APOBEC3B promotes hepatocarcinogenesis and metastasis through novel deaminase-independent activity.

Cytidine deaminase APOBEC3B (A3B) is known to play important roles in creating de novo genomic C-to-T mutations in cancers and contribute to induction of genomic instability. Our study evaluated the roles of A3B in the progression and metastasis of human hepatocellular carcinoma (HCC). Using whole-transcriptome and whole-exome sequencing, and quantitative PCR, we found that A3B was overexpressed in human HCCs and A3B expression was significantly correlated with the proportion of genomic C-to-A and G-to-T mutations. Upon clinicopathological correlation, higher A3B expression was associated with more aggressive tumor behavior. Wild-type A3B (wt-A3B) overexpression in HCC cells promoted cell proliferation, and cell migratory and invasive abilities in vitro, and tumorigenicity and metastasis in vivo. On the other hand, knockdown of A3B suppressed cell proliferation, migratory, and invasive abilities of HCC cells with high endogenous A3B level. However, to our surprise, overexpression of A3B deaminase-dead double mutant (E68A/E255Q) led to similar results as wt-A3B in HCC. Furthermore, overexpression of wt-A3B and mutant A3B both enhanced cell cycle progression in HCC cells. Altogether, our data demonstrated a novel deaminase-independent role of A3B in contributing to HCC tumorigenesis and metastasis.

Hepatic IFIT3 predicts interferon-α therapeutic response in patients of hepatocellular carcinoma.

Adjuvant interferon-α (IFN-α) therapy is used to control certain types of cancer in clinics. For hepatocellular carcinoma (HCC), IFN-α therapy is effective in only a subgroup of patients; therefore, identifying biomarkers to predict the response to IFN-α therapy is of high significance and clinical utility. As the induced IFN-stimulated gene expression following IFN-α treatment plays pivotal roles in IFN-α effects, we screened IFN-stimulated gene expression in HCC tissues and found that several IFN-stimulated genes were significantly decreased in HCC. Interestingly, expression of IFN-induced protein with tetratricopeptide repeats (IFIT) family members, including IFIT1, IFIT2, IFIT3, and IFIT5, was decreased in HCC tissues. We further analyzed the expression of IFIT family members in HCC and their roles in patients' responses to IFN-α therapy in two independent randomized controlled IFN-α therapy clinical trials of HCC patients. We found that higher expression of IFIT3, but not other IFITs, in HCC tissues predicts better response to IFN-α therapy, suggesting that IFIT3 may be a useful predictor of the response to IFN-α therapy in HCC patients. Mechanistically, IFIT3 enhanced the antitumor effects of IFN-α by promoting IFN-α effector responses both in vitro and in vivo. IFIT3 could bind signal transducer and activator of transcription 1 (STAT1) and STAT2 to enhance STAT1-STAT2 heterodimerization and nuclear translocation upon IFN-α treatment, thus promoting IFN-α effector signaling. CONCLUSION: Higher IFIT3 expression in HCC tissues predicts better response to IFN-α therapy in HCC patients; IFIT3 promotes IFN-α effector responses and therapeutic effects by strengthening IFN-α effector signaling in HCC. (Hepatology 2017;66:152-166).

Interaction between tumour-infiltrating B cells and T cells controls the progression of hepatocellular carcinoma.

OBJECTIVE: The nature of the tumour-infiltrating leucocytes (TILs) is known to impact clinical outcome in carcinomas, including hepatocellular carcinoma (HCC). However, the role of tumour-infiltrating B cells (TIBs) remains controversial. Here, we investigate the impact of TIBs and their interaction with T cells on HCC patient prognosis. DESIGN: Tissue samples were obtained from 112 patients with HCC from Singapore, Hong Kong and Zurich and analysed using immunohistochemistry and immunofluorescence. RNA expression of CD19, CD8A, IFNG was analysed using quantitative PCR. The phenotype of freshly isolated TILs was analysed using flow cytometry. A mouse model depleted of mature B cells was used for functional study. RESULTS: Tumour-infiltrating T cells and B cells were observed in close contact with each other and their densities are correlated with superior survival in patients with HCC. Furthermore, the density of TIBs was correlated with an enhanced expression of granzyme B and IFN-γ, as well as with reduced tumour viability defined by low expression of Ki-67, and an enhanced expression of activated caspase-3 on tumour cells. CD27 and CD40 costimulatory molecules and TILs expressing activation marker CD38 in the tumour were also correlated with patient survival. Mice depleted of mature B cells and transplanted with murine hepatoma cells showed reduced tumour control and decreased local T cell activation, further indicating the important role of B cells. CONCLUSIONS: The close proximity of tumour-infiltrating T cells and B cells indicates a functional interaction between them that is linked to an enhanced local immune activation and contributes to better prognosis for patients with HCC.

