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. 2014 Sep 1;135(5):1101-9.
doi: 10.1002/ijc.28759. Epub 2014 Feb 19.

PTPRG inhibition by DNA methylation and cooperation with RAS gene activation in childhood acute lymphoblastic leukemia

Jianqiao Xiao  1 , Seung-Tae Lee, Yuanyuan Xiao, Xiaomei Ma, E Andres Houseman, Ling-I Hsu, Ritu Roy, Margaret Wrensch, Adam J de Smith, Anand Chokkalingam, Patricia Buffler, John K Wiencke, Joseph L Wiemels
Affiliations

PTPRG inhibition by DNA methylation and cooperation with RAS gene activation in childhood acute lymphoblastic leukemia

Jianqiao Xiao et al. Int J Cancer. .

Abstract

While the cytogenetic and genetic characteristics of childhood acute lymphoblastic leukemias (ALL) are well studied, less clearly understood are the contributing epigenetic mechanisms that influence the leukemia phenotype. Our previous studies and others identified gene mutation (RAS) and DNA methylation (FHIT) to be associated with the most common cytogenetic subgroup of childhood ALL, high hyperdiploidy (having five more chromosomes). We screened DNA methylation profiles, using a genome-wide high-dimension platform of 166 childhood ALLs and 6 normal pre-B cell samples and observed a strong association of DNA methylation status at the PTPRG locus in human samples with levels of PTPRG gene expression as well as with RAS gene mutation status. In the 293 cell line, we found that PTPRG expression induces dephosphorylation of ERK, a downstream RAS target that may be critical for mutant RAS-induced cell growth. In addition, PTPRG expression is upregulated by RAS activation under DNA hypomethylating conditions. An element within the PTPRG promoter is bound by the RAS-responsive transcription factor RREB1, also under hypomethylating conditions. In conclusion, we provide evidence that DNA methylation of the PTPRG gene is a complementary event in oncogenesis induced by RAS mutations. Evidence for additional roles for PTPR family member genes is also suggested. This provides a potential therapeutic target for RAS-related leukemias as well as insight into childhood ALL etiology and pathophysiology.

Keywords: DNA methylation; PTPRG; RAS; childhood acute lymphoblastic leukemia.

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Conflict of interest statement

