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Background: Mounting evidence suggests that solid tumors display the features of collective invasion, however, the molecular mechanisms are far from clear. This study aims to verify the role and the underlying mechanisms of CD147 in collective invasion in hepatocellular carcinoma., Methods: Immunostaining was used to analyze human hepatocellular carcinoma specimens and three-dimensional cultures. Three-dimensional invasion model was established to mimic in vivo invasion. RNA-sequencing was used to identify downstream effectors., Results: Human hepatocellular carcinoma underwent collective invasion and CD147 was observed to be upregulated at the invasive front of tumor cell groups. CD147 was demonstrated to promote collective invasion using the modified three-dimensional invasion model, which recapitulated the main features of collective invasion. Through transcriptome analysis and enzyme activity assay, we found that CD147 enhanced cathepsin B expression and activity. Upregulated cathepsin B in hepatocellular carcinoma cells facilitated migration and invasion, which mediated CD147-induced invasive phenotype in hepatocellular carcinoma. In terms of mechanism, we found that CD147 promoted cathepsin B transcription by activating β-catenin signaling as a result of reduced GSK-3β expression. Furthermore, we found that elevated expression of CD147 as well as cathepsin B were correlated with poor prognosis in patients with hepatocellular carcinoma., Conclusions: CD147 promotes hepatocellular carcinoma cells collective invasion via upregulating cathepsin B expression and targeting CD147 would be valuable for the development of novel therapeutic modalities against invasion and metastasis of cancer.

Background: Frequent recurrent hepatic metastasis after hepatic metastasectomy is a major obstacle in the treatment of colorectal liver metastasis (CRLM). We performed the present systematic review to evaluate the short- and long-term outcomes after repeat hepatectomy for recurrent CRLM and determine factors associated with survival in these patients., Data Sources: An electronic search of PubMed database was undertaken to identify all relevant peer-reviewed papers published in English between January 2000 and July 2018. Hazard ratios (HR) with 95% confidence interval (95% CI) were calculated for prognostic factors of overall survival (OS)., Results: The search yielded 34 studies comprising 3039 patients, with a median overall morbidity of 23% (range 8%-71%), mortality of 0 (range 0-6%), and 5-year OS of 42% (range 17%-73%). Pooled analysis showed that primary T3/T4 stage tumor (HR = 1.94; 95% CI: 1.04-3.63), multiple tumors (HR = 1.49; 95% CI: 1.10-2.01), largest liver lesion ≥5 cm (HR = 1.89; 95% CI: 1.11-3.23) and positive surgical margin (HR = 1.80; 95% CI: 1.09-2.97) at initial hepatectomy, and high serum level of carcinoembryonic antigen (HR = 1.87; 95% CI: 1.27-2.74), disease-free interval ≤12 months (HR = 1.34; 95% CI: 1.10-1.62), multiple tumors (HR = 1.64; 95% CI: 1.32-2.02), largest liver lesion ≥5 cm (HR = 1.85; 95% CI: 1.34-2.56), positive surgical margin (HR = 2.25; 95% CI: 1.39-3.65), presence of bilobar disease (HR = 1.62; 95% CI: 1.19-2.20), and extrahepatic metastases (HR = 1.60; 95% CI: 1.23-2.09) at repeat hepatectomy were significantly associated with poor OS., Conclusions: Repeat hepatectomy is a safe and effective therapy for recurrent CRLM. Long-term outcome is predicted mainly by factors related to repeat hepatectomy., (Copyright © 2019 First Affiliated Hospital, Zhejiang University School of Medicine in China. Published by Elsevier B.V. All rights reserved.)

