Capacity Of Hepatocellular Carcinoma Cells Research Articles

Comprehensive analysis on clinical significance and therapeutic targets of LDL receptor related protein 11 (LRP11) in liver hepatocellular carcinoma.

LDL lipoprotein receptor-related protein 11 (LRP11) plays a role in several tumors. However, their roles in hepatocellular carcinoma remain unclear. The present study aimed to explore the expression profile and prognostic value of LRP11 in liver hepatocellular carcinoma (LIHC) patients using various cancer databases and bioinformatic tools. In bioinformatics analysis, The Cancer Genome Atlas datasets showed increased LRP11 expression in tumor tissues compared to that in non-tumor tissues in various cancers. Moreover, patients with high expression LRP11 correlated with poor prognosis and clinical features. The LRP11 expression positively correlated with the infiltration of immune cells such as macrophages, neutrophils, and myeloid-derived suppressor cells and a combination of high LRP11 expression and high immune infiltrates was associated with the worst survival in LIHC tumors. Our results also indicated that LRP11 expression was closely associated with immune-modulate function, such as antigen presentation. In DNA methylation profiling, hypomethylation of LRP11 is widely observed in tumors and has prognostic value in LIHC patients. Functional enrichment analysis revealed that LIHC-specific LRP11 interacting genes are involved in protein binding, intracellular processing, and G-protein-related signaling pathways. Analyses of drug sensitivity and immune checkpoint inhibitor predict a number of drugs that could potentially be used to target LRP11. In addition, in vitro experiments verified the promoting effect of LRP11 on the migration, invasion, and colony formation capacity of hepatocellular carcinoma cells. Collectively, our results aided a better understanding of the clinical significance of LRP11 in gene expression, functional interactions, and epigenetic regulation in LIHC and suggested that it may be a useful prognostic biomarker for LIHC patients.

CCDC167 promotes hepatocellular carcinoma cell proliferation and is associated with the immune microenvironment of hepatocellular carcinoma

Coiled-coil domain-containing (CCDC)family proteins play an important role in tumour progression, however, the role of CCDC167 in hepatocellular carcinoma remains unclear. To investigate the role of coiled-coil structural domain 167 (CCDC167) in hepatocellular carcinoma progression, we downloaded three hepatocellular carcinoma datasets (TCGA-LIHC, ICGC-LIRI and GSE76427) and analysed the mechanisms by which CCDC167 regulates hepatocellular carcinoma progression using MetaScape, CPTAC, HPA, TISCH and TISIDB.“ pRRophetic” package predicted the chemotherapeutic agents associated with CCDC167. The results showed that CCDC167 was highly expressed in hepatocellular carcinoma tissues and was associated with poor prognosis in patients with hepatocellular carcinoma. Knockdown of CCDC167 inhibits the proliferative capacity of hepatocellular carcinoma cells. CCDC167 was involved in the regulation of cell cycle, DNA replication and oxidative phosphorylation. Hepatocellular carcinoma samples with high expression of CCDC167 showed higher immune scores and C4 (lymphocyte depleted) subtypes. CCDC167 was associated with a variety of immune molecules (CD274, IL6R, and KDR), not only that, but CCDC167 was positively correlated with Treg infiltration and negatively correlated with T cells CD4 memory resting. High expression of CCDC167 increased immune escape in hepatocellular carcinoma patients. CCDC167 was associated with sensitivity to cisplatin in hepatocellular carcinoma patients. CCDC167 promoted the proliferative capacity of hepatocellular carcinoma cells. Our study suggests that CCDC167 is a novel and potentially valuable prognostic marker for hepatocellular carcinoma, and that CCDC167 iCCDC167 not only promotes hepatocellular carcinoma cell proliferation but is also associated with immune function in hepatocellular carcinoma patients.

Hepatitis C virus nonstructural protein 4B induces lipogenesis via the Hippo pathway

