Edmondson Grade Research Articles

MiR-3174 promotes proliferation and inhibits apoptosis by targeting FOXO1 in hepatocellular carcinoma

IntroductionMicroRNAs (miRNAs) have been confirmed to play a crucial part in oncogenesis. Several studies suggested that MiR-3174 act as a tumor promoter in various Malignant neoplasm. However, the biological function of miR-3174 in hepatocellular carcinoma (HCC) still highly unexplored. MethodsWe screened differentially over-expressed miRNAs by The Cancer Genome Atlas (TCGA) and the GEO databases. The expression of miR-3174 in HCC cells and tissues was detected by qRT-PCR. The cellular behaviors of transfected cells were respectively examined by colony formation assays, EdU Assays and flow cytometry. Forkhead box O1 transcription factor (FOXO1) was predicted and confirmed as a direct target of miR-3174 by bioinformatics analysis and dual-luciferase reporter gene assay. ResultsMiR-3174 was up-regulated in HCC tissues and cells, and the expression level of it was highly associated with tumor size and Edmondson grade. Our study pioneering validates that upregulated miR-3174 promote cell proliferation and inhibit cell apoptosis in vitro and in vivo. Meanwhile, our study verified that miR-3174 regulate Bim, P21, cyclin D1 and c-MYC expression by directly targeting FOXO1. ConclusionThe upregulated miR-3174 promote cell proliferation and inhibit cell apoptosis by downregulating FOXO1 expression in HCC. MiR-3174 may be a novel candidate for targeted delivery of miRNA therapeutics for HCC patients.

Low expression of citron kinase is associated with poor patient outcomes in hepatocellular carcinoma.

BackgroundCitron kinase (CIT) is a protein related to cytokinesis and is an important abscission regulator. However, the relationship between CIT and hepatocellular carcinoma (HCC) is unclear. The aim of this study was to investigate the expression CIT in HCC tissues, and explore the connection between this expression and clinicopathological characteristics of HCC.MethodsImmunohistochemistry staining on 235 HCC tissues and 96 non-tumorous liver tissues controls was performed to examine the CIT protein expression. We then analyzed the correlation between protein expression and clinicopathological parameters via χ2 tests, and we performed overall survival analyses via the Kaplan-Meier survival approach. Based on the online Oncomine Expression Array and UALCAN databases, we more broadly compared CIT mRNA expression between normal and HCC tissues. Finally, we compared CIT mRNA expression in these databases to protein expression in our study and explored potential sources for any observe differences.ResultsCompared to normal tissues, CIT expression was significantly lower in HCC tissues. Low CIT expression was found to be related to gender, tumor size, Edmondson Grade, Microvascular invasion, serum AFP levels and poor overall survival. Based on the online databases, CIT mRNA expression was found to be high in HCC tissues and decreased in normal tissues. We hypothesize that this unexpected result is due to a negative feedback loop whereby low protein CIT levels mediate increased CIT mRNA levels.ConclusionsLower CIT protein levels are associated with a poorer prognosis in HCC patients, and lower CIT protein levels may mediate a negative feedback loop leading to increased CIT mRNA levels.

High expression of FOXO3 is associated with poor prognosis in patients with hepatocellular carcinoma.

The role of forkhead box O3 (FOXO3) as a tumor suppressor gene and its association with the human lifespan is well documented. However, several studies have indicated that high expression of FOXO3 is also significantly associated with tumorigenesis. The aim of the present study was to determine the clinical significance of FOXO3 in the development and prognosis of hepatocellular carcinoma (HCC). mRNA expression data of FOXO3 from The Cancer Genome Atlas database was analyzed through the UALCAN online tool to compare the expression of FOXO3 between HCC and normal liver tissues. Subsequently, the expression of FOXO3 at the protein level was investigated via immunohistochemical staining of 314 HCC and 150 non-cancerous liver tissue samples. The association between protein expression and clinicopathological parameters was analyzed using the χ2 test, and the effect of FOXO3 expression on survival was assessed via Kaplan-Meier analysis. The expression of FOXO3 mRNA was significantly higher in HCC in comparison with healthy tissues. High FOXO3 protein expression was revealed in 43/150 non-cancerous liver tissues, and in 238/314 HCC samples. A significant association was demonstrated between FOXO3 expression and metastasis, Tumor-Node-Metastasis stage, Edmondson grade, α-fetoprotein level and overall survival. In conclusion, the high expression of FOXO3 predicts a poor prognosis in patients with HCC, indicating this protein as a potential therapeutic target in HCC.

