Research advances on amino acid starvation interventions for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common primary liver malignancy. Uncontrolled angiogenesis plays a critical role in hepatocellular tumor growth and metastasis. In this study, we aimed to investigate the effects of circular RNA hsa_circ_0000519 and the potential involvement of microRNA (miR)-1296 and E2F transcription factor 7 (E2F7) in HCC development. Hsa_circ_0000519 was highly expressed in HCC cells and hepatocellular tumor tissues, and correlated with poor prognosis of HCC patients. Knockdown of hsa_circ_0000519 significantly reduced HCC cell viability, suppressed cell proliferation, and induced cell cycle arrest in G0/G1. Downregulation of hsa_circ_0000519 also inhibited formation of capillary-like endothelial structures in vitro and impeded microvessel formation in mice bearing HCC tumors. The migration and invasive capacities of HCC cells were markedly reduced by hsa_circ_0000519 knockdown. Hsa_circ_0000519 possessed a binding site for microRNA (miR)-1296. Upregulation of hsa_circ_0000519 significantly decreased the miR-1296 expression in both HCC cells and mouse xenografts. Furthermore, E2F7 was a target of miR-1296. Hsa_circ_0000519 positively regulated E2F7 via acting as a miR-1296 sponge. Upregulation of E2F7 abolished the inhibitory effects of hsa_circ_0000519 knockdown on HCC cell proliferation and angiogenesis. In conclusion, hsa_circ_0000519 promoted tumor progression and angiogenesis in HCC through the miR-1296/E2F7 axis. These data suggest the potential clinical application of hsa_circ_0000519 in HCC treatment.

Autophagy is a conserved degradative process that is crucial for cellular homeostasis and cellular quality control via the selective removal of subcellular structures such as mitochondria. We demonstrate that a regulatory link exists between mitochondrial function and autophagy in Saccharomyces cerevisiae. During amino-acid starvation, the autophagic response consists of two independent regulatory arms-autophagy gene induction and autophagic flux-and our analysis indicates that mitochondrial respiratory deficiency severely compromises both. We show that the evolutionarily conserved protein kinases Atg1, target of rapamycin kinase complex I, and protein kinase A (PKA) regulate autophagic flux, whereas autophagy gene induction depends solely on PKA. Within this regulatory network, mitochondrial respiratory deficiency suppresses autophagic flux, autophagy gene induction, and recruitment of the Atg1-Atg13 kinase complex to the pre-autophagosomal structure by stimulating PKA activity. Our findings indicate an interrelation of two common risk factors-mitochondrial dysfunction and autophagy inhibition-for ageing, cancerogenesis, and neurodegeneration.

The immunobiology of hepatocellular carcinoma in humans and mice: Basic concepts and therapeutic implications

Rethinking future development of molecular therapies in hepatocellular carcinoma: A bottom-up approach