Hepatitis C virus (HCV) infection causes abnormal lipid metabolism in hepatocytes, which leads to hepatic steatosis and even hepatocellular carcinoma. HCV nonstructural protein 4B (NS4B) has been reported to induce lipogenesis, but the underlying mechanism is unclear. In this study, western blots were performed to investigate the effect of NS4B protein levels on key effectors of the Hippo and AKT signaling pathways. Yes-associated protein (YAP) and moesin-ezrin-radixin-like protein (Merlin) are effectors of the Hippo pathway. NS4B downregulated Merlin and phosphorylated YAP (p-YAP) protein expression while increasing the expression of the key AKT pathway proteins p-AKT and NF-κB. By observing the levels of AKT pathway proteins when Merlin was overexpressed or silenced, it was determined that Merlin mediates the AKT pathway. We suggest that HCV NS4B may mediate the AKT signaling pathway by inhibiting the Hippo pathway. Lipid droplets were observed in Huh7.5 cells overexpressing NS4B, and they increased significantly in number when Merlin was silenced. Overexpression of NS4B and Merlin silencing enhanced the expression of sterol regulatory element binding proteins (SREBPs), which have been demonstrated to be key regulatory factors controlling fatty acid synthesis. NS4B and Merlin silencing also enhanced the in vitro proliferative capacity of hepatocellular carcinoma cells. In conclusion, NS4B induces lipogenesis via the effect of the Hippo-YAP pathway on the AKT signaling pathway and thereby plays a significant role in the pathogenesis of HCV-associated diseases.

LncRNA BACE1-AS enhances the invasive and metastatic capacity of hepatocellular carcinoma cells through mediating miR-377-3p/CELF1 axis

AimsHepatocellular carcinoma (HCC) is a malignant cancer that threatened human life seriously. Long non-coding RNA (lncRNA) BACE1-AS has been reported as a key regulator in tumorigenesis. Yet the specific correlation between BACE1-AS and HCC still needs further investigation. The primary purpose of our study is to reveal the exact correlation between BACE1-AS and HCC. Main methodsBioinformatics via TCGA database revealed BACE1-AS closely related with HCC. qRT-PCR confirmed the abnormal BACE1-AS level in HCC tissues and cells. Databases prediction suggested that miR-377-3p might be a modulatory target of BACE1-AS and luciferase assay confirmed this hypothesis. Further study discovered that CELF1 also partook in the regulatory axis of BACE1-AS/miR-377-3p. Wound healing assays and transwell assays were utilized to investigate the impact of BACE1-AS, miR-377-3p and CELF1 in vitro. In vivo metastasis was examined by pulmonary metastasis model. Key findingsThis study found that BACE1-AS was overexpressed in HCC tissues and cell lines. Knockdown of BACE1-AS could restrain HCC progression in vitro, and inhibit pulmonary metastasis in vivo. MiR-377-3p was negatively modulated by BACE1-AS in HCC tumor tissues and cells. MiR-377-3p up-regulation inhibited HCC cells migration and invasion via inactivating EMT process. Moreover, CELF1 was identified as a downstream regulator of miR-377-3p and served as an oncogene in HCC cells. SignificanceOur findings supported that lncRNA BACE1-AS was up-regulated in HCC, promoting invasion and metastasis of hepatocellular carcinoma cells by modulating miR-377-3p/CELF1 axis via contributing to EMT pathway. BACE1-AS could be a potential biomarker in HCC for future treatment.

CDHR5 inhibits proliferation of hepatocellular carcinoma and predicts clinical prognosis.

As one of the most prevalent malignancies, hepatocellular carcinoma (HCC) ranks the third leading cause of cancer death worldwide. Due to the lack of biomarkers for early diagnosis, the clinical outcome of HCC remains unsatisfied with the current common therapeutic approaches, including surgery and chemotherapies. Thus, sensitive biomarkers and targeted therapies are in great need. In this study, we explored and verified whether CDHR5 (cadherin-related family member 5), a cadherin family protein, could serve as the potential biomarkers for HCC in the clinic. A retrospective study which contained 154 HCC patients was performed. Chi-square was utilized to analyze the relationship between CDHR5 expression and the clinicopathological features of HCC patients. The Kaplan-Meier method and Cox regression analyses were then used to evaluate the survival of HCC patients. In addition, cell proliferation assay and colony formation assay were performed to examine the effects of CDHR5 on the progression of HepG2 and Huh7 cells. IHC and RT-qPCR revealed that CDHR5 was downregulated in HCC tissues compared with adjacent liver tissues. In addition, CDHR5 expression was significantly correlated with tumor numbers, tumor size, and TNM stage. CDHR5 expression was then shown to be an independent risk factor for survival of HCC patients by survival analysis. In vitro experiments showed that CDHR5 suppressed the proliferation capacity of HCC cells. Taken together, our study not only identified CDHR5 as a novel prognostic biomarker in HCC but also provided evidence that CDHR5 can inhibit HCC cell proliferation.

Integration of metabolomics and expression of enolase-phosphatase 1 links to hepatocellular carcinoma progression.