Purpose: CD147 is a tumor-associated antigen that plays a key regulatory role in tumor invasion and distant metastasis. However, the exact role of CD147 phosphorylation, which is deregulated during cancer progression, is unknown. Here, the effects of CD147 phosphorylation on the malignant behavior of hepatocellular carcinoma (HCC) cells and its possible underlying mechanisms are explored., Methods: An in situ Duolink-proximity ligation assay (PLA) was used to detect CD147 phosphorylation. Tandem mass spectrometry was employed to identify the phosphorylation sites of CD147. The effects of CD147 phosphorylation on the malignant behavior of HCC cells were evaluated using scratch wound healing assays, transwell invasion assays and cell cycle assays. The genes regulated by CD147 phosphorylation were detected by RNA sequencing., Results: We identified phosphorylated serine-246 in the C terminus of CD147 in primary HCC tissues, whereas serine to alanine substitution mutation analysis suggested that CD147 is phosphorylated mainly at serine-252 in HCC-derived Huh-7 cells. Recovery expression of S246A/S252A mutants in CD147 knockout cells revealed significantly increased migration and invasion capacities compared to wildtype CD147 expressing cells. Cyclophilin A (CyPA) treatment decreased the phosphorylation level of CD147, whereas NIMA-related kinase 6 (NEK6) increased the CD147 phosphorylation level. Moreover, the CD147 phosphorylation level was found to be dramatically decreased in HCC tissues in patients with distant metastases, and a low phosphorylation level of CD147 was found to be associated with a high serum AFP level, recurrence and a poor overall survival., Conclusions: From our data we conclude that hypo-phosphorylated CD147 promotes the migration and invasion of HCC cells and correlates with an unfavorable prognosis in HCC patients, indicating that targeting the aberrantly hypo-phosphorylated form of CD147 may be instrumental for the development of novel therapeutic modalities directed against HCC metastasis.

Background: Combined hepatocellular-cholangiocarcinoma (cHCC-CC) is a rare primary liver malignancy. We conducted a systematic review and meta-analysis to assess the evidence available on the long-term outcomes of cHCC-CC patients after either hepatectomy or liver transplantation (LT)., Data Sources: Relevant studies published between January 2000 and January 2018 were identified by searching PubMed and Embase and reviewed systematically. Data were pooled using a random-effects model., Results: A total of 42 observational studies involving 1691 patients (1390 for partial hepatectomy and 301 for LT) were included in the analysis. The median tumor recurrence and 5-year overall survival (OS) rates were 65% (range 38%-100%) and 29% (range 0-63%) after hepatectomy versus 54% (range 14%-93%) and 41% (range 16%-73%) after LT, respectively. Meta-analysis found no significant difference in OS and tumor recurrence between LT and hepatectomy groups., Conclusion: Hepatectomy rather than LT should be considered as the prior treatment option for cHCC-CC., (Copyright © 2018 First Affiliated Hospital, Zhejiang University School of Medicine in China. Published by Elsevier B.V. All rights reserved.)

CD147, a type I transmembrane glycoprotein, is highly expressed in various cancer types and plays important roles in tumor progression, especially by promoting the motility and invasion of hepatocellular carcinoma (HCC) cells. These crucial roles make CD147 an attractive target for therapeutic intervention in HCC, but no small-molecule inhibitors of CD147 have been developed to date. To identify a candidate inhibitor, we used a pharmacophore model derived from the structure of CD147 to virtually screen over 300,000 compounds. The 100 highest-ranked compounds were subjected to biological assays, and the most potent one, dubbed AC-73 (ID number: AN-465/42834501), was studied further. We confirmed that AC-73 targeted CD147 and further demonstrated it can specifically disrupt CD147 dimerization. Moreover, molecular docking and mutagenesis experiments showed that the possible binding sites of AC-73 on CD147 included Glu64 and Glu73 in the N-terminal IgC2 domain, which two residues are located in the dimer interface of CD147. Functional assays revealed that AC-73 inhibited the motility and invasion of typical HCC cells, but not HCC cells that lacked the CD147 gene, demonstrating on-target action. Further, AC-73 reduced HCC metastasis by suppressing matrix metalloproteinase (MMP)-2 via down-regulation of the CD147/ERK1/2/signal transducer and activator of transcription 3 (STAT3) signaling pathway. Finally, AC-73 attenuated progression in an orthotopic nude mouse model of liver metastasis, suggesting that AC-73 or its derivatives have potential for use in HCC intervention. We conclude that the novel small-molecule inhibitor AC-73 inhibits HCC mobility and invasion, probably by disrupting CD147 dimerization and thereby mainly suppressing the CD147/ERK1/2/STAT3/MMP-2 pathways, which are crucial for cancer progression.

