PNU-74654 is primarily used in biomedical research as a selective antagonist of the Wnt signaling pathway, particularly targeting the T-cell factor (TCF)/β-catenin transcription complex. It is employed to study pathways involved in cell proliferation, differentiation, and tumorigenesis. Due to its ability to inhibit β-catenin-mediated transcription, it has been investigated in cancer research, especially in models of colorectal and other Wnt-driven cancers, to assess suppression of tumor growth and oncogenic signaling. Additionally, PNU-74654 serves as a tool compound for exploring stem cell regulation and developmental biology where Wnt signaling plays a critical role. Its application remains confined to preclinical research, aiding in the validation of Wnt pathway inhibitors as potential therapeutic agents.