Circulating tumor cells and CXCR4 in the prognosis of hepatocellular carcinoma.

BackgroundThis study was to determine circulating tumor cells (CTCs) and the expression of CXC chemokine receptor type 4 (CXCR4) in primary hepatocellular carcinoma (HCC) and the relationships with prognosis.MethodsWe used an advanced CanPatrolTM CTC-enrichment technique to collect CTCs for isolation and characterization from blood samples. The RNA in situ hybridization (RNA-ISH) method, which is based on branched DNA (bDNA) signal amplification technology, was used to determine the expression of CXCR4 according to epithelial-mesenchymal transition (EMT) markers in 99 patients with primary liver cancer in blood samples pre-operatively. The relationship between the EMT markers and HCC was determined.ResultsThe positive rates of CTCs and CXCR4 were 89.9% and 58.8%, respectively. CTCs were positively correlated with the Barcelona clinic liver cancer (BCLC) staging, tumor diameter and number, envelope, microsatellite damage, portal vein thrombosis, alpha-fetoprotein (AFP), and hepatitis B DNA, and negatively correlated with Edmondson grade. There were significant differences in the expression of CXCR4 between interstitial CTCs and mixed CTCs. A total of 99 patients underwent CTCs testing prior to surgery. The tumor-free survival time of HCC patients with interstitial CTCs <1 (13.3 months) was significantly longer than patients with interstitial CTCs ≥1 (5.0 months) pre-operatively.ConclusionsCTC-positivity was shown to be associated with HCC and can be used as an independent prognostic factor for HCC. High CXCR4 protein expression was more common in mixed CTCs.

Long non-coding RNA GAS6-AS1 acts as a ceRNA for microRNA-585, thereby increasing EIF5A2 expression and facilitating hepatocellular carcinoma oncogenicity

ABSTRACT Long non-coding RNA termed GAS6 antisense RNA 1 (GAS6-AS1) plays an essential role in gastric and non-small cell lung cancers. Nonetheless, the function of GAS6-AS1 in hepatocellular carcinoma (HCC) has not been so far studied in detail. In this study, reverse-transcription quantitative PCR was performed to measure GAS6-AS1 expression in HCC samples. A series of functional experiments, including MTT assay, colony formation assay, flow-cytometric analysis, and transwell migration and invasion assays, was performed to determine the influence of GAS6-AS1 knockdown on the malignant phenotype of HCC. The results showed that GAS6-AS1 was significantly upregulated in HCC tissue samples and cell lines. Increased GAS6-AS1 expression was associated with tumor size, Edmondson grade, and Tumor-Node-Metastasis (TNM) stage among patients with HCC. The overall survival of patients with HCC characterized with high expression of GAS6-AS1 was significantly shorter in comparison to that of patients with low level of GAS6-AS1. Functional experiments indicated that knockdown of GAS6-AS1 suppressed HCC cell proliferation, colony formation, migration, and invasion in vitro; promoted apoptosis in vitro; and decreased tumor growth in vivo. Of note, GAS6-AS1 was validated as a competing endogenous RNA (ceRNA) for microRNA-585 (miR-585) and consequently increased the expression of eukaryotic translation initiation factor 5A2 (EIF5A2). Finally, rescue experiments confirmed the association among GAS6-AS1, miR-585, and EIF5A2 in HCC cells. Our study provides substantial evidence that the GAS6-AS1/miR-585/EIF5A2 pathway plays an important role in HCC progression and that might be considered as a potential target for therapeutic approaches in HCC.

Gelsolin Promotes Cancer Progression by Regulating Epithelial-Mesenchymal Transition in Hepatocellular Carcinoma and Correlates with a Poor Prognosis.

