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RNA helicases are dysregulated in tumors. Here, we identified DHX37 as one of the top RNA helicase genes with upregulated expression in hepatocellular carcinoma (HCC). DHX37 promoted proliferation of liver cancer cells in vitro and in vivo. Epigenomic profiling of DHX37-knockdown and control HCC cells revealed that DHX37 is associated with superenhancer activity. Mechanistically, DHX37 interacted with pleiotropic regulator 1 (PLRG1) to transcriptionally activate cyclin D1 (CCND1) expression via co-occupation of its promoter and superenhancer elements. DHX37 and PLRG1 promoted liver cancer cell proliferation and contributed to the poor prognosis of patients with HCC. Importantly, CCND1 inhibitors were effective as antiproliferative agents for liver cancer. These results together demonstrate a cooperative mechanistic interaction between DHX37 and PLRG1 that regulates CCND1 expression and promotes liver cancer progression, advancing our understanding of the epigenetic and transcriptional dysregulations mediated by RNA helicases and superenhancers in HCC. Significance: This work characterizes a novel mechanism of superenhancer-driven cyclin D1 upregulation by DHX37 and PLRG1, implicating this pathway as a potential therapeutic target in hepatocellular carcinoma.

Long terminal repeat (LTR) retrotransposons are a major class of transposable elements, accounting for 8.67% of the human genome. LTRs can serve as regulatory sequences and drive transcription of tissue or cancer-specific transcripts. However, the role of these LTR-activated transcripts, especially long non-coding RNAs (lncRNA), in cancer development remains largely unexplored. Here, we identified a novel lncRNA derived from MER52A retrotransposons (lncMER52A) that was exclusively expressed in hepatocellular carcinoma (HCC). HCC patients with higher lncMER52A had advanced TNM stage, less differentiated tumors, and shorter overall survival. LncMER52A promoted invasion and metastasis of HCC cells in vitro and in vivo. Mechanistically, lncMER52A stabilized p120-catenin and triggered the activation of Rho GTPase downstream of p120-catenin. Furthermore, we found that chromatin accessibility was crucial for the expression of lncMER52A. In addition, YY1 transcription factor bound to the cryptic MER52A LTR promoter and drove lncMER52A transcription in HCC. In conclusion, we identified an LTR-activated lncMER52A, which promoted the progression of HCC cells via stabilizing p120-catenin and activating p120-ctn/Rac1/Cdc42 axis. LncMER52A could serve as biomarker and therapeutic target for patients with HCC.Significance:A novel long noncoding RNA lncMER52 modulates cell migration and invasion via posttranslational control of p120-catenin protein stability.

Differentiated expressed genes after lncMER52A knockdown.

Full scan of all the western blots in the main figures.

The list of 464 HCC-specific lncRNAs.

RNA helicases are dysregulated in tumors. Here, we identified DHX37 as one of the top RNA helicase genes with upregulated expression in hepatocellular carcinoma (HCC). DHX37 promoted proliferation of liver cancer cells in vitro and in vivo. Epigenomic profiling of DHX37-knockdown and control HCC cells revealed that DHX37 is associated with superenhancer activity. Mechanistically, DHX37 interacted with pleiotropic regulator 1 (PLRG1) to transcriptionally activate cyclin D1 (CCND1) expression via co-occupation of its promoter and superenhancer elements. DHX37 and PLRG1 promoted liver cancer cell proliferation and contributed to the poor prognosis of patients with HCC. Importantly, CCND1 inhibitors were effective as antiproliferative agents for liver cancer. These results together demonstrate a cooperative mechanistic interaction between DHX37 and PLRG1 that regulates CCND1 expression and promotes liver cancer progression, advancing our understanding of the epigenetic and transcriptional dysregulations mediated by RNA helicases and superenhancers in HCC.Significance:This work characterizes a novel mechanism of superenhancer-driven cyclin D1 upregulation by DHX37 and PLRG1, implicating this pathway as a potential therapeutic target in hepatocellular carcinoma.

Supplementary Table from RNA Helicase DHX37 Facilitates Liver Cancer Progression by Cooperating with PLRG1 to Drive Superenhancer-Mediated Transcription of Cyclin D1

Supplementary Data from RNA Helicase DHX37 Facilitates Liver Cancer Progression by Cooperating with PLRG1 to Drive Superenhancer-Mediated Transcription of Cyclin D1

Supplementary data-The Supplementary information includes: Figure S1. The genomic location, subcellular distribution, full length and coding potential of lncMER52A. Figure S2. The knockdown efficiency and specificity of lncMER52A siRNAs and Crispr-Cas9 mediated knockdown. The effects of lncMER52A on the migration, invasion and metastasis of HCC cells. Figure S3. The correlation of lncMER52A and p120-catenin in HCC tissues and paired normal livers. Figure S4. lncMER52A overexpression significantly inhibited the interaction between β-TrCP1 and p120-catenin. Figure S5. The effects of p120-catenin on the migration and invasion of HCC cells in vitro. Figure S6. H3K4me3 and H3K27ac levels at the promoter of lncMER52A in Huh7 and HepG2-C3A cells. Table S1. Primers for RACE assays. Table S2. Sequence of siRNAs and gRNAs. Table S3. Primers used in molecular cloning of vectors. Table S4. Primers used in the RNA pulldown assays. Table S5. Primers used in the northern blot assays. Table S6. Primers used in the qPCR experiments. Table S7. Primers used in the ChIP assays. Table S8. The correlation of lncMER52A expression with HCC. clinicopathological features.

