A genomic enhancer signature associates with hepatocellular carcinoma prognosis.

Background & Aims: Lifestyle and environmental-related exposures are important risk factors for hepatocellular carcinoma (HCC), suggesting that epigenetic dysregulation significantly underpins HCC. We profiled 30 surgically resected tumours and the matched adjacent normal tissues to understand the aberrant epigenetic events associated with HCC. Methods: We identified tumour differential enhancers and the associated genes by analysing H3K27 acetylation (H3K27ac) chromatin immunoprecipitation sequencing (ChIP-seq) and Hi-C/HiChIP data from the resected tumour samples of 30 patients with early-stage HCC. This epigenome dataset was analysed with previously reported genome and transcriptome data of the overlapping group of patients from the same cohort. We performed patient-specific differential expression testing using multiregion sequencing data to identify genes that undergo both enhancer and gene expression changes. Based on the genes selected, we identified two patient groups and performed a recurrence-free survival analysis. Results: We observed large-scale changes in the enhancer distribution between HCC tumours and the adjacent normal samples. Many of the gain-in-tumour enhancers showed corresponding upregulation of the associated genes and vice versa, but much of the enhancer and gene expression changes were patient-specific. A subset of the upregulated genes was activated in a subgroup of patients' tumours. Recurrence-free survival analysis revealed that the patients with a more robust upregulation of those genes showed a worse prognosis. Conclusions: We report the genomic enhancer signature associated with differential prognosis in HCC. Findings that cohere with oncofoetal reprogramming in HCC were underpinned by genome-wide enhancer rewiring. Our results present the epigenetic changes in HCC that offer the rational selection of epigenetic-driven gene targets for therapeutic intervention or disease prognostication in HCC. Impact and Implications: Lifestyle and environmental-related exposures are the important risk factors of hepatocellular carcinoma (HCC), suggesting that tumour-associated epigenetic dysregulations may significantly underpin HCC. We profiled tumour tissues and their matched normal from 30 patients with early-stage HCC to study the dysregulated epigenetic changes associated with HCC. By also analysing the patients' RNA-seq and clinical data, we found the signature genes - with epigenetic and transcriptomic dysregulation - associated with worse prognosis. Our findings suggest that systemic approaches are needed to consider the surrounding cellular environmental and epigenetic changes in HCC tumours.

Multi-region sampling with paired sample sequencing analyses reveals sub-groups of patients with novel patient-specific dysregulation in Hepatocellular Carcinoma.

BACKGROUND: Conventional differential expression (DE) testing compares the grouped mean value of tumour samples to the grouped mean value of the normal samples, and may miss out dysregulated genes in small subgroup of patients. This is especially so for highly heterogeneous cancer like Hepatocellular Carcinoma (HCC). METHODS: Using multi-region sampled RNA-seq data of 90 patients, we performed patient-specific differential expression testing, together with the patients' matched adjacent normal samples. RESULTS: Comparing the results from conventional DE analysis and patient-specific DE analyses, we show that the conventional DE analysis omits some genes due to high inter-individual variability present in both tumour and normal tissues. Dysregulated genes shared in small subgroup of patients were useful in stratifying patients, and presented differential prognosis. We also showed that the target genes of some of the current targeted agents used in HCC exhibited highly individualistic dysregulation pattern, which may explain the poor response rate. DISCUSSION/CONCLUSION: Our results highlight the importance of identifying patient-specific DE genes, with its potential to provide clinically valuable insights into patient subgroups for applications in precision medicine.

The aberrant upregulation of exon 10-inclusive SREK1 through SRSF10 acts as an oncogenic driver in human hepatocellular carcinoma.

