CRISPR screens identify cholesterol biosynthesis as a therapeutic target on stemness and drug resistance of colon cancer.

Cancer stem cells (CSCs) are responsible for tumor progression, recurrence, and drug resistance. To identify genetic vulnerabilities of colon cancer, we performed targeted CRISPR dropout screens comprising 657 Drugbank targets and 317 epigenetic regulators on two patient-derived colon CSC-enriched spheroids. Next-generation sequencing of pooled genomic DNAs isolated from surviving cells yielded therapeutic candidates. We unraveled 44 essential genes for colon CSC-enriched spheroids propagation, including key cholesterol biosynthetic genes (HMGCR, FDPS, and GGPS1). Cholesterol biosynthesis was induced in colon cancer tissues, especially CSC-enriched spheroids. The genetic and pharmacological inhibition of HMGCR/FDPS impaired self-renewal capacity and tumorigenic potential of the spheroid models in vitro and in vivo. Mechanistically, HMGCR or FDPS depletion impaired cancer stemness characteristics by activating TGF-ß signaling, which in turn downregulated expression of inhibitors of differentiation (ID) proteins, key regulators of cancer stemness. Cholesterol and geranylgeranyl diphosphate (GGPP) rescued the growth inhibitory and signaling effect of HMGCR/FDPS blockade, implying a direct role of these metabolites in modulating stemness. Finally, cholesterol biosynthesis inhibitors and 5-FU demonstrated antitumor synergy in colon CSC-enriched spheroids, tumor organoids, and xenografts. Taken together, our study unravels novel genetic vulnerabilities of colon CSC-enriched spheroids and suggests cholesterol biosynthesis as a potential target in conjunction with traditional chemotherapy for colon cancer treatment.

Squalene Epoxidase Induces Nonalcoholic Steatohepatitis Via Binding to Carbonic Anhydrase III and is a Therapeutic Target.

BACKGROUNDS & AIMS: Squalene epoxidase (SQLE) is the rate-limiting enzyme for cholesterol biosynthesis. We elucidated the functional significance, molecular mechanisms, and clinical impact of SQLE in nonalcoholic steatohepatitis (NASH). METHODS: We performed studies with hepatocyte-specific Sqle overexpression transgenic (Sqle tg) mice and mice given high-fat high-cholesterol (HFHC) or methionine- and choline-deficient (MCD) diet to induce NASH. SQLE downstream target carbonic anhydrase III (CA3) was identified using co-immunoprecipitation and Western Blot. Some mice were given SQLE inhibitor (terbinafine) and CA3 inhibitor (acetazolamide) to study the therapeutic effects in NASH. Human samples (N = 217) including 65 steatoses, 80 NASH, and 72 healthy controls were analyzed for SQLE levels in liver tissue and in serum. RESULTS: SQLE is highly up-regulated in human NASH and mouse models of NASH. Sqle tg mice triggered spontaneous insulin resistance, hepatic steatosis, liver injury, and accelerated HFHC or MCD diet-induced NASH development. Mechanistically, SQLE tg mice caused hepatic cholesterol accumulation, thereby triggering proinflammatory nuclear factor-κB signaling and steatohepatitis. SQLE directly bound to CA3, which induced sterol regulatory element-binding protein 1C activation, acetyl-CoA carboxylase, fatty acid synthase, and stearoyl-CoA desaturase1 expression and de novo hepatic lipogenesis. Combined targeting SQLE (terbinafine) and CA3 (acetazolamide) synergistically ameliorated NASH in mice with superior efficacy to either drug alone. Serum SQLE with CA3 could distinguish patients with NASH from steatosis and healthy controls (area under the receiver operating characteristic curve, 0.815; 95% confidence interval, 0.758-0.871). CONCLUSIONS: SQLE drives the initiation and progression of NASH through inducing cholesterol biosynthesis, and SQLE/CA3 axis-mediated lipogenesis. Combined targeting of SQLE and CA3 confers therapeutic benefit in NASH. Serum SQLE and CA3 are novel biomarkers for the noninvasive diagnosis of patients with NASH.

VSTM2A suppresses colorectal cancer and antagonizes Wnt signaling receptor LRP6.

