Irreversible repolarization of tumour-associated macrophages by low-Pi stress inhibits the progression of hepatocellular carcinoma

Numerous studies have shown the positive correlation between high levels of Pi and tumour progression. A critical goal of macrophage-based cancer therapeutics is to reduce anti-inflammatory macrophages (M2) and increase proinflammatory antitumour macrophages (M1). This study aimed to investigate the relationship between macrophage polarization and low-Pi stress. First, the spatial populations of M2 and M1 macrophages in 22 HCC patient specimens were quantified and correlated with the local Pi concentration. The levels of M2 and M1 macrophage markers expressed in the peritumour area were higher than the intratumour levels, and the expression of M2 markers was positively correlated with Pi concentration. Next, monocytes differentiated from THP-1 cells were polarized against different Pi concentrations to investigate the activation or silencing of the expression of p65, IκB-α and STAT3 as well as their phosphorylation. Results showed that low-Pi stress irreversibly repolarizes tumour-associated macrophages (TAMs) towards the M1 phenotype by silencing stat6 and activating p65. Moreover, HepG-2 and SMCC-7721 cells were cultured in conditioned medium to investigate the innate anticancer immune effects on tumour progression. Both cancer cell lines showed reduced proliferation, migration and invasion, as epithelial–mesenchymal transition (EMT) was inactivated. In vivo therapeutic effect on the innate and adaptive immune processes was validated in a subcutaneous liver cancer model by the intratumoural injection of sevelamer. Tumour growth was significantly inhibited by the partial deprivation of intratumoural Pi as the tumour microenvironment under low-Pi stress is more immunostimulatory. The anticancer immune response, activated by low-Pi stress, suggests a new macrophage-based immunotherapeutic modality.     

The role of macrophages polarization in predicting prognosis of radically resected gastric cancer patients

Tumour‐associated Macrophages (TAM) present two different polarizations: classical (M1) characterized by immunostimulation activity and tumour suppression; alternative (M2) characterized by tumour promotion and immune suppression. In this retrospective study, we evaluated the correlation between the two forms of TAM with survival time in radically resected gastric cancer patients. A total of 52 chemo‐ and radio‐naive patients were included. Two slides were prepared for each patient and double‐stained for CD68/NOS2 (M1) or CD68/CD163 (M2) and five representative high‐power fields per slide were evaluated for TAM count. The median value of the two macrophage populations density and the median value of M1/M2 ratio were used as cut‐off. Twenty‐seven patients with M1 density above‐the‐median had a significantly higher survival compared to those below the median. Twenty‐six patients with M1/M2 ratio above the median showed median OS of 27.2 months compared to 15.5 months of the patients below the median. No association between M2 macrophage density and patient's outcome was found. In multivariate analysis, M1/M2 was a positive independent predictor of survival. The M1 macrophage density and M1/M2 ratio, as confirmed in multivariate analysis, are factors that can help in predicting patients survival time after radical surgery for gastric cancer.     

Tumour‐associated macrophages correlate with microvascular bed extension in colorectal cancer patients

Tumour‐associated macrophages (TAMs) represent pivotal components of tumour microenvironment promoting angiogenesis, tumour progression and invasion. In colorectal cancer (CRC), there are no conclusive data about the role of TAMs in angiogenesis‐mediated tumour progression. In this study, we aimed to evaluate a correlation between TAMs, TAM immunostained area (TAMIA) microvascular density (MVD), endothelial area (EA) and cancer cells positive to VEGF‐A (CCP‐VEGF‐A) in primary tumour tissue of locally advanced CRC patients undergone to radical surgery. A series of 76 patients with CRC were selected and evaluated by immunohistochemistry and image analysis. An anti‐CD68 antibody was employed to assess TAMs and TAMIA expression, an anti‐CD34 antibody was utilized to detect MVD and EA expression, whereas an anti‐VEGF‐A antibody was used to detect CCP‐VEGF‐A; then, tumour sections were evaluated by image analysis methods. The mean ± S.D. of TAMs, MVD and CCP‐VEGF‐A was 65.58 ± 21.14, 28.53 ± 7.75 and 63% ± 37%, respectively; the mean ± S.D. of TAMIA and EA was 438.37 ± 124.14μ² and 186.73 ± 67.22μ², respectively. A significant correlation was found between TAMs, TAMIA, MVD and EA each other (r ranging from 0.69 to 0.84; P ranging from 0.000 to 0.004). The high level of expression of TAMs and TAMIA in tumour tissue and the significant correlation with both MVD and EA illustrate that TAMs could represent a marker that plays an important role in promoting angiogenesis‐mediated CRC. In this context, novel agents killing TAMs might be evaluated in clinical trials as a new anti‐angiogenic approach.     

