All Publications

  • Using mouse liver cancer models based on somatic genome editing to predict immune checkpoint inhibitor responses JOURNAL OF HEPATOLOGY Yuen, V., Chiu, D., Law, C., Cheu, J., Chan, C., Wong, B., Goh, C., Zhang, M., Xue, H., Tse, A., Zhang, Y., Lau, H., Lee, D., Au-Yeung, R. H., Wong, C., Chak-Lui, C. 2023; 78 (2): 376-389


    Tyrosine kinase inhibitors (TKIs) and immune checkpoint inhibitors (ICIs) are the only two classes of FDA-approved drugs for individuals with advanced hepatocellular carcinoma (HCC). While TKIs confer only modest survival benefits, ICIs have been associated with remarkable outcomes but only in the minority of patients who respond. Understanding the mechanisms that determine the efficacy of ICIs in HCC will help to stratify patients likely to respond to ICIs. This study aims to elucidate how genetic composition and specific oncogenic pathways regulate the immune composition of HCC, which directly affects response to ICIs.A collection of mouse HCCs with genotypes that closely simulate the genetic composition found in human HCCs were established using genome-editing approaches involving the delivery of transposon and CRISPR-Cas9 systems by hydrodynamic tail vein injection. Mouse HCC tumors were analyzed by RNA-sequencing while tumor-infiltrating T cells were analyzed by flow cytometry and single-cell RNA-sequencing.Based on the CD8+ T cell-infiltration level, we characterized tumors with different genotypes into cold and hot tumors. Anti-PD-1 treatment had no effect in cold tumors but was greatly effective in hot tumors. As proof-of-concept, a cold tumor (Trp53KO/MYCOE) and a hot tumor (Keap1KO/MYCOE) were further characterized. Tumor-infiltrating CD8+ T cells from Keap1KO/MYCOE HCCs expressed higher levels of proinflammatory chemokines and exhibited enrichment of a progenitor exhausted CD8+ T-cell phenotype compared to those in Trp53KO/MYCOE HCCs. The TKI sorafenib sensitized Trp53KO/MYCOE HCCs to anti-PD-1 treatment.Single anti-PD-1 treatment appears to be effective in HCCs with genetic mutations driving hot tumors, while combined anti-PD-1 and sorafenib treatment may be more appropriate in HCCs with genetic mutations driving cold tumors.Genetic alterations of different driver genes in mouse liver cancers are associated with tumor-infiltrating CD8+ T cells and anti-PD-1 response. Mouse HCCs with different genetic compositions can be grouped into hot and cold tumors based on the level of tumor-infiltrating CD8+ T cells. This study provides proof-of-concept evidence to show that hot tumors are responsive to anti-PD-1 treatment while cold tumors are more suitable for combined treatment with anti-PD-1 and sorafenib. Our study might help to guide the design of patient stratification systems for single or combined treatments involving anti-PD-1.

    View details for DOI 10.1016/j.jhep.2022.10.037

    View details for Web of Science ID 001029609600001

    View details for PubMedID 36455783

  • Hepatocellular Carcinoma Cells Up-regulate PVRL1, Stabilizing PVR and Inhibiting the Cytotoxic T-Cell Response via TIGIT to Mediate Tumor Resistance to PD1 Inhibitors in Mice GASTROENTEROLOGY Chiu, D., Yuen, V., Cheu, J., Wei, L., Ting, V., Fehlings, M., Sumatoh, H., Nardin, A., Newell, E. W., Ng, I., Yau, T., Wong, C., Wong, C. 2020; 159 (2): 609-623


