The overall survival of patients with hepatocellular carcinoma (HCC) remains poor, and the molecular mechanisms underlying HCC progression and aggressiveness are unclear. Here, we report that increased expression of p28 GANK (Gankyrin, PSMD10, or p28) in human HCC predicts poor survival and disease recurrence after surgery. Patients with HCC who have large tumors, with vascular invasion and intrahepatic or distant metastasis, expressed high levels of p28 GANK . Invasive tumors overexpressing p28 GANK were featured by active epithelial-mesenchymal transition (EMT), and exhibited increased angiogenesis associated with vascular endothelial growth factor overexpression, whereas silencing p28 GANK expression attenuated EMT and motility/invasion of tumor cells. The p28 GANK activates phosphoinositide 3-kinase (PI3K)-V-akt Murine Thymoma Viral Oncogene Homolog (AKT)-hypoxia-inducible factor 1a (HIF-1a) signaling to promote TWIST1, vascular endothelial growth factor, and metalloproteinase 2 expression. Suppression of the PI3K-AKT-HIF-1a pathway interfered with p28 GANK -mediated EMT and invasion. Consistently, we detected a significant correlation between p28GANK expression and p-AKT levels in a cohort of HCC biopsies, and the combination of these two parameters is a more powerful predictor of poor prognosis. Conclusion: These results present novel mechanistic insight into a critical role of p28 GANK in HCC progression and metastasis. (HEPATOLOGY 2011;53:181-192)

Background and Purpose: Necroptosis is a form of programmed, caspaseindependent, cell death, mediated by receptor-interacting protein kinases, RIPK1 and RIPK3, and the mixed lineage kinase domain-like (MLKL). Necroptosis contributes to the pathophysiology of various inflammatory, infectious, and degenerative diseases. Thus, identification of low MW inhibitors for necroptosis has broad therapeutic relevance. Here, we identified that the pan-Raf inhibitor TAK-632 was also an inhibitor of necroptosis. We have further generated a more selective, highly potent analogue of TAK-632 by targeting RIPK1 and RIPK3. Experimental Approach: Cell viability was measured by MTT, propidium staining, or CellTiter-Glo luminescent assays. Effects of TAK-632 on necroptosis signalling pathways were investigated by western blotting, immunoprecipitation, and in vitro kinase assays. Downstream targets of TAK-632 were identified by a drug affinity responsive target stability assay and a pull-down assay with biotinylated TAK-632. A mouse model of TNF-α-induced systemic inflammatory response syndrome (SIRS) was further used to explore the role of TAK-632 in protecting against necroptosisassociated inflammation in vivo.Key Results: TAK-632 protected against necroptosis in human and mouse cells but did not protect cells from apoptosis. TAK-632 directly bound with RIPK1 and RIPK3 to inhibit kinase activities of both enzymes. In vivo, TAK-632 alleviated TNF-induced SIRS. Furthermore, we performed a structure-activity relationship analysis of TAK-632 analogues and generated SZM594, a highly potent inhibitor of RIPK1/3. Abbreviations: DARTS, drug affinity responsive target stability assay; Nec-1, necrostatin-1; SAR, structure-activity relationship; SIRS, systemic inflammatory response syndrome Xiaofei Chen, Chunlin Zhuang and Yibin Ren are contributed equally to this work.

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BackgroundOur previous studies showed that ZBTB20, a new BTB/POZ-domain gene, could negatively regulate α feto-protein and other liver-specific genes, concerning such as bio-transformation, glucose metabolism and the regulation of the somatotropic hormonal axis. The aim of this study is to determine the potential clinical implications of ZBTB20 in hepatocellular carcinoma (HCC).MethodsQuantitative real-time RT-PCR and Western blot analyses were used to detect expression levels of ZBTB20 in 50 paired HCC tumorous and nontumorous tissues and in 20 normal liver tissues. Moreover, expression of ZBTB20 was assessed by immunohistochemistry of paired tumor and peritumoral liver tissue from 102 patients who had undergone hepatectomy for histologically proven HCC. And its relationship with clinicopathological parameters and prognosis was investigated.ResultsBoth messenger RNA and protein expression levels of ZBTB20 were elevated significantly in HCC tissues compared with the paired non-tumor tissues and normal liver tissues. Overexpressed ZBTB20 protein in HCC was significantly associated with vein invasion (P = 0.016). Importantly, the recurrence or metastasis rates of HCCs with higher ZBTB20 expression were markedly greater than those of HCCs with lower expression (P = 0.003, P = 0.00015, respectively). Univariate and multivariate analyses revealed that ZBTB20 overexpression was an independent prognostic factor for HCC. The disease-free survival period and over-all survival period in patients with overexpressed ZBTB20 in HCC was significantly reduced.ConclusionsThe expression of ZBTB20 is increased in HCC and associated with poor prognosis in patients with HCC, implicating ZBTB20 as a candidate prognostic marker in HCC.