Preoperative prealbumin-to-fibrinogen ratio to predict survival outcomes in hepatocellular carcinoma patients after hepatic resection

BackgroundThis study aimed to evaluate the clinical application of the preoperative prealbumin-to-fibrinogen ratio (PFR) in the clinical diagnosis of hepatocellular carcinoma (HCC) patients and its prognostic value.MethodsThe clinical and laboratory data of 269 HCC patients undergoing surgical treatment from January 2012 to January 2017 in Taizhou Hospital were retrospectively analysed. The Cox regression model was used to analyse the correlation between the PFR and other clinicopathological factors in overall survival (OS) and disease-free survival (DFS).ResultsCox regression analysis showed that the PFR (hazard ratio (HR)=2.123; 95% confidence interval (95% CI), 1.271-3.547; P=0.004) was an independent risk factor affecting the OS of HCC patients. Furthermore, a nomogram was built based on these risk factors. The C-index for the OS nomogram was 0.715.ConclusionsNomograms based on the PFR can be recommended as the correct and actual model to evaluate the prognosis of patients with HCC.     

缺氧相关长链非编码RNA作为肝癌预后预测标志物的潜在价值

肝细胞癌(hepatocellular carcinoma,简称肝癌)是一种常见的恶性肿瘤。缺氧是肝癌等实体肿瘤的一个重要特征,同时也是诱导肿瘤恶性进展的重要因素。然而,肝癌缺氧相关的长链非编码RNA(long non-coding RNA,lncRNA)的鉴定及其在临床生存预后等方面的价值仍未得到系统的研究。本研究旨在通过肝癌转录组的整合分析鉴定肝癌缺氧相关的lncRNA,并评估其在肝癌预后中的价值。基于癌症基因组图谱(The Cancer Genome Atlas,TCGA)计划的肝癌转录组数据的整合分析,初步鉴定到233个缺氧相关的候选lncRNA。进一步筛选具有预后价值的候选者,基于其中12个缺氧相关lncRNA(AC012676.1、PRR7-AS1、AC020915.2、AC008622.2、AC026401.3、MAPKAPK5-AS1、MYG1-AS1、AC015908.3、AC009275.1、MIR210HG、CYTOR和SNHG3)建立了肝癌预后风险模型。Cox比例风险回归分析显示,基于该模型计算的缺氧风险评分作为肝癌患者新的独立预后预测指标,优于传统的临床病理因...     

Mechanisms of portal vein tumour thrombus formation and development in patients with hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common and aggressive human malignancies worldwide. Portal vein tumour thrombus (PVTT) is considered one of most fearful complications of HCC and is strongly associated with a poor prognosis. Clarification of the mechanisms underlying the formation and development of PVTT is crucial for developing novel therapeutic strategies for HCC patients. Several studies have been made to uncover that tumour microenvironment, stem cells, abnormal gene expression and non-coding RNAs deregulation are associated with PVTT in patients with HCC in the last decade. However, the exact molecular mechanisms of PVTT in patients with HCC are still largely unknown. In the present review, we briefly summarized the molecular mechanisms underlying the formation and development of PVTT in HCC.     

Salvage liver transplantation for patients with recurrent hepatocellular carcinoma after curative resection

Objective

To summarize the experience with salvage liver transplantation (SLT) for patients with recurrent hepatocellular carcinoma (HCC) after primary hepatic resection in a single center.