HAb18G/CD147 belongs to the immunoglobulin superfamily and predominantly functions as an inducer of matrix metalloproteinase secretion for tumor invasion and metastasis. This study was designed to investigate the effects of HAb18G/CD147 knockout on hepatocellular carcinoma cells using zinc-finger nuclease (ZFNs)-targeted gene knockout approach. The HCC cell line SMMC-7721 was used for ZFNs-targeted cleavage of the HAb18G/CD147 gene. RT-PCR and Western blot assays were used to detect HAb18G/CD147 expression. HAb18G phenotypic changes following HAb18G/CD147 knockout in SMMC-K7721 cells were assessed using tumor cell adhesion, invasion, migration and colony formation and flow cytometric assays. These data demonstrated that tumor cell adhesion, invasion, migration, and colony formation capabilities of SMMC-K7721 were significantly reduced compared to parental cells or SMMC-7721 with re-expression of HAb18G/CD147 protein transfected with HAb18G/CD147 cDNA. Moreover, knockout of HAb18G/CD147 expression also induced SMMC-K7721 cells to undergo apoptosis compared to SMMC-7721 and SMMC-R7721 (P < 0.01). Molecularly, protein expression of p53 was induced in these cells, but re-expression of HAb18G/CD147 reduced p53 levels in SMMC-R7721 cells, possibly through inhibition of the PI3K-Akt-MDM2 signaling pathway. The findings provide a novel insight into the mechanisms underlying HAb18G/CD147-induced progression of HCC cells.

Metastasis is considered a dynamic process in tumor development that is related to abnormal migration and invasion. Tumor cells can move as individual cells in two interconvertible modes: mesenchymal-type and amoeboid. Previously, we reported that the interaction between CD147 and Annexin II can inhibit the amoeboid movement in hepatocellular carcinoma (HCC) cells. However, the mechanism of CD147 involved in mesenchymal movement is still unclear. Notably, our results show overexpression of CD147 led to mesenchymal-type movement in HCC cells. Evidence indicated that the mesenchymal-type cell movement induced by CD147 was Src dependent, as observed by confocal microscopy and Rac1 activity assay. The phosphorylation of Src (pY416-Src) can be up-regulated by CD147, and this regulation is mediated by focal adhesion kinase (FAK). Next, we identified DOCK8 as a GEF for Rac1, a key molecule driving mesenchymal-type movement. We also found that Src promotes STAT3 phosphorylation and STAT3 facilitates DOCK8 transcription, thus enhancing DOCK8 expression and Rac1 activation. This study provides a novel mechanism of CD147 regulating mesenchymal-type movement in HCC cells.

The purpose of the present study was to evaluate the preventive effects of hydrazinocurcumin (HZC) on diethylnitrosamine (DEN)-induced hepatocarcinogenesis in a male Sprague Dawley (SD) rat model. One hundred and twenty male SD rats used in this study were divided into six groups. Those receiving DEN with curcumin (CUR) or HZC were studied compared with the DEN-alone group. The study demonstrated that DEN induced severe histological and immunohistochemical changes in liver tissues, significantly increasing the levels of liver marker enzymes (alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), γ-glutamyltransferase (GGT) and total bilirubin level (TBL)). The hepatocarcinoma incidences were 100.0%, 36.7% and 20.0% in the DEN-alone, DEN-CUR and DEN-HZC groups, respectively. Although macroscopic and microscopic features suggested that both CUR and HZC were effective in inhibiting DEN- induced hepatocarcinogenesis, HZC was exerted a stronger influence. Immunohistochemical analysis with PCNA demonstrated significantly differences among the groups (all P < 0.05). Taken together, the results suggested application of CUR and HZC could prevent the occurrence of carcinogenesis and HZC may be a more potent compound for prevention of DEN-induced hepatocarcinogenesis in rats than CUR.

