Cells In Hepatocellular Carcinoma Tissues Research Articles

LMNB1/CDKN1A Signaling Regulates the Cell Cycle and Promotes Hepatocellular Carcinoma Progression.

Hepatocellular carcinoma (HCC) is one of the most aggressive malignancies in the world. Lamin B1 (LMNB1) is a key component of the nuclear skeleton structure. Recent studies have found that LMNB1 is overexpressed in tumor tissues and is associated with the prognosis of patients. However, the underlying mechanism remains unclear in HCC. This study aims to explore the clinical significance and molecular mechanisms of LMNB1 in HCC. The expression level of LMNB1 and its clinical values were analyzed with public databases, and the level of LMNB1 in HCC tissues and adjacent normal tissues was confirmed by qRT-PCR and IHC. Functional assays were conducted to explore the impact of LMNB1 knockdown on cell proliferation both in vivo and in vitro. Additionally, Genes and Genomes enrichment analysis, recovery analysis, and ChIP assays were employed to investigate its underlying molecular mechanisms. Finally, we carried out an analysis of the relationship between LMNB1 and immune cell infiltration in HCC. LMNB1 was found to be overexpressed in HCC and correlated with the pathological stage and unfavorable prognosis. Functional assays demonstrated that LMNB1 promotes HCC proliferation both in vitro and in vivo. Further analysis revealed that LMNB1 promotes the progression of HCC by regulating CDKN1A expression. Furthermore, the infiltration of immune cells in HCC tissues suggests a potential correlation between immune infiltration cell markers and the expression of LMNB1. LMNB1 emerged as a promising therapeutic target and prognostic biomarker for HCC, with its expression showing a correlation with several immune infiltration cell markers.

Integrated Analysis of Tumor Mutation Burden and Immune Infiltrates in Hepatocellular Carcinoma.

Tumor mutation burdens (TMBs) act as an indicator of immunotherapeutic responsiveness in various tumors. However, the relationship between TMBs and immune cell infiltrates in hepatocellular carcinoma (HCC) is still obscure. The present study aimed to explore the potential diagnostic markers of TMBs for HCC and analyze the role of immune cell infiltration in this pathology. We used OA datasets from The Cancer Genome Atlas database. First, the “maftools” package was used to screen the highest mutation frequency in all samples. R software was used to identify differentially expressed genes (DEGs) according to mutation frequency and perform functional correlation analysis. Then, the gene ontology (GO) enrichment analysis was performed with “clusterProfiler”, “enrichplot”, and “ggplot2” packages. Finally, the correlations between diagnostic markers and infiltrating immune cells were analyzed, and CIBERSORT was used to evaluate the infiltration of immune cells in HCC tissues. As a result, we identified a total of 359 DEGs in this study. These DEGs may affect HCC prognosis by regulating fatty acid metabolism, hypoxia, and the P53 pathway. The top 15 genes were selected as the hub genes through PPI network analysis. SRSF1, SNRPA1, and SRSF3 showed strong similarities in biological effects, NCBP2 was demonstrated as a diagnostic marker of HCC, and high NCBP2 expression was significantly correlated with poor over survival (OS) in HCC. In addition, NCBP2 expression was correlated with the infiltration of B cells (r = 0.364, p = 3.30 × 10−12), CD8+ T cells (r = 0.295, p = 2.71 × 10−8), CD4+ T cells, (r = 0.484, p = 1.37 × 10−21), macrophages (r = 0.551, p = 1.97 × 10−28), neutrophils (r = 0.457, p = 3.26 × 10−19), and dendritic cells (r = 0.453, p = 1.97 × 10−18). Immune cell infiltration analysis revealed that the degree of central memory T-cell (Tcm) infiltration may be correlated with the HCC process. In conclusion, NCBP2 can be used as diagnostic markers of HCC, and immune cell infiltration plays an important role in the occurrence and progression of HCC.

Identification of Key Functional Modules and Immunomodulatory Regulators of Hepatocellular Carcinoma.

