Aggressive Hepatocellular Carcinoma Research Articles

Predictors of poor outcome for transarterial chemoembolization (TACE) in hepatocellular carcinoma (HCC).

4100 Background: The use of TACE in select patients with BCLC stage B HCC has been shown to improve survival. Despite this, it remains unclear which patients will benefit from repeated TACE versus switching to systemic therapy upon disease progression. The purpose of this study is to identify prognostic factors that predict poor outcomes in patients who receive TACE. Methods: In this single-institutional retrospective analysis, patients with unresectable HCC were treated with TACE between 2007-2016. Relevant factors such as staging by BCLC stage B, Child-Pugh score, vascular invasion (VI), tumor thrombus (TT), AFP levels, and number of TACE treatments within six months from the initiation of TACE were analyzed using either Pearson’s chi-square test or the student's t-test. The Kaplan-Meier method was used for survival analysis. Results: Patients (n = 176) underwent TACE; 45% had stage I-II disease, 42% were BCLC stage B prior to TACE, 71% were Child-Pugh A, 21% had extrahepatic spread, 34.7% had VI, and 26% had TT. The median number of TACE treatments was 2 (range, 1- 6). The median overall survival (mOS) was 43 months (m) (95% CI 31.3-54.7) and mOS from start of TACE was 34m (95% CI 26.2-41.8). Elevated AFP (>400) correlated with decreased mOS (25m vs. 35m, p=0.041). Similarly, the presence of TT correlated with poor outcomes (25m vs. 37m, p=0.015). The mOS was also negatively impacted by having 3 or more TACE treatments within a 6 m period (25m vs. 38m, p = 0.09). AFP >400, TT, and interval between TACE were all independent factors in this multivariate analysis, resulting in a shorter mOS of approx. 2 years compared to 3 years in patients without these negative prognostic factors. There was a strong association with both elevated AFP and TT (Chi square p=0.009). Conclusions: Elevated AFP (>400), the presence of TT, and a need for 3 or more TACE treatments within 6 months appear to be independent predictors for shorter mOS in patients receiving TACE. Patients with these poor prognostic factors tend to have more aggressive HCC, and earlier initiation of systemic therapy might provide benefit to these patients. A larger study is needed for confirmation of these findings.

Nuclear GAPDH is vital for hypoxia-induced hepatic stellate cell apoptosis and is indicative of aggressive hepatocellular carcinoma behavior.

Background/aim: Hepatic stellate cells (HSCs) are critical determinants of liver tumor behavior such as vascular invasion, cell proliferation and migration. The apoptosis of HSCs can inhibit tumor growth and contribute to repressing hepatocellular carcinoma (HCC) progression. Our study aims to investigate the impact of nuclear glyceraldehyde-3-phosphate dehydrogenase (GAPDH) on HSCs under hypoxic conditions and the association of nuclear GAPDH with HCC patient outcomes and tumor progression.Patients and methods: Following stable cell passage, 0.3% O2 was used to induce hypoxia. Cell proliferation and apoptosis were analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assays and flow cytometry, respectively. Proteins expression were detected by extracting nuclear and cytoplasmic proteins and performing Western blots. GAPDH nuclear translocation was blocked by the agent deprenyl. Immunohistochemical staining for GAPDH was investigated in 137 HCC tissue samples from our center. An analysis of the clinicopathological features, Kaplan-Meier analysis and Cox proportional hazards regression analysis were applied.Results: MTT assays and flow cytometry analyses showed that the nuclear accumulation of GAPDH led to the apoptotic death of HSCs, while blockade of this process with deprenyl significantly decreased apoptosis. Western blots revealed that deprenyl inhibited the nuclear translocation of GAPDH. An analysis of the immunohistochemical staining of HSCs in HCC tissue samples (137) revealed that nuclear GAPDH expression was significantly positively correlated with HIF-1α expression. Overall survival (OS) and time-to-recurrence (TTR) estimated by Kaplan-Meier analyses showed that patients with high HIF-1α or low nuclear GAPDH levels in HSCs had significantly poorer prognosis compared with patients with low HIF-1α or high nuclear GAPDH expression in HSCs. Moreover, patients with combined high HIF-1α/low nuclear GAPDH expression in HSCs had the worst prognosis. The Cox regression analysis revealed that the combination of nuclear GAPDH/HIF-1α expression in HSCs was an independent prognostic factor for OS and TTR in HCC patients.Conclusions: These findings provide a novel mechanism underlying the involvement of intranuclear GAPDH in hypoxia-induced HSCs apoptosis and a correlation between nuclear GAPDH levels and the clinical prognosis, which may prompt the development of a novel therapeutic strategy for HCC.