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide. Therapeutic options for advanced HCC are limited, which is due to a lack of full understanding of pathogenesis. Cellular senescence is a state of cell cycle arrest, which plays important roles in the pathogenesis of HCC. Mechanisms underlying hepatocellular senescence are not fully understood. LncRNA NEAT1 acts as an oncogene and contributes to the development of HCC. Whether NEAT1 modulates hepatocellular senescence in HCC is unknown. The role of NEAT1 and KIF11 in cellular senescence and tumor growth in HCC was assessed both in vitro and in vivo. RNA pulldown, mass spectrometry, Chromatin immunoprecipitation (ChIP), luciferase reporter assays, RNA FISH and immunofluorescence (IF) staining were used to explore the detailed molecular mechanism of NEAT1 and KIF11 in cellular senescence of HCC. We found that NEAT1 was upregulated in tumor tissues and hepatoma cells, which negatively correlated with a senescence biomarker CDKN2A encoding p16INK4a and p14ARF proteins. NEAT1 was reduced in senescent hepatoma cells induced by doxorubicin (DOXO) or serum starvation. Furthermore, NEAT1 deficiency caused senescence in cultured hepatoma cells, and protected against the progression of HCC in a mouse model. During senescence, NEAT1 translocated into cytosol and interacted with a motor protein KIF11, resulting in KIF11 protein degradation and subsequent increased expression of CDKN2A in cultured hepatoma cells. Furthermore, KIF11 knockdown caused senescence in cultured hepatoma cells. Genetic deletion of Kif11 in hepatocytes inhibited the development of HCC in a mouse model. Conclusively, NEAT1 overexpression reduces senescence and promotes tumor progression in HCC tissues and hepatoma cells, whereas NEAT1 deficiency causes senescence and inhibits tumor progression in HCC. This is associated with KIF11-dependent repression of CDKN2A. These findings lay the foundation to develop potential therapies for HCC by inhibiting NEAT1 and KIF11 or inducing senescence.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-associated deaths worldwide. Although recent studies have proposed different biomarkers for HCC progression and therapy resistance, a better understanding of the molecular mechanisms underlying HCC progression and recurrence, as well as the identification of molecular markers with a higher diagnostic accuracy, are necessary for the development of more effective clinical management strategies. Here, we aimed to identify novel players in HCC progression. SPRYD4 mRNA and protein expression analyses were carried out on a normal liver-derived cell line (HL-7702) and four HCC-derived cell lines (HepG2, SMMC7721, Huh-7, BEL-7402) using qRT-PCR and Western blotting, respectively. Cell proliferation Cell Counting Kit-8 (CCK-8) assays, protein expression analyses for apoptosis markers using Western blotting, and Caspase-Glo 3/7 apoptosis assays were carried out onthe four HCC-derived cell lines. Expression comparison, functional annotation, gene set enrichment, correlation and survival analyses were carried out on patient data retrieved fromthe NCBI Gene module,the NCBI GEO database andthe TCGA database. Through a meta-analysis we found that the expression of SPRYD4was downregulated in primary HCC tissues compared to non-tumor tissues. We also found that the expression of SPRYD4 was downregulated in HCC-derived cells compared to normal liver-derived cells. Subsequently, we found that the expression of SPRYD4 was inversely correlated with a gene signature associated with HCC cell proliferation. Exogenous SPRYD4 expression was found to inhibit HCC cell proliferation by inducing apoptotic cell death. We also found that SPRYD4 expressionwas associated with a good prognosis and that its expression became downregulated when HCCs progressed towards more aggressive stages and higher grades. Finally, we found that SPRYD4 expression may serve as a biomarker for a good overall and relapse-free survival in HCC patients. Our data indicate that a decreased SPRYD4 expression may serve as an independent predictor for a poor prognosis in patients with HCC and that increased SPRYD4 expression may reduce HCC growth and progression through the induction of apoptotic cell death, thereby providing a potential therapeutic target.

Background and purpose: An increasing number of studies have revealed that microRNAs (miRNAs) are the main drivers of hepatocarcinogenesis including progression to later stages of liver cancer. Recently, miR-548b was identified as a cancer-related miRNA in glioma and tongue squamous cell carcinoma. Nonetheless, the expression pattern and specific roles of miR-548b in hepatocellular carcinoma (HCC) have not yet been clarified.Methods: Expression levels of miR-548b in HCC tissues and cell lines were measured by reverse-transcription quantitative PCR. In vitro and in vivo functional assays were performed to determine the effects of miR-548b on the malignant phenotypes of HCC cells. In addition, the molecular mechanisms by which miR-548b regulates the initiation and progression of HCC were investigated in detail.Results: miR-548b expression was weak in HCC tissues and cell lines. The low miR-548b expression significantly correlated with tumor size, TNM stage, and venous infiltration of HCC. In addition, exogenous miR-548b expression suppressed HCC cell proliferation, colony formation, and metastasis and induced apoptosis in vitro. Silencing of miR-548b exerted an opposite effect on these characteristics of HCC cells. Furthermore, miR-548b overexpression hindered tumor growth in vivo. Mechanistic analysis identified high-mobility group box 1 (HMGB1) as a direct target gene of miR-548b in HCC cells. Moreover, an HMGB1 knockdown reproduced the effects of miR-548b upregulation on HCC cells. Recovered HMGB1 expression reversed the effects of miR-548b on HCC cells. Notably, miR-548b overexpression deactivated the PI3K–AKT pathway in HCC cells in vitro and in vivo.Conclusion: Our findings provide the first evidence that miR-548b restrains HCC progression, at least partially, by downregulating HMGB1 and deactivating the PI3K–AKT pathway. Thus, miR-548b might be a novel target for the development of new therapies for HCC.