Gelsolin (GSN), a cytoskeletal protein, is frequently overexpressed in different cancers and promotes cell motility. The biological function of GSN in hepatocellular carcinoma (HCC) and its mechanism remain unclear. The expression of GSN was assessed in a cohort of 188 HCC patients. The effects of GSN on the migration and invasion of tumour cells were examined. Then, the role of GSN in tumour growth in vivo was determined by using a cancer metastasis assay. The possible mechanism by which GSN promotes HCC progression was explored. As a result, GSN was overexpressed in HCC tissues. High GSN expression was significantly correlated with late Edmondson grade, encapsulation, and multiple tumours. Patients with high GSN expression had worse overall survival (OS) and disease-free survival (DFS) than those with low GSN expression. GSN expression was identified as an independent risk factor in both OS (hazard risk (HR) = 1.620, 95% confidence interval (CI) = 1.105–2.373, P < 0.001) and DFS (HR = 1.744, 95% CI = 1.205–2.523, P=0.003). Moreover, GSN knockdown significantly inhibited the migration and invasion of HCC tumour cells, while GSN overexpression attenuated these effects by regulating epithelial-mesenchymal transition (EMT) In conclusion, GSN promotes cancer progression and is associated with a poor prognosis in HCC patients. GSN promotes HCC progression by regulating EMT.

Decreased expression of GBA3 correlates with a poor prognosis in hepatocellular carcinoma patients.

Beta-glucosidase (GBA), also known as acid β-glucosidase, exhibits an activity of glucosylceramidase (EC 3.2.1.45). Three main isoforms of β-glucosidases have been identified in mammals: GBA1, GBA2, and GBA3. The deficiency of these enzymes leads to glucosylceramide accumulation, resulting in Gaucher's disease. The present study is focused on the cytosolic β-glucosidase, GBA3, and its relationship with hepatocellular carcinoma (HCC). The expression of GBA3 mRNA in HCC was evaluated first using the TCGA database, and then by immunohistochemistry using tissue microarrays of 328 clinically characterized HCC samples and 151 non-tumor liver controls. Moreover, the presence of a correlation between GBA3 expression and clinicopathological characteristics of patients was examined. The obtained results indicated that the expression of GBA3 mRNA was significantly lower in HCC than in the adjacent non-tumor liver tissues. The expression of GBA3 was inversely related to the number of tumors (p=0.041), tumor size (p<0.001), Edmondson grade (p=0.007), microvascular invasion (p=0.049), patient status (p<0.001), and α-fetoprotein level (p<0.001). Patients exhibiting low GBA3 expression had a shorter survival time than those with high expression (p<0.001). In conclusion, the decreased GBA3 expression is strongly associated with a poor prognosis in HCC patients, and GBA3 may be a potential therapeutic target for HCC.

Correlation of PD-L1 and SOCS3 Co-expression with the Prognosis of Hepatocellular Carcinoma Patients.

Purpose: To investigate the correlation between the expression of PD-L1, SOCS3 and immune-related biomarkers CD276, CD4, CD8 in hepatocellular carcinoma (HCC) and further determine the relationship with clinicopathologic characteristics and the prognostic value of their co-expression in HCC patients.Methods: We assessed the expression of PD-L1, CD276, SOCS3, CD4 and CD8 by immunohistochemistry in tumor tissue from 74 HCC patients who underwent curative hepatectomy.Results: High expression of PD-L1 was significantly associated with high Edmondson grade (p<0.01) and elevated enzyme (p=0.037); high expression of CD276 was significantly correlated with high Edmondson grade (p=0.021); high expression of SOCS3 was significantly associated with age (p=0.026) and tumor size (p=0.041), while PD-L1 showed no significant correlation. The expression of PD-L1, CD276, SOCS3 protein and other clinicopathological factors (sex, vascular invasion, tumor number, tumor capsule, pT stage, liver cirrhosis, HBsAg, TBiL, AFP) showed no significant correlation (p>0.05). High expression of CD8 was respectively significantly associated with worse overall survival (OS) (p=0.002). There was no significantly difference between CD4 and CD8 high-expression and overall survival (OS) (p=0.100). Both high expression of PD-L1 (p=0.003) and low expression of SOCS3 (p=0.015) was significantly associated with worse overall survival (OS). But CD276 only had a trendency (p=0.166). Additionally, multivariate Cox regression models implied that PD-L1, SOCS3, as well as both CD4 and CD8 was an independent prognostic factor for OS (p<0.05). Furthermore, HCC patients with PD-L1 low-expression and SOCS3 high-expression had a better prognostic according to the different pT stages (p<0.05).Conclusions: We for the first time demonstrated that PD-L1 and SOCS3 were independent prognostic factor for HCC patients. Co-expression of low PD-L1 and high SOCS3 could be a better predictive marker for HCC patients.

