Post-transplant inflammatory cytokine signature adds value for predicting tumor recurrence after liver transplantation for hepatocellular carcinoma.

BACKGROUND: Cytokines are key regulators of post-transplant inflammation responses which reconstitute post-transplant hepatic and systemic environments to influence the likelihood of tumor relapse. This study investigated the prognostic value of post-transplant cytokines on tumor recurrence after liver transplantation (LT) for hepatocellular carcinoma (HCC). METHODS: A retrospective analysis was conducted in prospectively collected 150 adult HCC patients who received liver transplantation from 1997 to 2015. The post-transplant 41 inflammatory cytokines were quantified by multiplexing analysis and determined their prognostic value for predicting post-LT tumor recurrence by receiver operative characteristic analysis. A prediction model for post-LT tumor recurrence was generated by the logistic regression and internally validated Bootstrapping and compared with external prediction models. RESULTS: Post-transplant circulating CCL11, IFNα2, and IL17A cytokines were identified to be significant predictors of post-LT tumor recurrence and survival. A prediction score composed of the post-transplant 3-cytokine (P3C) signature, UCSF criteria, and pre-LT AFP was established. The P3C-UCSF-AFP score significantly predicted post-LT tumor recurrence and poor survival both in deceased donor liver transplantation (DDLT) and living donor liver transplantation (LDLT). The P3C-UCSF-AFP score was validated to significantly predict post-LT 2-year and 5-year tumor recurrence, outperforming the RETREAT score, French AFP model, up-to-seven, UCSF criteria, and Milan criteria. Importantly, the P3C-UCSF-AFP score could cost-effectively stratify high-risk recipients subjected to a refinement of post-recurrence survival. CONCLUSION: The integrated P3C-UCSF-AFP score not only compensated for the pre-LT unpredictability and predicted post-LT tumor recurrence accurately, but also guided the clinical refinements of post-LT surveillance and therapeutic strategies in transplant oncology.

Glutathione S-transferase A2 promotes hepatocellular carcinoma recurrence after liver transplantation through modulating reactive oxygen species metabolism.

Hepatocellular carcinoma (HCC) recurrence after liver transplantation remains a significant clinical problem. Ischemia-reperfusion injury (IRI) occurred inevitably at the early phase after liver transplantation (LT) spawns a significant risk of HCC recurrence. However, their linkage and IRI-derived risk factors for HCC recurrence remain exclusive. Understanding the mechanism of post-transplantation hepatic injury could provide new strategies to prevent the later event of HCC recurrence. We demonstrated that glutathione S-transferase A2 (GSTA2) expression was significantly associated with early phase hepatic and systemic injury and ROS level after liver transplantation. Early phase circulating GSTA2 (EPCGSTA2) protein was a significant predictor of HCC recurrence and survival. Heterogeneous single nucleotide polymorphism at G335C of GSTA2 was significantly associated with poor survival of HCC recipients. Enhancement of GSTA2 could protect HCC cells against H2O2-induced cell death by compensating for the elevated ROS stress. We also demonstrated that GSTA2 played crucial roles in regulating the ROS-associated JNK and AKT signaling pathways and ROS metabolism in HCCs in responding to a dynamic ROS environment. Functionally, endogenous or exogenous upregulation of GSTA2 could promote HCC growth and invasion through activating the epithelial-mesenchymal-transition process. Targeted inhibition of GSTA2 could suppress HCC growth and metastasis. In conclusion, GSTA2 could be a novel prognostic and therapeutic target to combat HCC recurrence after liver transplantation.

Type III TGF-ß Receptor Down-Regulation Promoted Tumor Progression via Complement Component C5a Induction in Hepatocellular Carcinoma.

Background and Aims-Transforming growth factor-beta (TGF-ß) signaling orchestrates tumorigenesis and one of the family members, TGF-ß receptor type III (TGFßR3), are distinctively under-expressed in numerous malignancies. Currently, the clinical impact of TGFßR3 down-regulation and the underlying mechanism remains unclear in hepatocellular carcinoma (HCC). Here, we aimed to identify the tumor-promoting roles of decreased TGFßR3 expression in HCC progression. Materials and Methods-For clinical analysis, plasma and liver specimens were collected from 100 HCC patients who underwent curative resection for the quantification of TGFßR3 by q-PCR and ELISA. To study the tumor-promoting mechanism of TGFßR3 downregulation, HCC mouse models and TGFßR3 knockout cell lines were applied. Results-Significant downregulation of TGFßR3 and its soluble form (sTGFßR3) were found in HCC tissues and plasma compared to healthy individuals (p < 0.01). Patients with <9.4 ng/mL sTGFßR3 exhibited advanced tumor stage, higher recurrence rate and shorter disease-free survival (p < 0.05). The tumor-suppressive function of sTGFßR3 was further revealed in an orthotopic mouse HCC model, resulting in 2-fold tumor volume reduction. In TGFßR3 knockout hepatocyte and HCC cells, increased complement component C5a was observed and strongly correlated with shorter survival and advanced tumor stage (p < 0.01). Interestingly, C5a activated the tumor-promoting Th-17 response in tumor associated macrophages. Conclusion-TGFßR3 suppressed tumor progression, and decreased expression resulted in poor prognosis in HCC patients through upregulation of tumor-promoting complement C5a.