TSC1/2 mutations define a molecular subset of HCC with aggressive behaviour and treatment implication.

OBJECTIVE: We investigated the mutational landscape of mammalian target of rapamycin (mTOR) signalling cascade in hepatocellular carcinomas (HCCs) with chronic HBV background, aiming to evaluate and delineate mutation-dependent mechanism of mTOR hyperactivation in hepatocarcinogenesis. DESIGN: We performed next-generation sequencing on human HCC samples and cell line panel. Systematic mutational screening of mTOR pathway-related genes was undertaken and mutant genes were evaluated based on their recurrence. Protein expressions of tuberous sclerosis complex (TSC)1, TSC2 and pRPS6 were assessed by immunohistochemistry in human HCC samples. Rapamycin sensitivity was estimated by colony-formation assay in HCC cell lines and the treatment was further tested using our patient-derived tumour xenograft (PDTX) models. RESULTS: We identified and confirmed multiple mTOR components as recurrently mutated in HBV-associated HCCs. Of significance, we detected frequent (16.2%, n=18/111) mutations of TSC1 and TSC2 genes in the HCC samples. The spectrum of TSC1/2 mutations likely disrupts the endogenous gene functions in suppressing the downstream mTOR activity through different mechanisms and leads to more aggressive tumour behaviour. Mutational disruption of TSC1 and TSC2 was also observed in HCC cell lines and our PDTX models. TSC-mutant cells exhibited reduced colony-forming ability on rapamycin treatment. With the use of biologically relevant TSC2-mutant PDTXs, we demonstrated the therapeutic benefits of the hypersensitivity towards rapamycin treatment. CONCLUSIONS: Taken together, our findings suggest the significance of previously undocumented mutation-dependent mTOR hyperactivation and frequent TSC1/2 mutations in HBV-associated HCCs. They define a molecular subset of HCC having genetic aberrations in mTOR signalling, with potential significance of effective specific drug therapy.

Novel pre-mRNA splicing of intronically integrated HBV generates oncogenic chimera in hepatocellular carcinoma.

BACKGROUND & AIMS: Hepatitis B virus (HBV) integration is common in HBV-associated hepatocellular carcinoma (HCC) and may play an important pathogenic role through the production of chimeric HBV-human transcripts. We aimed to screen the transcriptome for HBV integrations in HCCs. METHODS: Transcriptome sequencing was performed on paired HBV-associated HCCs and corresponding non-tumorous liver tissues to identify viral-human chimeric sites. Validation was further performed in an expanded cohort of human HCCs. RESULTS: Here we report the discovery of a novel pre-mRNA splicing mechanism in generating HBV-human chimeric protein. This mechanism was exemplified by the formation of a recurrent HBV-cyclin A2 (CCNA2) chimeric transcript (A2S), as detected in 12.5% (6 of 48) of HCC patients, but in none of the 22 non-HCC HBV-associated cirrhotic liver samples examined. Upon the integration of HBV into the intron of the CCNA2 gene, the mammalian splicing machinery utilized the foreign splice sites at 282nt. and 458nt. of the HBV genome to generate a pseudo-exon, forming an in-frame chimeric fusion with CCNA2. The A2S chimeric protein gained a non-degradable property and promoted cell cycle progression, demonstrating its potential oncogenic functions. CONCLUSIONS: A pre-mRNA splicing mechanism is involved in the formation of HBV-human chimeric proteins. This represents a novel and possibly common mechanism underlying the formation of HBV-human chimeric transcripts from intronically integrated HBV genome with functional impact. LAY SUMMARY: HBV is involved in the mammalian pre-mRNA splicing machinery in the generation of potential tumorigenic HBV-human chimeras. This study also provided insight on the impact of intronic HBV integration with the gain of splice sites in the development of HBV-associated HCC.

