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.

Prognostic implications of alpha-fetoprotein and C-reactive protein elevation in hepatocellular carcinoma following resection (PACE): a large cohort study of 2770 patients.

BACKGROUND: Routine clinical staging for hepatocellular carcinoma (HCC) incorporates liver function, general health, and tumor morphology. Further refinement of prognostic assessments and treatment decisions may benefit from the inclusion of tumor biological marker alpha-fetoprotein (AFP) and systemic inflammation indicator C-reactive protein (CRP). METHODS: Data from a multicenter cohort of 2770 HCC patients undergoing hepatectomy were analyzed. We developed the PACE risk score (Prognostic implications of AFP and CRP Elevation) after initially assessing preoperative AFP and CRP's prognostic value. Subgroup analyzes were performed in BCLC cohorts A and B using multivariable Cox analysis to evaluate the prognostic stratification ability of the PACE risk score and its complementary utility for BCLC staging. RESULTS: Preoperative AFP ≥ 400ng/mL and CRP ≥ 10 mg/L emerged as independent predictors of poorer prognosis in HCC patients who underwent hepatectomy, leading to the creation of the PACE risk score. PACE risk score stratified patients into low, intermediate, and high-risk groups with cumulative 5-year overall (OS) and recurrence-free survival (RFS) rates of 59.6%/44.9%, 43.9%/38.4%, and 20.6%/18.0% respectively (all P < 0.001). Increased PACE risk scores correlated significantly with early recurrence and extrahepatic metastases frequency (all P < 0.001). The multivariable analysis identified intermediate and high-risk PACE scores as independently correlating with poor postoperative OS and RFS. Furthermore, the PACE risk score proficiently stratified the prognosis of BCLC stages A and B patients, with multivariable analyses demonstrating it as an independent prognostic determinant for both stages. CONCLUSION: The PACE risk score serves as an effective tool for postoperative risk stratification, potentially supplementing the BCLC staging system.

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.

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.

miR-541 potentiates the response of human hepatocellular carcinoma to sorafenib treatment by inhibiting autophagy.

OBJECTIVE: Autophagy participates in the progression of hepatocellular carcinoma (HCC) and the resistance of HCC cells to sorafenib. We investigated the feasibility of sensitising HCC cells to sorafenib by modulating miR-541-initiated microRNA-autophagy axis. DESIGN: Gain- and loss-of-function assays were performed to evaluate the effects of miR-541 on the malignant properties and autophagy of human HCC cells. Autophagy was quantified by western blotting of LC3, transmission electron microscopy analyses and confocal microscopy scanning of mRFP-GFP-LC3 reporter construct. Luciferase reporter assays were conducted to confirm the targets of miR-541. HCC xenograft tumours were established to analyse the role of miR-541 in sorafenib-induced lethality. RESULTS: The expression of miR-541 was downregulated in human HCC tissues and was associated with malignant clinicopathologic phenotypes, recurrence and survival of patients with HCC. miR-541 inhibited the growth, metastasis and autophagy of HCC cells both in vitro and in vivo. Prediction software and luciferase reporter assays identified autophagy-related gene 2A (ATG2A) and Ras-related protein Rab-1B (RAB1B) as the direct targets of miR-541. Consistent with the effects of the miR-541 mimic, inhibition of ATG2A or RAB1B suppressed the malignant phenotypes and autophagy of HCC cells. Furthermore, siATG2A and siRAB1B partially reversed the enhancement of the malignant properties and autophagy in HCC cells mediated by the miR-541 inhibitor. More interestingly, higher miR-541 expression predicted a better response to sorafenib treatment, and the combination of miR-541 and sorafenib further suppressed the growth of HCC cells in vivo compared with the single treatment. CONCLUSIONS: Dysregulation of miR-541-ATG2A/RAB1B axis plays a critical role in patients' responses to sorafenib treatment. Manipulation of this axis might benefit survival of patients with HCC, especially in the context of the highly pursued strategies to eliminate drug resistance.

Trans-acting non-synonymous variant of FOXA1 predisposes to hepatocellular carcinoma through modulating FOXA1-ERα transcriptional program and may have undergone natural selection.

