Kwong-Fai Wong

Circulating miR-15b and miR-130b in serum as potential markers for detecting hepatocellular carcinoma: a retrospective cohort study

Objective Serum α-fetoprotein (AFP) is the most commonly used biomarker for screening hepatocellular carcinoma (HCC) but fails to detect about half of the patients. Thus, we investigated if circulating microRNAs (miRNAs) could outperform AFP for HCC detection. Design A retrospective cohort study. Setting Two clinical centres in China. Participants The exploration phase included 96 patients with HCC who received primary curative hepatectomy, and the validation phase included 29 hepatitis B carriers, 57 patients with HCC and 30 healthy controls. Main outcome measures Expression of miRNAs was measured by real-time quantitative reverse transcription–PCR. Areas under receiver operating characteristic curves were used to determine the feasibility of using serum miRNA concentration as a diagnostic marker for defining HCC. A multivariate logistic regression analysis was used to evaluate performances of combined serum miRNAs. Results In the exploration phase, miRNA profiling on resected tumour/adjacent non-tumour tissues identified miR-15b, miR-21, miR-130b and miR-183 highly expressed in tumours. These miRNAs were also detectable in culture supernatants of HCC cell lines and in serum samples of patients. Remarkably, these serum miRNAs were markedly reduced after surgery, indicating the tumour-derived source of these circulating miRNAs. In a cross-centre validation study, combined miR-15b and miR-130b demonstrated as a classifier for HCC detection, yielding a receiver operating characteristic curve area of 0.98 (98.2% sensitivity and 91.5% specificity). The detection sensitivity of the classifier in a subgroup of HCCs with low AFP (<20 ng/ml) was 96.7%. The classifier also identified early-stage HCC cases that could not be detected by AFP. Conclusion The combined miR-15b and miR-130b classifier is a serum biomarker with clinical value for HCC screening.

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.).

microRNA-122 as a regulator of mitochondrial metabolic gene network in hepatocellular carcinoma

Tumorigenesis involves multistep genetic alterations. To elucidate the microRNA (miRNA)–gene interaction network in carcinogenesis, we examined their genome‐wide expression profiles in 96 pairs of tumor/non‐tumor tissues from hepatocellular carcinoma (HCC). Comprehensive analysis of the coordinate expression of miRNAs and mRNAs reveals that miR‐122 is under‐expressed in HCC and that increased expression of miR‐122 seed‐matched genes leads to a loss of mitochondrial metabolic function. Furthermore, the miR‐122 secondary targets, which decrease in expression, are good prognostic markers for HCC. Transcriptome profiling data from additional 180 HCC and 40 liver cirrhotic patients in the same cohort were used to confirm the anti‐correlation of miR‐122 primary and secondary target gene sets. The HCC findings can be recapitulated in mouse liver by silencing miR‐122 with antagomir treatment followed by gene‐expression microarray analysis. In vitro miR‐122 data further provided a direct link between induction of miR‐122‐controlled genes and impairment of mitochondrial metabolism. In conclusion, miR‐122 regulates mitochondrial metabolism and its loss may be detrimental to sustaining critical liver function and contribute to morbidity and mortality of liver cancer patients.

Traditional Chinese herbal medicines for treatment of liver fibrosis and cancer: From laboratory discovery to clinical evaluation

Liver disease afflicts over 10% of the world population. This includes chronic hepatitis, alcoholic steatosis, fibrosis, cirrhosis and hepatocellular carcinoma (HCC), which are the most health‐threatening conditions drawing considerable attention from medical professionals and scientists. Patients with alcoholism or viral hepatitis are much more likely to have liver cell damage and cirrhosis, and some may eventually develop HCC, which is unfortunately, and very often, a fatal malignancy without cure. While liver surgery is not suitable in many of the HCC cases, patients are mostly given palliative support cares or transarterial chemoembolization or systemic chemotherapies. However, HCC is well known to be a highly chemoresistant tumour, and the response rate is <10–20%. To this end, alternative medicines are being actively sought from other sources with hopes to halt the diseases progression or even eliminate the tumours. Traditional Chinese herbal medicine has begun to gain popularity worldwide for promoting healthcare as well as disease prevention, and been used as conventional or complementary medicines for both treatable and incurable diseases in Asia and the West. In this article, we discuss the laboratory findings and clinical trial studies of Chinese herbal medicines (particularly small molecule compounds) for the treatment of liver disease ranging from fibrosis to liver cancer.

