Metastasis-associated in colon cancer-1 is associated with poor prognosis in hepatocellular carcinoma, partly by promoting proliferation through enhanced glucose metabolism.

Metastasis-associated in colon cancer-1 (MACC1) is a newly identified gene that is involved in the development and progression of hepatocellular carcinoma (HCC), however its investigation has not been comprehensive. In the present study, in vitro techniques, including immunohistochemistry, western blotting, reverse transcription quantitative polymerase chain reaction, metabolic assay, MTT assay, colony formation assay and prognostic analysis were used to confirm the involvement of MACC1 in HCC. Histological examination confirmed that the protein expression of MACC1 was upregulated in HCC and was associated with the hexokinase-2 (HK2) protein, which also indicates a poor prognosis. Knockdown of MACC1 induced the reduction of glycogen consumption and lactate production, which then lead to a marked reduction of proliferation in the MHCC-97H cells. However, the overexpression of MACC1 produced the opposite results in the HepG2 cells. These results suggested that MACC1 leads to a poor prognosis in HCC, partly by promoting proliferation via enhancement in glucose metabolism by HK2. Therefore, this pathway has the potential to become an important therapeutic target in HCC.

MACC1 suppresses cell apoptosis in hepatocellular carcinoma by targeting the HGF/c-MET/AKT pathway.

BACKGROUND & AIMS: To investigate the expression and prognostic value of MACC1 in patients with HCC and identify the mechanism by which MACC1 inhibits HCC cell apoptosis. METHODS: MACC1 and p-AKT expression was studied using immunohistochemistry of both HCC tissues and adjacent liver tissues. qRT-PCR and western immunoblotting were used to examine the expression of target genes at the mRNA and protein levels, respectively. The MTT assay was used to assess cell viability, and cell apoptosis was determined by DAPI staining, Annexin V/PI staining and Caspase 3/7 assay. Nude mice were used to perform in vivo experiments. RESULTS: The overexpression of MACC1 was found in HCC tissues and was correlated with poor postsurgical prognosis. There was a positive relationship between MACC1 and p-AKT expression in HCC tissues. In vitro experiments showed that MACC1 repressed HCC cell apoptosis and promoted cell growth. Knockdown of c-MET abolished the anti-apoptotic function of MACC1. Next, MACC1 was verified to activate PI3K/AKT signaling by sensitizing HGF/c-MET signaling in HCC. MACC1 overexpression enhanced the HGF-driven phosphorylation of BAD, Caspase 9 and FKHRL1 and inhibited their pro-apoptotic functions in HCC cells. Finally, MACC1 was shown to inhibit cell apoptosis and promote HCC growth in vivo. CONCLUSIONS: This investigation revealed that MACC1 overexpression predicted worse prognosis after liver resection, which was attributed to the repression of HCC cell apoptosis via a molecular mechanism in which MACC1 accelerated the activation of the HGF/c-MET/PI3K/AKT pathway and phosphorylated BAD, Caspase 9 and FKHRL1, ultimately preventing their nuclear translocation and their pro-apoptotic function.

[miR-143 inhibits proliferation and invasion of hepatocellular carcinoma cells via down-regulation of TLR2 expression].

OBJECTIVE: To detect the expression of microRNA-143 (miR-143) in hepatocellular carcinoma and investigate whether transfection of miR-143 could influence the biological behaviors of hepatocellular carcinoma cells. METHODS: Hepatocellular carcinoma tissues and its corresponding adjacent normal tissues were obtained from patients undergoing radical surgical resection. Real-time quantitative PCR was utilized to detect the relative expression of miR-143 in hepatocellular carcinoma tissues and corresponding adjacent normal tissues. MiR-143 mimics and negative control oligonucleotides were synthesized and transfected into HepG-2 cells in vitro. Proliferation, apoptosis and invasion of the transfected cells were measured by MTT assay, flow cytometry (FCM) combined with annexin V-FITC/PI staining, and Transwell(TM) assay, respectively. The protein levels of Toll-like receptor 2 (TLR2), nuclear factor kappa B (NF-κB), matrix metalloproteinase 2 (MMP-2) and MMP-9 were detected by Western blotting. RESULTS: Expression of miR-143 was significantly lower in hepatocellular carcinoma tissues than in paired adjacent normal tissues. The level of miR-143 was significantly up-regulated after transfection of miR-143 mimics. And proliferation and invasion were significantly inhibited, but apoptosis was promoted after transfection. Expressions of TLR2, NF-κB, MMP-2 and MMP-9 were reduced by miR-143 mimics' transfection. CONCLUSION: The miR-143 expression was low in hepatic carcinoma and its over-expression could down-regulate the expressions of TLR2, NF-κB, MMP-2 and MMP-9.