Hyperactivation of Wnt/ß-catenin signaling pathway is a critical step in colorectal tumorigenesis. In this study, we identified that V-set and transmembrane domain containing 2A (VSTM2A) was a top-downregulated secreted protein that negatively regulated Wnt singling pathways in colorectal cancer (CRC). We investigated the functional mechanisms and clinical implication of VSTM2A in CRC. Methods: Function of VSTM2A was investigated in vitro and in vivo. VSTM2A binding partner was identified by mass spectrometry, immunoprecipitation and Western blot. The clinical impact of VSTM2A was assessed in 355 CRC patients and TCGA cohort. Results: VSTM2A protein was prominently silenced in CRC tumor tissues and cell lines mediated by its promoter hypermethylation. VSTM2A DNA promoter hypermethylation and VSTM2A protein downregulation was associated with poor survival of CRC patients. Ectopic expression of VSTM2A inhibited colon cancer cell lines and organoid growth, induced CRC cells apoptosis, inhibited cell migration and invasion, and suppressed growth of xenograft tumors in nude mice. VSTM2A was released from CRC cells through a canonical secretion pathway. Secreted VSTM2A significantly suppressed Wnt signaling pathway in colon cancer cells. Wnt signaling co-receptor LDL receptor related protein 6 (LRP6) was identified as a cell membrane binding partner of VSTM2A. Using deletion/mutation and immunoprecipitation, we demonstrated that VSTM2A bound to LRP6 E1-4 domain with its IgV domain. VSTM2A suppressed LRP6 phosphorylation in a time and dose dependent manner, and induced LRP6 endocytosis and lysosome-mediated degradation, which collectively contributing to the inactivation of Wnt signaling. Conclusions: VSTM2A is a novel antagonist of canonical Wnt signaling by directly binding to LRP6 and induces LRP6 endocytosis and degradation. VSTM2A is a potential prognostic biomarker for the outcome of CRC patients.

APLN promotes hepatocellular carcinoma through activating PI3K/Akt pathway and is a druggable target.

Background: The pathogenesis of hepatocellular carcinoma (HCC) is a multistep process contributed by the accumulation of molecular alterations. We identified Apelin (APLN) as an outlier gene up-regulated in hepatocellular carcinoma (HCC) through RNA-Seq and microarray analysis. We aimed to investigate its function, mechanism of action and clinical implication in HCC. Methods: Gene expression and clinical implication of APLN were assessed in multiple human HCC cohorts. Ectopic expression and silencing of APLN were performed to determine its function. The therapeutic potential of APLN and its downstream pathway was investigated using in vitro and in vivo models. Results: APLN overexpression was commonly observed in more than 80% of HCCs and independently predicted poorer survival of patients in three independent HCC cohorts. Apelin up-regulation was mediated by active ß-catenin, which binds to the APLN promoter to induce transcription. Ectopic APLN expression in HCC cells promoted cell proliferation, accelerated G1/S progression and inhibited apoptosis, whilst APLN knockdown exerted opposite effects in vitro and inhibited HCC xenograft growth in mice. Mechanistically, APLN activated phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathway via APLN receptor, leading to increased expression of phospho-glycogen synthase kinase 3ß (p-GSK3ß) and cyclin D1. Pharmacological targeting of APLN by ML221 was safe and effective in inhibiting APLN-PI3K/Akt cascade and HCC growth in vitro and in vivo. Conclusions: Our findings unraveled an oncogenic role of APLN in HCC, and that targeting of APLN might be a promising for HCC treatment. APLN may serve as an independent prognostic factor for HCC patients.

CAB39L elicited an anti-Warburg effect via a LKB1-AMPK-PGC1α axis to inhibit gastric tumorigenesis.

Metabolic dysfunction is a hallmark of gastric cancer (GC). In this study, we reported the identification of Calcium Binding Protein 39-Like (CAB39L) as a novel regulator of tumor metabolism in GC. CAB39L mRNA was frequently silenced by promoter methylation in GC cell lines and tissues. Functional studies suggested that CAB39L functions as a tumor suppressor, as overexpression of CAB39L elicited suppression of multiple cancer phenotypes both in GC cells and an orthotopic mouse model; whilst its knockdown promoted tumorigenesis. Mechanistically, CAB39L interacted with LKB1-STRAD complex and induced LKB1, leading to the phosphorylation and activation of AMPKα/ß. LKB1-AMPK activation in GC cell lines was tumor suppressive, as metformin (an AMPK activator) inhibited GC cell growth in the CAB39L-silenced cells. Moreover, knockdown of LKB1 reversed growth inhibitory effect of CAB39L, indicating that tumor suppression by CAB39L depended on LKB1-AMPK. RNAseq and gene set enrichment analysis revealed that CAB39L was closely correlated with oxidative phosphorylation and mitochondrial biogenesis. Consistently, CAB39L-induced p-AMPK elicited PGC1α phosphorylation and increased the expression of genes involved in mitochondrial respiration complexes. Accordingly, CAB39L reversed the Warburg effect in GC, as evidenced by enhanced oxygen consumption rate and reduced extracellular acidification rate; inversely, CAB39L knockdown promoted a metabolic shift towards the Warburg phenotype. In GC patients, CAB39L promoter hypermethylation was correlated with poor prognosis. Our data demonstrate that CAB39L is a novel tumor suppressor which suppresses tumorigenesis by promoting LKB1-AMPK-PGC1α axis, thereby preventing a metabolic shift that drives carcinogenesis. CAB39L methylation is a potential prognostic biomarker for GC patients.

Sodium Channel Subunit SCNN1B Suppresses Gastric Cancer Growth and Metastasis via GRP78 Degradation.

