Yick-Pang Ching

Whole-genome sequencing and comprehensive molecular profiling identify new driver mutations in gastric cancer

Gastric cancer is a heterogeneous disease with diverse molecular and histological subtypes. We performed whole-genome sequencing in 100 tumor-normal pairs, along with DNA copy number, gene expression and methylation profiling, for integrative genomic analysis. We found subtype-specific genetic and epigenetic perturbations and unique mutational signatures. We identified previously known (TP53, ARID1A and CDH1) and new (MUC6, CTNNA2, GLI3, RNF43 and others) significantly mutated driver genes. Specifically, we found RHOA mutations in 14.3% of diffuse-type tumors but not in intestinal-type tumors (P < 0.001). The mutations clustered in recurrent hotspots affecting functional domains and caused defective RHOA signaling, promoting escape from anoikis in organoid cultures. The top perturbed pathways in gastric cancer included adherens junction and focal adhesion, in which RHOA and other mutated genes we identified participate as key players. These findings illustrate a multidimensional and comprehensive genomic landscape that highlights the molecular complexity of gastric cancer and provides a road map to facilitate genome-guided personalized therapy.

Human TRBP and PACT Directly Interact with Each Other and Associate with Dicer to Facilitate the Production of Small Interfering RNA

Mammalian Dicer interacts with double-stranded RNA-binding protein TRBP or PACT to mediate RNA interference and micro-RNA processing. TRBP and PACT are structurally related but exert opposite regulatory activities on PKR. It is not understood whether TRBP and PACT are simultaneously required for Dicer. Here we show that TRBP directly interacts with PACT in vitro and in mammalian cells. TRBP and PACT form a triple complex with Dicer and facilitate the production of small interfering RNA (siRNA) by Dicer. Knockdown of both TRBP and PACT in cultured cells leads to significant inhibition of gene silencing mediated by short hairpin RNA but not by siRNA, suggesting that TRBP and PACT function primarily at the step of siRNA production. Taken together, these findings indicate that human TRBP and PACT directly interact with each other and associate with Dicer to stimulate the cleavage of double-stranded or short hairpin RNA to siRNA. Our work significantly alters the current model for the assembly and function of the Dicer-containing complex that generates siRNA and micro-RNA in human.

Rho GTPase-Activating Protein Deleted in Liver Cancer Suppresses Cell Proliferation and Invasion in Hepatocellular Carcinoma

Deleted in liver cancer (DLC1) is a candidate tumor suppressor gene recently isolated from human hepatocellular carcinoma. Structurally, DLC1 protein contains a conserved GTPase-activating protein for Rho family protein (RhoGAP) domain, which has been thought to regulate the activity of Rho family proteins. Previous studies indicated that DLC1 was frequently inactivated in cancer cells. In the present study, we aimed to characterize the tumor suppressor roles of DLC1 in hepatocellular carcinoma. We showed that DLC1 significantly inhibited cell proliferation, anchorage-independent growth, and in vivo tumorigenicity when stably expressed in hepatocellular carcinoma cells. Moreover, DLC1 expression greatly reduced the motility and invasiveness of hepatocellular carcinoma cells. With RhoGAP-deficient DLC1 mutant (DLC1-K714E), we showed that the RhoGAP activity was essential for DLC1-mediated tumor suppressor function. Furthermore, the 292- to 648-amino acid region and the steroidogenic acute regulatory related lipid transfer domain played an auxiliary role to RhoGAP and tumor suppressor function of DLC1. Taken together, our findings showed that DLC1 functions as a tumor suppressor in hepatocellular carcinoma and provide the first evidence to support the hypothesis that DLC1 suppresses cancer cell growth by negatively regulating the activity of Rho proteins.

Physical exercise-induced hippocampal neurogenesis and antidepressant effects are mediated by the adipocyte hormone adiponectin.

Significance This study unmasks a previously unidentified functional role of adiponectin (a hormone secreted by adipocytes) in modulating hippocampal neurogenesis and alleviating depression-like behaviors. To our knowledge, this is the first report showing that adiponectin may be an essential factor that mediates the antidepressant effects of physical exercise on the brain by adiponectin receptor 1-mediated activation of AMP-activated protein kinase. Our results reveal a possible mechanism by which exercise increases hippocampal neurogenesis and also suggest a promising therapeutic treatment for depression. Adiponectin (ADN) is an adipocyte-secreted protein with insulin-sensitizing, antidiabetic, antiinflammatory, and antiatherogenic properties. Evidence is also accumulating that ADN has neuroprotective activities, yet the underlying mechanism remains elusive. Here we show that ADN could pass through the blood–brain barrier, and elevating its levels in the brain increased cell proliferation and decreased depression-like behaviors. ADN deficiency did not reduce the basal hippocampal neurogenesis or neuronal differentiation but diminished the effectiveness of exercise in increasing hippocampal neurogenesis. Furthermore, exercise-induced reduction in depression-like behaviors was abrogated in ADN-deficient mice, and this impairment in ADN-deficient mice was accompanied by defective running-induced phosphorylation of AMP-activated protein kinase (AMPK) in the hippocampal tissue. In vitro analyses indicated that ADN itself could increase cell proliferation of both hippocampal progenitor cells and Neuro2a neuroblastoma cells. The neurogenic effects of ADN were mediated by the ADN receptor 1 (ADNR1), because siRNA targeting ADNR1, but not ADNR2, inhibited the capacity of ADN to enhance cell proliferation. These data suggest that adiponectin may play a significant role in mediating the effects of exercise on hippocampal neurogenesis and depression, possibly by activation of the ADNR1/AMPK signaling pathways, and also raise the possibility that adiponectin and its agonists may represent a promising therapeutic treatment for depression.