Deregulation of alternative splicing is implicated as a relevant source of molecular heterogeneity in cancer. However, the targets and intrinsic mechanisms of splicing in hepatocarcinogenesis are largely unknown. Here, we report a functional impact of a Splicing Regulatory Glutamine/Lysine-Rich Protein 1 (SREK1) variant and its regulator, Serine/arginine-rich splicing factor 10 (SRSF10). HCC patients with poor prognosis express higher levels of exon 10-inclusive SREK1 (SREK1L). SREK1L can sustain BLOC1S5-TXNDC5 (B-T) expression, a targeted gene of nonsense-mediated mRNA decay through inhibiting exon-exon junction complex binding with B-T to exert its oncogenic role. B-T plays its competing endogenous RNA role by inhibiting miR-30c-5p and miR-30e-5p, and further promoting the expression of downstream oncogenic targets SRSF10 and TXNDC5. Interestingly, SRSF10 can act as a splicing regulator for SREK1L to promote hepatocarcinogenesis via the formation of a SRSF10-associated complex. In summary, we demonstrate a SRSF10/SREK1L/B-T signalling loop to accelerate the hepatocarcinogenesis.

Cis Selective RCM Study to the 14-Membered Cyclic Subunit of Bielschowskysin.

A concise, (Z)-selective ring-closing metathesis (RCM) route to the 14-membered carbocycle of bielschowskysin is detailed using naturally occurring chiral starting materials. Unproductive RCM substrates were attributed to alkyne chelation of the ruthenium catalyst and steric disadvantages within the cembranoid precursors, which was eventually circumvented by using cyclic diol benzylidene protection involving a C8-quaternary carbinol center.

Correlation of NUF2 Overexpression with Poorer Patient Survival in Multiple Cancers.

PURPOSE: NUF2 has been implicated in multiple cancers recently, suggesting NUF2 may play a role in the common tumorigenesis process. In this study, we aim to perform comprehensive meta-analysis of NUF2 expression in the cancer types included in the Cancer Genome Atlas (TCGA). MATERIALS AND METHODS: RNA-sequencing data in 31 cancer types in the TCGA data and 11 independent datasets were used to examine NUF2 expression. Silencing NUF2 using targeting shRNAs in hepatocellular carcinoma (HCC) cell lines was used to evaluate NUF2's role in HCC in vitro and in vivo. RESULTS: NUF2 up-regulation is significantly observed in 23 out of the 31 cancer types in the TCGA datasets and validated in 13 major cancer types using 11 independent datasets. NUF2 overexpression was clinically important as high NUF2 was significantly associated with tumor stages in eight different cancers. High NUF2 was also associated with significantly poorer patient overall survival and disease-free survival in eight and six cancers, respectively. We proceeded to validate NUF2 overexpression and its negative association with overall survival at the protein level in an independent cohort of 40 HCC patients. Compared to the non-targeting controls, NUF2 knockdown cells showed significantly reduced ability to grow, migrate into a scratch wound and invade the 8 µm porous membrane in vitro. Moreover, NUF2 knockdown cells also formed significantly smaller tumors than control cells in mouse xenograft assays in vivo. CONCLUSION: NUF2 up-regulation is a common feature of many cancers. The prognostic potential and functional impact of NUF2 up-regulation warrant further studies.

ß-Oxidation and autophagy are critical energy providers during acute glucose depletion in Saccharomyces cerevisiae.

The ability to tolerate and thrive in diverse environments is paramount to all living organisms, and many organisms spend a large part of their lifetime in starvation. Upon acute glucose starvation, yeast cells undergo drastic physiological and metabolic changes and reestablish a constant-although lower-level of energy production within minutes. The molecules that are rapidly metabolized to fuel energy production under these conditions are unknown. Here, we combine metabolomics and genetics to characterize the cells' response to acute glucose depletion and identify pathways that ensure survival during starvation. We show that the ability to respire is essential for maintaining the energy status and to ensure viability during starvation. Measuring the cells' immediate metabolic response, we find that central metabolites drastically deplete and that the intracellular AMP-to-ATP ratio strongly increases within 20 to 30 s. Furthermore, we detect changes in both amino acid and lipid metabolite levels. Consistent with this, both bulk autophagy, a process that frees amino acids, and lipid degradation via ß-oxidation contribute in parallel to energy maintenance upon acute starvation. In addition, both these pathways ensure long-term survival during starvation. Thus, our results identify bulk autophagy and ß-oxidation as important energy providers during acute glucose starvation.

Bacterial Glycogen Provides Short-Term Benefits in Changing Environments.

