John M. Luk

Whole genome sequencing identifies recurrent mutations in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most deadly cancers worldwide and has no effective treatment, yet the molecular basis of hepatocarcinogenesis remains largely unknown. Here we report findings from a whole-genome sequencing (WGS) study of 88 matched HCC tumor/normal pairs, 81 of which are Hepatitis B virus (HBV) positive, seeking to identify genetically altered genes and pathways implicated in HBV-associated HCC. We find beta-catenin to be the most frequently mutated oncogene (15.9%) and TP53 the most frequently mutated tumor suppressor (35.2%). The Wnt/beta-catenin and JAK/STAT pathways, altered in 62.5% and 45.5% of cases, respectively, are likely to act as two major oncogenic drivers in HCC. This study also identifies several prevalent and potentially actionable mutations, including activating mutations of Janus kinase 1 (JAK1), in 9.1% of patients and provides a path toward therapeutic intervention of the disease.

MicroRNA-375 targets hippo-signaling effector YAP in liver cancer and inhibits tumor properties

UNLABELLED Hepatocellular carcinoma (HCC) is a malignant form of liver cancer that ranks the second leading cause of cancer-related deaths in China and many Asia regions. The dismal outcome reflects the need for a better understanding of the transcriptional control of oncogenic signaling pathway. Our recent findings have identified yes-associated protein (YAP) is a potent oncogenic driver and independent prognostic risk factor of HCC. The present study aims to elucidate the transcriptional regulation of YAP targeted by microRNA (miRNA). miR-375 is a putative target and was found significantly down-regulated in the tumor versus adjacent non-tumor tissues of HCC patients (n=48). As determined by luciferase reporter assay, we found ectopic expression of miR-375 could diminish the transcriptional activity of YAP. Furthermore, immunoblotting revealed miR-375 suppressed endogenous YAP protein level. Functional assays showed that miR-375 was able to inhibit proliferation and invasion of HCC cells. CONCLUSION miR-375 is an important regulator of YAP oncogene, implicating a potential therapeutic role in HCC treatment.

Oncofetal Gene SALL4 in Aggressive Hepatocellular Carcinoma

BACKGROUND Hepatocellular carcinoma is the third leading cause of cancer-related deaths worldwide. In the heterogeneous group of hepatocellular carcinomas, those with characteristics of embryonic stem-cell and progenitor-cell gene expression are associated with the worst prognosis. The oncofetal gene SALL4, a marker of a subtype of hepatocellular carcinoma with progenitor-like features, is associated with a poor prognosis and is a potential target for treatment. METHODS We screened specimens obtained from patients with primary hepatocellular carcinoma for the expression of SALL4 and carried out a clinicopathological analysis. Loss-of-function studies were then performed to evaluate the role of SALL4 in hepatocarcinogenesis and its potential as a molecular target for therapy. To assess the therapeutic effects of a peptide that targets SALL4, we used in vitro functional and in vivo xenograft assays. RESULTS SALL4 is an oncofetal protein that is expressed in the human fetal liver and silenced in the adult liver, but it is reexpressed in a subgroup of patients who have hepatocellular carcinoma and an unfavorable prognosis. Gene-expression analysis showed the enrichment of progenitor-like gene signatures with overexpression of proliferative and metastatic genes in SALL4-positive hepatocellular carcinomas. Loss-of-function studies confirmed the critical role of SALL4 in cell survival and tumorigenicity. Blocking SALL4-corepressor interactions released suppression of PTEN (the phosphatase and tensin homologue protein) and inhibited tumor formation in xenograft models in vivo. CONCLUSIONS SALL4 is a marker for a progenitor subclass of hepatocellular carcinoma with an aggressive phenotype. The absence of SALL4 expression in the healthy adult liver enhances the potential of SALL4 as a treatment target in hepatocellular carcinoma. (Funded by the Singapore National Medical Research Council and others.).