IntroductionHCC is the leading cause of cancer related mortality world wide. Emerging evidence suggests aberrant activation of an embryological trans-differentiation programme termed epithelial-mesenchymal transition (EMT) is critical in promoting metastasis...

Hepatocellular Carcinoma Radiation Therapy: Review of Evidence and Future Opportunities

PRR34-AS1 sponges miR-498 to facilitate TOMM20 and ITGA6 mediated tumor progression in HCC

PurposeThe aim of this study was to investigate the role of Yes1 associated transcriptional regulator (YAP1) in the pathology of hepatocellular carcinoma (HCC) and its potential as a therapeutic target.MethodsYAP1 expression in HCC and adjacent tissues was determined via immunohistochemistry; in HCC and human normal liver cell lines, expression was examined via Western blotting. The effects of YAP1 knockdown and overexpression were detected following transfection of HCC cells with siRNA-YAP1 recombinants or pcDNA3.1-YAP1 plasmids. A tumor xenograft model was constructed by implanting YAP1-knockdown lentivirus-infected Hep-3B cells into nude mice, and the animals were treated with sorafenib.ResultsIn patients with HCC, YAP1 was upregulated in tumor tissue compared with adjacent tissue, and its high expression in the tumor was associated with increased Edmonson grade. In vitro, YAP1 expression was increased in Hep-3B, SK-HEP-1 and Huh7 cells, while it was similar in SMMC-7721 cells and LO2 cells. Meanwhile, YAP1 increased cell proliferation and invasion, promoted the progression of epithelial–mesenchymal transition, and inhibited cell apoptosis in HCC cells; furthermore, YAP1 knockdown combined with the administration of sorafenib decreased cell viability and increased cell apoptosis compared with YAP1 knockdown or treatment with sorafenib alone. In vivo, YAP1 knockdown inhibited tumor growth and metastasis, whereas it promoted apoptosis; meanwhile, YAP1 knockdown synergized with sorafenib to suppress tumor progression in HCC mice.ConclusionYAP1 is upregulated in both HCC tumor tissues and cell lines. Moreover, it promotes cell proliferation and invasion and promoted the progression of epithelial–mesenchymal transition in vitro. Furthermore, targeting YAP1 inhibits HCC progression and improves sensitivity to sorafenib in vitro and in vivo.

Percutaneous radiofrequency ablation of tumor feeding artery before target tumor ablation may reduce local tumor progression in hepatocellular carcinoma