Despite the advances in the treatment of hepatocellular carcinoma (HCC), the prognosis of HCC patients remains unsatisfactory due to postsurgical recurrence and treatment resistance. Therefore, it is important to reveal the mechanisms underlying HCC and identify potential therapeutic targets against HCC, which could facilitate the development of novel therapies. Based on 12 HCC samples and 12 paired paracancerous normal tissues, we identified differentially expressed mRNAs and lncRNAs using the “limma” package in R software. Moreover, we used the weighted gene coexpression network analysis (WGCNA) to analyze the expression data and screened hub genes. Furthermore, we performed pathway enrichment analysis based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. In addition, the relative abundance of a given gene set was estimated by single-sample Gene Set Enrichment Analysis. We identified 687 differentially expressed mRNAs and 260 differentially expressed lncRNAs. A total of 6 modules were revealed by WGCNA, and MT1M and MT1E genes from the red module were identified as hub genes. Moreover, pathway analysis revealed the top 10 enriched KEGG pathways of upregulated or downregulated genes. Additionally, we also found that CD58 might act as an immune checkpoint gene in HCC via PD1/CTLA4 pathways and regulate the levels of tumor-infiltrating immune cells in HCC tissues, which might be an immunotherapeutic target in HCC. Our research identified key functional modules and immunomodulatory regulators for HCC, which might offer novel diagnostic biomarkers and/or therapeutic targets for cancer immunotherapy.

Cancer cell-intrinsic STING is associated with CD8 + T-cell infiltration and might serve as a potential immunotherapeutic target in hepatocellular carcinoma.

The activation of stimulator of interferon genes (STING) pathway triggers the antitumor immunity by CD8 + T cells. However, the differentiated antitumor effects of STING activation in different cell types is still unclear. We aimed to investigate the expression and potential prognostic value of cancer cell-intrinsic STING in hepatocellular carcinoma (HCC), and whether STING could be a potential immunotherapeutic target of HCC was then evaluated. We separately assessed the expression of STING in cancer cells and infiltrating immune cells in HCC tissues. The independent clinicopathological factors associated with survival outcomes were evaluated by the multivariable analysis. The HCC orthotopic mice model were used to confirm the immunotherapeutic effects of STING agonists, and CD8 + T-cell infiltration level was analyzed through immunofluorescence and flow cytometry. The expression of cancer cell-intrinsic STING was significantly reduced in HCC compared with adjacent tissues. Patients with low levels of cancer cell-intrinsic STING expression was associated with increased tumor volume (P = 0.009), higher serum AFP levels (P = 0.028), and decreased CD8 + T-cell infiltration (P = 0.002). Low levels of cancer cell-intrinsic STING expression indicated a poor overall survival (OS) and disease-free survival (DFS). Multivariate analysis demonstrated that low levels of cancer cell-intrinsic STING expression was an independent prognostic factor. Additionally, cancer cell-intrinsic STING expression was positively related with CD8 + T-cell infiltration levels in HCC patients (r = 0.308; P = 0.001). When mice with orthotopic HCC tumors treated with STING agonists, tumor growth was significantly reduced with enhanced levels of CD8 + T-cell infiltration. Cancer cell-intrinsic STING might affect HCC tumor progression through enhancing CD8 + T-cell infiltration and can be an immunotherapeutic target for HCC.

Increased infiltration of regulatory T cells in hepatocellular carcinoma of patients with hepatitis B virus pre-S2 mutant

Hepatocellular carcinoma (HCC) is a frequent and deadly human cancer worldwide that is intimately associated with chronic hepatitis B virus (HBV) infection. Pre-S2 mutant is a HBV oncoprotein that plays important roles in HCC development and is linked to poor prognosis in HCC patients. However, the profiles of tumor-infiltrating lymphocytes in HCC tissues of pre-S2 mutant-positive patients remain unknown. In this study, we performed fluorescent immunohistochemistry staining to detect the infiltration of ‘anti-tumor’ cytotoxic T lymphocytes (CTLs) and ‘pro-tumor’ regulatory T cells (Tregs) in pre-S2 mutant-positive and -negative HCC patients. We showed that pre-S2 mutant-positive patients had a significantly higher infiltration of CD4+CD25+ cells and forkhead box P3 (Foxp3)-expressing cells but similar CTLs and lower granzyme B-expressing cells in HCC tissues compared with pre-S2 mutant-negative patients. Moreover, the percentage of pre-S2 plus pre-S1 + pre-S2 deletion (pre-S2 mutant) was positively correlated with the density of CD4+CD25+ cells and Foxp3-expressing cells but negatively with granzyme B-expressing cells in HCC tissues. Considering that increased intratumoral Tregs have been shown to promote tumor immune evasion, our data may provide new insights into the pathogenesis of HBV pre-S2 mutant-induced HCC and suggest that therapeutics targeting Tregs may be a promising strategy for treating pre-S2 mutant-positive high-risk patient population.

