Circular RNAs (circRNAs) represent a class of endogenous noncoding RNAs that have recently been recognized as important regulators of gene expression and pathological networks. However, their ...transcriptional activities and functional mechanisms in cancer remain largely unknown. Here, we present results from a global circRNA expression and functional analysis of patients with hepatocellular carcinoma (HCC). Using a circRNA microarray, we identified 226 differentially expressed circRNAs, of which 189 were significantly upregulated and 37 were downregulated. High expression of circRNA_100338, one of the upregulated circRNAs in HCC, is closely correlated with a low cumulative survival rate and metastatic progression in HCC patients with hepatitis B. Furthermore, our in silico and experimental analyses identified miR-141-3p as a direct target of circRNA_100338. Thus, circRNA_100338 functions as an endogenous sponge for miR-141-3p in HCC. In addition, we identified the crucial antagonistic roles of circRNA_100338 and miR-141-3p in the regulation of invasive potential in liver cancer cells. Overall, the differential expression of multiple circRNAs in HCC tissues and their clinical significance in hepatitis B-related HCC patients as revealed by our study suggests that circRNA_100338 is a potentially valuable biomarker for HCC diagnosis and target for HCC therapeutics.
Exosomes play crucial roles in regulating the crosstalk between normal and cancer cells in the tumor microenvironment, and in regulating cancer proliferation, migration and invasion through their ...cargo molecules.
We analyzed the pro-invasiveness of exosomal circRNA-100,338 in HCC using the transwell invasion assay. The co-culture of human umbilical vein endothelial cells (HUVEC) and exosomes derived from HCC cell lines were used to evaluate the impact of HCC derived exosomes on HUVEC. Nude mice models were used to validate the findings in vitro. Clinically, quantitative RT-PCR was used to quantify the expression of serum exosomal circRNA-100,338 in HCC patients at both pre-surgery within one week and post-surgery within three weeks.
We aim to investigate the pro-invasive role of exosomal circRNA-100,338 in HCC metastasis. We for the first time demonstrated that circRNA-100,338 was highly expressed in both highly metastatic HCC cells and their secreted exosomes. The transwell invasion assay showed that the overexpression or knockdown of exosomal circRNA-100,338 significantly enhanced or reduced the invasive abilities of HCC cells. Subsequently, in vitro and in vivo assays showed that exosomal circRNA-100,338 affected the cell proliferation, angiogenesis, permeability, and vasculogenic mimicry (VM) formation ability of human umbilical vein endothelial cells (HUVEC), and tumor metastasis. Furthermore, we also observed that the persistent high expression of exosomal circRNA-100,338 in serum of HCC patients who underwent curative hepatectomy may be a risk indicator of pulmonary metastasis and poor survival.
Our findings indicated that metastatic ability of HCC cells could be enhanced by transferring exosomal circRNA-100,338 to recipient HUVECs, which could affect proangiogenic activity by regulating angiogenesis.
