In Vivo Genome-Wide CRISPR Activation Screening Identifies Functionally Important Long Noncoding RNAs in Hepatocellular Carcinoma.

BACKGROUND & AIMS: Long noncoding RNAs (lncRNAs) are found to have profound impacts on diverse cellular processes. Although high-throughput sequencing studies have shown the differential lncRNA expression profiles between hepatocellular carcinoma (HCC) and nontumor livers, the functional impacts of lncRNAs on HCC development await further investigation. Herein, we sought to address the functional roles of lncRNAs in HCC pathogenesis by in vivo functional screening. METHODS: We performed genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/dead CRISPR-associated protein 9 (dCas9) lncRNA activation screening in HCC xenografts. We characterized the clinical relevance of positively selected lncRNAs using transcriptomic data sets. We used CRISPR-based gene activation and knockdown approaches to show the functional roles of positively selected lncRNAs including Cancer Susceptibility 11 (CASC11) in HCC. RNA sequencing and chromatin isolation by RNA purification sequencing were used to investigate the molecular mechanisms of CASC11 in HCC progression. RESULTS: The in vivo functional screening identified 1603 positively selected lncRNAs, 538 of which were overexpressed in HCC patients. Systematic transcriptomic data analysis and clinical investigation showed that patients with high expression of these lncRNA candidates correlated with aggressive tumor behaviors. Overexpression of these lncRNAs aggravated HCC cell growth. Detailed characterization of a lncRNA candidate, CASC11, showed its pivotal role in cell proliferation and tumor growth. Mechanistically, chromatin isolation by RNA purification sequencing showed that CASC11 was bound to the CASC11/MYC proto-oncogene shared promoter region on chromosome 8q24. CASC11 modulated the transcriptional activity of MYC in a cis-regulatory manner, which affected the expression of MYC downstream target genes, consequently promoting G1/S progression. CONCLUSIONS: Our study showed the power of in vivo CRISPR screening, which comprehensively investigated the functionality of lncRNAs in HCC progression, providing a rationale for targeting these lncRNAs clinically.

Adaptive and Constitutive Activations of Malic Enzymes Confer Liver Cancer Multilayered Protection Against Reactive Oxygen Species.

BACKGROUND AND AIMS: HCC undergoes active metabolic reprogramming. Reactive oxygen species (ROS) are excessively generated in cancer cells and are neutralized by NADPH. Malic enzymes (MEs) are the less studied NADPH producers in cancer. APPROACH AND RESULTS: We found that ME1, but not ME3, was regulated by the typical oxidative stress response pathway mediated by kelch-like ECH associated protein 1/nuclear factor erythroid 2-related factor (NRF2). Surprisingly, ME3 was constitutively induced by superenhancers. Disruption of any ME regulatory pathways decelerated HCC progression and sensitized HCC to sorafenib. Therapeutically, simultaneous blockade of NRF2 and a superenhancer complex completely impeded HCC growth. We show that superenhancers allow cancer cells to counteract the intrinsically high level of ROS through constitutively activating ME3 expression. When HCC cells encounter further episodes of ROS insult, NRF2 allows cancer cells to adapt by transcriptionally activating ME1. CONCLUSIONS: Our study reveals the complementary regulatory mechanisms which control MEs and provide cancer cells multiple layers of defense against oxidative stress. Targeting both regulatory mechanisms represents a potential therapeutic approach for HCC treatment.

Histone chaperone FACT complex mediates oxidative stress response to promote liver cancer progression.