Background and aims The nuclear factor kappa B (NF-κB) signaling pathway is important for linking inflammation and tumorigenesis. Here, we characterized an NF-κB signaling activation-induced long intergenic noncoding (LINC) RNA in hepatocellular carcinoma (HCC), LINC00665, that contributes to the enhanced cell proliferation of HCC cells both in vitro and in vivo. Approach and results LINC00665 physically interacts with the double-stranded RNA (dsRNA)-activated protein kinase (PKR), enhances its activation, and maintains its protein stability by blocking ubiquitin/proteasome-dependent degradation, resulting in a positive feedback regulation of NF-κB signaling in HCC cells. Notably, patients with HCC and higher LINC00665 have poorer outcomes in the clinic. Conclusions Our findings indicate that LINC00665 is involved in the NF-κB signaling activation in HCC cells and that the inflammatory LINC00665/PKR/NF-κB loop plays important oncogenic roles in hepatic cancer progression and may be a potential therapeutic target.

Background and aims Alternative splicing (AS) is a key step that increases the diversity and complexity of the cancer transcriptome. Recent evidence has highlighted that AS has an increasingly crucial role in cancer. Nonetheless, the mechanisms underlying AS and its dysregulation in hepatocellular carcinoma (HCC) remain elusive. Here, we report that the expression of RNA-binding protein p54nrb /non-POU domain-containing octamer-binding protein (NONO) is frequently increased in patients with HCC and is associated with poor outcomes. Approach and results Knockdown of NONO significantly abolished liver cancer cell proliferation, migration, and tumor formation. RNA-sequencing revealed that NONO regulates MYC box-dependent interacting protein 1 (or bridging integrator 1 [BIN1]; also known as amphiphysin 2 3P9) exon 12a splicing. In the normal liver, BIN1 generates a short isoform (BIN1-S) that acts as a tumor suppressor by inhibiting the binding of c-Myc to target gene promoters. In HCC, NONO is highly up-regulated and produces a long isoform (BIN1-L, which contains exon 12a) instead of BIN1-S. High levels of BIN1-L promote carcinogenesis by binding with the protein polo-like kinase 1 to enhance its stability through the prevention of ubiquitin/proteasome-dependent cullin 3 degradation. Further analysis revealed that NONO promotes BIN1 exon 12a inclusion through interaction with DExH-box helicase 9 (DHX9) and splicing factor proline and glutamine-rich (SFPQ). Notably, frequent coexpression of DHX9-NONO-SFPQ is observed in patients with HCC. Conclusions Taken together, our findings identify the DHX9-NONO-SFPQ complex as a key regulator manipulating the oncogenic splicing switch of BIN1 and as a candidate therapeutic target in liver cancer.

Long terminal repeat (LTR) retrotransposons are a major class of transposable elements, accounting for 8.67% of the human genome. LTRs can serve as regulatory sequences and drive transcription of tissue or cancer-specific transcripts. However, the role of these LTR-activated transcripts, especially long non-coding RNAs (lncRNA), in cancer development remains largely unexplored. Here, we identified a novel lncRNA derived from MER52A retrotransposons (lncMER52A) that was exclusively expressed in hepatocellular carcinoma (HCC). HCC patients with higher lncMER52A had advanced TNM stage, less differentiated tumors, and shorter overall survival. LncMER52A promoted invasion and metastasis of HCC cells in vitro and in vivo. Mechanistically, lncMER52A stabilized p120-catenin and triggered the activation of Rho GTPase downstream of p120-catenin. Furthermore, we found that chromatin accessibility was crucial for the expression of lncMER52A. In addition, YY1 transcription factor bound to the cryptic MER52A LTR promoter and drove lncMER52A transcription in HCC. In conclusion, we identified an LTR-activated lncMER52A, which promoted the progression of HCC cells via stabilizing p120-catenin and activating p120-ctn/Rac1/Cdc42 axis. LncMER52A could serve as biomarker and therapeutic target for patients with HCC. Significance: A novel long noncoding RNA lncMER52 modulates cell migration and invasion via posttranslational control of p120-catenin protein stability.