Methods

A total of 376 adult patients with HCC underwent orthotopic liver transplantation (OLT) at Organ Transplantation Center, the First Affiliated Hospital of Sun Yat-sen University, between 2004 and 2008. Among these patients, 36 underwent SLT after primary liver curative resection due to intrahepatic recurrence. During the same period, one hundred and forty-seven patients with HCC within Milan criteria underwent primary OLT (PLTW group), the intra-operative and post-operative parameters were compared between these two groups. Furthermore, we compared tumor recurrence and patient survival of patients with SLT to 156 patients with HCC beyond Milan criteria (PLTB group). Cox Hazard regression was made to identify the risk factors for tumor recurrence.

Results

The median interval between initial liver resection and SLT was 35 months (1–63 months). The intraoperative blood loss (P<0.05) and transfusion volume (P<0.05) were larger in the SLT group than in the PLTW group. The operation time was longer in the SLT group (P<0.05). The post-operative complications incidence, tumor recurrence rate, patients'' survival rate, and tumor-free survival rate were comparable between these two groups (all P>0.05). When compared to those patients with HCC beyond Milan criteria undergoing primary OLT, patients undergoing SLT achieved a better survival and a lower tumor recurrence. Cox Proportional Hazards model showed that vascular invasion, including macrovascular and microvascular invasion, as well as AFP level >400 IU/L were risk factors for tumor recurrence after LT.

Conclusions

In comparison with primary OLT, although SLT is associated with increased operation difficulties, it provides a good option for patients with HCC recurrence after curative resection.     

Recent progress in the emerging role of exosome in hepatocellular carcinoma

Exosomes are small membrane vesicles 50‐150 nm in diameter released by a variety of cells, which contain miRNAs, mRNAs and proteins with the potential to regulate signalling pathways in recipient cells. Exosomes deliver nucleic acids and proteins to participate in orchestrating cell‐cell communication and microenvironment modulation. In this review, we summarize recent progress in our understanding of the role of exosomes in hepatocellular carcinoma (HCC). This review focuses on recent studies on HCC exosomes, considering biogenesis, cargo and their effects on the development and progression of HCC, including chemoresistance, epithelial‐mesenchymal transition, angiogenesis, metastasis and immune response. Finally, we discuss the clinical application of exosomes as a therapeutic agent for HCC.     

miR-125a/b inhibits tumor-associated macrophages mediated in cancer stem cells of hepatocellular carcinoma by targeting CD90

Cancer stem cells promote tumorigenesis and progression of hepatocellular carcinoma (HCC). Recently, emerging evidence indicates tumor-associated macrophages (TAMs) play an important role in tumor progression. However, TAMs often occurs with unknown mechanisms. As an important mediator in intercellular communications, exosomes secreted by host cells mediate the exchange of genetic materials and proteins, which involves tumor aggressiveness. The aim of the study was to investigate whether exosomes derived from TAMs mediate stem cell properties in HCC. TAMs were isolated from the tissues of HCC. microRNA (miRNA) expression profiles of TAMs were analyzed using miRNA microarray. In vitro cell coculture was further conducted to investigate the crosstalk between TAMs and tumor cells mediated by TAMs exosomes. In this study, we showed that TAMs exosomes promote HCC cell proliferation and stem cell properties. Using miRNA profiles assay, we identified significantly lower levels of miR-125a and miR-125b in exosomes and cell lysate isolated from TAMs. Functional studies revealed that the HCC cells were treated with TAM exosomes or transfected with miR-125a/b suppressed cell proliferation and stem cell properties by targeting CD90, a stem cell marker of HCC stem cells. The study indicated that miR-125a/b targeting CD90 played important roles in cancer stem cells of HCC.     

