Background & Aims Hepatocellular carcinoma (HCC) is an aggressive malignancy; its mechanisms of development and progression are poorly understood. We used an integrative approach to identify HCC ...driver genes, defined as genes whose copy numbers associate with gene expression and cancer progression. Methods We combined data from high-resolution, array-based comparative genomic hybridization and transcriptome analysis of HCC samples from 76 patients with hepatitis B virus infection with data on patient survival times. Candidate genes were functionally validated using in vitro and in vivo models. Results Unsupervised analyses of array comparative genomic hybridization data associated loss of chromosome 8p with poor outcome (reduced survival time); somatic copy number alterations correlated with expression of 27.3% of genes analyzed. We associated expression levels of 10 of these genes with patient survival times in 2 independent cohorts (comprising 319 cases of HCC with mixed etiology) and 3 breast cancer cohorts (637 cases). Among the 10-gene signature, a cluster of 6 genes on 8p, ( DLC1 , CCDC25 , ELP3 , PROSC , SH2D4A , and SORBS3 ) were deleted in HCCs from patients with poor outcomes. In vitro and in vivo analyses indicated that the products of PROSC , SH2D4A , and SORBS3 have tumor-suppressive activities, along with the known tumor suppressor gene DLC1. Conclusions We used an unbiased approach to identify 10 genes associated with HCC progression. These might be used in assisting diagnosis and to stage tumors based on gene expression patterns.
Radiofrequency ablation (RFA) is one of the curative therapies for hepatocellular carcinoma (HCC), however, accelerated progression of residual HCC after incomplete RFA has been reported more ...frequently. The underlying molecular mechanism of this phenomenon remains to be elucidated. In this study, we used an incomplete RFA orthotopic HCC nude mouse model to study the invasive and metastatic potential of residual cancer as well as the correlated mechanism.
The incomplete RFA orthotopic nude mouse models were established using high metastatic potential HCC cell line HCCLM3 and low metastatic potential HCC cell line HepG2, respectively. The changes in cellular morphology, motility, metastasis and epithelial-mesenchymal transition (EMT), and HCC cell molecular markers after in vitro and in vivo incomplete RFA intervention were observed.
Pulmonary and intraperitoneal metastasis were observed in an in vivo study. The underlying pro-invasive mechanism of incomplete RFA appeared to be associated with promoting EMT, including down-regulation of E-cadherin and up-regulation of N-cadherin and vimentin. These results were in accordance with the in vitro response of HCC cells to heat intervention. Further studies demonstrated that β-catenin was a pivotal factor during this course and blocking β-catenin reduced metastasis and EMT phenotype changes in heat-treated HCCLM3 cells in vitro.
Incomplete RFA enhanced the invasive and metastatic potential of residual cancer, accompanying with EMT-like phenotype changes by activating β-catenin signaling in HCCLM3 cells.
To investigate the prognostic value of tumor-infiltrating lymphocytes (TILs), especially regulatory T cells (Tregs), in hepatocellular carcinoma (HCC) patients after resection.
CD3+, CD4+, CD8+, ...Foxp3-positive, and granzyme B-positive TILs were assessed by immunohistochemistry in tissue microarrays containing HCC from 302 patients. Prognostic effects of low- or high-density TIL subsets were evaluated by Cox regression and Kaplan-Meier analysis using median values as cutoff.
CD3+, CD4+, CD8+ TILs were associated with neither overall survival (OS) nor disease-free survival (DFS). The presence of low intratumoral Tregs in combination with high intratumoral activated CD8+ cytotoxic cells (CTLs), a balance toward CTLs, was an independent prognostic factor for both improved DFS (P = .001) and OS (P < .0001). Five-year OS and DFS rates were only 24.1% and 19.8% for the group with intratumoral high Tregs and low activated CTLs, compared with 64.0% and 59.4% for the group with intratumoral low Tregs and high activated CTLs, respectively. Either intratumoral Tregs alone (P = .001) or intratumoral activated CTLs (P = .001) alone is also an independent predictor for OS. In addition, high Tregs density was associated with both absence of tumor encapsulation (P = .032) and presence of tumor vascular invasion (P = .031).
Tregs are associated with HCC invasiveness, and intratumoral balance of regulatory and cytotoxic T cells is a promising independent predictor for recurrence and survival in HCC. A combination of depletion of Tregs and concomitant stimulation of effector T cells may be an effective immunotherapy to reduce recurrence and prolong survival after surgery.
Hepatocellular carcinoma is a common and aggressive cancer that occurs mainly in men. We examined microRNA expression patterns, survival, and response to interferon alfa in both men and women with ...the disease.
