Procarcinogen Activation and Mutational Signatures Model the Initiation of Carcinogenesis in Human Urothelial Tissues In Vitro

Abstract

Disparity between genome-wide mutations in bladder and other cancers where smoking is a risk factor raises questions about carcinogenesis in different epithelia. To develop an experimental model of bladder carcinogenesis, we clonally expanded in vitro differentiated normal human urothelial (NHU) cells following exposure to an exemplar procarcinogen and used whole-genome DNA sequencing to derive mutational signatures. Benzo[a]pyrene (BaP) was activated by endogenous cytochrome P450 (cytochrome P450 family 1 subfamily A member 1 [CYP1A1]) to create genomically modified NHU cells. Comparison with the Catalogue of Somatic Mutations in Cancer (COSMIC) showed that mutations induced by BaP in NHU cells were similar to smoking-associated signatures in bladder and other cancers, including single- and doublet-base substitution signatures characterised by C > A transversions (COSMIC_SBS4 and COSMIC_DBS2, respectively), and an insertion/deletion signature of C deletions in homopolymer regions (COSMIC ID3). Our study provides the first direct evidence that BaP is activated locally in the urothelium, initiating the well-described smoking-associated mutational signatures. An absence of other common bladder cancer (BLCA)-associated genomic signatures points strongly to other primary causes of BLCA, which the new experimental approach described here is well placed to investigate. Mutational signatures ignore whether genes are affected, but tissue-specific drivers (KMT2D, KMT2C, and CDKN1A) were significantly overmutated in this model, providing insight on the emergent selection pressures. PATIENT SUMMARY: In a carefully controlled laboratory setting, we exposed normal human urothelial tissues to a procarcinogen (benzo[a]pyrene) found in cigarette smoke. We show that the urothelial tissues activated the carcinogen and led to mutations forming across the genome in a characteristic pattern. This particular "mutational signature" is found in bladder tumours and other smoking-induced cancers (eg, lung); however, our study highlights that there are other unknown mutational processes in bladder cancer that is not the direct result of smoke carcinogens, and this will require further investigation.

Keywords: APOBEC; BaP; Benzo[a]pyrene; Bladder; Bladder cancer; CYP1A1; Cytochrome P450; DNA mutation; Urothelium; Whole-genome DNA sequencing.

Fig. 1

Schematic of the experimental approach wherein functionally differentiated NHU cell sheets (generating a transepithelial electrical resistance of >2000 Ω.cm²; Supplementary Fig. 1) were exposed to benzo[a]pyrene (BaP) for 7 d before single-cell clones were expanded as colonies in feeder-supported cultures. Expanded clones were analysed by wgDNAseq and mutational signatures were derived. Graph shows mean percentage of mutations within each class of single-base substitution (SBS). Error bars = standard deviation (n = 3, control, and n = 4, BaP-exposed independent clones). The control SBS percentages (ANOVA p = 0.0002) showed significant enrichment of C > T and T > C transitions, which are more common than transversions in virtually all DNA sequences. In the control SBS, C > A transversions were not significantly enriched. The BaP SBS percentages showed significant enrichment of C > A transversions. Multiple comparisons were performed using Tukey’s post hoc test. ANOVA = analysis of variance; NHU = normal human urothelial; wgDNAseq = whole-genome DNA sequencing. * C > T and T > C changes were significantly enriched (p < 0.01) in comparison with C > G, T > A, and T > G. *** p < 0.0001 for C > A changes in comparison with all other types.

Fig. 2

Mutational signatures derived from BaP-exposed functionally differentiated NHU cells. (A) Single-base substitution (SBS) signature of 96 subtypes based around six substitution classes (referred to by the pyrimidine of the mutated Watson-Crick base pair) and framed by their 3′ and 5′ flanking nucleotides. The SBS signature shows an enrichment of diverse C > A transversions. (B) Doublet-base substitution (DBS) signature of 78 strand-agnostic mutation types show an enrichment mainly of CC > AA. TG > AT and TG > CA substitutions were additionally observed and were previously reported following BPDE exposure of iPS cells . (C) Insertions/deletion (ID) signature reveals BaP-caused single C/G deletions most commonly in homopolymer runs of two to four cytosines/guanines (n = 4 independent BaP-exposed clones normalised to n = 3 independent control clones; bars indicate the mean and error bars denote the standard deviation). Data in this figure are expressed as percentages; for counts and normalisation data, see Supplementary Figures 2–4. BaP = benzo[a]pyrene; BPDE = benzo(a)pyrene diol epoxide; iPS = induced pluripotent stem; NHU = normal human urothelial.