Purpose: Hepatocellular Carcinoma (HCC) is one of the most malignant cancers worldwide. Due to the male prevalence of HCC, androgen signals have been suspected to be involved in the disease process. Although it has been known for the past five decades that androgens promote HCC progression, failure of antiandrogens in HCC clinical trials puzzled the field. Increasing evidences implied that androgen/AR signals might function differently during cancer progression. Therefore, it is necessary to examine the roles of AR in late stage HCC progression. Methods: Immunohistological staining on patient HCC tissues were performed. Using conditional knockout of AR specifically in the hepatocyte (h-ARKO), we characterized the pathophysiological function of AR in HCC. Cancer survival, pathological malignancy, and cancer metastasis was measured in the late stage of cancer development (40∼60-wks). Results: To our surprise, the h-ARKO mice exhibited poorer survival, malignant histological tumor patterns, and higher lung metastasis rates. Moreover, we found less collagen deposition, and higher cellular proteolysis activity in the h-ARKO mice tumors compared to wild type mice implicating invasive characteristic in the h-ARKO tumors. We further validated the mechanism that AR could suppress cancer progression through multiple pathways. We found AR functions as p38 phosphorylation suppressor while p38 phosphorylation increasing by cancer malignancy so that to obtain cell anoikis resistance. Therefore, androgen/AR signals enhance cell anoikis. We also found AR suppressed cancer cells invasion via suppression of MMP9 transcription. We further demonstrated that MMP9 suppression was through NFκB deactivation in the HCC cells. This is the first report that AR functions as cancer suppressor in late stage of cancer development. Furthermore, we demonstrated a potential application of this finding for HCC therapy. We introduced the combination therapy concept that using low doses of Sorafenib (multiple kinase inhibitor that pass clinical trials) and expression of AR in the HCC cells or metastasis mice model. It's exciting that AR could synergistically facilitate Sorafenib effects to increase cell anoikis and suppress cell invasion. Combination therapy in the mice metastasis model pertain more metastasis-free and better cancer survival in the mice. Conclusion: This study revealed the biphasic, yet, opposite functions of AR in HCC progression. It not only explained the failure of antiandrogen in HCC therapy, but also implicated a critical timing of using anti-Androgen/AR signals. Last, our hypothetical preclinical trial shed a light on future medicine for late stage HCC therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2917.

Reduced Expression of Fibroblast Growth Factor Receptor 2IIIb in Hepatocellular Carcinoma Induces a More Aggressive Growth

Hepatocellular carcinoma (HCC) is one of the most serious malignant cancers and has a high fatality rate. However, clinical strategies for the effective treatment of HCC remain lacking. Long non-coding RNAs (lncRNAs) with aberrant expression have been closely correlated with the occurrence and development of HCC. Here we investigated the underlying mechanism of the lncRNA CERS6-AS1 in HCC progression. The expression and prognosis of CERS6-AS1 in HCC patients was explored using The Cancer Genome Atlas. PCR analysis was utilized to measure the expression of CERS6-AS1 in tissues and cell lines. Transwell, wound healing, proliferation and glycolysis assays were conducted to evaluate the function of CERS6-AS1 on HCC cell functions. Bioinformation methods and luciferase assays were used to screen and verify potential target miRNAs and genes. A subcutaneous tumorigenesis model was constructed in nude mice to assess the effect of CERS6-AS1 on tumorigenesis in vivo. CERS6-AS1 was highly expressed in HCC tissues and cell lines. Upregulated CERS6-AS1 expression was remarkably correlated with poor prognosis of HCC patients. High CERS6-AS1 expression facilitated cell growth, invasion and glycolysis of HCC cells. Bioinformatics analyses combining with PCR analysis identified miR-30b-3p as the potential target of CERS6-AS1, and MDM2 mRNA was verified as the target of miR-30b-3p. The expression of miR-30b-3p was negatively correlated with CERS6-AS1, whereas MDM2 was positively associated with CERS6-AS1. Mechanistic studies showed that CERS6-AS1 may sponge miR-30b-3p to elevate MDM2, thus promoting the MDM2-mediated ubiquitin-dependent degradation of the p53 tumor suppressor. MDM2 overexpression or miR-30b-3p inhibitors blocked the inhibitory effect of CERS6-AS1 knockdown on proliferation, migration and glycolysis. CERS6-AS1 depletion reduced tumor formation in the in vivo mouse model. The CERS6-AS1/miR-30b-3p/MDM2/p53 signaling axis may play key roles in regulating HCC progression. CERS6-AS1 may exert as a novel biomarker or therapeutic target for HCC.