Intratumoral CD45+CD71+ erythroid cells induce immune tolerance and predict tumor recurrence in hepatocellular carcinoma.

CD45+CD71+ erythroid cells generated through splenic extramedullary erythropoiesis have recently been found to suppress anti-infection and tumor immunity in neonates and adults with malignances. However, their role in tumor microenvironment has not been investigated. In the present study, we found that the number of CD45+CD71+ erythroid cells was significantly elevated in hepatocellular carcinoma (HCC) tissues compared to that in paratumor region and circulation. Additionally, they were more abundant in HCC tissues compared to some immune suppressive cells as well as CD45-CD71+ erythroid cells. CD45+CD71+ erythroid cells suppressed T cells through generation of reactive oxygen species, IL-10, and TGF-β in a paracrine and cell-cell contact manner, and their suppressive effect was stronger than that of myeloid-derived suppressor cells. The abundance of CD45+CD71+ erythroid cells in tumor tissue, as illustrated via immunofluorescence, predicted disease-free survival and overall survival, and its prognostic value was better than that of Cancer of the Liver Italian Program score. This study demonstrated that accumulation of intratumoral CD45+CD71+ erythroid cells in HCC tissues could play a superior immunosuppressive role in tumor microenvironment and may serve as a valuable biomarker to predict recurrence of HCC.

Increased Expression of Programmed Death Ligand 1 in Hepatocellular Carcinoma of Patients with Hepatitis B Virus Pre-S2 Mutant.

PurposeChronic hepatitis B virus (HBV) infection is a major risk factor for hepatocellular carcinoma (HCC), a leading cause of cancer-related death worldwide. The HCC patients who harbor HBV pre-S2 mutant, an oncoprotein that plays key roles in HCC development, have been closely associated with a worse prognosis after curative surgical resection, suggesting an urgent need for alternative therapeutic options to improve their survival. In this study, we aimed to evaluate the expression profiles of programmed death 1 (PD-1) and programmed death ligand 1 (PD-L1), two of the most well-studied immune checkpoint molecules that promote tumor immune evasion, in tumor of the pre-S2 mutant-positive/high HCC patients.MethodsWe classified 40 HBV-related HCC patients into the pre-S2-positive/high and -negative/low groups by a next-generation sequencing-based approach. The fluorescent immunohistochemistry staining was performed to detect the expression of PD-1 and PD-L1 in HCC tissues of patients.ResultsWe showed that patients with either deletion spanning pre-S2 gene segment or high percentage of pre-S2 plus pre-S1+pre-S2 deletion (the pre-S2 mutant-positive/high group) exhibited a significantly higher density of PD-L1-positive cells in HCC tissues than those without. Moreover, the percentage of pre-S2 plus pre-S1+pre-S2 deletion displayed a high positive correlation with the density of PD-L1-positive cells in HCC tissues.ConclusionThe increased expression of PD-L1 in tumor tissues of the pre-S2 mutant-positive HCC patients suggest that pre-S2 mutant may play a potential role in dysregulation of tumor immune microenvironment in the progression of HBV-related HCC, implicating for the development of future therapeutic strategies.

Clinical significance of herpes virus entry mediator expression in hepatitis B virus-related hepatocellular carcinoma.

Herpes virus entry mediator (HVEM) is overexpressed in several malignancies, including hepatocellular carcinoma (HCC). However, to the best of our knowledge, the clinical significance of HVEM in hepatitis B virus (HBV)-related HCC remains unclear. Thus, the present study aimed to explore the clinical significance of HVEM in HBV-related HCC. In the present study, HVEM expression was evaluated in HCC cell lines and HCC frozen samples. The prognostic value of HVEM was assessed in a cohort of 221 patients with HBV-related HCC, following radical resection. B- and T-lymphocyte attenuator (BTLA) expression in subsets of CD8+ T cells was determined via flow cytometry analysis. The results demonstrated high HVEM expression in HCC cell lines, and in HCC tissues compared with paired non-cancerous liver tissues. HVEM expression was demonstrated to be significantly associated with tumor encapsulation and vascular invasion. Furthermore, tumor HVEM status was significantly associated with infiltration of regulatory T cells, but not with CD8+ T cells. Notably, high HVEM expression in HCC was determined to be an independent predictor of an unfavorable outcome of patients with HCC following radical resection. Higher BTLA expression (the receptor of HVEM) was observed in both HCC-infiltrating CD8+ effector memory (CCR7- CD45RA-) and CD45RA+ effector memory (CCR7- CD45RA+) T cells in HCC tissues and blood compared with those in paired peritumor tissues or peripheral blood. Taken together, the results of the present study suggest that HVEM may serve a critical role in HBV-related HCC, most likely by promoting tumor progression and tumor immune evasion, thus the HVEM/BLTA signaling pathway may be a potential target in tumor immunotherapy.