Background Human cancer cell lines are fundamental models for cancer research and therapeutic strategy development. However, there is no characterization of circular RNAs (circRNAs) in a large number of cancer cell lines. Methods Here, we apply four circRNA identification algorithms to heuristically characterize the expression landscape of circRNAs across ~ 1000 human cancer cell lines from CCLE polyA-enriched RNA-seq data. By using integrative analysis and experimental approaches, we explore the expression landscape, biogenesis, functional consequences, and drug response of circRNAs across different cancer lineages. Results We revealed highly lineage-specific expression patterns of circRNAs, suggesting that circRNAs may be powerful diagnostic and/or prognostic markers in cancer treatment. We also identified key genes involved in circRNA biogenesis and confirmed that TGF-β signaling may promote biogenesis of circRNAs. Strikingly, we showed that clinically actionable genes are more likely to generate circRNAs, potentially due to the enrichment of RNA-binding protein (RBP) binding sites. Among these, circMYC can promote cell proliferation. We observed strong association between the expression of circRNAs and the response to drugs, especially those targeting chromatin histone acetylation. Finally, we developed a user-friendly data portal, CircRNAs in cancer cell lines (CircRiC, https://hanlab.uth.edu/cRic), to benefit the biomedical research community. Conclusions Our study provides the characterization of circRNAs in cancer cell lines and explored the potential mechanism of circRNA biogenesis as well as its therapeutic implications. We also provide a data portal to facilitate the related biomedical researches. Electronic supplementary material The online version of this article (10.1186/s13073-019-0663-5) contains supplementary material, which is available to authorized users.

Alternative splicing is an important RNA processing event that contributes to RNA complexity and protein diversity in cancer. Accumulating evidence demonstrates the essential roles of some alternatively spliced genes in carcinogenesis. However, the potential roles of alternatively spliced genes in hepatocellular carcinoma (HCC) are still largely unknown. Here we showed that the HnRNP Associated with Lethal Yellow Protein Homolog (RALY) gene is upregulated and associated with poor outcomes in HCC patients. RALY acts as a tumor-promoting factor by cooperating with splicing factor 3b subunit 3 (SF3B3) and modulating the splicing switch of Metastasis Associated 1 (MTA1) from MTA-S to MTA1-L. Normally, MTA1-S inhibits cell proliferation by reducing the transcription of cholesterol synthesis genes. In HCC, RALY and SF3B3 cooperate to regulate the MTA1 splicing switch, leading to a reduction in the MTA1-S level, and alleviating the inhibitory effect of MTA1-S on cholesterol synthesis genes, thus promoting HCC cell proliferation. In conclusion, our results revealed that the RALY-SF3B3/MTA1/cholesterol synthesis pathway contributes essentially to hepatic carcinogenesis and could serve as a promising therapeutic target for HCC.

Background Heat shock proteins (HSPs), a representative family of chaperone genes, play crucial roles in malignant progression and are pursued as attractive anti-cancer therapeutic targets. Despite tremendous efforts to develop anti-cancer drugs based on HSPs, no HSP inhibitors have thus far reached the milestone of FDA approval. There remains an unmet need to further understand the functional roles of HSPs in cancer. Methods We constructed the network for HSPs across ~ 10,000 tumor samples from The Cancer Genome Atlas (TCGA) and ~ 10,000 normal samples from Genotype-Tissue Expression (GTEx), and compared the network disruption between tumor and normal samples. We then examined the associations between HSPs and cancer hallmarks and validated these associations from multiple independent high-throughput functional screens, including Project Achilles and DRIVE. Finally, we experimentally characterized the dual function effects of HSPs in tumor proliferation and metastasis. Results We comprehensively analyzed the HSP expression landscape across multiple human cancers and revealed a global disruption of the co-expression network for HSPs. Through analyzing HSP expression alteration and its association with tumor proliferation and metastasis, we revealed dual functional effects of HSPs, in that they can simultaneously influence proliferation and metastasis in opposite directions. We experimentally characterized the dual function of two genes, DNAJC9 and HSPA14, in lung cancer cells. We further demonstrated the generalization of this dual direction of associations between HSPs and cancer hallmarks, suggesting the necessity to more carefully evaluate HSPs as therapeutic targets and develop highly specific HSP inhibitors for cancer intervention. Conclusions Our study furnishes a holistic view of functional associations of HSPs with cancer hallmarks to aid the development of HSP inhibitors as well as other drugs in cancer therapy.

Additional file 1: Fig. S1. HSP co-expression network in normal tissue and matched cancer types. Fig. S2. Clinically relevant HSPs across human cancers. Fig. S3. Associations between HSPs and cell proliferation (ki67) in cancer cells. Fig. S4. Correlation between HSPs and EMT score. Fig. S5. Dual functional effects of HSPs on proliferation and EMT. Fig. S6. HSPs associated with cancer hallmark across cancer types. Fig. S7. Associations between HSPs and hallmarks across breast cancer subtypes. Table S1. Samples across TCGA cancer types. Table S2. Samples across GTEx tissues. Table S3. HSP genes investigated in this study.

