Innovative In-Vitro Systems For Genome-Wide Modeling Of Mutational Spectra Of Human Cancer Risk Agents

Maria ZHIVAGUI, International Agency for Research on Cancer, France
ARDIN M. 1 , CAHAIS V. 2 , GUYTON K. 3 , BARUPAL D. 4 , MCKAY J. 5 , BYRNES G. 6 , STRAIF K. 3 , HERCEG Z. 2 , OLIVIER M. 1 , HOLLSTEIN M. 1,7 , KORENJAK M. 1 , ZAVADIL J. 1

1 Molecular Mechanisms and Biomarkers Group, International Agency for Research on Cancer, Lyon, France
2 Epigenetics Group, International Agency for Research on Cancer, Lyon, France
3 IARC Monographs Section, International Agency for Research on Cancer, Lyon, France
4 Biomarkers Group, International Agency for Research on Cancer, Lyon, France
5 Genetic Susceptibility Group, International Agency for Research on Cancer, Lyon, France
6 Biostatistics Group, International Agency for Research on Cancer, Lyon, France
7 Faculty of Medicine and Health, University of Leeds, Leeds, United Kingdom

Purpose
Covalent interaction of carcinogenic compounds with DNA (adduct formation) and other DNA-altering factors can produce characteristic mutation patterns. Thus, identification of mutational signatures of chemical compounds in human tumors provides insights on cancer etiology. Given the complexity of human exposures, it remains a challenging task to extract individual mutational signatures attributable to particular agents. To address this question, we developed robust in-vitro models allowing for individual exposures to be tested in a controlled experimental setting.
 
Methods
Here we present two experimental cell systems that take advantage of selective barrier bypass, clonal expansion and immortalization upon carcinogen exposure: primary mouse embryonic fibroblasts (MEF) and HepaRG human liver cell line. MEFs have been successfully used in the past to reveal mutational effects of known carcinogens on the TP53 sequence. In our models, we investigate exome-wide mutational profiles. A list of tested chemical agents has been defined in collaboration with the IARC Monographs Section, and by extensive semi-automated data mining to prioritize compounds with evidence for mutagenicity, DNA adducts and epidemiological studies.
 
Results
Primary MEF cultures were exposed to the potential carcinogens acrylamide (Group 2A) and ochratoxin A (Group 2B), whereas HepaRG cells were treated with aristolochic acid I (Group 1). Upon treatment, MEFs underwent senescence followed by senescence bypass leading to immortalized clones which were subject to sequencing and mutational signature analysis. Meta-analysis with in-vivo mutational spectra in rodent tumor collection of the US National Toxicology Program and with public human cancer genomics data will be performed to ensure high-confidence identification and cross-validation of the determined mutational signatures.
 
Conclusions
This simple and powerful experimental strategy can facilitate the interpretation of mutation fingerprints identified in human tumors, elucidate cancer etiology, and ultimately support IARC’s carcinogen classification by providing mechanistic evidence.
 
Funding sources
IARC; ITMO CANCER – INSERM Plan Cancer 2015