HIF-1 promotes murine breast cancer brain metastasis by increasing production of integrin β3–containing extracellular vesicles
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
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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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 8

ITGB3+ EVs promote interaction of BC cells with ECs and increase endothelial permeability.

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ITGB3+ EVs promote interaction of BC cells with ECs and increase endothe...
(A–E) As shown in the schematic created with BioRender (biorender.com) (A), hCMEC/D3 ECs were seeded on 6-well plates, cultured to confluence, and treated for 24 hours with EVs from MDA231-BrM2 cells that were exposed to 20% or 1% O2 (B and C) or subclones that were exposed to 1% O2 (D and E). GFP+ MDA231-BrM2 cells were then added onto the hCMEC/D3 monolayer and incubated for 1 hour, and non-adherent cells were removed by washing with PBS. Adherent BC cells were imaged by fluorescence microscopy (B and D; scale bars: 10 μm) and quantified (C and E; mean ± SD, n = 3). *P < 0.05 or ***P < 0.001 vs. no EVs (C) or shNTC EVs (E); ##P < 0.01 vs. 20% O2 EVs (C) (by 1-way ANOVA with Tukey’s multiple-comparison test). (F) hCMEC/D3 ECs were seeded on Boyden chamber filters, cultured to confluence, and incubated with EVs from MDA231-BrM2 subclones for 24 hours. FITC-dextran was added to the upper chamber, and fluorescence in the lower chamber was measured 20 minutes later using a plate reader. Data are shown as mean ± SD (n = 3). ****P < 0.0001 vs. no EVs; ####P < 0.0001 vs. 20% O2 shNTC-EVs; &&&&P < 0.0001 vs. 1% O2 shNTC-EVs (2-way ANOVA followed by Tukey’s multiple-comparison test).