Globally, hepatocellular carcinoma (HCC) accounts for 70%‐85% of primary liver cancers and ranks as the second leading cause of male cancer death. Serum alpha‐fetoprotein (AFP), normally highly ...expressed in the liver only during fetal development, is reactivated in 60% of HCC tumors and associated with poor patient outcome. We hypothesize that AFP+ and AFP− tumors differ biologically. Multivariable analysis in 237 HCC cases demonstrates that AFP level predicts poor survival independent of tumor stage (P < 0.043). Using microarray‐based global microRNA (miRNA) profiling, we found that miRNA‐29 (miR‐29) family members were the most significantly (P < 0.001) down‐regulated miRNAs in AFP+ tumors. Consistent with miR‐29's role in targeting DNA methyltransferase 3A (DNMT3A), a key enzyme regulating DNA methylation, we found a significant inverse correlation (P < 0.001) between miR‐29 and DNMT3A gene expression, suggesting that they might be functionally antagonistic. Moreover, global DNA methylation profiling reveals that AFP+ and AFP− HCC tumors have distinct global DNA methylation patterns and that increased DNA methylation is associated with AFP+ HCC. Experimentally, we found that AFP expression in AFP− HCC cells induces cell proliferation, migration, and invasion. Overexpression of AFP, or conditioned media from AFP+ cells, inhibits miR‐29a expression and induces DNMT3A expression in AFP− HCC cells. AFP also inhibited transcription of the miR‐29a/b‐1 locus, and this effect is mediated through c‐MYC binding to the transcript of miR‐29a/b‐1. Furthermore, AFP expression promotes tumor growth of AFP− HCC cells in nude mice. Conclusion: Tumor biology differs considerably between AFP+ HCC and AFP− HCC; AFP is a functional antagonist of miR‐29, which may contribute to global epigenetic alterations and poor prognosis in HCC. (Hepatology 2014;60:872–883)
Intrahepatic cholangiocellular carcinoma (ICC) is the second most common type of primary liver cancer. However, its tumor heterogeneity and molecular characteristics are largely unknown. In this ...study, we conducted transcriptomic profiling of 23 ICC and combined hepatocellular cholangiocarcinoma tumor specimens from Asian patients using Affymetrix messenger RNA (mRNA) and NanoString microRNA microarrays to search for unique gene signatures linked to tumor subtypes and patient prognosis. We validated the signatures in an additional 68 ICC cases derived from Caucasian patients. We found that both mRNA and microRNA expression profiles could independently classify Asian ICC cases into two main subgroups, one of which shared gene expression signatures with previously identified hepatocellular carcinoma (HCC) with stem cell gene expression traits. ICC‐specific gene signatures could predict survival in Asian HCC cases and independently in Caucasian ICC cases. Integrative analyses of the ICC‐specific mRNA and microRNA expression profiles revealed that a common signaling pathway linking miR‐200c signaling to epithelial‐mesenchymal transition (EMT) was preferentially activated in ICC with stem cell gene expression traits. Inactivation of miR‐200c resulted in an induction of EMT, whereas activation of miR‐200c led to a reduction of EMT including a reduced cell migration and invasion in ICC cells. We also found that miR‐200c and neural cell adhesion molecule 1 (NCAM1) expression were negatively correlated and their expression levels were predictive of survival in ICC samples. NCAM1, a known hepatic stem/progenitor cell marker, was experimentally demonstrated to be a direct target of miR‐200c. Conclusion: Our results indicate that ICC and HCC share common stem‐like molecular characteristics and poor prognosis. We suggest that the specific components of EMT may be exploited as critical biomarkers and clinically relevant therapeutic targets for an aggressive form of stem cell‐like ICC. (HEPATOLOGY 2012;56:1792–1803)
MicroRNAs (miRNAs) have been used as cancer‐related biomarkers. Hepatocellular carcinoma (HCC) is an aggressive cancer with a dismal outcome largely due to metastasis and postsurgical recurrence. We ...investigated whether the expression of certain miRNAs are associated with HCC metastasis. We examined the miRNA expression profiles of 482 cancerous and noncancerous specimens from radical resection of 241 patients with HCC. Using a supervised algorithm and a clinically well‐defined cohort of 131 cases, we built a unique 20‐miRNA metastasis signature that could significantly predict (P < 0.001) primary HCC tissues with venous metastases from metastasis‐free solitary tumors with 10‐fold cross‐validation. However, significant miRNAs could not be identified from the corresponding noncancerous hepatic tissues. A survival risk prediction analysis revealed that a majority of the metastasis‐related miRNAs were associated with survival. Furthermore, the 20‐miRNA tumor signature was validated in 110 additional cases as a significant independent predictor of survival (P = 0.009) and was significantly associated with both survival and relapse in 89 cases of early stage HCC (P = 0.022 and 0.002, respectively). These 20 miRNAs may provide a simple profiling method to assist in identifying patients with HCC who are likely to develop metastases/recurrence. In addition, functional analysis of these miRNAs may enhance our biological understanding of HCC metastasis. (HEPATOLOGY 2008.)
