MinireviewExperimental investigations of carcinogen-induced mutation spectra: Innovation, challenges and future directions
Introduction
The previous decade has witnessed a broad implementation of massively parallel sequencing technologies into biomedical sciences, and associated in-depth investigations of human cancer genomes have generated vast, publicly available data collections [1]. This work has substantially expanded our knowledge about the accumulation and functional impact of somatic mutations in tumor genomes. A special type of computational analysis of large sets of mutated genomes has focused on the extraction of mutational signatures, i.e. characteristic somatic DNA mutation patterns attributable to particular mutagenic sources, reflecting their contributions to cancer development, and often shaped further by the activity of the DNA repair machinery [2,3]. The current catalog of mutational signatures reflects an expansion from the initially considered patterns of single-base substitutions (SBS) (from 30 to ∼50), and contains also 11 signatures based on doublet-base substitutions (DBS) and 17 based on small insertions-deletions (indels) [2]. The original as well as the updated patterns can be accessed on the COSMIC web site (https://cancer.sanger.ac.uk/cosmic/signatures). Recently genome-scale sequencing studies also revealed extensive mutagenesis occurring in cancer-associated genes and mutational signatures in various normal tissues in the absence of overt neoplastic disease as well as in the normal cells of cancer patients [[4], [5], [6], [7], [8], [9], [10], [11]]. These studies present a paradigm shift that poses new challenges and opportunities with regards to the precise identification of the causal events underlying cancer development.
The power of mutational signature analysis lies in its ability to point to the effects of specific etiologic agents and to reveal exposure history imprinted into the exposed cell genome. These mutagenic factors can be of endogenous nature, such as DNA repair deficiency or deregulation of endogenous mutagenic enzymes, or of exogenous nature, originating from environmental, life-style or therapeutic exposures to mutagenic agents, with both types of activities often acting in cooperation to leave mixed mutation fingerprints. A number of the known mutational signatures have already been associated with particular etiologies, although a large proportion of signatures remain unexplained. Furthermore, as highly similar mutation spectra can arise in a given tumor from various convergent mutagenic processes, or the results of certain background mutagenic processes do not yield a robust signal, it remains a challenge to disentangle the resulting signatures by the currently applied de-convolution procedures such as non-negative matrix factorization (NMF), the most commonly used method of unscrambling mutational signatures from the somatic mutation mixtures of complex origins [12,13]. It is plausible that a number of signatures may have eluded discovery at the level of the pan-cancer genome signature extraction analyses conducted to-date [2], while novel mutational signatures are being uncovered within more focused studies including also experimental modeling [6,11,14–16].
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Experimental approaches to mutational signature modeling
Experimental models of chemical exposure and genetic perturbation provide highly controlled approaches allowing to systematically address some of the challenges associated with the discovery of mutational signatures linked to specific causal factors or agents. In the last few years these invaluable models have been combined with massively-parallel sequencing at the exome and genome-scale levels to generate large amounts of experimental data using various in vitro and in vivo exposure systems,
Clonal expansion systems based on primary cells and cell lines
Current experimental exposure mutagenesis models have been developed based on the combination of clonally expanded cell lines coupled with genome sequencing technologies, allowing modeling of exposure-specific mutational signatures. Primary mouse embryonic fibroblasts harboring a knocked-in human TP53 gene (Hupki MEFs), thanks to the general inherent propensity of MEF cells to bypass a biological barrier (senescence) and immortalize in a clonal manner, have emerged as a robust and suitable in
Induced pluripotent stem cells
A recent study [35] based on human induced pluripotent stem cells (iPSC) and their intrinsic clonal expansion stands out for addressing for the first time the mutagenic effects of a larger number (∼80) of environmental and therapeutic exposures by whole-genome sequencing. About half of the exposure experiments yielded SBS signatures, with some corroborating the previous cell line-based studies (platinum complexes [15], aflatoxin [28] or aristolochic acid [36]), while other exposures generated
3-D exposure cell systems
Numerous 3-D cell culture and organoid-based models have been developed over the past two decades. These developments are driven by the promise that such 3-D models resemble and/or mimic real tissue arrangements, and they may thus significantly accelerate translational research in cancer biology, tissue engineering, and regenerative medicine. In most cases, however, the 3-D cell culture models represent solely the epithelial cell layer and lack the native microenvironment and the usual
Retrospective use of rodent bioassay material
In contrast to the cell-based model systems discussed above, in vivo animal bioassays permit studies of mutation spectra at an organismal level. However, since such studies are based on exposure-mediated tumor induction and cancer burden in animals, they have been linked to ethical concerns. Apart from de novo cancer-formation rodent models used to derive mutational signatures of real-life or research carcinogens and their matches with human cancer signature data [28,37,47,48], additional
Non-mammalian exposure systems
The analyses of genetic and environmental signatures using simpler model organisms including bacteria, yeast, Drosophila, and the nematode C. elegans were thoroughly summarized in the beginnings of the experimental signature modeling research field [54,55]. A recent analysis of close to 3000 genomes of more than 50 C. elegans strains, each deficient for a specific DNA repair gene, compared the derived mutation profiles to those of wild-type counterparts in the context of a dozen of distinct
Prioritization of cancer risk agents for mutagenicity testing
A key aspect for the characterization of mutation spectra associated with exogenous exposures using any of the discussed experimental models is the selection of the tested compounds. The agents prioritized for systematic mutational signature analysis should be selected based on evidence for DNA damage induction and mutagenicity, following the recently established framework for key characteristics of carcinogens [63,64]. Ideally they would consist of chemicals introducing different types of
Future directions
At the current stage of development of the experimental signature modeling field, choosing a reliable, uniform, one-size-fits-all approach remains a challenge. However, the field of experimental signature modeling has solidified in the last several years, providing extensive insights. Summary frameworks are being put in place, recommending experimental designs, protocols and analytical procedures, and discussing advantages and limitations associated with various methods and systems [20,27,34].
Role of funding sources
Experimental studies discussed here and co-authored by M.K. and J.Z. were possible thanks to the funding support from ITMO CANCER INCa – INSERM, Plan Cancer 2015 Grant No. ENV201507 to J.Z.
Disclosure
Views expressed in this review are those of the authors and not necessarily reflect the views, the decisions, policy of the International Agency for Research on Cancer/World Health Organization.
Acknowledgments
The authors gratefully acknowledge the relevant contributions of all team-mates and collaborators and colleagues in the field of experimental modeling of mutational signatures.
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Present address: Department of Pharmacology and Toxicology, American University of Beirut, Beirut 1107 2020, Lebanon.