Interleukin 17A Promotes Hepatocellular Carcinoma Metastasis via NF-kB Induced Matrix Metalloproteinases 2 and 9 Expression

Background  IL-17A is a pro-inflammatory cytokine that plays important role in inflammatory disease pathology and tumor microenvironment. The aim of this study is to investigate the effect of IL-17A on the progression of hepatocellular carcinoma (HCC). Methodology and Principal Finding Expression pattern of IL-17A in clinical HCC samples (n = 43) was determined by immunohistochemistry staining. Transcript levels of MMP2, MMP9 and IL-17A were measured in another 50 pairs (including tumor and related non-tumor tissues) HCC samples. Cell growth, focus formation, cell migration, invasion and western blot assays were used to characterize the functional and signaling mechanisms in IL-17A-treated HCC. Association study was used to identify clinical significance of IL-17A in HCC. Compared with paired non-tumor tissue, higher frequency of IL-17A-positive cells was detected in tumor tissues in HCCs with metastasis, and the frequency of IL-17A-positive cells was also significantly associated with poor prognosis of HCC (P = 0.01). Functional study found that IL-17A could promote HCC cell migration and invasion. Further molecular analysis also showed that IL-17A could upregulate MMP2 and MMP9 expression via NF-κB signaling activation. Conclusions  IL-17A could promote HCC metastasis by the upregulation of MMP2 and MMP9 expression via activating NF-κB signaling pathway.

Predicting prognosis in hepatocellular carcinoma after curative surgery with common clinicopathologic parameters

BackgroundSurgical resection is one important curative treatment for hepatocellular carcinoma (HCC), but the prognosis following surgery differs substantially and such large variation is mainly unexplained. A review of the literature yields a number of clinicopathologic parameters associated with HCC prognosis. However, the results are not consistent due to lack of systemic approach to establish a prediction model incorporating all these parameters.MethodsWe conducted a retrospective analysis on the common clinicopathologic parameters from a cohort of 572 ethnic Chinese HCC patients who received curative surgery. The cases were randomly divided into training (n = 272) and validation (n = 300) sets. Each parameter was individually tested and the significant parameters were entered into a linear classifier for model building, and the prediction accuracy was assessed in the validation setResultsOur findings based on the training set data reveal 6 common clinicopathologic parameters (tumor size, number of tumor nodules, tumor stage, venous infiltration status, and serum α-fetoprotein and total albumin levels) that were significantly associated with the overall HCC survival and disease-free survival (time to recurrence). We next built a linear classifier model by multivariate Cox regression to predict prognostic outcomes of HCC patients after curative surgery This analysis detected a considerable fraction of variance in HCC prognosis and the area under the ROC curve was about 70%. We further evaluated the model using two other protocols; leave-one-out procedure (n = 264) and independent validation (n = 300). Both were found to have excellent prediction power. The predicted score could separate patients into distinct groups with respect to survival (p-value = 1.8e-12) and disease free survival (p-value = 3.2e-7).ConclusionThis described model will provide valuable guidance on prognosis after curative surgery for HCC in clinical practice. The adaptive nature allows easy accommodation for future new biomarker inputs, and it may serve as the foundation for future modeling and prediction for HCC prognosis after surgical treatment.

Tripterygium wilfordii bioactive compounds as anticancer and anti‐inflammatory agents

1. Bioactive compounds from medicinal plants with anticancer and anti‐inflammatory effects have become key resources in drug discovery fields for the treatment of various malignancies and immunological disorders.

Targeting YAP and Hippo signaling pathway in liver cancer.

Importance of the field: The Hippo signaling pathway plays pivotal roles in controlling both cell growth and organ size, emerging as a new paradigm in tumor suppression. Yes-associated protein (YAP) functions as a potent transcription co-activator and is a major downstream target tightly regulated by the Hippo pathway. Inactivation of the Hippo signaling induces YAP-mediated activation of various target genes that functionally causes cellular proliferation and outgrowth of organ size. Recently, YAP has been implicated as a bona fide oncogene in solid tumors, but little is known about its exact molecular mechanism in carcinogenesis. Areas covered in this review: We discuss the latest important findings in the Hippo signaling pathway and the possible means of developing potential cancer therapeutics by targeting multiple sites along the Hippo pathway. What the reader will gain: An overview of the emerging roles of YAP and Hippo signaling in oncogenesis and the possible ways of developing cancer therapies against the pathway components, downstream targets or interconnected pathways. Take home message: YAP is a key oncogenic driver in liver carcinogenesis and deregulation of the Hippo pathway causes tumor formation and malignancy. Targeting YAP and cognate downstream signaling targets may have clinical utility in cancer therapies.