Changing nutritional conditions challenge microbes and shape their evolutionary optimization. Here, we used real-time metabolomics to investigate the role of glycogen in the dynamic physiological adaptation of Escherichia coli to fluctuating nutrients following carbon starvation. After the depletion of environmental glucose, we found significant metabolic activity remaining, which was linked to rapid utilization of intracellular glycogen. Glycogen was depleted by 80% within minutes of glucose starvation and was similarly replenished within minutes of glucose availability. These fast time scales of glycogen utilization correspond to the short-term benefits that glycogen provided to cells undergoing various physiological transitions. Cells capable of utilizing glycogen exhibited shorter lag times than glycogen mutants when starved between periods of exposure to different carbon sources. The ability to utilize glycogen was also important for the transition between planktonic and biofilm lifestyles and enabled increased glucose uptake during pulses of limited glucose availability. While wild-type and mutant strains exhibited comparable growth rates in steady environments, mutants deficient in glycogen utilization grew more poorly in environments that fluctuated on minute scales between carbon availability and starvation. Taken together, these results highlight an underappreciated role of glycogen in rapidly providing carbon and energy in changing environments, thereby increasing survival and competition capabilities under fluctuating and nutrient-poor conditions.IMPORTANCE Nothing is constant in life, and microbes in particular have to adapt to frequent and rapid environmental changes. Here, we used real-time metabolomics and single-cell imaging to demonstrate that the internal storage polymer glycogen plays a crucial role in such dynamic adaptations. Glycogen is depleted within minutes of glucose starvation and similarly is replenished within minutes of glucose availability. Cells capable of utilizing glycogen exhibited shorter lag times than glycogen mutants when starved between periods of exposure to different carbon sources. While wild-type and mutant strains exhibited comparable growth rates in steady environments, mutants deficient in glycogen utilization grew more poorly in environments that fluctuated on minute scales between carbon availability and starvation. These results highlight an underappreciated role of glycogen in rapidly providing carbon and energy in changing environments, thereby increasing survival and competition capabilities under fluctuating and nutrient-poor conditions.

Clinical and metabolomics analysis of hepatocellular carcinoma patients with diabetes mellitus.

INTRODUCTION: Diabetes and cancer are among the most frequent causes of death worldwide. Recent epidemiological findings have indicated a link between diabetes and cancer in several organs, particularly the liver. A number of epidemiological studies have demonstrated that diabetes is an established independent risk factor for hepatocellular carcinoma (HCC). However, the metabolites connecting diabetes and HCC remains less well understood. OBJECTIVES: The study aimed to identify clinical and metabolomics differences of HCC from patients with/without diabetes using comprehensive global metabolomics analysis. METHODS: Metabolite profiling was conducted with the Metabolon platform for 120 human diabetes/non-diabetes HCC tumor/normal tissues. Standard statistical analyses were performed using the Partek Genomics Suite on log-transformed data. Principal component analysis (PCA) was conducted using all and dysregulated metabolites. RESULTS: We identified a group of metabolites that are differentially expressed in the tumor tissues of diabetes HCC compared to non-diabetes HCC patients. Meanwhile, we also identified a group of metabolites that are differentially expressed in the matched normal liver tissues of diabetes HCC compared to non-diabetes HCC patients. Some metabolites are consistently dysregulated in the tumor or matched normal tissues of HCC with or without diabetes. However, some metabolites, including 2-hydroxystearate, were only overexpressed in the tumor tissues of HCC with diabetes and associated with the glucose level. CONCLUSION: Metabolic profiling identifies distinct dysregulated metabolites in HCC patients with/without diabetes.

Relevance of a TCGA-derived Glioblastoma Subtype Gene-Classifier among Patient Populations.

