Promoting colonization in metastatic HCC cells by modulation of autophagy.
BACKGROUND
Autophagy is an important adaptive survival mechanism, which has been postulated to be involved in cancer metastasis. The purpose of this study was to investigate autophagy in metastasis of hepatocellular carcinoma (HCC).


METHODS
Immunohistochemical analysis of autophagic activity in metastatic and paired primary HCC tissues using LC3 as autophagosome marker was performed in samples from 216 HCC patients diagnosed with metastasis (including 158 intravascular, 42 intrabiliary, 8 lymph node, 4 bone and 4 lung metastases). Then a mouse model of pulmonary metastasis was established using a highly metastatic HCC cell line (HCCLM3). Autophagy in pulmonary metastases and paired primary tumors were analyzed by LC3 immunohistochemistry, transmission electron microscopy (TEM) and western blot analysis. Further, mouse model of pulmonary metastasis and in vitro cell migration, invasion and detachment models were established using a stable GFP-LC3-expressing HCCLM3 cell line (HCCLM3-GFP-LC3). Autophagic alterations during metastatic colonization, migration, invasion and detachment were determined by GFP-LC3 analysis and western blot analysis.


RESULTS
LC3 immunohistochemistry of metastases and primary tumors from HCC patients revealed significantly higher LC3 expression in metastases than primary HCC, which suggested a higher level of autophagy in HCC metastases. Further immunohistochemical, TEM, western blot and in vivo GFP-LC3 analyses of lung metastases and primary tumors in mouse model of pulmonary metastasis confirmed that metastatic colonies displayed higher level of autophagy than primary tumors and the early metastatic colonies displayed highest level. The dynamic monitoring of autophagy in cell migration, invasion and detachment showed that autophagy did not significantly alter in those processes.


CONCLUSIONS
Autophagy is activated in metastatic colonization but not in invasion, migration and detachment of HCC cells. Autophagy may play a role in HCC metastasis via promoting metastatic colonization of HCC cells.
Department of Liver Surgery, Liver Cancer Institute, Zhongshan Hospital, Fudan University, Key Laboratory of Carcinogenesis and Cancer Invasion of Ministry of Education, Shanghai, China.
Yuan-Fei
YF
Peng
Ying-Hong
YH
Shi
Ying-Hao
YH
Shen
Zhen-Bin
ZB
Ding
Ai-Wu
AW
Ke
Jian
J
Zhou
Shuang-Jian
SJ
Qiu
Jia
J
Fan
D000818:Animals; D001343:Autophagy; D006528:Carcinoma, Hepatocellular / Q000378:metabolism / Q000473:pathology* / Q000648:ultrastructure; D002448:Cell Adhesion; D045744:Cell Line, Tumor; D002465:Cell Movement; D003710:Demography; D005260:Female; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D008113:Liver Neoplasms / Q000378:metabolism / Q000473:pathology* / Q000648:ultrastructure; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008875:Middle Aged; D009361:Neoplasm Invasiveness; D009362:Neoplasm Metastasis; D014410:Tumor Stem Cell Assay; D015854:Up-Regulation
C477012:MAP1LC3A protein, human; D008869:Microtubule-Associated Proteins; D049452:Green Fluorescent Proteins