Glutathione pathway genetic polymorphisms and lung cancer survival after platinum-based chemotherapy

Abstract

Background: Lung cancer is commonly treated with platinum compounds. The "glutathione pathway" participates in the metabolism of platinum compounds. We set out to test the hypotheses that single nucleotide polymorphisms (SNPs) or copy number polymorphisms for genes within the glutathione pathway might influence survival in lung cancer patients treated with these drugs.

Methods: Germline DNA samples from 973 lung cancer patients were genotyped for 290 glutathione pathway SNPs. GSTT1 copy number was also assayed. We determined the association of these polymorphisms with survival for lung cancer patients, followed by functional genomic validation.

Results: We observed suggestive associations between survival and GSTT1 copy number (P = 0.017), and GSTA5, GSTM4, and ABCC4 SNPs, adjusted for covariates (P = 0.018, 0.002, and 0.002, respectively) or not (P = 0.005, 0.011, and 0.002). One hundred lymphoblastoid cell lines were then treated with cisplatin, and IC(50) values were significantly associated with the GSTM4 SNP (P = 0.019). Furthermore, GSTM4, GSTT1, and ABCC4 overexpression significantly decreased cisplatin sensitivity in lung cancer and HEK293T cell lines.

Conclusions: These results suggest that GSTM4 polymorphisms are biomarkers for the prediction of cisplatin response. ABCC4 polymorphisms, as well as GSTT1 copy number, may also help to predict cisplatin response, but further validation is required. These results represent a step toward the individualized chemotherapy of lung cancer.

Figure 1. The “Glutathione Pathway”

The figure shows a schematic representation of the “glutathione pathway.” Glutathione (GSH) is synthesized from glutamate, cysteine, and glycine by γ-glutamylcysteine synthetase and glutathione synthetase. Glutathione redox state is regulated, in part, by glutathione peroxidases, forming oxidized glutathione (GSSG), and by a reaction catalyzed by glutathione reductase. Glutathione is conjugated to substrates both through the action of the glutathione S-transferases and through non-enzymatic reactions. Glutathione conjugates can be excreted from the cells by members of the ABCC transporter family.

Figure 2. Lung Cancer Patient Survival Curves

Kaplan-Meier survival curves for lung cancer patients are shown for the GSTT1 CNP and for SNPs in GSTM4, GSTA5, and ABCC4. The x-axis on each graph indicates years since lung cancer diagnosis, while the y-axis indicates the percentage of patients surviving. The p-values unadjusted for covariates (p) and after adjustment for covariates (p adj) are indicated for each variant. For GSTT1, 0, 1, and 2 indicate the number of copies of GSTT1. For GSTM4, GSTA5, and ABCC4, CC indicates two copies of the common allele, CR refers to heterozygotes, and RR indicates patients having two copies of the rare allele.

Figure 3. Association of SNPs and Cisplatin IC₅₀ Values in Lymphoblastoid Cells

The plots show log cisplatin log IC₅₀ values by copy number or SNP genotype in 100 lymphoblastoid cell lines from Caucasian-American subjects. Each dot represents an individual sample. The green line across the center of each diamond represents the group mean, while the vertical span of each diamond represents the 95% confidence interval for each group. Mean values and standard deviation lines are shown as blue lines. The p-value for each ANOVA is listed at the bottom left of the plot.

Figure 4. Effect Overexpression on Cisplatin IC₅₀

Relative cisplatin IC₅₀ values, after overexpression of the gene indicated, are shown in the cell lines indicated. The IC₅₀ value after transfection with the empty vector control for each experiment was defined as 100%, and IC₅₀ values are reported as a percentage of that for the control. All experiments were performed at least six times. * indicates p<0.05

Figure 5. GSTM4 and GSTA5 Haplotype Structures

The SNPs that we identified in both GSTM4 and GSTA5 are linked to other SNPs in these genes. The haplotype structures of the two genes were obtained from the HapMap. SNP pairs with high D′ values are shown in red, while SNP pairs with lower D′ values are shown in lighter red, grey, or white. The SNPs which we identified are “boxed” in red, while a SNP in GSTM4 that we did not identify but which is discussed in the text is indicated with a “broken” red box.