MRC2 expression correlates with TGFß1 and survival in hepatocellular carcinoma.

MRC2 (Mannose Receptor C Type 2) is a constitutively recycling endocytic receptor belonging to the mannose receptor family, which has been found to be closely involved with cancer metastasis. This study attempted to determine MRC2 expression on hepatocellular carcinoma (HCC) and its significance on postsurgical prognosis of HCCs. The expression of both MRC2 and transforming growth factor (TGFß1) was detected in tumor tissues and adjacent liver tissues from 96 HCCs by immunohistochemistry staining, and it was found that MRC2 expression in HCC tissues was significantly higher than in adjacent liver tissues. HCCs with higher MRC2 expression had worse prognosis after liver resection. Univariate analysis showed that advanced TNM staging of HCC, higher Edmonson-Steiner classification, intrahepatic metastases, portal vein invasion, higher MRC2 and higher TGFß1 were the poor prognostic factors. Furthermore, multivariate analysis revealed that intrahepatic metastases, higher MRC2 and higher TGFß1 were the independent prognostic factors. TGFß1 treatment up-regulated MRC2 expression, cell migration and invasion of Huh7 cells notably. In addition, knockdown of MRC2 repressed the effect of TGFß1 on cell migration and invasion. These data suggest that MRC2 overexpression predicts poor prognosis of HCCs after liver resection and MRC2 potentially contributed to TGFß1-driven up-regulation of cell migration and invasion in HCC.

Fbxw7 is an independent prognostic marker and induces apoptosis and growth arrest by regulating YAP abundance in hepatocellular carcinoma.

BACKGROUND: The E3 ubiquitin ligase Fbxw7 functions as a general tumor suppressor by targeting several well-known oncoproteins for ubiquitination and proteasomal degradation. However, the clinical significance of Fbxw7 and the mechanisms involved in the anti-cancer effect of Fbxw7 in HCC are not clear. METHOD: The Fbxw7 and YAP expression in 60 samples of surgical resected HCC and matched normal tumor-adjacent tissues were assessed using IHC or immunoblotting. Flow cytometry, caspase 3/7 activity assay, BrdU cell proliferation assay and MTT assay were used to detect proliferation and apoptosis of HCC cells. The regulatory effect of Fbxw7 on YAP in HCC cells was confirmed by qRT-PCR, immunoblotting and immunofluorescence. Co-immunoprecipitation was used to analyze interaction between YAP and Fbxw7. Nude mice subcutaneous injection, Ki-67 staining and TUNEL assay were used to evaluate tumor growth and apoptosis in vivo. RESULTS: In this study, we found that Fbxw7 expression was impaired in HCC tissues and loss of Fbxw7 expression was correlated with poor clinicopathological features including large tumor size, venous infiltration, high pathological grading and advanced TNM stage. Additionally, we demonstrated that patients with positive Fbxw7 expression had a better 5-year survival and Fbxw7 was an independent factor for predicting the prognosis of HCC patients. We confirmed that Fbxw7 inhibited HCC by inducing both apoptosis and growth arrest. Elevated YAP expression was observed in the same cohort of HCC tissues. Pearson's correlation coefficient analysis indicated that Fbxw7 was inversely associated with YAP protein expression in HCC tissues. We also found that Fbxw7 regulated YAP protein abundance by targeting YAP for ubiquitination and proteasomal degradation in HCC. Furthermore, restoring YAP expression partially abrogated Fbxw7 induced HCC cell apoptosis and growth arrest in vitro and in vivo. CONCLUSION: These results indicate that Fbxw7 may serve as a prognostic marker and that YAP may be a potential target of Fbxw7 in HCC.

