Exploring ID4 as a Driver of Aggression and a Therapeutic Target in Triple-Negative Breast Cancer

Abstract

Basal-like breast cancer (BLBC) is characterized by an aggressive clinical course, high genomic instability, and limited therapeutic options. The Inhibitor of Differentiation 4 (ID4) protein has been identified as a critical regulator of BLBC, where its overexpression correlates with poor prognosis. However, the mechanistic contributions of ID4 to BLBC tumorigenesis remain incompletely understood. In this study, we employed an integrative approach combining CRISPR-Cas9-mediated ID4 knockout, small-molecule inhibition, in vivo tumor modeling, and in silico transcriptional analyses to investigate the functional role of ID4 in BLBC. CRISPR-Cas9-mediated knockout of ID4 in MDA-MB-231 cells resulted in significant reductions in proliferation, colony formation, and Ki67 expression, indicating a loss of aggressive phenotypic traits. In vivo xenograft studies further revealed that ID4-silenced cells exhibited markedly delayed tumor formation and a significant reduction in metastatic potential compared to controls. Kaplan-Meier survival analysis of basal-like tumors from The Cancer Genome Atlas (TCGA) dataset demonstrated that patients with low ID4 expression had improved relapse-free survival. Gene set enrichment analysis (GSEA) of BLBC tumors stratified by ID4 expression revealed a shift toward luminal-like transcriptional programs in the ID4-low subgroup, including increased estrogen response and inflammatory signaling pathways. Furthermore, transcription factor activity analysis identified the activation of MYC, JUN, and STAT in ID4-low tumors, suggesting a transition toward a more differentiated phenotype. Finally, pharmacological inhibition of ID4 using the small-molecule degrader AGX51 effectively reduced proliferation in TNBC cells, highlighting ID4 as a potential therapeutic target. Together, these findings establish ID4 as a key driver of BLBC aggressiveness and suggest that its inhibition may represent a viable therapeutic strategy. This study provides compelling evidence supporting the development of ID4-targeted therapies for TNBC patients, with the potential to improve clinical outcomes in this challenging disease subset.