OBJECTIVE: Facilitates Chromatin Transcription (FACT) complex is a histone chaperone participating in DNA repair-related and transcription-related chromatin dynamics. In this study, we investigated its oncogenic functions, underlying mechanisms and therapeutic implications in human hepatocellular carcinoma (HCC). DESIGN: We obtained HCC and its corresponding non-tumorous liver samples from 16 patients and identified FACT complex as the most upregulated histone chaperone by RNA-Seq. We further used CRISPR-based gene activation and knockout systems to demonstrate the functions of FACT complex in HCC growth and metastasis. Functional roles and mechanistic insights of FACT complex in oxidative stress response were investigated by ChIP assay, flow cytometry, gene expression assays and 4sU-DRB transcription elongation assay. Therapeutic effect of FACT complex inhibitor, Curaxin, was tested in both in vitro and in vivo models. RESULTS: We showed that FACT complex was remarkably upregulated in HCC and contributed to HCC progression. Importantly, we unprecedentedly revealed an indispensable role of FACT complex in NRF2-driven oxidative stress response. Oxidative stress prevented NRF2 and FACT complex from KEAP1-mediated protein ubiquitination and degradation. Stabilised NRF2 and FACT complex form a positive feedback loop; NRF2 transcriptionally activates the FACT complex, while FACT complex promotes the transcription elongation of NRF2 and its downstream antioxidant genes through facilitating rapid nucleosome disassembly for the passage of RNA polymerase. Therapeutically, Curaxin effectively suppressed HCC growth and sensitised HCC cell to sorafenib. CONCLUSION: In conclusion, our findings demonstrated that FACT complex is essential for the expeditious HCC oxidative stress response and is a potential therapeutic target for HCC treatment.

Genome-wide CRISPR/Cas9 library screening identified PHGDH as a critical driver for Sorafenib resistance in HCC.

Sorafenib is the standard treatment for advanced hepatocellular carcinoma (HCC). However, the development of drug resistance is common. By using genome-wide CRISPR/Cas9 library screening, we identify phosphoglycerate dehydrogenase (PHGDH), the first committed enzyme in the serine synthesis pathway (SSP), as a critical driver for Sorafenib resistance. Sorafenib treatment activates SSP by inducing PHGDH expression. With RNAi knockdown and CRISPR/Cas9 knockout models, we show that inactivation of PHGDH paralyzes the SSP and reduce the production of αKG, serine, and NADPH. Concomitantly, inactivation of PHGDH elevates ROS level and induces HCC apoptosis upon Sorafenib treatment. More strikingly, treatment of PHGDH inhibitor NCT-503 works synergistically with Sorafenib to abolish HCC growth in vivo. Similar findings are also obtained in other FDA-approved tyrosine kinase inhibitors (TKIs), including Regorafenib or Lenvatinib. In summary, our results demonstrate that targeting PHGDH is an effective approach to overcome TKI drug resistance in HCC.

Aberrant Super-Enhancer Landscape in Human Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) cells exploit an aberrant transcriptional program to sustain their infinite growth and progression. Emerging evidence indicates that the continuous and robust transcription of oncogenes in cancer cells is often driven by super-enhancers (SEs). In this study, we systematically compared the SE landscapes between normal liver and HCC cells and revealed that the cis-acting SE landscape was extensively reprogrammed during liver carcinogenesis. HCC cells acquired SEs at multiple prominent oncogenes to drive their vigorous expression. We identified sphingosine kinase 1 (SPHK1) as an SE-associated oncogene, and we used this gene as an example to illustrate the impact of SEs on the activation of oncogenes in HCC. Concurrently, we also showed that the critical components of the trans-acting SE complex, namely, cyclin-dependent kinase 7 (CDK7), bromodomain-containing protein 4 (BRD4), E1A binding protein P300 (EP300), and mediator complex subunit 1 (MED1), were frequently overexpressed in human HCCs and were associated with the poor prognosis of patients with HCC. Using the CRISPR/Cas9 gene-editing system and specific small-molecule inhibitors, we further demonstrated that HCC cells were highly sensitive to perturbations of the SE complex. The inactivation of CDK7, BRD4, EP300, and MED1 selectively repressed the expression of SE-associated oncogenes in HCC. Finally, we demonstrated that THZ1, which is a small-molecule inhibitor of CDK7, exerted a prominent anticancer effect in both in vitro and in vivo HCC models. Conclusion: The SE landscape and machinery were significantly altered in human HCCs. HCC cells are highly susceptible to perturbations of the SE complex due to the resulting selective suppression of SE-associated oncogenes. Our results suggest that targeting SE complex is a promising therapeutic strategy for HCC treatment.