Reprogramming of cellular metabolism is one of the hallmarks for cancer, in which tumor cells rewire their metabolic fluxes to generate sufficient energy and biosynthetic intermediates. Therefore, elucidating the correlation between cellular metabolism and hepatocellular carcinoma (HCC) progression may provide insights into novel approaches to cancer therapy.Methods: We assembled an integrated pathway-level metabolic profiling by mining metabolomic, transcriptomic and proteomic data of three HCC cell lines with increasing metastatic potentials. Immunohistochemical staining was performed in a tissue microarray from 185 HCC clinical specimens. Kaplan-Meier survival and Cox regression analyses were applied to test the association between gene expression and survival outcome. In vitro assays were conducted to investigate the functional role of enolase-phosphatase 1 (ENOPH1) in HCC malignant behaviors. Reversed genetics analysis was performed to determine the function of ENOPH1 in HCC metastasis. An intrahepatic mouse model further confirmed the role of ENOPH1 in metastasis.Results: We have determined that HCC cell metastasis is associated with alterations in metabolite levels and expressions of metabolic enzymes in the cysteine/methionine metabolism pathway, and show that one of metabolic enzymes, enolase-phosphatase 1 (ENOPH1), is persistently upregulated with an increase in metastatic potential. The upregulation of ENOPH1 expression was observed as an independent prognostic factor for HCC patients. ENOPH1 overexpression promoted cell migration and invasion, whereas ENOPH1 downregulation inhibited cell migration and invasion. Furthermore, an enhanced phosphorylation of AKT with ENOPH1 upregulation was observed. ENOPH1-mediated malignant capacity in HCC cells can be rescued by an AKT inhibitor.Conclusion: Taken together, our findings illustrate that ENOPH1 promotes HCC progression and could serve as a novel biomarker and therapeutic target for HCC.

PO-012 Pre-clinical insight into how platelet count affects the activity of HDACi resminostat in combination with the multi-kinase inhibitor sorafenib in HCC

Introduction In a recent phase I/II clinical trial in hepatocellular carcinoma (HCC), (NCT02400788), the combination of HDAC-inhibitor resminostat (YHI1001) with a multi-kinase inhibitor sorafenib was clinically beneficial compared to sorafenib mono-treatment in patients with platelet counts ≥1.5 × 10 5 /µl, but not below that threshold. In HCC, platelets have been linked to bad prognosis, tumour growth and metastasis as well as to resistance against the standard treatment with sorafenib. This begs the question how platelets affect HCC cells and modulate their drug response. Material and methods Several HCC cell lines were used to compare phenotypical features, susceptibility to platelets and drug-mediated effects. To determine effects on cancer cell features, in vitro cell growth and transwell invasion assays were performed. Furthermore, the underlying mechanism of a platelet-modulated drug response on the molecular level was explored. Results and discussions In HCC cells, the anti-proliferative potency of sorafenib was counteracted by platelet factors and the mesenchymal phenotype. However, resminostat alone and in combination with sorafenib effectively triggered an anti-proliferative response independently of platelets or the mesenchymal phenotype. Therefore, resminostat determined the anti-proliferative response of the drug combination. Moreover, recent reports highlight HCC cell subpopulations which express cancer stem cell genes and harbour clonogenic growth and cell invasive capacities as critical for metastasis. Intriguingly, we found that platelets induced the cell-invasive capacity in HCC cells with detectable levels of several cancer stem cell markers and which featured a mixed epithelial-mesenchymal phenotype. Importantly, only the combination of resminostat with sorafenib, but not the mono-treatments, significantly reversed the platelet-induced cell invasion. Conclusion Our pre-clinical data provide evidence on how platelets mediate pro-tumorigenic effects and modulate the therapeutic response to the resminostat/sorafenib drug combination. Platelet factors negatively modulated the drug response to sorafenib. This was overcome by the anti-proliferative activities of resminostat. Importantly, providing an explanation for the clinical benefit of the combination therapy, the platelet-induced invasive capacity was reversed only by the combination of resminostat with sorafenib, but not the mono-treatments.

Upregulation of lncRNA Sox2ot indicates a poor prognosis for patients with hepatocellular carcinoma and promotes cell invasion.