Expression of Pregnancy Up-regulated Non-ubiquitous Calmodulin Kinase (PNCK) in Hepatocellular Carcinoma.

Pregnancy up-regulated non-ubiquitous calmodulin kinase (PNCK) is a member of calmodulin kinase, and overexpression of PNCK with involvement in carcinogenesis have been reported in HER-2 amplified breast cancer, clear cell renal cell carcinoma and nasopharygeal carcinoma. However, the expression of PNCK and its clinical implication have not been elucidated in hepatocellular carcinoma (HCC). We investigated PNCK expression at both the protein and mRNA level using immunohistochemistry (IHC) and microarray gene expression profiling in HCC tissue samples, and evaluated its association with clinicopathological parameters and their potential prognostic significance. High PNCK protein expression and high PNCK mRNA level was observed in 61.7% and 34.7% of total HCC cases, respectively. PNCK mRNA level was higher in tumor tissues than in background non-tumor tissues, and significantly correlated with protein expression by IHC. High PNCK expression was associated with higher Edmondson grade, intrahepatic metastasis, microvascular invasion and higher AFP levels. Patients with high PNCK expression showed shorter recurrence-free survival and disease-specific survival, and high mRNA expression of PNCK was an independent prognostic factor in disease-specific survival. Up-regulation of PNCK expression as well as its association with poor prognosis was demonstrated in HCC. PNCK might be a prognostic biomarker of HCC, and could be a potential candidate therapeutic target.

Decreased DC-SIGNR expression in hepatocellular carcinoma predicts poor patient prognosis.

Dendritic cell-specific intercellular adhesion molecule-grabbing non-integrin-related protein (DC-SIGNR) is a transmembrane receptor primarily involved in pathogen recognition by the innate immune system, with particular importance for viral recognition. DC-SIGNR may also be associated with tumorigenesis. The aim of the present study was to investigate the association between DC-SIGNR expression, development of hepatocellular carcinoma (HCC), and clinicopathological features. Immunohistochemistry was used to assess DC-SIGNR protein expression in HCC and paired non-cancerous tissue samples. DC-SIGNR expression was lower in HCC tissues compared with adjacent non-tumor tissue samples. The expression of DC-SIGNR was associated with small tumor size, low Edmondson grade and high patient long term survival rates. Bioinformatics analyses were performed on several datasets to assess the potential function of DC-SIGNR and related genes; the data revealed that DC-SIGNR mRNA expression was lower in HCC tissues compared with non-cancerous controls, and analyses of ten-year survival rates indicated patients with low DC-SIGNR expression exhibited shorter average survival times. In conclusion, decreased DC-SIGNR expression in HCC tissues may be a relevant predictive biomarker of clinical prognosis, in addition to being a viable therapeutic target for HCC treatment.

Tissue and serum metabolomic phenotyping for diagnosis and prognosis of hepatocellular carcinoma.

More than two-thirds of patients with hepatocellular carcinoma (HCC) cannot receive curative therapy and have poor survival due to late diagnosis and few prognostic directions. In our study, nontargeted and targeted metabolomics analyses were conducted by liquid chromatography-mass spectrometry to characterize metabolic features of HCC and identify diagnostic and prognostic biomarker candidate incorporating liver tissue and serum metabolites. A total of 552 subjects, including 432 with liver tissue and 120 with serum specimens, were recruited in China. In the discovery cohort, a series of 138 metabolites were identified to discriminate HCC tissues from matched nontumor tissues. Retinol presented with the highest area under the curve (AUC) of 0.991 and associated with Edmondson grade. In the validation cohort, all metabolites in retinol metabolism pathway were examined and the levels of retinol and retinal in tumor tissue and serum decreased in the order of normal to cirrhosis to HCC of Edmondson Grades I to IV. Retinol and retinal levels could also differentiate between HCC and cirrhosis, with AUCs of 0.996 and 0.994, respectively, in tissue and 0.812 and 0.744, respectively, in serum. The AUC of the combined retinol and retinal panel in serum was 0.852. Univariate and multivariate Cox regression identified this panel as an independent predictor for HCC and showed that low expression of retinol and retinal correlated with decreased survival time. In conclusion, the retinol metabolic signature had considerable diagnostic and prognostic value for identifying HCC patients who would benefit from prompt therapy and optimal prognostic direction.