HELLS Regulates Chromatin Remodeling and Epigenetic Silencing of Multiple Tumor Suppressor Genes in Human Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the third most lethal cancer worldwide. Increasing evidence shows that epigenetic alterations play an important role in human carcinogenesis. Deregulation of DNA methylation and histone modifications have recently been characterized in HCC, but the significance of chromatin remodeling in liver carcinogenesis remains to be explored. In this study, by systematically analyzing the expression of chromatin remodeling genes in human HCCs, we found that helicase, lymphoid-specific (HELLS), an SWI2/SNF2 chromatin remodeling enzyme, was remarkably overexpressed in HCC. Overexpression of HELLS correlated with more aggressive clinicopathological features and poorer patient prognosis compared to patients with lower HELLS expression. We further showed that up-regulation of HELLS in HCC was conferred by hyperactivation of transcription factor specificity protein 1 (SP1). To investigate the functions of HELLS in HCC, we generated both gain-of-function and loss-of-function models by the CRISPR activation system, lentiviral short hairpin RNA, and the CRISPR/Cas9 genome editing system. We demonstrated that overexpression of HELLS augmented HCC cell proliferation and migration. In contrast, depletion of HELLS reduced HCC growth and metastasis both in vitro and in vivo. Moreover, inactivation of HELLS led to metabolic reprogramming and reversed the Warburg effect in HCC cells. Mechanistically, by integrating analysis of RNA sequencing and micrococcal nuclease sequencing, we revealed that overexpression of HELLS increased nucleosome occupancy, which obstructed the accessibility of enhancers and hindered formation of the nucleosome-free region (NFR) at the transcription start site. Though this mechanism, up-regulation of HELLS mediated epigenetic silencing of multiple tumor suppressor genes including E-cadherin, FBP1, IGFBP3, XAF1 and CREB3L3 in HCC. Conclusion: Our data reveal that HELLS is a key epigenetic driver of HCC; by altering the nucleosome occupancy at the NFR and enhancer, HELLS epigenetically suppresses multiple tumor suppressor genes to promote HCC progression.

Hepatitis transactivator protein X promotes extracellular matrix modification through HIF/LOX pathway in liver cancer.

Hepatocellular carcinoma (HCC), accounting for 90% of primary liver cancer, is a lethal malignancy that is tightly associated with chronic hepatitis B virus (HBV) infection. HBV encodes a viral onco-protein, transactivator protein X (HBx), which interacts with proteins of hepatocytes to promote oncogenesis. Our current study focused on the interaction of HBx with a transcription factor, hypoxia-inducible factor-1α (HIF-1α), which is stabilized by low O2 condition (hypoxia) and is found to be frequently overexpressed in HCC intra-tumorally due to poor blood perfusion. Here, we showed that overexpression of HBx by tetracycline-inducible systems further stabilized HIF-1α under hypoxia in HBV-negative HCC cell lines. Reversely, knockdown of HBx reduced HIF-1α protein stabilization under hypoxia in HBV-positive HCC cell lines. More intriguingly, overexpression of HBx elevated the mRNA and protein expression of a family of HIF-1α target genes, the lysyl oxidase (LOX) family in HCC. The LOX family members function to cross-link collagen in the extracellular matrix (ECM) to promote cancer progression and metastasis. By analyzing the collagens under scanning electron microscope, we found that collagen fibers were significantly smaller in size when incubated with conditioned medium from HBx knockdown HCC cells as compared to control HCC cells in vitro. Transwell invasion assay further revealed that less cells were able to invade through the matrigel which was pre-treated with conditioned medium from HBx knockdown HCC cells as compared to control HCC cells. Orthotopic and subcutaneous HCC models further showed that knockdown of HBx in HCC cells reduced collagen crosslinking and stiffness in vivo and repressed HCC growth and metastasis. Taken together, our in vitro and in vivo studies showed the HBx remodeled the ECM through HIF-1α/LOX pathway to promote HCC metastasis.

