Boshi Sun

Gankyrin promotes tumor growth and metastasis through activation of IL‐6/STAT3 signaling in human cholangiocarcinoma

Although gankyrin is involved in the tumorigenicity and metastasis of some malignancies, the role of gankyrin in cholangiocarcinoma (CCA) is unclear. In this study we investigated the expression of gankyrin in human CCA tissues and cell lines. The effects of gankyrin on CCA tumor growth and metastasis were determined both in vivo and in vitro. The results showed that gankyrin was overexpressed in CCA tissues and cell lines. Gankyrin expression was associated with CCA histological differentiation, TNM stage, and metastasis. The multivariate Cox analysis revealed that gankyrin was an independent prognostic indicator for overall survival. Gankyrin overexpression promoted CCA cell proliferation, migration, and invasion, while gankyrin knockdown inhibited CCA tumor growth, metastasis, and induced Rb‐dependent senescence and G1 phase cell cycle arrest. Gankyrin increased the phosphorylation of signal transducer and activator of transcription 3 (STAT3) and promoted the nuclear translocation of p‐STAT3. Suppression of STAT3 signaling by small interfering RNA (siRNA) or STAT3 inhibitor interfered with gankyrin‐mediated carcinogenesis and metastasis, while interleukin (IL)‐6, a known upstream activator of STAT3, could restore the proliferation and migration of gankyrin‐silenced CCA cells. The IL‐6 level was decreased by gankyrin knockdown, while increased by gankyrin overexpression. Gankyrin regulated IL‐6 expression by way of facilitating the phosphorylation of Rb; meanwhile, rIL‐6 treatment increased the expression of gankyrin, suggesting that IL‐6 was regulated by a positive feedback loop involving gankyrin in CCA. In the xenograft experiments, gankyrin overexpression accelerated tumor formation and increased tumor weight, whereas gankyrin knockdown showed the opposite effects. The in vivo spontaneous metastasis assay revealed that gankyrin promoted CCA metastasis through IL‐6/STAT3 signaling pathway. Conclusion: Gankyrin is crucial for CCA carcinogenesis and metastasis by activating IL‐6/STAT3 signaling pathway through down‐regulating Rb protein. (Hepatology 2014;59:935–946)

Reciprocal activation between ATPase inhibitory factor 1 and NF‐κB drives hepatocellular carcinoma angiogenesis and metastasis

Hepatocellular carcinoma (HCC) is a highly vascularized tumor with frequent extrahepatic metastasis. Active angiogenesis and metastasis are responsible for rapid recurrence and poor survival of HCC. However, the mechanisms that contribute to tumor metastasis remain unclear. Here we evaluate the effects of ATPase inhibitory factor 1 (IF1), an inhibitor of the mitochondrial H(+)‐adenosine triphosphate (ATP) synthase, on HCC angiogenesis and metastasis. We found that increased expression of IF1 in human HCC predicts poor survival and disease recurrence after surgery. Patients with HCC who have large tumors, with vascular invasion and metastasis, expressed high levels of IF1. Invasive tumors overexpressing IF1 were featured by active epithelial‐mesenchymal transition (EMT) and increased angiogenesis, whereas silencing IF1 expression attenuated EMT and invasion of HCC cells. Mechanistically, IF1 promoted Snai1 and vascular endothelial growth factor (VEGF) expression by way of activating nuclear factor kappa B (NF‐κB) signaling, which depended on the binding of tumor necrosis factor (TNF) receptor‐associated factor 1 (TRAF1) to NF‐κB‐inducing kinase (NIK) and the disruption of NIK association with the TRAF2‐cIAP2 complex. Suppression of the NF‐κB pathway interfered with IF1‐mediated EMT and invasion. Chromatin immunoprecipitation assay showed that NF‐κB can bind to the Snai1 promoter and trigger its transcription. IF1 was directly transcribed by NF‐κB, thus forming a positive feedback signaling loop. There was a significant correlation between IF1 expression and pp65 levels in a cohort of HCC biopsies, and the combination of these two parameters was a more powerful predictor of poor prognosis. Conclusion: IF1 promotes HCC angiogenesis and metastasis by up‐regulation of Snai1 and VEGF transcription, thereby providing new insight into HCC progression and IF1 function. (Hepatology 2014;60:1659–1673)

Nutlin-3 overcomes arsenic trioxide resistance and tumor metastasis mediated by mutant p53 in Hepatocellular Carcinoma