Celastrol suppresses growth and induces apoptosis of human hepatocellular carcinoma through the modulation of STAT3/JAK2 signaling cascade In Vitro and In Vivo

Cumulative evidences(s) have established that the constitutive activation of STAT3 plays a pivotal role in the proliferation, survival, metastasis, and angiogenesis and thus can contribute directly to the pathogenesis of hepatocellular carcinoma (HCC). Thus, novel agents that can inhibit STAT3 activation have potential for both prevention and treatment of HCCs. The effect of celastrol on STAT3 activation, associated protein kinases, STAT3-regulated gene products, cellular proliferation, and apoptosis was investigated. The in vivo effect of celastrol on the growth of human HCC xenograft tumors in athymic nu/nu mice was also examined. We observed that celastrol inhibited both constitutive and inducible STAT3 activation, and the suppression was mediated through the inhibition of activation of upstream kinases c-Src, as well as Janus-activated kinase-1 and -2. Vanadate treatment reversed the celastrol-induced modulation of STAT3, suggesting the involvement of a tyrosine phosphatase. The inhibition of STAT3 activation by celastrol led to the suppression of various gene products involved in proliferation, survival, and angiogenesis. Celastrol also inhibited the proliferation and induced apoptosis in HCC cells. Finally, when administered intraperitoneally, celastrol inhibited STAT3 activation in tumor tissues and the growth of human HCC xenograft tumors in athymic nu/nu mice without any side effects. Overall, our results suggest for the first time that celastrol exerts its antiproliferative and proapoptotic effects through suppression of STAT3 signaling in HCC both in vitro and in vivo. Cancer Prev Res; 5(4); 631–43. ©2012 AACR.

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.

miR-122 Targets Pyruvate Kinase M2 and Affects Metabolism of Hepatocellular Carcinoma

In contrast to normal differentiated cells that depend on mitochondrial oxidative phosphorylation for energy production, cancer cells have evolved to utilize aerobic glycolysis (Warburg’s effect), with benefit of providing intermediates for biomass production. MicroRNA-122 (miR-122) is highly expressed in normal liver tissue regulating a wide variety of biological processes including cellular metabolism, but is reduced in hepatocellular carcinoma (HCC). Overexpression of miR-122 was shown to inhibit cancer cell proliferation, metastasis, and increase chemosensitivity, but its functions in cancer metabolism remains unknown. The present study aims to identify the miR-122 targeted genes and to investigate the associated regulatory mechanisms in HCC metabolism. We found the ectopic overexpression of miR-122 affected metabolic activities of HCC cells, evidenced by the reduced lactate production and increased oxygen consumption. Integrated gene expression analysis in a cohort of 94 HCC tissues revealed miR-122 level tightly associated with a battery of glycolytic genes, in which pyruvate kinase (PK) gene showed the strongest anti-correlation coefficient (Pearson r = −0.6938, p = <0.0001). In addition, reduced PK level was significantly associated with poor clinical outcomes of HCC patients. We found isoform M2 (PKM2) is the dominant form highly expressed in HCC and is a direct target of miR-122, as overexpression of miR-122 reduced both the mRNA and protein levels of PKM2, whereas PKM2 re-expression abrogated the miR-122-mediated glycolytic activities. The present study demonstrated the regulatory role of miR-122 on PKM2 in HCC, having an implication of therapeutic intervention targeting cancer metabolic pathways.

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.

Diverse modes of genomic alteration in hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is a heterogeneous disease with high mortality rate. Recent genomic studies have identified TP53, AXIN1, and CTNNB1 as the most frequently mutated genes. Lower frequency mutations have been reported in ARID1A, ARID2 and JAK1. In addition, hepatitis B virus (HBV) integrations into the human genome have been associated with HCC.ResultsHere, we deep-sequence 42 HCC patients with a combination of whole genome, exome and transcriptome sequencing to identify the mutational landscape of HCC using a reasonably large discovery cohort. We find frequent mutations in TP53, CTNNB1 and AXIN1, and rare but likely functional mutations in BAP1 and IDH1. Besides frequent hepatitis B virus integrations at TERT, we identify translocations at the boundaries of TERT. A novel deletion is identified in CTNNB1 in a region that is heavily mutated in multiple cancers. We also find multiple high-allelic frequency mutations in the extracellular matrix protein LAMA2. Lower expression levels of LAMA2 correlate with a proliferative signature, and predict poor survival and higher chance of cancer recurrence in HCC patients, suggesting an important role of the extracellular matrix and cell adhesion in tumor progression of a subgroup of HCC patients.ConclusionsThe heterogeneous disease of HCC features diverse modes of genomic alteration. In addition to common point mutations, structural variations and methylation changes, there are several virus-associated changes, including gene disruption or activation, formation of chimeric viral-human transcripts, and DNA copy number changes. Such a multitude of genomic events likely contributes to the heterogeneous nature of HCC.

Regulators of mammalian Hippo pathway in cancer.

Hippo pathway, originally discovered in Drosophila, is responsible for organ size control. The pathway is conserved in mammals and has a significant role in restraining cancer development. Regulating the Hippo pathway thus represents a potential therapeutic approach to treat cancer, which however requires deep understanding of the targeted pathway. Despite our limited knowledge on the pathway, there are increasing discoveries of new molecules that regulate and modulate the Hippo downstream signaling particularly in various solid malignancies, from extracellular stimuli or via pathway crosstalk. Herein, we discuss the roles of newly identified and key regulators that connect with core components (MST1/2, LATS1/2, SAV1, and MOB1) and downstream effector (YAP) in the Hippo pathway having an important role in cancer development and progression. Understanding of the mammalian Hippo pathway regulation may shed new insights to allow us selecting the right oncogenic targets and designing effective drugs for cancer treatments.