Conflict of interest statement: None declared

Figures

Figure 1
Figure 1. Association of CpG methylation in FHIT and PTPRG genes with RAS status
Mean differences in DNA methylation beta values between RAS mutant and RAS wild type for each CpG site within FHIT and PTPRG regions is shown in the top panel. The −Log10(Pvalue) is shown for the comparison of DNA methylation status [Student’s t-test of arcsine(beta values)] between RAS mutant positive patients versus negative are shown in the lower panel. The locations of CpG sites in relation to chromosome 3 location (hg19) and gene annotation is noted. Data is derived from Illumina HM450K methylation screening of 166 patients.
Figure 2
Figure 2. Relationship between methylation levels of the two top CpG loci (by P-value, for RAS mutations and PTPRG DNA methylation) and gene expression of PTPRG
Eighty-seven leukemia bone marrow samples were assayed with the Affymetrix ST 1.0 expression microarray as well as Illumina HM450 DNA methylation array. The DNA methylation status of the indicated Illumina loci (x-axis) were graphed against the expression of the gene (y-axis). A regression line was graphed, and the line equation and Pearson R2 is indicated.
Figure 3
Figure 3. RRE sequences in the PTPRG promoter and cloning of different promoter fragments
A. A portion of the PTPRG promoter fragment showing a Putative RRE (bold and underlined) in the PTPRG promoter 290 base pairs upstream of the ATG translation start. B. Cloning of the 5 different promoter fragments into the luciferase reporter plasmid. Fragment A, the full length promoter; Fragment B, the promoter lacking the RRE; Fragment C, the full length promoter with a mutated RRE (see Materials and Methods); Fragment D, short promoter containing only the RRE and its flanking sequences; Fragment E, fragment D minus the RRE and its upstream flanking sequence. C. Promoter activity of the PTPRG promoter fragments. Promoter activity was detected using the luciferase reporter system in 293 cells. Each of the 5 different fragments and the Luc-Min P plasmid was co-transfected into 293 cells with the plasmid vector, pMSCV/neo (Vector), pMSCV/Ras Wt (Wt Ras), and pMSCV/Ras Mut (Mut Ras) respectively. Luciferase activity (y-axis) was measured as described in Materials and Methods.
Figure 3
Figure 3. RRE sequences in the PTPRG promoter and cloning of different promoter fragments
A. A portion of the PTPRG promoter fragment showing a Putative RRE (bold and underlined) in the PTPRG promoter 290 base pairs upstream of the ATG translation start. B. Cloning of the 5 different promoter fragments into the luciferase reporter plasmid. Fragment A, the full length promoter; Fragment B, the promoter lacking the RRE; Fragment C, the full length promoter with a mutated RRE (see Materials and Methods); Fragment D, short promoter containing only the RRE and its flanking sequences; Fragment E, fragment D minus the RRE and its upstream flanking sequence. C. Promoter activity of the PTPRG promoter fragments. Promoter activity was detected using the luciferase reporter system in 293 cells. Each of the 5 different fragments and the Luc-Min P plasmid was co-transfected into 293 cells with the plasmid vector, pMSCV/neo (Vector), pMSCV/Ras Wt (Wt Ras), and pMSCV/Ras Mut (Mut Ras) respectively. Luciferase activity (y-axis) was measured as described in Materials and Methods.
Figure 4
Figure 4. Inhibition of ERK1/2 phosphorylation by PTPRG
Phosphorylated ERK1/2 protein in 293 cells transfected with different plasmids was individually detected using the Bio-Plex Phospho 5-plex panel kit. Phosphorylated proteins were quantitated using the Bio-plex 200 Luminex apparatus and are expressed as fluorescence intensity. Commercial phosphatase-treated Hela cell lysates were used as a negative control. Transfection Conditions in 293 cells are the following: Vector control, the empty vector pMSCVneo; PTPRG, plasmid pMSCV/PTPRG which expresses the human PTPRG gene; RAS Mutant, plasmid pMSCV/RAS Mut which expresses the KRAS mutant gene; RAS Wt, plasmid pMSCV/RAS Wt which expresses the Wt KRAS gene; PTPRG/RAS Mutant, co-transfection of pMSCV/PTPRG and pMSCV/RAS Mut; PTPRG/RAS Wt, co-transfection of pMSCV/PTPRG and pMSCV/RAS Wt. Error bars indicate standard deviations. “*”, P-value was calculated using the Student T test for RAS mutant and PTPRG/RAS Mutant (P < 0.001).
Figure 5
Figure 5. Chromatin Immunoprecipitation of RREB1 in cell line 697
RREB1 was immunoprecipitated from chromatin prepared from 697 cells treated with Azacytidine or buffer (+, − as noted). All bars represent an average of four experiments, and the fold-enrichment determined by quantitative PCR over the input is noted.
Figure 6
Figure 6. PTPRG expression in response to DNA demethylation and KRAS expression
293 cells were treated with 5-Aza-d-C, or DMSO and transfected with either the marker plasmid, pMSCV/neo, or the plasmid expressing the wt KRAS, pMSCV/RAS Wt. mRNA expression was detected by quantitative real-time RT-PCR and protein expression was detected by Western blotting. A. relative expression of PTPRG detected by Quantitative real-time RT-PCR. Error bars indicate standard deviations. PMSCV, 293 cells transfected with the marker plasmid, pMSCV/neo; RAS, 293 cells transfected with pMSCV/RAS Wt, or RAS mut, as indicated; Aza-pMSSCV, 293 cells treated with 5-Aza-d-C and transfected with pMSCV/neo; Aza-Ras Wt; 293 cells treated with 5-Aza-d-C and transfected with pMCV/RAS Wt; Aza-Ras, 293 cells treated with 5-Aza-d-C and transfected with pMCV/RAS Mut. B. Western blot analysis of PTPRG protein, with the same sample order as in A. The PTPRG and actin protein bands are indicated by the arrows, actin was detected together with PTPRG to show equal loading.
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