Abstract Background: The 14-3-3 protein family comprises seven isoforms (γ, ζ, β, ε, η, τ, σ) and is highly expressed in various malignancies, including breast cancer. These scaffold proteins interact with p53 in response to DNA damage, thereby regulating apoptosis and cell cycle progression. Pan-14-3-3 inhibition using difopein has been shown to enhance apoptosis when combined with chemotherapeutic agents, primarily in tumors harboring wild-type TP53. However, its effects in TP53-mutant tumors remain poorly understood. TP53 mutations are present in approximately 30% of primary breast cancers and are associated with poor prognosis, but no mutation-based therapeutic strategies have been established. The current study investigated transcriptomic responses to pan-14-3-3 inhibition under DNA-damaging conditions using RNA sequencing in TP53 wild-type and mutant breast cancer cell lines. Methods: TP53 wild-type MCF-7 and TP53-mutant MDA-MB-231 breast cancer cells were treated with 1 μM difopein for 24 hours, followed by 3 μM doxorubicin for 2 hours to induce DNA damage-associated phosphorylation. Total RNA was extracted from biological triplicates and sequenced using the DNBSEQ-G400RSQ platform. Differential gene expression analysis was performed by comparing the difopein + doxorubicin group with the doxorubicin-only control. Differentially expressed genes (DEGs) were identified, and pathway enrichment analysis was conducted using Metascape to determine pathways significantly modulated by pan-14-3-3 inhibition under DNA-damaging conditions. Results: In TP53 wild-type MCF-7 cells, 112 DEGs were identified. Upregulated genes included TNS4, STBD1, PPARA, and ZSCAN26, whereas FHL1, HERPUD1, and TXNIP were downregulated. In gene ontology (GO) analysis “positive regulation of apoptotic process” was the most significantly enriched biological process. Protein-protein interaction (PPI) network analysis identified ATF3, ATF4, and CEBPB as central hubs using the MCODE algorithm. TRRUST transcription factor analysis highlighted KLF6 and TP53, and transcription factor target analysis identified STAT3 as the most significantly enriched regulator. In contrast, in TP53-mutant MDA-MB-231 cells, 43 DEGs were identified. Upregulated genes included PPIAP22, STXBP4, and MSI2, whereas SGK1, MIDN, and EPHB3 were downregulated. In GO analysis “NGF-stimulated transcription” was the most significantly enriched process. PPI network analysis identified RIPK4, RGS16, and RGS2 as central nodes. TRRUST analysis highlighted RBMX and TP53, and transcription factor target analysis identified GTF2A2 as the most significantly enriched regulator. Conclusion: Pan-14-3-3 inhibition under DNA-damaging conditions elicited distinct transcriptomic responses depending on TP53 status. In TP53 wild-type cells, the response was characterized by activation of pro-apoptotic transcriptional programs involving ATF3, ATF4, and CEBPB, consistent with enhanced apoptotic signaling. In contrast, TP53-mutant cells exhibited a survival-adaptive response involving ceRNA regulation (PPIAP22), SGK1 suppression, and reprogramming of transcriptional and proteasomal machinery. These findings highlight subtype-specific vulnerabilities and suggest that TP53 status may inform therapeutic strategies targeting 14-3-3 proteins. Further functional validation, including apoptosis assays, is currently underway to elucidate the mechanistic consequences of these transcriptomic changes. Citation Format: E. HIRAOKA, H. SHIGEMATSU, T. MARINO, T. MOMOKO, S. KANAKO, F. MUTSUMI, I. HARUKA, A. AI, S. SHINSUKE, O. MORIHITO. Tp53-dependent and -independent transcriptomic responses to pan-14-3-3 inhibition under dna damage in breast cancer cells abstract. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS2-12-26.
HIRAOKA et al. (Tue,) studied this question.
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