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Studies have reported dysregulation of TIPRL, LC3 and CD133 in liver cancer tissues. However, their respective relationships to liver cancer and roles as biomarkers for prognosis and diagnosis of liver cancer have never been studied. Here we report that the level of TIPRL is significantly correlated with levels of LC3 (Spearman r = 0.9) and CD133 (r = 0.7) in liver cancer tissues. We observed significant upregulations of TIPRL, LC3 and CD133 in hepatocellular carcinomas (HCCs) compared with adjacent normal tissues. Importantly, TIPRL, tested among additional variables, showed a significant impact on the prognosis of HCC patients. TIPRL knockdown significantly reduced expressions of LC3, CD133, stemness-related genes, as well as viability and stemness of liver cancer cell-lines, which were promoted by ectopic TIPRL expression. Either alone or as a combination, TIPRL, LC3 and CD133 showed significant values of area under the curve (AUC) and sensitivity/specificity in early liver cancer tissues. Furthermore, the statistical association and the diagnostic efficacies of TIPRL, LC3 and CD133 in HCC tissues were confirmed in a different IHC cohort. This data demonstrates that the complex involvement of TIPRL/LC3/CD133 in liver cancer aggressiveness can together or individually serve as potential biomarkers for the early detection of liver cancer.

We fabricated a spheroid-forming unit (SFU) for efficient and economic production of cell spheroids. We optimized the protocol for generating large and homogenous liver cancer cell spheroids using Huh7 hepatocellular carcinoma (HCC) cells. The large Huh7 spheroids showed apoptotic and proliferative signals in the centre and at the surface, respectively. In particular, hypoxia-induced factor-1 alpha (HIF-1α) and ERK signal activation were detected in the cell spheroids. To diminish core necrosis and increase the oncogenic character, we co-cultured spheroids with 2% human umbilical vein endothelial cells (HUVECs). HUVECs promoted proliferation and gene expression of HCC-related genes and cancer stem cell markers in the Huh7 spheroidsby activating cytokine signalling, mimicking gene expression in liver cancer. HUVECs induced angiogenesis and vessel maturation in Huh7 spheroids in vivo by activating epithelial-mesenchymal transition and angiogenic pathways. The large Huh7 cell spheroids containing HUVECs survived at higher concentrations of anti-cancer drugs (doxorubicin and sorafenib) than did monolayer cells. Our large cell spheroid provides a useful in vitro HCC model to enable intuitive observation for anti-cancer drug testing.

Tumor metastasis is the leading cause of cancer death. In the metastatic process, EMT is a unique phenotypic change that plays an important role in cell invasion and changes in cell morphology. Despite the clinical significance, the mechanism underlying tumor metastasis is still poorly understood. Here we report a novel mechanism by which secreted plasma glutamate carboxypeptidase(PGCP) negatively involves Wnt/β-catenin signaling by DKK4 regulation in liver cancer metastasis. Pathway analysis of the RNA sequencing data showed that PGCP knockdown in liver cancer cell lines enriched the functions of cell migration, motility and mesenchymal cell differentiation. Depletion of PGCP promoted cell migration and invasion via activation of Wnt/β-catenin signaling pathway components such as phospho-LRP6 and β-catenin. Also, addition of DKK4 antagonized the Wnt/β-catenin signaling cascade in a thyroxine (T4)-dependent manner. In an in vivo study, metastatic nodules were observed in the lungs of the mice after injection of shPGCP stable cell lines. Our findings suggest that PGCP negatively associates with Wnt/β-catenin signaling during metastasis. Targeting this regulation may represent a novel and effective therapeutic option for liver cancer by preventing metastatic activity of primary tumor cells.