Long non-coding RNA Sox2 overlapping transcript (lncRNA Sox2ot) expression has been demonstrated to be upregulated in a number of types of tumor, and may act as an oncogene. The present study aimed to evaluate the clinical role of lncRNA Sox2ot and its association with the epithelial-mesenchymal transition in hepatocellular carcinoma (HCC). Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to determine the expression of lncRNA Sox2ot in 86 cases of HCC tissues and adjacent non-tumor tissues. Kaplan-Meier and log-rank test was used to analyze the association between lncRNA Sox2ot expression and disease-free (DFS) or overall (OS) survival time. In addition, the capacity of HCC cells with lncRNA Sox2ot knockdown for invasion was evaluated via transwell cell invasion assays. RT-qPCR and western blot analyses were also performed to determine the mRNA and protein expression of Twist1, E-cadherin and N-cadherin in the HCC cells. It was indicated that lncRNA Sox2ot expression levels were significantly higher in HCC tissues compared with adjacent non-tumor tissues. Increased expression levels of lncRNA Sox2ot were associated with the tumor size, the tumor number and vein invasion in patients with HCC. An association was observed between lncRNA Sox2ot and the DFS and OS of patients with HCC. Furthermore, it was determined that cell invasion was inhibited following the siRNA knockdown of lncRNA Sox2ot in MHCC97H and SMCC-7721 cells via transwell cell invasion assays. Furthermore, it was demonstrated that knockdown of lncRNA Sox2ot downregulated the mRNA and protein expression of Twist1 and N-cadherin, but upregulated the E-cadherin expression levels in MHCC97H and SMCC-7721 cells. Thus, it was indicated that lncRNA Sox2ot may be a novel predictive biomarker and a potential therapeutic target for HCC.

Overexpression of CXCR7 induces angiogenic capacity of human hepatocellular carcinoma cells via the AKT signaling pathway.

Angiogenesis is essential for tumor growth, especially in hepatocellular carcinoma(HCC). The hypervascularity is associated with poor prognosis and highly invasive HCC. The C‑X‑C chemokine receptor type7(CXCR7) has been implied overexpressed in many tumor types. Our study aimed to investigate the CXCR7 function in HCC. The tube formation, Transwell migration assay of human umbilical vein endothelial cells(HUVECs) and chicken chorioallantoic membrane(CAM) assay were used. We confirmed that CXCR7 induces angiogenic capacity. Moreover, overexpressing CXCR7 increased the phosphorylated (but not total) AKT expression in HCC cells. Furthermore, overexpressing CXCR7 increased the expression of tumor necrosis factor(TNF)‑α, interleukin(IL)‑6 and IL‑8 in HCC cells. Additionally, inhibition of AKT by LY294002 abrogated CXCR7‑induced angiogenic capacity in HCC cells. Our study suggested that CXCR7 plays an important pro‑angiogenic role in HCC via activation of the AKT pathway. So CXCR7 may be a potential target for anti‑angiogenic therapy in HCC.

URG4/URGCP enhances the angiogenic capacity of human hepatocellular carcinoma cells in vitro via activation of the NF-κB signaling pathway.

BackgroundAngiogenesis is essential for tumor growth. Hepatocellular carcinoma (HCC) is characterized by hypervascularity; high levels of angiogenesis are associated with poor prognosis and a highly invasive phenotype in HCC. Up-regulated gene-4 (URG4), also known as upregulator of cell proliferation (URGCP), is overexpressed in multiple tumor types and has been suggested to act as an oncogene. This study aimed to elucidate the effect of URG4/URGCP on the angiogenic capacity of HCC cells in vitro.MethodsExpression of URG4/URGCP in HCC cell lines and normal liver epithelial cell lines was examined by Western blotting and quantitative real-time PCR. URG4/URGCP was stably overexpressed or transiently knocked down using a shRNA in two HCC cell lines. The human umbilical vein endothelial cell (HUVEC) tubule formation and Transwell migration assays and chicken chorioallantoic membrane (CAM) assay were used to examine the angiogenic capacity of conditioned media from URG4/URGCP-overexpressing and knockdown cells. A luciferase reporter assay was used to examine the transcriptional activity of nuclear factor kappa – light – chain - enhancer of activated B cells (NF-κB). NF-κB was inhibited by overexpressing degradation-resistant mutant inhibitor of κB (IκB)-α. Expression of vascular endothelial growth factor C (VEGFC), tumor necrosis factor-α (TNFα), interleukin (IL)-6, IL-8 and v-myc avian myelocytomatosis viral oncogene homolog (MYC) were examined by quantitative real-time PCR; VEGFC protein expression was analyzed using an ELISA.ResultsURG4/URGCP protein and mRNA expression were significantly upregulated in HCC cell lines. Overexpressing URG4/URGCP enhanced - while silencing URG4/URGCP decreased - the capacity of HCC cell conditioned media to induce HUVEC tubule formation and migration and neovascularization in the CAM assay. Furthermore, overexpressing URG4/URGCP increased - whereas knockdown of URG4/URGCP decreased - VEGFC expression, NF-κB transcriptional activity, the levels of phosphorylated (but not total) IκB kinase (IKK) and IκB-α, and expression of TNFα, IL-6, IL-8 and MYC in HCC cells. Additionally, inhibition of NF-κB activity in HCC cells abrogated URG4/URGCP-induced NF-κB activation and angiogenic capacity.ConclusionsThis study suggests that URG4/URGCP plays an important pro-angiogenic role in HCC via a mechanism linked to activation of the NF-κB pathway; URG4/URGCP may represent a potential target for anti-angiogenic therapy in HCC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1378-7) contains supplementary material, which is available to authorized users.