    Immune checkpoint inhibitors are effective in the treatment of some hepatocellular carcinomas (HCCs), but these tumors do not always respond to inhibitors of programmed cell death 1 (PDCD1, also called PD1). We investigated mechanisms of resistance of liver tumors in mice to infiltrating T cells.Mice were given hydrodynamic tail vein injections of clustered regularly interspaced short palindromic repeats-Cas9 (CRISPR-Cas9) and transposon vectors to disrupt Trp53 and overexpress C-Myc (Trp53KO/C-MycOE mice). Pvrl1 and Pvrl3 were knocked down in Hepa1-6 cells by using short hairpin RNAs. Hepa1-6 cells were injected into livers of C57BL/6 mice; some mice were given intraperitoneal injections of antibodies against PD1, T-cell immunoreceptor with Ig and ITIM domains (TIGIT), or CD8 before the cancer cells were injected. Liver tissues were collected from mice and analyzed by histology, immunohistochemistry, and quantitative real-time polymerase chain reaction; tumors were analyzed by mass cytometry using markers to detect T cells and other lymphocytes. We obtained HCC and nontumorous liver tissues and clinical data from patients who underwent surgery in Hong Kong and analyzed the tissues by immunohistochemistry.Trp53KO/C-MycOE mice developed liver tumors in 3-5 weeks; injections of anti-PD1 did not slow tumor development. Tumors from mice given anti-PD1 had larger numbers of memory CD8+ T cells (CD44+CD62L-KLRG1int) and T cells that expressed PD1, lymphocyte activating 3 (LAG3), and TIGIT compared with mice not given the antibody. HCC tissues from patients had higher levels of PVRL1 messenger RNA and protein than nontumorous tissues. Increased PVRL1 was associated with shorter times of disease-free survival. Knockdown of Pvrl1 in Hepa1-6 cells caused them to form smaller tumors in mice, infiltrated by higher numbers of CD8+ T cells that expressed the inhibitory protein TIGIT; these effects were not observed in mice with depletion of CD8+ T cells. In Hepa1-6 cells, PVRL1 stabilized cell surface PVR, which interacted with TIGIT on CD8+ T cells; knockdown of Pvrl1 reduced cell-surface levels of PVR but not levels of Pvr messenger RNA. In Trp53KO/C-MycOE mice and mice with tumors grown from Hepa1-6 cells, injection of the combination of anti-PD1 and anti-TIGIT significantly reduced tumor growth, increased the ratio of cytotoxic to regulatory T cells in tumors, and prolonged survival.PVRL1, which is up-regulated by HCC cells, stabilizes cell surface PVR, which interacts with TIGIT, an inhibitory molecule on CD8+ effector memory T cells. This suppresses the ant-tumor immune response. Inhibitors of PVRL1/TIGIT, along with anti-PD1 might be developed for treatment of HCC.

    View details for DOI 10.1053/j.gastro.2020.03.074

    View details for Web of Science ID 000561847000008

    View details for PubMedID 32275969

  • Hypoxia regulates the mitochondrial activity of hepatocellular carcinoma cells through HIF/HEY1/PINK1 pathway CELL DEATH & DISEASE Chiu, D., Tse, A., Law, C., Xu, I., Lee, D., Chen, M., Lai, R., Yuen, V., Cheu, J., Ho, D., Wong, C., Zhang, H., Ng, I., Wong, C. 2019; 10: 934


    Hypoxia is commonly found in cancers. Hypoxia, due to the lack of oxygen (O2) as the electron recipient, causes inefficient electron transfer through the electron transport chain at the mitochondria leading to accumulation of reactive oxygen species (ROS) which could create irreversible cellular damages. Through hypoxia-inducible factor 1 (HIF-1) which elicits various molecular events, cells are able to overcome low O2. Knowledge about the new molecular mechanisms governed by HIF-1 is important for new therapeutic interventions targeting hypoxic tumors. Using hepatocellular carcinoma (HCC) as a model, we revealed that the HIF-1 and the Notch signaling pathways cross-talk to control mitochondrial biogenesis of cancer cells to maintain REDOX balance. From transcriptome sequencing, we found that HEY1, a transcriptional repressor, in the NOTCH pathway was consistently induced by hypoxia in HCC cell lines. We identified a strong hypoxia response element (HRE) in HEY1 by chromatin immunoprecipitation (ChIP) and luciferase reporter assays. Transcriptome and ChIP sequencing further identified PINK1, a gene essential for mitochondrial biogenesis, as a novel transcriptional target of HEY1. HCC cells with HEY1 knockdown re-expressed PINK1. HEY1 and PINK1 expressions inversely correlated in human HCC samples. Overexpression of HEY1 and under-expression of PINK1 were detected in human HCC and associated with poor clinical outcomes. Functionally, we found that overexpression of HEY1 or knockdown of PINK1 consistently reduced mitochondrial cristae, mitochondrial mass, oxidative stress level, and increased HCC growth.

    View details for DOI 10.1038/s41419-019-2155-3

    View details for Web of Science ID 000502975000001

    View details for PubMedID 31819034

    View details for PubMedCentralID PMC6901483

  • Hypoxia inducible factor HIF-1 promotes myeloid-derived suppressor cells accumulation through ENTPD2/CD39L1 in hepatocellular carcinoma NATURE COMMUNICATIONS Chiu, D., Tse, A., Xu, I., Di Cui, J., Lai, R., Li, L., Koh, H., Tsang, F., Wei, L., Wong, C., Ng, I., Wong, C. 2017; 8: 517


    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.

