Circulating microRNA-200 Family as Diagnostic Marker in Hepatocellular Carcinoma

Goals

In this clinical study, we aimed to evaluate the role of circulating microRNA-200 family as a non-invasive tool to identify patients with cirrhosis-associated hepatocellular carcinoma (HCC).

Background

Prognosis of HCC remains poor with increasing incidence worldwide, mainly related to liver cirrhosis. So far, no reliable molecular targets exist for early detection of HCC at surgically manageable stages. Recently, we identified members of the microRNA-200 family as potential diagnostic markers of cirrhosis-associated HCC in patient tissue samples. Their value as circulating biomarkers for HCC remained undefined.

Methods

Blood samples and clinicopathological data of consecutive patients with liver diseases were collected prospectively. Expression of the microRNA-200 family was investigated by qRT-PCR in blood serum samples of 22 HCC patients with and without cirrhosis. Serum samples of patients with non-cancerous chronic liver cirrhosis (n = 22) and of healthy volunteers (n = 15) served as controls.

Results

MicroRNA-141 and microRNA-200a were significantly downregulated in blood serum of patients with HCC compared to liver cirrhosis (p<0.007) and healthy controls (p<0.002). MicroRNA-141 and microRNA-200a could well discriminate patients with cirrhosis-associated HCC from healthy volunteers with area under the receiver-operating characteristic curve (AUC) values of 0.85 and 0.82, respectively. Additionally, both microRNAs could differentiate between HCC and non-cancerous liver cirrhosis with a fair accuracy.

Conclusions

Circulating microRNA-200 family members are significantly deregulated in patients with HCC and liver cirrhosis. Further studies are necessary to confirm the diagnostic value of the microRNA-200 family as accurate serum marker for cirrhosis-associated HCC.     

The G1613A mutation in the HBV genome affects HBeAg expression and viral replication through altered core promoter activity

Infection of hepatitis B virus (HBV) causes acute and chronic hepatitis and is closely associated with the development of cirrhosis and hepatocellular carcinoma (HCC). Previously, we demonstrated that the G1613A mutation in the HBV negative regulatory element (NRE) is a hotspot mutation in HCC patients. In this study, we further investigated the functional consequences of this mutation in the context of the full length HBV genome and its replication. We showed that the G1613A mutation significantly suppresses the secretion of e antigen (HBeAg) and enhances the synthesis of viral DNA, which is in consistence to our clinical result that the G1613A mutation associates with high viral load in chronic HBV carriers. To further investigate the molecular mechanism of the mutation, we performed the electrophoretic mobility shift assay with the recombinant RFX1 protein, a trans-activator that was shown to interact with the NRE of HBV. Intriguingly, RFX1 binds to the G1613A mutant with higher affinity than the wild-type sequence, indicating that the mutation possesses the trans-activating effect to the core promoter via NRE. The trans-activating effect was further validated by the enhancement of the core promoter activity after overexpression of RFX1 in liver cell line. In summary, our results suggest the functional consequences of the hotspot G1613A mutation found in HBV. We also provide a possible molecular mechanism of this hotspot mutation to the increased viral load of HBV carriers, which increases the risk to HCC.     

Hepatocellular carcinoma and liver cirrhosis TP53 mutation analysis reflects a moderate dietary exposure to aflatoxins in Espírito Santo State, Brazil

The close relationship between aflatoxins and 249^ser TP53 gene mutation (AGG to AGT, Arg to Ser) in hepatocellular carcinoma (HCC) makes this mutation an indirect indicator of dietary contamination with this toxin. We have examined the prevalence of codon 249 TP53 mutation in 41 HCC and 74 liver cirrhosis (without HCC) cases diagnosed at the HUCAM University Hospital in Vitoria, Espírito Santo State, Brazil. DNA was extracted from paraffin sections and from plasma. The mutation was detected by DNA amplification, followed by restriction endonuclease digestion and confirmed by direct sequencing. DNA restriction showed 249^ser mutation in 16 HCC and 13 liver cirrhosis, but sequencing confirmed mutations in only 6 HCC and 1 liver cirrhosis. In addition, sequencing revealed 4 patients with mutations at codon 250 (250^ser and 250^leu) in HCC cases. The prevalence of TP53 mutation was 10/41 (24.3 %) in HCC and 1/74 (1.4 %) in liver cirrhosis. No relationship between the presence of mutations and the etiology of HCC was observed. TP53 exon 7 mutations, which are related to aflatoxins exposure, were found at 14.6 % (249^ser), 7.3 % (250^leu) and 2.4 % (250^ser) in 41 cases of HCC and 1.4 % in 74 liver cirrhosis (without HCC) cases, suggesting a moderate dietary exposure to aflatoxins in the Espírito Santo State, Brazil.     