Glioblastoma multiforme (GBM), a deadly cancer, is the most lethal and common malignant brain tumor, and the leading cause of death in adult brain tumors. While genomic data continues to rocket, clinical application and translation to patient care are lagging behind. Big data now deposited in the TCGA network offers a window to generate novel clinical hypotheses. We hypothesized that a TCGA-derived gene-classifier can be applied across different gene profiling platforms and population groups. This gene-classifier validated three robust GBM-subtypes across six different platforms, among Caucasian, Korean and Chinese populations: Three Caucasian-predominant TCGA-cohorts (Affymetrix U133A = 548, Agilent Custom-Array = 588, RNA-seq = 168), and three Asian-cohorts (Affymetrix Human Gene 1.0ST-Array = 61, Illumina = 52, Agilent 4 × 44 K = 60). To understand subtype-relevance in patient therapy, we investigated retrospective TCGA patient clinical sets. Subtype-specific patient survival outcome was similarly poor and reflected the net result of a mixture of treatment regimens with/without surgical resection. As a proof-of-concept, in subtype-specific patient-derived orthotopic xenograft (PDOX) mice, Classical-subtype demonstrated no survival difference comparing radiation-therapy versus temozolomide monotherapies. Though preliminary, a PDOX model of Proneural/Neural-subtype demonstrated significantly improved survival with temozolomide compared to radiation-therapy. A larger scale study using this gene-classifier may be useful in clinical outcome prediction and patient selection for trials based on subtyping.

Genome-wide CRISPR knockout screens identify NCAPG as an essential oncogene for hepatocellular carcinoma tumor growth.

Hepatocellular carcinoma (HCC) is a common and deadly cancer with limited treatment options. Through genome-wide growth depletion screens using clustered regularly interspaced short palindromic repeats and expression profiling of primary HCC tumors, we identified 13 clinically relevant target genes with therapeutic potential. Subsequent functional annotation analysis revealed significant enrichment of these 13 genes in the cell cycle, cell death, and survival pathways. Non-structural maintenance of chromosomes condensin I complex subunit G (NCAPG) was ranked the highest among the depletion screens and multiple HCC expression datasets. Transient inhibition of NCAPG using specific small interfering RNAs resulted in a significant reduction in cell growth, migration, and the down-regulation of mitochondrial gene expression in vitro. Small homologous RNA-mediated knockdown of NCAPG significantly impaired cell viability, caused aberrant mitotic division, fragmented the mitochondrial network, and increased cell death in vitro. HCC cells with a reduced expression of NCAPG formed significantly smaller xenograft tumors in vivo. Importantly, high NCAPG expression was significantly associated with poorer overall and disease-free survival in HCC patients. High NCAPG expression is a novel prognostic biomarker to predict HCC early recurrence after surgical resection. In conclusion, NCAPG is an essential gene for HCC tumor cell survival. It represents a promising novel target for treating HCC and a prognostic biomarker for clinical management of HCC.-Wang, Y., Gao, B., Tan, P. Y., Handoko, Y. A., Sekar, K., Deivasigamani, A., Seshachalam, V. P., OuYang, H.-Y., Shi, M., Xie, C., Goh, B. K. P., Ooi, L. L., Hui, K. M. Genome-wide CRISPR knockout screens identify NCAPG as an essential oncogene for hepatocellular carcinoma tumor growth.

NUF2 is a valuable prognostic biomarker to predict early recurrence of hepatocellular carcinoma after surgical resection.

Early tumor recurrence after curative surgical resection poses a great challenge to the clinical management of hepatocellular carcinoma (HCC). We conducted whole genome expression microarrays on 64 primary HCC tumors with clinically defined recurrence status and cross-referenced with RNA-seq data from 18 HCC tumors in the Cancer Genome Atlas project. We identified a 77-gene signature, which is significantly associated with early recurrent (ER) HCC tumors. This ER-associated signature shows significant enrichment in genes involved in cell cycle pathway. We performed receiver operating characteristic (ROC) analysis to evaluate the prognostic biomarker potential of these 77 genes and Pearson correlation analysis to identify 11 close clusters. The one gene with the best area under the ROC curve in each of the 11 clusters was selected for validation using reverse-transcription quantitative PCR in an independent cohort of 24 HCC tumors. NUF2 was identified to be the minimal biomarker sufficient to discriminate ER tumors from LR tumors. NUF2 in combination with liver cirrhosis could significantly improve the detection of ER tumors with an AUROC of 0.82 and 0.85 in the test and validation cohort, respectively. In conclusion, NUF2 in combination with liver cirrhosis is a promising prognostic biomarker for early HCC recurrence.

Synthesis and degradation of FtsZ quantitatively predict the first cell division in starved bacteria.