EYA4 serves as a prognostic biomarker in hepatocellular carcinoma and suppresses tumour angiogenesis and metastasis

Eye absent homolog 4 (EYA4) has been demonstrated to be down‐regulated in hepatocellular carcinoma (HCC), but its biological function and the mechanism in HCC angiogenesis and metastasis remain largely unknown. Herein, we showed that EYA4 expression was frequently low in HCC tissue samples compared with matched adjacent non‐tumourous tissues. In the analysis of 302 HCC specimens, we revealed that decreased expression of EYA4 correlated with tumour differentiation status. Univariate and multivariate analyses identified EYA4 as an independent risk factor for recurrence‐free survival (RFS) and overall survival (OS) among the 302 patients. Functional assays showed that forced expression of EYA4 suppressed HCC cell migration, invasion and capillary tube formation of endothelial cells in vitro, as well as in vivo tumour angiogenesis and metastasis in a mouse model. Furthermore, mechanism study exhibited that EYA4 could inhibit HCC angiogenesis and metastasis by inhibiting c‐JUN/VEGFA pathway. Together, we provide proof that EYA4 is a novel tumour suppressor in HCC and a new prognostic biomarker and therapeutic target in HCC.     

Derepression of c‐Fos caused by MicroRNA‐139 down‐regulation contributes to the metastasis of human hepatocellular carcinoma

This study investigates whether the anti‐metastasis effect of microRNA‐139 (miR‐139) on hepatocellular carcinoma (HCC) is mediated through regulating c‐fos expression. The expression levels of miR‐139 and c‐fos in human HCC cell sublines with high (MHCC97H) and low (MHCC97L) spontaneous metastatic potentials were quantified using QPCR or Western blot. miR‐139 mimics was transfected into MHCC97H cells to overexpress miR‐139, and miR‐139 inhibitor was transfected into MHCC97L cells to down‐express miR‐139. The effect of overexpression or down‐expression of miR‐139 on c‐fos expression of MHCC97H and MHCC97L cells was evaluated using QPCR and Western blot. The 3′ untranslated region segments of FOS containing the miR‐139 binding sites were amplified by PCR, and the luciferase activity in the transfected cells was assayed. In comparison with the expression level of miR‐139 in MHCC97L cells, the expression level in MHCC97H cells was significantly decreased, whereas c‐Fos was significantly up‐regulated in MHCC97H. The overexpression of miR‐139 significantly inhibited the expression of c‐fos in MHCC97H cells, and the down‐expression of miR‐139 significantly promoted the expression of c‐fos in MHCC97L cells. miR‐139 suppressed the luciferase activity of the pGL‐FOS by approximately 40% compared with the negative control. In vitro cell migration analysis demonstrated that depletion of c‐fos or overexpression of miR‐139 in MHCC97H cells reduced cell migration, whereas overexpression of c‐fos or depletion of miR‐139 in MHCC97L cells increased cell migration. Thus, we got the conclusion that miR‐139 expression is down‐regulated in human HCC cell sublines with high spontaneous metastatic potentials (MHCC97H). Derepression of c‐Fos caused by miR‐139 down‐regulation contributes to the metastasis of HCC. Copyright © 2012 John Wiley & Sons, Ltd.     

Diagnostic and prognostic value of circular RNAs in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the sixth most common malignant tumour, which has posed a heavy health and financial burden worldwide. Due to limited symptoms at the early stage and the limitation in current biomarkers, HCC patients are usually diagnosed at the advanced stage with a pessimistic overall survival rate. Circular RNAs (circRNAs) are a subclass of single-stranded RNAs characterized by a covalently closed loop structure without 3’- or 5’-end. With advances in high-throughput sequencing technology and bioinformatics, accumulating studies have demonstrated the promotor or suppressor roles of circRNAs in the carcinogenesis, progression, and metastasis of HCC. Moreover, circRNAs are characteristic of higher abundance, stability and conservation compared with linear RNAs. Therefore, circRNAs have emerged as one of the most promising diagnostic and prognostic biomarkers for HCC with reliable accuracy, sensitivity and specificity. In this review, we briefly introduce the characteristics of circRNAs and summarize the roles of circRNAs in the biological procedures of HCC. Furthermore, we provide an overview on the potential diagnostic and prognostic value of circRNAs as biomarkers for patients with HCC. Finally, we discuss future perspectives of circRNAs in cancer research.     

