Neuronal architect Pax6 orchestrates stemness and brain metastatic progression

At the time of breast cancer (BC) diagnosis, many patients have already seeded disseminated tumor cells in the brain, where these cells can persist in a dormant, clinically silent state for years before reactivating to form devastating brain metastasis (BrM). The molecular programs and signals that enable dormant cells to awaken in the neural microenvironment remain an area of limited understanding. Yet this transition is thought to rely heavily on the acquisition of stem cell-like properties. To decipher its mechanism, we performed integrative analyses of publicly available paired primary tumor and brain metastatic transcriptomic patient datasets to identify genes that may be involved in neuronal mimicry. We identified Pax6 as one of the most consistently upregulated genes in metastatic lesions, which was further confirmed through BCBrM tissue microarray. Pax6 is a key neuronal developmental transcription factor that orchestrates CNS specification and sustains neural stem cell identity during embryogenesis. Functional studies using CRISPR/Cas9-mediated Pax6 knockout (KO) in BCBrM cell lines revealed profound reductions in proliferation, migration, and wound closure. Pax6 loss also diminished core stemness programs, including suppression of OCT4, NANOG, and KLF4 expression, diminished CD44+/CD24- tumor initiating cell populations, and impaired mammosphere formation and side populations, a phenotype consistent with a collapse of stem-like potential. Consistently, genes previously linked to BrM were elevated upon Pax6 overexpression (OE) in primary BC cells and were downregulated in Pax6 KO models, further supporting Pax6 as an upstream regulator of BrM - associated transcriptional programs. To critically examine if the observation holds true in preclinical setting, we performed intracardiac injection of Pax6 KO BrM cells in athymic nude mice. We observed that Pax6 KO cells failed to form significant brain macro metastasis, demonstrating the importance of Pax6 in establishing BrM outgrowth. Conversely, Pax6 OE in primary BC cells induced a BrM-like phenotype with increased cell proliferation, migration, and induction of stemness-associated markers, supporting a causal role of Pax6 in promoting metastatic reprogramming. Together, these findings identify Pax6 as a developmental stem cell regulator hijacked by BC cells to escape dormancy and acquire the functional plasticity necessary for growth within the brain microenvironment. By showing that Pax6 plays a key role in driving BCBrM, this work highlights a potential targetable weakness and suggests that therapeutic targeting of Pax6 or its associated pathways may offer a new therapeutic strategy to limit brain metastatic progression and improve clinical outcomes.