There remains a paucity of functional biomarkers in gastric cancer. Here, we report the identification of the sodium channel subunit SCNN1B as a candidate biomarker in gastric cancer. SCNN1B mRNA expression was silenced commonly by promoter hypermethylation in gastric cancer cell lines and primary tumor tissues. Tissue microarray analysis revealed that high expression of SCNN1B was an independent prognostic factor for longer survival in gastric cancer patients, especially those with late-stage disease. Functional studies demonstrated that SCNN1B overexpression was sufficient to suppress multiple features of cancer cell pathophysiology in vitro and in vivo Mechanistic investigations revealed that SCNN1B interacted with the endoplasmic reticulum chaperone, GRP78, and induced its degradation via polyubiquitination, triggering the unfolded protein response (UPR) via activation of PERK, ATF4, XBP1s, and C/EBP homologous protein and leading in turn to caspase-dependent apoptosis. Accordingly, SCNN1B sensitized gastric cancer cells to the UPR-inducing drug tunicamycin. GRP78 overexpression abolished the inhibitory effect of SCNN1B on cell growth and migration, whereas GRP78 silencing aggravated growth inhibition by SCNN1B. In summary, our results identify SCNN1B as a tumor-suppressive function that triggers UPR in gastric cancer cells, with implications for its potential clinical applications as a survival biomarker in gastric cancer patients. Cancer Res; 77(8); 1968-82. ©2017 AACR.

SLC25A22 Promotes Proliferation and Survival of Colorectal Cancer Cells With KRAS Mutations and Xenograft Tumor Progression in Mice via Intracellular Synthesis of Aspartate.

BACKGROUND & AIMS: Many colorectal cancer (CRC) cells contain mutations in KRAS. Analyses of CRC cells with mutations in APC or CTNNB1 and KRAS identified SLC25A22, which encodes mitochondrial glutamate transporter, as a synthetic lethal gene. We investigated the functions of SLC25A22 in CRC cells with mutations in KRAS. METHODS: We measured levels of SLC25A22 messenger RNA and protein in paired tumor and nontumor colon tissues collected from 130 patients in Hong Kong and 17 patients in China and compared protein levels with patient survival times. Expression of SLC25A22 was knocked down in KRAS mutant CRC cell lines (DLD1, HCT116, LOVO, SW480, SW620, and SW1116) and CRC cell lines without mutations in KRAS (CACO-2, COLO205, HT29, and SW48); cells were analyzed for colony formation, proliferation, glutaminolysis and aspartate synthesis, and apoptosis in Matrigel and polymerase chain reaction array analyses. DLD1 and HCT116 cells with SLC25A22 knockdown were grown as xenograft tumors in nude mice; tumor growth and metastasis were measured. SLC25A22 was expressed ectopically in HCT116 cells, which were analyzed in vitro and grown as xenograft tumors in nude mice. RESULTS: Levels of SLC25A22 messenger RNA and protein were increased in colorectal tumor tissues compared with matched nontumor colon tissues; increased protein levels were associated with shorter survival times of patients (P = .01). Knockdown of SLC25A22 in KRAS mutant CRC cells reduced their proliferation, migration, and invasion in vitro, and tumor formation and metastasis in mice, compared with cells without SLC25A22 knockdown. Knockdown of SLC25A22 reduced aspartate biosynthesis, leading to apoptosis, decreased cell proliferation in KRAS mutant CRC cells. Incubation of KRAS mutant CRC cells with knockdown of SLC25A22 with aspartate increased proliferation and reduced apoptosis, which required GOT1, indicating that oxaloacetate is required for cell survival. Decreased levels of oxaloacetate in cells with knockdown of SLC25A22 reduced regeneration of oxidized nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate. Reduced oxidized nicotinamide adenine dinucleotide inhibited glycolysis and decreased levels of adenosine triphosphate, which inactivated mitogen-activated protein kinase kinase and extracellular signal-regulated kinase signaling via activation of AMP-activated protein kinase. An increased ratio of oxidized nicotinamide adenine dinucleotide phosphate to reduced nicotinamide adenine dinucleotide phosphate induced oxidative stress and glutathione oxidation, which suppressed cell proliferation. Asparagine synthetase mediated synthesis of asparagine from aspartate to promote cell migration. CONCLUSIONS: SLC25A22 promotes proliferation and migration of CRC cells with mutations KRAS, and formation and metastasis of CRC xenograft tumors in mice. Patients with colorectal tumors that express increased levels of SLC25A22 have shorter survival times than patients whose tumors have lower levels. SLC25A22 induces intracellular synthesis of aspartate, activation of mitogen-activated protein kinase kinase and extracellular signal-regulated kinase signaling and reduces oxidative stress.

Dysregulated Lysine Acetyltransferase 2B Promotes Inflammatory Bowel Disease Pathogenesis Through Transcriptional Repression of Interleukin-10.

