Yingying Lee

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 β-catenin/T cell factor signalling through phosphorylation of glycogen synthase kinase-3β (GSK-3β) 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-3β/β-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-3β phosphorylation at Ser9, active dephosphorylated β-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.

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-3β phosphorylation by CCRK activated a β-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.

W1919 Psychological Stress in Early Life Leads to Abnormal Postprandial Ghrelin Profile and Early Satiety in Adulthood: Validation of a Rat Model for Functional Dyspepsia (FD)

G A A b st ra ct s rats is clearly evident in H&E-stained sections whereby villi height is lower and muscularis atrophy is evident. Conclusions: Our data indicate that SMA blood flow is compromised in rats after T3 SCI at a time when gastric emptying and motility are also reduced. Duodenal administration of a liquid meal was insufficient in triggering visceral hyperemia. We suggest that impaired systemic vascular flow diminishes compensatory mesenteric blood flow to support the GI tract during digestion. The resulting GI ischemia may be an underlying pathology leading to gastric dysfunction seen following SCI. Support: NINDS 049177

Dissecting the pleiotropic actions of HBx mutants against hypoxia in hepatocellular carcinoma

Error-prone integration of the hepatitis B virus X protein (HBx) into the hepatocellular genome generates a multitude of mutants exerting diverse effects on the development and progression of hepatocellular carcinoma (HCC). A recent study by Lai and colleagues revealed the disparate regulatory activity of clinically-predominant HBx mutants towards hypoxia-inducible factor-1α (HIF-1α), a central regulator of tumor angiogenesis, proliferation, metastasis and differentiation. These findings have shed insight into specific viral contribution of hypoxic response during hepatocarcinogenesis.

Loss of tumor suppressor IGFBP4 drives epigenetic reprogramming in hepatic carcinogenesis

Abstract Genomic sequencing of hepatocellular carcinoma (HCC) uncovers a paucity of actionable mutations, underscoring the necessity to exploit epigenetic vulnerabilities for therapeutics. In HCC, EZH2-mediated H3K27me3 represents a major oncogenic chromatin modification, but how it modulates the therapeutic vulnerability of signaling pathways remains unknown. Here, we show EZH2 acts antagonistically to AKT signaling in maintaining H3K27 methylome through epigenetic silencing of IGFBP4. ChIP-seq revealed enrichment of Ezh2/H3K27me3 at silenced loci in HBx-transgenic mouse-derived HCCs, including Igfbp4 whose down-regulation significantly correlated with EZH2 overexpression and poor survivals of HCC patients. Functional characterizations demonstrated potent growth- and invasion-suppressive functions of IGFBP4, which was associated with transcriptomic alterations leading to deregulation of multiple signaling pathways. Mechanistically, IGFBP4 stimulated AKT/EZH2 phosphorylation to abrogate H3K27me3-mediated silencing, forming a reciprocal feedback loop that suppressed core transcription factor networks (FOXA1/HNF1A/HNF4A/KLF9/NR1H4) for normal liver homeostasis. Consequently, the in vivo tumorigenicity of IGFBP4-silenced HCC cells was vulnerable to pharmacological inhibition of EZH2, but not AKT. Our study unveils chromatin regulation of a novel liver tumor suppressor IGFBP4, which constitutes an AKT-EZH2 reciprocal loop in driving H3K27me3-mediated epigenetic reprogramming. Defining the aberrant chromatin landscape of HCC sheds light into the mechanistic basis of effective EZH2-targeted inhibition.

Abstract 4519: Nanoscale ChIP-seq analysis in primary hepatocellular carcinoma tissues reveals tumor-specific chromatin deregulation for oncogene activation