Immunohistochemical assessment of proliferating cell nuclear antigen in primary hepatocellular carcinoma and dysplastic nodules

A complementary way for the assessment of HCC prognosis is represented by the analysis of molecular markers. Thus, immunohistochemical assessment of proliferation can describe tumor aggressiveness, probability of local recurrence or metastasis potential, being very useful for the assessment of recurrence-free survival and survival until death. The aim of our study was to assess proliferating cell nuclear antigen activity in HCC and dysplastic nodules as compared with surrounding nonneoplasic areas. Immunohistochemical techniques were thus performed on the samples obtained by ultrasound-guided liver biopsies or intraoperative biopsies, in 32 patients with HCC, as well as in 3 patients with dysplastic nodules ocurring in liver cirrhosis. Expression of PCNA within extranodular areas of the HCC patients in the absence or presence of cirrhosis, was increasing from 40% to 70%, respectively. PCNA expression further increased within intranodular areas of dysplastic nodules and HCC, to 100% and 96.88%, respectively. A progressive increase of the mean values of PCNA-LI was also observed from extranodular areas without or with cirrhosis, towards intranodular areas of dysplastic nodules and HCC (4.2%, 6.8%, 31.9%, respectively). Dysplastic nodules can thus be considered lesions with a high-proliferation rate, representing an early stage of hepatocarcinogenesis. This supported the current recommendations for borderline hepatocellular nodules identified by ultrasound, which indicate an aggressive treatment similar to malignant lesions. In summary, we demonstrated a progressively increasing rate of cellular proliferation, from extranodular non-neoplasic areas to intranodular areas (dysplastic nodules and HCC), as reflected by an increased expression of proliferating cell nuclear antigen labelling index.     

Polymorphisms of hemochromatosis, and alpha-1 antitrypsin genes in Egyptian HCV patients with and without hepatocellular carcinoma

Hereditary hemochromatosis and alpha-1antitrypsin deficiency are genetic diseases characterized by endoplasmic reticulum (ER) stress with subsequent development of liver disease. Our aim was to estimate the frequency of hemochromatosis gene (HFE) mutant alleles (C282Y and H63D) and alpha-1 antitrypsin S/Z variants among Egyptian HCV cirrhotic patients and in hepatocellular carcinoma patients and to evaluate their effects on disease progression. HFE and alpha-1 antitrypsin polymorphisms were characterized in 200 Egyptian patients with HCV infection (100 patients complicated with cirrhosis, 100 patients with HCC) and 100 healthy subjects who had no history of any malignancy. The frequencies of HD genotype of H63D mutation were significantly increased in HCC patients compared to control group and to cirrhosis group. Also, the frequencies of DD genotype were significantly increased In HCC group compared to control group and to cirrhosis group. Our results suggested that Carriers of the D allele of H63D mutation were significantly more likely to develop HCC.     

