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. 2009 Nov 9;4(11):e7765.
doi: 10.1371/journal.pone.0007765.

Gemcitabine and arabinosylcytosin pharmacogenomics: genome-wide association and drug response biomarkers

Liang Li  1 , Brooke L Fridley, Krishna Kalari, Gregory Jenkins, Anthony Batzler, Richard M Weinshilboum, Liewei Wang
Affiliations

Gemcitabine and arabinosylcytosin pharmacogenomics: genome-wide association and drug response biomarkers

Liang Li et al. PLoS One. .

Abstract

Cancer patients show large individual variation in their response to chemotherapeutic agents. Gemcitabine (dFdC) and AraC, two cytidine analogues, have shown significant activity against a variety of tumors. We previously used expression data from a lymphoblastoid cell line-based model system to identify genes that might be important for the two drug cytotoxicity. In the present study, we used that same model system to perform a genome-wide association (GWA) study to test the hypothesis that common genetic variation might influence both gene expression and response to the two drugs. Specifically, genome-wide single nucleotide polymorphisms (SNPs) and mRNA expression data were obtained using the Illumina 550K(R) HumanHap550 SNP Chip and Affymetrix U133 Plus 2.0 GeneChip, respectively, for 174 ethnically-defined "Human Variation Panel" lymphoblastoid cell lines. Gemcitabine and AraC cytotoxicity assays were performed to obtain IC(50) values for the cell lines. We then performed GWA studies with SNPs, gene expression and IC(50) of these two drugs. This approach identified SNPs that were associated with gemcitabine or AraC IC(50) values and with the expression regulation for 29 genes or 30 genes, respectively. One SNP in IQGAP2 (rs3797418) was significantly associated with variation in both the expression of multiple genes and gemcitabine and AraC IC(50). A second SNP in TGM3 (rs6082527) was also significantly associated with multiple gene expression and gemcitabine IC50. To confirm the association results, we performed siRNA knock down of selected genes with expression that was associated with rs3797418 and rs6082527 in tumor cell and the knock down altered gemcitabine or AraC sensitivity, confirming our association study results. These results suggest that the application of GWA approaches using cell-based model systems, when combined with complementary functional validation, can provide insights into mechanisms responsible for variation in cytidine analogue response.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Genome-wide SNP association with drug IC50.
(A). Genome-wide SNP-gemcitabine IC50 association data. (B). Genome-wide SNP-AraC IC50 association data. Genome-wide SNPs were obtained for all 171 cell lines using Illumina 550K Beadchips. Genome-wide SNP association analysis was then performed with gemcitabine IC50 as the drug-response phenotype (n = 171). The y-axis is the −log10 (p-value) for the SNP. SNPs are plotted on the x-axis on the basis of their chromosomal locations. The “cut-off” p-value of 0.001 is highlighted with a red line.
Figure 2
Figure 2. Top candidate SNPs that overlapped between gemcitabine and AraC.
49 SNPs were common (p<0.001) among 442 unique SNPs associated with gemcitabine and 504 associated with AraC.
Figure 3
Figure 3. Strategy applied in these studies.
A genome-wide SNP association study was performed with a drug-related phenotype, gemcitabine or AraC cytotoxicity (IC50). SNPs associated with cytotoxicity (p<10−3) were used to perform an association study with 54,000 expression probesets to identify SNPs that were associated with both gene expression (p<10−6) and with gemcitabine or AraC IC50. Finally, an association study was performed with gene expression and gemcitabine or AraC IC50 to identify SNPs that might be associated with drug IC50 values through an influence on gene expression.
Figure 4
Figure 4. Association among SNPs in IQGAP2 and TGM3 with expression and gemcitabine or AraC cytotoxicity.
(A) SNP rs3797418 (A/C) association with VAV3 expression, gemcitabine IC50 values and VAV3 expression association with gemcitabine IC50 in individual ethnic groups and all of the cell lines. (B) SNP rs6082527 (A/G) association with GPM6A gene expression, gemcitabine IC50 values and GPM6A gene expression association with gemcitabine IC50 values. (C). SNP rs3797418 (A/C) association with VAV3 expression, AraC IC50 values and VAV3 expression association with AraC IC50 in individual ethnic groups and all of the cell lines. Each dot represents one sample. Genotypes for each SNP are plotted against gene expression levels (upper panels) as well as gemcitabine or AraC IC50 values (middle panels). In the lower panel, correlations were determined between gene expression levels and gemcitabine or AraC IC50 values.
Figure 5
Figure 5. Functional characterization of candidate genes with siRNA knock down of VAV3 (A) and GPM6A (B).
Knock down of VAV3 and GPM6A expression in human SU86 and Hup-T3 pancreatic cancer cell lines as well as lymphoblastoid cell lines showed increased resistance to gemcitabine and AraC after siRNA knock down as determined by MTS assays. Error bars represent SEM values for 3 independent experiments. Quantitative RT-PCR was performed to assess VAV3 and GPM6A gene expression levels after knock down with specific siRNAs. Results are expressed as % of control. Error bars represent SEM values for 3 independent experiments. *  = p<0.05.
Figure 6
Figure 6. Linkage disequilibrium within ∼300 Kb surrounding the rs3797418 and rs6082527 SNPs.
Red indicates combinations where D′ = 1 and linkage of disequilibrium (LOD) ≥2; light red, combinations where D′<1 and LOD ≥2; red squares, combinations where D′ = 1 and LOD <2; white squares, combinations with D′<1 and LOD <2. SNPs are arranged in order from 5′ to 3′ in each gene, as shown in the gene structure above each plot.
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