RNA‐binding proteins (RBPs) play fundamental roles in the RNA life cycle. The aberrant expression of RBPs is often observed in human disease, including cancer. In this study, we screened for the expression levels of 1542 human RBPs in The Cancer Genome Atlas liver hepatocellular carcinoma samples and found 92 consistently upregulated RBP genes in HCC compared with normal samples. Additionally, we undertook a Kaplan–Meier analysis and found that high expression of 15 RBP genes was associated with poor prognosis in patients with HCC. Furthermore, we found that eIF3c promotes HCC cell proliferation in vitro as well as tumorigenicity in vivo. Gene Set Enrichment Analysis showed that high eIF3c expression is positively associated with KRAS, vascular endothelial growth factor, and Hedgehog signaling pathways, all of which are closely associated with specific cancer‐related gene sets. Our study provides the basis for further investigation of the molecular mechanism by which eIF3c promotes the development and progression of HCC.

Alternative splicing (AS) is a key step that increases the diversity and complexity of the cancer transcriptome. Recent evidence has highlighted that AS has an increasingly crucial role in cancer. Nonetheless, the mechanisms underlying AS and its dysregulation in hepatocellular carcinoma (HCC) remain elusive. Here, we report that the expression of RNA-binding protein p54Knockdown of NONO significantly abolished liver cancer cell proliferation, migration, and tumor formation. RNA-sequencing revealed that NONO regulates MYC box-dependent interacting protein 1 (or bridging integrator 1 [BIN1]; also known as amphiphysin 2 3P9) exon 12a splicing. In the normal liver, BIN1 generates a short isoform (BIN1-S) that acts as a tumor suppressor by inhibiting the binding of c-Myc to target gene promoters. In HCC, NONO is highly up-regulated and produces a long isoform (BIN1-L, which contains exon 12a) instead of BIN1-S. High levels of BIN1-L promote carcinogenesis by binding with the protein polo-like kinase 1 to enhance its stability through the prevention of ubiquitin/proteasome-dependent cullin 3 degradation. Further analysis revealed that NONO promotes BIN1 exon 12a inclusion through interaction with DExH-box helicase 9 (DHX9) and splicing factor proline and glutamine-rich (SFPQ). Notably, frequent coexpression of DHX9-NONO-SFPQ is observed in patients with HCC.Taken together, our findings identify the DHX9-NONO-SFPQ complex as a key regulator manipulating the oncogenic splicing switch of BIN1 and as a candidate therapeutic target in liver cancer.

Hepatocellular carcinoma (HCC) is a highly lethal cancer and its underlying etiology remains understudied. The immense diversity and complexity of the cancer transcriptome hold the potential to yield tumor-specific transcripts (TSTs). Here, we showed that hundreds of TSTs are frequently expressed in HCC by an assembling spliced junction analysis of RNA sequencing raw data from approximately 1,000 normal and HCC tissues. Many of the TSTs were found to be unannotated and noncoding RNAs. We observed that intergenic TSTs are generated from transcription initiation sites frequently harboring long terminal repeat (LTR) elements. The strong presence of TSTs indicates significantly poor prognoses in HCC. Functional screening revealed a noncoding TST (termed TST1), which acted as a regulator of HCC cell proliferation and tumorigenesis. TST1 is generated from an LTR12C promoter regulated by DNA methylation and retinoic-acid-related drugs. Additionally, we observed that TSTs may be detected in the blood extracellular vesicles of patients with HCC. Conclusion: Our findings suggest an abundance of TSTs in HCC and their potential in clinical settings. The identification and characterization of TSTs may help toward the development of strategies for cancer diagnosis and treatment.

Figure S1. Expression landscape of circRNAs across cancer cell lines. Figure S2. Potential regulation of EMT on biogenesis of circRNAs. Figure S3. Enrichment of circRNAs in clinically actionable genes. Figure S4. Characterization of functional effects and drug response of circMYC in cancer. Figure S5. Therapeutic liability of circRNAs in CTRP. Figure S6. Examples of modules in CircRiC. Table S1. Summary of circRNAs identified across different cancer cell lines. (PDF 7931 kb)

Circular RNAs (circRNAs) are an intriguing class of widely prevalent endogenous RNAs, the vast majority of which have not been characterized functionally. Here, we identified a novel oncogenic circRNA originating from the back‐splicing of Exon2 and Exon3 of a tumor suppressor gene, ARHGAP35 (also known as P190‐A), termed as circARHGAP35. have observe that circARHGAP35 and linear ARHGAP35 have antithetical expression and functions. Interestingly, circARHGAP35 contains a 3867 nt long ORF with an m6A‐modified start codon and encodes a truncated protein comprising four FF domains and lacking the Rho GAP domain. Mechanistically, circARHGAP35 protein promotes cancer cell progression by interacting with TFII‐I protein in the nucleus. The RNA binding protein, HNRNPL, facilitates the formation of circARHGAP35. Clinically, circARHGAP35 is associated with poor survival in cancer patients. Our findings characterize an oncogenic circRNA and demonstrate a novel mechanism of oncogene activation in cancer by circRNA through the production of a truncated protein., Herein, the role of circular RNAs (circRNAs) in cancer is investigated. circARHGAP35 and linear ARHGAP35 are observed to have antithetical expression and functions in cancer. circARHGAP35 translates into an oncogenic large protein that binds to TFII‐I and promotes cancer progression, while its linear counterpart encodes a tumor suppressor. N6‐methyladenosine (m6A) modification of circARHGAP35 promotes its protein translation, and HNRNPL boosts the biogenesis of circARHGAP35.