Prognostic significance and therapeutic potential of eukaryotic translation initiation factor 5A (eIF5A) in hepatocellular carcinoma

Using comparative proteomic and genomic approaches, the authors identified eukaryotic translation initiation factor 5A (eIF5A) as an oncofetal molecule highly abundant in mouse embryonic livers and human hepatocellular carcinoma (HCC) cell lines. To evaluate the oncogenic role and prognostic significance of eIF5A in HCC, we investigate the expression patterns of the two isoforms (eIF5A1 and eIF5A2) in a cohort of 258 HCC cases by cDNA microarray. Both eIF5A isoforms were expressed in the tumors, and clinically correlated eIF5A1 with more numbers of tumor nodules and eIF5A2 with tumor venous infiltration in HCC. In a separate cohort of 50 HCCs, high level of eIF5A2, but not eIF5A1, was associated with elevated levels of deoxyhypusine synthase and deoxyhypusine hydroxylase that catalyze post‐translational hypusination of eIF5A protein. Interestingly, N1‐guanyl‐1,7‐diaminoheptane (GC7), which is an inhibitor for the first step of eIF5A hypusination, was shown to significantly impair the cell proliferation and invasion of primary HCC cells (HepG2 and Hep3B). To further demonstrate the tumorigenic role associated with eIF5A, a drastic reduction of cell proliferation was associated with suppression of eIF5A2 by transfecting Hep3B, H2‐P and H2‐M HCC cells expressing high level of this isoform using small interfering RNA (siRNA) against eIF5A2. For these assays, a milder response was usually observed in normal hepatocyte cell line. Therefore, these findings suggest that eIF5A plays an important role in HCC tumorigenesis and metastasis, and targeting eIF5A hypusination by GC7 inhibitor or eIF5A2 by RNA interference (RNAi) may offer new therapeutic alternatives to HCC patients.

Two-tiered Approach Identifies a Network of Cancer and Liver Disease-related Genes Regulated by miR-122

MicroRNAs function as important regulators of gene expression and are commonly linked to development, differentiation, and diseases such as cancer. To better understand their roles in various biological processes, identification of genes targeted by microRNAs is necessary. Although prediction tools have significantly helped with this task, experimental approaches are ultimately required for extensive target search and validation. We employed two independent yet complementary high throughput approaches to map a large set of mRNAs regulated by miR-122, a liver-specific microRNA implicated in regulation of fatty acid and cholesterol metabolism, hepatitis C infection, and hepatocellular carcinoma. The combination of luciferase reporter-based screening and shotgun proteomics resulted in the identification of 260 proteins significantly down-regulated in response to miR-122 in at least one method, 113 of which contain predicted miR-122 target sites. These proteins are enriched for functions associated with the cell cycle, differentiation, proliferation, and apoptosis. Among these miR-122-sensitive proteins, we identified a large group with strong connections to liver metabolism, diseases, and hepatocellular carcinoma. Additional analyses, including examination of consensus binding motifs for both miR-122 and target sequences, provide further insight into miR-122 function.

Inhibition of STAT3 dimerization and acetylation by garcinol suppresses the growth of human hepatocellular carcinoma in vitro and in vivo

BackgroundConstitutive activation of signal transducer and activator of transcription 3 (STAT3) has been linked with proliferation, survival, invasion and angiogenesis of a variety of human cancer cells, including hepatocellular carcinoma (HCC). Thus, novel agents that can suppress STAT3 activation have potential for both prevention and treatment of HCC. Here we report, garcinol, a polyisoprenylated benzophenone, could suppress STAT3 activation in HCC cell lines and in xenografted tumor of HCC in nude mice model.Experimental designDifferent HCC cell lines have been treated with garcinol and the inhibition of STAT3 activation, dimerization and acetylation have been checked by immunoblotting, immuno-fluorescence, and DNA binding assays. Xenografted tumor model has been generated in nude mice using HCC cell line and effect of garcinol in the inhibition of tumor growth has been investigated.ResultsGarcinol could inhibit both constitutive and interleukin (IL-6) inducible STAT3 activation in HCC cells. Computational modeling showed that garcinol could bind to the SH2 domain of STAT3 and suppress its dimerization in vitro. Being an acetyltransferase inhibitor, garcinol also inhibits STAT3 acetylation and thus impairs its DNA binding ability. The inhibition of STAT3 activation by garcinol led to the suppression of expression of various genes involved in proliferation, survival, and angiogenesis. It also suppressed proliferation and induced substantial apoptosis in HCC cells. Remarkably, garcinol inhibited the growth of human HCC xenograft tumors in athymic nu/nu mice, through the inhibition of STAT3 activation.ConclusionOverall, our results suggest that garcinol exerts its anti-proliferative and pro-apoptotic effects through suppression of STAT3 signaling in HCC both in vitro and in vivo.