EGFR activation limits the response of liver cancer to lenvatinib.

Hepatocellular carcinoma (HCC)-the most common form of liver cancer-is an aggressive malignancy with few effective treatment options1. Lenvatinib is a small-molecule inhibitor of multiple receptor tyrosine kinases that is used for the treatment of patients with advanced HCC, but this drug has only limited clinical benefit2. Here, using a kinome-centred CRISPR-Cas9 genetic screen, we show that inhibition of epidermal growth factor receptor (EGFR) is synthetic lethal with lenvatinib in liver cancer. The combination of the EGFR inhibitor gefitinib and lenvatinib displays potent anti-proliferative effects in vitro in liver cancer cell lines that express EGFR and in vivo in xenografted liver cancer cell lines, immunocompetent mouse models and patient-derived HCC tumours in mice. Mechanistically, inhibition of fibroblast growth factor receptor (FGFR)  by lenvatinib treatment leads to feedback activation of the EGFR-PAK2-ERK5 signalling axis, which is blocked by EGFR inhibition. Treatment of 12 patients with advanced HCC who were unresponsive to lenvatinib treatment with the combination of lenvatinib plus gefitinib (trial identifier NCT04642547) resulted in meaningful clinical responses. The combination therapy identified here may represent a promising strategy for the approximately 50% of patients with advanced HCC who have high levels of EGFR.

Dual Inhibition of CDK4/6 and XPO1 Induces Senescence With Acquired Vulnerability to CRBN-Based PROTAC Drugs.

BACKGROUND & AIMS: Despite the increasing number of treatment options available for liver cancer, only a small proportion of patients achieve long-term clinical benefits. Here, we aim to develop new therapeutic approaches for liver cancer. METHODS: A compound screen was conducted to identify inhibitors that could synergistically induce senescence when combined with cyclin-dependent kinase (CDK) 4/6 inhibitor. The combination effects of CDK4/6 inhibitor and exportin 1 (XPO1) inhibitor on cellular senescence were investigated in a panel of human liver cancer cell lines and multiple liver cancer models. A senolytic drug screen was performed to identify drugs that selectively killed senescent liver cancer cells. RESULTS: The combination of CDK4/6 inhibitor and XPO1 inhibitor synergistically induces senescence of liver cancer cells in vitro and in vivo. The XPO1 inhibitor acts by causing accumulation of RB1 in the nucleus, leading to decreased E2F signaling and promoting senescence induction by the CDK4/6 inhibitor. Through a senolytic drug screen, cereblon (CRBN)-based proteolysis targeting chimera (PROTAC) ARV-825 was identified as an agent that can selectively kill senescent liver cancer cells. Up-regulation of CRBN was a vulnerability of senescent liver cancer cells, making them sensitive to CRBN-based PROTAC drugs. Mechanistically, we find that ubiquitin specific peptidase 2 (USP2) directly interacts with CRBN, leading to the deubiquitination and stabilization of CRBN in senescent liver cancer cells. CONCLUSIONS: Our study demonstrates a striking synergy in senescence induction of liver cancer cells through the combination of CDK4/6 inhibitor and XPO1 inhibitor. These findings also shed light on the molecular processes underlying the vulnerability of senescent liver cancer cells to CRBN-based PROTAC therapy.

An inferior vena cava-priority approach in laparoscopic isolated hepatic caudate lobectomy.