Summary Animal models of liver cancer are instrumental in the study of hepatocarcinogenesis and development of novel therapeutic approaches. Here, we describe steps to establish liver cancer in a rat model, via chronic administration of diethylnitrosamine. This causes liver tumors with a sequential progression of hepatitis, cirrhosis, and tumor formation, which closely mimics the development of human liver cancer. This protocol was optimized to significantly increase the incidence of liver tumor formation and reduce the duration of the procedure. For complete details on the use and execution of this protocol, please refer to Chen et al. (2020)., Graphical Abstract, Highlights • Detailed protocol for a carcinogen-induced rat hepatocellular carcinoma model • Undergoes a sequential progression of hepatitis, cirrhosis, and tumor formation • Optimized to significantly increase the incidence of liver tumor formation, Animal models of liver cancer are instrumental in the study of hepatocarcinogenesis and development of novel therapeutic approaches. Here, we describe steps to establish liver cancer in a rat model, via chronic administration of diethylnitrosamine, This causes liver tumors with a sequential progression of hepatitis, cirrhosis, and tumor formation, which closely mimics the development of human liver cancer. This protocol was optimized to significantly increase the incidence of liver tumor formation and reduce the duration of the procedure.

Summary Hepatocellular carcinoma (HCC) initiation is characterized by stepwise accumulation of molecular alterations, during which the early events are largely unknown. Here, we presented a comprehensive genomic and transcriptomic landscape at stages of hepatitis, cirrhosis, and HCC by using a diethylnitrosamine-induced rat HCC model. We observed the early occurrence of gene instability and aberrant cancer associated signaling pathways in liver hepatitis. We further characterized the progressive molecular changes during hepatocarcinogenesis, wherein the intense rivalry between tumor-suppressive and oncogenic strengths occurred in cirrhosis stage. Despite the significant pathological difference, mutation signatures and expression landscape are highly similar between hepatitis and cirrhosis stages. Furthermore, we identified PI3K-Akt signaling pathway as a key pathway in the process of hepatocarcinogenesis through integrative analysis, and PIK3CD is a potential biomarker indicating HCC recurrence. The dynamic immune response during hepatocarcinogenesis, such as continuous decline of monocytes, suggests an immunological intervention strategy beyond chemoprevention for liver cancer., Graphical Abstract, Highlights • Genome instability and cancer-related signaling aberrance occurred in liver hepatitis • The rivalry between tumor-suppressive and oncogenic strengths peaked in liver cirrhosis • PI3K-Akt plays a key role in the process of hepatocarcinogenesis • Hepatocarcinogenesis witnessed dynamic changes of monocytes and natural killer cells, Genomics; Cancer Systems Biology; Cancer; Transcriptomics

Hepatocellular carcinoma (HCC) is the most common form of liver cancer and is typically diagnosed at advanced stages. Identification and characterisation of genes within amplified and deleted chromosomal loci can provide new insights into the pathogenesis of cancer and lead to new approaches for diagnosis and therapy. In our previous study, we found a recurrent region of copy number amplification at 19p13.2 in hepatocellular carcinoma (HCC). In the present study, we performed integrated copy number analysis and expression profiling at this locus and a putative cancer gene, SMARCA4/BRG1, was uncovered in this region. BRG1 is a part of the large ATP-dependent chromatin remodelling complex SWI/SNF. The function of BRG1 in various cancers is unclear, including its role in HCC tumorigenesis. Here, we found that BRG1 is upregulated in HCC and that its level significantly correlates with cancer progression in HCC patients. Importantly, we also found that nuclear expression of BRG1 predicts early recurrence for HCC patients. Furthermore, we demonstrated that BRG1 promotes HCC cell proliferation in vitro and in vivo. BRG1 was observed not only to facilitate S-phase entry but also to attenuate cell apoptosis. Finally, we discovered that one of the mechanisms by which BRG1 promotes cell proliferation is the upregulation of SMAD6. These findings highlight the important role of BRG1 in the regulation of HCC proliferation and provide valuable information for cancer prognosis and treatment.

