppressor gene (TSG) such as TP53, retinoblastoma 1, CDKN2A, DAPK1 and others [16,26,27]. Interestingly, we found that GRAF transcript was further down-regulated during CML progression. p210 Bcr-Abl, containing a centrally located Rho-specific guanine nucleotide exchange factors (RhoGEF) domain, affects the actin cytoskeleton assembly and thereby the cellular adhesion and migration by RhoA signaling pathway [28]. Further studies are required to elucidate the function of GRAF and RhoA in the pathogenesis and progression of CML.
Our preliminary results showed that MDS with 5q deletion might have lower expression of GRAF than those without 5q deletion. Deleted 5q is a one of common chromosomal abnormalities in AML and MDS. Although GRAF maps telomeric to the previously delineated commonly deleted 5(q31) region, Borkhardt et al found that one allele of GRAF was consistently lost in all studied 10 patients with 5q deletion and with either MDS or AML [9]. Besides GRAF deletion, abnormal methylation of GRAF promoter was also observed in AML and MDS [10]. These results suggested that haploinsufficiency (i.e., decreased GRAF mRNA expression) caused by deletion of GRAF allele or promoter methylation might be instrumental in the development and progression of hematopoietic malignancies.
In conclusion, GRAF mRNA is decreased in myeloid malignancies. Whether the GRAF expression level could improve the stratification or prognostication of patients with myeloid diseases should be further addressed in future studies.