MicroRNA-203 impacts on the growth, aggressiveness and prognosis of hepatocellular carcinoma by targeting MAT2A and MAT2B genes.

Hepatocellular carcinoma (HCC) is characterized by the down-regulation of the liver-specific methyladenosyltransferase 1A (MAT1A) gene, encoding the S-adenosylmethionine synthesizing isozymes MATI/III, and the up-regulation of the widely expressed methyladenosyltransferase 2A (MAT2A), encoding MATII isozyme, and methyladenosyltransferase 2B (MAT2B), encoding a β-subunit without catalytic action that regulates MATII enzymatic activity. Different observations showed hepatocarcinogenesis inhibition by miR-203. We found that miR-203 expression in HCCs is inversely correlated with HCC proliferation and aggressiveness markers, and with MAT2A and MAT2B levels. MiR-203 transfection in HepG2 and Huh7 liver cancer cells targeted the 3’-UTR of MAT2A and MAT2B, inhibiting MAT2A and MAT2B mRNA levels and MATα2 and MATβ2 protein expression. These molecular events were paralleled by an increase in SAM content and were associated with growth restraint and apoptosis, inhibition of cell migration and invasiveness, and suppression of the expression of CD133 and LIN28B stemness markers. In contrast, MAT2B transfection in the same cell lines led to a rise of both MATβ2 and MATα2 expression, associated with increases in cell growth, migration, invasion and overexpression of stemness markers and p-AKT. Altogether, our results indicate that the miR-203 oncosuppressor activity may at least partially depend on its inhibition of MAT2A and MAT2B and show, for the first time, an oncogenic activity of MAT2B linked to AKT activation.

MicroRNA-34a suppresses aggressiveness of hepatocellular carcinoma by modulating E2F1, E2F3, and Caspase-3.

Background: Accumulating evidence suggests an antineoplastic role of MicroRNA-34a (miR-34a) in human cancer. However, its precise biological functions stay largely elusive.Purpose: Our study was aimed to investigate the impact of miR-34a on hepatocellular carcinoma (HCC) and its underlying apoptosis related mechanisms in vitro, as well as the association of miR-34a, E2F1 and E2F3 expression with patient survival of HCC using publicly accessed datasets.Methods: The HBV-expressing Hep3B and SNU-449 cell lines with or without enforced expression of miR-34a were in vitro cultured for cell proliferation, colony formation, wound healing, cell invasion, and 3D spheroid formation. Quantitative reverse transcription PCR (RT-qPCR) was performed for E2F1, E2F3 expression. Caspase-3 (CASP3) activity was determined using a CaspACETM Assay System. Kaplan–Meier survival curves were used to analyze the associations of miR-34a, E2F1 and E2F3 expression and overall survival in HCC. Meta-analysis was performed to examine the differential expression of E2F1 and E2F3 between primary HCC vs normal tissues.Results: The results in vitro showed that enforced miR-34a expression significantly inhibited cell proliferation, migration, and invasion of both Hep3B and SNU-449. RT-qPCR results demonstrated that miR-34a could significantly suppress E2F1 and E2F3 expression, particularly in SNU-449. CASP3 activity in both Hep3B and SNU-449 increased in miR-34a treatment group. Overexpressed E2F1 and E2F3 were observed in primary HCC vs normal tissues. Survival analyses showed that HCC patients with either high miR-34a, or low E2F1, or low E2F3 expression had better survival than their opposite counterparts, respectively.Conclusion: Our study suggested thatmiR-34a can modulate the expression of E2F1, E2F3, and CASP3 activity, thereby repressing tumor aggressiveness and expediting apoptosis in liver cancer cells

HMGB1 links chronic liver injury to progenitor responses and hepatocarcinogenesis.