PURPOSE: Laparoscopic isolated caudate lobectomy is still a challenging operation for surgeons. The access route of the operation plays a vital role during laparoscopic caudate lobectomy. There are few references regarding this technique. Here, we introduce a preferred inferior vena cava (IVC) approach in laparoscopic caudate lobectomy. METHODS: Twenty-one consecutive patients with caudate hepatic tumours between June 2016 and December 2021 were included in this study. All of them received laparoscopic caudate lobectomy involving an IVC priority approach. The IVC priority approach refers to prioritizing the dissection of the IVC from the liver parenchyma before proceeding with the conventional left or right approach. It emphasizes the importance of the IVC dissection during process. Clinical data, intraoperative parameters and postoperative results were evaluated. Sixteen patients were performed pure IVC priority approach, while 5 patients underwent a combined approach. We subsequently compared the intraoperative and postoperative between the two groups. RESULTS: All 21 patients were treated with laparoscopic technology. The operative time was 190.95 ± 92.65 min. The average estimated blood loss was 251.43 ± 247.45 ml, and four patients needed blood transfusions during the perioperative period. The average duration of hospital stay was 8.43 ± 2.64 (range from 6.0 to 16.0) days. Patients who underwent the pure inferior vena cava (IVC) approach required a shorter hepatic pedicle clamping time (26 vs. 55 min, respectively; P < 0.001) and operation time (150 vs. 380 min, respectively; P = 0.002) than those who underwent the combined approach. Hospitalization (7.0 vs. 9.0 days, respectively; P = 0.006) was shorter in the pure IVC group than in the combined group. CONCLUSIONS: Laparoscopic caudate lobectomy with an IVC priority approach is safe and feasible for patients with caudate hepatic tumours.

Serum thrombospondin-1 serves as a novel biomarker and agonist of gemcitabine-based chemotherapy in intrahepatic cholangiocarcinoma.

BACKGROUND: At present, the first-line treatment for advanced intrahepatic cholangiocarcinoma (ICC) is gemcitabine combined with cisplatin, but a considerable portion of ICC patients exhibit resistance to gemcitabine. Therefore, finding sensitisers for gemcitabine chemotherapy in ICC patients and predicting molecular markers for chemotherapy efficacy have become urgent needs. METHODS: In this study, PDX models were established to conduct gemcitabine susceptibility tests. The selected PDX tissues of the chemotherapy-sensitive group and drug-resistant group were subjected to transcriptome sequencing and protein chip technology to identify the key genes. Sixty-one ICC patients treated with gemcitabine chemotherapy were recruited for clinical follow-up validation. RESULTS: We found that thrombospondin-1 (TSP1) can predict gemcitabine chemosensitivity in ICC patients. The expression level of TSP1 could reflect the sensitivity of ICC patients to gemcitabine chemotherapy. Functional experiments further confirmed that TSP1 can increase the efficacy of gemcitabine chemotherapy for ICC. A mechanism study showed that TSP1 may affect the intake of oleic acid by binding to the CD36 receptor. CONCLUSIONS: In summary, we found a key molecule-TSP1-that can predict and improve the sensitivity of ICC patients to gemcitabine chemotherapy, which is of great significance for the treatment of advanced cholangiocarcinoma.

Development of preoperative prognostic models including radiological features for survival of singular nodular HCC patients.