BackgroundArsenic trioxide has been demonstrated as an effective anti-cancer drug against leukemia and solid tumors both in vitro and in vivo. However, recent phase II trials demonstrated that single agent arsenic trioxide was poorly effective against hepatocellular carcinoma (HCC), which might be due to drug resistance.MethodsMutation detection of p53 gene in arsenic trioxide resistant HCC cell lines was performed. The therapeutic effects of arsenic trioxide and Nutlin-3 on HCC were evaluated both in vitro and in vivo. A series of experiments including MTT, apoptosis assays, co-Immunoprecipitation, siRNA transfection, lentiviral infection, cell migration, invasion, and epithelial-mesenchy-mal transition (EMT) assays were performed to investigate the underlying mechanisms.ResultsThe acquisition of p53 mutation contributed to arsenic trioxide resistance and enhanced metastatic potential of HCC cells. Mutant p53 (Mutp53) silence could re-sensitize HCC resistant cells to arsenic trioxide and inhibit the metastatic activities, while mutp53 overexpression showed the opposite effects. Neither arsenic trioxide nor Nutlin-3 could exhibit obvious effects against arsenic trioxide resistant HCC cells, while combination of them showed significant effects. Nutlin-3 can not only increase the intracellular arsenicals through inhibition of p-gp but also promote the p73 activation and mutp53 degradation mediated by arsenic trioxide. In vivo experiments indicated that Nutlin-3 can potentiate the antitumor activities of arsenic trioxide in an orthotopic hepatic tumor model and inhibit the metastasis to lung.ConclusionsAcquisitions of p53 mutations contributed to the resistance of HCC to arsenic trioxide. Nutlin-3 could overcome arsenic trioxide resistance and inhibit tumor metastasis through p73 activation and promoting mutant p53 degradation mediated by arsenic trioxide.

FCN2 inhibits epithelial–mesenchymal transition-induced metastasis of hepatocellular carcinoma via TGF-β/Smad signaling

Hepatocellular carcinoma (HCC) is currently still a major cause of cancer-related deaths. Identifying early metastatic biomarkers and therapeutic targets for HCC is of great importance. Emerging evidence suggest that epithelial-mesenchymal transitions (EMTs) play important roles in tumor metastasis and recurrence. Understanding molecular mechanisms that regulate the EMT process is crucial for improving HCC. In this study, we find Ficolin-2 (FCN2) plays an essential role in metastasis and EMT of HCC. FCN2 expression is downregulated in HCC cells and tissues. Low level of FCN2 in HCCs is correlated with aggressive metastatic features, and would be a prognostic factor for overall disease-free survival of HCC patients. Ectopic expression of FCN2 markedly inhibits HCC cells migration, invasion as well as EMT in vitro and in vivo. Moreover, TGF-β is found contribute to the function of FCN2 in suppressing metastasis and EMT of HCC. Collectively, our data suggest that FCN2 may have prognostic value in HCC metastasis. Additionally, the FCN2/ TGF-β/EMT axis identified in this study provides novel insight into the mechanisms of HCC metastasis, which may facilitate the development of new therapeutics against HCC.

EF24 inhibits tumor growth and metastasis via suppressing NF-kappaB dependent pathways in human cholangiocarcinoma

A synthetic monoketone analog of curcumin, termed 3, 5-bis (2-flurobenzylidene) piperidin-4-one (EF24), has been reported to inhibit the growth of a variety of cancer cells both in vitro and in vivo. However, whether EF24 has anticancer effects on cholangiocarcinoma (CCA) cells and the mechanisms remain to be investigated. The aim of our study was to evaluate the molecular mechanisms underlying the anticancer effects of EF24 on CCA tumor growth and metastasis. Cell proliferation, apoptosis, migration, invasion, tumorigenesis and metastasis were examined. EF24 exhibited time- and dose-dependent inhibitory effects on HuCCT-1, TFK-1 and HuH28 human CCA cell lines. EF24 inhibited CCA cell proliferation, migration, and induced G2/M phase arrest. EF24 induced cell apoptosis along with negative regulation of NF-κB- X-linked inhibitor of apoptosis protein (XIAP) signaling pathway. XIAP inhibition by lentivirus mediated RNA interference enhanced EF24-induced apoptosis, while XIAP overexpression reduced it in CCA cells. In vivo, EF24 significantly suppressed the growth of CCA tumor xenografts and tumor metastasis while displaying low toxicity levels. Our findings indicate that EF24 is a potent antitumor agent that inhibits tumor growth and metastasis by inhibiting NF-κB dependent signaling pathways. EF24 may represent a novel approach for CCA treatment.

A preliminary study of ALPPS procedure in a rat model

Associating Liver Partition and Portal vein ligation for Staged hepatectomy (ALPPS) has been reported to be a novel surgical technique that provides fast and effective growth of liver remnant. Despite occasional reports on animal studies, the mechanisms of rapid liver regeneration in ALPPS remains unclear. In the present study, we intend to develop a reproducible rat model to mimick ALPPS and to explore the underlying mechanisms. Rats assigned to the portal vein ligation (PVL), left lateral lobe (LLL) resection, transection and sham groups served as controls. Results indicated that the regeneration rate in the remnant liver after ALPPS was two times relative to PVL, whereas rats with transection alone showed minimal volume increase. The expression levels of Ki-67 and PCNA were about ten-fold higher after ALPPS compared with the transection and LLL resection groups, and four-fold higher compared with the PVL group. The levels of TNF-α, IL-6 and HGF in the regenerating liver remnant were about three-fold higher after ALPPS than the controls. There was a more significant activation of NF-κB p65, STAT3 and Yap after ALPPS, suggesting synergistic activation of the pathways by PVL and transection, which might play an important role in liver regeneration after ALPPS.

