Larry Lai Wei

Hypoxia induces myeloid‐derived suppressor cell recruitment to hepatocellular carcinoma through chemokine (C‐C motif) ligand 26

A population of stromal cells, myeloid‐derived suppressor cells (MDSCs), is present in tumors. Though studies have gradually revealed the protumorigenic functions of MDSCs, the molecular mechanisms guiding MDSC recruitment remain largely elusive. Hypoxia, O2 deprivation, is an important factor in the tumor microenvironment of solid cancers, whose growth often exceeds the growth of functional blood vessels. Here, using hepatocellular carcinoma as the cancer model, we show that hypoxia is an important driver of MDSC recruitment. We observed that MDSCs preferentially infiltrate into hypoxic regions in human hepatocellular carcinoma tissues and that hypoxia‐induced MDSC infiltration is dependent on hypoxia‐inducible factors. We further found that hypoxia‐inducible factors activate the transcription of chemokine (C‐C motif) ligand 26 in cancer cells to recruit chemokine (C‐X3‐C motif) receptor 1‐expressing MDSCs to the primary tumor. Knockdown of chemokine (C‐C motif) ligand 26 in cancer cells profoundly reduces MDSC recruitment, angiogenesis, and tumor growth. Therapeutically, blockade of chemokine (C‐C motif) ligand 26 production in cancer cells by the hypoxia‐inducible factor inhibitor digoxin or blockade of chemokine (C‐X3‐C motif) receptor 1 in MDSCs by chemokine (C‐X3‐C motif) receptor 1 neutralizing antibody could substantially suppress MDSC recruitment and tumor growth. Conclusion: This study unprecedentedly reveals a novel molecular mechanism by which cancer cells direct MDSC homing to primary tumor and suggests that targeting MDSC recruitment represents an attractive therapeutic approach against solid cancers. (Hepatology 2016;64:797‐813)

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.

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.

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.

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.

RhoE/ROCK2 regulates chemoresistance through NF-κB/IL-6/ STAT3 signaling in hepatocellular carcinoma

Small Rho GTPase (Rho) and its immediate effector Rho kinase (ROCK) are reported to regulate cell survival, but the detailed molecular mechanism remains largely unknown. We had previously shown that Rho/ROCK signaling was highly activated in hepatocellular carcinoma (HCC). In this study, we further demonstrated that downregulation of RhoE, a RhoA antagonist, and upregulation of ROCK enhanced resistance to chemotherapy in HCC in both in vitro cell and in vivo murine xenograft models, whereas a ROCK inhibitor was able to profoundly sensitize HCC tumors to cisplatin treatment. Specifically, the ROCK2 isoform but not ROCK1 maintained the chemoresistance in HCC cells. Mechanistically, we demonstrated that activation of ROCK2 enhanced the phosphorylation of JAK2 and STAT3 through increased expression of IL-6 and the IL-6 receptor complex. We also identified IKKβ as the direct downstream target of Rho/ROCK, and activation of ROCK2 significantly augmented NF-κB transcription activity and induced IL-6 expression. These data indicate that Rho/ROCK signaling activates a positive feedback loop of IKKβ/NF-κB/IL-6/STAT3 which confers chemoresistance to HCC cells and is a potential molecular target for HCC therapy.

Abstract 1390: Deregulation of RNA N6-adenosine methylation contribute to liver carcinogenesis

