Cheuk-Ting Law

Transketolase counteracts oxidative stress to drive cancer development

Significance Excessive accumulation of oxidative stress is harmful to cancer cells. Our study demonstrates the important roles of a pentose phosphate pathway (PPP) enzyme, transketolase (TKT), in redox homeostasis in cancer development. We highlight the clinical relevance of TKT expression in cancers. We also show that TKT overexpression in cancer cells is a response of Nuclear Factor, Erythroid 2-Like 2 (NRF2) activation, a sensor to cellular oxidative stress. TKT locates at an important position that connects PPP with glycolysis to affect production of antioxidant NADPH. Our preclinical study shows that targeting TKT leads to elevation of oxidative stress, making cancer cells more vulnerable to therapeutic treatment, such as Sorafenib. Using TKT as an example, our study suggests that targeting enzymes for antioxidant production represents a direction for cancer treatment. Cancer cells experience an increase in oxidative stress. The pentose phosphate pathway (PPP) is a major biochemical pathway that generates antioxidant NADPH. Here, we show that transketolase (TKT), an enzyme in the PPP, is required for cancer growth because of its ability to affect the production of NAPDH to counteract oxidative stress. We show that TKT expression is tightly regulated by the Nuclear Factor, Erythroid 2-Like 2 (NRF2)/Kelch-Like ECH-Associated Protein 1 (KEAP1)/BTB and CNC Homolog 1 (BACH1) oxidative stress sensor pathway in cancers. Disturbing the redox homeostasis of cancer cells by genetic knockdown or pharmacologic inhibition of TKT sensitizes cancer cells to existing targeted therapy (Sorafenib). Our study strengthens the notion that antioxidants are beneficial to cancer growth and highlights the therapeutic benefits of targeting pathways that generate antioxidants.

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

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)

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).

NDUFA4L2 Fine-tunes Oxidative Stress in Hepatocellular Carcinoma.

Purpose: Hepatocellular carcinoma (HCC) lacks effective curative therapy. Hypoxia is commonly found in HCC. Hypoxia elicits a series of protumorigenic responses through hypoxia-inducible factor-1 (HIF1). Better understanding of the metabolic adaptations of HCC cells during hypoxia is essential to the design of new therapeutic regimen. Experimental Design: Expressions of genes involved in the electron transport chain (ETC) in HCC cell lines (20% and 1% O2) and human HCC samples were analyzed by transcriptome sequencing. Expression of NDUFA4L2, a less active subunit in complex I of the ETC, in 100 pairs of HCC and nontumorous liver tissues were analyzed by qRT-PCR. Student t test and Kaplan–Meier analyses were used for clinicopathologic correlation and survival studies. Orthotopic HCC implantation model was used to evaluate the efficiency of HIF inhibitor. Results: NDUFA4L2 was drastically overexpressed in human HCC and induced by hypoxia. NDUFA4L2 overexpression was closely associated with tumor microsatellite formation, absence of tumor encapsulation, and poor overall survival in HCC patients. We confirmed that NDUFA4L2 was HIF1-regulated in HCC cells. Inactivation of HIF1/NDUFA4L2 increased mitochondrial activity and oxygen consumption, resulting in ROS accumulation and apoptosis. Knockdown of NDUFA4L2 markedly suppressed HCC growth and metastasis in vivo. HIF inhibitor, digoxin, significantly suppressed growth of tumors that expressed high level of NDUFA4L2. Conclusions: Our study has provided the first clinical relevance of NDUFA4L2 in human cancer and suggested that HCC patients with NDUFA4L2 overexpression may be suitable candidates for HIF inhibitor treatment. Clin Cancer Res; 22(12); 3105–17. ©2016 AACR.

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.

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

BACKGROUND & AIMSnHepatocellular 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.nnnMETHODSnGene 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.nnnRESULTSnWe 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.nnnCONCLUSIONnThis 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.

Abstract 1022: Chromatin remodeler HELLS is an epigenetic driver for hepatocellular carcinoma progression

Hepatocellular carcinoma (HCC) is the major type of liver cancer and the second leading cause of cancer death worldwide. Every year, more than six hundred thousand people are newly diagnosed. For the advanced HCC patients, only one FDA-approved drug is currently available. Therefore, there is an urge to delineate the molecular mechanism of HCC progression for developing new therapeutics. Increasing evidence showed that epigenetic alterations play an important role in the carcinogenesis process. The epigenetic regulatory mechanism is accomplished by DNA methylation, histone modification, and chromatin remodeling. 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, we employed RNA-seq to investigate the expression of chromatin remodelers in human HCCs. We found that HELicase, Lymphoid-Specific (HELLS), a SWI2/SNF2 chromatin remodeling enzyme, was remarkably overexpressed in HCC. Overexpression of HELLS was correlated with more aggressive clinicopathological features and poorer patient prognosis. We further showed that up-regulation of HELLS in HCC was conferred by hyper-activation of transcription factor SP1. To investigate the functions of HELLS in HCC, we generated both gain- and loss-of-function models by CRISPR activation system, lentiviral shRNA, and CRISPR/Cas9 genome editing system. We demonstrated that overexpression of HELLS augmented HCC cell proliferation and migration. In contrast, depletion of HELLS reduced HCC cell growth and motility. Moreover, inactivation of HELLS induced apoptosis in HCC cells. Coherently, ablation of HELLS also mitigated tumorigenicity and lung metastasis in vivo as demonstrated with both subcutaneous and orthotopic tumor implantation models. Mechanistically, by using RNA-seq and MNase-seq, we revealed that HELLS controls the nucleosome occupancy at gene enhancer and transcription start site (TSS). Overexpression of HELLS increased nucleosome occupancy that obstructed the accessibility of enhancers and hindered the formation of nucleosome-free region (NFR) at TSS of its target genes, thereby blocks the binding of transcription factors for activating gene expression. Consequently, though this mechanism, up-regulation of HELLS mediated epigenetic silencing of multiple tumor suppressor genes including E-Cadherin, FBP1, IGFBP3, XAF1 and CREB-H in HCC. In conclusion, our data unravel that HELLS is a key epigenetic driver of HCC. By altering the nucleosome occupancy at NFR and enhancer, HELLS epigenetically suppresses numerous tumor suppressor genes to promote HCC progression. Citation Format: Cheuk-Ting Law, Larry L. Wei, Felice HC Tsang, Iris MJ Xu, Robin KH Lai, Daniel WH Ho, Joyce MF Lee, Carmen CL Wong, Irene OL NG, Jack CM Wong, The State Key Laboratory for Liver Research. Chromatin remodeler HELLS is an epigenetic driver for hepatocellular carcinoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1022. doi:10.1158/1538-7445.AM2017-1022