Comparison of the effect on the prognosis of HCC in terms of different surgical approaches for hepatic inflow occlusion

Background: To investigate the effect of surgical approaches for hepatic inflow occlusion performed during hepatectomy on postoperative tumor-free survival (TFS) in patients treated with R0 resection. Methods: In total, 343 hepatocellular carcinoma (HCC) patients who underwent hepatectomy (R0) with different surgical approaches for hepatic inflow occlusion were analyzed retrospectively. Results: In total, 68.80% of the patients underwent hepatic inflow occlusion, including 46.65% with a routine Pringle maneuver and 22.16% with selective hemi-hepatic vascular exclusion (SHVE), during surgery. The TFS did not significantly differ among the Pringle group, the SHVE group and the no hepatic inflow occlusion group. After further stratifying the patients according to the Barcelona clinic liver cancer (BCLC) staging system, the patients with BCLC stage A disease in the SHVE group and no hepatic inflow occlusion group displayed better TFS than those in the Pringle maneuver group (P=0.04; P=0.002), but the patients with BCLC stage B–C disease did not show significant differences among the groups. Furthermore, all 214 patients with BCLC stage A disease were classified into two subgroups according to the microvascular invasion (MVI) status. Interestingly, among the patients with MVI, those in the SHVE group and no hepatic inflow occlusion group had significantly longer TFS than those in the Pringle group (P=0.025; P=0.006); however, the patients without MVI did not show differences among the groups. Additionally, the multivariate analysis revealed that a tumor size ≥5 cm, an absent capsule, a low Edmondson grade, MVI positivity and performance of the Pringle maneuver were independent risk factors of the prognosis in patients with BCLC stage A disease. Conclusions: Hepatic inflow occlusion and the surgical approach used to address this issue may have an impact on HCC prognosis in patients with BCLC stage A disease, especially among those with MVI positivity. No hepatic inflow occlusion or an SHVE approach rather than the Pringle maneuver should be considered first during hepatectomy for patients with BCLC stage A disease.

Comparison of the effect on the prognosis of HCC in terms of different surgical approaches for hepatic inflow occlusion.

BackgroundTo investigate the effect of surgical approaches for hepatic inflow occlusion performed during hepatectomy on postoperative tumor-free survival (TFS) in patients treated with R0 resection.MethodsIn total, 343 hepatocellular carcinoma (HCC) patients who underwent hepatectomy (R0) with different surgical approaches for hepatic inflow occlusion were analyzed retrospectively.ResultsIn total, 68.80% of the patients underwent hepatic inflow occlusion, including 46.65% with a routine Pringle maneuver and 22.16% with selective hemi-hepatic vascular exclusion (SHVE), during surgery. The TFS did not significantly differ among the Pringle group, the SHVE group and the no hepatic inflow occlusion group. After further stratifying the patients according to the Barcelona clinic liver cancer (BCLC) staging system, the patients with BCLC stage A disease in the SHVE group and no hepatic inflow occlusion group displayed better TFS than those in the Pringle maneuver group (P=0.04; P=0.002), but the patients with BCLC stage B–C disease did not show significant differences among the groups. Furthermore, all 214 patients with BCLC stage A disease were classified into two subgroups according to the microvascular invasion (MVI) status. Interestingly, among the patients with MVI, those in the SHVE group and no hepatic inflow occlusion group had significantly longer TFS than those in the Pringle group (P=0.025; P=0.006); however, the patients without MVI did not show differences among the groups. Additionally, the multivariate analysis revealed that a tumor size ≥5 cm, an absent capsule, a low Edmondson grade, MVI positivity and performance of the Pringle maneuver were independent risk factors of the prognosis in patients with BCLC stage A disease.ConclusionsHepatic inflow occlusion and the surgical approach used to address this issue may have an impact on HCC prognosis in patients with BCLC stage A disease, especially among those with MVI positivity. No hepatic inflow occlusion or an SHVE approach rather than the Pringle maneuver should be considered first during hepatectomy for patients with BCLC stage A disease.