In natural environments, microbes are typically non-dividing and gauge when nutrients permit division. Current models are phenomenological and specific to nutrient-rich, exponentially growing cells, thus cannot predict the first division under limiting nutrient availability. To assess this regime, we supplied starving Escherichia coli with glucose pulses at increasing frequencies. Real-time metabolomics and microfluidic single-cell microscopy revealed unexpected, rapid protein, and nucleic acid synthesis already from minuscule glucose pulses in non-dividing cells. Additionally, the lag time to first division shortened as pulsing frequency increased. We pinpointed division timing and dependence on nutrient frequency to the changing abundance of the division protein FtsZ. A dynamic, mechanistic model quantitatively relates lag time to FtsZ synthesis from nutrient pulses and FtsZ protease-dependent degradation. Lag time changed in model-congruent manners, when we experimentally modulated the synthesis or degradation of FtsZ. Thus, limiting abundance of FtsZ can quantitatively predict timing of the first cell division.

CDK1-mediated BCL9 phosphorylation inhibits clathrin to promote mitotic Wnt signalling.

Uncontrolled cell division is a hallmark of cancer. Deregulation of Wnt components has been linked to aberrant cell division by multiple mechanisms, including Wnt-mediated stabilisation of proteins signalling, which was notably observed in mitosis. Analysis of Wnt components revealed an unexpected role of B-cell CLL/lymphoma 9 (BCL9) in maintaining mitotic Wnt signalling to promote precise cell division and growth of cancer cell. Mitotic interactome analysis revealed a mechanistic role of BCL9 in inhibiting clathrin-mediated degradation of LRP6 signalosome components by interacting with clathrin and the components in Wnt destruction complex; this function was further controlled by CDK1-driven phosphorylation of BCL9 N-terminal, especially T172. Interestingly, T172 phosphorylation was correlated with cancer patient prognosis and enriched in tumours. Thus, our results revealed a novel role of BCL9 in controlling mitotic Wnt signalling to promote cell division and growth.

Insights into the etiology-associated gene regulatory networks in hepatocellular carcinoma from The Cancer Genome Atlas.

BACKGROUND AND AIM: Hepatitis B virus (HBV), hepatitis C virus, alcohol consumption, and non-alcoholic fatty liver disease are the major known risk factors for hepatocellular carcinoma (HCC). There have been very few studies comparing the underlying biological mechanisms associated with the different etiologies of HCC. In this study, we hypothesized the existence of different regulatory networks associated with different liver disease etiologies involved in hepatocarcinogenesis. METHODS: Using upstream regulatory analysis tool in ingenuity pathway analysis software, upstream regulators (URs) were predicted using differential expressed genes for HCC to facilitate the interrogation of global gene regulation. RESULTS: Analysis of regulatory networks for HBV HCC revealed E2F1 as activated UR, regulating genes involved in cell cycle and DNA replication, and HNF4A and HNF1A as inhibited UR. In hepatitis C virus HCC, interferon-γ, involved in cellular movement and signaling, was activated, while IL1RN, mitogen-activated protein kinase 1 involved in interleukin 22 signaling and immune response, was inhibited. In alcohol consumption HCC, ERBB2 involved in inflammatory response and cellular movement was activated, whereas HNF4A and NUPR1 were inhibited. For HCC derived from non-alcoholic fatty liver disease, miR-1249-5p was activated, and NUPR1 involved in cell cycle and apoptosis was inhibited. The prognostic value of representative genes identified in the regulatory networks for HBV HCC can be further validated by an independent HBV HCC dataset established in our laboratory with survival data. CONCLUSIONS: Our study identified functionally distinct candidate URs for HCC developed from different etiologic risk factors. Further functional validation studies of these regulatory networks could facilitate the management of HCC towards personalized medicine.

Simultaneous silencing of ACSL4 and induction of GADD45B in hepatocellular carcinoma cells amplifies the synergistic therapeutic effect of aspirin and sorafenib.

