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 2

ITGB3 is a direct HIF-1 target gene.

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ITGB3 is a direct HIF-1 target gene. (A and B) ITGB3 mRNA and protein w...
(A and B) ITGB3 mRNA and protein were analyzed by RT-qPCR (A) and immunoblot assays (B) in 4T1 and 4T1-BR5 cells exposed to 20% or 1% O2. ITGB3 mRNA was quantified relative to 18S rRNA and normalized to mean for 4T1 cells at 20% O2; mean ± SD (n = 3). ****P < 0.0001 vs. 4T1 at 20% O2; ####P < 0.0001 vs. 4T1 at 1% O2 (unpaired 2-tailed Student’s t test). (C–F) 4T1-BR5 (C and D) or MDA231-BrM2 (E and F) subclones were exposed to 20% or 1% O2, and ITGB3 mRNA or protein levels were analyzed by RT-qPCR (C and E) and immunoblot assays (D and F); mean ± SD (n = 3). ****P < 0.0001 vs. NTC at 20% O2; ####P < 0.0001 vs. NTC at 1% O2 (2-way ANOVA with Tukey’s multiple-comparison test). (G and H) Flow cytometry histograms showing anti-ITGB3 antibody binding to 4T1-BR5 (G) or MDA231-BrM2 (H) cells that were exposed to 20% or 1% O2; mean ± SD (n = 3). ****P < 0.0001 vs. 20% O2. (I) ChIP-Seq analysis revealed 3 matches to the HIF consensus binding site 5′-(A/G)CGTG-3′ or its complement (underlined) under the HIF-1α peak in the ITGB3 gene in MDA231-BrM2 cells using the Integrative Genomics Viewer (IGV) genome browser. (J) MDA231-BrM2 cells were exposed to 20% or 1% O2, and ChIP-qPCR was performed using antibodies against HIF-1α, HIF-1β, or HIF-2α. Primers flanking the nucleotide sequence shown in I were used for qPCR, and results were normalized to the mean result at 20% O2; mean ± SD (n = 3). ****P < 0.0001 vs. 20% O2; ns, not significant (unpaired 2-tailed Student’s t test).