BACKGROUND AND AIMS: Accumulating evidence supports epigenetic modifications in mediating intestinal immunity in inflammatory bowel disease [IBD] pathogenesis. This study aimed to identify key dysregulated epigenetic modulators and the molecular downstream pathways in IBD. METHODS: Expression of 116 well-defined epigenetic modulators was profiled and validated in 96 intestinal tissues from patients with Crohn's disease [CD], ulcerative colitis [UC], and healthy controls using quantitative reverse transcriptase polymerase chain reaction [QRT-PCR], western blot, and immunohistochemistry. Dysregulation of histone modifications and IBD-related cytokines were examined by chromatin immunoprecipitation, luciferase activity, and gene expression analyses in normal colonic epithelial cell line, NCM460, upon small-molecule inhibition or RNA interference, followed by validation in primary colonic tissues. RESULTS: Targeted expression profiling uncovered seven differentially expressed epigenetic modulators, of which the down-regulation of lysine acetyltransferase 2B [KAT2B] mRNA and protein was the most significant and was consequently validated in inflamed CD and UC compared with healthy colonic tissues. KAT2B protein localised abundantly in nuclei of normal colonic epithelium but diminished in paired inflamed CD and UC tissues. Pharmacological inhibition of KAT2B by anacardic acid in NCM460 cells reduced the levels of histone H4 lysine 5 acetylation [H4K5ac] and interleukin-10 [IL-10] in a dose-dependent manner. Knockdown of KAT2B reduced the IL-10 promoter occupancy of KAT2B and H4K5ac, resulting in transcriptional silencing. IL-10 level was also diminished in inflamed IBD tissues. CONCLUSIONS: Our findings demonstrated a novel epigenetic mechanism of IL-10 dysregulation in IBD. Down-regulation of KAT2B may disrupt the innate and adaptive inflammatory responses due to the suppression of this crucial anti-inflammatory cytokine.

Carbonic anhydrase IV inhibits colon cancer development by inhibiting the Wnt signalling pathway through targeting the WTAP-WT1-TBL1 axis.

OBJECTIVE: We found that carbonic anhydrase IV (CA4), a member of the carbonic anhydrases, is silenced in colorectal cancer (CRC). We analysed its epigenetic inactivation, biological effects and prognostic significance in CRC. DESIGN: The biological functions of CA4 were determined by in vitro and in vivo tumorigenicity assays. The CA4 co-operator was identified by immunoprecipitation and mass spectrometry. CA4 downstream effectors and signalling pathways were elucidated by promoter luciferase assay, electrophoretic mobility shift assay and chromatin immunoprecipitation. The clinical impact of CA4 was assessed in 115 patients with CRC. RESULTS: CA4 was silenced in all nine CRC cell lines and 92.6% of CRC tumours. The promoter hypermethylation contributed to the inactivation of CA4, and it was detected in 75.7% of the patients with CRC. After a median follow-up of 49.3 months, multivariate analysis showed that the patients with CA4 hypermethylation had a recurrence of Stage II/III CRC. The re-expression of CA4 inhibited cell proliferation, induced apoptosis and cell cycle arrest in the G1 phase. CA4 inhibited the activity of the Wnt signalling pathway and mediated the degradation of ß-catenin. CA4 interacted with Wilms' tumour 1-associating protein (WTAP) and induced WTAP protein degradation through polyubiquitination. Moreover, CA4 promoted the transcriptional activity of Wilms' tumour 1 (WT1), an antagonist of the Wnt pathway, which resulted in the induction of transducin ß-like protein 1 (TBL1) and the degradation of ß-catenin. CONCLUSIONS: CA4 is a novel tumour suppressor in CRC through the inhibition of the Wnt signalling pathway by targeting the WTAP-WT1-TBL1 axis. CA4 methylation may serve as an independent biomarker for the recurrence of CRC.

MDGA2 is a novel tumour suppressor cooperating with DMAP1 in gastric cancer and is associated with disease outcome.

BACKGROUND: Using the promoter methylation assay, we have shown that MDGA2 (MAM domain containing glycosylphosphatidylinositol anchor 2) is preferentially methylated in gastric cancer. We analysed its biological effects and prognostic significance in gastric cancer. METHODS: MDGA2 methylation status was evaluated by combined bisulfite restriction analysis and bisulfite genomic sequencing. The effects of MDGA2 re-expression or knockdown on cell proliferation, apoptosis and the cell cycle were determined. MDGA2 interacting protein was identified by mass spectrometry and MDGA2-related cancer pathways by reporter activity and PCR array analyses. The clinical impact of MDGA2 was assessed in 218 patients with gastric cancer. RESULTS: MDGA2 was commonly silenced in gastric cancer cells (10/11) and primary gastric cancers due to promoter hypermethylation. MDGA2 significantly inhibited cell proliferation by causing G1-S cell cycle arrest and inducing cell apoptosis in vitro, and suppressed xenograft tumour growth in both subcutaneous and orthotopic xenograft mouse models (both p<0.001). The anti-tumorigenic effect of MDGA2 was mediated through direct stabilising of DNA methyltransferase 1 associated protein 1 (DMAP1), which played a tumour suppressive role in gastric cancer. This interaction activated their downstream key elements of p53/p21 signalling cascades. Moreover, promoter methylation of MDGA2 was detected in 62.4% (136/218) of gastric cancers. Multivariate analysis showed that patients with MDGA2 hypermethylation had a significantly decreased survival (p=0.005). Kaplan-Meier survival curves showed that MDGA2 hypermethylation was significantly associated with shortened survival in patients with early gastric cancer. CONCLUSIONS: MDGA2 is a critical tumour suppressor in gastric carcinogenesis; its hypermethylation is an independent prognostic factor in patients with gastric cancer.

