Adaptive response analysis of colorectal cancer cells to low-dose oxaliplatin as a tool to deciphering mechanisms of synergistic drug interaction.

Abstract

3091 Background: Using an in vitro dose matrix approach, we previously showed in multiple colorectal cancer cell lines a striking cytotoxic synergism between oxaliplatin at very low concentrations and the ATR inhibitor VE-822. We confirmed this finding in vivo, and, surprisingly, in this setting the oxaliplatin-induced cell addiction to VE-822 persists over several days after oxaliplatin elimination. We tried to elucidate the molecular mechanism of the latter phenomenon. Methods: We evaluated by RNAseq the gene expression changes induced in vitro by low-dose oxaliplatin in the colorectal cancer cell line HCT-116 after 24 and 48 hours of treatment. In order to untangle the functional significance of the adaptive response to oxaliplatin, we performed on the RNAseq data an extensive gene set enrichment analysis (GSEA) using gene set from all Molecular Signature Database v7.4 collections with the exception of C7. For ontology-based gene set collections, we clusterized the enriched gene sets using the semantic similarity methodology in order to increase the readability of global functional response. Results: Extensive GSEA showed that after 24 hours of oxaliplatin treatment cancer cells upregulate several gene sets involved in aspecific responses to cellular stress or to various type of extracellular stimulations, including other organisms, oxygen-containing compounds, abiotic stimuli and hypoxia. In addition, several gene sets involved in proteolysis and autophagy are upregulated, suggesting a rewiring of cell machinery. After 48 hours of oxaliplatin treatment, we observed the activation of ribosome function, mitochondrial assembly and synthesis of aminoacids and ribonucleosides. Finally, a widespread negative enrichment of gene sets involved in DNA repair-related was detected both at 24 and at 48 hours, with a far greater negative enrichment at 48 hours, which suggest a commitment of cancer cell to a major limitation of DNA repairing capability lasting several days following a DNA damaging insult. Analysis of leading edge genes from the DNA repair gene sets showed a profound repression both at 24 and 48 hours of the transcripts of BRCA1, BRCA2, ATM, CHK1, WEE1, BARD1, BRIP1, NEHJ1, RAD51, XRCC2, CLSPN, GEN1, DNA2, EXO1, TOPBP1, POLE, RMI1. Interestingly, ATR mRNA was minimally repressed both at 24 and at 48 hours, which could explain the long-standing in vivo dependence of cancer cell to ATR after a brief oxaliplatin exposure. Conclusions: Extensive GSEA was able to elucidate the molecular mechanism underlying synergistic interaction between oxaliplatin and VE-822. The impact of profiling cancer cell adaptive responses by extensive GSEA should be further evaluated in the setting of rational development of drug combinations.