c‐Myc‐mediated SNRPB upregulation functions as an oncogene in hepatocellular carcinoma

Dysregulation of genes involved in alternative splicing contributes to hepatocarcinogenesis. SNRPB, a component of spliceosome, is implicated in human cancers, yet its clinical significance and biological function in hepatocellular carcinoma (HCC) remains unknown. Here, we show that SNRPB expression is increased in HCC tissues, compared with the nontumorous tissues, at both messenger RNA and protein levels in two independent cohorts. High expression of SNRPB is significantly associated with higher pathological grade, vascular invasion, serum alpha‐fetoprotein level, tumor metastasis, and poor disease‐free and overall survivals. Luciferase reporter and chromatin immunoprecipitation assays demonstrate that SNRPB upregulation in HCC is mediated by c‐Myc. Positive correlation is found between SNRPB and c‐Myc expression in clinical samples. In vitro studies show that ectopic expression of SNRPB promotes HCC cell proliferation and migration, whereas knockdown of SNRPB results in the opposite phenotypes. Collectively, our data suggest SNRPB function as an oncogene and serve as a potential prognostic factor in HCC.     

Development of a preoperative prognostic scoring system to predict benefits of hepatic resection in advanced hepatocellular carcinoma patients

Objective: The present study aimed to identify risk factors for overall survival in advanced hepatocellular carcinoma (HCC) patients and establish a scoring system to select patients who would benefit from hepatic resection.Methods: Survival curves were analyzed using the Kaplan–Meier method and log-rank test. The prognostic scoring system was developed from training cohort using a Cox-regression model and validated in a external validation cohortResults: There were 401 patients in the training cohort, 163 patients in the external validation cohorts. The training cohort median survival in all patients was 12 ± 1.07 months, rate of overall survival was 49.6% at 1 year, 25.0% at 3 years, and 18.0% at 5 years. A prognostic scoring system was established based on age, body mass index, alkaline phosphatase, tumor number and tumor capsule. Patients were classified as low- risk group(≤3.5) or high-risk group(>3.5). High-risk patients had a median survival of 9 months, compared with 23 months in low-risk patients. The area under the receiver operating characteristic curve (AUC) of the prognostic scoring system was 0.747 (0.694–0.801), which is significantly better than AFP, Child-Pugh and ALBI. The AUC of validation cohorts was 0.716 (0.63–0.803).Conclusion: A prognostic scoring system for hepatic resection in advanced HCC patients has been developed based entirely on preoperative variables. Patients classified as low risk using this system may experience better prognosis after hepatic resection.     

Prognostic genes of hepatocellular carcinoma based on gene coexpression network analysis

Hepatocellular carcinoma (HCC) is the most common subtype in liver cancer whose prognosis is affected by malignant progression associated with complex gene interactions. However, there is currently no available biomarkers associated with HCC progression in clinical application. In our study, RNA sequencing expression data of 50 normal samples and 374 tumor samples was analyzed and 9225 differentially expressed genes were screened. Weighted gene coexpression network analysis was then conducted and the blue module we were interested was identified by calculating the correlations between 17 gene modules and clinical features. In the blue module, the calculation of topological overlap was applied to select the top 30 genes and these 30 genes were divided into the green group (11 genes) and the yellow group (19 genes) through searching whether these genes were validated by in vitro or in vivo experiments. The genes in the green group which had never been validated by any experiments were recognized as hub genes. These hub genes were subsequently validated by a new data set GSE76427 and KM Plotter Online Tool, and the results indicated that 10 genes (FBXO43, ARHGEF39, MXD3, VIPR1, DNASE1L3, PHLDA1, CSRNP1, ADR2B, C1RL, and CDC37L1) could act as prognosis and progression biomarkers of HCC. In summary, 10 genes who have never been mentioned in HCC were identified to be associated with malignant progression and prognosis of patients. These findings may contribute to the improvement of the therapeutic decision, risk stratification, and prognosis prediction for HCC patients.