Aflatoxin-induced TP53 R249S mutation in hepatocellular carcinoma in Thailand: association with tumors developing in the absence of liver cirrhosis

Primary Liver Cancer (PLC) is the leading cause of death by cancer among males in Thailand and the 3(rd) among females. Most cases are hepatocellular carcinoma (HCC) but cholangiocarcinomas represent between 4 and 80% of liver cancers depending upon geographic area. Most HCC are associated with chronic infection by Hepatitis B Virus while a G → T mutation at codon 249 of the TP53 gene, R249S, specific for exposure to aflatoxin, is detected in tumors for up to 30% of cases. We have used Short Oligonucleotide Mass Analysis (SOMA) to quantify free circulating R249S-mutated DNA in plasma using blood specimens collected in a hospital case:control study. Plasma R249S-mutated DNA was detectable at low concentrations (≥ 67 copies/mL) in 53 to 64% of patients with primary liver cancer or chronic liver disease and in 19% of controls. 44% of patients with HCC and no evidence of cirrhosis had plasma concentrations of R249S-mutated DNA ≥ 150 copies/mL, compared to 21% in patients with both HCC and cirrhosis, 22% in patients with cholangiocarcinoma, 12% in patients with non-cancer chronic liver disease and 3% of subjects in the reference group. Thus, plasma concentrations of R249S-mutated DNA ≥ 150 copies/mL tended to be more common in patients with HCC developing without pre-existing cirrhosis (p = 0.027). Overall, these results support the preferential occurrence of R249S-mutated DNA in HCC developing in the absence of cirrhosis in a context of HBV chronic infection.     

Retraction Note: miR-195-5p/NOTCH2-mediated EMT modulates IL-4 secretion in colorectal cancer to affect M2-like TAM polarization

Background

Hepatocellular carcinoma (HCC) generally arises from a background of liver cirrhosis (LC). Patients with cirrhosis and suspected HCC are recommended to undergo serum biomarker tests and imaging diagnostic evaluation. However, the performance of routine diagnostic methods in detecting early HCC remains unpromising.

Methods

Here, we conducted a large-scale, multicenter study of 1675 participants including 490 healthy controls, 577 LC patients, and 608 HCC patients from nine clinical centers across nine provinces of China, profiled gene mutation signatures of cell-free DNA (cfDNA) using Circulating Single-Molecule Amplification and Resequencing Technology (cSMART) through detecting 931 mutation sites across 21 genes.

Results

An integrated diagnostic model called “Combined method” was developed by combining three mutation sites and three serum biomarkers. Combined method outperformed AFP in the diagnosis of HCC, especially early HCC, with sensitivities of 81.25% for all stages and 66.67% for early HCC, respectively. Importantly, the integrated model exhibited high accuracy in differentiating AFP-negative, AFP-L3-negative, and PIVKA-II-negative HCCs from LCs.

     

SAMD9L Inactivation Promotes Cell Proliferation via Facilitating G1-S Transition in Hepatitis B Virus-associated Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is a highly malignant cancer with poor prognosis, and driver genes harboring genetic lesions and/or expression dysregulation contribute to hepatocarcinogenesis. Sterile Alpha Motif Domain-containing 9-like (SAMD9L) was a novel identified mutated gene in our previous study on exome sequencing of hepatitis B virus (HBV)-associated HCC, but its expression and role in HCC remain unknown. Here, we demonstrated that SAMD9L was frequently inactivated by somatic mutations, and that its expression was deregulated in HCC patients with hepatitis B virus (HBV) infection. SAMD9L knockdown significantly promoted cell proliferation, colony formation of SK-hep-1, QGY-7701, BEL-7721 and MHCC-97H HCC cells. Furthermore, SK-hep-1 and MHCC-97H cells with stable SAMD9L knockdown exhibited enhanced tumorigenicity in athymic mice. Interestingly, SAMD9L silence facilitated G1-S transition of cell cycle progression and led to the elevated activity of Wnt/β-catenin pathway. Collectively, these findings highlight a novel tumor-suppressive role of SAMD9L inactivation by somatic mutation and decreased expression in human HBV-related HCC.     

A garlic derivative, S-allylcysteine (SAC), suppresses proliferation and metastasis of hepatocellular carcinoma

Background

Hepatocellular carcinoma (HCC) is highly malignant and metastatic. Currently, there is no effective chemotherapy for patients with advanced HCC leading to an urgent need to seek for novel therapeutic options. We aimed to investigate the effect of a garlic derivative, S-allylcysteine (SAC), on the proliferation and metastasis of HCC.

