Netrin-1 induces epithelial-mesenchymal transition and promotes hepatocellular carcinoma invasiveness.
BACKGROUND
Hypoxia is often found in solid tumors and is associated with tumor progression and poor clinical outcomes. We elucidated the mechanism by which netrin-1 released under hypoxic stress can induce epithelial-mesenchymal transition (EMT) to promote invasion in hepatocellular carcinoma (HCC) cells.


METHODS
The expression of netrin-1 and the dependent receptors UNC5H and deleted in colorectal cancer (DCC) in HCC was examined by immunohistochemistry or western blot. The HepG2 cells were cultured in 21% O2 (normoxia) or 1% O2 (hypoxia) for 24 h. The release of netrin-1 from hypoxic cells was detected by ELISA. Expression of E-cadherin and vimentin were examined by western blot. Inverted microscopy or confocal microscopy was used to show the cell morphology or cytoskeletal rearrangements. Cell invasion induced by hypoxia was analyzed by Transwell chamber. Cytokine IL-8 and IL-10 mRNA levels were assessed by real-time PCR.


RESULTS
The expression of netrin-1 was increased in HCC tissue and cell lines. The dependent receptors UNC5H and DCC were decreased in most HCC cell lines. Hypoxia induced netrin-1 release in a time-dependent manner. EMT induction was found to occur in hypoxic HCC cells in a process that was dependent on the extracellular release of netrin-1. Moreover, overexpression of netrin-1 resulted in EMT induction in normoxic tumor cells. Cytoskeletal rearrangements were found to occur and cell invasion was increased in cells with netrin-1 overexpression. Lastly, mRNA of IL-8 and IL-10 were also increased after recombinant human netrin-1 treatment.


CONCLUSIONS
These results suggest that in hypoxic HCC cells, netrin-1 activates downstream signaling pathways to induce EMT activation with subsequent production of multiple inflammatory mediators which in turn promotes cancer invasion.
Department of Gastroenterology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430030, People's Republic of China, yanwei@tjh.tjmu.edu.cn.
Wei
W
Yan
Ping
P
Han
Zhenzhen
Z
Zhou
Wei
W
Tu
Jiazhi
J
Liao
Peiyuan
P
Li
Mei
M
Liu
Dean
D
Tian
Yu
Y
Fu
D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D058750:Epithelial-Mesenchymal Transition / Q000235:genetics / Q000502:physiology*; D015972:Gene Expression Regulation, Neoplastic / Q000502:physiology; D056945:Hep G2 Cells; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism*; D009361:Neoplasm Invasiveness / Q000235:genetics*; D009414:Nerve Growth Factors / Q000235:genetics / Q000378:metabolism*; D000075388:Netrin-1; D010100:Oxygen; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*
C000617351:NTN1 protein, human; D009414:Nerve Growth Factors; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D025521:Tumor Suppressor Proteins; D000075388:Netrin-1; D010100:Oxygen