CXC chemokine receptor 3 promotes steatohepatitis in mice through mediating inflammatory cytokines, macrophages and autophagy.

BACKGROUND & AIMS: CXC chemokine receptor 3 (CXCR3) is involved in virus-related chronic liver inflammation. However, the role of CXCR3 in non-alcoholic steatohepatitis (NASH) remains unclear. We aimed to investigate the role of CXCR3 in NASH. METHODS: Human liver tissues were obtained from 24 non-alcoholic fatty liver disease (NAFLD) patients and 20 control subjects. CXCR3 knockout (CXCR3(-/-)), obese db/db mice and their wild-type (WT) littermates were used in both methionine-and-choline-deficient (MCD) diet and high-fat high-carbohydrate high-cholesterol (HFHC) diet-induced NASH models. In addition, MCD-fed WT mice were administrated with CXCR3 specific antagonists. RESULTS: CXCR3 was significantly upregulated in liver tissues of patients with NAFLD and in dietary-induced NASH animal models. Compared with WT littermates, CXCR3(-/-) mice were more resistant to both MCD and HFHC diet-induced steatohepatitis. Induction of CXCR3 in dietary-induced steatohepatitis was associated with the increased expression of hepatic pro-inflammatory cytokines, activation of NF-κB, macrophage infiltration and T lymphocytes accumulation (Th1 and Th17 immune response). CXCR3 was also linked to steatosis through inducing hepatic lipogenic genes. Moreover, CXCR3 is associated with autophagosome-lysosome impairment and endoplasmic reticulum (ER) stress in steatohepatitis as evidenced by LC3-II and p62/SQSTM1 accumulation and the induction of GRP78, phospho-PERK and phospho-eIF2α. Inhibition of CXCR3 using CXCR3 antagonist significantly suppressed MCD-induced steatosis and hepatocytes injury in AML-12 hepatocytes. Blockade of CXCR3 using CXCR3 antagonists in mice reversed the established steatohepatitis. CONCLUSIONS: CXCR3 plays a pivotal role in NASH development by inducing production of cytokines, macrophage infiltration, fatty acid synthesis and causing autophagy deficiency and ER stress.

Histone Deacetylase HDAC8 Promotes Insulin Resistance and ß-Catenin Activation in NAFLD-Associated Hepatocellular Carcinoma.

The growing epidemic of obesity, which causes nonalcoholic fatty liver disease (NAFLD) and the more severe phenotype nonalcoholic steatohepatitis (NASH), has paralleled the increasing incidence of hepatocellular carcinoma (HCC). Accumulating evidence demonstrates that overnutrition and metabolic pathways can trigger modifications of DNA and histones via deregulation of chromatin modifiers, resulting in aberrant transcriptional activity. However, the epigenetic regulation of HCC development in NAFLD remains obscure. Here, we uncover key epigenetic regulators using both dietary and genetic obesity-promoted HCC models through quantitative expression profiling and characterize the oncogenic activities of histone deacetylase HDAC8 in NAFLD-associated hepatocarcinogenesis. HDAC8 is directly upregulated by the lipogenic transcription factor SREBP-1 where they are coexpressed in dietary obesity models of NASH and HCC. Lentiviral-mediated HDAC8 attenuation in vivo reversed insulin resistance and reduced NAFLD-associated tumorigenicity. HDAC8 modulation by genetic and pharmacologic approaches inhibited p53/p21-mediated apoptosis and G2-M phase cell-cycle arrest and stimulated ß-catenin-dependent cell proliferation. Mechanistically, HDAC8 physically interacted with the chromatin modifier EZH2 to concordantly repress Wnt antagonists via histone H4 deacetylation and H3 lysine 27 trimethylation. In human NAFLD-associated HCC, levels of SREBP-1, HDAC8, EZH2, H4 deacetylation, H3K27me3, and active ß-catenin were all correlated positively in tumors compared with nontumor tissues. Overall, our findings show how HDAC8 drives NAFLD-associated hepatocarcinogenesis, offering a novel epigenetic target to prevent or treat HCC in obese patients.

A CCRK-EZH2 epigenetic circuitry drives hepatocarcinogenesis and associates with tumor recurrence and poor survival of patients.