LncRNA XIST regulates proliferation and migration of hepatocellular carcinoma cells by acting as miR-497-5p molecular sponge and targeting PDCD4

BackgroundMicroRNAs (miRNAs) play a pivotal role in hepatocellular carcinoma (HCC) progression and have been confirmed to participate in the carcinogenesis and development of HCC. However, the relationship between miR-497-5p and HCC remains unclear.MethodsKaplan–Meier curve analysis and the log-rank test were used to investigate the efficacy of miR-497-5p on overall survival (OS) and disease-free survival (DFS) in patients with HCC. According to in vitro experiments, programmed cell death 4 (PDCD4) was a target of miR-497-5p by the dual-luciferase activity assay. The efficacy of PDCD4 on cell proliferation and metastasis in HCC was examined by transwell assays, CCK-8 assays and reverse transcription quantitative PCR (RT-qPCR). Additionally, we conducted a luciferase activity reporter assay to confirm the interaction between lncRNA XIST and miR-49-5p. Then, to evaluate the relationship between lncRNA XIST and miR-497-5p, several mechanistic experiments, including qRT-PCR, Western blotting, transwell assays and tumor xenograft assays, were performed.ResultsmiR-497-5p was upregulated in HCC tissues, and high expression of miR-497-5p resulted in increases in tumor size and tumor number and a higher tumor-node-metastasis (TNM) stage and Edmondson grade in patients with HCC. Silencing miR-497-5p inhibited the proliferation and migration of HCC cells. PDCD4, which was downregulated in HCC tissues, was shown to be a target of miR-497-5p and was negatively correlated with the expression of miR-497-5p. lncRNA XIST was found to act as a miR-497-5p sponge and to regulate the level of PDCD4, which is targeted by miR-497-5p. lncRNA XIST was observed to be downregulated in the HCC tissues and positively correlated with the expression of PDCD4.ConclusionsOur findings reveal that the XIST/miR-497-5p/PDCD4 axis participates in HCC development and that XIST could be used as a biomarker of HCC.

A preliminary study of serum marker alpha-enolase in the diagnosis of hepatocellular carcinoma

Objective: To investigate the diagnostic value of serum α-enolase (ENO1) in the primary hepatocellular carcinoma. Methods: From May 2012 to March 2017, 163 cases with liver diseases who met the inclusion and exclusion criteria were admitted to the Infectious Diseases Department of the General Hospital of Ningxia Medical University. Among them, 28 cases were of chronic hepatitis B (CHB), 31 cases with liver cirrhosis (LC), 104 cases with hepatocellular carcinoma (HCC), and 18 healthy volunteers (NC). Patient data and serum samples were collected and liver disease related indicators were measured to detect ENO1 levels with enzyme-linked immunosorbent assay (ELISA). The measured indicators were expressed in median. Mann-Whitney U nonparametric test was used to analyze the differences between the data. A Spearman's correlation analysis was used for bivariate correlation analysis. The sensitivity and specificity of ENO1 and alpha-fetoprotein in the diagnosis of liver cancer were analyzed by ROC curve. Results: Serum level of ENO1 in CHB group, LC group and HCC group was significantly higher than normal group. Serum level of ENO1 in HCC group was higher than CHB group (P = 0.001) and LC group (P < 0.01). Area under the curve (AUC) for serum ENO1 and alpha-fetoprotein were 0.782 (cut-off value 75.96, P = 0.000 1) and 0.800 (cut-off value 27.02, P = 0.000 1), respectively. There was a positive correlation between ENO1 and AFP (P = 0.001). The combined detection had significantly improved the detection efficiency (AUC = 0.835). Serum ENO1 was statistically significant (P < 0.05) in HCC tumor size (AUC = 0.663), tumor metastasis (AUC = 0.681), TNM stage (AUC = 0.710, stage I vs. II), and Edmondson grade (AUC = 0.685) (P < 0.05) and the elevated levels of ENO1 had significantly reduced (P < 0.05) the survival time. Conclusion: ENO1 can be a new candidate marker for the diagnosis of early stage HCC and its progression.