Non-coding RNAs in hepatocellular carcinoma: molecular functions and pathological implications.

Hepatocellular carcinoma (HCC) is a leading lethal malignancy worldwide. However, the molecular mechanisms underlying liver carcinogenesis remain poorly understood. Over the past two decades, overwhelming evidence has demonstrated the regulatory roles of different classes of non-coding RNAs (ncRNAs) in liver carcinogenesis related to a number of aetiologies, including HBV, HCV and NAFLD. Among the ncRNAs, microRNAs, which belong to a distinct class of small ncRNAs, have been proven to play a crucial role in the post-transcriptional regulation of gene expression. Deregulation of microRNAs has been broadly implicated in the inactivation of tumour-suppressor genes and activation of oncogenes in HCC. Modern high-throughput sequencing analyses have unprecedentedly identified a very large number of non-coding transcripts. Divergent groups of long ncRNAs have been implicated in liver carcinogenesis through interactions with DNA, RNA or proteins. Overall, ncRNAs represent a burgeoning field of cancer research, and we are only beginning to understand the importance and complicity of the ncRNAs in liver carcinogenesis. In this Review, we summarize the common deregulation of small and long ncRNAs in human HCC. We also comprehensively review the pathological roles of ncRNAs in liver carcinogenesis, epithelial-to-mesenchymal transition and HCC metastasis and discuss the potential applications of ncRNAs as diagnostic tools and therapeutic targets in human HCC.

RNA N6-methyladenosine methyltransferase-like 3 promotes liver cancer progression through YTHDF2-dependent posttranscriptional silencing of SOCS2.

Epigenetic alterations have contributed greatly to human carcinogenesis. Conventional epigenetic studies have predominantly focused on DNA methylation, histone modifications, and chromatin remodeling. Recently, diverse and reversible chemical modifications of RNAs have emerged as a new layer of epigenetic regulation. N6-methyladenosine (m6A) is the most abundant chemical modification of eukaryotic messenger RNA (mRNA) and is important for the regulation of mRNA stability, splicing, and translation. Using transcriptome sequencing, we discovered that methyltransferase-like 3 (METTL3), a major RNA N6-adenosine methyltransferase, was significantly up-regulated in human hepatocellular carcinoma (HCC) and multiple solid tumors. Clinically, overexpression of METTL3 is associated with poor prognosis of patients with HCC. Functionally, we proved that knockdown of METTL3 drastically reduced HCC cell proliferation, migration, and colony formation in vitro. Knockout of METTL3 remarkably suppressed HCC tumorigenicity and lung metastasis in vivo. On the other hand, using the CRISPR/dCas9-VP64 activation system, we demonstrated that overexpression of METTL3 significantly promoted HCC growth both in vitro and in vivo. Through transcriptome sequencing, m6A sequencing, and m6A methylated RNA immuno-precipitation quantitative reverse-transcription polymerase chain reaction, we identified suppressor of cytokine signaling 2 (SOCS2) as a target of METTL3-mediated m6A modification. Knockdown of METTL3 substantially abolished SOCS2 mRNA m6A modification and augmented SOCS2 mRNA expression. We also showed that m6A-mediated SOCS2 mRNA degradation relied on the m6A reader protein YTHDF2-dependent pathway. CONCLUSION: METTL3 is frequently up-regulated in human HCC and contributes to HCC progression. METTL3 represses SOCS2 expression in HCC through an m6A-YTHDF2-dependent mechanism. Our findings suggest an important mechanism of epigenetic alteration in liver carcinogenesis. (Hepatology 2018;67:2254-2270).

Hypoxia inducible factor HIF-1 promotes myeloid-derived suppressor cells accumulation through ENTPD2/CD39L1 in hepatocellular carcinoma.