Interplay of pioneer transcription factor forkhead box A1 (FOXA1) and estrogen receptor has been implicated in sexual dimorphism in hepatocellular carcinoma (HCC), but etiological relevance of its polymorphism was unknown. In the case control study (1152 patients versus1242 controls), we observed significant increase in HCC susceptibility in hepatitis B virus carriers associated with a non-synonymous Thr83Ala variant of FOXA1 (odds ratio [OR], 1.28; 95% confidence interval [CI], 1.11-1.48, for Ala83-containing genotype, after validation in an independent population with 933 patients versus 1030 controls), a tightly linked (CGC)5/6or7 repeat polymorphism at its promoter (OR 1.32; 95% CI 1.10-1.60, for (CGC)6or7-repeat-containing genotype), and their combined haplotype (OR 1.50; 95% CI 1.24-1.81, for (CGC)6or7-Ala83 haplotype). The susceptible FOXA1-Ala83 impairs its interaction with ERα, attenuates transactivation toward some of their dual target genes, such as type 1 iodothyronine deiodinase, UDP glucuronosyltransferase 2 family, polypeptide B17 and sodium/taurocholate cotransporting polypeptide, but correlates with strengthened cellular expression of α-fetoprotein (AFP) and elevated AFP serum concentration in HCC patients (n = 1096). The susceptible FOXA1 cis-variant with (CGC)6or7 repeat strengthens the binding to transcription factor early growth response 1 and enhances promoter activity and gene expression. Evolutionary population genetics analyses with public datasets reveal significant population differentiation and unique haplotype structure of the derived protective FOXA1-Thr83 and suggest that it may have undergone positive natural selection in Chinese population. These findings epidemiologically highlight the functional significance of FOXA1-ERα transcriptional program and regulatory network in liver cancer development.

Plasma circular RNA panel to diagnose hepatitis B virus-related hepatocellular carcinoma: A large-scale, multicenter study.

To explore whether plasma circular RNAs (circRNAs) can diagnose hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC), microarray and qPCR were used to identify plasma circRNAs that were increased in HBV-related HCC patients compared to controls (including healthy controls, chronic hepatitis B and HBV-related liver cirrhosis). A logistic regression model was constructed using a training set (n = 313) and then validated using another two independent sets (n = 306 and 526, respectively). Area under the receiver operating characteristic curve (AUC) was used to evaluate diagnostic accuracy. We identified a plasma circRNA panel (CircPanel) containing three circRNAs (hsa_circ_0000976, hsa_circ_0007750 and hsa_circ_0139897) that could detect HCC. CircPanel showed a higher accuracy than AFP (alpha-fetoprotein) to distinguish individuals with HCC from controls in all three sets (AUC, 0.863 [95% confidence interval, CI: 0.819-0.907] vs. 0.790 [0.738-0.842], p = 0.036 in training set; 0.843 [0.796-0.890] vs. 0.747 [0.691-0.804], p = 0.011 in validation set 1 and 0.864 [0.830-0.898] vs. 0.769 [0.728-0.810], p < 0.001 in validation set 2). CircPanel also performed well in detecting Small-HCC (solitary, ≤3 cm), AFP-negative HCC and AFP-negative Small-HCC.

Characterization of breast lesions using diffusion kurtosis model-based imaging: An initial experience.

OBJECTIVE: To investigate the characterization of breast lesions using diffusion kurtosis model-based imaging. METHODS: This prospective study included 120 consecutive patients underwent preoperative DCE-MRI examinations and multi-b-value diffusion-weighted imaging (DWI). Among them, 88 malignant lesions and 44 benign lesions were detected, 56 normal fibroglandular breast tissue were selected as normal control. Conventional apparent diffusion coefficient (ADC), DKI-based parameters mean kurtosis (MK) and mean diffusivity (MD) were analyzed by lesions types and histological subtypes using one-way ANOVA and receiver operating characteristic (ROC) curve. RESULTS: (1) The malignant group showed significantly lower ADC and MD (1.07±0.32×10-3 mm2/s and 1.30±0.40×10-3 mm2/s, respectively) and higher MK (0.87±0.18) than those in the benign group (1.29±0.26×10-3 mm2/s, 1.62±0.31×10-3 mm2/s and 0.67±0.18) and control group (1.67±0.33×10-3 mm2/s, 2.24±0.28×10-3 mm2/s and 0.52±0.08) with all P < 0.001. (2) Areas under ROC curve (AUC) for diagnosing malignant lesions were 0.936 for MD, 0.911 for MK and 0.897 for ADC, respectively. AUC for MD was significantly higher than that for ADC (P = 0.015). The optimal cut-off value, sensitivity, specificity, positive predictive value, negative predictive value and accuracy were as follow: ADC = 1.18×10-3mm2/s, 78.3%, 93.2%, 81.2%, 81.6%, 81.4%; MD = 1.48×10-3mm2/s, 82.2%, 98.3%, 84.4%, 87.8%, 86.2%; MK = 0.78, 91.5%, 85.3%, 89.0%, 85.8%, 87.2%. (3) Invasive ductal carcinoma (IDC), ductal carcinoma in situ (DCIS) and mucinous adenocarcinoma also showed significant differences among ADC, MD and MK (with P < 0.05). CONCLUSIONS: MR-DKI parameters enable to improve breast lesion characterization and have diagnostic potential applying to different pathological subtypes of breast cancers.