BACKGROUND: Early singular nodular hepatocellular carcinoma (HCC) is an ideal surgical indication in clinical practice. However, almost half of the patients have tumor recurrence, and there is no reliable prognostic prediction tool. Besides, it is unclear whether preoperative neoadjuvant therapy is necessary for patients with early singular nodular HCC and which patient needs it. It is critical to identify the patients with high risk of recurrence and to treat these patients preoperatively with neoadjuvant therapy and thus, to improve the outcomes of these patients. The present study aimed to develop two prognostic models to preoperatively predict the recurrence-free survival (RFS) and overall survival (OS) in patients with singular nodular HCC by integrating the clinical data and radiological features. METHODS: We retrospective recruited 211 patients with singular nodular HCC from December 2009 to January 2019 at Eastern Hepatobiliary Surgery Hospital (EHBH). They all met the surgical indications and underwent radical resection. We randomly divided the patients into the training cohort (n =132) and the validation cohort (n = 79). We established and validated multivariate Cox proportional hazard models by the preoperative clinicopathologic factors and radiological features for association with RFS and OS. By analyzing the receiver operating characteristic (ROC) curve, the discrimination accuracy of the models was compared with that of the traditional predictive models. RESULTS: Our RFS model was based on HBV-DNA score, cirrhosis, tumor diameter and tumor capsule in imaging. RFS nomogram had fine calibration and discrimination capabilities, with a C-index of 0.74 (95% CI: 0.68-0.80). The OS nomogram, based on cirrhosis, tumor diameter and tumor capsule in imaging, had fine calibration and discrimination capabilities, with a C-index of 0.81 (95% CI: 0.74-0.87). The area under the receiver operating characteristic curve (AUC) of our model was larger than that of traditional liver cancer staging system, Korea model and Nomograms in Hepatectomy Patients with Hepatitis B Virus-Related Hepatocellular Carcinoma, indicating better discrimination capability. According to the models, we fitted the linear prediction equations. These results were validated in the validation cohort. CONCLUSIONS: Compared with previous radiography model, the new-developed predictive model was concise and applicable to predict the postoperative survival of patients with singular nodular HCC. Our models may preoperatively identify patients with high risk of recurrence. These patients may benefit from neoadjuvant therapy which may improve the patients' outcomes.

HBV/Pregenomic RNA Increases the Stemness and Promotes the Development of HBV-Related HCC Through Reciprocal Regulation With Insulin-Like Growth Factor 2 mRNA-Binding Protein 3.

BACKGROUND AND AIMS: HBV-pgRNA (pregenomic RNA) has been proposed for predicting the response of nucleos(t)ide analogue (NA) treatment, guiding discontinuation of NA therapy and monitoring the emergence of viral mutations. However, the contributions of HBV-pgRNA to HCC remain open for study. APPROACH AND RESULTS: Double-center cohorts of serum samples with undetectable serum HBV-DNA (below the lower limit of detection) were obtained from long-term NA-treated (≥48 weeks) HBV-related HCC patients. The correlation between serum pgRNA concentration and the prognosis of HCC were analyzed. The role pgRNA played in HCC development was assessed both in vitro and in vivo. Our findings revealed that for patients who underwent long-term NA therapy with undetectable serum HBV-DNA, patients with high serum pgRNA expression had a poorer overall survival rate and higher cumulative recurrence rate after hepatectomy. Experiments demonstrated that pgRNA promotes proliferation, stemness, and tumorigenicity of HCC cells. Mechanistically, we found that pgRNA could up-regulate the expression of insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3), a well-proven oncoprotein, at the posttranscriptional level. Furthermore, interferon (IFN)-α-2a could degrade the stability of pgRNA through increasing its N6-methyladenosine (m6A) RNA modification. Collectively, our findings uncover that serum pgRNA could serve as a potential biomarker for predicting the prognosis and recurrence of HCC in patients who received long-term NA therapy with undetectable serum HBV-DNA; and the pgRNA-IGF2BP3 axis plays an important role in the development of HBV-related HCC. Moreover, IFN-α-2a could reduce the stability of pgRNA by increasing its m6A RNA modification level, thereby suppressing the development of HBV-related HCC. CONCLUSIONS: In conclusion, our studies reveal a significance and mechanism of HBV-pgRNA in increasing stemness features and offer a potential prognostic marker and a therapeutic target for HBV-related HCC.

Laparoscopic right hemihepatectomy following a novel optimized portal vein embolization: a video case report.