Carbonic anhydrase 2 (CA2) plays vital role in the regulation of ion transport and pH balance and is involved in many biological processes; however, its role in cancer remains obscure. In this study, we identified a novel function of CA2 in facilitating hepatocellular carcinoma (HCC) metastasis. CA2 expression was elevated in Na+-K+-ATPase α1 (ATP1A1)-downregulated HCC cells and was inversely correlated with that of ATP1A1 in HCC. ATP1A1 acted as an oncoprotein whereas CA2 overexpression inhibited cell migration and invasion by reversing epithelial-mesenchymal transition (EMT) in HCC. CA2 downregulation promoted HCC metastasis and invasion whereas ATP1A1 downregulation inhibited HCC metastasis. Because of the opposing effects of CA2 and ATP1A1 in HCC, we examined the role of their correlation in HCC metastasis. CA2 attenuated ATP1A1-triggered tumor growth in vivo and ATP1A1-induced metastasis in vitro. Taken together, the present results suggest that CA2 serves as a suppressor of HCC metastasis and EMT and is correlated with favorable overall survival (OS) in HCC patients.

Background & Aims The pathogenesis of intrahepatic cholangiocarcinoma (ICC), the second most common hepatic cancer, is poorly understood, and the incidence of ICC is increasing worldwide. We searched for mutations in human ICC tumor samples and investigated how they affect ICC cell function. Methods We performed whole exome sequencing of 7 pairs of ICC tumors and their surrounding nontumor tissues to detect somatic alterations. We then screened 124 pairs of ICC and nontumor samples for these mutations, including 7 exomes. We compared mutations in PTPN3 with tumor recurrence in 124 patients and PTPN3 expression levels with recurrence in 322 patients (the combination of both in 86 patients). The functional effects of PTPN3 variations were determined by RNA interference and transgenic expression in cholangiocarcinoma cell lines (RBE, HCCC-9810, and Huh28). Results Based on exome sequencing, pathways that regulate protein phosphorylation were among the most frequently altered in ICC samples and genes encoding protein tyrosine phosphatases (PTPs) were among the most frequently mutated. We identified mutations in 9 genes encoding PTPs in 4 of 7 ICC exomes. In the prevalence screen of 124 paired samples, 51.6% of ICCs contained somatic mutations in at least 1 of 9 PTP genes; 41.1% had mutations in PTPN3. Transgenic expression of PTPN3 in cell lines increased cell proliferation, colony formation, and migration. PTPN3L232R and PTPN3L384H, which were frequently detected in ICC samples, were found to be gain-of-function mutations; their expression in cell lines further increased cell proliferation, colony formation, and migration. ICC-associated variants of PTPN3 altered phosphatase activity. Patients whose tumors contained activating mutations or higher levels of PTPN3 protein than nontumor tissues had higher rates of disease recurrence than patients whose tumors did not have these characteristics. Conclusions Using whole exome sequencing of ICC samples from patients, we found that more than 40% contain somatic mutations in PTPN3. Activating mutations in and high expression levels of PTPN3 were associated with tumor recurrence.

Background/Aims Sphingosine kinase 1 (SphK1), which phosphorylates sphingosine to sphingosine-1-phosphate (S1P), is overexpressed in various types of cancers, and may act as an oncogene in tumorigenesis. However, little is known about the precise role of the SphK1/S1P pathway in human liver cancer, especially regarding the metastasis of hepatocellular carcinoma (HCC). Materials and methods The expression of SphK1 was detected by quantitative reverse-transcription PCR. In addition, transwell cell migration and invasion assay were carried out for functional analysis. Furthermore, the level of S1P was quantified by ELISA and Rac1/Cdc42 GTPase activation was assessed by western blot analysis. Results The levels of SphK1 mRNA are commonly up-regulated in HCC patients and human liver cancer cell migration and invasion can be promoted by the overexpression of SphK1. In addition, inhibition of SphK1 with either a SphK1 inhibitor or siRNA reduced human liver cancer cell migration and invasion. Furthermore, overexpression of SphK1 increased S1P levels, and the exogenous addition of S1P increased liver cell migration and invasion through the EDG1 receptor. Discussion and conclusion The results from this study provide strong evidence of a role for the SphK1/S1P/EDG1 pathway in liver metastasis, thus making it an attractive therapeutic target for the development of new anti-HCC drugs.

A powerful way to identify driver genes with causal roles in carcinogenesis is to detect genomic regions that undergo frequent alterations in cancers. Here we identified 1,241 regions of somatic copy number alterations in 58 paired hepatocellular carcinoma (HCC) tumors and adjacent nontumor tissues using genome-wide single nucleotide polymorphism (SNP) 6.0 arrays. Subsequently, by integrating copy number profiles with gene expression signatures derived from the same HCC patients, we identified 362 differentially expressed genes within the aberrant regions. Among these, 20 candidate genes were chosen for further functional assessments. One novel tumor suppressor (tripartite motif-containing 35 [TRIM35]) and two putative oncogenes (hairy/enhancer-of-split related with YRPW motif 1 [HEY1] and small nuclear ribonucleoprotein polypeptide E [SNRPE]) were discovered by various in vitro and in vivo tumorigenicity experiments. Importantly, it was demonstrated that decreases of TRIM35 expression are a frequent event in HCC and the expression level of TRIM35 was negatively correlated with tumor size, histological grade, and serum alpha-fetoprotein concentration. Conclusion: These results showed that integration of genomic and transcriptional data offers powerful potential for identifying novel cancer genes in HCC pathogenesis. (HEPATOLOGY 2011;) © 147.