Long non-coding RNA HOTTIP is frequently up-regulated in hepatocellular carcinoma and is targeted by tumour suppressive miR-125b.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is one of the most common human cancers. Recently, emerging evidence has suggested the role of long non-coding RNAs (lncRNAs) in human carcinogenesis. In this study, we aimed to investigate the expression and functional implications of lncRNAs in human HCC. METHODS: Eighty-eight well-annotated lncRNAs were profiled in primary HCC by quantitative RT-PCR. Functional relevance of lncRNAs was elucidated in HCC cell lines and nude mice models. The regulatory relationship between miRNA and lncRNA was predicted in silico and further validated by luciferase reporter assay and expression analysis. RESULTS: In our profiling study, HOTTIP was identified as the most significantly up-regulated lncRNA in human HCCs, even in early stage of HCC formation. Functionally, knock-down of HOTTIP attenuated HCC cell proliferation in vitro and markedly abrogated tumourigenicity in vivo. In addition, knock-down of HOTTIP also inhibited migratory ability of HCC cells and significantly abrogated lung metastasis in orthotopic implantation model in nude mice. HOTTIP is an antisense lncRNA mapped to the distal end of the HOXA gene cluster. Knock-down of HOTTIP significantly suppressed the expression of a number of HOXA genes. Furthermore, we identified miR-125b as a post-transcriptional regulator of HOTTIP. Ectopic expression of miR-125b reduced HOTTIP-coupled luciferase activity and suppressed the endogenous level of HOTTIP. Moreover, in human HCCs, HOTTIP expression negatively correlated with that of miR-125b. CONCLUSIONS: HOTTIP is a novel oncogenic lncRNA, which negatively regulated by miR-125b. Overexpression of HOTTIP contributes to hepatocarcinogenesis by regulating the expression of its neighbouring protein-coding genes.

C-terminal truncated hepatitis B virus x protein is associated with metastasis and enhances invasiveness by C-Jun/matrix metalloproteinase protein 10 activation in hepatocellular carcinoma.

UNLABELLED: Random integration of hepatitis B virus (HBV) DNA into the host genome is frequent in human hepatocellular carcinoma (HCC) and this leads to truncation of the HBV DNA, particularly at the C-terminal end of the HBV X protein (HBx). In this study, we investigated the frequency of this natural C-terminal truncation of HBx in human HCCs and its functional significance. In 50 HBV-positive patients with HCC, full-length HBx was detected in all nontumorous livers. However, full-length HBx was found in only 27 (54%) of the HCC tumors, whereas natural carboxylic acid (COOH)-truncated HBx was found in the remaining 23 (46%) tumors. Upon clinicopathological analysis, the presence of natural COOH-truncated HBx significantly correlated with the presence of venous invasion, a hallmark of metastasis (P = 0.005). Inducible stable expression of the COOH-truncated HBx protein (with 24 amino acids truncated at the C-terminal end) enhanced the cell-invasive ability of HepG2 cells, as compared to full-length HBx, using the Matrigel cell-invasion assay. It also resulted in increased C-Jun transcriptional activity and enhanced transcription of matrix metalloproteinase 10 (MMP10), whereas activation of the MMP10 promoter by COOH-truncated HBx was abolished when the activator protein 1-binding sites on the MMP10 promoter were mutated. Furthermore, silencing of MMP10 by short interfering RNA in HBxΔC1-expressing HepG2 cells resulted in significant reduction of cell invasiveness. CONCLUSIONS: Our data suggest that COOH truncation of HBx, particularly with 24 amino acids truncated at the C-terminal end, plays a role in enhancing cell invasiveness and metastasis in HCC by activating MMP10 through C-Jun.

Chemokine-driven lymphocyte infiltration: an early intratumoural event determining long-term survival in resectable hepatocellular carcinoma.