Cell death is a key driver of disease progression and carcinogenesis in chronic liver disease (CLD), highlighted by the well-established clinical correlation between hepatocellular death and risk for the development of cirrhosis and hepatocellular carcinoma (HCC). Moreover, hepatocellular death is sufficient to trigger fibrosis and HCC in mice. However, the pathways through which cell death drives CLD progression remain elusive. Here, we tested the hypothesis that high-mobility group box 1 (HMGB1), a damage-associated molecular pattern (DAMP) with key roles in acute liver injury, may link cell death to injury responses and hepatocarcinogenesis in CLD. While liver-specific HMGB1 deficiency did not significantly affect chronic injury responses such as fibrosis, regeneration, and inflammation, it inhibited ductular/progenitor cell expansion and hepatocyte metaplasia. HMGB1 promoted ductular expansion independently of active secretion in a nonautonomous fashion, consistent with its role as a DAMP. Liver-specific HMGB1 deficiency reduced HCC development in 3 mouse models of chronic injury but not in a model lacking chronic liver injury. As with CLD, HMGB1 ablation reduced the expression of progenitor and oncofetal markers, a key determinant of HCC aggressiveness, in tumors. In summary, HMGB1 links hepatocyte death to ductular reaction, progenitor signature, and hepatocarcinogenesis in CLD.

A hypothesis on paradoxical privileged portal vein metastasis of hepatocellular carcinoma. Can organ evolution shed light on patterns of human pathology, and vice versa?

Unlike other carcinomas, hepatocellular carcinoma (HCC) metastasizes to distant organs relatively rarely. In contrast, it routinely metastasizes to liver vasculature/liver, affecting portal veins 3–10 times more often than hepatic veins. This portal metastatic predominance is traditionally rationalized within the model of a reverse portal flow, due to accompanying liver cirrhosis. However, this intuitive model is not coherent with facts: 1) reverse portal flow occurs in fewer than 10% of cirrhotic patients, while portal metastasis occurs in 30–100% of HCC cases, and 2) portal vein prevalence of HCC metastasis is also characteristic of HCC in non-cirrhotic livers. Therefore, we must assume that the route for HCC metastatic dissemination is the same as for other carcinomas: systemic dissemination via the draining vessel, i.e., via the hepatic vein. In this light, portal prevalence versus hepatic vein of HCC metastasis appears as a puzzling pattern, particularly in cases when portal HCC metastases have appeared as the sole manifestation of HCC. Considering that other GI carcinomas (colorectal, pancreatic, gastric and small bowel) invariably disseminate via portal vein, but very rarely form portal metastasis, portal prevalence of HCC metastasis appears as a paradox. However, nature does not contradict itself; it is rather our wrong assumptions that create paradoxes. The ‘portal paradox’ becomes a logical event within the hypothesis that the formation of the unique portal venous system preceded the appearance of liver in evolution of chordates. The analysis suggests that the appearance of the portal venous system, supplying hormones and growth factors of pancreatic family, which includes insulin, glucagon, somatostatin, and pancreatic polypeptide (HGFPF) to midgut diverticulum in the early evolution of chordates (in an Amphioxus-like ancestral animal), promoted differentiation of enterocytes into hepatocytes and their further evolution to the liver of vertebrates. These promotional-dependent interactions are conserved in the vertebrate lineage. I hypothesize that selective homing and proliferation of malignant hepatocytes (i.e., HCC cells) in the portal vein environment are due to a uniquely high concentration of HGFPF in portal blood. HGFPF are also necessary for liver function and renewal and are significantly extracted by hepatocytes from passing blood, creating a concentration gradient of HGFPF between the portal blood and hepatic vein outflow, making post-liver vasculature and remote organs less favorable spaces for HCC growth. It also suggested that the portal vein environment (i.e., HGFPF) promotes the differentiation of more aggressive HCC clones from already-seeded portal metastases, explaining the worse outcome of HCC with the portal metastatic pattern. The analysis also offers new hypothesis on the phylogenetic origin of the hepatic diverticulum of cephalochordates, with certain implications for the modeling of the chordate phylogeny.

A comprehensive study of epigenetic alterations in hepatocellular carcinoma identifies potential therapeutic targets