Sorafenib is currently the only US Food and Drug Administration (FDA)-approved molecular inhibitor for the systemic therapy of advanced hepatocellular carcinoma (HCC). Aspirin has been studied extensively as an anti-inflammation, cancer preventive and therapeutic agent. However, the potential synergistic therapeutic effects of sorafenib and aspirin on advanced HCC treatment have not been well studied. Drug combination studies and their synergy quantification were performed using the combination index method of Chou-Talalay. The synergistic therapeutic effects of sorafenib and aspirin were evaluated using an orthotopic mouse model of HCC and comprehensive gene profiling analyses were conducted to identify key factors mediating the synergistic therapeutic effects of sorafenib and aspirin. Sorafenib was determined to act synergistically on HCC cells with aspirin in vitro. Using Hep3B and HuH7 HCC cells, it was demonstrated that sorafenib and aspirin acted synergistically to induce apoptosis. Mechanistic studies demonstrated that combining sorafenib and aspirin yielded significant synergistically anti-tumor effects by simultaneously silencing ACSL4 and the induction of GADD45B expression in HCC cells both in vitro and in the orthotopic HCC xenograft mouse model. Importantly, clinical evidence has independently corroborated that survival of HCC patients expressing ACSL4highGADD45Blow was significantly poorer compared to patients with ACSL4lowGADD45Bhigh, thus demonstrating the potential clinical value of combining aspirin and sorafenib for HCC patients expressing ACSL4highGADD45Blow. In conclusion, sorafenib and aspirin provide synergistic therapeutic effects on HCC cells that are achieved through simultaneous silencing of ACSL4 and induction of GADD45B expression. Targeting HCC with ACSL4highGADD45Blow expression with aspirin and sorafenib could provide potential synergistic therapeutic benefits.

Frontiers of high-throughput metabolomics.

Large scale metabolomics studies are increasingly used to investigate genetically different individuals and time-dependent responses to environmental stimuli. New mass spectrometric approaches with at least an order of magnitude more rapid analysis of small molecules within the cell's metabolome are now paving the way towards true high-throughput metabolomics, opening new opportunities in systems biology, functional genomics, drug discovery, and personalized medicine. Here we discuss the impact and advantages of the progress made in profiling large cohorts and dynamic systems with high temporal resolution and automated sampling. In both areas, high-throughput metabolomics is gaining traction because it can generate hypotheses on molecular mechanisms and metabolic regulation. We conclude with the current status of the less mature single cell analyses where high-throughput analytics will be indispensable to resolve metabolic heterogeneity in populations and compartmentalization of metabolites.

MELK is an oncogenic kinase essential for early hepatocellular carcinoma recurrence.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths worldwide. Many kinases have been found to be intimately involved in oncogenesis and the deregulation of kinase function has emerged as a major mechanism by which cancer cells evade normal physiological constraints on growth and survival. Previously, we have performed gene expression profile analysis on HCC samples and have identified a host of kinases that are remarkably overexpressed in HCC. Among these, the Maternal Embryonic Leucine Zipper Kinase (MELK) is highly overexpressed in HCC and its overexpression strongly correlates with early recurrence and poor patients' survival. Silencing MELK inhibited cell growth, invasion, stemness and tumorigenicity of HCC cells by inducing apoptosis and mitosis. We further showed that the overexpression of MELK in HCC samples strongly correlated with the cell cycle- and mitosis-related genes which are directly regulated as part of the forkhead transcription factor FoxM1-related cell division program. Together, our data establish MELK as an oncogenic kinase involved in the pathogenesis and recurrence of HCC and could provide a promising molecular target to develop therapeutic strategies for patients with advanced HCC.

The microtubule-associated protein PRC1 promotes early recurrence of hepatocellular carcinoma in association with the Wnt/ß-catenin signalling pathway.