Several lines of evidence suggest that HAb18G/CD147 interacts with the integrin variants α3β1 and α6β1. However, the mechanism of the interaction remains largely unknown. In this study, mammalian protein-protein interaction trap (MAPPIT), a mammalian two-hybrid method, was used to study the CD147-integrin β1 subunit interaction. CD147 in human hepatocellular carcinoma (HCC) cells was interfered with by small hairpin RNA. Nude mouse xenograft model and metastatic model of HCC were used to detect the role of CD147 in carcinogenesis and metastasis. We found that the extracellular membrane-proximal domain of HAb18G/CD147 (I-type domain) binds at the metal ion-dependent adhesion site in the βA domain of the integrin β1 subunit, and Asp(179) in the I-type domain of HAb18G/CD147 plays an important role in the interaction. The levels of the proteins that act downstream of integrin, including focal adhesion kinase (FAK) and phospho-FAK, were decreased, and the cytoskeletal structures of HCC cells were rearranged bearing the HAb18G/CD147 deletion. Simultaneously, the migration and invasion capacities, secretion of matrix metalloproteinases, colony formation rate in vitro, and tumor growth and metastatic potential in vivo were decreased. These results indicate that the interaction of HAb18G/CD147 extracellular I-type domain with the integrin β1 metal ion-dependent adhesion site motif activates the downstream FAK signaling pathway, subsequently enhancing the malignant properties of HCC cells.

Unlabelled: Tumor cells can move as individual cells in two interconvertible modes: mesenchymal mode and amoeboid mode. Cytoskeleton rearrangement plays an important role in the interconversion. Previously, we reported that HAb18G/CD147 and annexin II are interacting proteins involved in cytoskeleton rearrangement, yet the role of their interaction is unclear. In this study we found that the depletion of HAb18G/CD147 produced a rounded morphology, which is associated with amoeboid movement, whereas the depletion of annexin II resulted in an elongated morphology, which is associated with mesenchymal movement. The extracellular portion of HAb18G/CD147 can interact with a phosphorylation-inactive mutant of annexin II and inhibit its phosphorylation. HAb18G/CD147 inhibits Rho signaling pathways and amoeboid movement by inhibiting annexin II phosphorylation, promotes membrane localization of WAVE2 and Rac1 activation by way of the integrin-FAK-PI3K/PIP3 signaling pathway, and promotes the formation of lamellipodia and mesenchymal movement., Conclusion: These results suggest that the interaction of HAb18G/CD147 with annexin II is involved in the interconversion between mesenchymal and amoeboid movement of hepatocellular carcinoma cells., (Copyright © 2011 American Association for the Study of Liver Diseases.)

βig-h3 is a TGF-β (transforming growth factor β)-induced ECM (extracellular matrix) protein that induces the secretion of MMPs (matrix metalloproteinases). However, the mechanism of induction is yet to be established. In this study, siRNAs (small interfering RNAs) targeted against βig-h3 were transfected into SMMC-7721 cells [a HCC (human hepatocellular carcinoma) cell line] to knockdown the expression of βig-h3. We found that NiCl2, a potent blocker of extracellular Ca2+ entry, reduced βig-h3-induced secretion of MMP-2 and -9. Further investigation suggested that reduction in the levels of βig-h3 decreased the secretion of MMP-2 and -9 that was enhanced by an increase in the concentration of extracellular Ca2+. SNAP (S-nitroso-N-acetylpenicillamine), a NO (nitric oxide) donor, and 8-Br-cGMP (8-bromo-cGMP) inhibited thapsigargin-induced Ca2+ entry and MMP secretion in the invasive potential of human SMMC-7721 cells. Further, the inhibitory effects of 8-Br-cGMP and SNAP could be significantly enhanced by down-regulating βig-h3. βig-h3 attenuates the negative regulation of NO/cGMP-sensitive store-operated Ca2+ entry. Our findings suggest that the expression of βig-h3 might play an important role in the regulation of store-operated Ca2+ entry to increase the invasive potential of HCC cells.

