Dynamics and evolution of β-catenin-dependent Wnt signaling revealed through massively parallel clonogenic screening

• Published in: Integrative biology (Cambridge) 2014-07, Vol.6 (7), p.673-684
• Main Authors: Shah, Pavak K, Walker, Matthew P, Sims, Christopher E, Major, Michael B, Allbritton, Nancy L
• Format: Article
• Language: English
• Subjects: beta catenin; beta Catenin - metabolism; carcinogenesis; Cell Line, Tumor; cell lines; Clone Cells; embryogenesis; Humans; Image Processing, Computer-Assisted; Kinetics; Luminescent Proteins - chemistry; Luminescent Proteins - metabolism; Microscopy, Fluorescence - methods; phenotype; phenotypic variation; Red Fluorescent Protein; screening; signal transduction; therapeutics; tissue repair; transcription (genetics); Wnt Proteins - metabolism; Wnt Signaling Pathway
• ISSN: 1757-9694; 1757-9708; 1757-9708
• DOI: 10.1039/c4ib00050a

Wnt/β-catenin signaling is of significant interest due to the roles it plays in regulating development, tissue regeneration and disease. Transcriptional reporters have been widely employed to study Wnt/β-catenin signal transduction in live cells and whole organisms and have been applied to understanding embryonic development, exploring oncogenesis and developing therapeutics. Polyclonal heterogeneity in reporter cell lines has historically been seen as a challenge to be overcome in the development of novel cell lines and reporter-based assays, and monoclonal reporter cell lines are commonly employed to reduce this variability. A375 cell lines infected with a reporter for Wnt/β-catenin signaling were screened over short (25) generational timescales. To characterize phenotypic divergence over these time-scales, a microfabricated cell array-based screen was developed enabling characterization of 1119 clonal colonies in parallel. This screen revealed phenotypic divergence after 25 generations. Not only were reporter dynamics observed to diverge widely, but monoclonal cell lines were observed with seemingly opposite signaling phenotypes. Additionally, these observations revealed a generational-dependent trend in Wnt signaling in A375 cells that provides insight into the pathway's mechanisms of positive feedback and self-inhibition.