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.

Effect of Huaier granule on recurrence after curative resection of HCC: a multicentre, randomised clinical trial.

OBJECTIVE: There is little evidence that adjuvant therapy after radical surgical resection of hepatocellular carcinoma (HCC) improves recurrence-free survival (RFS) or overall survival (OS). We conducted a multicentre, randomised, controlled, phase IV trial evaluating the benefit of an aqueous extract of Trametes robinophila Murr (Huaier granule) to address this unmet need. DESIGN AND RESULTS: A total of 1044 patients were randomised in 2:1 ratio to receive either Huaier or no further treatment (controls) for a maximum of 96 weeks. The primary endpoint was RFS. Secondary endpoints included OS and tumour extrahepatic recurrence rate (ERR). The Huaier (n=686) and control groups (n=316) had a mean RFS of 75.5 weeks and 68.5 weeks, respectively (HR 0.67; 95% CI 0.55 to 0.81). The difference in the RFS rate between Huaier and control groups was 62.39% and 49.05% (95% CI 6.74 to 19.94; p=0.0001); this led to an OS rate in the Huaier and control groups of 95.19% and 91.46%, respectively (95% CI 0.26 to 7.21; p=0.0207). The tumour ERR between Huaier and control groups was 8.60% and 13.61% (95% CI -12.59 to -2.50; p=0.0018), respectively. CONCLUSIONS: This is the first nationwide multicentre study, involving 39 centres and 1044 patients, to prove the effectiveness of Huaier granule as adjuvant therapy for HCC after curative liver resection. It demonstrated a significant prolongation of RFS and reduced extrahepatic recurrence in Huaier group. TRIAL REGISTRATION: NCT01770431; Post-results.

Circular RNA cSMARCA5 inhibits growth and metastasis in hepatocellular carcinoma.

BACKGROUND & AIMS: In recent years, circular RNAs (circRNAs) have been shown to have critical regulatory roles in cancer biology. However, the contributions of circRNAs to hepatocellular carcinoma (HCC) remain largely unknown. METHODS: cSMARCA5 (a circRNA derived from exons 15 and 16 of the SMARCA5 gene, hsa_circ_0001445) was identified by RNA-sequencing and validated by quantitative reverse transcription PCR. The role of cSMARCA5 in HCC progression was assessed both in vitro and in vivo. circRNAs in vivo precipitation, luciferase reporter assay, biotin-coupled microRNA capture and fluorescence in situ hybridization were conducted to evaluate the interaction between cSMARCA5 and miR-17-3p/miR-181b-5p. RESULTS: The expression of cSMARCA5 was lower in HCC tissues, because of the regulation of DExH-Box Helicase 9, an abundant nuclear RNA helicase. The downregulation of cSMARCA5 in HCC was significantly correlated with aggressive characteristics and served as an independent risk factor for overall survival and recurrence-free survival in patients with HCC after hepatectomy. Our in vivo and in vitro data indicated that cSMARCA5 inhibits the proliferation and migration of HCC cells. Mechanistically, we found that cSMARCA5 could promote the expression of TIMP3, a well-known tumor suppressor, by sponging miR-17-3p and miR-181b-5p. CONCLUSION: These results reveal an important role of cSMARCA5 in the growth and metastasis of HCC and provide a fresh perspective on circRNAs in HCC progression. LAY SUMMARY: Herein, we studied the role of cSMARCA5, a circular RNA, in hepatocellular carcinoma. Our in vitro and in vivo data showed that cSMARCA5 inhibits the growth and migration of hepatocellular carcinoma cells, making it a potential therapeutic target.