Background & Objective: The blood supply of liver metastatic tumors affects the selection of administration approach and target vessels of interventional therapy. This study was to investigate the blood supply of liver metastatic tumors with both CT arterial portography (CTAP) via the superior mesenterica arterial (SMA) and multi-slice spiral CT perfusion imaging., Methods: Twenty patients with liver metastatic tumors received CTAP via SMA and multi-slice spiral CT perfusion imaging. The CT values of 33 metastatic lesions and 20 specimens of normal liver tissue from the patients were measured through the time-density curves (TDC) of region of interest (ROI). The blood flow (BF), blood volume (BV), mean transit time (MTT), capillary vessel permeability surface (PS) of the portal vein were calculated with single-blood perfusion model., Results: The diameters of the 33 metastatic lesions were 0.8-3.5 cm. The increment of CT values after enhancement was no more than 10 Hu in all lesions. At portal venous phase of SMA-digital subtraction angiography (DSA), no tumor vessel was found in the lesions. The BF, BV, MTT, and PS of the portal vein were (17.15+/-11.38) ml x min-(1) x 100 ml-(1), (1.62+/-0.97) ml x 100 g-(1), (9.47+/-2.78) s, and (3.97+/-3.44) ml x min-(1) x 100 ml-(1), respectively, in metastatic lesions, and were (133.43+/-67.17) ml x min-(1) x 100 ml-(1), (16.56+/-11.15) ml x 100 g-(1), (9.52+/-2.84) s, (35.16+/-14.03) ml x min-(1) x 100 ml-(1), respectively, in normal liver tissues. The differences in BF, BV and PS between the 2 groups were significant (P<0.001). However, the difference in MTT was not significant (P=0.96)., Conclusions: CTAP via SMA can be used to measure accurately portal venous flow. The portal vein is not involved in the blood supply of liver metastatic tumors.

Background & Objective: Hepatic metastases are common for patients with malignant tumors, especially for the gastrointestinal malignancies. Early diagnosis confers better prognosis. This study was designed to investigate the hepatic hemodynamic changes by multi-slice helical perfusion CT in rats with liver micrometastatases of Walker-256 tumor cells., Methods: Liver micrometastatases were produced in 22 SD rats by injecting 2 x 10(7) Walker-256 cells into the spleens. The ten experimental control rats were injected with normal saline solution. Ten rats were randomized into empty self control group from the experimental group before injecting tumor cells, which were studied by CT perfusion technique before being injected tumor cells. The time-density curves of the aorta, portal vein, and liver were used to calculate liver perfusion parameters by gradient method designed for the dual blood supply. These liver perfusion parameters were hepatic arterial perfusion (HAP), portal vein perfusion (PVP), hepatic perfusion index (HPI) and total hepatic blood flow. All the parameters were compared between the groups. H&E staining method was used to confirm the micrometastases pathologically., Results: In the experimental group, 19 rats were found with micrometastases, of which the diameter was 0.5 mm to 6.6 mm. HAP was (97.67+/-31.42) ml x min(-1) x (100 ml) (-1) in metastasis group, and (43.35+/-17.39)ml x min(-1) x (100 ml) (-1) in control group, and (40.77+/-18.91) ml x min(-1) x (100 ml) (-1) in empty self control group. PVP was (295.49+/-61.85) ml x min(-1) x (100 ml) (-1) in metastasis group, and (385.7+/-71.25) ml x min(-1) x (100 ml) (-1) in control group, and (362.73+/-78.56) ml x min(-1) x (100 ml) (-1) in empty self control group, It was found that the HAP was higher in the rats with micrometastases than in those of the empty control group and also those of control group (F=47.84, P<0.000,1). While the PVP was lower in the rats with micrometastases than in those of the two control groups (F=14.10, P<0.000,1). For the total hepatic blood flow, no significant difference was found among the three groups (F=1.39, P=0.255)., Conclusion: Higher HAP and lower PVP was noted in the rats with micrometastases. Perfusion CT technique can be used to evaluate the hepatic hemodynamic changes and thus has a potential clinical value for early diagnosis of liver micrometastases.