John Epps

Background: Triple-negative breast cancer (TNBC) lacks estrogen, progesterone, and HER2 receptors, severely limiting pharmaceutical interventions.³ TNBC accounts for 15% to 20% of all breast cancer, but disproportionately drives poor health outcomes due to its high propensity to metastasize to the brain.³ Brain metastasis leads to a range of grim intracranial pathologies with a high likelihood of recurrence due to the aggressiveness of breast cancer stem cells.² The truncated glioma-associated oncogene homolog 1 (tGLI1) is an understudied tumor-specific gain-of-function splice variant of GLI1 that acts as a key regulator of breast cancer brain metastasis (BCBM) in TNBC and HER2 phenotypes.⁴ tGLI1 has an in-frame deletion of 41 codons that alters its molecular shape, enabling it to outcompete GLI1 and bind to a wider range of receptor binding sites, activating oncogenic pathways not present in normal tissue.^3,4

Objective: This narrative review explores the biochemical pathways that tGLI1 acts on to drive BCBM and a first-in-class direct tGLI1 inhibitor designed to cross the blood-brain barrier.

Search Methods: The PubMed database with a publication window from 2018 to 2022 was used to find primary laboratory studies evaluating tGLI1-related pathology and therapy with selective keywords including: “triple-negative breast cancer”, “tGLI1”, “brain metastasis”, “cancer stem cells”, and “tumor microenvironment”.

Results: Mechanistic analysis reveals that tGLI1 participates in a reciprocal activation loop with the transcription factor STAT3, synergistically overexpressing downstream angiogenic targets, including R-Ras2, Cep70, and UPF3A.³ High co-activation yields a median metastasis-free survival of 1.25 years in TNBC patients.³ Although combined inhibition of upstream JAK2 and SMO pathways reduces lung and liver metastasis in vivo, it fails to prevent brain metastasis due to poor blood-brain barrier penetration.² Importantly, tGLI1 preferentially drives brain tropism and enables intracranial growth by promoting the stemness genes Nanog, SOX2, and OCT4.⁴ Additionally, tGLI1-positive cancer cells shed extracellular vesicles with miR-1290 into the brain-tumor microenvironment, reprogramming astrocytes into a reactive state characterized by high levels of glial fibrillary acidic protein expression. These reactive astrocytes suppress the transcription factor FOXA2, activating the promoter CNTF to release vascularization growth factors, facilitating tumor survival.⁵ 1,527 compounds were screened to find an inhibitory compound to this cascade. The antifungal ketoconazole (KCZ) was identified as a selective inhibitor of tGLI1 DNA-binding.¹ To address hepatotoxicity and improve blood-brain barrier penetration, a novel hydrophobic derivative, KCZ-7, was engineered with a toluene moiety.¹ Western blot and mouse model analyses confirmed that KCZ-7 achieved significantly higher concentrations in the brain compared to the original KCZ, leading to a marked reduction in brain metastasis and tumor size. However, its overall efficacy was not significantly higher than KCZ.¹

Conclusions: Expression of tGLI1 preferentially promotes BCBM and facilitates intracranial growth, angiogenesis, and proliferation. Direct tGLI1 inhibitors, like KCZ-7, offer a promising clinical strategy to cross the blood-brain barrier and disrupt the supportive tumor microenvironment. Future research is needed to further investigate possible combination therapies and higher efficacy KCZ derivatives to improve patient outcomes.

Works Cited:

1. Doheny, D., Manore, S., Sirkisoon, S. R., Zhu, D., Aguayo, N. R., Harrison, A., Najjar, M., Anguelov, M., Cox, A. O., Furdui, C. M., Watabe, K., Hollis, T., Thomas, A., Strowd, R., & Lo, H. W. (2022). An FDA-Approved Antifungal, Ketoconazole, and Its Novel Derivative Suppress tGLI1-Mediated Breast Cancer Brain Metastasis by Inhibiting the DNA-Binding Activity of Brain Metastasis-Promoting Transcription Factor tGLI1. Cancers, 14(17), 4256. https://doi.org/10.3390/cancers14174256
2. Doheny, D., Sirkisoon, S., Carpenter, R. L., Aguayo, N. R., Regua, A. T., Anguelov, M., Manore, S. G., Arrigo, A., Jalboush, S. A., Wong, G. L., Yu, Y., Wagner, C. J., Chan, M., Ruiz, J., Thomas, A., Strowd, R., Lin, J., & Lo, H. W. (2020). Combined inhibition of JAK2-STAT3 and SMO-GLI1/tGLI1 pathways suppresses breast cancer stem cells, tumor growth, and metastasis. Oncogene, 39(42), 6589–6605. https://doi.org/10.1038/s41388-020-01454-1
3. Sirkisoon, S. R., Carpenter, R. L., Rimkus, T., Anderson, A., Harrison, A., Lange, A. M., Jin, G., Watabe, K., & Lo, H. W. (2018). Interaction between STAT3 and GLI1/tGLI1 oncogenic transcription factors promotes the aggressiveness of triple-negative breast cancers and HER2-enriched breast cancer. Oncogene, 37(19), 2502–2514. https://doi.org/10.1038/s41388-018-0132-4
4. Sirkisoon, S. R., Carpenter, R. L., Rimkus, T., Doheny, D., Zhu, D., Aguayo, N. R., Xing, F., Chan, M., Ruiz, J., Metheny-Barlow, L. J., Strowd, R., Lin, J., Regua, A. T., Arrigo, A., Anguelov, M., Pasche, B., Debinski, W., Watabe, K., & Lo, H. W. (2020). TGLI1 transcription factor mediates breast cancer brain metastasis via activating metastasis-initiating cancer stem cells and astrocytes in the tumor microenvironment. Oncogene, 39(1), 64–78. https://doi.org/10.1038/s41388-019-0959-3
5. Sirkisoon, S. R., Wong, G. L., Aguayo, N. R., Doheny, D. L., Zhu, D., Regua, A. T., Arrigo, A., Manore, S. G., Wagner, C., Thomas, A., Singh, R., Xing, F., Jin, G., Watabe, K., & Lo, H. W. (2022). Breast cancer extracellular vesicles-derived miR-1290 activates astrocytes in the brain metastatic microenvironment via the FOXA2→CNTF axis to promote progression of brain metastases. Cancer letters, 540, 215726. https://doi.org/10.1016/j.canlet.2022.215726