We analyzed three independent cohorts that included a total of 455 patients with hepatocellular carcinoma who had undergone radical tumor resection between 1999 and 2003. MicroRNA-expression profiling was performed in a cohort of 241 patients with hepatocellular carcinoma to identify tumor-related microRNAs and determine their association with survival in men and women. In addition, to validate our findings, we used quantitative reverse-transcriptase-polymerase-chain-reaction assays to measure microRNAs and assess their association with survival and response to therapy with interferon alfa in 214 patients from two independent, prospective, randomized, controlled trials of adjuvant interferon therapy.
In patients with hepatocellular carcinoma, the expression of miR-26a and miR-26b in nontumor liver tissue was higher in women than in men. Tumors had reduced levels of miR-26 expression, as compared with paired noncancerous tissues, which indicated that the level of miR-26 expression was also associated with hepatocellular carcinoma. Moreover, tumors with reduced miR-26 expression had a distinct transcriptomic pattern, and analyses of gene networks revealed that activation of signaling pathways between nuclear factor kappaB and interleukin-6 might play a role in tumor development. Patients whose tumors had low miR-26 expression had shorter overall survival but a better response to interferon therapy than did patients whose tumors had high expression of the microRNA.
The expression patterns of microRNAs in liver tissue differ between men and women with hepatocellular carcinoma. The miR-26 expression status of such patients is associated with survival and response to adjuvant therapy with interferon alfa.
microRNAs (miRNAs) have been reported to regulate angiogenesis by down-regulating the expression of pro-angiogenic or anti-angiogenic factors. The aims of this study were to investigate whether ...miR-26a inhibited angiogenesis by down-regulating vascular endothelial growth factor A (VEGFA) and its clinical relevance in hepatocellular carcinoma (HCC).
The expression of miR-26a was modified in HepG2 and HCCLM3 cell lines respectively, and a panel of angiogenic factors was measured by real-time PCR in the cells. A luciferase reporter assay was used to validate the target gene of miR-26a. Specific inhibitors of signal transduction pathway and siRNA approaches were used to explore the regulatory mechanism of miR-26a. Migration and tube forming assays were conducted to show the changes of angiogenesis induced by miR-26a and its target genes. Finally animal studies were used to further validate those findings.
Ectopic expression of miR-26a exhibited decreased levels of VEGFA in HepG2 cells. Migration and tube forming of human umbilical vein endothelial cells (HUVECs) were decreased in the conditioned medium from ectopic expression of miR-26a in HepG2 cells compared to control HepG2 cells. The pro-angiogenic effects of the conditioned medium of HepG2 cells on HUVECs were specifically decreased by LY294002, YC-1, and bevacizumab. Integrated analysis disclosed PIK3C2α as a downstream target gene of miR-26a. Ectopic expression of miR-26a suppressed ectopic and orthotopic tumor growth and vascularity in nude mice. The results in HCCLM3 were consistent with those in HepG2. miR-26a expression was inversely correlated with VEGFA expression in HCC patients.
miR-26a modulated angiogenesis of HCC through the PIK3C2α/Akt/HIF-1α/VEGFA pathway. The expression of VEGFA was inversely correlated with miR-26a expression in HCC tumors.
Screening for hepatocellular carcinoma (HCC) has been conducted for over 20 years, but there is no conclusive evidence that screening may reduce HCC mortality. The aim of this study was to assess the ...effect of screening on HCC mortality in people at increased risk.
This study included 18,816 people, aged 35-59 years with hepatitis B virus infection or a history of chronic hepatitis in urban Shanghai, China. Participants were randomly allocated to a screening (9,373) or control (9,443) group. Controls received no screening and continued to use health-care facilities. Screening group participants were invited to have an AFP test and ultrasonography examination every 6 months. Screening was stopped in December 1997; by that time screening group participants had been offered five to ten times. All participants were followed up until December 1998. The primary outcome measure was HCC mortality.
The screened group completed 58.2 percent of the screening offered. When the screening group was compared to the control group, the number of HCC was 86 versus 67; subclinical HCC being 52 (60.5%) versus 0; small HCC 39 (45.3%) versus 0; resection achieved 40 (46.5%) versus 5 (7.5%); 1-, 3,-, and 5-year survival rate 65.9%, 52.6%, 46.4% versus 31.2%, 7.2%, 0, respectively. Thirty-two people died from HCC in the screened group versus 54 in the control group, and the HCC mortality rate was significantly lower in the screened group than in controls, being 83.2/100,000 and 131.5/100,000, respectively, with a mortality rate ratio of 0.63 (95%CI 0.41-0.98).
Our finding indicated that biannual screening reduced HCC mortality by 37%.
