ABSTRACT
Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, and metastasis is the major cause of the high mortality of HCC. In this study, we identified that AnnexinA7 (ANXA7) and Sorcin (SRI) are overexpressed and interacting proteins in HCC tissues and cells. In vitro functional investigations revealed that the interaction between ANXA7 and SRI regulated epithelial-mesenchymal transition (EMT), and then affected migration, invasion, and proliferation in HCC cells. Furthermore overexpression/knockdown of ANXA7 was remarkably effective in promoting/inhibiting tumorigenicity and EMT in vivo. Altogether, our study unveiled a mechanism that ANXA7 promotes EMT by interacting with SRI and further contributes to the aggressiveness in HCC, which provides a novel potential therapeutic target for preventing recurrence and metastasis in HCC.
Subject(s)
Annexin A7/metabolism , Calcium-Binding Proteins/metabolism , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Epithelial-Mesenchymal Transition/genetics , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Signal Transduction/genetics , Animals , Annexin A7/genetics , Carcinoma, Hepatocellular/genetics , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Female , Humans , Liver Neoplasms/genetics , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Neoplasm Invasiveness , Transfection , Tumor Burden/genetics , Xenograft Model Antitumor AssaysABSTRACT
We report for the first time that Guanine nucleotide-binding protein subunit beta-2-like 1 (RACK1) formed a complex with Annexin A7. Hca-F and Hca-P are a pair of syngeneic mouse hepatocarcinoma cell lines established and maintained in our laboratory. Our previous study showed that both Annexin A7 and RACK1 were expressed higher in Hca-F (lymph node metastasis >70%) than Hca-P (lymph node metastasis <30%). Suppression of Annexin A7 expression in Hca-F cells induced decreased migration and invasion ability. In this study, knockdown of RACK1 by RNA interference (RNAi) had the same impact on metastasis potential of Hca-F cells as Annexin A7 down-regulation. Furthermore, by co-immunoprecipitation and double immunofluorescence confocal imaging, we found that RACK1 was in complex with Annexin A7 in control cells, but not in the RACK1-down-regulated cells, indicating the abolishment of RACK1-Annexin A7 interaction in Hca-F cells by RACK1 RNAi. Taken together, these results suggest that RACK1-Annexin A7 interaction may be one of the means by which RACK1 and Annexin A7 influence the metastasis potential of mouse hepatocarcinoma cells in vitro.
Subject(s)
Annexin A7/genetics , Cell Movement/genetics , Cell Proliferation , Neuropeptides/genetics , RNA Interference , Animals , Annexin A7/metabolism , Blotting, Western , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Lymphatic Metastasis , Mice , Microscopy, Confocal , Neuropeptides/metabolism , Protein Binding , Receptors for Activated C Kinase , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
In the tumor malignancy progression, lymph node metastasis (LNM) is recognized as an important factor. In this study, RNA interference (RNAi) was employed to down-regulate ANXA7 gene in Hca-F cells, a hepatocarcinoma cell line with high LNM rate. There was no significant effect on cell proliferation ability, but cell division, motility, and invasion abilities were markedly inhibited. By contrast, up-regulating the expression of ANXA7 gene in Hca-P cells with lower LNM rate, cell migration ability was improved and the percentage of cells in S phase was significantly decreased in vitro. Here, we reported that the expression of Ech1, GSN and JNK1 genes, which were relevant to tumor lymphatic metastasis, had been inhibited due to down-regulation ANXA7 gene and promoted due to up-regulation ANXA7 gene by western blot analysis. These results indicated that ANXA7 is a critical factor in the development of lymphatic metastasis in hepatocarcinoma progression.
Subject(s)
Annexin A7/genetics , Carcinoma, Hepatocellular/pathology , Gene Expression Regulation, Neoplastic , Liver Neoplasms/pathology , Animals , Blotting, Western , Carbon-Carbon Double Bond Isomerases/genetics , Carcinoma, Hepatocellular/genetics , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation , Down-Regulation , Gelsolin/genetics , Liver Neoplasms/genetics , Lymphatic Metastasis , Mice , Mitogen-Activated Protein Kinase 8/genetics , Neoplasm Invasiveness/genetics , RNA Interference , Up-RegulationABSTRACT
Hca-P and Hca-F is a pair of synogenetic mouse hepatocarcinoma ascites cell lines, possessing different capacity of lymphatic metastasis. Receptor of activated C-kinase 1 (Rack1), together with Jnk1 and gelsolin (Gsn) were previously identified as differentially expressed proteins for lymphatic metastatic potential between the two cell lines. As an intracellular scaffold protein, Rack1 could recruit such signaling molecules as integrins, Src, PKC which are involved in many important biological processes and play key roles in cancer progression. In our present studies, pCDNA3.1(+)-Rack1, a eukaryotic expression plasmid, was constructed and stably transfected into Hca-P cells with a low metastatic potential. CCK8 assay and transwell system were used to evaluate the effects of Rack1 on proliferation, migration and invasion of Hca-P cells in vitro. Then, LY294002, an inhibitor of PI3K, was added into the culture medium of pCDNA3.1(+)-Rack1-Hca-P cells and their biological behaviors observed further. Moreover, the expression of Jnk1, Rac1 and Gsn of pCDNA3.1(+)-Rack1-Hca-P cells were detected by western blot after pretreated with various doses of LY294002. As a result, the proliferation, migration and invasion of pCDNA3.1(+)-Rack1-Hca-P cells were significantly enhanced and could be inhibited by LY294002. In addition, the expression of Gsn, Rac1 and Jnk1 of pCDNA3.1(+)-Rack1-Hca-P cells also decreased after pretreated with LY294002. The expression of Gsn can be inhibited by NSC33766 (an inhibitor of Rac1). Taken together, Rack1/PI3K/Rac1 signaling pathway may play a crucial role in malignant biological behaviors of mouse hepatocarcinoma cells with lymphatic metastasis potential. It may be a potential target for therapy of cancer lymphatic metastasis.