TRAIL (TNF-related apoptosis inducing ligand) is a promising anti-cancer drug target that selectively induces apoptosis in cancer cells. However, many cancer cells are resistant to TRAIL-induced apoptosis. Therefore, reversing TRAIL resistance is an important step for the development of effective TRAIL-based anti-cancer therapies. We previously reported that knockdown of the TOR signaling pathway regulator-like (TIPRL) protein caused TRAIL-induced apoptosis by activation of the MKK7-c-Jun N-terminal Kinase (JNK) pathway through disruption of the MKK7-TIPRL interaction. Here, we identified Taraxacum officinale F.H. Wigg (TO) as a novel TRAIL sensitizer from a set of 500 natural products using an ELISA system and validated its activity by GST pull-down analysis. Furthermore, combination treatment of Huh7 cells with TRAIL and TO resulted in TRAIL-induced apoptosis mediated through inhibition of the MKK7-TIPRL interaction and subsequent activation of MKK7-JNK phosphorylation. Interestingly, HPLC analysis identified chicoric acid as a major component of the TO extract, and combination treatment with chicoric acid and TRAIL induced TRAIL-induced cell apoptosis via JNK activation due to inhibition of the MKK7-TIPRL interaction. Our results suggest that TO plays an important role in TRAIL-induced apoptosis, and further functional studies are warranted to confirm the importance of TO as a novel TRAIL sensitizer for cancer therapy. © 2015 Wiley Periodicals, Inc., (© 2015 Wiley Periodicals, Inc.)

Induction of apoptosis through activation of the TRAIL pathway is considered to be a promising anticancer strategy due to its ability to selectively induce apoptosis in cancer cells. However, the ability of cancer cells to acquire TRAIL resistance has limited the clinical translation of this approach. We previously reported that the TOR signaling pathway regulator-like (TIPRL) protein contributes to the resistance to TRAIL-induced apoptosis by inhibiting the MKK7-c-Jun N-terminal kinase (JNK) pathway via MKK7‑TIPRL interaction. In the present study, we identified Tussilago farfara L. (TF) as a novel TRAIL sensitizer among 500 natural products using an ELISA system that specifically detects the MKK7-TIPRL interaction, and we validated candidates by GST-pull down assay. Co-treatment of Huh7 cells with TF and TRAIL induced apoptosis via inhibition of the MKK7-TIPRL interaction and an increase in MKK7/JNK phosphorylation. This is the first report to describe TF as a novel TRAIL sensitizer, unveiling a potentially novel therapeutic strategy in cancer therapy.

Background & Aims: The TOR signaling pathway regulator-like (TIPRL) protein, the mammalian ortholog of yeast TIP41, was identified in an expression profiling screen for factors that regulate human liver carcinogenesis. We investigated the role of human TIPRL protein in hepatocellular carcinoma (HCC)., Methods: We measured the level of TIPRL in HCC and adjacent nontumor tissues from patients. We used small interfering RNAs and zebrafish to study the function of TIPRL. We used annexin V propidium iodide staining and immunoblot analyses to measure apoptosis and activation of apoptotic signaling pathways. We used confocal microscopy, coimmunoprecipitation, and glutathione-S transferase pull-down analyses to determine interactions among mitogen-activated protein kinase kinase 7 (MKK7 or MAP2K7), TIPRL, and the protein phosphatase type 2A (PP2Ac). We studied the effects of TIPRL in tumor xenografts in mice., Results: Levels of TIPRL were higher in HCC tissues and cell lines than nontumor tissues and primary hepatocytes. Knockdown of tiprl expression in zebrafish led to large amounts of apoptosis throughout the embryos. Incubation of HCC cells, but not primary human hepatocytes, with small interfering RNA against TIPRL (siTIPRL) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) caused prolonged activation (phosphorylation) of MKK7 and c-Jun N-terminal kinase (JNK) and led to apoptosis, indicated by cleavage of procaspase-8,-3 and of poly-(adenosine diphosphate-ribose) polymerase. TIPRL bound to MKK7 and PP2Ac and promoted the interaction between MKK7 and PP2Ac. In mice, injection of HCC xenograft tumors with siTIPRL and TRAIL led to tumor apoptosis and regression., Conclusions: TIPRL is highly up-regulated in human HCC samples and cell lines, compared with noncancerous liver tissues. TIPRL prevents prolonged activation of MKK7 and JNK and TRAIL-induced apoptosis by mediating the interaction between MKK7 and PP2Ac., (Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Two thousand sixty-eight multi-purpose expression clones for the 326 candidate genes related to gastric or liver cancers were constructed using the Gateway system. These clones can be expressed as His, Glutathione-S-transferase (GST) or Enhanced version of the green fluorescent protein (EGFP) fusion proteins in E. coli, insect cells or mammalian cells. For the 246 E. coli expression clones, the GST fusion proteins had greater expression efficiency and solubility than the His fusion proteins. Approximately 20% of the expressed proteins had unexpected molecular weights. A detailed sequence analysis of these clones revealed frameshift mutations resulting from insertion, deletion or substitution of nucleotides. The results indicate that these changes in the candidate genes may affect the occurrence of gastric or liver cancers. In addition, when 105 proteins, which were expressed in E. coli at very low or undetectable levels, were expressed in insect cells, 76% of the proteins were expressed very well and most were soluble. We also found that most of the 30 proteins prepared using EGFP mammalian expression clones were localized to cellular compartments expected by Gene ontology (GO) and this localization was unaffected if the EGFP-fusion was at the N-terminal or C-terminal region of the protein. Antibody production and subcellular localization analysis of the candidate genes as well as a screen of genes involved in carcinogenesis pathways are currently in progress using these expression clones. These studies provide a valuable resource for developing a better understanding of the molecular mechanism of carcinogenesis in both gastric and liver cancer and would be very helpful in diagnosis and therapeutic predictions.