To investigate the role of macrophages in tumor progression under sorafenib treatment and to explore whether combination of drugs that deplete macrophages improved the antitumor effect of sorafenib.
...Tumor growth, lung metastasis, and tumor angiogenesis were observed in HCCLM3-R and SMMC7721, two human hepatocellular carcinoma xenograft nude mouse models, when treated with sorafenib (30 mg/kg daily, n = 6 per group) or a vehicle as control. Macrophage infiltration was measured in the peripheral blood and in sorafenib-treated tumor by immunohistochemistry and flow cytometry with F4/80 antibody and CD11b antibody. The effect of macrophage depletion on tumor angiogenesis and metastasis after sorafenib treatment, using two drug target macrophages, zoledronic acid (ZA) and clodrolip, was measured in the two models of hepatocellular carcinoma.
Although sorafenib significantly inhibited tumor growth and lung metastasis, it induced a significant increase in peripheral recruitment and intratumoral infiltration of F4/80- and CD11b-positive cells, which was accompanied with elevation of colony-stimulating factor-1, stromal-derived factor 1alpha, and vascular endothelial growth factor in the tumor and elevation of plasma colony-stimulating factor-1 and mouse vascular endothelial growth factor in peripheral blood, suggesting the role of macrophages in tumor progression under sorafenib treatment. Depletion of macrophages by clodrolip or ZA in combination with sorafenib significantly inhibited tumor progression, tumor angiogenesis, and lung metastasis compared with mice treated with sorafenib alone. ZA was more effective than clodrolip.
Macrophages may have an important role in tumor progression under sorafenib treatment. ZA is promising when combined with sorafenib to enhance its antitumor effect.
To investigate prognostic values of the intratumoral and peritumoral expression of macrophage colony-stimulating factors (M-CSF) in hepatocellular carcinoma (HCC) patients after curative resection.
...Expression of M-CSF and density of macrophages (M Phi) were assessed by immunohistochemistry in tissue microarrays containing paired tumor and peritumoral liver tissue from 105 patients who had undergone hepatectomy for histologically proven HCC. Prognostic value of these and other clinicopathologic factors was evaluated.
Neither intratumoral M-CSF nor M Phi density was associated with overall survival (OS) or disease-free survival (DFS). High peritumoral M-CSF and M Phi density, which correlated with large tumor size, presence of intrahepatic metastasis, and high TNM stage, were independent prognostic factors for both OS (P = .001 and P < .001, respectively) and DFS (P = .001 and P = .003, respectively) and affected incidence of early recurrence. In a small HCC subset, peritumoral M-CSF was also correlated with both OS and DFS (P = .038 and P = .001, respectively). The combination of peritumoral M-CSF and M Phi had a better power to predict the patients' death and disease recurrence (P < .001 for both).
High peritumoral M-CSF and M Phi were associated with HCC progression, disease recurrence, and poor survival after hepatectomy, highlighting the importance of peritumoral tissue in the recurrence and metastasis of HCC. M-CSF and M Phi may be targets of postoperative adjuvant therapy.
High frequency of recurrence is the major cause of the poor outcomes for patients with hepatocellular carcinoma (HCC). microRNA (miR)-182-5p emerged as a high-priority miRNA in HCC and was found to ...be related to HCC metastasis. Whether the expression of miR-182-5p in tumor tissue correlated with early recurrence in HCC patients underwent curative surgery was unknown.
Real-time PCR (RT-PCR) and in situ hybridization (ISH) were conducted to assess the expression of miR-182-5p in HCC cells and tissues. Cell Counting Kit-8 (CCK-8), transwell assays were performed to detected cells proliferation and migration ability. Flow cytometry assays were used to detect cell apoptosis rate, and xenograft model was employed to study miR-182-5p in HCC growth and lung metastasis. The target of miR-182-5p was validated with a dual-luciferase reporter assay and western blotting. Immunohistochemistry, immumoblotting, and immunoprecipitation were performed to test relative protein expression.
We showed that high expression of miR-182-5p in tumor tissues correlated with poor prognosis as well as early recurrence in HCC patients underwent curative surgery. miR-182-5p enhanced motility and invasive ability of HCC cells both in vitro and in vivo. miR-182-5p directly targets 3'-UTR of FOXO3a and repressed FOXO3a expression, activating AKT/FOXO3a pathway to promote HCC proliferation. Notably, miR-182-5p activated Wnt/β-catenin signaling by inhibiting the degradation of β-catenin and enhancing the interaction between β-catenin and TCF4 which was mediated by repressed FOXO3a.
Consistently, miR-182-5p can be a potential predictor of early recurrence for HCC patients underwent curative surgery, and FOXO3a plays a key mediator in miR-182-5p induced HCC progression.
PDF