Oleocanthal inhibits growth and metastasis by blocking activation of STAT3 in human hepatocellular carcinoma

We explored the anti-cancer capacity of (−)-oleocanthal in human hepatocellular carcinoma (HCC). (−)-Oleocanthal inhibited proliferation and cell cycle progression and induced apoptosis in HCC cells in vitro and suppressed tumor growth in an orthotopic HCC model. (−)-Oleocanthal also inhibited HCC cell migration and invasion in vitro and impeded HCC metastasis in an in vivo lung metastasis model. ( )-Oleocanthal acted by inhibiting epithelial-mesenchymal transition (EMT) through downregulation Twist, which is a direct target of STAT3. (−)-Oleocanthal also reduced STAT3 nuclear translocation and DNA binding activity, ultimately downregulating its downstream effectors, including the cell cycle protein Cyclin D1, the anti-apoptotic proteins Bcl-2 and survivin, and the invasion-related protein MMP 2. Overexpression of constitutively active STAT3 partly reversed the anti cancer effects of (−)-oleocanthal, which inhibited STAT3 activation by decreasing the activities of JAK1 and JAK2 and increasing the activity of SHP-1. These data suggest that (−)-oleocanthal may be a promising candidate for HCC treatment.

N‐myc downstream‐regulated gene 2 inhibits human cholangiocarcinoma progression and is regulated by leukemia inhibitory factor/MicroRNA‐181c negative feedback pathway

Increasing evidence supports a role for N‐myc downstream‐regulated gene 2 (NDRG2) deregulation in tumorigenesis. We investigated the roles and mechanisms of NDRG2 in human cholangiocarcinoma (CCA) progression. In the present study, expression of NDRG2, microRNA (miR)‐181c and leukemia inhibitory factor (LIF) in human CCA and adjacent nontumor tissues were examined. The effects of NDRG2 on CCA tumor growth and metastasis were determined both in vivo and in vitro. The role of the NDRG2/LIF/miR‐181c signaling pathway in cholangiocarcinogenesis and metastasis were investigated both in vivo and in vitro. The results showed that human CCA tissues exhibited decreased levels of NDRG2 and increased levels of miR‐181c and LIF compared with nontumor tissues. NDRG2 could inhibit CCA cell proliferation, chemoresistance, and metastasis both in vitro and in vivo. We found that NDRG2 is a target gene of miR‐181c, and the down‐regulation of NDRG2 was attributed to miR‐181c overexpression in CCA. Furthermore, miR‐181c can be activated by LIF treatment, whereas NDRG2 could inhibit LIF transcription through disrupting the binding between Smad, small mothers against decapentaplegic complex and LIF promoter. Down‐regulation of NDRG2 and overexpression of miR‐181c or LIF are significantly associated with a poorer overall survival (OS) in CCA patients. Finally, we found that a combination of NDRG2, miR‐181c, and LIF expression is a strong predictor of prognosis in CCA patients. Conclusion: These results establish the counteraction between NDRG2 and LIF/miR‐181c as a key mechanism that regulates cholangiocarcinogenesis and metastasis. Our results elucidated a novel pathway in NDRG2‐mediated inhibition of cholangiocarcinogenesis and metastasis and suggest new therapeutic targets, including NDRG2, LIF, miR‐181c, and transforming growth factor beta, in CCA prevention and treatment. (Hepatology 2016;64:1606‐1622)

Deregulated AJAP1|[sol]||[beta]|-catenin|[sol]|ZEB1 signaling promotes hepatocellular carcinoma carcinogenesis and metastasis

Adherens junctions-associated protein 1 (AJAP1) is an integral membrane protein that is thought to function as a tumor suppressor in various malignancies. Downregulation of AJAP1 mRNA levels may predict recurrence in hepatocellular carcinoma (HCC) patients, but the underlying molecular mechanism is unknown. This was addressed in the present study by examining the role of AJAP1 in HCC cell proliferation, migration, and invasion in vitro as well as in human specimens and mouse xenograft model. We found that AJAP1 expression was reduced in HCC cells and human HCC tissue, which was associated with metastasis. AJAP1 overexpression inhibited HCC progression and metastasis, while its silencing had the opposite effect both in vitro and in vivo. Furthermore, AJAP1 blocked epithelial–to–mesenchymal transition by interacting with β-catenin and inhibiting its nuclear translocation, which suppressed zinc finger E-box binding homeobox 1 (ZEB1) transcription. These results indicate that AJAP1 inhibits HCC metastasis, and is thus a potential therapeutic target for HCC treatment.