Abstract 1380: Expression and functions of ASH1L in liver cancer

Hepatocellular carcinoma (HCC) is a common cancer and ranks the third lethal cancer worldwide. However, the detail molecular mechanisms underlying the initiation and progression of HCC remain poorly understood. Liver carcinogenesis is a multistep process that is driven by the accumulation of genetic and epigenetic alterations. Mutational landscape and driver mutations have recently been delineated by high-throughput sequencing studies. On the other hand, our current knowledge about epigenetic deregulation in human HCC is limited. Histone modification is a major component of epigenetic regulation, and deregulation of histone medication could alter local chromatin structure and gene expression. We hypothesized that deregulation of histone methyltransferases might contribute to HCC development. In this study, we showed that trithorax-group protein ASH1L was frequently up-regulated in human HCC. Overexpression of ASH1L was detected in 40% of primary HCCs and significantly associated with larger tumor size, present of venous invasion and tumor microsatellite formation. Up-regulation of ASH1L also correlated with increased Ki67 expression. ASH1L is a histone methyltransferase specific for catalyzing transcriptional-active H3K4 and H3K36 methylation. We showed that stable knockdown of ASH1L significantly suppressed HCC cell proliferation and colony formation. We further demonstrated that knockdown of ASH1L inhibited HCC cell migration. In addition, we investigated the underlying mechanisms contribute to ASH1L up-regulation. We showed that up-regulation of ASH1L in human HCC was contributed by chromosome amplification at chromosome 1q22. Furthermore, we showed that ASH1L was negatively regulated by miR-142. Therefore, down-regulation of miR-142 contributed to ASH1L up-regulation in HCC. In summary, our findings suggested that ASH1L was frequently up-regulated in human HCC due to chromosome amplification and miRNA deregulation. Citation Format: Lai Wei, Felice H. Tsang, Daniel Ho, Cheuk-Ting Law, Mengnuo Chen, Long-Hin Tsang, Carmen Cl Wong, Irene Ol Ng, Chun-Ming Wong. Expression and functions of ASH1L in liver cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1380. doi:10.1158/1538-7445.AM2017-1380

Abstract LB-310: NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 4-like 2 (NDUFA4L2) reduces oxidative stress to promote hepatocellular carcinoma

Background & Aims: Liver is a major metabolic organ, yet the detailed metabolic alterations driving hepatocellular carcinoma (HCC) remain elusive. The rapid growing nature of HCC results in oxygen deprivation or hypoxia in regions of tumors with insufficient blood supply. Hypoxia unbalances the electron flow through the electron transport chain (ETC) resulting in reactive oxygen species (ROS) accumulation. Here we aim at delineating the mechanisms by which HCC evades oxidative stress. Methods: We performed transcriptome sequencing to study the gene expression profile in both HCC patients and HCC cell line. The mRNA expression of 100 paired HCC and corresponding non-tumorous tissues were analyzed. Stable RNAi knockdown by shRNA and genetic knockout by TALEN were established in HCC cells for functional characterization. Results: We demonstrated that HCC cells specifically utilized the mitochondrial protein NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 4-like 2 gene (NDUFA4L2), in the complex I of the ETC, to survive hypoxia. NDUFA4L2 was drastically over-expressed in human HCC and closely associated with poor clinical outcomes in HCC patients. We confirmed that NDUFA4L2 was regulated by HIF-1α in HCC cells. Inactivation of HIF-1α/NDUFA4L2 in different HCC cell lines increased mitochondrial activity and oxygen consumption, resulting in ROS accumulation and ROS-mediated apoptosis in HCC cells. Knockdown of NDUFA4L2 markedly suppressed HCC growth and metastasis in vitro and in vivo. In addition, HIF inhibitors, digoxin and sorafenib, significantly suppressed growth of tumors that expressed high level of NDUFA4L2 in orthotopic HCC model. Conclusions: Our results have unprecedentedly uncovered the clinical relevance and oncogenic roles of NDUFA4L2 in HCC. Citation Format: Robin Kit-Ho Lai, Irix Ming-Jing Xu, David Kung-Chun Chiu, Aki Pui-Wah Tse, Larry Lai Wei, Cheuk-Ting Law, Derek Lee, Chun-Ming Wong, Maria Pik Wong, Irene Oi-Lin Ng, Carmen Chak Lui Wong. NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 4-like 2 (NDUFA4L2) reduces oxidative stress to promote hepatocellular carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-310.