BACKGROUND: This article is the first report of laparoscopic major hepatectomy of Hepatocellular carcinoma (HCC) following optimized portal vein embolization (oPVE). CASE PRESENTATION: The patient was diagnosed with a single 3 × 3.5 cm HCC located in segment 5 and 8 detected by enhanced computed tomography and magnetic resonance imaging. The lesion was adjacent to the right anterior and posterior portal veins, making it difficult to confirm the adequate liver functional remnant volume, surgical margin and R0 resection. In addition, the liver cirrhosis induced by a long history of chronic hepatitis B virus increased the potential risk of postoperative liver failure and refractory ascites. Therefore, we conducted a laparoscopic surgery following oPVE, by which the safe tumor margin was ensured and the outcome of the surgery was improved. The patient was discharged on the seventh day after the surgery. The AFP gradually decreased to a normal level during the 90-day follow-up. CONCLUSION: This case report demonstrates that, in experienced hands for selected patients, laparoscopic hepatectomy after portal vein embolization is feasible and may be an alternative to open liver resection.

NQO1 promotes an aggressive phenotype in hepatocellular carcinoma via amplifying ERK-NRF2 signaling.

Patients with hepatocellular carcinoma (HCC) are usually diagnosed at the later stages and have poor survival outcomes. New molecules are urgently needed for the prognostic predication and individual treatment. Our study showed that high levels of NQO1 expression frequently exist in HCC with an obvious cancer-specific pattern. Patients with NQO1-high tumors are significantly associated with poor survival outcomes and serve as independent predictors. Functional experiments showed that NQO1 promotes the growth and aggressiveness of HCC in both in vitro and in vivo models, and the underlying mechanism involved NQO1-derived amplification of ERK/p38-NRF2 signaling. Combined block of ERK and NRF2 signaling generated stronger growth inhibition compared with any single block, especially for HCC with high-NQO1. Therefore, NQO1 is a potential biomarker for HCC early diagnosis and prognosis prediction, and also attractive for cancer-specific targets for HCC treatment.

Phosphoglucomutase 1 inhibits hepatocellular carcinoma progression by regulating glucose trafficking.

Glycogen metabolism commonly altered in cancer is just beginning to be understood. Phosphoglucomutase 1 (PGM1), the first enzyme in glycogenesis that catalyzes the reversible conversion between glucose 1-phosphate (G-1-P) and glucose 6-phosphate (G-6-P), participates in both the breakdown and synthesis of glycogen. Here, we show that PGM1 is down-regulated in hepatocellular carcinoma (HCC), which is associated with the malignancy and poor prognosis of HCC. Decreased PGM1 expression obstructed glycogenesis pathway, which leads to the increased flow of glucose into glycolysis, thereby promoting tumor cell proliferation and HCC development. The loss of forkhead box protein J2 (FOXJ2), at least partly due to low genomic copy number in HCC, releases cellular nucleic acid-binding protein (CNBP), a nucleic acid chaperon, to bind to and promote G-quadruplex formation in PGM1 promoter and therefore decreases PGM1 expression. In addition, integrated analyses of PGM1 and FOXJ2 expression provide a better prediction for the malignance and prognosis of HCC. This study establishes a tumor-suppressive role of PGM1 by regulating glucose trafficking and uncovers a novel regulatory mechanism of PGM1 expression.

Circulating Tumor Cells Expressing Krüppel-Like Factor 8 and Vimentin as Predictors of Poor Prognosis in Pancreatic Cancer Patients.

BACKGROUND: Circulating tumor cells (CTCs) with an epithelial-mesenchymal transition phenotype in peripheral blood may be a useful marker of carcinomas with poor prognosis. The aim of this study was to determine the prognostic significance of CTCs expressing Krüppel-like factor 8 (KLF8) and vimentin in pancreatic cancer (PC). METHODS: CTCs were isolated by immunomagnetic separation from the peripheral blood of 40 PC patients before undergoing surgical resection. Immunocytochemistry was performed to identify KLF8+ and vimentin+ CTCs. The associations between CTCs and time to recurrence (TTR), clinicopathologic factors, and survival were assessed. Univariate and multivariate analyzes were performed to identify risk factors. RESULTS: Patients with CTCs (n = 30) had a higher relapse rate compared to those without (n = 10) (70.0% vs 20.0%; P < 0.01). The proportion of KLF8+/vimentin+ CTCs to total CTCs was inversely related to TTR (r = -0.646; P < 0.01); TTR was reduced in patients with > 50% of CTCs identified as KLF8+/vimentin+ (P < 0.01). Independent risk factors for recurrence were perineural invasion and > 50% KLF8+/vimentin+ CTCs (both P < 0.05). CONCLUSION: Poor prognosis can be predicted in PC patients when > 50% of CTCs are positive for KLF8 and vimentin.

