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Iron dyshomeostasis is associated with hepatocellular carcinoma (HCC) development. However, the role of iron in HCC metastasis is unknown. This study aimed to elucidate the underlying mechanisms of iron’s enhancement activity on HCC metastasis. In addition to the HCC cell lines and clinical samples in vitro, iron deficient (ID) mouse models were generated using iron-free diet and transferrin receptor protein knock out, followed by administration of HCC tumors through either orthotopic or ectopic route. Clinical metastatic HCC samples showed significant ID status, accompanied by overexpression of sphingosine-1-phosphate transporter spinster homologue 2 (SPNS2). Mechanistically, ID increased SPNS2 expression, leading to HCC metastasis in both cell cultures and mouse models. ID not only altered the anti-tumor immunity, which was indicated by phenotypes of lymphatic subsets in the liver and lung of tumor-bearing mice, but also promoted HCC metastasis in a cancer cell autonomous manner through the SPNS2. Since germline knockout of globe SPNS2 showed significantly reduced HCC metastasis, we further developed hepatic-targeting recombinant adeno-associated virus vectors to knockdown SPNS2 expression and to inhibit iron-regulated HCC metastasis. Our observation indicates the role of iron in HCC pulmonary metastasis and suggests SPNS2 as a potential therapeutic target for the prevention of HCC pulmonary metastasis.

Background & Aims Early recurrence of hepatocellular carcinoma (HCC) after curative resection is common. However, the association between genetic mechanisms and early HCC recurrence, especially in Chinese patients, remains largely unknown. Methods We performed whole-genome sequencing (49 cases), whole-exome sequencing (18 cases), and deep targeted sequencing (115 cases) on 182 primary HCC samples. Focusing on WNK2, we used Sanger sequencing and qPCR to evaluate all the coding exons and copy numbers of that gene in an additional 554 HCC samples. We also explored the functional effect and mechanism of WNK2 on tumor growth and metastasis. Results We identified 5 genes (WNK2, RUNX1T1, CTNNB1, TSC1, and TP53) harboring somatic mutations that correlated with early tumor recurrence after curative resection in 182 primary HCC samples. Focusing on WNK2, the overall somatic mutation and copy number loss occurred in 5.3% (39/736) and 27.2% (200/736), respectively, of the total 736 HCC samples. Both types of variation were associated with lower WNK2 protein levels, higher rates of early tumor recurrence, and shorter overall survival. Biofunctional investigations revealed a tumor-suppressor role of WNK2: its inactivation led to ERK1/2 signaling activation in HCC cells, tumor-associated macrophage infiltration, and tumor growth and metastasis. Conclusions Our results delineate genomic events that characterize Chinese HCCs and identify WNK2 as a driver of early HCC recurrence after curative resection. Lay summary We applied next-generation sequencing and conducted an in-depth genomic analysis of hepatocellular carcinomas from a Chinese patient cohort. The results delineate the genomic events that characterize hepatocellular carcinomas in Chinese patients and identify WNK2 as a driver associated with early tumor recurrence after curative resection.

Major treatments for small hepatocellular carcinoma (SHCC) include percutaneous ethanol injection (PEI), percutaneous acetic acid injection (PAI), radiofrequency ablation (RFA), or surgical resection (SR). We aimed to compare these therapies concerning with effectiveness and safety.Cochrane Library, PubMed, and Embase were searched for randomized controlled studies (RCTs) from inception to 30 April 2017. Odds ratios (OR) for proportion dead (PD), local recurrence (LR) and adverse events (AEs).Fourteen RCTs were identified. Compared with SR, PEI (OR 2.79, CrI 1.25, 6.45, p 0.01) provided a significantly increased risk of PD. Similarly, PEI (OR 4.29, CrI 1.18, 18.35, p 0.01) yielded more LR than SR. Also, SR significantly conferred more AEs than RFA (OR 0.10; CrI 0.02, 0.35, p 0.01), PEI (OR 0.06; CrI 0.01, 0.31, p 0.01). Besides, RFA conferred the highest efficacy for survival, time to recurrence, and new development of HCC.SR was superior to PEI. Although SR achieved highest cumulative ranking probabilities in clinical efficacy, it obtained a low benefit-to-risk ratio for patients. RFA was superior to the other ablative therapies. For tumor sizes 2 cm or ≤ 2 cm in diameter, SR conferred non-significant effects compared with other therapies for SHCC.