Metastasis-related recurrence often occurs in hepatocellular carcinoma (HCC) patients who receive curative therapies. At present, it is challenging to identify patients with high risk of recurrence, ...which would warrant additional therapies. In this study, we sought to analyze a recently developed metastasis-related gene signature for its utility in predicting HCC survival, using 2 independent cohorts consisting of a total of 386 patients who received radical resection. Cohort 1 contained 247 predominantly HBV-positive cases analyzed with an Affymetrix platform, whereas cohort 2 contained 139 cases with mixed etiology analyzed with the NCI Oligo Set microarray platform. We employed a survival risk prediction algorithm with training, test, and independent cross-validation strategies and found that the gene signature is predictive of overall and disease-free survival. Importantly, risk was significantly predicted independently of clinical characteristics and microarray platform. In addition, survival prediction was successful in patients with early disease, such as small (<5 cm in diameter) and solitary tumors, and the signature predicted particularly well for early recurrence risk (<2 years), especially when combined with serum alpha fetoprotein or tumor staging. In conclusion, we have shown in 2 independent cohorts with mixed etiologies and ethnicity that the metastasis gene signature is a useful tool to predict HCC outcome, suggesting the general utility of this classifier. We recommend the use of this classifier as a molecular diagnostic test to assess the risk that an HCC patient will develop tumor relapse within 2 years after surgical resection, particularly for those with early-stage tumors and solitary presentation.
Background & Aims Cancer progression/metastases and embryonic development share many properties including cellular plasticity, dynamic cell motility, and integral interaction with the ...microenvironment. We hypothesized that the heterogeneous nature of hepatocellular carcinoma (HCC), in part, may be owing to the presence of hepatic cancer cells with stem/progenitor features. Methods Gene expression profiling and immunohistochemistry analyses were used to analyze 235 tumor specimens derived from 2 recently identified HCC subtypes (EpCAM+ α-fetoprotein AFP+ HCC and EpCAM− AFP− HCC). These subtypes differed in their expression of AFP, a molecule produced in the developing embryo, and EpCAM, a cell surface hepatic stem cell marker. Fluorescence-activated cell sorting was used to isolate EpCAM+ HCC cells, which were tested for hepatic stem/progenitor cell properties. Results Gene expression and pathway analyses revealed that the EpCAM+ AFP+ HCC subtype had features of hepatic stem/progenitor cells. Indeed, the fluorescence-activated cell sorting–isolated EpCAM+ HCC cells displayed hepatic cancer stem cell–like traits including the abilities to self-renew and differentiate. Moreover, these cells were capable of initiating highly invasive HCC in nonobese diabetic, severe combined immunodeficient mice. Activation of Wnt/β-catenin signaling enriched the EpCAM+ cell population, whereas RNA interference-based blockage of EpCAM, a Wnt/β-catenin signaling target, attenuated the activities of these cells. Conclusions Taken together, our results suggest that HCC growth and invasiveness is dictated by a subset of EpCAM + cells, opening a new avenue for HCC cancer cell eradication by targeting Wnt/β-catenin signaling components such as EpCAM.
MicroRNAs (miRNAs) are endogenous small noncoding RNAs that regulate gene expression with functional links to tumorigenesis. Hepatocellular carcinoma (HCC) is the most common type of liver cancer, ...and it is heterogeneous in clinical outcomes and biological activities. Recently, we have identified a subset of highly invasive epithelial cell adhesion molecule (EpCAM)+ HCC cells from alpha‐fetoprotein (AFP)+ tumors with cancer stem/progenitor cell features, that is, the abilities to self‐renew, differentiate, and initiate aggressive tumors in vivo. Here, using a global microarray‐based miRNA profiling approach followed by validation with quantitative reverse transcription polymerase chain reaction, we have demonstrated that conserved miR‐181 family members were up‐regulated in EpCAM+AFP+ HCCs and in EpCAM+ HCC cells isolated from AFP+ tumors. Moreover, miR‐181 family members were highly expressed in embryonic livers and in isolated hepatic stem cells. Importantly, inhibition of miR‐181 led to a reduction in EpCAM+ HCC cell quantity and tumor initiating ability, whereas exogenous miR‐181 expression in HCC cells resulted in an enrichment of EpCAM+ HCC cells. We have found that miR‐181 could directly target hepatic transcriptional regulators of differentiation (for example, caudal type homeobox transcription factor 2 CDX2 and GATA binding protein 6 GATA6) and an inhibitor of Wnt/β‐catenin signaling (nemo‐like kinase NLK). Taken together, our results define a novel regulatory link between miR‐181s and human EpCAM+ liver cancer stem/progenitor cells and imply that molecular targeting of miR‐181 may eradicate HCC. (HEPATOLOGY 2009.)