Hepatocellular Carcinoma Xenografts Established From Needle Biopsies Preserve the Characteristics of the Originating Tumors.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer‐related deaths worldwide. Treatment options for patients with advanced‐stage disease are limited. A major obstacle in drug development is the lack of an in vivo model that accurately reflects the broad spectrum of human HCC. Patient‐derived xenograft (PDX) tumor mouse models could overcome the limitations of cancer cell lines. PDX tumors maintain the genetic and histologic heterogeneity of the originating tumors and are used for preclinical drug development in various cancers. Controversy exists about their genetic and molecular stability through serial passaging in mice. We aimed to establish PDX models from human HCC biopsies and to characterize their histologic and molecular stability during serial passaging. A total of 54 human HCC needle biopsies that were derived from patients with various underlying liver diseases and tumor stages were transplanted subcutaneously into immunodeficient, nonobese, diabetic/severe combined immunodeficiency gamma‐c mice; 11 successfully engrafted. All successfully transplanted HCCs were Edmondson grade III or IV. HCC PDX tumors retained the histopathologic, transcriptomic, and genomic characteristics of the original HCC biopsies over 6 generations of retransplantation. These characteristics included Edmondson grade, expression of tumor markers, tumor gene signature, tumor‐associated mutations, and copy number alterations. Conclusion: PDX mouse models can be established from undifferentiated HCCs, with an overall success rate of approximately 20%. The transplanted tumors represent the entire spectrum of the molecular landscape of HCCs and preserve the characteristics of the originating tumors through serial passaging. HCC PDX models are a promising tool for preclinical personalized drug development.

High expression of agrin is associated with tumor progression and poor prognosis in hepatocellular carcinoma.

The heparan sulfate proteoglycan agrin is known to accumulate in the context of hepatocellular carcinoma (HCC). Agrin is important for neoangiogenesis in HCC tissues, and is incorporated into newly formed vasculature, but exactly how agrin contributes to the pathology of HCC remains to be fully defined. We therefore examined the clinical relevance of agrin as it pertains to HCC progression and prognosis using tissue sections from a total of 313 HCC patients. We found that agrin expression was detectable in more HCC samples (25.4% vs. 77.1%; P < 0.05) compared to normal tissue controls. Agrin expression was notably linked to tumor size (P = 0.041) and metastasis (P = 0.034). The recurrence free survival rate of agrin-positive HCC patients was considerably lower than that of agrin-negative patients (P = 0.001). We further confirmed HCC survival to be independently correlated with tumor size, metastasis, microvascular invasion and edmondson Grade via a Cox regression analysis. Upregulation of Agrin may play a crucial role in HCC progression. Together our results suggest that Agrin has the potential to be used as a prognostic indicator in predicting HCC patient outcomes.

Endoplasmic reticulum resident oxidase ERO1-Lalpha promotes hepatocellular carcinoma metastasis and angiogenesis through the S1PR1/STAT3/VEGF-A pathway

Mounting evidence demonstrates that expression of ERO1α, an endoplasmic reticulum (ER)-resident oxidase, is a poor prognosis factor in a variety of human cancers. However, the clinical relevance of ERO1α and its molecular mechanisms underlying tumor progression have not been determined for hepatocellular carcinoma (HCC). ERO1α expression levels in HCC tissues and cells were detected by quantitative real-time PCR and western blotting. ERO1α shRNAs and overexpression vector were transfected into HCC cells to downregulate or upregulate ERO1α expression. In vitro and in vivo assays were performed to investigate the function of ERO1α in invasion, metastasis, and angiogenesis of HCC. We found high ERO1α expression in HCC tissues and cells that was significantly associated with metastasis and poor clinicopathologic features of vascular invasion, advanced Edmondson Grade, and TNM stage. Loss-of-function and gain-of-function studies showed that ERO1α prompted migration, invasion, epithelial–mesenchymal transition (EMT), and angiogenesis of HCC cells both in vitro and in vivo. Further studies verified a positive correlation between ERO1α and S1PR1, upregulated in metastatic HCC tissues compared with HCC tissues without metastasis. S1PR1 knockdown markedly diminished the effects of ERO1α on HCC cell migration, invasion and vascular endothelial growth factor (VEGF) expression. Most importantly, ERO1α knockdown significantly repressed the death of HCC xenograft mouse models by reducing tumor distant metastasis, and host angiogenesis by suppressing the expression of S1PR1, p-STAT3, and VEGF-A in HCC cells. Our findings suggest that ERO1α is significantly correlated with reduced survival and poor prognosis, and promotes HCC metastasis and angiogenesis by triggering the S1PR1/STAT3/VEGF-A signaling pathway. ERO1α might be a novel candidate in HCC prognosis and therapy.