Methodology/Principal Findings

A series of in vitro experiments including MTT, colony-forming, wound-healing, invasion, apoptosis and cell cycle assays were performed to examine the anti-proliferative and anti-metastatic effects of SAC on a metastatic HCC cell line MHCC97L. The therapeutic values of SAC single and combined with cisplatin treatments were examined in an in vivo orthotopic xenograft liver tumor model. The result showed that the proliferation rate and colony-forming abilities of MHCC97L cells were suppressed by SAC together with significant suppression of the expressions of proliferation markers, Ki-67 and proliferating cell nuclear antigen (PCNA). Moreover, SAC hindered the migration and invasion of MHCC97L cells corresponding with up-regulation of E-cadherin and down-regulation of VEGF. Furthermore, SAC significantly induced apoptosis and necrosis of MHCC97L cells through suppressing Bcl-xL and Bcl-2 as well as activating caspase-3 and caspase-9. In addition, SAC could significantly induce the S phase arrest of MHCC97L cells together with down-regulation of cdc25c, cdc2 and cyclin B1. In vivo xenograft liver tumor model demonstrated that SAC single or combined with cisplatin treatment inhibited the progression and metastasis of HCC tumor.

Conclusions/Significance

Our data demonstrate the anti-proliferative and anti-metastatic effects of SAC on HCC cells and suggest that SAC may be a potential therapeutic agent for the treatment of HCC patients.     

Serum MicroRNAs as Potential Biomarkers for Early Diagnosis of Hepatitis C Virus-Related Hepatocellular Carcinoma in Egyptian Patients

Circulating microRNAs are deregulated in liver fibrosis and hepatocellular carcinoma (HCC) and are candidate biomarkers. This study investigated the potential of serum microRNAs; miR-19a, miR-296, miR-130a, miR-195, miR-192, miR-34a, and miR-146a as early diagnostic biomarkers for hepatitis C virus (HCV)-related HCC. As how these microRNAs change during liver fibrosis progression is not clear, we explored their serum levels during fibrosis progression in HCV-associated chronic liver disease (CLD) and if they could serve as non-invasive biomarkers for fibrosis progression to HCC. 112 Egyptian HCV-HCC patients, 125 non-malignant HCV-CLD patients, and 42 healthy controls were included. CLD patients were subdivided according to Metavir fibrosis-scoring. Serum microRNAs were measured by qRT-PCR custom array. Serum microRNAs were deregulated in HCC versus controls, and except miR-130a, they were differentially expressed between HCC and CLD or late fibrosis (F3-F4) subgroup. Serum microRNAs were not significantly different between individual fibrosis-stages or between F1-F2 (early/moderate fibrosis) and F3-F4. Only miR-19a was significantly downregulated from liver fibrosis (F1-F3) to cirrhosis (F4) to HCC. Individual microRNAs discriminated HCC from controls, and except miR-130a, they distinguished HCC from CLD or F3-F4 patients by receiver-operating-characteristic analysis. Multivariate logistic analysis revealed a panel of four microRNAs (miR-19a, miR-195, miR-192, and miR-146a) with high diagnostic accuracy for HCC (AUC = 0.946). The microRNA panel also discriminated HCC from controls (AUC = 0.949), CLD (AUC = 0.945), and F3-F4 (AUC = 0.955). Studied microRNAs were positively correlated in HCC group. miR-19a and miR-34a were correlated with portal vein thrombosis and HCC staging scores, respectively. In conclusion, studied microRNAs, but not miR-130a, could serve as potential early biomarkers for HCC in high-risk groups, with miR-19a as a biomarker for liver fibrosis progression to cirrhosis to HCC. We identified a panel of four serum microRNAs with high accuracy in HCC diagnosis. Additional studies are required to confirm this panel and test its prognostic significance.     

