Serum starvation drives ALIX-dependent extracellular vesicle biogenesis and determines tumor progression
Xueqiang Peng, Jiaxing Liu, Guolong Zeng, Yafei Xiao, Zhixiong Hao, Guangpeng He, Hongyuan Jin, Yu Gao, Shilei Tang, Shibo Wei, Yan Li, Yifan Yu, Liang Yang, Hangyu Li
Xueqiang Peng, Jiaxing Liu, Guolong Zeng, Yafei Xiao, Zhixiong Hao, Guangpeng He, Hongyuan Jin, Yu Gao, Shilei Tang, Shibo Wei, Yan Li, Yifan Yu, Liang Yang, Hangyu Li
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Research Article Cell biology Oncology

Serum starvation drives ALIX-dependent extracellular vesicle biogenesis and determines tumor progression

Abstract

Tumor cells are constantly confronted with nutrient deprivation; however, the effect of serum starvation on the remodeling of endosomal compartments and extracellular vesicles (EVs) in tumor cells remains unclear. Here, we found that serum starvation pronouncedly promotes multivesicular body (MVB) biogenesis, EV formation, and cargo selection. Specifically, by generating a constitutively active Rab5Q79L mutant to induce the enlargement of MVB, we revealed for the first time to our knowledge that ANXA3 is sorted into intraluminal vesicles (ILVs) of MVB. Mechanistically, we confirmed that serum starvation regulates the endosomal sorting complex required for transport–associated (ESCRT-associated) protein ALG-2 interacting protein X (ALIX), which recruits ESCRT-III to MVB and binds to annexin A3 (ANXA3) to mediate its sorting into ILVs of MVB. Our study highlights that serum starvation promotes an ALIX-dependent ESCRT-III recruitment pathway, which loads protumor ANXA3 cargo to exert a profound effect on tumor progression.

Authors

Xueqiang Peng, Jiaxing Liu, Guolong Zeng, Yafei Xiao, Zhixiong Hao, Guangpeng He, Hongyuan Jin, Yu Gao, Shilei Tang, Shibo Wei, Yan Li, Yifan Yu, Liang Yang, Hangyu Li

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Figure 4

ANXA3 is enriched in starvation-induced EVs.

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ANXA3 is enriched in starvation-induced EVs. (A) Western blot analysis o...
(A) Western blot analysis of ANXA3 in Huh7-EVs and HeLa-EVs, with TSG101 and CD63 as EV markers. Right, quantification relative to the 10% FBS group. n = 3. (B) IEM showing ANXA3 localization in EVs using gold-labeled anti-ANXA3 antibody. Gold particles were mainly detected on EVs, indicating specific ANXA3 enrichment. Scale bar: 100 nm. (C) Polar-SIM images of ANXA3 (red) and CD63-GFP (green) in cells cultured in 10% FBS, under serum starvation, or recultured in 10% FBS after starvation. Nuclei were stained with DAPI (blue). Scale bar: 10 μm. Right, percentage of ANXA3+ MVB among CD63+ MVB. n = 20 cells. (D) Confocal images of ANXA3 (red) and LAMP1 (green); nuclei were stained with DAPI (blue). Bottom, Pearson’s correlation analysis of colocalization. n = 10 cells. (E) Dual-fluorescence imaging of Rab5Q79L (green) and CD63 (red) in HeLa-Rab5Q79L cells showing CD63 accumulation within Rab5Q79L+ endosomal lumens. Scale bar: 10 μm. (F) TEM of HeLa-Rab5Q79L cells showing abnormally enlarged MVB. Scale bars: 500 nm (left) and 2000 nm (right). (G) Polar-SIM images of ANXA3 (red) and Rab5Q79L (green) in HeLa-Rab5Q79L cells under the indicated conditions; nuclei were stained with DAPI (blue). Scale bar: 10 μm. Right, quantification of the percentage of ANXA3+ area within Rab5Q79L+ endosomes under the indicated conditions. n = 31, 27, 35 Rab5Q79L+ endosomes. Data are presented as mean ± SD. Statistical analysis was performed using ordinary 1-way ANOVA followed by Tukey’s multiple-comparison test. Comparisons between 2 groups were done with 2-tailed Student’s t test. **P < 0.01, ***P < 0.001.