A causal link has recently been established between epigenetic alterations and hepatocarcinogenesis, indicating that epigenetic inhibition may have therapeutic potential. We aimed to identify and target epigenetic modifiers that show molecular alterations in hepatocellular carcinoma (HCC). We studied the molecular-clinical correlations of epigenetic modifiers including bromodomains, histone acetyltransferases, lysine methyltransferases and lysine demethylases in HCC using The Cancer Genome Atlas (TCGA) data of 365 patients with HCC. The therapeutic potential of epigenetic inhibitors was evaluated in vitro and in vivo. RNA sequencing analysis and its correlation with expression and clinical data in the TCGA dataset were used to identify expression programs normalized by Jumonji lysine demethylase (JmjC) inhibitors. Genetic alterations, aberrant expression, and correlation between tumor expression and poor patient prognosis of epigenetic enzymes are common events in HCC. Epigenetic inhibitors that target bromodomain (JQ-1), lysine methyltransferases (BIX-1294 and LLY-507) and JmjC lysine demethylases (JIB-04, GSK-J4 and SD-70) reduce HCC aggressiveness. The pan-JmjC inhibitor JIB-04 had a potent antitumor effect in tumor bearing mice. HCC cells treated with JmjC inhibitors showed overlapping changes in expression programs related with inhibition of cell proliferation and induction of cell death. JmjC inhibition reverses an aggressive HCC gene expression program that is also altered in patients with HCC. Several genes downregulated by JmjC inhibitors are highly expressed in tumor vs. non-tumor parenchyma, and their high expression correlates with a poor prognosis. We identified and validated a 4-gene expression prognostic signature consisting of CENPA, KIF20A, PLK1, and NCAPG. The epigenetic alterations identified in HCC can be used to predict prognosis and to define a subgroup of high-risk patients that would potentially benefit from JmjC inhibitor therapy. In this study, we found that mutations and changes in expression of epigenetic modifiers are common events in human hepatocellular carcinoma, leading to an aggressive gene expression program and poor clinical prognosis. The transcriptional program can be reversed by pharmacological inhibition of Jumonji enzymes. This inhibition blocks hepatocellular carcinoma progression, providing a novel potential therapeutic strategy.

Sox9 Enhances Sorafenib Resistance Through Upregulating ABCG2 Expression in Hepatocellular Carcinoma

Sorafenib is a multikinase blocker and the only suggested drug treatment for aggressive hepatocellular carcinoma (HCC) patients. However, drug resistance to sorafenib often occurs after a period of time and cause further tumor aggression. Cancer stem cells are recently found in HCC and regarded to be involved in this process. Sox9 is highly expressed in HCC cancer stem cells and promotes cell proliferation and self-renewal. Meanwhile, HCC patients with higher Sox9 expression show poorer prognosis. Whether Sox9 is involved in sorafenib resistance in HCC is still unclear. Here, we found that sorafenib treatment increased Sox9 expression in HCC cell lines. Overexpression of exogenous Sox9 in HCC increased sorafenib resistance both in vitro and in vivo, and such phenomenon could be inhibited when Sox9 was down-regulated. We checked the down-stream gene expression profile through Sox9 RNA interference and found ATP binding cassette subfamily G member 2 (ABCG2) expression was closely affected by Sox9. Furthermore, we overexpressed Sox9 in combination with ABCG2 inhibition in HCC cell lines, and confirmed that the drug resistance to sorafenib could be ameliorated. In the cohort of patients who took sorafenib, we found those with lower Sox9 expression had longer OS and PFS. Cox analysis shows that Sox9 expression is an independent risk factor of HCC and logistic regression analysis reveals that Sox9 expression, tumor capsule deficiency, tumor diameters and microvascular invasion are risk factors of prognosis of HCC patients. Conclusion: These findings demonstrate that Sox9 enhances sorafenib resistance of HCC and meanwhile Sox9 may regulate such process through modulating ABCG2 expression. Funding: Natural Science Foundation of China. Declaration of Interest: We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled ”Sox9 enhances sorafenib resistance through upregulating ABCG2 expression in hepatocellular carcinoma”. Ethical Approval: The animal experiments in this study conformed to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines and were approved by the Institutional Animal Care and Use Committee of Second Military Medical University (Shanghai,China).

Novel urokinase-plasminogen activator inhibitor SPINK13 inhibits growth and metastasis of hepatocellular carcinoma in vivo

Advanced hepatocellular carcinoma (HCC) is a highly aggressive malignancy that is a serious threat to the public health system of China. Urokinase-plasminogen activator (uPA) can promote the invasive growth and metastasis of HCC cells by activating matrix metalloproteinases (MMPs), leading to the breakage of the extra-cellular matrix. uPA is a promising target for advanced HCC treatment. In this stuy the expression of uPA was examined by quantitative polymerase chain reaction in hepatic cell lines. Protein interaction between uPA and SPINK13 was identified by immunoprecipitation. In vitro biochemical assay was used to examine the inhibitory effect of the SPINK13 on the direct cleaving of the recombinant pro-MMP9 by uPA. The antitumor effect of SPINK13 was examined by transwell assay or the nude mice tumor model.The expression of uPA was much higher in highly aggressive HCC cell lines than in lowly aggressive HCC cell lines or non-tumor hepatic cell lines. SPINK13 interacted with uPA in HCC cells and directly inhibited the cleaving of MMP9 by uPA. Treatment of the recombinant SPINK13 protein inhibited the invasion of HCC cells in several experiments, such as transwell experiments or the intrahepatic growth model. The results of the study indicated that SPINK13 could function as a promising therapeutic approach for patients with advanced HCC.