Reactive oxygen species (ROS) have been implicated in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resistance of many cancers. We evaluated the role of peroxiredoxin (Prx) I in TRAIL resistance governed by coupling of nicotinamide adenosine dinucleotide phosphate oxidase (Nox)-derived ROS signaling with the p38 mitogen-activated protein kinase (MAPK)/caspase-signaling cascade in liver cancer cells. Upregulated Prx I expression was found in neoplastic regions of human patient liver, and Prx I knockdown resulted in accelerated TRAIL-induced cell death in SK-Hep-1 human hepatoma cells. The TRAIL cytotoxicity by Prx I knockdown was dependent on activation of caspase-8/3 cascades, which was ablated by addition of inhibitors for p38 MAPK, ROS or Nox, suggesting the association with Nox-driven redox signaling. Furthermore, we found that Nox4 was constitutively expressed in both SK-Hep-1 cells and tumor regions of patient livers, knockdown of Nox4 expression could alleviate ROS generation and TRAIL-mediated cytotoxicity. In accordance with previous findings, increased activation of both p38 MAPK and caspase cascades by Prx I knockdown was inhibited by either Nox4 knockdown or SB203580 addition. Collectively, these data suggest that Prx I functions to block propagation of Nox-derived ROS signaling to the p38 MAPK/caspase/cell death cascade during TRAIL treatment and also provides a molecular mechanism by which Prx I contributes to TRAIL resistance in liver cancers.

The activity of beta-catenin/TCF, the key component of Wnt signaling pathway, is frequently deregulated in HCC, resulting in the activation of genes whose dysregulation has significant consequences on tumor development. Therefore, identifying the target genes of Wnt signaling is important for understanding beta-catenin-mediated carcinogenesis. We analyzed the transcriptome profile of human hepatoma cell lines using cDNA microarrays representing 15,127 unique, liver-enriched gene loci to identify the target genes of beta-catenin-mediated transcription (p<0.005). This analysis yielded 130 potential Wnt-associated classifier genes, and we found 33 of them contain consensus TCF-binding sites in presumptive transcriptional regulatory sequences. These genes were, then, tested for their Wnt-dependence of expression in experimental models of Wnt activation. Genes such as RPL29, NEDD4L, FUT8, LYZ, STMN2, STARD7 and KIAA0998 were proven to be up-regulated upon Wnt/beta-catenin activation. Gene ontology analysis of the 33 candidate genes indicated the presence of functional categories relevant to Wnt pathway such as cell growth, proliferation, adhesion and signal transduction. In conclusion, we identified a number of candidate Wnt/beta-catenin target genes that can be useful for studying the role of altered Wnt signaling in liver cancer development, and showed that some of them might be direct targets of Wnt signaling in hepatoma cells.