Myeloid-derived suppressor cells (MDSCs) possess immunosuppressive activities, which allow cancers to escape immune surveillance and become non-responsive to immune checkpoints blockade. Here we report hypoxia as a cause of MDSC accumulation. Using hepatocellular carcinoma (HCC) as a cancer model, we show that hypoxia, through stabilization of hypoxia-inducible factor-1 (HIF-1), induces ectoenzyme, ectonucleoside triphosphate diphosphohydrolase 2 (ENTPD2/CD39L1), in cancer cells, causing its overexpression in HCC clinical specimens. Overexpression of ENTPD2 is found as a poor prognostic indicator for HCC. Mechanistically, we demonstrate that ENTPD2 converts extracellular ATP to 5'-AMP, which prevents the differentiation of MDSCs and therefore promotes the maintenance of MDSCs. We further find that ENTPD2 inhibition is able to mitigate cancer growth and enhance the efficiency and efficacy of immune checkpoint inhibitors. Our data suggest that ENTPD2 may be a good prognostic marker and therapeutic target for cancer patients, especially those receiving immune therapy.Myeloid-derived suppressor cells (MDSCs) promote tumor immune escape. Here, the authors show that in hepatocellular carcinoma, hypoxia induces the expression of ENTPD2 on cancer cells leading to elevated extracellular 5'-AMP, which in turn promote the maintenance of MDSCs by preventing their differentiation.

Histone methyltransferase G9a promotes liver cancer development by epigenetic silencing of tumor suppressor gene RARRES3.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is a major leading cause of cancer mortality worldwide. Epigenetic deregulation is a common trait of human HCC. G9s is an important epigenetics regulator however, its role in liver carcinogenesis remains to be investigated. METHODS: Gene expressions were determined by RNA-Seq and qRT-PCR. G9a knockdown and knockout cell lines were established by lentiviral-based shRNA and CRISPR/Cas9 gene editing system. Tumor-promoting functions of G9a was studied in both HCC cell lines and nude mice model. The downstream targets of G9a were identified by RNA-Seq and confirmed by ChIP assay. The therapeutic value of G9a inhibitors was evaluated both in vitro and in vivo. RESULTS: We identified G9a as a frequently upregulated histone methyltransferase in human HCCs. Upregulation of G9a was significantly associated with HCC progression and aggressive clinicopathological features. Functionally, we demonstrated that inactivation of G9a by RNAi knockdown, CRISPR/Cas9 knockout, and pharmacological inhibition remarkably abolished H3K9 di-methylation and suppressed HCC cell proliferation and metastasis in both in vitro and in vivo models. Mechanistically, we showed that the frequent upregulation of G9a in human HCCs was attributed to gene copy number gain at chromosome 6p21. In addition, we identified miR-1 as a negative regulator of G9a. Loss of miR-1 relieved the post-transcriptional repression on G9a and contributed to its upregulation in human HCC. Utilizing RNA sequencing, we identified the tumor suppressor RARRES3 as a critical target of G9a. Epigenetic silencing of RARRES3 contributed to the tumor-promoting function of G9a. CONCLUSION: This study shows a frequent deregulation of miR-1/G9a/RARRES3 axis in liver carcinogenesis, highlighting the pathological significance of G9a and its therapeutic potential in HCC treatment. Lay summary: In this study, we identified G9a histone methyltransferase was frequently upregulated in human HCC and contributes to epigenetic silencing of tumor suppressor gene RARRES3 in liver cancer. Targeting G9a may be a novel approach for HCC treatment.

Folate cycle enzyme MTHFD1L confers metabolic advantages in hepatocellular carcinoma.