Repression of the long noncoding RNA-LET by histone deacetylase 3 contributes to hypoxia-mediated metastasis.

Recently, long noncoding RNAs (lncRNAs) were found to be dysregulated in a variety of tumors. However, it remains unknown how and through what molecular mechanisms the expression of lncRNAs is controlled. In this study, we found that the lncRNA Low Expression in Tumor (lncRNA-LET) was generally downregulated in hepatocellular carcinomas, colorectal cancers, and squamous-cell lung carcinomas. We demonstrated that hypoxia-induced histone deacetylase 3 repressed lncRNA-LET by reducing the histone acetylation-mediated modulation of the lncRNA-LET promoter region. Interestingly, the downregulation of lncRNA-LET was found to be a key step in the stabilization of nuclear factor 90 protein, which leads to hypoxia-induced cancer cell invasion. Moreover, the relationship among hypoxia, histone acetylation disorder, low lncRNA-LET expression level, and metastasis was found in clinical hepatocellular carcinoma samples. These results advance our understanding of the role of lncRNA-LET as a regulator of hypoxia signaling and offer new avenues for therapeutic intervention against cancer progression.

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

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

A novel HBx genotype serves as a preoperative predictor and fails to activate the JAK1/STATs pathway in hepatocellular carcinoma.

BACKGROUND & AIMS: Genetic variability in the hepatitis B virus X gene (HBx) is frequently observed and is associated with hepatocellular carcinoma (HCC) progression. However, a genotype classification based on the full-length HBx sequence and the impact of genotypes on hepatitis B virus (HBV)-related HCC prognosis remain unclear. We therefore aimed to perform this genotype classification and assess its clinical impact. METHODS: We classified the genotypes of the full-length HBx gene through sequencing and a cluster analysis of HBx DNA from a cohort of patients with HBV-related HCC, which served as the primary cohort (n = 284). Two independent HBV-related HCC cohorts, a validation cohort (n = 171) and a serum cohort (n = 168), were used to verify the results. Protein microarray assay analysis was performed to explore the underlying mechanism. RESULTS: In the primary cohort, the HBx DNA was classified into 3 genotypes: HBx-EHBH1, HBx-EHBH2, and HBx-EHBH3. HBx-EHBH2 (HBx-E2) indicated better recurrence-free survival and overall survival for patients with HCC. HBx-E2 was significantly correlated with the absence of liver cirrhosis, a small tumor size, a solitary tumor, complete encapsulation and Barcelona Clinic Liver Cancer (BCLC) stage A-0 tumors. Additionally, HBx-E2 served as a significant prognostic factor for patients with BCLC stage B HCC after hepatectomy. Mechanistically, HBx-E2 is unable to promote proliferation in HCC cells and normal hepatocytes. It also fails to activate the Janus kinase 1 (JAK1)/signal transducer and activator of transcription 3 (STAT3)/STAT5 pathway. CONCLUSION: Our study identifies a novel HBx genotype that is unable to promote the proliferation of HCC cells and suggests a potential marker to preoperatively predict the prognosis of patients with BCLC stage B, HBV-associated, HCC. LAY SUMMARY: We classified a novel genotype of the full-length hepatitis B virus X gene (HBx), HBx-E2. This genotype was identified in tumor and nontumor tissues from patients with hepatitis B virus-related hepatocellular carcinoma. HBx-E2 could preoperatively predict the prognosis of patients with intermediate stage hepatocellular carcinoma, after resection.