Low mir-372 expression correlates with poor prognosis and tumor metastasis in hepatocellular carcinoma

BackgroundRecent studies have shown that miR-372 plays important roles in hepatocellular carcinoma (HCC) progression. However, results have been conflicting regarding its expression levels and role in HCC.MethodsRT-PCR and in situ hybridization was used to evaluate miR-372 expression in HCC tissues and cell lines. The methylation status of neighboring CpG islands upstream of the miR-372 promoter was analyzed by methylation-specific PCR (MSP). Transfection of miR-372 mimic into HCC cell lines was used to evaluate cellular proliferation and invasion. Prognostic significance was analyzed by the Kaplan–Meier survival method and Cox regression.ResultsmiR-372 was expressed at lower levels in HCC tissues compared with controls and was related to tumor metastasis and poor prognosis. Hypermethylation of miR-372 was detected in HCC cell lines and tissues, and miR-372 expression was restored upon 5-aza-dCyd treatment. Upregulated expression by mir-372 mimic transfection inhibited proliferation and invasion capacity in HCC cells.ConclusionsmiR-372 may play an important role in hepatic carcinogenesis and may serve as a new target or method to detect and treat HCC in the future.

Positive association of long telomeres with the invasive capacity of hepatocellular carcinoma cells

Invasion, the representative feature of malignant tumors, leads to an increase in mortality. The malignant liver tumor – hepatocellular carcinoma (HCC) – has an enhanced invasive capacity that results in increased patient mortality. Moreover, this enhanced invasive capacity is due to the up-regulation of invasion promoters such as zinc finger protein SNAI1 (Snail) and matrix metalloproteinases (MMPs), and the down-regulation of invasion suppressor molecules such as E-cadherin. Telomerase reverse transcriptase (TERT), which encodes the catalytic subunit of telomerase, is highly expressed in a variety of invasive cancers, including HCC. Telomerase activation induces telomere elongation, thereby leading to cell immortalization during malignant tumor progression. However, the relationship between telomere length and invasion is yet to be experimentally corroborated. In this paper, we revealed that invasive HCC cells passing through the Matrigel display significantly longer telomeres than non-invasive HCC cells. Moreover, we established a method that can distinguish and sort cells containing long telomeres and short telomeres. Using this system, we observed that the HCC cells containing long telomeres had a high-level expression of invasion-promoting genes and a low-level expression of invasion-suppressing E-cadherin. Furthermore, HCC cells containing long telomeres exhibited a higher invasive capacity than HCC cells containing short telomeres. Taken together, our findings suggest that long telomeres are positively associated with the invasive capacity of HCC cells and may be a potent target for malignant liver cancer treatment.

Expression and prognostic significance of the αB-crystallin gene in human hepatocellular carcinoma

The aim of this study was to characterize expression of the alpha B-crystallin gene in human hepatocellular carcinomas, to investigate the relationship between expression of this gene and the prognosis of human hepatocellular carcinoma. Real-time polymerase chain reaction, reverse transcriptase-polymerase chain reaction and immunohistochemistry were used to characterize expression of the alpha B-crystallin gene in human hepatocellular carcinoma. Kaplan-Meier survival and Cox regression analyses were performed to evaluate the prognosis of human hepatocellular carcinoma. We characterized alpha B-crystallin gene expression in human hepatocellular carcinoma. Statistical analysis of hepatocellular carcinoma patients showed that patients expressing alpha B-crystallin have different survival rates relative to those not expressing this gene (P = .041). After 18 months, the survival rate of patients expressing alpha B-crystallin declined, but survival in the alpha B-crystallin-negative group remained stable. COX multi-factor analysis showed that alpha B-crystallin (P = .007) and venous invasion (P = .037) were independent prognosis factors for hepatocellular carcinoma. Expression of the alpha B-crystallin gene, which is related with the transferability and invasive capacity of hepatocellular carcinoma cells, can be used as a prognostic indicator in human hepatocellular carcinomas. It may also be involved in the malignant transformation of hepatocytes.