AMPK promotes p53 acetylation via phosphorylation and inactivation of SIRT1 in liver cancer cells

AMP-activated protein kinase (AMPK), a biologic sensor for cellular energy status, has been shown to act upstream and downstream of known tumor suppressors. However, whether AMPK itself plays a tumor suppressor role in cancer remains unclear. Here, we found that the α2 catalytic subunit isoform of AMPK is significantly downregulated in hepatocellular carcinoma (HCC). Clinicopathologic analysis revealed that underexpression of AMPK-α2 was statistically associated with an undifferentiated cellular phenotype and poor patient prognosis. Loss of AMPK-α2 in HCC cells rendered them more tumorigenic than control cells both in vitro and in vivo. Mechanistically, ectopic expression of AMPK enhanced the acetylation and stability of p53 in HCC cells. The p53 deacetylase, SIRT1, was phosphorylated and inactivated by AMPK at Thr344, promoting p53 acetylation and apoptosis of HCC cells. Taken together, our findings suggest that underexpression of AMPK is frequently observed in HCC, and that inactivation of AMPK promotes hepatocarcinogenesis by destabilizing p53 in a SIRT1-dependent manner.

P21-Activated Protein Kinase Is Overexpressed in Hepatocellular Carcinoma and Enhances Cancer Metastasis Involving c-Jun NH2-Terminal Kinase Activation and Paxillin Phosphorylation

Hepatocellular carcinoma (HCC) is one of the major malignancies in the world. The prognosis of HCC is poor, due to frequent intrahepatic metastasis and tumor recurrence. P21-activated protein kinase (Pak1), a main downstream effector of small Rho GTPases, Rac1 and Cdc42, plays an important role in the regulation of cell morphogenesis, motility, mitosis, and angiogenesis. Here, we show that Pak1 gene was overexpressed in human HCCs. Overexpression of Pak1 in human HCCs was associated with more aggressive tumor behavior in terms of more metastatic phenotype and more advanced tumor stages. In addition, HCC cell line stably expressing Pak1 displayed increased cell motility rates and, conversely, knockdown of endogenous Pak1 expression by small interfering RNA reduced the migration rates of HCC cells. In an established metastatic HCC cell line, we found that Pak1 was overexpressed compared with its primary HCC cell line and this overexpression was associated with higher cell motility. Importantly, we found that c-Jun NH(2)-terminal kinase (JNK) was activated in HCC cell lines overexpressing Pak1. Inhibition of the JNK activity by chemical inhibitor significantly reduced the migration rates of HCC cells via attenuation of paxillin phosphorylation at Ser(178). In conclusion, our results document that Pak1 is overexpressed in HCCs and plays an important role in the metastasis of HCC. The mechanism by which Pak1 induces cancer metastasis may involve activation of JNK and phosphorylation of paxillin.

Tissue factor pathway inhibitor-2 as a frequently silenced tumor suppressor gene in hepatocellular carcinoma†

In HCC, inactivation of tumor suppressor genes plays a significant role in carcinogenesis. Apart from deletions and mutations, growing evidence has indicated that epigenetic alterations including aberrant promoter methylation and histone deacetylation are also implicated in inactivation of tumor suppressor genes. The goal of this study was to identify epigenetically silenced candidate tumor suppressor genes in human HCC by comparing the changes in oligonucleotide microarray gene expression profiles in HCC cell lines upon pharmacological treatment with the demethylating agent 5‐Aza‐2′‐deoxycytidine (5‐Aza‐dC). By analyzing the gene expression profiles, we selected tissue factor pathway inhibitor‐2 (TFPI‐2), a Kunitz‐type serine protease inhibitor, for validation and further characterization. Our results showed that TFPI‐2 was frequently silenced in human HCC and HCC cell lines. TFPI‐2 was significantly underexpressed in approximately 90% of primary HCCs when compared with their corresponding nontumorous livers. TFPI‐2 promoter methylation was detected in 80% of HCC cell lines and 47% of human HCCs and was accompanied by reduced TFPI‐2 messenger RNA expression. In addition, TFPI‐2 expression in HCC cell lines can be robustly restored by combined treatment with 5‐Aza‐dC and histone deacetylase inhibitor trichostatin A. These findings indicate that TFPI‐2 is frequently silenced in human HCC via epigenetic alterations, including promoter methylation and histone deacetylation. Moreover, ectopic overexpression of TFPI‐2 significantly suppressed the proliferation and invasiveness of HCC cells. Conclusion: Our findings suggest that TFPI‐2 is a candidate tumor suppressor gene in human HCC. (HEPATOLOGY 2007;45:1129–1138.)