Clinical significance and functional role of transmembrane protein 47 (TMEM47) in chemoresistance of hepatocellular carcinoma.

Chemoresistance is the main cause of chemotherapy failure in patients with hepatocellular carcinoma (HCC). The gene encoding transmembrane protein 47 (TMEM47) was previously identified to be significantly upregulated in HCC cell lines with acquired chemoresistance. The aim of the present study was to characterize the clinical significance and function of TMEM47 in HCC chemoresistance. The results demonstrated that the TMEM47 expression levels in the tumors of patients not responding to cisplatin­based transarterial chemoembolization (TACE) treatment was significantly higher compared with those in patients who responded to TACE treatment. Moreover, analyses from clinical samples and HCC cell lines indicated that TMEM47 expression may be upregulated in HCC in response to cisplatin treatment. Furthermore, the TMEM47 mRNA expression levels were positively correlated with the degree of cisplatin resistance of HCC cells. Overexpression of TMEM47 in HCC cells significantly promoted cisplatin resistance. The present study also demonstrated that targeted inhibition of TMEM47 could significantly reduce cisplatin resistance of cisplatin­resistant HCC cells via enhancing caspase­mediated apoptosis. In addition, targeted inhibition of TMEM47 enhanced the sensitivity of cisplatin­resistant cells to cisplatin via suppressing cisplatin­induced activation of the genes involved in drug efflux and metabolism. The present study also validated that TMEM47 expression was significantly correlated with multidrug resistance­associated protein 1 in patients with HCC who received TACE treatment. In conclusion, the findings of the present study demonstrated that TMEM47 may be a useful biomarker for predicting the response to chemotherapy and a potential therapeutic target for overcoming HCC chemoresistance.

NLRP3 inflammasome induced liver graft injury through activation of telomere-independent RAP1/KC axis.

Acute-phase inflammation plays a critical role in liver graft injury. Inflammasomes, multi-molecular complexes in the cytoplasm, are responsible for initiating inflammation. Here, we aimed to explore the role of inflammasomes in liver graft injury and further to investigate the regulatory mechanism. In a clinical liver transplant cohort, we found that intragraft expression of nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasomes was significantly up-regulated post-transplantation. Importantly, overexpression of NLRP3 was strongly associated with poor liver function characterized by high levels of ALT, AST, and urea, as well as neutrophil infiltration after transplantation. The significant correlation between NLRP3 and IL-1ß mRNA levels led us to focus on one of the associated upstream regulators, telomere-independent repressor activator protein 1 (RAP1), which was further proved to be co-localized with NLRP3 in neutrophils. In the liver of a mouse model (hepatic ischaemia/reperfusion and hepatectomy model) and isolated neutrophils from RAP1-/- mice, the expression levels of NLRP3 and keratinocyte chemoattractant (KC) were significantly down-regulated in contrast to those in wild types. The levels of ALT and AST, as well as the neutrophil infiltration, were also decreased by RAP1 deficiency. In our clinical validation, intragraft KC expression was associated with NLRP3 and co-localized with RAP1 in neutrophils. Furthermore, NLRP3 inflammasomes were up-regulated by recombinant KC in the isolated neutrophils and liver of the mouse model. Our data demonstrated that NLRP3 inflammasomes, activated by the RAP1/KC axis, played a critical role in initiating inflammation during the early stage of liver graft injury. Targeting RAP1/KC/NLRP3 inflammasomes may offer a new therapeutic strategy against liver graft injury. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

CXCL10/CXCR3 signaling mobilized-regulatory T cells promote liver tumor recurrence after transplantation.