Antiviral Therapy Reduces Hepatocellular Carcinoma Recurrence in Patients With Low HBV-DNA Levels: A Randomized Controlled Trial.

BACKGROUND: Despite antiviral treatment has been shown to reduce hepatocellular carcinoma (HCC) recurrence after curative treatment for hepatitis B virus (HBV)-related HCC in patients with high preoperative HBV-DNA levels, it is still unclear whether antiviral therapy is useful in reducing recurrence in patients with low preoperative HBV-DNA levels. METHODS: In this randomized controlled trial, 200 patients who underwent curative resection for HCC with low baseline HBV-DNA levels were randomly assigned to receive preemptive antiviral therapy or not. The primary endpoints were recurrence-free survival. This study was censored on March 31, 2015 when all surviving patients had a minimum follow-up of 60 months. The analysis was done on an intention-to-treat basis. 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 85.9%, 55.2%, and 52.0% and 80.6%, 40.9%, and 32.3%, respectively. The corresponding overall survival rates for the 2 groups were 94.0%, 75.7%, and 64.1% and 90.0%, 62.4%, and 43.7%, respectively. The recurrence-free survival and overall survival for the antiviral group were significantly better than the control group (P = 0.016, P = 0.004, respectively). After adjusting for confounding prognostic factors in a Cox model, the relative risks of recurrence and death for antiviral treatment were 0.601 [95% confidence interval (CI), 0.409-0.884; P = 0.010] and 0.509 (95% CI, 0.333-0.778; P = 0.002), respectively. Antiviral therapy was an independent protective factor of late tumor recurrence (hazard ratio [HR] = 0.316, 95% CI 0.157-0.637; P = 0.001) but not of early tumor recurrence (HR = 0.782, 95% CI, 0.493-1.240; P = 0.296). CONCLUSIONS: In patients with low preoperative HBV-DNA levels, antiviral therapy significantly reduced HCC recurrence after R0 hepatic resection.

Choline Kinase α Mediates Interactions Between the Epidermal Growth Factor Receptor and Mechanistic Target of Rapamycin Complex 2 in Hepatocellular Carcinoma Cells to Promote Drug Resistance and Xenograft Tumor Progression.

BACKGROUND & AIMS: Choline kinase α (CHKA) catalyzes conversion of choline to phosphocholine and can contribute to carcinogenesis. Little is known about the role of CHKA in the pathogenesis of hepatocellular carcinoma (HCC). METHODS: We performed whole-exome and transcriptome sequence analyses of 9 paired HCC and non-tumor-adjacent tissues. We performed tissue chip analyses of 120 primary HCC and non-tumor-adjacent tissues from patients who received surgery in Shanghai, China from January 2006 through December 2009; 48 sets of specimens (HCC and non-tumor-adjacent tissues) were also analyzed. CHKA gene copy number was quantified and findings were validated by quantitative reverse transcription polymerase chain reaction analysis. CHKA messenger RNA and protein levels were determined by polymerase chain reaction, immunohistochemical, and immunoblot analyses. CHKA was examined in 2 hepatocyte cell lines and 7 HCC-derived cell lines, and knocked down with small interfering RNAs in 3 HCC cell lines. Cells were analyzed in proliferation, wound healing, migration, and invasion assays. Cells were injected into tail veins of mice and tumor growth and metastasis were quantified. Immunoprecipitation and immunofluorescence assays were conducted to determine interactions between CHKA and the epidermal growth factor receptor (EGFR) and the mechanistic target of rapamycin complex 2. RESULTS: Levels of CHKA messenger RNA were frequently increased in HCC tissues compared with nontumor tissues; increased expression was associated with amplification at the CHKA loci. Tumors that expressed high levels of CHKA had more aggressive phenotypes, and patients with these tumors had shorter survival times after surgery compared to patients whose tumors expressed low levels of CHKA. HCC cell lines that stably overexpressed CHKA had higher levels of migration and invasion than control HCC cells, and formed larger xenograft tumors with more metastases in mice compared to HCC cells that did not overexpress CHKA. CHKA was required for physical interaction between EGFR and mechanistic target of rapamycin complex 2. This complex was required for HCC cells to form metastatic xenograft tumors in mice and to become resistant to EGFR inhibitors. CONCLUSIONS: We found levels of CHKA to be increased in human HCCs compared to nontumor tissues, and increased expression to be associated with tumor aggressiveness and reduced survival times of patients. Overexpression of CHKA in HCC cell lines increased their invasiveness, resistance to EGFR inhibitors, and ability to form metastatic tumors in mice by promoting interaction of EGFR with mechanistic target of rapamycin complex 2.