OBJECTIVE: Hepatocellular carcinoma (HCC) is a heterogeneous disease with poor prognosis and limited methods for predicting patient survival. The nature of the immune cells that infiltrate tumours is known to impact clinical outcome. However, the molecular events that regulate this infiltration require further understanding. Here the ability of immune genes expressed in the tumour microenvironment to predict disease progression was investigated. METHODS: Using quantitative PCR, the expression of 14 immune genes in resected tumour tissues from 57 Singaporean patients was analysed. The nearest-template prediction method was used to derive and test a prognostic signature from this training cohort. The signature was then validated in an independent cohort of 98 patients from Hong Kong and Zurich. Intratumoural components expressing these critical immune genes were identified by in situ labelling. Regulation of these genes was analysed in vitro using the HCC cell line SNU-182. RESULTS: The identified 14 immune-gene signature predicts patient survival in both the training cohort (p=0.0004 and HR=5.2) and the validation cohort (p=0.0051 and HR=2.5) irrespective of patient ethnicity and disease aetiology. Importantly, it predicts the survival of patients with early disease (stages I and II), for whom classical clinical parameters provide limited information. The lack of predictive power in late disease stages III and IV emphasises that a protective immune microenvironment has to be established early in order to impact disease progression significantly. This signature includes the chemokine genes CXCL10, CCL5 and CCL2, whose expression correlates with markers of T helper 1 (Th1), CD8(+) T and natural killer (NK) cells. Inflammatory cytokines (tumour necrosis factor α, interferon γ) and Toll-like receptor 3 ligands stimulate intratumoural production of these chemokines which drive tumour infiltration by T and NK cells, leading to enhanced cancer cell death. CONCLUSION: A 14 immune-gene signature, which identifies molecular cues driving tumour infiltration by lymphocytes, accurately predicts survival of patients with HCC especially in early disease.

MicroRNA expression, survival, and response to interferon in liver cancer.

BACKGROUND: Hepatocellular carcinoma is a common and aggressive cancer that occurs mainly in men. We examined microRNA expression patterns, survival, and response to interferon alfa in both men and women with the disease. METHODS: We analyzed three independent cohorts that included a total of 455 patients with hepatocellular carcinoma who had undergone radical tumor resection between 1999 and 2003. MicroRNA-expression profiling was performed in a cohort of 241 patients with hepatocellular carcinoma to identify tumor-related microRNAs and determine their association with survival in men and women. In addition, to validate our findings, we used quantitative reverse-transcriptase-polymerase-chain-reaction assays to measure microRNAs and assess their association with survival and response to therapy with interferon alfa in 214 patients from two independent, prospective, randomized, controlled trials of adjuvant interferon therapy. RESULTS: In patients with hepatocellular carcinoma, the expression of miR-26a and miR-26b in nontumor liver tissue was higher in women than in men. Tumors had reduced levels of miR-26 expression, as compared with paired noncancerous tissues, which indicated that the level of miR-26 expression was also associated with hepatocellular carcinoma. Moreover, tumors with reduced miR-26 expression had a distinct transcriptomic pattern, and analyses of gene networks revealed that activation of signaling pathways between nuclear factor kappaB and interleukin-6 might play a role in tumor development. Patients whose tumors had low miR-26 expression had shorter overall survival but a better response to interferon therapy than did patients whose tumors had high expression of the microRNA. CONCLUSIONS: The expression patterns of microRNAs in liver tissue differ between men and women with hepatocellular carcinoma. The miR-26 expression status of such patients is associated with survival and response to adjuvant therapy with interferon alfa.

Role of cadherin-17 in oncogenesis and potential therapeutic implications in hepatocellular carcinoma.

Cadherin is an important cell adhesion molecule that plays paramount roles in organ development and the maintenance of tissue integrity. Dysregulation of cadherin expression is often associated with disease pathology including tissue dysplasia, tumor formation, and metastasis. Cadherin-17 (CDH17), belonging to a subclass of 7D-cadherin superfamily, is present in fetal liver and gastrointestinal tract during embryogenesis, but the gene becomes silenced in healthy adult liver and stomach tissues. It functions as a peptide transporter and a cell adhesion molecule to maintain tissue integrity in epithelia. However, recent findings from our group and others have reported aberrant expression of CDH17 in major gastrointestinal malignancies including hepatocellular carcinoma (HCC), stomach and colorectal cancers, and its clinical association with tumor metastasis and advanced tumor stages. Furthermore, alternative splice isoforms and genetic polymorphisms of CDH17 gene have been identified in HCC and linked to an increased risk of HCC. CDH17 is an attractive target for HCC therapy. Targeting CDH17 in HCC can inhibit tumor growth and inactivate Wnt signaling pathway in concomitance with activation of tumor suppressor genes. Further investigation on CDH17-mediated oncogenic signaling and cognate molecular mechanisms would shed light on new targeting therapy on HCC and potentially other gastrointestinal malignancies.