BACKGROUND & AIMS: Liver graft injury and tumor recurrence are the major challenges of liver transplantation for the patients with hepatocellular carcinoma (HCC). Here, we aimed to explore the role and mechanism of liver graft injury mobilizing regulatory T cells (Tregs), which lead to late phase tumor recurrence after liver transplantation. METHODS: The correlation among tumor recurrence, liver graft injury and Tregs mobilization were studied in 257 liver transplant recipients with HCC and orthotopic rat liver transplantation models. The direct roles of CXCL10/CXCR3 signaling on Tregs mobilization and tumor recurrence were investigated in CXCL10-/- and CXCR3-/- mice models with hepatic IR injury. RESULTS: Clinically, patients received the graft with graft weight ratio (GWR) <60% had higher HCC recurrence after liver transplantation than the recipients with GWR ⩾60% graft. More circulating Tregs and higher intragraft TLR4/CXCL10/CXCR3 levels were detected in recipients with GWR <60% graft. These results were further validated in rat transplantation model. Foxp3+ cells and expressions of TLR4, CXCL10, TGFß, CTLA-4 and CD274 were increased in rat liver tumor tissues from small-for-size graft group. In mouse model, the mobilization and recruitment of Tregs were decreased in TLR4-/-, CXCL10-/- and CXCR3-/- mice compared to wild-type mice. Moreover, less CXCR3+ Tregs were recruited into liver in CXCL10-/- mice after hepatic IR injury. The knockout of CXCL10 and depletion of Tregs inhibited tumor recurrence after hepatic IR injury. CONCLUSION: CXCL10/CXCR3 signaling upregulated at liver graft injury directly induced the mobilization and intragraft recruitment of Tregs, which further promoted HCC recurrence after transplantation. LAY SUMMARY: There were positive correlation among tumor recurrence, circulating Tregs and liver graft injury after human transplantation for HCC patients. The knockout of CXCL10 decreased hepatic recruitment of CXCR3+ Tregs and late phase tumor recurrence after hepatic IR injury.

Repressor and activator protein accelerates hepatic ischemia reperfusion injury by promoting neutrophil inflammatory response.

Repressor and activator protein (Rap1) directly regulates nuclear factor-κB (NF-κB) dependent signaling, which contributes to hepatic IRI. We here intended to investigate the effect of Rap1 in hepatic ischemia reperfusion injury (IRI) and to explore the underlying mechanisms. The association of Rap1 expression with hepatic inflammatory response were investigated in both human and rat liver transplantation. The effect of Rap1 in hepatic IRI was studied in Rap1 knockout mice IRI model in vivo and primary cells in vitro. Our results showed that over expression of Rap1 was associated with severe liver graft inflammatory response, especially in living donor liver transplantation. The results were also validated in rat liver transplantation model. In mice hepatic IRI model, the knockout of Rap1 reduced hepatic damage and hepatic inflammatory response. In primary cells, the knockout of Rap1 suppressed neutrophils migration activity and adhesion in response to liver sinusoidal endothelial cells through down-regulating neutrophils F-Actin expression and CXCL2/CXCR2 pathway. In addition, the knockout of Rap1 also decreased production of pro-inflammatory cytokines/chemokines in primary neutrophils and neutrophils-induced hepatocyte damage. In conclusion, Rap1 may induce hepatic IRI through promoting neutrophils inflammatory response. Rap1 may be the potential therapeutic target of attenuating hepatic IRI.

Early-phase circulating miRNAs predict tumor recurrence and survival of hepatocellular carcinoma patients after liver transplantation.

Post-liver transplantation tumor recurrence is a major challenge for hepatocellular carcinoma (HCC) recipients. We aimed to identify early-phase circulating microRNAs after liver transplantation for predicting tumor recurrence and survival of HCC recipients. Circulating microRNA profiles at early-phase (2-hour after portal vein reperfusion) after liver transplantation were compared between HCC recipients with (n=4) and without tumor recurrence (n=8) by microarray analyses. Candidate microRNAs were validated in 62 HCC recipients by quantitative RT-PCR. The prognostic values of microRNAs for tumor recurrence and survival were examined. Simulated in vitro ischemia-reperfusion injury models were employed to characterize the possible mechanism of up-regulation of circulating microRNAs. Our results showed that up-regulation of circulating miR-148a, miR-1246 or miR-1290 at early-phase was significantly associated with HCC recurrence after liver transplantation. Among them, miR-148a (p=0.030) and miR-1246 (p=0.009) were significant predictors of HCC recurrence. MiR-1246 was an independent predictor of overall (p=0.023) and disease-free survival (p=0.020) of HCC recipients. The level of early-phase circulating miR-1246 was positively correlated with serum AST and ALT levels in HCC recipients after liver transplantation. The expression of hepatic miR-1246 was positively correlated with TNFα mRNA. In vitro experiments indicated that injury-induced activation and differentiation of macrophages significantly elevated the expression and secretion of miR-1246. In conclusion, early-phase circulating miR-1246 is an indicator of hepatic injury and a novel prognostic biomarker for tumor recurrence and survival of HCC recipients after liver transplantation.