Toll-like receptors (TLRs) play a central role in sensing and initiating innate antiviral response. In this study, we first investigated the expression of TLR1-10 mRNA transcripts in peripheral blood mononuclear cells (PBMCs) from chronic HBV-infected (CHB) patients and healthy donors by quantitative real-time PCR. The expression of TLR1, TLR2, TLR4 and TLR6 transcripts was significantly lower in PBMCs from CHB patients, and the down-regulation of TLR2 was related to HBV genotype C. Flow cytometric analysis showed that the expression of TLR2 on PBMCs was significantly decreased in CHB patients. Furthermore, impaired cytokine production was observed in PBMCs from CHB patients after challenged with TLR2 and TLR4 ligands and was correlated with the levels of plasma hepatitis B virus surface antigen (HBsAg). In conclusion, our study reveals a possible interaction between HBsAg, TLR signaling and the innate immune response, which may partially explain the mechanism of HBV infection induced immuno-tolerance.

Speckle-type POZ protein (SPOP) is an E3 ubiquitin ligase adaptor that is frequently mutated in human cancers. Our previous findings have indicated that SPOP is mutated and functions as a novel tumor suppressor in hepatoblastoma (HB). However, the biological roles and clinical significance of this SPOP in hepatocellular carcinoma (HCC) remain unknown. In this study, we found that the expression level of SPOP was downregulated in HCC primary tumors by quantitative real-time PCR and the protein level of SPOP was also reduced in 72 pairs of HCC tissue microarrays by immunohistochemical analyses. Moreover, SPOP expression was observed to negatively correlate with the tumor grade and intrahepatic metastasis of HCC patients. Furthermore, we revealed that SPOP not only inhibits cell proliferation but also inhibits the motility of liver cancer cells. Finally, we discovered that one of the mechanisms through which SPOP inhibits liver cancer cell migration involves the disruption of ZEB2 expression and the associated epithelial-mesenchymal transition program. Together, our findings emphasize the critical role of SPOP in the regulation of proliferation and migration in liver cancer.

Cancer cells possess a unique metabolic phenotype that allows them to preferentially utilize glucose through aerobic glycolysis. This phenomenon is referred to as the “Warburg effect.” Accumulating evidence suggests that microRNAs (miRNAs), a class of small noncoding regulatory RNAs, interact with oncogenes/tumor suppressors and induce such metabolic reprograming in cancer cells. To systematically study the metabolic roles of miRNAs in cancer cells, we developed a gain-of-function miRNA screen in HeLa cells. Subsequent investigation of the characterized miRNAs indicated that miR-199a-5p acts as a suppressor for glucose metabolism. Furthermore, miR-199a-5p is often down-regulated in human liver cancer, and its low expression level was correlated with a low survival rate, large tumor size, poor tumor differentiation status, high tumor-node-metastasis stage and the presence of tumor thrombus of patients. MicroRNA-199a-5p directly targets the 3′-untranslated region of hexokinase 2 (HK2), an enzyme that catalyzes the irreversible first step of glycolysis, thereby suppressing glucose consumption, lactate production, cellular glucose-6-phosphate and adenosine triphosphate levels, cell proliferation, and tumorigenesis of liver cancer cells. Moreover, HK2 is frequently up-regulated in liver cancer tissues and associated with poor patient outcomes. The up-regulation of hypoxia-inducible factor-1α under hypoxic conditions suppresses the expression of miR-199a-5p and promotes glycolysis, whereas reintroduction of miR-199a-5p interferes with the expression of HK2, abrogating hypoxia-enhanced glycolysis. Conclusion: miR-199a-5p/HK2 reprograms the metabolic process in liver cancer cells and provides potential prognostic predictors for liver cancer patients. (Hepatology 2015;62:1132-1144)