BACKGROUND & AIMS: Aberrant chromatin modification is a key feature of hepatocellular carcinoma (HCC), which is characterized by strong sexual dimorphism. Both enhancer of zeste homolog 2 (EZH2) and cell cycle-related kinase (CCRK) contribute to hepatocarcinogenesis, yet whether the two oncogenic factors have functional crosstalk is unknown. METHODS: Cellular proliferation and tumorigenicity upon transgenic expression and RNA interference were determined by colony formation and soft agar assays, xenograft, orthotopic and diethylnitrosamine-induced HCC models. Gene regulation was assessed by chromatin immunoprecipitation, site-directed mutagenesis, luciferase reporter, co-immunoprecipitation and expression analyses. Protein levels in clinical specimens were correlated with clinicopathological parameters and patient survival rates. RESULTS: Ectopic CCRK expression in immortalized human liver cells increased EZH2 and histone H3 lysine 27 trimethylation (H3K27me3) to stimulate proliferation and tumor formation. Conversely, knockdown of CCRK reduced EZH2/H3K27me3 levels and decreased HCC cell growth, which could be rescued by EZH2 over-expression. Mechanistically, GSK-3ß phosphorylation by CCRK activated a ß-catenin/TCF/E2F1/EZH2 transcriptional feedback loop to epigenetically enhance androgen receptor (AR) signaling. Simultaneously, the phosphorylation of AKT/EZH2 by CCRK facilitated the co-occupancy of CCRK promoter by EZH2-AR and its subsequent transcriptional activation, thus forming a self-reinforcing circuitry. Lentiviral-mediated knockdown of CCRK, which abrogated the phosphorylation-transcriptional network, prevented diethylnitrosamine-induced tumorigenicity. More importantly, the hyperactivation of the CCRK-EZH2 circuitry in human HCCs correlated with tumor recurrence and poor survival. CONCLUSIONS: These findings uncover an epigenetic vicious cycle in hepatocarcinogenesis that operates through reciprocal regulation of CCRK and EZH2, providing novel therapeutic strategy for HCC.

microRNA-29b prevents liver fibrosis by attenuating hepatic stellate cell activation and inducing apoptosis through targeting PI3K/AKT pathway.

microRNA-29b (miR-29b) is known to be associated with TGF-ß-mediated fibrosis, but the mechanistic action of miR-29b in liver fibrosis remains unclear and is warranted for investigation. We found that miR-29b was significantly downregulated in human and mice fibrotic liver tissues and in primary activated HSCs. miR-29b downregulation was directly mediated by Smad3 through binding to the promoter of miR-29b in hepatic stellate cell (HSC) line LX1, whilst miR-29b could in turn suppress Smad3 expression. miR-29b transduction in the liver of mice prevented CCl4 induced-fibrogenesis, concomitant with decreased expression of α-SMA, collagen I and TIMP-1. Ectopic expression of miR-29b in activated HSCs (LX-1, HSC-T6) inhibited cell viability and colony formation, and caused cell cycle arrest in G1 phase by downregulating cyclin D1 and p21cip1. Further, miR-29b induced apoptosis in HSCs mediated by caspase-9 and PARP. miR-29b inhibited its downstream effectors of PIK3R1 and AKT3 through direct targeting their 3'UTR regions. Moreover, knockdown of PIK3R1 or AKT3 suppressed α-SMA and collagen I and induced apoptosis in both HSCs and in mice. In conclusion, miR-29b prevents liver fibrogenesis by inhibiting HSC activation and inducing HSC apoptosis through inhibiting PI3K/AKT pathway. These results provide novel mechanistic insights for the anti-fibrotic effect of miR-29b.

CXCL10 plays a key role as an inflammatory mediator and a non-invasive biomarker of non-alcoholic steatohepatitis.

BACKGROUND & AIMS: Perpetuate liver inflammation is crucial in the pathogenesis of non-alcoholic steatohepatitis (NASH). Expression of CXCL10, a pro-inflammatory cytokine, correlates positively with obesity and type 2 diabetes. Whether CXCL10 plays a role in NASH was unknown. We aimed to investigate the functional and clinical impact of CXCL10 in NASH. METHODS: Cxcl10 gene-deleted (Cxcl10(-/-)) and C57BL/6 wild type (WT) mice were fed a methionine- and choline-deficient (MCD) diet for 4 or 8 weeks. In other experiments, we injected neutralizing anti-CXCL10 mAb into MCD-fed WT mice. Human serum was obtained from 147 patients with biopsy-proven non-alcoholic fatty liver disease and 73 control subjects. RESULTS: WT mice, fed the MCD diet, developed steatohepatitis with higher hepatic CXCL10 expression. Cxcl10(-/-) mice were refractory to MCD-induced steatohepatitis. We further revealed that CXCL10 was associated with the induction of important pro-inflammatory cytokines (TNF-α, IL-1ß, and MCP-1) and activation of the NF-κB pathway. CXCL10 was linked to steatosis through upregulation of the lipogenic factors SREBP-1c and LXR, and also to oxidative stress (upregulation of CYP2E1 and C/EBPß). Blockade of CXCL10 protected against hepatocyte injury in vitro and against steatohepatitis development in mice. We further investigated the clinical impact of CXCL10 and found circulating and hepatic CXCL10 levels were significantly higher in human NASH. Importantly, the circulating CXCL10 level was correlated with the degree of lobular inflammation and was an independent risk factor for NASH patients. CONCLUSIONS: We demonstrate for the first time that CXCL10 plays a pivotal role in the pathogenesis of experimental steatohepatitis. CXCL10 maybe a potential non-invasive biomarker for NASH patients.