Impact of Cytomegalovirus Infection on the Outcome of Patients With Cirrhosis: A Preliminary Study.

The aims of this study were to evaluate whether cytomegalovirus (CMV) infection is associated with hepatocellular carcinoma (HCC) and liver-related mortality in cirrhotic patients. In cirrhotic patients, the determinants of HCC and liver-related death are imperfectly known. CMV infection, by its prooncogenic and proinflammatory properties, may favor both the development of HCC and deleterious systemic inflammation. In the 1178 patients included between June 2008 and December 2012 in the CIrrhose et Risque de Carcinome Hépatocellulaire dans le grand-Est (CIRCE) study, a French multicenter case-control study designed to identify risk factors of HCC among cirrhotic patients, we identified 432 patients with interpretable CMV serological status at baseline. They included 159 cases with HCC and 273 controls. We measured factors associated with HCC at baseline and subsequent HCC in controls, and predictors of overall and liver-related death in the whole study population. During a median follow-up of 31 months, 25 cases of HCC developed in controls, and 209 deaths (163 liver-related) were recorded. There were 247 (57.2%) CMV-seropositive patients. CMV seropositivity was not associated with more frequent HCC at baseline or during follow-up, but among CMV-positive patients with HCC, the proportion of multinodular, infiltrative, or metastatic tumors at diagnosis was higher (73.8% vs. 57.3%; P=0.029), inducing higher mortality (74% vs. 52% at 3 years; P=0.004). By Cox-regression adjusted for age, gender, Model for End-stage Liver Disease (MELD) score, HCC at baseline, and diabetes, CMV seropositivity independently predicted all-cause (hazard ratio=1.45; 95% confidence interval, 1.08-1.94; P=0.013) and liver-related mortality (hazard ratio=1.56; 95% confidence interval, 1.04-2.30; P=0.031). In this preliminary study, CMV-seropositive cirrhotic patients were at higher risk of liver-related death caused by more aggressive HCCs or severe cirrhosis complications. These findings warrant confirmation.

MicroRNA-944 inhibits the malignancy of hepatocellular carcinoma by directly targeting IGF-1R and deactivating the PI3K/Akt signaling pathway.

Purpose: Recent studies have identified microRNA-944 (miR-944) as a cancer-related miRNA, but its expression and precise functions in hepatocellular carcinoma (HCC) remain unknown.Patients and methods: miR-944 expression in HCC tissues and cell lines were detected by RT-qPCR. A series of functional assays were utilized to examine the influence of miR-944 on the malignant phenotypes of HCC cells in vitro and in vivo. More importantly, the associated mechanisms underlying the activity of miR-944 in HCC cells were investigated using bioinformatics, luciferase reporter assays, RT-qPCR, and western blot analysis.Results: In this study, we report for the first time, a significant downregulation of miR-944 in HCC tissues and cell lines and the correlation between its downregulation and malignant clinical parameters, including Edmondson-Steiner grade, TNM stage, and venous infiltration. Low miR-944 expression predicted poorer overall survival rate and disease-free survival rate in patients with HCC. Functionally, exogenous miR-944 expression attenuated cell proliferation, clone formation, metastasis, and epithelial-mesenchymal transition and increased apoptosis in HCC, whereas miR-944 knockdown produced the opposite results. In addition, ectopic miR-944 expression hindered HCC tumor growth in vivo. Mechanistically, insulin-like growth factor 1 receptor (IGF-1R) was demonstrated to be the direct target gene of miR-944 in HCC cells. Furthermore, the expression level of miR-944 was inversely correlated with IGF-1R expression in HCC tissues. Rescue experiments showed that IGF-1R was at least partially responsible for the effects of miR-944 on the malignant phenotypes of HCC cells. In addition, the PI3K/Akt pathway was notably deactivated, both in vitro and in vivo, upon miR-944 upregulation.Conclusion: This study provides the first evidence that miR-944 directly targets IGF-1R and inhibits the aggressiveness of HCC, in vitro and in vivo, by decreasing PI3K/Akt signaling. Hence, targeting miR-944 may open a new avenue for the treatment of patients with HCC.