Lycium barbarum polysaccharides protect mice liver from carbon tetrachloride-induced oxidative stress and necroinflammation

ETHNOPHARMACOLOGICAL RELEVANCE Lycium barbarum has been used as a traditional Chinese medicine to nourish liver, kidneys and the eyes. AIM OF THE STUDY We investigated the protective mechanisms of Wolfberry, Lycium barbarum polysaccharides (LBP) in carbon tetrachloride (CCl(4))-induced acute liver injury. MATERIALS AND METHODS Mice were intraperitoneally injected with a 50 μl/kg CCl(4) to induce acute hepatotoxicity (8h) and were orally fed with LBP 2 h before the CCl(4) injection. There were six experimental groups of mice (n=7-8 per group), namely: control mice (vehicle only; 1 mg/kg LBP or 10 mg/kg LBP), CCl(4)-treated mice and CCl(4)+LBP treated mice (1 mg/kg LBP or 10 mg/kg LBP). RESULTS Pre-treatment with LBP effectively reduced the hepatic necrosis and the serum ALT level induced by CCl(4) intoxication. LBP remarkably inhibited cytochrome P450 2E1 expression and restored the expression levels of antioxidant enzymes. It also decreased the level of nitric oxide metabolism and lipid peroxidation induced by CCl(4). LBP attenuated hepatic inflammation via down-regulation of proinflammatory mediators and chemokines. Furthermore, LBP promoted liver regeneration after CCl(4) treatment. The protective effects of LBP against hepatotoxicity were partly through the down-regulation of nuclear factor kappa-B activity. CONCLUSION LBP is effective in reducing necroinflammation and oxidative stress induced by a chemical toxin, thus it has a great potential use as a food supplement in the prevention of hepatic diseases.

dp5/HRK is a c-jun target gene and required for apoptosis induced by potassium deprivation in cerebellar granule neurons

In cerebellar granule neurons, a BH3-only Bcl-2 family member, death protein 5/harakiri, is up-regulated in a JNK-dependent manner during apoptosis induced by potassium deprivation. However, it is not clear whether c-Jun is directly involved in the induction of dp5. Here, we showed that the up-regulation of dp5, but not fas ligand and bim, after potassium deprivation was suppressed by the expression of a dominant negative form of c-Jun. Deletion analysis of the 5′-flanking sequence of the dp5 gene revealed that a major responsive element responsible for the induction by potassium deprivation is an ATF binding site located at -116 to -109 relative to the transcriptional start site. Mutation of this site completely abolished promoter activation. Furthermore, a gel shift assay showed that a specific complex containing c-Jun and ATF2 recognized this site and increased in potassium-deprived cerebellar granule neurons. Chromatin immunoprecipitation demonstrated that c-Jun was able to bind to this site in vivo. Finally, we demonstrated that knockdown of Dp5 by small interfering RNA rescued neurons from potassium deprivation-induced apoptosis. Taken together, these results suggest that dp5 is a target gene of c-Jun and plays a critical role in potassium deprivation-induced apoptosis in cerebellar granule neurons.

The retroviral oncoprotein Tax targets the coiled-coil centrosomal protein TAX1BP2 to induce centrosome overduplication

Emerging evidence suggests that supernumerary centrosomes drive genome instability and oncogenesis. Human T-cell leukaemia virus type I (HTLV-I) is etiologically associated with adult T-cell leukaemia (ATL). ATL cells are aneuploid, but the causes of aneuploidy are incompletely understood. Here, we show that centrosome amplification is frequent in HTLV-I-transformed cells and that this phenotype is caused by the viral Tax oncoprotein. We also show that the fraction of Tax protein that localizes to centrosomes interacts with TAX1BP2, a novel centrosomal protein composed almost entirely of coiled-coil domains. Overexpression of TAX1BP2 inhibited centrosome duplication, whereas depletion of TAX1BP2 by RNAi resulted in centrosome hyperamplification. Our findings suggest that the HTLV-I Tax oncoprotein targets TAX1BP2 causing genomic instability and aneuploidy.