Background & Aims We combined gene expression and metabolic profiling analyses to identify factors associated with outcomes of patients with hepatocellular carcinoma (HCC). Methods We compared ...metabolic and gene expression patterns between paired tumor and nontumor tissues from 30 patients with HCC, and validated the results using samples from 356 patients with HCC. A total of 469 metabolites were measured using liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry. Metabolic and genomic data were integrated, and Kaplan–Meier and Cox proportional hazards analyses were used to associate specific patterns with patient outcomes. Associated factors were evaluated for their effects on cancer cells in vitro and tumor formation in nude mice. Results We identified 28 metabolites and 169 genes associated with aggressive HCC. Lipid metabolites of stearoyl-CoA-desaturase (SCD) activity were associated with aberrant palmitate signaling in aggressive HCC samples. Expression of gene products associated with these metabolites, including SCD, were associated independently with survival times and tumor recurrence in the test and validation sets. Combined expression of SCD and α-fetoprotein were associated with outcomes of patients with early-stage HCC. Levels of monounsaturated palmitic acid, the product of SCD activity, were increased in aggressive HCCs; monounsaturated palmitic acid increased migration and invasion of cultured HCC cells and colony formation by HCC cells. HCC cells that expressed small interfering RNA against SCD had decreased cell migration and colony formation in culture and reduced tumorigenicity in mice. Conclusions By using a combination of gene expression and metabolic profile analysis, we identified a lipogenic network that involves SCD and palmitate signaling and was associated with HCC progression and patient outcomes. The microarray platform and data have been submitted to the Gene Expression Omnibus public database at NCBI following MIAME guidelines. Accession numbers: GPL4700 (platform), and GSE6857 (samples).
Background: Combined therapy with tyrosine kinase inhibitors (TKIs) and anti-PD-1 antibodies has shown high tumor response rates for patients with unresectable hepatocellular carcinoma (HCC). ...However, using this treatment strategy to convert initially unresectable HCC to resectable HCC was not reported. Methods: Consecutive patients with unresectable HCC who received first-line therapy with combined TKI/anti-PD-1 antibodies were analyzed. Tumor response and resectability were evaluated via imaging every 2 months (±2 weeks) using RECIST v1.1. Resectability criteria were (1) R0 resection could be achieved with sufficient remnant liver volume and function; (2) intrahepatic lesions were evaluated as partial responses or stable disease for at least 2 months; (3) no severe or persistent adverse effects occurred; and (4) hepatectomy was not contraindicated. Results: Sixty-three consecutive patients were enrolled. Of them, 10 (15.9%) underwent R0 resection in 3.2 months (range: 2.4–8.3 months) after the initiation of combination therapy. At baseline, these 10 patients had a median largest tumor diameter of 9.3 cm, 7 had Barcelona Clinic Liver Cancer stage C (vascular invasion) disease, 2 had stage B, and 1 had stage A. Before surgery, 6 patients were evaluated as a partial response, 3 stable disease, and 1 partial response in the intrahepatic lesion but a new metastatic lesion in the right adrenal gland. Six patients (60%) achieved a pathological complete response. One patient died from immune-related adverse effects 2.4 months after hepatectomy. After a median follow-up of 11.2 months (range: 7.8–15.9 months) for other 9 patients, 8 survived without disease recurrence, and 1 experienced tumor recurrence. Conclusions: Combination of TKI/anti-PD-1 antibodies is a feasible conversion therapy for patients with unresectable HCC to become resectable. This study represents the largest patient cohort on downstaging role of combinational systemic therapy on TKI and PD-1 antibody for HCC.
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