03:18 PM Abstract No. 210 Association between modified BALAD score and locoregional therapy response in transplant waitlist patients with hepatocellular carcinoma: prospective, observational analysis at a high-volume transplant center

The BALAD score, a risk index combining threshold values for bilirubin, albumin, α-fetoprotein (AFP), des-γ-carboxy prothrombin (DCP), and Lens culinaris agglutinin-reactive α-fetoprotein (AFP-L3), has been useful in predicting survival and recurrence risk in hepatocellular carcinoma (HCC). This study seeks to correlate BALAD score with durable locoregional treatment response to doxorubicin-eluting bead transarterial chemoembolization (DEB-TACE). A single-center, prospective analysis was performed on patients undergoing liver transplant evaluation with HCC who were selected for down-staging or bridge-to-transplantation LRT with DEB-TACE. Blood was collected immediately prior to DEB-TACE in all patients. Plasma levels of AFP, DCP, and AFP-L3 were measured using the μTASWako i30 autoanalyzer. DEB-TACE was performed with 100-300μ LC beads mixed with 50mg Doxorubicin to near-complete stasis. Tumor response to DEB-TACE was calculated by mRECIST at 30-day and 3-month follow-up imaging. Cohort demographics were median age of 60, 69% male, 78% hepatitis C cirrhosis etiology, 88% within Milan criteria with an average MELD-Na score of 10. Median BALAD components were as follows: bilirubin 1.5 mg/dL, albumin 3.2 g/dL, AFP 7 ng/mL, AFP L3% 8, and DCP 2.8 ng/mL. Median follow up imaging for durable tumor response was 112 days. Fifty-percent of cohort had a complete response to DEB-TACE. Univariate analysis revealed BALAD score was associated with response to DEB-TACE (P=0.04), with 73% of patients with BALAD ≥ 2 not responding to DEB-TACE. BALAD component analysis revealed tumor biomarker thresholds poorly modeled to the dataset. The BALAD biomarker cutoffs were modified from AFP 400ng/mL to 20ng/mL, AFP-L3% from 15% to 10%, and DCP 1.9ng/mL to 7.5ng/mL decreasing the corresponding P-value (0.04 to 0.02) between BALAD and treatment response. Waitlist dropout after DEB-TACE for patients with modified-BALAD ≤ 1 was 5%. By combining metrics for liver synthetic function with biomarkers of HCC aggressiveness, the BALAD can identify HCC resistance to embolization therapy and risk of waitlist dropout.

Somatic mutations of PREX2 gene in patients with hepatocellular carcinoma

Characterized with a high recurrence rate and low detection rate, prevention is the best approach to reduce mortality in hepatocellular carcinoma (HCC). The overexpression of Phosphatidylinositol-3,4,5-Trisphosphate Dependent Rac Exchange Factor 2 (PREX2) is observed in various tumors, including HCC; and the frequent PREX2 mutations in melanoma are associated with invasiveness. We sought to identify somatic mutations and the functional changes in mutational signatures of PREX2. Genomic DNA sequencing was performed in 68 HCC samples with three types of hepatitis viral infection status: HBs Ag-positive, anti-HCV Ab-positive, and negative for any hepatitis B or C markers. Stabilities and interactions of proteins as well as cell proliferation and migration were evaluated. Fourteen non-silent point mutations in PREX2 were detected, with 16 of 68 HCC patients harboring at least one non-silent mutation. All mutant forms of PREX2, except for K400f, had an extended half-life compared with wild-type PREX2. Moreover, only the half-life of S1113R was twice that of the wild-type. PREX2 mutant-S1113R also promoted migration and activated the AKT pathway as well as impaired HectH9-mediated ubiquitination. Our study identified a gain-of-function mutation of PREX2 – S1113R in HCC. Such mutation enhanced PREX2 protein stability, promoted cell proliferation, and was associated with aggressiveness of HCC.