Portal inflow and pressure changes in right liver living donor liver transplantation including the middle hepatic vein.

The middle hepatic vein may be included in right liver living donor liver transplantation (LDLT) to optimize hepatic venous outflow. We studied the graft's ability to relieve portal hypertension and accommodate portal hyperperfusion with portal manometry and ultrasonic flowmetry. Surgical outcomes with respect to portal hemodynamometry were also investigated. The ages of the recipients and donors for 46 consecutive LDLT procedures were 50 (range, 16-66 years) and 31 years (range, 18-54 years), respectively. The graft to standard liver volume ratio was 47.4% (range, 32.4%-69.0%). The hospital mortality rate was 4.4% as 2 recipients died from a subarachnoid hemorrhage and sepsis. The portal pressure dropped by 8 mm Hg (range, -7 to 19 mm Hg) from 23 (range, 8-37 mm Hg) to 14 mm Hg (range, 10-26 mm Hg) after graft implantation. The portal inflow positively correlated with the portal pressure before native liver hepatectomy (R(2) = 0.305, P = 0.001) and not with the graft size. The portal inflow increased from 81 mL/minute/100 g (range, 35-210 mL/minute/100 g) before donor right hepatectomy to 318 mL/minute/100 g (range, 102-754 mL/minute/100 g) after graft implantation. The graft portal inflow had a positive linear correlation with the recipient portal pressure before native liver total hepatectomy (R(2) = 0.261, P = 0.001) but not after graft implantation, and it had a negative correlation with the graft to standard liver volume ratio (R(2) = 0.247, P = 0.001). Only 1 of the graft biopsies showed moderate sinusoidal congestion. Twelve recipients had Clavien grade 2+ complications that were not related to the portal inflow and pressure or graft size. Right liver LDLT including the middle hepatic vein effectively lowered the recipient portal pressure by allowing unimpeded venous outflow.

Proteomics of hepatocellular carcinoma: serum vimentin as a surrogate marker for small tumors (

Small hepatocellular carcinomas (HCCs) can be effectively cured by surgery with good clinical outcomes. However, the conventional AFP marker is ineffective in detecting small tumors. Here we employed a proteomic profiling approach to identify a candidate marker for HCC (

Genetic and epigenetic inactivation of T-cadherin in human hepatocellular carcinoma cells.

T-cadherin is an atypical cadherin and growing evidence has indicated that T-cadherin exerts tumor-suppressive effects on cancers of epithelial cell type and also causes positive effects on tumor angiogenesis. Human hepatocellular carcinoma (HCC) is a hypervascular tumor and T-cadherin has been shown to be overexpressed in intratumoral endothelial cells of HCCs. However, the expression status and functions of T-cadherin in hepatocytes or HCC cells remain unclear. Here, we demonstrated that T-cadherin was underexpressed in HCC cells (26.5%, 13/49 cases), but was frequently (77.6%, 38/49) overexpressed in intratumoral endothelial cells immunohistochemically. Semiquantitative RT-PCR analysis also showed that the T-cadherin gene was underexpressed in 7 of 11 HCC cell lines. Loss of heterozygosity analysis revealed that 32-38% of the 42 human HCC samples had allelic losses at this locus. Upon pharmacological treatment with demethylating agent 5-aza-2'-deoxycytidine or histone deacetylase inhibitor trichostatin A, T-cadherin promoter hypermethylation and/or histone deacetylation was frequently observed in HCC samples and cell lines. Functionally, enforced expression of T-cadherin induced G(2)/M cell cycle arrest, reduced cell proliferation in low serum medium, suppressed anchorage-independent growth in soft agar and increased sensitivity to TNFalpha-mediated apoptosis in HCC cells. Intriguingly, we found that T-cadherin significantly suppressed the activity of c-Jun, a crucial oncoprotein constitutively activated in HCC cells. To conclude, T-cadherin was differentially expressed in human HCCs. The underexpression of T-cadherin in HCC cells suggests it may be another critical event in addition to T-cadherin-mediated angiogenesis during HCC development.

