Modeling Cancer Driver-Like Events Using In Vitro Carcinogen Exposure And Immortalization Assays
Hana HUSKOVA, International Agency for Research on Cancer, France
ARDIN M. 1
, VARGOVA K. 2
, PANDEY M. 1
, OLIVIER M. 1
, HOLLSTEIN M. 1,3
, STOPKA T. 2
, HERCEG Z. 4
, ZAVADIL J. 1
, KORENJAK M. 1
1 Molecular Mechanisms and Biomarkers Group, International Agency for Research on Cancer, Lyon, France
2 Institute of Pathological Physiology, First Faculty of Medicine, Charles University in Prague, Prague, Czech Republic
3 Faculty of Medicine and Health, University of Leeds, Leeds, United Kingdom
4 Epigenetics Group, International Agency for Research on Cancer, Lyon, France
Cancer genomes harbor mutational spectra that document exposures to external factors and endogenous events underlying the tumor development. Information on candidate cancer driver alterations is accessible from public compendia of somatic mutations, yet much of this knowledge remains hypothetical and of little mechanistic insight. Simple, robust and rapid systems are thus needed for well-controlled experimental investigations of functional impact of carcinogenic exposures on the genome and on cancer cell growth.
We devised barrier bypass-clonal expansion (BBCE) assays based on cultured primary mouse embryonic fibroblasts which upon carcinogen exposure circumvent senescence, a selective pressure barrier, and immortalize. Exomes of the resulting clones are sequenced and analyzed to decipher both the mutational signatures and the putative functional driver events selected and enriched for during the outgrowth phase.
Using the BBCE assays, we tested the global mutagenic effects of several known human carcinogens. We obtained 25 independently arising cell lines, altogether harboring 16,061 acquired mutations of which 7,615 were non-synonymous. As in human cancers, the alterations affected pathways regulating DNA damage response, DNA repair, cell cycle, cell death, transcription and chromatin structure, and multiple developmental signaling pathways. Forty-eight genes listed in the COSMIC Cancer Gene Census were recurrently mutated across the BBCE cell line panel, including well-established oncogenes (HRAS, KRAS, ABL1, EGFR) and tumor suppressors (APC, ATM, BRCA2, PTCH1, TP53). A number of epigenetic and chromatin regulators also acquired recurrent mutations. Data will be presented describing a customized pipeline to prioritize candidate driver events, followed by systematic genome editing or pharmacological manipulation of select genes and assessment of the resulting phenotypic and molecular traits.
In summary, our BBCE approach may yield new mechanistic insights into driver-like events underlying cancer development.
International Agency for Research on Cancer; ITMO CANCER – INSERM Plan Cancer 2015.