Current status of imaging biomarkers predicting the biological nature of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is heterogeneous in terms of its biological nature. Various factors related to its biological nature, including size, multifocality, macroscopic morphology, grade of differentiation, macro/microvascular invasion, bile duct invasion, intra-tumoral fat and molecular factors, and their value as prognostic imaging biomarkers have been reported. And recently, genome-based molecular HCC classification correlated with clinical outcome has been elucidated. The imaging biomarkers suggesting a less aggressive nature of HCC are smaller size, solitary tumor, smooth margin suggesting small nodular type with indistinct margin and simple nodular type with distinct margin, capsule, imaging biomarkers predicting early or well-differentiated grade, intra-tumoral fat detection, and low fluorodeoxyglucose (FDG) accumulation. The imaging biomarkers suggesting an aggressive HCC nature are larger size, multifocality, non-smooth margin suggesting simple nodular type with extranodular growth, confluent multinodular, and infiltrative type, imaging biomarkers predicting poor differentiation, macrovascular tumor thrombus, predicting microvascular invasion imaging biomarkers, bile duct dilatation or tumor thrombus, and high FDG accumulation. In the genome-based molecular classification, CTNNB-1 mutated HCC shows a less aggressive nature, while CK19/EpCAM positive HCC and macrotrabecular massive HCC show an aggressive one. Better understanding of these imaging biomarkers can contribute to devising more appropriate treatment plans for HCC.

Uridine-cytidine kinase 2 upregulation predicts poor prognosis of hepatocellular carcinoma and is associated with cancer aggressiveness.

Patients with advanced hepatocellular carcinoma (HCC) continue to have a dismal prognosis. Potential biomarkers to determine prognosis and select targeted therapies are urgently needed for patients with HCC. This study aimed to elucidate the role of UCK2 in HCC prognosis and tumor progression. We performed a screen of public databases to identify functional genes associated with HCC tumorigenesis, progression, and outcome. We identified uridine-cytidine kinase 2 (UCK2) as a gene of interest for further study. UCK2 promoting HCC aggressiveness was demonstrated by evaluation of clinical samples, in vitro experiments, in vivo tumorigenicity, and transcript analysis. UCK2 expression was generally elevated in HCC and was significantly correlated with poor survival and inferior clinicopathological characteristics of HCC patients. A multivariate analysis revealed that high UCK2 expression was an independent factor for poor prognosis. In HCC cell lines, UCK2 knockdown suppressed cell migration and invasion and inhibited cell proliferation, while UCK2 overexpression had an opposite effect. Animal model experiments confirmed that knockdown of UCK2 suppressed tumor growth in vivo. The bioinformatics analysis demonstrated that UCK2 might associated with metabolsim, splicesome, and adherens junction. UCK2 is highly associated with HCC malignant behavior and is a potential prognostic predictor for HCC patients in the clinic.

Immunonutritional consequences of different serine-type protease inhibitors in a C57BL/6 hepatocarcinoma model.

Imbalances in innate immunity and the activity of innate immune cells are implicated in the development of hepatocellular carcinoma (HCC). Plant seeds are good sources of protease inhibitors, which can have a significant influence on human health disorders, especially in the field of cancer prevention. To elucidate the impact and preventive effects of immunonutritional serine-type protease inhibitors (STPIs) on HCC, it was used an established model of chemically induced liver injury. Injured livers induced Akt as well as hepatic infiltration of NKG2D+ and CD74+ cells. Feeding STPIs reduced size and number of intrahepatic nodes of mononuclear. These animals showed an inverse association of the severity of HCC with bioactive hepcidin levels, which was significantly correlated with the hepatic myeloperoxidase activity. According to their origin, administration of STPIs significantly induce increased numbers of F4/80+ cells in injured livers that can be responsible for the biological effects detected on the parenchyma and inflammatory markers under DEN/TAA treatment. These findings can have direct implications in HCC immunotherapy where enhanced response(s) in inflammation-driven cancer patients could help promoting inflammation-driven processes and favor tumor growth. Altogether, this study demonstrates that oral administration of STPIs modulate innate immunity response influencing HCC aggressiveness and progression. These results represent a path forward to develop durable, long-lasting response against hepatocarcinoma and open a future research path in the development of coadjutant intervention strategies to pharmacological therapies.

An arsenic trioxide nanoparticle prodrug (ATONP) potentiates a therapeutic effect on an aggressive hepatocellular carcinoma model via enhancement of intratumoral arsenic accumulation and disturbance of the tumor microenvironment

An arsenic trioxide nanoparticle prodrug has a therapeutic effect on a transgenic liver cancer model by disturbing the tumor micro-environment and increasing the delivery efficiency.