Dysregulated cancer cell transdifferentiation into erythrocytes is an additional metabolic stress in hepatocellular carcinoma.

A number of human and canine hepatocellular carcinoma tissues showed clear signs of hypoxia indicated by HIF1α-activation and the presence of large clusters of cells resembling erythrocytes at different stages of nuclear elimination without any defined endothelial cell lining or blood vessel walls. Differentiated erythrocytic identity of such cells in hepatocellular carcinoma tissues was apparent from their non-nucleated and evolving basophilic to eosinophilic staining characteristics. In addition to the fully differentiated non-nucleated mesenchymal cell clusters, the onset of erythroblastic transdifferentiation was apparent from the activation of Glycophorin A, a marker of erythrocytic progenitors, in some epithelial cancer cells. Activation of canonical Wnt signalling in such tumours was apparent from the expression of Wnt2 ligand and active β-catenin translocation into the nucleus indicating Wnt signalling to be one of the key signalling pathways participating in such cell transdifferentiation. Sonic hedgehog and bone morphogenetic protein signalling along with Sulf1/Sulf2 activation was also observed in such hepatocellular carcinoma tissue samples. The presence of stem cell markers and the cell signalling pathways associated with erythropoiesis, and the detection of messenger RNAs for both α and β haemoglobins, support the assumption that hepatocellular carcinoma cells have the potential to undergo cell fate change despite this process being dysregulated as indicated by the lack of simultaneous generation of endothelial cell lining. Lack of blood vessel walls or endothelial cell lining around erythrocytic clusters was confirmed by non-detection of multiple blood vessel markers such as vWF, CD146 and smooth muscle α-actin that were clearly apparent in normal and unaffected adjacent regions of hepatocellular carcinoma livers. In addition to the activation of Glycophorin A, transdifferentiation of some hepatocellular carcinoma hepatocytes into other cell fates was further confirmed by the activation of some stem cell markers, for example, NANOG and OCT4 transcription factors, not only by reverse transcription polymerase chain reaction but also by their restricted expression in such cells at protein level.

Increased Expression of Programmed Death-Ligand 1 in Infiltrating Immune Cells in Hepatocellular Carcinoma Tissues after Sorafenib Treatment

Objective: Programmed death-ligand 1 (PD-L1) expression in the tumor microenvironment (TME) has been reported to be related to prognosis in patients with hepatocellular carcinoma (HCC) after hepatectomy. The impact of sorafenib on PD-L1 expression in the TME of advanced HCC is unclear. Patients and Methods: Patients with HCC who received sorafenib for advanced disease at National Taiwan University Hospital, Taipei, Taiwan, and who had paired HCC tissues obtained before and after sorafenib treatment were included in the study group. HCC patients not treated with sorafenib who had paired primary and recurrent or metastatic tissues were identified as the reference group. The membrane PD-L1 staining, detected by immunohistochemistry (IHC) using SP142 antibody, was semiquantitatively scored in tumor cells (TCs) or tumor-infiltrating immune cells (ICs). Additional IHC assays were employed to characterize the PD-L1-expressing ICs. Results: Twenty-three advanced HCC patients with pre- and post-sorafenib paired HCC tissues were included in the study group. The median duration of sorafenib treatment was 4.3 months (range: 1.3–18.7). PD-L1 expression in ICs was significantly higher in post-sorafenib HCC tissues than in pre-sorafenib HCC tissues (pre-sorafenib vs. post-sorafenib IHC 0/1/2/3: 11/5/5/2 vs. 5/5/2/11, p = 0.016). However, PD-L1 expression in TCs was not significantly different between pre- and post-sorafenib tissues (IHC 0/1/2/3: 19/2/0/2 vs. 14/5/0/4, p = 0.094). In the reference group of 44 patients not treated with sorafenib, PD-L1 expression in ICs and TCs was not significantly different between the paired primary and metastatic HCC tissues. By performing IHC double staining with PD-L1 and CD68, we found the PD-L1-expressing ICs were mainly CD68-positive macrophages. PD-L1 expression levels of pre- and post-sorafenib tissues were not associated with patients’ overall survival or duration of sorafenib treatment. Conclusions: PD-L1 expression in ICs was significantly increased in post-sorafenib HCC tissues. The mechanisms and clinical significance of this observation warrants further investigation.

