Identification of C/EBPδ-Modifying Compounds as Potential Anticancer Agents Using a High-Throughput Drug Screen

CCAAT/enhancer-binding protein delta, commonly referred to as C/EBPδ, has been implicated in diverse roles in cancer progression. Studies have indicated that C/EBPδ can foster tumor growth, contribute to the development of drug resistance, and promote the formation of metastases in certain types of cancer. Conversely, our previous work has demonstrated that the re-expression of C/EBPδ can actually limit the characteristics associated with tumor progression in pancreatic ductal adenocarcinoma, or PDAC. Consequently, the pharmacological manipulation of C/EBPδ activity, either through activation or inhibition, may hold clinical significance. The ability to modulate C/EBPδ is also highly desirable for preclinical investigations aimed at understanding its function in various cancer contexts. However, it is noteworthy that only a limited number of molecules capable of modifying C/EBPδ activity have been identified to date.

In this study, we present the results of a high-throughput screening of chemical compounds, utilizing a newly developed enhanced green fluorescent protein, or eGFP, reporter system. The objective of this screen was to identify additional compounds that could either enhance or diminish the transcriptional activity of C/EBPδ. Through the screening of 1402 small molecule inhibitors, we successfully identified a total of 22 potent inducers and 18 inhibitors of C/EBPδ-mediated eGFP fluorescence. To gain further insight into the mechanisms of action of these compounds, we performed pathway enrichment analysis. This analysis revealed a general trend: the inhibition of the cell cycle tends to induce an increase in C/EBPδ activity, while compounds that target the PI3K/Akt/mTOR signaling pathway tend to reduce C/EBPδ activity.

We further validated the potential importance of cell cycle-mediated regulation of C/EBPδ by demonstrating that four of the most effective C/EBPδ activators identified in our screen—specifically R547, PHA793387, AZD5438, and AT7519, all of which are multi-cyclin-dependent kinase, or CDK, inhibitors—were able to limit the clonal expansion of PDAC cells. Beyond providing a valuable collection of compounds that can modulate C/EBPδ for use in preclinical studies, this research contributes to a broader understanding of the molecular mechanisms that regulate C/EBPδ, both in general and specifically within the context of PDAC.