Tropomodulin 3 modulates EGFR-PI3K-AKT signaling to drive hepatocellular carcinoma metastasis.

The mechanism of hepatocellular carcinoma (HCC) metastasis remains poorly understood. Tropomodulin 3 (TMOD3) is a member of the pointed end capping protein family that contributes to invasion and metastasis in several types of malignancies. It has been found to be crucial for the membranous skeleton and embryonic development, although, its role in HCC progression remains largely unclear. We observed increased levels of Tmod3 in HCCs, especially in extrahepatic metastasis. High Tmod3 expression correlated with aggressive carcinoma and poor patient with HCC survival. Loss-of-function studies conducted by us determined Tmod3 as an oncogene that promoted HCC growth and metastasis. Mechanistically, Tmod3 increases transcription of matrix metalloproteinase-2, -7, and -9 which required PI3K-AKT. Interaction between Tmod3 and epidermal growth factor receptor (EGFR) that supports the activation of EGFR phosphorylation, is essential for signaling activation of PI3K-AKT viral oncogene homolog. These findings reveal that Tmod3 enhances aggressive behavior of HCC both in vitro and in vivo by interacting with EFGR and by activating the PI3K-AKT signaling pathway.

Long Stress Induced Non-Coding Transcripts 5 (LSINCT5) Promotes Hepatocellular Carcinoma Progression Through Interaction with High-Mobility Group AT-hook 2 and MiR-4516.

BACKGROUND Long non-coding RNAs (lncRNAs) have been implicated in various human cancer types. However, the underlying mechanisms involved in hepatocellular carcinoma (HCC) progression remain poorly understood. MATERIAL AND METHODS In this study, lncRNA array was used to identify HCC related lncRNAs. RNA immunoprecipitation (RIP) followed mass spectrometry was used to explore lncRNA binding proteins. Western blot, quantitative PCR, tumor sphere formation, migration and viability assay were performed to evaluate the oncogenic role of lncRNAs. RESULTS We identified a novel lncRNA named long stress induced non-coding transcripts 5 (LSINCT5) which facilitates HCC progression. LSINCT5 was significantly upregulated in both HCC specimens and cell lines and correlates with poor survival. In vitro experiments showed that LSINCT5 promoted migration and viability of HepG2 and Huh7 cells. The in vivo xenograft mouse model also confirmed an oncogenic role for LSINCT5. RIP in combination with mass spectrometry identified HMGA2 as the LSINCT5 binding partner. LSINCT5 could bind to HMGA2 and decrease proteasome-mediated HMGA2 degradation leading to EMT activation. LSINCT5 also served as a competing endogenous RNA (ceRNA) for miR-4516, resulting in increased STAT3/BclxL expression and attenuated apoptosis. CONCLUSIONS Our data have collectively established a lncRNA LSINCT5 mediated process during HCC carcinogenesis and might have provided novel insight into therapeutic targeting.

Systemic genome screening identifies the outcome associated focal loss of long noncoding RNA PRAL in hepatocellular carcinoma.

UNLABELLED: Systemic analyses using large-scale genomic profiles have successfully identified cancer-driving somatic copy number variations (SCNVs) loci. However, functions of vast focal SCNVs in "protein-coding gene desert" regions are largely unknown. The integrative analysis of long noncoding RNA (lncRNA) expression profiles with SCNVs in hepatocellular carcinoma (HCC) led us to identify the recurrent deletion of lncRNA-PRAL (p53 regulation-associated lncRNA) on chromosome 17p13.1, whose genomic alterations were significantly associated with reduced survival of HCC patients. We found that lncRNA-PRAL could inhibit HCC growth and induce apoptosis in vivo and in vitro through p53. Subsequent investigations indicated that the three stem-loop motifs at the 5' end of lncRNA-PRAL facilitated the combination of HSP90 and p53 and thus competitively inhibited MDM2-dependent p53 ubiquitination, resulting in enhanced p53 stability. Additionally, in vivo lncRNA-PRAL delivery efficiently reduced intrinsic tumors, indicating its potential therapeutic application. CONCLUSIONS: lncRNA-PRAL, one of the key cancer-driving SCNVs, is a crucial stimulus for HCC growth and may serve as a potential target for antitumor therapy.