Introduction: Emerging evidence that epigenetics converts alterations in nutrient and metabolism into heritable pattern of gene expression has profound implications in understanding human physiology and diseases. Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of metabolic syndrome including obesity and diabetes which elevate the risk of hepatocellular carcinoma (HCC). The metabolites derived from excessive insulin, glucose and lipid may perturb epigenetic gene regulation through DNA methylation, histone modifications, and RNA interference, leading to activation of pro-inflammatory signaling and oncogenic pathways [1] . Transcriptional and chromatin patterns are mostly interrogated by chromatin immunoprecipitation (ChIP). However, conventional ChIP protocols necessitate the use of large numbers of cells and usually limit to the studies in cell lines, which may display epigenetic patterns distinct from primary tumors. Aims and methods: To study chromatin deregulation in primary human NAFLD-related HCC tumor and adjacent non-tumor tissues, we performed nanoscale ChIP sequencing (nano ChIP-seq) [2] , which allows genome-wide location of histone modifications for scanty materials, of histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 27 acetylation (H3K27ac) for active promoters and enhancers, respectively. We also integrated the nano ChIP-seq profiles with transcriptomic data from TCGA and in-house cohorts. Results and summary: We found that primary human HCC tumors were associated with increased H3K4me3 occupancy, which occurred predominantly nearby the transcription start sites. H3K4me3-occupied promoters were enriched with signal transducer and activator of transcription (STAT) binding sites, which is in accordance with the crucial role of interleukin 6/STAT3 signaling in NAFLD-HCC initiation and progression. Moreover, tumor-specific H3K4me3 and H3K27ac profiles correlated with gene over-expression, including novel STAT3-driven oncogenes e.g. UBE2C and PLK1 in HCC. In summary, our findings demonstrated the feasibility of profiling chromatin using limited HCC tissues and identified aberrant promoter and enhancer regulatory elements associated with NAFLD-related hepatocarcinogenesis. Acknowledgements: This project was supported by the University Grants Committee through the Collaborative Research Fund C4017-14G, Theme Based Research Scheme T12-403/11-1 and General Research Fund 14102914. References: 1. Tian, Y., et al., Epigenetic regulation of hepatocellular carcinoma in non-alcoholic fatty liver disease. Semin Cancer Biol, 2013. 23(6 Pt B): p. 471-82. 2. Muratani, M., et al., Nanoscale chromatin profiling of gastric adenocarcinoma reveals cancer-associated cryptic promoters and somatically acquired regulatory elements. Nat Commun, 2014. 5: p. 4361. Citation Format: Feng Wu, Yingying Lee, Sau Dan Lee, Patrick Tan, Nathalie Wong, Kevin Yuk-Lap Yip, Kai F To, Alfred Sze Lok Cheng. Nanoscale ChIP-seq analysis in primary hepatocellular carcinoma tissues reveals tumor-specific chromatin deregulation for oncogene activation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4519.

Decreased Basal and Postprandial Plasma 5-Hydroxytryptamine (5-HT) in Patients With Functional Dyspepsia (FD): A Pilot Study

BACKGROUND: Increased postprandial 5-HT levels have been observed in patients with diarrhea-predominant irritable bowel syndrome (IBS). However, it is unclear whether patients with FD have 5-HT dysfunction. AIM: To compare the plasma 5-HT profile in FD patients and healthy controls. METHODS: Consecutive patients with FD (Rome III criteria) were recruited. Patients with GERD and IBS as predominant symptoms were excluded. After an overnight fast, they underwent caloric drinking test (Ensure O,1.06 kcal/ml at 30ml/min) and 13C-octanoic acid breath test for gastric emptying time measurement. Serial blood samples were collected at 0, 30, 60, 90, 120 min postprandially for plasma 5-HT assay. Healthy controls without history of peptic ulcer and dyspepsia were recruited for the same protocol. RESULTS: 29 FD patients (M:F, 8:21; mean age: 45±2) and 20 controls were studied. 18 (62%) patients had postprandial distress syndrome (PDS) and 11 (40%) had both PDS and epigastric pain syndrome. 7 (24%) patients had concomitant IBS. There was no significant difference in total calorie intake (FD: 762±59 kcal; Control: 940±89 kcal, p= 0.11) and gastric emptying rate (T1/2, FD: 76±4min.; Control: 80±5min., p=0.52). However, FD patients had significantly lower basal (FD: 139±17 ng/109platelets, Control: 238.0±11.7 ng/109platelets, p<0.001) and postprandial plasma serotonin contents at 30min (FD: 127±18 ng/109platelets, Control: 206±10 ng/109platelets, p<0.001), 60 min (FD: 119±18 ng/ 109platelets, Control: 193 ±19 ng/109platelets, p=0.006), 90 min (FD: 110±17 ng/109platelets, Control: 171 ± 23 ng/109platelets, p=0.038) and AUC over the period of 120 min (FD: 14410±1968 ng/109platelets.min, control: 23160±1537 ng/109platelets.min, p=0.0062). Repeated measures of ANOVA revealed high correlation between FD and serotonin profile across 120 min (p=0.0021 for FD, time <0.0001) CONCLUSION: In contrast to IBS, FD patients have decreased basal and postprandial plasma 5-HT concentrations. These findings suggest (1) IBS and FD have different pathophysiological mechanism in particular the 5HT dysfunction and (2) Modulation of 5-HT system may have therapeutic value in treatment of FD.

W1920 Neonatal Maternal Separation Stress Leads to Altered Ghrelin Profile in Response to Acute Psychological Stress in Adulthood

G A A b st ra ct s rats is clearly evident in H&E-stained sections whereby villi height is lower and muscularis atrophy is evident. Conclusions: Our data indicate that SMA blood flow is compromised in rats after T3 SCI at a time when gastric emptying and motility are also reduced. Duodenal administration of a liquid meal was insufficient in triggering visceral hyperemia. We suggest that impaired systemic vascular flow diminishes compensatory mesenteric blood flow to support the GI tract during digestion. The resulting GI ischemia may be an underlying pathology leading to gastric dysfunction seen following SCI. Support: NINDS 049177