The Carcinogenome Project: In-vitro Gene Expression Profiling of Chemical Perturbations to Predict Long-Term Carcinogenicity

Abstract

Background: Most chemicals in commerce have not been evaluated for their carcinogenic potential. The current de-facto gold-standard approach to carcinogen testing adopts the two-year rodent bioassay, a time consuming and costly procedure. Alternative approaches, such as high-throughput in-vitro assays, show promise in addressing the limitations in carcinogen screening.\n\nObjectives: We developed a screening process for predicting chemical carcinogenicity and genotoxicity and characterizing modes of actions (MoAs) using in-vitro gene expression assays.\n\nMethods: We generated a large toxicogenomics resource comprising ~6,000 expression profiles corresponding to 330 chemicals profiled in HepG2 cells at multiple doses and in replicates. Predictive models of carcinogenicity were built using a Random Forest classifier. Differential pathway enrichment analysis was performed to identify pathways associated with carcinogen exposure. Signatures of carcinogenicity and genotoxicity were compared with external data sources including Drugmatrix and the Connectivity Map.\n\nResults: Among profiles with sufficient bioactivity, our classifiers achieved 72.2% AUC for predicting carcinogenicity and 82.3% AUC for predicting genotoxicity. Our analysis showed that chemical bioactivity, as measured by the strength and reproducibility of the transcriptional response, is not significantly associated with long-term carcinogenicity, as evidenced by the many carcinogenic chemicals that did not elicit substantial changes in gene expression at doses up to 40 M. However, sufficiently high transcriptional bioactivity is necessary for a chemical to be used for prediction of carcinogenicity. Pathway enrichment analysis revealed several pathways consistent with literature review of pathways that drive cancer, including DNA damage and DNA repair. These data are available for download via https://clue.io/CRCGN_ABC, and a web portal for interactive query and visualization of the data and results is accessible at https://carcinogenome.org.\n\nConclusions: We demonstrated a short-term in-vitro screening approach using gene expression profiling to predict long-term carcinogenicity and infer MoAs of chemical perturbations.