CXCR2-CXCL1 axis is correlated with neutrophil infiltration and predicts a poor prognosis in hepatocellular carcinoma.

Background & aimsInflammation is a hallmark of cancer, yet the mechanisms that regulate immune cell infiltration into tumors remain poorly characterized. This study attempted to characterize the composition, distribution, and prognostic value of CXCR2+ cells in hepatocellular carcinoma (HCC) and to examine the CXCR2 ligands that are responsible for local immune infiltration in different areas of HCC tumors.MethodsImmunohistochemistry and immunofluorescene were used to identify CXCR2+ cells in HCC tissues. Kaplan–Meier analysis and Cox regression models were applied to estimate recurrence-free survival (RFS) and overall survival (OS) for 259 HCC patients. The expression levels of CXCR2 ligands (CXCL-1, −2, −5, and −8) were measured by real-time PCR and compared with local immune cell density. The combined prognostic value of the CXCR2–CXCL1 axis was further evaluated.ResultsIn HCC tissues, CXCR2+ cells were mainly neutrophils that were enriched in the peri-tumoral stroma (PS) region. Kaplan–Meier survival analysis showed that increased CXCR2+PS cells were associated with reduced RFS and OS (P = 0.015 for RFS; P = 0.002 for OS). Multivariate Cox proportional hazards analysis identified CXCR2+PS cell density as an independent prognostic factor for OS (hazard ratio [HR] = 1.737, 95 % confidence interval [CI] = 1.167–2.585, P = 0.006). Furthermore, we detected a positive correlation between the density of CD15+ neutrophils and CXCL1 levels in both the peri-tumoral stroma and intra-tumoral regions. The combination of CXCR2 and CXCL1 expression levels represented a powerful predictor of a poor prognosis for patients with HCC.ConclusionsOur data showed that the CXCR2+ cell density was an independent prognostic factor for predicting OS for HCC patients. The CXCR2–CXCL1 axis can regulate neutrophil infiltration into HCC tumor tissues and might represent a useful target for anti-HCC therapies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-015-0247-1) contains supplementary material, which is available to authorized users.

Monocyte/macrophage-elicited natural killer cell dysfunction in hepatocellular carcinoma is mediated by CD48/2B4 interactions

Defects in natural killer (NK) cell functions are necessary for tumor immune escape, but their underlying regulatory mechanisms in human cancers remain largely unknown. Here we show, in detailed studies of NK cells in 294 untreated patients with hepatocellular carcinoma (HCC), that accumulation of functional NK cells in HCC tissues could predict improved survival of patients. However, in patients with advanced-stage HCC, NK cells were significantly decreased in number with impaired tumor necrosis factor alpha (TNF-α) and interferon-gamma (IFN-γ) production. High infiltration of peritumoral stroma monocytes/macrophages was positively correlated with impaired functional activities of NK cells in intratumoral areas. Further kinetic experiments revealed that soon after exposure to tumor-derived monocytes, NK cells underwent a rapid, transient activation, but then they became exhausted, and eventually died. The monocytes from HCC tissues, but not from nontumoral liver, strongly express CD48 proteins; and such monocyte-induced NK cell dysfunction was markedly attenuated by blocking CD48 receptor 2B4 on NK cells, but not by blockade of NKG2D and NKp30. These data reveal that human NK cells are regulated by a fine-tuned collaborative action between different types of immune cells, which may reflect a novel immune-escape mechanism by which tumors dynamically regulate their functions at distinct tumor microenvironments.