MACC1 expression correlates with PFKFB2 and survival in hepatocellular carcinoma.

OBJECTIVE: To validate the relationship between MACC1 and 6-phosphofructo-2-kinase/fructose 2, 6 bisphosphatase (PFKFB2) expression as well as its clinicopathological features and prognostic significance in hepatocellular carcinoma. METHODS: By using immunohistochemistry, we investigated the MACC1 and PFKFB2 protein expression in 60 pairs of hepatocellular carcinoma and corresponding non-tumor tissues. Using the Mann-Whitney U test, the Chi-square test, Kaplan-Meier survival analysis, Cox proportional hazard regression analysis and Spearman analysis, we studied the relationship between MACC1 and PFKFB2 protein expression and postoperative overall survival (OS) of the HCC patients. RESULTS: MACC1 and PFKFB2 positive staining rates were significantly higher in hepatocellular carcinoma than in the corresponding nontumor tissues (P=0.012 and 0.04, respectively). The clinicopathological features evaluation revealed that positive expression of MACC1 was associated with a high Edmondson classification (P=0.007) and advanced TNM stage (P=0.027). Similar findings were evident for PFKFB2 expression (P=0.002 and P=0.027). MACC1 and PFKFB2 positive expression was associated with a lower OS rate (P=0.004 and 0.03, respectively). Kaplan-Meier survival and Cox proportional hazard regression analyses revealed MACC1 positive expression to be a prognostic factor for postoperative OS, but PFKFB was not. CONCLUSION: Highly expressed MACC1 and PFKFB2 protein were associated with TNM stage, Edmondson -Steier classification and overall survival. MACC1 may affect tumor metabolism partly through expression and phophorylation of PFKFB2.

Caveolin-1 is up-regulated by GLI1 and contributes to GLI1-driven EMT in hepatocellular carcinoma.

Caveolin-1 (Cav-1) has been recently identified to be over-expressed in hepatocellular carcinoma (HCC) and promote HCC cell motility and invasion ability via inducing epithelial-mesenchymal transition (EMT). However, the mechanism of aberrant overexpression of Cav-1 remains vague. Here, we observed that Cav-1 expression was positively associated with GLI1 expression in HCC tissues. Forced expression of GLI1 up-regulated Cav-1 in Huh7 cells, while knockdown of GLI1 decreased expression of Cav-1 in SNU449 cells. Additionally, silencing Cav-1 abolished GLI1-induced EMT of Huh7 cells. The correlation between GLI1 and Cav-1 was confirmed in tumor specimens from HCC patients and Cav-1 was found to be associated with poor prognosis after hepatic resection. The relationship between protein expression of GLI1 and Cav-1 was also established in HCC xenografts of nude mice. These results suggest that GLI1 may be attributed to Cav-1 up-regulation which plays an important role in GLI1-driven EMT phenotype in HCC.

Histone acetyltransferase PCAF up-regulated cell apoptosis in hepatocellular carcinoma via acetylating histone H4 and inactivating AKT signaling.

BACKGROUND: PCAF is an important intrinsic histone acetyltransferases. This study tried to establish the effect of PCAF on HCC cell apoptosis. METHOD: Both in vitro and in vivo experiments including IHC, DAPI staining, caspase 3/7 activity assay, BrdU assay, MTT assay, western immunoblotting and co-immunoprecipitation were used here. RESULTS: PCAF was found to be expressed at the low level in most of HCC cell lines. PCAF overexpression induced cell apoptosis and growth arrest with increased Histone H4 acetylation and inactivation of AKT signaling in Huh7 and HepG2 cells. The opposite results were obtained by silencing PCAF in Hep3B cells. The co-immunoprecipitation assay confirmed that PCAF protein was bound with histone H4 protein in the nucleus of Hep3B cells. Finally, the in vivo experiment confirmed the findings mentioned-above. CONCLUSION: These data identified PCAF promotes cell apoptosis and functions as a HCC repressor through acetylating histone H4 and inactivating AKT signaling.