Long noncoding RNA DANCR increases stemness features of hepatocellular carcinoma by derepression of CTNNB1.

UNLABELLED: Tumor cells with stemness (stem-cell) features contribute to initiation and progression of hepatocellular carcinoma (HCC), but involvement of long noncoding RNAs (lncRNAs) remains largely unclear. Genome-wide analyses were applied to identify tumor-associated lncRNA-DANCR. DANCR expression level and prognostic values of DANCR were assayed in two HCC cohorts (China and Korea, n = 135 and 223). Artificial modulation of DANCR (down- and overexpression) was done to explore the role of DANCR in tumorigenesis and colonization, and tumor-bearing mice were used to determine therapeutic effects. We found that lncRNA-DANCR is overexpressed in stem-like HCC cells, and this can serve as a prognostic biomarker for HCC patients. Experiments showed that DANCR markedly increased stemness features of HCC cells to promote tumorigenesis and intra-/extrahepatic tumor colonization. Conversely, DANCR knockdown attenuated the stem-cell properties and in vivo interference with DANCR action led to decreased tumor cell vitality, tumor shrinkage, and improved mouse survival. Additionally, we found that the role of DANCR relied largely on an association with, and regulation of, CTNNB1. Association of DANCR with CTNNB1 blocked the repressing effect of microRNA (miR)-214, miR-320a, and miR-199a on CTNNB1. This observation was confirmed in vivo, suggesting a novel mechanism of tumorigenesis involving lncRNAs, messenger RNAs, and microRNAs. CONCLUSIONS: These studies reveal a significance and mechanism of DANCR action in increasing stemness features and offer a potential prognostic marker and a therapeutic target for HCC.

Double-negative feedback loop between microRNA-422a and forkhead box (FOX)G1/Q1/E1 regulates hepatocellular carcinoma tumor growth and metastasis.

UNLABELLED: Growing evidence indicates that the aberrant expression of microRNAs (miRNAs) contributes to tumor development; however, the function of miRNAs in human hepatocellular carcinoma (HCC) remains largely undefined. In this study, we report that microRNA-422a (miR-422a) is significantly down-regulated in HCC tumor samples and cell lines compared with normal controls, and its expression level is negatively correlated with pathological grading, recurrence, and metastasis. The restoration of miR-422a expression in HCC tumor cells significantly inhibited cell proliferation and migration in vitro. At the same time, the overexpression of miR-422a in HCC tumor cells significantly inhibits tumor growth and liver metastasis in xenograft tumor models. A mechanistic study identified three genes, forkhead box G1 (FOXG1), FOXQ1, and FOXE1, as miR-422a targets in the regulation of HCC development. We also investigated the function of the three targets themselves in HCC tumorigenesis using RNAi manipulation and demonstrated that the knockdown of these targets led to significant inhibition of tumor cell proliferation and migration both in vitro and in vivo. More interestingly, a potential miR-422a promoter region was identified. Both the promoter activity and miR-422a expression were negatively regulated by the three targets, indicating that a double-negative feedback loop exists between miR-422a and its targets. Moreover, we explored the therapeutic potential of miR-422a in HCC treatment and found that the therapeutic delivery of miR-422a significantly inhibited tumor development in a xenograft tumor model and a diethylnitrosamine-induced primary HCC model. CONCLUSION: Our findings show the critical roles of miR-422a and its targets--FOXG1, FOXQ1, and FOXE1--in the regulation of HCC development and provide new potential candidates for HCC therapy.