EXO1 overexpression is associated with poor prognosis of hepatocellular carcinoma patients

ABSTRACTThe roles of exonuclease 1 (EXO1) in hepatocellular carcinoma (HCC) tumorigenesis and progression remain unclear. This study aimed to assess the prognostic value and therapeutic potential of EXO1 in HCC. Exo1 gene copy numbers were obtained from three Oncomine microarray datasets (n = 447). EXO1 mRNA expression was validated by semi-quantitative PCR and QuantiGene® 2.0 assays. Cell growth curve and colony formation were performed to asses the cell proliferation. Clonogenic assay, flow cytometry, and immunofluorescence were adopted to acess the effects of EXO1 knockdown and radiation on cell survival, cell cycle distribution and DNA repair. Western blots were performed to reveal the related mechanism. A significant copy number variation (CNV) of the Exo1 gene was found in HCC specimens in three separate sets of published microarray data. In the 143 cases treated by our team, EXO1 expression levels were elevated (86.71%, 124/143). In addition, EXO1 overexpression was correlated with larger tumor size (P = 0.002), increased lymph node metastasis (P=0.033) and lower Edmondson grade (P = 0.018). High EXO1 expression unfavorably affected overall survival (OS) (P = 0.009). Both univariate and multivariate Cox regression analyses identified EXO1 as an independent predictor of OS (univariate, P = 0.012; multivariate, P = 0.039). Silencing of EXO1 in vitro reduced cell proliferation. EXO1 knockdown further suppressed clonogenic cell survival, abrogated radiation-induced G2/M phase arrest, and enhanced γ-H2AX foci after exposure to irradiation. The accumulation of ataxiatelangiectasia mutated (ATM) might partially regulate the EXO1 related radiosensitivity. In summary, EXO1 could be a promising prognostic marker, with a potential therapeutic value in HCC.

Heat Shock Protein 90α-Dependent B-Cell-2-Associated Transcription Factor 1 Promotes Hepatocellular Carcinoma Proliferation by Regulating MYC Proto-Oncogene c-MYC mRNA Stability.

B‐cell lymphoma 2 (Bcl‐2)‐associated transcription factor 1 (Bclaf1) is known to be involved in diverse biological processes, but, to date, there has been no evidence for any functional role of Bclaf1 in hepatocellular carcinoma (HCC) progression. Here, we demonstrate that Bclaf1 is frequently up‐regulated in HCC and that Bclaf1 up‐regulation is associated with Edmondson grade, lower overall survival rates, and poor prognosis. Overexpression of Bclaf1 in HCC cell lines HepG2 and Huh7 promoted proliferation considerably, whereas Bclaf1 knockdown had the opposite effect. Xenograft tumors grown from Bclaf1 knockdown Huh7 cells had smaller tumor volumes than tumors grown from control cells. Furthermore, our study describes MYC proto‐oncogene (c‐Myc) as a downstream target of Bclaf1, given that Bclaf1 regulates c‐MYC expression posttranscriptionally by its RS domain. To exert this function, Bclaf1 must interact with the molecular chaperone, heat shock protein 90 alpha (Hsp90α). In HCC tissue samples, Hsp90α levels were also increased significantly and Hsp90α‐Bclaf1 interaction was enhanced. Bclaf1 interacts with the C‐terminal domain of Hsp90α, and this interaction is disrupted by the C‐terminal domain inhibitor, novobiocin (NB), resulting in proteasome‐dependent degradation of Bclaf1. Moreover, NB‐induced disruption of Hsp90α‐Bclaf1 interaction dampened the production of mature c‐MYC mRNA and attenuated tumor cell growth in vitro and in vivo. Conclusion: Our findings suggest that Bclaf1 affects HCC progression by manipulating c‐MYC mRNA stability and that the Hsp90α/Bclaf1/c‐Myc axis might be a potential target for therapeutic intervention in HCC.