Primary liver cancer, particularly the most common type hepatocellular carcinoma (HCC), is the third leading cause of cancer death worldwide. Case is even worse in Asia due to extremely high prevalence of HBV/HCV infection, which enhances the risk of HCC. However, the exact mechanism of how HCC develops is still not clearly defined. Previous studies have shown strong evidence for epigenetic alterations in human carcinogenesis, which mainly focus on abnormal DNA methylation, histone modifications, and chromatin remodeling. Recent studies suggest that diverse chemical modifications on RNAs, also known as “epi-transcriptome”, constitute another layer of gene expression regulation. N-6-methyladenosine (m6A) is the most abundant modification on eukaryotic mRNA, which has been found to functionally influence mRNA stability, alternative splicing, and translation efficiency. However, the roles of m6A deregulation in human carcinogenesis remain unclear. Through whole-transcriptome sequencing, we identified that METTL3, the major component of m6A methyltransferase, was significantly up-regulated in human HCC. Up-regulation of METTL3 negatively associated with poor patient overall and disease free survival. Functionally knockdown/knockout of METTL3 dramatically inhibited HCC proliferation, migration and colony formation abilities in vitro and significantly suppressed in vivo tumorigenicity. In contrast, overexpression of METTL3 using CRISPR/dCas9 SAM system modestly but significantly enhanced HCC proliferation, migration, anchorage-independent growth in vitro and tumor formation in vivo. In order to find the downstream mechanisms which might attributed to METTL3 oncogenic function we performed RNA-Seq on two independent METTL3 knockdown cell lines to interrogated their transcriptome changes. Based on RNA-Seq data, we identified a prominent tumor suppressor SOCS2 as a potential down-stream target of METTL3. With m6A-RNA immunoprecipitation (RIP) assay, we confirmed that SOCS2 mRNA was subject to m6A modification in cancer cells. Inactivation of METTL3 by RNAi or treatment of methylation inhibitor (DAA) impaired m6A mediated mRNA degradation and thereby stabilized SOSC2 mRNA. We further confirm SOCS2 tumor suppressor function in HCC. Downregulation of SOCS2 was associated with poor patient overall survival and disease free survival of HCC patients. Knockdown of SOCS2 significantly promoted HCC proliferation, migration in HCC cells. In conclusion, our findings suggested that deregulation of METTL3 and its associated m6A modification could contribute to human carcinogenesis by imposing an epigenetic control on the stability and expressions of critical tumor suppressor genes. Citation Format: Mengnuo Chen, Larry L. Wei, Cheuk Ting Law, Felice HC Tsang, Iris MJ Xu, Joyce Lee MF Lee, Carmen Wong CL Wong, Irene OL Ng, Chun-Ming Wong. Deregulation of RNA N6-adenosine methylation contribute to liver carcinogenesis [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 1390. doi:10.1158/1538-7445.AM2017-1390

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 4399: Hypoxia as a driver of myeloid-derived suppressor cell recruitment in hepatocellular carcinoma via CCL26/CX3CR1

Background and Objective: Myeloid-derived suppressor cells (MDSCs) accumulate in tumors and highly pro-tumorigenic. These MDSCs exhibit inhibitory functions against effector T cells and natural killer cells in tumor sites, as well as secrete pro-angiogenic factors or differentiate to endothelial cells to promote angiogenesis and metastasis. While it is appreciated that depletion of MDSCs could bring tumoricidal effects, there are significant gaps in knowledge about the underlying mechanisms responsible for MDSC recruitment to tumor sites. Hypoxia, O2 deprivation, is an important factor in the tumor microenvironment of HCC that modifies the stromal components. Using hepatocellular carcinoma (HCC) as a model, we investigated whether hypoxia is a driver of MDSC recruitment in HCC. Experimental Procedures: Gene profiling of HCC cells exposed to hypoxia and normoxia were analyzed by transcriptome sequencing to identify potential hypoxia-induced chemokines for MDSC recruitment. MDSCs were isolated from HCC-bearing mice by magnetic bead sorting for different functional assays. Boyden chambers were used to evaluate the invasive ability of MDSCs. Flow cytometry was used to detect the frequencies of tumor-associated MDSCs in orthotopic HCC mouse models. Results: We observed that MDSCs preferentially infiltrated into hypoxic regions in human HCC tissues and hypoxia-induced MDSC infiltration was dependent on hypoxia-inducible factors (HIFs). HIFs activated the transcription of chemokine (C-C motif) ligand 26 (CCL26) in HCC cells to recruit chemokine (C-X3-C motif) receptor 1 (CX3CR1)-expressing MDSCs to the primary tumor. Knockdown of CCL26 in HCC cells profoundly reduces MDSC recruitment, angiogenesis, and tumor growth. Therapeutically, blockade of CCL26 production in HCC cells by HIF inhibitor, digoxin, or blockade of CX3CR1 in MDSCs by CX3CR1 neutralizing antibody could substantially suppress MDSC recruitment and tumor growth. Conclusion: This study unprecedentedly reveals a novel molecular mechanism by which HCC cells direct MDSC homing to primary tumor and suggests that targeting MDSC recruitment represents an attractive therapeutic approach against HCC. Citation Format: David Kung-Chun Chiu, Iris Ming-Jing Xu, Robin Kit-Ho Lai, Aki Pui-Wah Tse, Larry Lai Wei, Hui-Yu Koh, Regina Cheuk-Lam Lo, Chun-Ming Wong, Irene Oi-Lin Ng, Carmen Chak-Lui Wong. Hypoxia as a driver of myeloid-derived suppressor cell recruitment in hepatocellular carcinoma via CCL26/CX3CR1. [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 4399.

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.