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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.

Neuropilin-1 (NRP1) is a type I transmembrane glycoprotein that was originally found playing a role in neuronal axon guidance and embryonic angiogenesis. NRP1 can promote various aspects of tumorigenesis, such as angiogenesis, cell survival, migration, invasion and chemo-resistance, but little is known about the nanomechanica signature of NRP1 inregulating hepatocellular carcinoma(HCC) cells. Our study found that NRP1 was overexpressed in HCC and high expression of NRP1 was associated with poor prognosis in HCC patients.but there are no correlation between NRP1 and liver cirrhosis. In vivo and vitro experiments knockdown of NRP1 reduced proliferation and invasion of HCC. We quantified the Young's modulus, RMS residuals of HCC cell lines and normal liver cell line L02 using Atomic force microscopy (AFM). AFM data indicated that Young's modulus decreased in highly metastatic HCC cells, which is consistent with the verification results of NRP1 knockdown. Our analyses revealed that down regulation of NRP1 negatively correlated with increased Young's modulus in cells exhibiting differing degrees of invasiveness. Our molecular data showed that NRP1 activated AKT, ERK1/2, and epithelial-mesenchymal transition (EMT)-related signals in HCC cells and promoted tumor apoptosis and invasion. As an important biophysical property of cells, the membrane stiffness of HCC cells was further quantified by AFM force measurements, which indicated that these cells became much stiffer after NRP1 knockdown. These results collectively illuminated that Neuropilin-1 alterrated namomechanica signature in hepatocellular carcinoma cells and can be a potential therapeutic agent for HCC. Funding Statement: The present study was supported by the National Natural Science Fund of China (grant nos. 31570509 and 81702326), Science and Technology Major Project of Gansu province(1602FKDA001), the Talent innovation and Entrepreneurship Program of Lanzhou City (grant no. 2016-RC57) and the science and technology project of Chenguan District (2017SHFZ0014); Declaration of Interests: The authors declare no conflicts of interest. Ethics Approval Statement: The study was approved by the Zhongshan Hospital Ethics Committee and informed consent was obtained from each patient under Institutional Reviewer Board protocols. The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a prior approval by the institution's human research committee.

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.

Background: Due to the phenotypic and molecular diversity of hepatocellular carcinomas (HCC), it is a challenge to determine patients' prognosis. We aimed to identify new biomarkers of resected patients. Methods: We collected 274 HCC samples from patients at Zhongshan hospital, Fudan university. Gene expression analyses were performed using NanoString technology and compared with patient survival times. Using the coefficient and regression formula of the multivariate Cox model, we identified a "9-gene signature" associated with survival times. This molecular signature was then validated in three groups of patients from internal cohort (n=69), TCGA dataset (n=369) and HCC genomic consortium (n=80). Results: We identified 9-gene signature consisting of ZC2HC1A, MARCKSL1, PTGS1, CDKN2B, CLEC10A, PRDX3, PRKCH, MPEG1 and LMO2. The 9-gene signature was used, combined with clinical parameters, to fit a multivariable Cox model to the training data (concordance index, ci=0.85), which was successfully validated (ci=0.86 for internal cohort; ci=0.78 for external cohort). The signature showed improved performance compared with clinical parameters alone (ci=0.70). Furthermore, the signature more accurately predicted patient outcomes than gene signatures reported previously. It was also used to stratify early-stage, HBV or HCV-infected patients into groups with low and high risk of survival, leading to significant differences in survival in training and validation (P

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.