Molecular pathogenesis of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of cancer death worldwide. Hepatocarcinogenesis is a multistep process evolving from normal through chronic hepatitis/cirrhosis and dysplastic nodules to HCC. With advances in molecular methods, there is a growing understanding of the molecular mechanisms in hepatocarcinogenesis. Hepatocarcinogenesis is strongly linked to increases in allelic losses, chromosomal changes, gene mutations, epigenetic alterations and alterations in molecular cellular pathways. Some of these alterations are accompanied by a stepwise increase in the different pathological disease stages in hepatocarcinogenesis. Overall, a detailed understanding of the underlying molecular mechanisms involved in the progression of HCC is of fundamental importance to the development of effective prevention and treatment regimes for HCC.

Down-regulation of retinol binding protein 5 is associated with aggressive tumor features in hepatocellular carcinoma.

PURPOSE: Acyclic retinoid (ACR) has been shown to be a promising chemopreventive agent for hepatocellular carcinoma (HCC) after curative resection. The effects of retinoid are mediated by retinol-binding proteins (RBPs) through regulating cell proliferation and differentiation. PATIENTS AND METHODS: This study investigated the clinical significance of RBP5 in HCC. RBP5 mRNA level was examined by real-time quantitative PCR on 52 matched tumor and adjacent non-tumor liver tissues, and on ten normal livers. Expression of RBP5 protein was examined using Western blotting analysis and immunohistochemistry. RESULTS: Down-regulation of RBP5 was found in HCC tissues at both mRNA and protein levels. Decreased RBP5 level was closely related to poor differentiation (P=0.02) and large tumor size (P=0.01). Low level of RPB5 was associated with poor overall survival (P=0.02), and was an independent prognostic factor for HCC. CONCLUSIONS: Our study revealed that RBP5 down-regulation in HCC was associated with aggressive tumor features, suggesting an important role of RPB5 in HCC progression.

Gene expression patterns in human liver cancers.

Hepatocellular carcinoma (HCC) is a leading cause of death worldwide. Using cDNA microarrays to characterize patterns of gene expression in HCC, we found consistent differences between the expression patterns in HCC compared with those seen in nontumor liver tissues. The expression patterns in HCC were also readily distinguished from those associated with tumors metastatic to liver. The global gene expression patterns intrinsic to each tumor were sufficiently distinctive that multiple tumor nodules from the same patient could usually be recognized and distinguished from all the others in the large sample set on the basis of their gene expression patterns alone. The distinctive gene expression patterns are characteristic of the tumors and not the patient; the expression programs seen in clonally independent tumor nodules in the same patient were no more similar than those in tumors from different patients. Moreover, clonally related tumor masses that showed distinct expression profiles were also distinguished by genotypic differences. Some features of the gene expression patterns were associated with specific phenotypic and genotypic characteristics of the tumors, including growth rate, vascular invasion, and p53 overexpression.

Identify metastasis-associated genes in hepatocellular carcinoma through clonality delineation for multinodular tumor.

Disease recurrence and metastasis are frequently observed in many successfullytreated localized cancers, including hepatocellular carcinoma in which intrahepatic and extrahepatic recurrence (metastasis) are frequently observed after curative resection. The present study aimed at identifying metastasis-associated genes through delineation of the clonality for multinodular liver cancer. The clonal relationship of 22 tumor foci from six patients was investigated by the genome-wide expression profile via cDNA microarray consisting of 23,000 genes. Tumor molecular properties including p53 protein overexpression and gene mutation, hepatitis B virus integration pattern, and genetic alteration examined by comparative genomic hybridization were compared. Results indicated that gene expression patterns could serve as the molecular fingerprint for clonality identification. Together with the molecular data from p53, hepatitis B virus integration and comparative genomic hybridization profiles, tumor nodules from five patients were confirmed with clonal relationship, and the expression profiles of the primary nodules were compared with their corresponding intrahepatic metastatic nodules. A total of 90 clones were found to be correlated with intrahepatic metastasis by Student's t test (P < 0.05). With reference to the primary tumor, 63 clones (39 known genes and 24 express sequence tags) were down-regulated whereas 27 clones (14 known genes and 13 express sequence tags) were up-regulated in the metastatic nodules. These metastasis-associated genes may provide clues to reveal patients with increased risk of developing metastasis, and to identify novel therapeutic targets for the treatment of metastasis.