Expression of estrogen receptors (ER)-alpha and -beta, as well as androgen receptor (AR), in hepatocellular carcinoma (HCC) is thought to be correlated with prognosis, survival, and male prevalence of HCC. These hypotheses are based on investigations of European patients; however the expression patterns of these receptors in Asian patients are largely unknown. In this study, we collected liver carcinoma and peritumor tissues from 32 patients (9 females and 23 males) in South Korea. The expression of ERs and ARs was studied using RT-PCR. Wild-type ER-alpha and AR were expressed in all of the samples investigated, and their expression was independent of the causal virus or patient sex. Expression of the ER-alpha variant was independent of sex (100% female vs. 91.3% male) and HCV and HBV status (91.3% vs. 100%). Wild-type ER-beta was expressed more often in HCV patients than in HBV patients (95.7% vs. 44.4%; p < 0.05). In conclusion, the stronger ER-alpha variant expression in HCC tissues implies that this variant has an important role in HCC development. However, at least in Korean patients, expression of the ER-alpha variant (vER-alpha) is not related to male HCC prevalence. In addition, the predominant expression of ER-beta in HCV patients suggests that it plays an important role in HCV-induced liver disease.

Ras-related, estrogen-regulated, and growth-inhibitory gene (RERG) is a novel gene that was first reported in breast cancer. However, the functions of RERG are largely unknown in other tumor types. In this study, RERG expression was analyzed in hepatocellular carcinomas of human patients using reverse transcriptase PCR analysis. In addition, the possible regulation of RERG expression by histone deacetyltransferases (HDACs) was studied in several cell lines. Interestingly, the expression of RERG gene was increased in hepatocellular carcinoma (HCC) of male patients (57.9%) but decreased in HCC of females (87.5%) comparison with paired peri-tumoral tissues. Moreover, RERG gene expression was increased in murine hepatoma Hepa1-6 cells, human breast tumor MDA-MB-231 cells, and mouse normal fibroblast NIH3T3 cells after treated by HDAC inhibitor, trichostatin A. Our results suggest that RERG may function in a gender-dependent manner in hepatic tumorigenesis and that the expression of this gene may be regulated by an HDAC-related signaling pathway.

Migration is an essential feature of metastatic cancer cells. To understand how motility is regulated in hepatocellular carcinoma, we analyzed gene expression profiles of mouse model cell lines we established from transgenic mice carrying SV40 large T antigen. A non-motile HC9 cell line was isolated from mouse liver tumors, and two additional cell lines, HCM1 and HCM4, were derived from HC9 cells. We found that both HCM1 and HCM4 cells were substantially more migratory than HC9, and that HCM1 generated tumor nodules in nude mice. In contrast to HCM4 cells that exhibited mesenchymal cell-type gene expression similar to HC9 cells, HCM1 cells appeared to have undergone a mesenchymal-amoeboidal transition. Thus, HCM1 and HCM4 cells have distinct migration and gene expression patterns, and together with HC9 cells, they can serve as model cell lines for understanding how migration is acquired and controlled in hepatocellular carcinoma.

Liver cancer is one of the leading causes of cancer death worldwide. To identify novel target genes that are related to liver carcinogenesis, we examined new genes that are differentially expressed in human hepatocellular carcinoma (HCC) cell lines and tissues based on the expressed sequence tag (EST) frequency. Eleven libraries were constructed from seven HCC cell lines and three normal liver tissue samples obtained from Korean patients. An analysis of gene expression profiles for HCC was performed using the frequency of ESTs obtained from these cDNA libraries. Genes were identified (n=120) as being either up- or down-regulated in human liver cancer cells. Among these, 14 genes (FTL, K-ALPHA1, LDHA, RPL4, ENO1, ANXA2, RPL9, RPL10, RPL13A, GNB2L1, AMBP, GC, A1BG, and SERPINC1), in addition to previously well-known liver cancer related genes, were confirmed to be differentially expressed in seven liver cancer cell lines and 17 HCC tissues by semi-quantitative RT-PCR. In addition, 73 genes, in which there was a significant difference (P>0.99) between HBV- and HCV-associated HCC cells, were selected. Of these, expression patterns of 14 (RPLP0, AKR1C, KRT8, GPX4, RPS15, ID1, RPS21, VIM, EEF1G, EIF4A1, HLA-C, FN1, CD44, and RPS10) were confirmed by semi-quantitative RT-PCR in four of HBV- and three of HCV-associated HCC cell lines. Among those genes, an immunohistochemical analysis for ANXA2 showed that it is expressed at high levels in HCC. Using an analysis of EST frequency, the newly identified genes, especially ANXA2, represent potential biomarkers for HCC and useful targets for elucidating the molecular mechanisms associated with HCC involving virological etiology.