Cancer cells preferentially utilize glucose and glutamine, which provide macromolecules and antioxidants that sustain rapid cell division. Metabolic reprogramming in cancer drives an increased glycolytic rate that supports maximal production of these nutrients. The folate cycle, through transfer of a carbon unit between tetrahydrofolate and its derivatives in the cytoplasmic and mitochondrial compartments, produces other metabolites that are essential for cell growth, including nucleotides, methionine, and the antioxidant NADPH. Here, using hepatocellular carcinoma (HCC) as a cancer model, we have observed a reduction in growth rate upon withdrawal of folate. We found that an enzyme in the folate cycle, methylenetetrahydrofolate dehydrogenase 1-like (MTHFD1L), plays an essential role in support of cancer growth. We determined that MTHFD1L is transcriptionally activated by NRF2, a master regulator of redox homeostasis. Our observations further suggest that MTHFD1L contributes to the production and accumulation of NADPH to levels that are sufficient to combat oxidative stress in cancer cells. The elevation of oxidative stress through MTHFD1L knockdown or the use of methotrexate, an antifolate drug, sensitizes cancer cells to sorafenib, a targeted therapy for HCC. Taken together, our study identifies MTHFD1L in the folate cycle as an important metabolic pathway in cancer cells with the potential for therapeutic targeting.

Up-regulation of histone methyltransferase SETDB1 by multiple mechanisms in hepatocellular carcinoma promotes cancer metastasis.

UNLABELLED: Epigenetic deregulation plays an important role in liver carcinogenesis. Using transcriptome sequencing, we examined the expression of 591 epigenetic regulators in hepatitis B-associated human hepatocellular carcinoma (HCC). We found that aberrant expression of epigenetic regulators was a common event in HCC. We further identified SETDB1 (SET domain, bifurcated 1), an H3K9-specific histone methyltransferase, as the most significantly up-regulated epigenetic regulator in human HCCs. Up-regulation of SETDB1 was significantly associated with HCC disease progression, cancer aggressiveness, and poorer prognosis of HCC patients. Functionally, we showed that knockdown of SETDB1 reduced HCC cell proliferation in vitro and suppressed orthotopic tumorigenicity in vivo. Inactivation of SETDB1 also impeded HCC cell migration and abolished lung metastasis in nude mice. Interestingly, SETDB1 protein was consistently up-regulated in all metastatic foci found in different organs, suggesting that SETDB1 was essential for HCC metastatic progression. Mechanistically, we showed that the frequent up-regulation of SETDB1 in human HCC was attributed to the recurrent SETDB1 gene copy gain at chromosome 1q21. In addition, hyperactivation of specificity protein 1 transcription factor in HCC enhanced SETDB1 expression at the transcriptional level. Furthermore, we identified miR-29 as a negative regulator of SETDB1. Down-regulation of miR-29 expression in human HCC contributed to SETDB1 up-regulation by relieving its post-transcriptional regulation. CONCLUSION: SETDB1 is an oncogene that is frequently up-regulated in human HCCs; the multiplicity of SETDB1 activating mechanisms at the chromosomal, transcriptional, and posttranscriptional levels together facilitates SETDB1 up-regulation in human HCC.

MicroRNA-142-3p and microRNA-142-5p are downregulated in hepatocellular carcinoma and exhibit synergistic effects on cell motility.

MicroRNAs (miRNAs), an important class of small non-coding RNAs, regulate gene expression at the post-transcriptional level. miRNAs are involved in a wide range of biological processes and implicated in different diseases, including cancers. In this study, miRNA profiling and qRT-PCR validation revealed that miR-142-3p and miR-142-5p were significantly downregulated in hepatocellular carcinoma (HCC) and their expression levels decreased as the disease progressed. The ectopic expression of miR-142 significantly reduced HCC cell migration and invasion. Overexpression of either miR-142-3p or miR-142-5p suppressed HCC cell migration, and overexpression of both synergistically inhibited cell migration, which indicated that miR-142-3p and miR-142-5p may cooperatively regulate cell movement. miR-142-3p and miR-142-5p, which are mature miRNAs derived from the 3'- and 5'-strands of the precursor miR-142, target distinct pools of genes because of their different seed sequences. Pathway enrichment analysis showed a strong association of the putative gene targets of miR-142-3p and miR-142-5p with several cell motility-associated pathways, including those regulating actin cytoskeleton, adherens junctions, and focal adhesion. Importantly, a number of the putative gene targets were also significantly upregulated in human HCC cells. Moreover, overexpression of miR-142 significantly abrogated stress fiber formation in HCC cells and led to cell shrinkage. This study shows that mature miR-142 pairs collaboratively regulate different components of distinct signaling cascades and therefore affects the motility of HCC cells.

