Mutation signatures implicate aristolochic acid in bladder cancer development

Abstract

Background: Aristolochic acid (AA) is a natural compound found in many plants of the Aristolochia genus, and these plants are widely used in traditional medicines for numerous conditions and for weight loss. Previous work has connected AA-mutagenesis to upper-tract urothelial cell carcinomas and hepatocellular carcinomas. We hypothesize that AA may also contribute to bladder cancer.

Methods: Here, we investigated the involvement of AA-mutagenesis in bladder cancer by sequencing bladder tumor genomes from two patients with known exposure to AA. After detecting strong mutational signatures of AA exposure in these tumors, we exome-sequenced and analyzed an additional 11 bladder tumors and analyzed publicly available somatic mutation data from a further 336 bladder tumors.

Results: The somatic mutations in the bladder tumors from the two patients with known AA exposure showed overwhelming AA signatures. We also detected evidence of AA exposure in 1 out of 11 bladder tumors from Singapore and in 3 out of 99 bladder tumors from China. In addition, 1 out of 194 bladder tumors from North America showed a pattern of mutations that might have resulted from exposure to an unknown mutagen with a heretofore undescribed pattern of A > T mutations. Besides the signature of AA exposure, the bladder tumors also showed the CpG > TpG and activated-APOBEC signatures, which have been previously reported in bladder cancer.

Conclusions: This study demonstrates the utility of inferring mutagenic exposures from somatic mutation spectra. Moreover, AA exposure in bladder cancer appears to be more pervasive in the East, where traditional herbal medicine is more widely used. More broadly, our results suggest that AA exposure is more extensive than previously thought both in terms of populations at risk and in terms of types of cancers involved. This appears to be an important public health issue that should be addressed by further investigation and by primary prevention through regulation and education. In addition to opportunities for primary prevention, knowledge of AA exposure would provide opportunities for secondary prevention in the form of intensified screening of patients with known or suspected AA exposure.

Figure 1

Mutation spectra of AA-bladder cancers and AA-UTUCs. (A,B) Somatic mutation proportions in trinucleotide contexts for bladder cancers (130T and 136T) from Taiwan with known AA exposure (A) and AA-UTUCs (9T and 20T) (B). The height of each bar (the y axis) represents the proportion of somatic mutations that fall into a particular trinucleotide mutational class, adjusted for the frequency of the trinucleotide in the exome (Table S3 in Additional file 1). Along the x axis the mutations are organized first by the nucleotide mutation itself: C > A (turquoise bars), C > G (orange bars), C > T (blue bars), A:T > T:A (red bars), T > C (green bars), T > G (brown bars). For each mutation, the 16 trinucleotide contexts are ordered by the flanking 5′ then 3′ nucleotides. Numbers in parentheses indicate counts of mutations for each single nucleotide substitution (for example, C > A, C > G). Cosine similarities for A:T > T:A mutations were as follows: between 9T and 20T (two UTUCs), 0.989; between 20T and 130T (a UTUC and a bladder cancer), 0.992; between 20T and 136T (a UTUC and a bladder cancer), 0.982. (C,D) Strand bias showing counts of A > T mutations (y axis) on the transcribed (T) and non-transcribed (N) strands. Many fewer somatic A > T mutations were observed on the transcribed than on the non-transcribed strand in both AA-bladder cancers and AA-UTUCs. P-values were computed by one-sided binomial tests compared with the null hypotheses of equal proportions of mutations on the transcribed and non-transcribed strands.

Figure 2

Mutation spectra and strand bias of examples of cancers with and without evidence for AA exposure. (A,B) Bladder cancers (33324197T, B77). (C) Bladder cancer with statistically significant over-representation of A:T > T:A mutations and strand bias but with a pattern of trinucleotide contexts for A:T > T:A mutations notably dissimilar from tumors with known AA exposure (TCGA-K4-A6FZ-01A-11D-A31L-08). (D) HCC (HK41T). (E) For comparison, a bladder cancer (TCGA-FD-A6TE-01A-12D-A339-08) without evidence of AA exposure. Plotting conventions are as for Figure 1. In total, 6 of 349 bladder cancers showed evidence of AA exposure (Figure S1 in Additional file 1).

Figure 3

Inferred mutation signatures and their contributions to somatic mutations in bladder cancers, UTUCs, and HCCs. (A) EMu discerned three mutation signatures. The height of each bar (the y axis) represents the proportion of mutations in the inferred signature that fall into a particular trinucleotide mutation class, adjusted for the frequency of the trinucleotide in the exome. (B) The contributions of each mutation signature (that is, inferred mutational process) to the somatic mutations in each tumor in Table 1. BGI-Bladder: tumors reported in reference 36.