Abstract 79: Pax6 regulates stemness and enhances brain metastases in breast cancer

Abstract Brain metastasis (BrM) remains the most severe complication of breast cancer (BC), the most diagnosed cancer in females, associated with an abysmal prognosis. Our poor understanding of the molecular mechanisms driving the disease is one of the major reasons for the dearth of specific therapies. This highlights an urgent clinical need to decipher how BC cells form BrM. BCBrM is preceded by a period of dormancy, during which stem cell-like features of cancer cells may play a role in reactivating them to form metastatic lesions. Cancer stem cells, known as “brain tumor-initiating cells” contribute to tumor formation in the brain and could also lead to radio and chemoresistance in the treatment of brain metastases. We, therefore, conducted a bioinformatic analysis on publicly available transcriptomic datasets of matched primary and brain metastatic patient samples and found that Pax6, a transcription factor, was upregulated in the metastatic samples compared to the primary tumors. In tissue microarray (TMA) belonging to BrM patients, Pax6 expression was evident in the BrM lesions and nearly absent in normal brain tissue. Pax6 was found to be upregulated in BCBrM cell lines compared to primary BC cell lines. Pax6, a master regulator of transcription, essential for normal development of the eyes and central nervous system, has been implicated in cancer progression. Pax6 can promote proliferation, migration, and resistance to apoptosis. To delineate the essentiality of Pax6 in BCBrM, we performed CRISPR/Cas9-mediated Pax6 knockout (KO) in the BCBrM cell lines, which decreased proliferation, wound healing, and migration properties. Intracardiac injection of these Pax6 KO BCBrM cells into immunocompromised mice resulted in a significant reduction of brain metastatic tumor lesions compared to controls. Conversely, overexpression of Pax6 in primary BC cells induced a BrM-like phenotype, enhancing proliferation, migration, and wound healing. Since Pax6 regulates stem cell features we also found that Pax6 KO reduced the expression of stemness-promoting factors such as OCT4, NANOG, and KLF4. Further, to confirm the stemness features we found that in serum-free conditions, Pax6 KO cells significantly lacked mammosphere-forming abilities and side populations. These findings suggest that Pax6 mediates stemness in cancer cells and enhances the metastatic potential of BCBrM cells. Unraveling the role of Pax6 in BCBrM could potentially open new therapeutic avenues for the treatment of BCBrM patients which eventually will improve outcomes of these patients. Citation Format: Laiba Anwar. Pax6 regulates stemness and enhances brain metastases in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 79.