MiR-200b/200c/429 subfamily negatively regulates Rho/ROCK signaling pathway to suppress hepatocellular carcinoma metastasis.

MiR-200 family is an important regulator of epithelial-mesenchymal transition and has been implicated in human carcinogenesis. However, their expression and functions in human cancers remain controversial. In the work presented here, we showed that miR-200 family members were frequently down-regulated in hepatocellular carcinoma (HCC). Although all five members of miR-200 family inhibited ZEB1/2 expression in HCC cell lines, we showed that overexpression only of the miR-200b/200c/429 subfamily, but not the miR-200a/141 subfamily, resulted in impeded HCC cell migration. Further investigations led to the identification of RhoA and ROCK2 as specific down-stream targets of the miR-200b/200c/429 subfamily. We demonstrated that the miR-200b/200c/429 subfamily inhibited HCC cell migration through modulating Rho/ROCK mediated cell cytoskeletal reorganization and cell-substratum adhesion. Re-expression of miR-200b significantly suppressed lung metastasis of HCC cells in an orthotopic liver implantation model in vivo. In conclusion, our findings identified the miR-200b/200c/429 subfamily as metastasis suppressor microRNAs in human HCC and highlighted the functional discrepancy among miR-200 family members.

Switching of pyruvate kinase isoform L to M2 promotes metabolic reprogramming in hepatocarcinogenesis.

Hepatocellular carcinoma (HCC) is an aggressive tumor, with a high mortality rate due to late symptom presentation and frequent tumor recurrences and metastasis. It is also a rapidly growing tumor supported by different metabolic mechanisms; nevertheless, the biological and molecular mechanisms involved in the metabolic reprogramming in HCC are unclear. In this study, we found that pyruvate kinase M2 (PKM2) was frequently over-expressed in human HCCs and its over-expression was associated with aggressive clinicopathological features and poor prognosis of HCC patients. Furthermore, knockdown of PKM2 suppressed aerobic glycolysis and cell proliferation in HCC cell lines in vitro. Importantly, knockdown of PKM2 hampered HCC growth in both subcutaneous injection and orthotopic liver implantation models, and reduced lung metastasis in vivo. Of significance, PKM2 over-expression in human HCCs was associated with a down-regulation of a liver-specific microRNA, miR-122. We further showed that miR-122 interacted with the 3UTR of the PKM2 gene. Re-expression of miR-122 in HCC cell lines reduced PKM2 expression, decreased glucose uptake in vitro, and suppressed HCC tumor growth in vivo. Our clinical data and functional studies have revealed a novel biological mechanism involved in HCC metabolic reprogramming.

Lysyl oxidase-like 2 is critical to tumor microenvironment and metastatic niche formation in hepatocellular carcinoma.

