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).
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.
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.
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.
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. (Hepatology 2016;63:474–487)
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‐β), 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‐β 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. (Hepatology 2014;60:1645–1658)
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.
Graphical abstract Deregulation of G9a in human HCC. G9a was frequently up-regulated in human HCC and implicated in HCC tumorigenesis and metastasis. The frequent up-regulation of G9a in human HCC ...was attributed to gene copy number gain at 6p21 and loss of miR-1. The oncogenic function of G9a was at least partially attributed to the epigenetic silencing of tumor suppressor RARRES3.
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.
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. (HEPATOLOGY 2013)
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