Aldolase B inhibits metastasis through Ten-Eleven Translocation 1 and serves as a prognostic biomarker in hepatocellular carcinoma.

BACKGROUND: Downregulation of Aldolase B (ALDOB) has been reported in hepatocellular carcinoma. However, its clinical significance and its role in pathogenesis of HCC remain largely unknown. METHODS: We analyzed the expression of ALDOB and its clinical features in a large cohort of 313 HCC patients using tissue microarray and immunohistochemistry. Moreover, the function of stably overexpressed ALDOB in HCC cells was explored in vitro and in vivo. Gene expression microarray analysis was performed on ALDOB-overexpressing SMMC7721 cells to elucidate its mechanism of action. RESULTS: ALDOB downregulation in HCC was significantly correlated with aggressive characteristics including absence of encapsulation, increased tumor size (>5 cm) and early recurrence. ALDOB downregulation was indicative of a shorter recurrence-free survival (RFS) and overall survival (OS) for all HCC patients and early-stage HCC patients (BCLC 0-A and TNM I stage patients). Multiple analyses revealed that ALDOB downregulation was an independent risk factor of RFS and OS. Stable expression of ALDOB in HCC cell lines reduced cell migration in vitro and inhibited lung metastasis, intrahepatic metastasis, and reduced circulating tumor cells in vivo. Mechanistically, we found that cells stably expressing ALDOB show elevated Ten-Eleven Translocation 1 (TET1) expression. Moreover, ALDOB expressing cells have higher levels of methylglyoxal than do control cells, which can upregulate TET1 expression. CONCLUSION: The downregulation of ALDOB could indicate a poor prognosis for HCC patients, and therefore, ALDOB might be considered a prognostic biomarker for HCC, especially at the early stage. In addition, ALDOB inhibits the invasive features of cell lines partly through TET1 expression.

Antiviral therapy improves postoperative survival in patients with hepatocellular carcinoma: a randomized controlled trial.

OBJECTIVE: A randomized controlled trial was conducted to find out whether antiviral therapy in patients with hepatitis B-related hepatocellular carcinoma (HCC) improves long-term survival after hepatic resection. BACKGROUND: Despite advances in surgery and in multidisciplinary treatment, there is still no effective adjuvant treatment to prevent HCC recurrence after R0 resection for HCC. Whether antiviral therapy is useful in reducing postoperative HCC recurrence is unclear. METHODS: Between May 2007 and April 2008, patients who received R0 hepatic resection for HBV-related HCC were randomly assigned to receive no treatment (the control group, n = 100) or antiviral therapy (adefovir 10 mg/d, the antiviral group, n = 100). RESULTS: The baseline clinical, laboratory, and tumor characteristics of the 2 groups were comparable. The 1-, 3-, and 5-year recurrence-free survival rates for the antiviral group and the control group were 85.0%, 50.3%, 46.1% and 84.0%, 37.9%, 27.1%, respectively. The corresponding overall survival rates for the 2 groups were 96.0%, 77.6%, 63.1% and 94.0%, 67.4%, 41.5%, respectively. The recurrence-free survival and overall survival for the antiviral group were significantly better than the control group (P = 0.026, P = 0.001). After adjusting for the confounding prognostic factors in a Cox model, the relative risks of recurrence and death for antiviral treatment were 0.651 [95% confidence interval (CI): 0.451-0.938; P = 0.021] and 0.420 (95% CI: 0.271-0.651; P < 0.001). Antiviral therapy was an independent protective factor of late tumor recurrence (HR = 0.348, 95% CI: 0.177-0.687; P = 0.002) but not of early tumor recurrence [hazard ratio (HR) = 0.949, 95% CI: 0.617-1.459; P = 0.810]. CONCLUSIONS: In patients with hepatitis B-related HCC, adefovir antiviral therapy reduced late HCC recurrence and significantly improved overall survival after R0 hepatic resection.