乙型肝炎病毒变异与肝细胞癌发生关系

乙型肝炎病毒（hepatitisBvirus,HBV）基因组复制时,以病毒前基因组RNA作为模板合成子代病毒DNA,催化该过程的逆转录酶缺乏校对功能,所以HBV易出现变异。近年来,各国学者通过比较肝细胞癌（hepatocellular carcinoma,HCC）患者和非HCC患者的HBV基因序列,发现HBV基本核心启动子区的A1762T／G1764A变异或T1753V变异、增强子Ⅰ区的G1053A或G1229A变异、前S蛋白的F141L变异、前s2区基因缺失变异和x基因的截短变异,分别是HCC的易患因素,而前c区常见的G1896A变异,与HCC的发生无关。增强子Ⅱ区的C1653T变异在c基因HBV感染中可能与发生HCC有关,而在A基因型可能无关。     

PET probe detecting non-small cell lung cancer susceptible to epidermal growth factor receptor tyrosine kinase inhibitor therapy

Tyrosine kinase inhibitors for epidermal growth factor receptor (EGFR-TKIs) are used as molecular targeted therapy for non-small cell lung cancer (NSCLC) patients. The therapy is applied to the patients having EGFR-primary L858R mutation, but drug tolerance caused by EGFR-secondary mutation is occurred within one and half years. For the non-invasive detection of the EGFR-TKIs treatment positive patients by positron emission tomograpy (PET) imagaing, fluorine-18 labeled thienopyrimidine derivative, [¹⁸F]FTP2 was newly synthesized. EGFR inhibition assay, cell uptake study, and blocking study indicated [¹⁸F]FTP2 binds with high and selective affinity for EGFR with L858R mutation, and not with L858R/T790M dual mutations. On animal PET study using tumor bearing mice, H3255 cells expressing L858R mutated EGFR was more clearly visualized than H1975 cells expressing L858R/T790M dual mutated EGFR. [¹⁸F]FTP2 has potential for detecting NSCLC which is susceptible to EGFR-TKI treatment.     

Cyclin-dependent kinase inhibitor 3 (CDKN3) novel cell cycle computational network between human non-malignancy associated hepatitis/cirrhosis and hepatocellular carcinoma (HCC) transformation

The relationship of cyclin-dependent kinase inhibitor 3 (CDKN3) with tumours has previously been presented in a number of publications. However, the molecular network and interpretation of CDKN3 through the cell cycle between non-malignancy associated hepatitis/cirrhosis and hepatocellular carcinoma (HCC) have remained to be elucidated. Here, we have constructed and analysed significant high expression gene CDKN3 activated and inhibited cell cycle networks from 25 HCC versus 25 non-malignancy associated hepatitis/cirrhosis patients (viral infection HCV or HBV) in GEO Dataset GSE10140-10141, by combination of a gene regulatory network inference method based on linear programming, and decomposition procedure using CapitalBio MAS 3.0 software, based on integration of public databases including Gene Ontology, KEGG, BioCarta, GenMapp, Intact, UniGene, OMIM, and others. Comparing the same and differently activated and inhibited CDKN3 networks with GO analysis, between non-malignancy associated hepatitis/cirrhosis and HCC, our results suggest a CDKN3 cell cycle network (i) with stronger DNA replication and with weaker ubiquitin-dependent protein catabolism as common characteristics in both non-malignancy associated hepatitis/cirrhosis and HCC; (ii) with more cell division and weaker mitotic G2 checkpoint in non-malignancy associated hepatitis/cirrhosis; (iii) with stronger cell cycle and weaker cytokinesis, as a result forming multinucleate cells in HCC. Thus, it is useful to identify CDKN3 cell cycle networks for comprehension of molecular mechanism between non-malignancy associated hepatitis/cirrhosis and HCC transformation.     