Odd-skipped related 1 is a novel tumour suppressor gene and a potential prognostic biomarker in gastric cancer.

We report that the odd-skipped related 1 (OSR1) gene encoding a zinc-finger transcription factor was preferentially methylated in gastric cancer by genome-wide methylation screening. OSR1 expression was frequently silenced or down-regulated in gastric cancer cell lines. OSR1 expression was also significantly down-regulated at both mRNA and protein levels in primary gastric cancer tissues compared with adjacent normal tissues. The silencing or down-regulation of OSR1 was closely associated with promoter hypermethylation. Overexpression of OSR1 significantly inhibited cell growth, arrested the cell cycle, and induced apoptosis in the gastric cancer cell lines AGS, MKN28, and MGC803. Conversely, knockdown of OSR1 by OSR1-short hairpin RNA significantly enhanced cell growth, promoted the cell cycle, and inhibited apoptosis in the normal gastric epithelial cell line GES1. The dual-luciferase reporter assay revealed that OSR1 activated p53 transcription and repressed the T-cell factor (TCF)/lymphoid enhancer factor (LEF). Complementary DNA expression array and western blotting showed that OSR1 increased the expression of nuclear p53, p21, Fas, and death receptor-5, and suppressed the expression of cyclin D1 and cyclin-dependent kinase 4 in the p53 signalling pathway. In addition, OSR1 suppressed the expression of cytoplasmic ß-catenin, TCF-1, and LEF1 in the Wnt/ß-catenin signalling pathway. OSR1 methylation was detected in 51.8% of primary gastric cancer patients (85 of 164) by bisulphite genomic sequencing. Multivariate Cox regression analysis showed that OSR1 methylation was an independent predictor of poor survival. Kaplan-Meier survival curves revealed that OSR1 methylation was associated with shortened survival in TNM stage I-III patients. In conclusion, OSR1 acts as a functional tumour suppressor through the transcriptional activation of p53 and repression of TCF/LEF in gastric cancer. Detection of OSR1 methylation may serve as a potential biomarker of the early stage of gastric cancer.

Cell cycle-related kinase mediates viral-host signalling to promote hepatitis B virus-associated hepatocarcinogenesis.

BACKGROUND: Androgen receptor (AR) signalling contributes to male predominance in hepatocellular carcinoma (HCC), which is more pronounced in HBV-endemic areas. Cell cycle-related kinase (CCRK) is essential for AR-induced hepatocarcinogenesis but its molecular function in HBV-associated HCC remains obscure. OBJECTIVE: To determine the molecular function of CCRK in HBV-associated HCC. DESIGN: Transcriptional regulation was assessed by chromatin immunoprecipitation, promoter mutation and luciferase reporter assays. Hepatocellular proliferation and tumourigenesis were examined by colony formation, soft agar assays and using HBV X protein (HBx) transgenic mice with low-dose exposure to diethylnitrosamine. Protein expressions were examined in clinical samples and correlated with patient survival by log-rank Mantel-Cox test. RESULTS: Overexpression of CCRK, but not its kinase-defective mutant, activated ß-catenin/T cell factor signalling through phosphorylation of glycogen synthase kinase-3ß (GSK-3ß) at Ser9, led to upregulation of AR transcriptional activity and, subsequently, expression of HBx. The viral transactivator in turn induced CCRK expression through enhanced AR signalling, thus forming a positive regulatory loop. RNA interference silencing of CCRK, which suppressed the CCRK/GSK-3ß/ß-catenin/AR regulatory loop, significantly suppressed HBx-induced hepatocellular proliferation (p=0.001) and transformation (p<0.001) and remarkably reduced >80% diethylnitrosamine-mediated hepatocarcinogenesis in HBx transgenic mice. Finally, patients with HBV-associated HCC with concordant overexpression of CCRK, GSK-3ß phosphorylation at Ser9, active dephosphorylated ß-catenin and AR phosphorylation at Ser81 had poorer overall (HR=31.26, p<0.0001) and disease-free (HR=3.60, p<0.01) survival rates. CONCLUSIONS: Our findings highlight the critical role of CCRK in a self-reinforcing circuitry that regulates HBV-associated hepatocarcinogenesis. Further characterisation of this intricate viral-host signalling may provide new prognostic biomarkers and therapeutic targets for HCC treatment.

Helicobacter pylori-induced STAT3 activation and signalling network in gastric cancer.