Upregulation of CXCR2 in tumor cells has been documented in several types of cancer. As one of its ligands, CXCL5 is associated with neutrophil infiltration and poor prognosis in hepatocellular carcinoma (HCC). However, little is known about the role of the CXCR2/CXCL5 axis in the invasion and metastasis of HCC cells. In this study, we examined CXCR2 expression in human HCC cell lines and in three independent cohorts of HCC patients. The molecular effects of high expression levels of CXCR2 and CXCL5 in HCC cells were determined using qRT-PCR, western blot analysis, immunofluorescence, matrigel invasion assay, and xenograft mouse models. We found that high levels of CXCR2 correlated with progression and poor prognosis in human HCC. CXCR2/CXCL5 together promoted cell spreading by inducing the epithelial-mesenchymal transition (EMT) through activation of the PI3K/Akt/GSK-3β/Snail signaling pathway. In clinical HCC samples, high expression of both CXCR2 and CXCL5 showed a significant correlation with the activation of PI3K/Akt/GSK-3β/Snail signaling and EMT phenotype. In conclusion, our data showed that the CXCR2/CXCL5 axis contributes to EMT of HCC cells through activating PI3K/Akt/GSK-3β/Snail signaling, and it may serve as a potential therapeutic target.

Calpain small subunit 1 (Capn4) has been identified as a major gene that promotes metastasis of hepatocellular carcinoma (HCC). However, the mechanism by which Capn4 promotes progression of HCC is not understood. In this study, we found that Capn4 expression was increased in highly metastatic HCC cell lines and in tumour tissue from HCC patients compared to healthy patient tissue. Over-expression of Capn4 in HCC cells enhanced tumour cell growth in vitro and increased invasiveness, tumourigenicity and lung metastasis in vivo. Protein microarray analyses showed that expression of multiple proteins was regulated by Capn4. Interestingly, Capn4 was found to physically associate with FAK and promoted hyperactivity of the FAK-Src signalling pathway via increased phosphorylation of specific tyrosine residues of FAK, Src and p130Cas. Knock-down of Capn4 expression suppressed the malignant behaviour of HCC cells and inhibited the FAK-Src signalling pathway. Furthermore, Capn4-mediated invasion and metastasis of HCC cells required up-regulation of matrix metalloproteinase-2 (MMP2) through activation of this signalling pathway. Our clinical data revealed that Capn4 expression correlated well with the levels of phospho-FAK, and over-expression of both Capn4 and phospho-FAK correlates with the poorest survival outcomes in HCC. In conclusion, our data showed that Capn4 can contribute to HCC growth and metastasis via activation of the FAK-Src signalling pathway and MMP2.

CXCL5 (epithelial neutrophil-activating peptide-78) is a member of a proangiogenic subgroup of the CXC-type chemokine family of small, secreted proteins. Recently, evidence that CXCL5 is involved in carcinogenesis and cancer progression has emerged. To investigate the role of CXCL5 in tumor growth, invasion, and prognosis of hepatocellular carcinoma (HCC), we examined CXCL5 messenger RNA (mRNA) and protein levels in HCC cell lines with various metastatic potentials and in three independent cohorts of 919 HCC patients. We found that CXCL5 expression was increased in the highly metastatic HCC cell lines and in tumor tissues from patients with recurrent HCC compared to controls. CXCL5 activated the PI3K-Akt and ERK1/2 signaling pathways in HCC cells and promoted proliferation, migration, and invasion. Furthermore, we found that CXCL5 had a direct chemoattractant effect on neutrophils in vitro. In animal studies, the up-regulation of CXCL5 in HCC cells promoted tumor growth, lung metastasis, and intratumoral neutrophil infiltration. Conversely, down-regulation of CXCL5 in HCC cells reduced tumor growth, metastasis, and intratumoral neutrophil infiltration. Immunohistochemical analysis in HCC samples showed that overexpression of CXCL5 was well correlated with intratumoral neutrophil infiltration, shorter overall survival, and tumor recurrence. Multivariate analysis revealed that CXCL5 overexpression alone, or combined with the presence of intratumoral neutrophils, was an independent prognostic indicator for overall survival and cumulative recurrence. Conclusion: CXCL5 promotes HCC cell proliferation, invasion, and intratumoral neutrophil infiltration. CXCL5 overexpression, alone or combined with intratumoral neutrophil presence, is a novel prognostic predictor, and CXCL5 is a potential therapeutic target for HCC. (HEPATOLOGY 2012;56:2242–2254)