Background & Aims Cancer cells alter glucose metabolism to support their uncontrolled proliferation. Changes in microRNAs (miRNAs) have been associated with colorectal cancer (CRC) development and progression, but it is not clear whether they regulate metabolism in CRC cells. We aimed to identify miRNAs that alter glucose metabolism in CRC cells and to determine their effects on tumor development. Methods CRC tissues and matched nontumor tissues were collected from 78 patients for messenger RNA (mRNA) analysis and from 112 patients for immunohistochemical analysis at the Fudan University Shanghai Cancer Center from 2005 through 2007. We integrated data on 100 miRNAs previously identified as potential regulators of glucose metabolism in a high-throughput screen with data on 66 miRNAs that often are deregulated in CRC cells. miRNAs with the potential to regulate glucose metabolism in CRC cells were blocked with mimics, and effects on lactate production were measured in CRC cell lines. miRNAs and their targets were overexpressed from lentivirals in CRC cell lines (LoVo and HCT-116) or knocked down with small interfering RNAs. The cells were analyzed in proliferation and colony formation assays and for growth as xenograft tumors in mice. Results We identified 3 miRNAs that significantly inhibited lactate production in 3 CRC cell lines; miR124-3p (miR124) had the strongest effect. By using complementary DNA microarray analyses, we identified 67 mRNAs that were reduced in CRC cell lines that overexpressed miR124; the mRNAs encoding phosphoribosyl pyrophosphate synthetase 1 ( PRPS1 ) and ribose-5-phosphate isomerase-A ( RPIA ) were found to be direct targets of miR124. Knockdown of PRPS1 and RPIA, as well as overexpression of miR124, each reduced glucose consumption and adenosine triphosphate in level CRC cells. Conversely, overexpression of PRPS1 or RPIA restored glycometabolism to these cells. RPIA and PRPS1 contribute to nucleotide metabolism and supply precursors for DNA and RNA biosynthesis. CRC cells that overexpressed miR124 or with knockdown of RPIA or PRPS1 had reduced DNA synthesis and proliferation, whereas cells incubated with an inhibitor of miR124 had significantly increased DNA synthesis and proliferation and formed more colonies. LoVo cells that overexpressed miR124 formed smaller xenograft tumors that controlled cells in mice, and had lower levels of PRPS1 and RPIA mRNA and protein. Compared with normal colorectal tissues, levels of miR124 were reduced significantly in CRC tissues from patients, whereas levels of PRPS1 and RPIA increased, which was associated with reduced patient survival times. Conclusions miR124 inhibits DNA synthesis and proliferation by reducing levels of pentose phosphate pathway enzymes in CRC cells. Expression of miR124 and its targets correlate with survival times and might be used in prognosis.

Tripartite motif-containing protein 35 (TRIM35) is a member of RBCC family, which has a highly conserved order consisting of a RING domain followed by one or two B-Box domains and then a coiled-coil domain. We previously identified TRIM35 as a novel tumor suppressor in human hepatocellular carcinoma (HCC). However, the molecular mechanism that TRIM35 uses to suppress tumorigenicity is largely unknown. Pyruvate kinase isoform M2 (PKM2) has been demonstrated to have a central role in metabolic reprogramming during cancer progression. Phosphorylation of PKM2 tyrosine residue 105 (Y105) regulates PKM2 to provide a metabolic advantage to tumor cells, thereby promoting tumor growth. In the present work, mass spectrometry analysis demonstrated an interaction between TRIM35 and PKM2. Co-IP experiments confirmed that TRIM35 interacts with PKM2 and that the coiled-coil domain is required for such an interaction. Furthermore, the coiled-coil domain mediates decreases in the Warburg effect and in the cell proliferation of HCC cells. In addition, TRIM35 suppresses the tumorigenicity of HCC cells through the blockade of PKM2 Y105 phosphorylation. Collectively, our data reveal a new function for TRIM35, which is to regulate the Warburg effect and tumorigenicity through interaction with PKM2 in HCC.

It is widely accepted that chronic hepatitis B virus (HBV) infection is the result of an ineffective antiviral immune response against HBV infection. Our previous study found that the hepatitis B surface Ag (HBsAg) was related to decreased cytokine production induced by the TLR2 ligand (Pam3csk4) in PBMCs from chronic hepatitis B patients. In this study, we further explored the mechanism involved in the inhibitory effect of HBsAg on the TLR2 signaling pathway. The results showed that both Pam3csk4-triggered IL-12p40 mRNA expression and IL-12 production in PMA-differentiated THP-1 macrophage were inhibited by HBsAg in a dose-dependent manner, but the production of IL-1β, IL-6, IL-8, IL-10, and TNF-α was not influenced. The Pam3csk4-induced activation of NF-κB and MAPK signaling were further examined. The phosphorylation of JNK-1/2 and c-Jun was impaired in the presence of HBsAg, whereas the degradation of IκB-α, the nuclear translocation of p65, and the phosphorylation of p38 and ERK-1/2 were not affected. Moreover, the inhibition of JNK phosphorylation and IL-12 production in response to Pam3csk was observed in HBsAg-treated monocytes/macrophages (M/MΦs) from the healthy donors and the PBMCs and CD14-positive M/MΦs from chronic hepatitis B patients. Taken together, these results demonstrate that HBsAg selectively inhibits Pam3csk4- stimulated IL-12 production in M/MΦs by blocking the JNK–MAPK pathway and provide a mechanism by which HBV evades immunity and maintains its persistence.

Cancer is an ancient disease of multi-cellular organisms acquired in the history of evolution. For more than one century, cancer was defined as a disease of autonomous, abnormal growth of cells from an organ or tissue. Therefore, the majority of research and resources were focused on the cancer, especially cancer cells. In recent decades, an increasing body of evidence has emerged to indicate that carcinogenesis and cancer progressions involve systemic dysregulation, which may be the important driving force in cancer development as well as its progression. As we have emphasized in Chapter 1, we defined cancer as a systems disease which is characterized by abnormal cell growth in a defined tissue or organ and progressive systemic dysregulation.