PIN1 overexpression and beta-catenin gene mutations are distinct oncogenic events in human hepatocellular carcinoma.

The peptidyl-proplyl-isomerase, PIN1, upregulates beta-catenin by inhibiting its interaction with APC. beta-catenin accumulation occurs in about 70% of hepatocellular carcinoma (HCC), of which only 20% are due to beta-catenin mutations. The role of PIN1 in beta-catenin upregulation in HCC was investigated. PIN1 was shown to be overexpressed in more than 50% of HCC. All cases with PIN1 overexpression also showed beta-catenin accumulation, with 68% of cases showing concomitant beta-catenin and cyclin D1 accumulation. PIN1 was shown to contribute to beta-catenin and cyclin D1 overexpression directly by in vitro cell-line transfection experiments. Finally, we showed that PIN1 overexpression and beta-catenin gene mutations appeared to be mutually exclusive events, leading to beta-catenin accumulation in HCC. These results showed that PIN1 overexpression leading to beta-catenin accumulation might be a critical event in hepatocarcinogenesis, and that PIN1 is a potential target for therapeutic intervention in HCC.

Clinicopathological significance of loss of heterozygosity on chromosome 13q in hepatocellular carcinoma.

PURPOSE: Allelic loss is the most frequently genetic alteration found in hepatocellular carcinoma (HCC). Previous genome-wide studies have indicated that chromosome 13q is one of the most frequently affected chromosomes. However, reports on detailed deletion mapping as well as detailed clinicopathological correlation are scanty. EXPERIMENTAL DESIGN: We performed high-density allelotyping on chromosome 13q in HCC from 60 patients and investigated the correlation between allelic losses on chromosome 13q and the clinicopathological features. RESULTS: Allelic loss at one or more of the 29 microsatellite markers was found in 28 (47%) of the 60 HCCs. Allelic losses were more frequently found in tumors with larger size or in tumors at more advanced tumor stages (P = 0.015 and 0.012, respectively). These two clinicopathological features were also significantly associated with the accumulation of allelic losses in terms of fractional allelic loss index (P = 0.028 and 0.018, respectively). In addition, subchromosomal regions located at 13q12.3-14.1 and 13q32 were found to be significantly associated with advanced tumor stages and larger tumor size, respectively (P = 0.008 and 0.007). CONCLUSIONS: The overall findings suggested that allelic losses on 13q might play an important role in contributing to a more aggressive tumor behavior. Putative tumor suppressor genes might be harbored at 13q12.3-14.1 and 13q32, and inactivation of these genes via allelic losses might enhance tumor progression in HCC.

uGPA: unified Gene Pathway Analyzer package for high-throughput genome-wide screening data provides mechanistic overview on human diseases.

BACKGROUND: Gene set or pathway analysis (GPA) can provide a comprehensive mechanistic overview in delineating molecular pathoetiology of human diseases. Existing tools that can handle for GPA on both mutational and gene expressional aspects are limited. This leads to the development of uGPA (unified Gene Pathway Analyzer). METHODS: uGPA package provides a unified solution to knowledge base-driven competitive GPA that can analyze genome-wide screening data derived from multiple platforms (next generation sequencing or microarray) and sample sources (genomic or transcriptomic) on both mutational and gene expressional perspectives. It allows for a quick assessment of perspective outline of gene sets on the studied disease through enrichment test modeled by cumulative hypergeometric distribution and reports the gene components detected with events (mutation or differential gene expression) in the gene sets. RESULTS: uGPA package has been successfully verified to be applicable on input data generated from NGS-based platforms (transcriptome sequencing and whole-exome sequencing). Genome-wide screening data from other platforms should also apply. CONCLUSIONS: uGPA package delivers a simple, flexible and platform-independent functionality to multiple aspects of GPA. It equips clinicians and scientists with a useful tool in studying molecular mechanism of human diseases using modern high-throughput genome-wide screening strategy, which assists personalized therapy.