Overexpression of hepatitis C virus NS5A protein induces chromosome instability via mitotic cell cycle dysregulation

Hepatocellular carcinoma (HCC) is a common primary cancer associated with high incidences of genetic variations including chromosome instability. Moreover, it has been demonstrated that hepatitis C virus (HCV) is one of the major causes of HCC. However, no previous work has assessed whether HCV proteins are associated with the induction of chromosome instability. Here, we found that liver cell lines constitutively expressing full-length or truncated versions of the HCV genome show a high incidence of chromosome instability. In particular, the overexpression of HCV NS5A protein in cultured liver cells was found to promote chromosome instability and aneuploidy. Further experiments showed that NS5A-induced chromosome instability is associated with aberrant mitotic regulations, such as, an unscheduled delay in mitotic exit and other mitotic impairments (e.g. multi-polar spindles). Thus, our results indicate that HCV NS5A protein may be directly involved in the induction of chromosome instability via mitotic cell cycle dysregulation, and provide novel insights into the molecular mechanisms of HCV-associated hepatocarcinogenesis.     

The PNPLA3 rs738409 148M/M genotype is a risk factor for liver cancer in alcoholic cirrhosis but shows no or weak association in hepatitis C cirrhosis

Background

An isoleucine>methionine mutation at position 148 in the PNPLA3 gene (p.I148M, rs738409) has recently been identified as a susceptibility factor for liver damage in steatohepatitis. Here, we studied whether the PNPLA3 rs738409 polymorphism also affects predisposition to hepatocellular carcinoma (HCC).

Methods

We compared distributions of PNPLA3 genotypes in 80 and 81 Caucasian patients with alcoholic and hepatitis C virus (HCV)-associated HCC to 80 and 81 age- and sex-matched patients with alcohol-related and HCV-related cirrhosis without HCC, respectively. PNPLA3 genotypes in 190 healthy individuals from the same population served as reference. Potential confounders obesity, diabetes, HCV genotype and HBV co-infection were controlled by univariate and multivariate logistic regression with forward variable selection.

Results

PNPLA3 genotypes were in Hardy-Weinberg equilibrium for all study groups. The frequency of the 148M allele was significantly (p<0.001) increased in alcoholic cirrhosis with (53.7%) and without HCC (36.2%) but was not different between healthy controls (22.9%) and patients with cirrhosis (25.3%; p = 0.545) and HCC (30.2%; p = 0.071) due to hepatitis C. HCC risk was highest in 148M/M homozygous patients with alcoholic liver disease (odds ratio (OR) 16.8 versus healthy controls; 95% confidence interval (CI) 6.68–42.43, p<0.001). Finally, multivariate regression confirmed 148M/M homozygosity (OR 2.8; 95%-CI: 1.24–6.42; p = 0.013) as HCC risk factor in alcoholic cirrhosis. In HCV-related cirrhosis only HCV genotype 1 was confirmed as a HCC risk factor (OR 4.2; 95%-CI: 1.50–11.52; p = 0.006).

Conclusion

The PNPLA3 148M variant is a prominent risk factor for HCC in patients with alcoholic cirrhosis, while its effects are negligible in patients with cirrhosis due to HCV. This polymorphism provides an useful tool to identify individuals with particularly high HCC risk in patients with alcoholic liver disease that should be taken into account in future HCC prevention studies.     

The role of the IGF axis in hepatocarcinogenesis.

Primary hepatocellular carcinoma (HCC) is one of the most common forms of malignant cancer with the fourth highest mortality rate worldwide. Major risk factors for the development of HCC include chronic infections with the hepatitis B or C virus, alcohol consumption, exposure to dietary aflatoxin B1, hereditary liver disease or liver cirrhosis of any etiology. Recent studies have discovered changes in the insulin-like growth factor (IGF) axis that affect the molecular pathogenesis of HCC, including the autocrine production of IGFs, IGF binding proteins (IGFBPs), IGFBP proteases, and IGF receptor expression. Characteristic alterations detected in HCC and hepatoma cell lines comprise the overexpression of IGF-II and the IGF-I receptor emerging as critical events in malignant transformation and growth of tumors. Simultaneous reduction of IGFBP expression and the increase in proteolytic cleavage of IGFBPs result in an excess of bioactive IGFs. Finally, defective functions of the IGF-II/mannose 6-phosphate receptor involved in degradation of IGF II, the activation of the growth inhibitor TGF-beta1, and the lysosomal targeting of cathepsin proteases capable to degrade extracellular matrix proteins may contribute to the development of HCC.