BACKGROUND: Helicobacter pylori (H. pylori) is the most important gastric carcinogen. However, the mechanisms of H. pylori induced gastric carcinogenesis through STAT3 activation are largely unknown. We evaluated the effects of H. pylori infection on STAT3 activation and dissected the signalling network of STAT3 in H. pylori- infected gastric carcinogenesis. METHODS: The expression of phospho-STAT3 (pSTAT3) was evaluated by immunohistochemistry and western blot. Gene expression array and chromatin immunoprecipitation were used to dissect the STAT3 signalling network on H. pylori co-cultured AGS. RESULTS: pSTAT3 was significantly higher in H. pylori -positive gastritis than in H. pylori -negative gastritis ( P = 0.003). In addition, 98% of H. pylori positive intestinal metaplasia specimens showed STAT3 activation, whereas pSTAT3 was significantly decreased in all 43 specimens one year after H. pylori eradication ( P < 0.001). Moreover, pSTAT3 was only detected in the H. pylori -infected gastric tissues of mice but not in control mice. We further identified 6 candidates ( BRUNOL4, FGFR1, SHOX2, JAK3, MAPK8, and PDPN ) were directly up-regulated by H. pylori induced STAT3 activation. CONCLUSION: H. pylori infection triggers the activation of STAT3 and de-regulates multitude of tumorigenic genes which may contribute to the initiation and progression of gastric cancer.

Inhibitory role of Smad7 in hepatocarcinogenesis in mice and in vitro.

Smad7 is a principal inhibitor of the TGFß-Smad signalling pathway. We have investigated the functional significance of Smad7 in hepatocellular carcinoma (HCC). Smad7 knockout (KO) and wild-type (WT) mice were injected with diethylnitrosamine (DEN) to induce HCC. The effects of Smad7 on cellular features were examined in HCC cells, using a Smad7 over-expression or deletion approach. Signalling pathway components modulated by Smad7 in HCC were evaluated using luciferase reporter assay and co-immunoprecipitation. Smad7 was down-regulated in human HCCs compared with the adjacent normal tissues (p < 0.001). Smad7 KO mice were more susceptible to DEN-induced HCC than WT mice (78% versus 22%, p < 0.05). HCCs from KO mice displayed a greater proliferation activity (p < 0.05) and a reduced apoptotic index compared with WT littermates (p < 0.05). Deletion of Smad7 promoted cell proliferation in primary cultured HCC cells. In addition, over-expression of Smad7 in HCC cell lines markedly suppressed cell growth (p < 0.0001) and colony formation (p < 0.01). Cell cycle analysis revealed an increase in the G1 phase and a reduction in the S-phase populations, accompanied by up-regulation of p27(Kip1) and down-regulation of cyclin D1. Smad7 increased cell apoptosis (p < 0.01) by mediating an intrinsic [caspase-9, caspase-3 and poly(ADP-ribose) polymerase] apoptotic pathway. Moreover, Smad7 inhibited NF-κB signalling by interacting with TAB2, an upstream activator of NF-κB, and inhibited TGFß signalling by suppressing phosphorylation of Smad3. In conclusion, loss of Smad7 enhances susceptibility to HCC. Smad7 suppresses HCC cell growth by inhibiting proliferation and G1 -S phase transition and inducing apoptosis through attenuation of NF-κB and TGFß signalling. Smad7 acts as a potential tumour suppressor in liver.

Helicobacter pylori causes epigenetic dysregulation of FOXD3 to promote gastric carcinogenesis.

BACKGROUND & AIMS: Deregulation of forkhead box (Fox) proteins, an evolutionarily conserved family of transcriptional regulators, leads to tumorigenesis. Little is known about their regulation or functions in the pathogenesis of gastric cancer. Promoter hypermethylation occurs during Helicobacter pylori-induced gastritis. We investigated whether the deregulated genes contribute to gastric tumorigenesis. METHODS: We used integrative genome-wide scans to identify concomitant hypermethylated genes in mice infected with H pylori and human gastric cancer samples. We also analyzed epigenetic gene silencing in gastric tissues from patients with H pylori infection and gastritis, intestinal metaplasia, gastric tumors, or without disease (controls). Target genes were identified by chromatin immunoprecipitation microarrays and expression and luciferase reporter analyses. RESULTS: Methylation profile analyses identified the promoter of FOXD3 as the only genomic region with increased methylation in mice and humans during progression of H pylori-associated gastric tumors. FOXD3 methylation also correlated with shorter survival times of patients with gastric cancer. Genome demethylation reactivated FOXD3 expression in gastric cancer cell lines. Transgenic overexpression of FOXD3 significantly inhibited gastric cancer cell proliferation and invasion, and reduced growth of xenograft tumors in mice, at least partially, by promoting tumor cell apoptosis. FOXD3 bound directly to the promoters of, and activated transcription of, genes encoding the cell death regulators CYFIP2 and RARB. Levels of FOXD3, CYFIP2, and RARB messenger RNAs were reduced in human gastric tumor samples, compared with control tissues. CONCLUSIONS: FOXD3-mediated transcriptional control of tumor suppressors is deregulated by H pylori infection-induced hypermethylation; this could perturb the balance between cell death and survival. These findings identify a pathway by which epigenetic changes affect gastric tumor suppression.