MiR-HCC2 Up-regulates BAMBI and ELMO1 Expression to Facilitate the Proliferation and EMT of Hepatocellular Carcinoma Cells.

MicroRNAs (miRNAs) are a class of gene expression regulators that participate in the occurrence and development of hepatocellular carcinoma (HCC), although the underlying mechanism by which they function in HCC has not been fully elucidated. Here, small RNA deep sequencing was used to identify aberrantly expressed miRNAs in HCC tissues, and a novel miRNA named miR-HCC2 was identified. RT-qPCR analysis demonstrated that miR-HCC2 displayed higher expression in HCC tissues than in adjacent non-tumor tissues. We documented that miR-HCC2 facilitated the growth, migration and invasion of HCC cells by accelerating cell cycle progression, incressing the expression of epithelial-to-mesenchymal transition (EMT)-associated marker vimentin but decreasing the expression of E-cadherin. MiR-HCC2 directly targeted the 3′ UTR of BAMBI and ELMO1 and up-regulated their expression. Both BAMBI and ELMO1 had the same patterns of expression with miR-HCC2 in HCC tissues. Additionally, blocking BAMBI or ELMO1 counteracted the phenotypic alterations elicited by miR-HCC2. Collectively, our investigation identified miR-HCC2 as a new positive modulator of HCC aggressiveness that may serve as a potential biomarker for the development of diagnostic and therapeutic approaches for HCC.

Tanshinol inhibits the growth, migration and invasion of hepatocellular carcinoma cells via regulating the PI3K-AKT signaling pathway.

BackgroundTanshinol is an active constituent of Salvia miltiorrhiza and possess anti-inflammatory, antioxidant, and anti-bacterial activity. Herein, we explored the role of tanshinol on the growth and aggressiveness of hepatocellular carcinoma (HCC) cells in vitro and in vivo.Materials and methodsThe proliferation of a panel of HCC cell lines was measured using MTT assay. The expressions of phosphatidylinositol 3 kinase (PI3K) and protein kinase B (AKT) were detected by immunofluorescence staining and immunohistochemical assay. The levels of Bcl-2 and Bax were determined using immunoblotting assay. The secretions of matrix metalloproteinase-2 (MMP-2) and MMP-9 were detected by ELISA. The migration and invasion abilities of HepG2 cell were determined using wound healing and Transwell invasion assays. The apoptosis of HepG2 cell induced by tanshinol was analyzed by Annexin V/propidium iodide staining. A xenograft model was constructed to investigate the inhibitory effect of tanshinol on HepG2 cell growth in vivo. To further investigate the role of tanshinol on the metastasis of HepG2 cell in vivo, an experimental metastasis assay was performed.ResultsTanshinol inhibited the growth and colony formation of HCC cell in vitro. Tanshinol also induced the apoptosis of HepG2 cell and inhibited the migration and invasion of HepG2 cell. In in vivo experiments, tanshinol suppressed the tumor growth and metastasis of HepG2 cell. Furthermore, the phosphorylation of PI3K and AKT was decreased by tanshinol in vitro and in vivo.ConclusionTanshinol exerts its anti-cancer effects via regulating the PI3K-AKT signaling pathway in HCC.

LOXL2 promotes vasculogenic mimicry and tumour aggressiveness in hepatocellular carcinoma.

Lysyl oxidase‐like 2 (LOXL2) has shown to promote metastasis and poor prognosis in hepatocellular carcinoma (HCC). Also, we have previously reported that vasculogenic mimicry (VM) is associated with invasion, metastasis and poor survival in HCC patients. In the present study, we investigated molecular function of LOXL2 in HCC and VM. We used the immunohistochemical and CD31/periodic acid‐Schiff double staining to detect the relationship between LOXL2 and VM formation. We performed the gain and loss of function studies and analysed the migratory, invasion and tube formation in HCC cell lines. We analysed the function of LOXL2 in VM formation and HCC metastasis both in vitro and in vivo. We have showed that LOXL2 was overexpression in HCC and was positively correlated with tumour grade, metastasis, VM formation and poor survival in 201 HCC patients. Secondly, our studies have showed that LOXL2 overexpression in HCC cells significantly promoted migration, invasion and tube formation. Finally, we found that LOXL2 may increase SNAIL expression, thereby enabling VM. Our study indicated that LOXL2 may promote VM formation and tumour metastasis by collaborating with SNAIL in HCC. What's more, the overexpression of LOXL2 indicated a poor prognosis in HCC patients.