Expression of Potential Cancer Stem Cell Marker ABCG2 is Associated with Malignant Behaviors of Hepatocellular Carcinoma

Background. Despite improvement in treatment, the prognosis of hepatocellular carcinoma (HCC) remains disastrous. Cancer stem cells (CSCs) may be responsible for cancer malignant behaviors. ATP-binding cassette, subfamily G, member 2 (ABCG2) is widely expressed in both normal and cancer stem cells and may play an important role in cancer malignant behaviors. Methods. The expression of ABCG2 in HCC tissues and SMMC-7721 cells was examined, and the relevance of ABCG2 expression with clinical characteristics was analyzed. ABCG2+ and ABCG2− cells were sorted, and the potential of tumorigenicity was determined. Expression level of ABCG2 was manipulated by RNA interference and overexpression. Malignant behaviors including proliferation, drug resistance, migration, and invasion were studied in vitro. Results. Expression of ABCG2 was found in a minor group of cells in HCC tissues and cell lines. ABCG2 expression showed tendencies of association with unfavorable prognosis factors. ABCG2 positive cells showed a superior tumorigenicity. Upregulation of ABCG2 enhanced the capacity of proliferation, doxorubicin resistance, migration, and invasion potential, while downregulation of ABCG2 significantly decreased these malignant behaviors. Conclusion. Our results indicate that ABCG2 is a potential CSC marker for HCC. Its expression level has a close relationship with tumorigenicity, proliferation, drug resistance, and metastasis ability.

Increased CD4+CD69+CD25‐ T cells in patients with hepatocellular carcinoma are associated with tumor progression

A new subset of Treg cells, CD4(+) CD69(+) CD25(-) T cells, has been identified in mice. Herein, we aimed to identify this subset of T cells and to evaluate its function in patients with hepatocellular carcinoma (HCC). We detected CD4(+) CD69(+) CD25(-) T cells and its expression of CCR6 and transforming growth factor-β1 (TGF-β1) in peripheral blood of 91 HCC patients, 38 chronic hepatitis patients and 34 healthy donors by flow cytometry. CD4(+) CD69(+) CD25(-) T cells in HCC tissues were also analyzed. CD4(+) CD69(+) CD25(-) T cells were significantly increased in peripheral blood of HCC patients compared with healthy persons and chronic hepatitis patients (8.74% ± 0.42% vs 4.55% ± 0.33% and 5.15% ± 0.36%, P < 0.0001). The percentage of peripheral CD4(+) CD69(+) CD25(-) T cells was significantly higher in HCC patients with Tumor Node Metastasis (TNM) stage III plus IV (P < 0.05). Patients with large tumor size and tumor vascular invasion were inclined to obtain high percentage of CD4(+) CD69(+) CD25(-) T cells (P < 0.05). The frequency of membrane-bound TGF-β1 positive cells in CD4(+) CD69(+) CD25(-) T cells from HCC patients was higher than that from the other two groups (P < 0.0001). A considerable proportion of CD4(+) CD69(+) CD25(-) T cells were present in HCC tissues, which has significant correlation with tumor size and TNM stage. Few CD4(+) CD69(+) CD25(-) T cells express CCR6 both in peripheral blood and tumor tissues from HCC patients. Increased CD4(+) CD69(+) CD25(-) T cells in HCC patients are significantly correlated with tumor size, vascular invasion and TNM stage. Thus, increased CD4(+) CD69(+) CD25(-) T cells exert a critical role in HCC progression and might be a clinically aggressive phenotype of HCC.

Oxidative stress is closely associated with tumor angiogenesis of hepatocellular carcinoma

Oxidative stress (OS) plays an important role in the progression of chronic liver disease and hepatocarcinogenesis. However, the role of OS in the progression of hepatocellular carcinoma (HCC) is unclear. The aim of this study was to assess whether OS promotes angiogenesis in HCC. The expressions of vascular endothelial growth factor (VEGF), VEGF receptor2 (VEGFR2), and phosphorylated Akt were assessed, and microvessel density (MVD) and the cancer-associated fibroblast (CAF) population were examined by immunohistological staining in 55 HCC samples. The OS level in these tissues was assessed using 8-hydroxy-2'-deoxyguanosine (8-OHdG) and 4-hydroxy-2-nonenal (4-HNE) immunostaining, and an 8-OHdG enzyme-linked immunosorbent assay (ELISA). The expression and activation of angiogenic factors and the effect of growth stimulation of human umbilical vein endothelial cells (HUVECs) were also assessed in vitro, using HLE hepatoma-derived cells and conditioned medium with or without treatment with hydrogen peroxide (H₂O₂); a phosphoinositide 3-kinase (PI3K) inhibitor, wortmannin; and an anti-oxidative agent, N-acetyl-L-cysteine (NAC). A higher OS grade was significantly associated with higher MVD, VEGF expression, Akt activity, and OS grade of CAFs, but not with the percentage of the CAF population in HCC tissues. Additionally, cancer cells constituted a major population of OS marker-positive cells in HCC tissues. In vitro, H₂O₂ treatment induced up-regulation of VEGF at both the mRNA and protein levels, activated Akt, and resulted in the proliferation of HUVECs; the addition of wortmannin and NAC counteracted the effects of OS. OS enhances the malignant potential of HCC through the stimulation of angiogenesis by activation of the Akt-VEGF pathway.

