GABARAPL1 is required for the secretion of pro-angiogenic extracellular vesicles during hypoxia

• Published in: Journal of extracellular vesicles Vol. 7; p. 50
• Main Authors: Keulers, Tom G H, Zonneveld, Marijke I, Libregts, Sten F H M, Wauben, Marca H M, Rouschop, Kasper M A
• Format: Journal Article
• Language: English
• Published: Abingdon John Wiley & Sons, Inc 01-01-2018
• Subjects: Angiogenesis; Animal models; Cancer; Cell signaling; Doxycycline; Extracellular vesicles; Flow cytometry; GABARAP protein; Growth factors; Hypoxia; miRNA; mRNA; Radiation; Secretion; Solid tumors; Therapeutic applications; Tumor microenvironment; Xenografts
• ISSN: 2001-3078

Background: Hypoxia is a hallmark of solid tumours and is associated with tumour progression and therapy resistance. In response to hypoxia, tumour cells secrete pro-angiogenic factors to induce blood vessel formation and restore oxygen supply to the tumour. Extracellular vesicles (EVs) are emerging as mediators of intercellular communication in the tumour microenvironment and mediate intercellular communication by shuttling biological information such as miRNA's, mRNA, proteins and growth factors to recipient cells. Previously, we demonstrated that the expression of GABARAPL1, a member of the LC3/GABARAP protein family, is induced during hypoxia. Now, we demonstrate that GABARAPL1 is required for secretion of pro-angiogenic EVs during hypoxia. Methods: Ht29 and U87 doxycycline-inducible GABARAPL1 knockdown cell lines were exposed to hypoxia (16 h, <0.02% O2). EVs were isolated by sucrose density gradient isolation and analysed by western blot, qNANO or high-resolution flow cytometry. Angiogenic potential of cells was assessed by tube formation assays. Xenografts were implanted subcutaneously at the lateral flanks of NMRInu/nu mice and tumour size was measured by calliper. Results: GABARAPL1 is expressed on the EV surface and can be targeted with antibodies. This results in blockade of pro-angiogenic responses in vitro. Silencing GABARAPL1 with inducible knockdown models perturbs EV secretion and results in decreased tumour growth due to decreased vascularisation and enhanced necrosis. Additionally, targeting GABARAPL1 directly after tumour irradiation resulted in enhanced tumour regrowth delay. Furthermore, we demonstrate that GABARAPL1+ EVs are detectable and increased in blood of cancer patients. Summary/conclusion: Here, we reveal that hypoxic tumour cells secrete a unique EV subset, marked by GABARAPL1 expression. These EVs control tumour progression, are targetable and are therefore interesting to pursue as biomarker and therapeutic target.