Background Rejection of transplanted liver occurs when the host generates alloantigen-reactive T cells, and CD4 + T-cell subsets, including Th1, Th2, T17 and iTreg, could be involved in changing the dynamics of graft rejection onset. In the current immunosuppressive strategies, rejection is treated as an undifferentiated process that is uniform, which results in the failure of tolerance induction. Here, we established a rejection model to observe the reciprocal interaction of CD4 + T-cell subsets in the complex networks of the immune system. Methods Orthotopic liver transplantation (OLT) from male inbred Lewis rats ( n = 15) to male inbred Brown Norway (BN) rats was performed by Kamada's two-cuff technique without reconstruction of the hepatic artery. OLT from BN to BN rats ( n = 5) was performed as a control. Recipients were sacrificed on postoperative days 3, 5, 7, 9, 11, 13 and 15. Recipient spleens and grafts were harvested, fixed in 10% neutral formalin, and embedded in paraffin. Meanwhile, hematoxylin and eosin and immunohistochemical staining was done, acute rejection was graded by the Banff scheme, and the number of T-bet + , GATA-3 + , RORγt + and FOXP3 + cells in the spleen and grafts were counted. Results In recipient spleens, the T-bet + and RORγt + cells were increased more significantly in the mild acute rejection (AR) group than in the control group ( P = 0.016, P = 0.009, respectively), while both cell types were decreased in the moderate AR group. Compared with the control group, the RORγt + cells did not differ significantly in the severe AR group, while the T-bet + cells were significantly decreased ( P = 0.465, P = 0.009, respectively). The GATA-3 + cells were significantly decreased in the mild AR group compared with the control group ( P = 0.009). With regard to the FOXP3 + cells, there was no significant difference between the control and mild AR groups ( P = 0.754), while they were significantly decreased in the moderate and severe AR groups ( P = 0.028, P = 0.009, respectively). The ratio of T-bet + /GATA-3 + cells was more associated with AR than was the RORγt + /FOXP3 + cell ratio in the early stage. In the graft, the T-bet + and RORγt + cells were significantly increased in mild, moderate and severe AR groups compared with the control group ( P + cell was not significantly increased in the mild AR group compared with the control group, while it was significantly increased in the moderate and severe AR groups ( P = 0.028, P = 0.009, respectively). Concerning the FOXP3 + cells, no significant difference was found between the control and mild AR groups ( P = 0.347), while they were significantly increased in the moderate and severe AR groups ( P Conclusions Our study revealed the dynamic changes of T-bet + , GATA-3 + , RORγt + and FOXP3 + cells in the spleen and grafts of recipient rats. It seems that the ratio of T-bet + /GATA-3 + cells was more associated with AR than was RORγt + /FOXP3 + cells in the early stage of rejection, while the ratio of RORγt + /FOXP3 + cells was associated more with the later but more persistent stage of rejection. This study could make a contribution to the optimal selection of immunosuppressive regimens according to the dynamic changes in CD4 + T-cell subsets at different stages of rejection.

Ten eleven translocation (TET) enzymes convert 5-methylcytosine (5-mC) to 5-hydroxy-methylcytosine (5-hmC) and have crucial roles in biological and pathological processes by mediating DNA demethylation, however, the functional role of this epigenetic mark and the related enzymes in hepatocellular carcinoma (HCC) progression remains unknown. Here, we demonstrated that TET-family enzymes downregulation was one likely mechanism underlying 5-hmC loss in HCC. We found that miR-29a overexpression increased DNA methylation of suppressor of cytokine signaling 1 (SOCS1) promoter was associated with HCC metastasis in vitro and in vivo. Furthermore, miR-29a silenced anti-metastatic SOCS1 through direct TET-family targeting, resulting in SOCS1 promoter demethylation inhibition. Chromatin immunoprecipitation analyses confirmed that TET1 regulated SOCS1 expression through binding to the promoter region of SOCS1. Finally, miR-29a overexpression correlated with poor clinical outcomes and TET–SOCS1–matrix metalloproteinase (MMP) 9 axis silencing in HCC patients. In conclusion, our findings demonstrate that 5-hmC loss is an epigenetic hallmark of HCC, and miR-29a is an important epigenetic modifier, promoting HCC metastasis through TET–SOCS1–MMP9 axis silencing. The results offer a new strategy for epigenetic cancer therapy.

Calpain small subunit 1 (Capn4) has been identified as a major gene that promotes metastasis of hepatocellular carcinoma (HCC). However, the mechanism by which Capn4 promotes progression of HCC is not understood. In this study, we found that Capn4 expression was increased in highly metastatic HCC cell lines and in tumour tissue from HCC patients compared to healthy patient tissue. Over-expression of Capn4 in HCC cells enhanced tumour cell growth in vitro and increased invasiveness, tumourigenicity and lung metastasis in vivo. Protein microarray analyses showed that expression of multiple proteins was regulated by Capn4. Interestingly, Capn4 was found to physically associate with FAK and promoted hyperactivity of the FAK-Src signalling pathway via increased phosphorylation of specific tyrosine residues of FAK, Src and p130Cas. Knock-down of Capn4 expression suppressed the malignant behaviour of HCC cells and inhibited the FAK-Src signalling pathway. Furthermore, Capn4-mediated invasion and metastasis of HCC cells required up-regulation of matrix metalloproteinase-2 (MMP2) through activation of this signalling pathway. Our clinical data revealed that Capn4 expression correlated well with the levels of phospho-FAK, and over-expression of both Capn4 and phospho-FAK correlates with the poorest survival outcomes in HCC. In conclusion, our data showed that Capn4 can contribute to HCC growth and metastasis via activation of the FAK-Src signalling pathway and MMP2.