OBJECTIVES: Hepatocellular carcinoma (HCC) is the second leading cause of cancer mortality worldwide. Alterations in microtubule-associated proteins (MAPs) have been observed in HCC. However, the mechanisms underlying these alterations remain poorly understood. Our aim was to study the roles of the MAP protein regulator of cytokinesis 1 (PRC1) in hepatocarcinogenesis and early HCC recurrence. DESIGN: PRC1 expression in HCC samples was evaluated by microarray, immunoblotting and immunohistochemistry analysis. Molecular and cellular techniques including siRNA-mediated and lentiviral vector-mediated knockdown were used to elucidate the functions and mechanisms of PRC1. RESULTS: PRC1 expression was associated with early HCC recurrence and poor patient outcome. In HCC, PRC1 exerted an oncogenic effect by promoting cancer proliferation, stemness, metastasis and tumourigenesis. We further demonstrated that the expression and distribution of PRC1 is dynamically regulated by Wnt3a signalling. PRC1 knockdown impaired transcription factor (TCF) transcriptional activity, decreased Wnt target expression and reduced nuclear ß-catenin levels. Mechanistically, PRC1 interacts with the ß-catenin destruction complex, regulates Wnt3a-induced membrane sequestration of this destruction complex, inhibits adenomatous polyposis coli (APC) stability and promotes ß-catenin release from the APC complex. In vivo, high PRC1 expression correlated with nuclear ß-catenin and Wnt target expression. PRC1 acted as a master regulator of a set of 48 previously identified Wnt-regulated recurrence-associated genes (WRRAGs) in HCC. Thus, PRC1 controlled the expression and function of WRRAGs such as FANCI, SPC25, KIF11 and KIF23 via Wnt signalling. CONCLUSIONS: We identified PRC1 as a novel Wnt target that functions in a positive feedback loop that reinforces Wnt signalling to promote early HCC recurrence.

N-Terminal-Based Targeted, Inducible Protein Degradation in Escherichia coli.

Dynamically altering protein concentration is a central activity in synthetic biology. While many tools are available to modulate protein concentration by altering protein synthesis rate, methods for decreasing protein concentration by inactivation or degradation rate are just being realized. Altering protein synthesis rates can quickly increase the concentration of a protein but not decrease, as residual protein will remain for a while. Inducible, targeted protein degradation is an attractive option and some tools have been introduced for higher organisms and bacteria. Current bacterial tools rely on C-terminal fusions, so we have developed an N-terminal fusion (Ntag) strategy to increase the possible proteins that can be targeted. We demonstrate Ntag dependent degradation of mCherry and beta-galactosidase and reconfigure the Ntag system to perform dynamic, exogenously inducible degradation of a targeted protein and complement protein depletion by traditional synthesis repression. Model driven analysis that focused on rates, rather than concentrations, was critical to understanding and engineering the system. We expect this tool and our model to enable inducible protein degradation use particularly in metabolic engineering, biological study of essential proteins, and protein circuits.

EDIL3 is a novel regulator of epithelial-mesenchymal transition controlling early recurrence of hepatocellular carcinoma.

BACKGROUND & AIMS: Patients with advanced hepatocellular carcinoma (HCC) continue to have a dismal prognosis. Early recurrence, metastases and angiogenesis are the major obstacles to improve the outcome of HCC. Epithelial-mesenchymal transition (EMT) is a key contributor to cancer metastasis and recurrence, which are the major obstacles to improve prognosis of HCC. METHODS: Combining gene expression profiles of HCC samples with or without early recurrence and established cell lines with epithelial or mesenchymal phenotype, EDIL3 was identified as a novel regulator of EMT. The expression of EDIL3 was evaluated by quantitative PCR, Western blotting or immunohistochemistry. The effects of EDIL3 on the angiogenesis and metastasis of HCC cells were examined by wound healing, Matrigel invasion and tube formation assay in vitro and orthotopic xenograft mouse model of HCC in vivo. The signaling pathways of EDIL3 mediated were investigated through microarray and Western blotting analysis. RESULTS: EDIL3 was identified as a novel regulator of EMT, which contributes to angiogenesis, metastasis and recurrence of HCC. EDIL3 induces EMT and promotes HCC migration, invasion and angiogenesis in vitro. Mechanistically, overexpression of EDIL3, which was regulated by the downregulation of miR-137 in HCC, triggered the activation of ERK and TGF-ß signaling through interactions with αvß3 integrin. Blocking ERK and TGF-ß signaling overcomes EDIL3 induced angiogenesis and invasion. Using the orthotopic xenograft mouse model of HCC, we demonstrated that EDIL3 enhanced the tumorigenic, metastatic and angiogenesis potential of HCC in vivo. CONCLUSIONS: EDIL3-mediated activation of TGF-ß and ERK signaling could provide therapeutic implications for HCC.