METTL14 suppresses the metastatic potential of hepatocellular carcinoma by modulating N6 -methyladenosine-dependent primary MicroRNA processing.

N6 -Methyladenosine (m6 A) modification has been implicated in many biological processes. However, its role in cancer has not been well studied. Here, we demonstrate that m6 A modifications are decreased in hepatocellular carcinoma, especially in metastatic hepatocellular carcinoma, and that methyltransferase-like 14 (METTL14) is the main factor involved in aberrant m6 A modification. Moreover, METTL14 down-regulation acts as an adverse prognosis factor for recurrence-free survival of hepatocellular carcinoma and is significantly associated with tumor metastasis in vitro and in vivo. We confirm that METTL14 interacts with the microprocessor protein DGCR8 and positively modulates the primary microRNA 126 process in an m6 A-dependent manner. Further experiments show that microRNA 126 inhibits the repressing effect of METTL14 in tumor metastasis. CONCLUSION: These studies reveal an important role of METTL14 in tumor metastasis and provide a fresh view on m6 A modification in tumor progression. (Hepatology 2017;65:529-543).

The peripheral blood transcriptome identifies dysregulation of inflammatory response genes in polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a common endocrine disorder in women, resulting in ovulation failure and other metabolic problems. However, the underlying mechanisms of it remain largely uncertain due to the complexity of clinical manifestations. This systemic disorder is involved in endocrine, metabolism, immune system and many organs, and few studies have explored peripheral blood transcriptome in patients with PCOS. We performed gene expression profiling of peripheral blood from 8 PCOS patients and eight healthy women with microarray. The significance analysis of microarray (SAM) software was employed to screen the differentially expressed genes (DEGs) and gene ontology (GO) was used for functional enrichment analysis. In total, 181 DEGs with fold-changes >2.0 and q-values <0.05 were identified between the two groups. Among them, 149 were up-regulated and 32 down-regulated in PCOS. Unsupervised clustering of expressed genes could readily differentiate PCOS from control. More importantly, inflammatory response pathway including 14 dysregulated genes was highly enriched in PCOS. Furthermore, 10 DEGs were validated using quantitative reverse-transcription PCR (qRT-PCR) assays. Our study provides independent evidence for the involvement of systemic inflammatory response in PCOS and it may facilitate a greater understanding of this complex disease.

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.

Utility of colon leakage score in left-sided colorectal surgery.

BACKGROUND: The aim of the study was to evaluate the clinical utility of the colon leakage score (CLS) in predicting the risk of anastomotic leakage (AL) after left-sided colorectal surgery. MATERIALS AND METHODS: This investigation was designed as a retrospective study of 304 patients who underwent left-sided colorectal surgery. The patients were classified into two groups as those who developed AL and those who did not develop AL, and the CLSs of the two groups were compared. The predictive value and the cutoff value of the CLS were assessed by receiver operating characteristic and logistic regression analysis. RESULTS: A significant difference was noted in the mean CLSs of the groups with and without AL (P < 0.001). The values of the area under the receiver operating characteristic curve (0.965; confidence interval, 0.913-1.00) and the odds ratio (2.9; confidence interval, 1.59-4.83; P < 0.001) indicated that CLS was a good predictor of AL. A CLS of 11 was found to be the best cutoff value, with a sensitivity and specificity of 84.6% and 87.2%, respectively. CONCLUSIONS: Our findings indicate that CLS can effectively predict the risk of AL after left-sided colorectal surgery and that a CLS of 11 can be used as a cutoff value for the risk level.