Hepatocellular carcinoma (HCC) is a prototype of inflammation‐associated cancer. Oncoprotein Gankyrin, which mostly increases in HCC, plays a critical role in HCC development and metastasis. However, the exact mechanism of Gankyrin up‐regulation in HCC remains unclear. A Gankyrin luciferase reporter was developed to screen a potential regulator for Gankyrin from a list of proinflammatory cytokines, and interleukin (IL)‐1β was found as one of its activators. In clinical premalignant and malignant liver disease samples, enhanced IL‐1β/interleukin‐1 receptor‐associated kinase 1 (IRAK‐1) signaling accompanied by increased Gankyrin was observed. Lower expression of Gankyrin and phospho‐IRAK‐1 are favorable prognostic markers for HCC. A similar correlation was observed in the diethylnitrosamine (DEN) model of rat hepatocarcinogenesis. The results from Gankyrin reporter activity, real‐time polymerase chain reaction, or immunoblotting further confirmed the up‐regulation of Gankyrin by IL‐1β/IRAK‐1 inflammatory signaling. Moreover, a series of Gankyrin's truncated reporters were constructed, and electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) were performed to analyze the properties of Gankyrin promoter. Mechanistically, the core promoter of Gankyrin contains the binding site of nuclear factor Y (NF‐Y) family members, which can recruit histone acetyltransferase coactivator E1A‐binding protein p300 (p300) or CREB‐binding protein (CBP) to promote Gankyrin transcription. Conversely, knockdown of NF‐Y, p300, or CBP inhibits Gankyrin expression. IL‐1β stimulation causes sequential phosphorylation of IRAK‐1, c‐Jun N‐terminal kinase (JNK), and p300 and enhances recruitment of the p300/CBP/NF‐Y complex to Gankyrin promoter. Inhibition of phospho‐JNK impairs IL‐1β/IRAK‐1 signaling‐mediated up‐regulation of Gankyrin. Conclusion: The finding of IL‐1β/IRAK‐1 signaling promoting Gankyrin expression through JNK and NF‐Y/p300/CBP complex provides a fresh view on inflammation‐enhanced hepatocarcinogenesis. (Hepatology 2015;61:585‐597)

Hepatocellular carcinoma (HCC) is the global leading cause of cancer-related deaths due to the deficiency of targets for precision therapy. A new modality of epigenetic regulation has emerged involving RNA–RNA crosstalk networks where two or more competing endogenous RNAs (ceRNAs) bind to the same microRNAs. However, the contribution of such mechanisms in HCC has not been well studied. Herein, potential HMGB1-driven RNA–RNA crosstalk networks were evaluated at different HCC stages, identifying the mTORC2 component RICTOR as a potential HMGB1 ceRNA in HBV+ early stage HCC. Indeed, elevated HMGB1 mRNA was found to promote the expression of RICTOR mRNA through competitively binding with the miR-200 family, especially miR-429. Functional assays employing overexpression or interference strategies demonstrated that the HMGB1 and RICTOR 3′untranslated regions (UTR) epigenetically promoted the malignant proliferation, self-renewal, and tumorigenesis in HCC cells. Intriguingly, interference against HMGB1 and RICTOR in HCC cells promoted a stronger anti-PD-L1 immunotherapy response, which appeared to associate with the production of PD-L1+ exosomes. Mechanistically, the HMGB1-driven RNA-RNA crosstalk network facilitated HCC cell glutamine metabolism via dual mechanisms, activating a positive feedback loop involving mTORC2-AKT-C-MYC to upregulate glutamine synthetase (GS) expression, and inducing mTORC1 signaling to derepress SIRT4 on glutamate dehydrogenase (GDH). Meanwhile, this crosstalk network could impede the efficacy of immunotherapy through mTORC1-P70S6K dependent PD-L1 production and PD-L1+ exosomes activity. In conclusion, our study highlights the non-coding regulatory role of HMGB1 with implications for RNA-based therapeutic targeting together with a prediction of anti-PD-L1 immunotherapy in HCC.

It has been shown that oncoprotein p28GANK , which is consistently overexpressed in human hepatocellular carcinoma (HCC), plays a critical role in tumorigenesis of HCC. However, the underlying mechanism remains unclear. Here, we demonstrated that p28 GANK inhibits apoptosis in HCC cells induced by the endoplasmic reticulum (ER) stress. During ER stress, p28 GANK enhances the unfolded protein response, promotes ER recovery from translational repression, and thereby facilitates cell's ability to cope with the stress conditions. Furthermore, p28GANK upregulates glucose-regulated protein 78 (GRP78), a key ER chaperone protein, which subsequently enhances the ER folding capacity and promotes recovery from ER stress. We also demonstrated that p28 GANK increases p38 mitogen-activated protein kinase and Akt phosphorylation, and inhibits nuclear factor kappa B (NF-κB) activation under ER stress, which in turn contributes to GRP78 upregulation. Taken together, our results indicate that p28 GANK inhibits ER stress-induced apoptosis in HCC cells, at least in part, by enhancing the adaptive response and GRP78 expression. We propose that p28 GANK has potential implications for HCC progression under the ER stress conditions.

p28GANK (also known as PSMD10 or gankyrin) is a novel oncoprotein that is highly expressed in hepatocellular carcinoma (HCC). Through its interaction with various proteins, p28GANK mediates the degradation of the tumor suppressor proteins Rb and p53. Although p53 was reported to downregulate β-catenin, whether p28 GANK is involved in the regulation of β-catenin remains uncertain. Here we report that both growth factors and Ras upregulate p28 GANK expression through the activation of the phosphoinositide 3-kinase-AKT pathway. Upregulation of p28 GANK expression subsequently enhanced the transcription activity of β-catenin. This effect was observed in p53-deficient cells, suggesting a p53-independent mechanism for the p28 GANK -mediated activation of β-catenin. p28 GANK overexpression also reduced E-cadherin protein levels, leading to increased release of free β-catenin into the cytoplasm from the cadherin-bound pool. Interestingly, exogenous expression of p28 GANK resulted in elevated expression of the endogenous protein. We also observed that both β-catenin and c-Myc were transcriptional activators of p28 GANK , and a correlation between p28 GANK overexpression and c-Myc, cyclin D1 and β-catenin activation in primary human HCC. Together, these results suggest that p28 GANK expression is regulated by a positive feedback loop involving β-catenin, which may play a critical role in tumorigenesis and the progression of HCC.