UNLABELLED: Poor prognosis of cancers, including hepatocellular carcinoma (HCC), is mainly associated with metastasis; however, the underlying mechanisms remain poorly understood. This article investigates the role of lysyl oxidase-like 2 (LOXL-2) in the biology of HCC metastasis. First, we showed that HCC metastasis relies on a collagen-modifying enzyme, LOXL2, which was significantly overexpressed in tumorous tissues and sera of HCC patients, indicating that LOXL2 may be a good diagnostic marker for HCC patients. Second, we delineated a complex, interlinked signaling network that involves multiple regulators, including hypoxia, transforming growth factor beta (TGF-ß), and microRNAs (miRNAs), converging to control the expression of LOXL2. We found not only that LOXL2 was regulated by hypoxia/hypoxia-inducible factor 1 alpha (HIF-1α), but also that TGF-ß activated LOXL2 transcription through mothers against decapentaplegic homolog 4 (Smad4), whereas two frequently underexpressed miRNA families, miR-26 and miR-29, cooperatively suppressed LOXL2 transcription through interacting with the 3' untranslated region of LOXL2. Third, we demonstrated the imperative roles of LOXL2 in modifying the extracellular matrix components in the tumor microenvironment and metastatic niche of HCC. LOXL2 promoted intrahepatic metastasis by increasing tissue stiffness, thereby enhancing the cytoskeletal reorganization of HCC cells. Furthermore, LOXL2 facilitated extrahepatic metastasis by enhancing recruitment of bone-marrow-derived cells to the metastatic site. CONCLUSION: These findings integrate the clinical relevance, molecular regulation, and functional implications of LOXL2 in HCC metastasis.

Regulation of hepatocarcinogenesis by microRNAs.

Liver cancer (hepatocellular carcinoma, HCC) is a major malignancy worldwide. Etiologically, hepatocarcinogenesis is closely associated with HBV and HCV infections; however, its underlying molecular mechanism is not completely understood. MicroRNAs are a class of small non-coding RNAs that negatively regulate gene expression by interacting with the 3'UTR of protein-coding mRNA. MicroRNAs are implicated in nearly all major biological and cellular events, and recent findings further link microRNA deregulation to human carcinogenesis. In this review, we will focus on the aberrant expression of miRNAs in liver cancer and the pathological implications and molecular functions of some well-characterized oncogenic and tumor suppressive miRNAs. Finally, the clinical prospect of miRNAs as a novel diagnostic and therapeutic intervention will be discussed.

Histone lysine methyltransferase, suppressor of variegation 3-9 homolog 1, promotes hepatocellular carcinoma progression and is negatively regulated by microRNA-125b.

UNLABELLED: Hepatocellular carcinoma (HCC) is a major liver malignancy. We previously demonstrated that deregulation of epigenetic regulators is a common event in human HCC. Suppressor of variegation 3-9 homolog 1 (SUV39H1), the prototype of histone methyltransferase, is the major enzyme responsible for histone H3 lysine 9 trimethylation, which, essentially, is involved in heterochromatin formation, chromosome segregation, and mitotic progression. However, the implication of SUV39H1 in hepatocarcinogenesis remains elusive. In this study, we found that SUV39H1 was frequently up-regulated in human HCCs and was significantly associated with increased Ki67 expression (P < 0.001) and the presence of venous invasion (P = 0.017). To investigate the role of SUV39H1 in HCC development, both gain- and loss-of-function models were established. SUV39H1 overexpression remarkably enhanced HCC cell clonogenicity, whereas knockdown of SUV39H1 substantially suppressed HCC cell proliferation and induced cell senescence. In addition, ectopic expression of SUV39H1 increased the migratory ability of HCC cells, whereas a reduced migration rate was observed in SUV39H1 knockdown cells. The significance of SUV39H1 in HCC was further demonstrated in a nude mice model; SUV39H1 knockdown drastically inhibited in vivo tumorigenicity and abolished pulmonary metastasis of HCC cells. We also identified microRNA-125b (miR-125b) as a post-transcriptional regulator of SUV39H1. Ectopic expression of miR-125b inhibited SUV39H1 3'-untranslated-region-coupled luciferase activity and suppressed endogenous SUV39H1 expression at both messenger RNA and protein levels. We have previously reported frequent down-regulation of miR-125b in HCC. Interestingly, miR-125b level was found to be inversely correlated with SUV39H1 expression (P = 0.001) in clinical specimens. Our observations suggested that miR-125b down-regulation may account for the aberrant SUV39H1 level in HCC. CONCLUSION: Our study demonstrated that SUV39H1 up-regulation contributed to HCC development and metastasis. The tumor-suppressive miR-125b served as a negative regulator of SUV39H1.