Relationship and clinical significance of TGF-beta1 expression with Treg cell infiltration in hepatocellular carcinoma

There are few studies about origins of regulatory T (Treg) cells increased in primary hepatocellular carcinoma (HCC) tissue. Studies showed that Treg cells could be induced by transforming growth factor-beta1 (TGF-beta1), but the relation between TGF-beta1 expression and Treg cell infiltration is unclear in HCC tissue. This study was to investigate the expression of TGF-beta1 and correlations with the amount of Treg cells in HCC, and to evaluate their clinical values in predicting the prognosis of HCC. Envision immunohistochemistry was used to detect the expression of TGF-beta1 and Foxp3 in 102 specimens of HCC tissue and paired adjacent non-tumor liver tissue. Of the 102 specimens of HCC, 41 showed low TGF-beta1 expression and 61 (59.8%) showed high expression; of the 102 specimens of adjacent non-tumor tissue, 22 showed low TGF-beta1 expression and 80 (78.4%) showed high expression. The high expression rate of TGF-beta1 was significantly lower in HCC than in adjacent non-tumor tissues (P = 0.001). Average Foxp3+ cell density in HCC was 2.98 cells/HP, but there was very few or no expression of Foxp3 in adjacent non-tumor liver tissue. Expression of TGF-beta1 was positively correlated with expression of Foxp3 in HCC tissues (r = 0.228, P = 0.021). The expression of TGF-beta1 was significantly higher in HCC tissues with high preoperative AFP concentration than in those with low preoperative AFP concentration (P = 0.023). TGF-beta1 and Foxp3 expression had no correlations with tumor diameter, tumor capsule, liver cirrhosis, and so on. The 5-year survival rate was not different between HCC tissues with high and low TGF-beta1 expression (P = 0.790); however, it was significantly lower in HCC tissues with high Treg cell infiltration than in those low infiltration (25% vs. 44%, P = 0.007). Cox multivariate analysis showed that the number of Treg cells and tumor capsule were independent prognostic factors (P = 0.021, P = 0.001). Expression of TGF-beta1 may relate to the infiltration of Treg cells in HCC tissues, but the relation need to be further investigated. The number of Treg cells in HCC tissues could be used as a potential immunological prognostic indicator for HCC patients after resection.

Novel role for epidermal growth factor-like domain 7 in metastasis of human hepatocellular carcinoma

Epidermal growth factor-like domain 7 (Egfl7) is a recently identified secreted protein that is believed to be primarily expressed in endothelial cells (ECs). Although its expression was reported elevated during tumorigenesis, whether and how Egfl7 contributes to human malignancies remains unknown. In the present study overexpression of Egfl7 was found predominantly in hepatocellular carcinoma (HCC) cells in HCC tissues and closely correlated with poor prognosis of HCC. The expression of Egfl7 in cancer cells was further verified with HCC cell lines including HepG2, MHCC97-L, and HCCLM3, and the Egfl7 expression levels positively correlated with metastatic potential of HCC cell lines was tested. To functionally characterize Egfl7 in HCC, we depleted its expression in HCCLM3 cells by using small interfering RNA. Interestingly, reduction of Egfl7 expression resulted in significant inhibition of migration but not growth of HCCLM3 cells. Biochemical analysis indicated that Egfl7 could facilitate the phosphorylation of focal adhesion kinase (FAK) and therefore promote the migration of HCCLM3 cells. In addition, this effect was almost completely blocked by inhibition of epidermal growth factor receptor (EGFR), indicating that the activation of FAK by Egfl7 is mediated through EGFR. Finally, we used a mouse model to demonstrate that down-regulation of Egfl7 was associated with suppression of intrahepatic and pulmonary metastases of HCC. Collectively, our study shows for the first time that overexpression of Egfl7 in HCC and Egfl7 promotes metastasis of HCC by enhancing cell motility through EGFR-dependent FAK phosphorylation. Our study suggests Egfl7 as a novel prognostic marker for metastasis of HCC and a potential therapeutic target.