Abstract 3767: Transcriptional and proteomic profiling of drug-tolerant cancer cell subpopulations using colonies that emerge in the presence of anticancer agents

Abstract

Abstract Background: Cancer relapse after curative treatment is thought to originate from drug-tolerant cancer cell subpopulations. To isolate and characterize the drug-tolerant cell subpopulations, we collected individual colonies that emerged in the presence of anticancer drugs and performed proteomic profiling using reverse-phase protein arrays (RPPAs). Materials and Methods: Cancer cells (HCT116, HeLa, HT29, MCF7, and MKN45) were sparsely disseminated (10-100 cells/cm2) into the presence of anticancer drugs (cisplatin, docetaxel, gefitinib, and sorafenib) of six serially diluted concentrations on the basis of 50% colony inhibitory concentration (CI50) for each drug. A total of 2,400 individual colony lysates (20 colony lysates per drug concentration) were printed on glass slides using a dedicated microarrayer followed by immunochemical detection of 44 functionally-diverse proteins. A transcriptional profiling followed by gene ontology (GO) analysis was also performed. Results: CI50 of cisplatin and docetaxel were >10 times lower than the 50% growth inhibitory concentration (GI50) in all cell lines. This indicates that colony formation is functionally different from exponential cell growth, and drug-tolerant colony initiation can be suppressed by traditional anticancer drugs with very low doses. Colony lysate arrays revealed that the colonies expressing stemness, pluripotent, and mesenchymal markers tended to show low levels of epithelial proteins. However, individual protein expression levels were not well associated with drug concentrations, suggesting that these protein phenotypes of colonies were not necessarily induced by anticancer drugs. Instead, they may have emerged spontaneously. To identify molecular fractions important to drug-tolerant phenotypes, we screened compounds inhibiting chromatin formation, transcription, and protein biosynthesis. A transcriptional inhibitor eliminated drug-tolerant colonies within a short (four-hour) temporal treatment, suggesting that putative drug-tolerance inducing factors were controlled at the transcriptional level. Analysis of cDNA microarray using drug-free and cisplatin-resistant colonies followed by GO analysis revealed that most expressed genes in cisplatin-resistant colonies were specifically associated with embryonic development. Conclusion: These findings suggest that the regulatory system at the protein level is unlikely to be directly associated with drug-tolerant colony formation. Instead, transcriptional regulation appears to play a crucial role in forming drug-tolerant colonies. Citation Format: Kohei Kume, Satoshi Nishizuka, Miyuki Ikeda, Sawako Miura, Fumitaka Endo, Katagiri Hirokatsu, Kaoru Ishida, Kei Sato, Chihaya Maesawa, Go Wakabayashi. Transcriptional and proteomic profiling of drug-tolerant cancer cell subpopulations using colonies that emerge in the presence of anticancer agents. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3767. doi:10.1158/1538-7445.AM2014-3767