HIF-1 promotes murine breast cancer brain metastasis by increasing production of integrin β3–containing extracellular vesicles
Yongkang Yang, … , Kathleen L. Gabrielson, Gregg L. Semenza
Yongkang Yang, … , Kathleen L. Gabrielson, Gregg L. Semenza
Published July 15, 2025
Citation Information: J Clin Invest. 2025;135(14):e190470. https://doi.org/10.1172/JCI190470.
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Research Article Oncology Vascular biology

HIF-1 promotes murine breast cancer brain metastasis by increasing production of integrin β3–containing extracellular vesicles

Abstract

Brain metastasis is a major cause of breast cancer (BC) mortality, but the cellular and molecular mechanisms have not been fully elucidated. BC cells must breach the blood-brain barrier in order to colonize the brain. Here, we determined that integrin β3 (ITGB3) expression mediated by hypoxia-inducible factor 1 (HIF-1) plays a critical role in metastasis of BC cells to the brain. Hypoxia stimulated BC cell migration and invasion ex vivo and brain colonization in vivo. Knockdown of either HIF-1α or ITGB3 expression impaired brain colonization by human or mouse BC cells injected into the cardiac left ventricle. Exposure of BC cells to hypoxia increased expression of ITGB3 and its incorporation into small extracellular vesicles (EVs). EVs harvested from the conditioned medium of hypoxic BC cells showed increased retention in the brain after intracardiac injection that was HIF-1α and ITGB3 dependent. EVs from hypoxic BC cells showed binding to brain endothelial cells (ECs), leading to increased EC–BC cell interaction, increased vascular endothelial growth factor receptor 2 signaling, increased EC permeability, and increased transendothelial migration of BC cells. Taken together, our studies implicate HIF-1–stimulated production of ITGB3+ EVs as a key mechanism by which hypoxia promotes BC brain metastasis.

Authors

Yongkang Yang, Chelsey Chen, Yajing Lyu, Olesia Gololobova, Xin Guo, Tina Yi-Ting Huang, Vijay Ramu, Varen Talwar, Elizabeth E. Wicks, Shaima Salman, Daiana Drehmer, Dominic Dordai, Qiaozhu Zuo, Kenneth W. Witwer, Kathleen L. Gabrielson, Gregg L. Semenza

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Figure 3

ITGB3 expression is required for hypoxia-induced migration and invasion of brain-metastatic BC cells.

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ITGB3 expression is required for hypoxia-induced migration and invasion ...
(A–D) 4T1-BR5 subclones that were stably transduced with a lentivirus encoding an NTC shRNA or shRNA targeting ITGB3 or ITGAV were subjected to RT-qPCR (A and C) or immunoblot assays (B and D). Data are shown as mean ± SD (n = 3). **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. shNTC; ns, not significant vs. shNTC (unpaired 2-tailed Student’s t test). (E–H) 4T1-BR5 subclones were seeded on top of uncoated (E and F) or Matrigel-coated (G and H) Boyden chamber inserts and incubated at 20% or 1% O2 for 16 (E and F) or 24 (G and H) hours. Cells on the underside of the insert were stained with crystal violet and imaged by light microscopy (E and G; scale bars: 100 μm). The stained area was quantified using ImageJ (NIH) and expressed as mean ± SD (n = 3). **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. shNTC at 20% O2; ####P < 0.0001 vs. shNTC at 1% O2 (2-way ANOVA with Tukey’s multiple-comparison test).