    View details for DOI 10.1038/s41467-017-00530-7

    View details for Web of Science ID 000410237600019

    View details for PubMedID 28894087

    View details for PubMedCentralID PMC5593860

  • Hypoxia induces myeloid-derived suppressor cell recruitment to hepatocellular carcinoma through chemokine (C-C motif) ligand 26 HEPATOLOGY Chiu, D., Xu, I., Lai, R., Tse, A., Wei, L., Koh, H., Li, L., Lee, D., Lo, R., Wong, C., Ng, I., Wong, C. 2016; 64 (3): 797–813


    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.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).

    View details for DOI 10.1002/hep.28655

    View details for Web of Science ID 000383285900012

    View details for PubMedID 27228567

  • Neutrophil-activating therapy for the treatment of cancer. Cancer cell Linde, I. L., Prestwood, T. R., Qiu, J., Pilarowski, G., Linde, M. H., Zhang, X., Shen, L., Reticker-Flynn, N. E., Chiu, D. K., Sheu, L. Y., Van Deursen, S., Tolentino, L. L., Song, W., Engleman, E. G. 2023


    Despite their cytotoxic capacity, neutrophils are often co-opted by cancers to promote immunosuppression, tumor growth, and metastasis. Consequently, these cells have received little attention as potential cancer immunotherapeutic agents. Here, we demonstrate in mouse models that neutrophils can be harnessed to induce eradication of tumors and reduce metastatic seeding through the combined actions of tumor necrosis factor, CD40 agonist, and tumor-binding antibody. The same combination activates human neutrophils invitro, enabling their lysis of human tumor cells. Mechanistically, this therapy induces rapid mobilization and tumor infiltration of neutrophils along with complement activation in tumors. Complement component C5a activates neutrophils to produce leukotriene B4, which stimulates reactive oxygen species production via xanthine oxidase, resulting in oxidative damage and Tcell-independent clearance of multiple tumor types. These data establish neutrophils as potent anti-tumor immune mediators and define an inflammatory pathway that can be harnessed to drive neutrophil-mediated eradication of cancer.

    View details for DOI 10.1016/j.ccell.2023.01.002

    View details for PubMedID 36706760

  • CFI-402257, a TTK inhibitor, effectively suppresses hepatocellular carcinoma PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chan, C., Chiu, D., Yuen, V., Law, C., Wong, B., Thu, K., Cescon, D., Soria-Bretones, I., Cheu, J., Lee, D., Tse, A., Zhang, M., Tan, K., Ng, I., Khong, P., Yau, T., Bray, M., Mak, T., Wong, C. 2022; 119 (32): e2119514119


    Deregulation of cell cycle is a typical feature of cancer cells. Normal cells rely on the strictly coordinated spindle assembly checkpoint (SAC) to maintain the genome integrity and survive. However, cancer cells could bypass this checkpoint mechanism. In this study, we showed the clinical relevance of threonine tyrosine kinase (TTK) protein kinase, a central regulator of the SAC, in hepatocellular carcinoma (HCC) and its potential as therapeutic target. Here, we reported that a newly developed, orally active small molecule inhibitor targeting TTK (CFI-402257) effectively suppressed HCC growth and induced highly aneuploid HCC cells, DNA damage, and micronuclei formation. We identified that CFI-402257 also induced cytosolic DNA, senescence-like response, and activated DDX41-STING cytosolic DNA sensing pathway to produce senescence-associated secretory phenotypes (SASPs) in HCC cells. These SASPs subsequently led to recruitment of different subsets of immune cells (natural killer cells, CD4+ T cells, and CD8+ T cells) for tumor clearance. Our mass cytometry data illustrated the dynamic changes in the tumor-infiltrating immune populations after treatment with CFI-402257. Further, CFI-402257 improved survival in HCC-bearing mice treated with anti-PD-1, suggesting the possibility of combination treatment with immune checkpoint inhibitors in HCC patients. In summary, our study characterized CFI-402257 as a potential therapeutic for HCC, both used as a single agent and in combination therapy.

    View details for DOI 10.1073/pnas.2119514119

    View details for Web of Science ID 000926104800001

    View details for PubMedID 35914158

    View details for PubMedCentralID PMC9371652

  • Polo-like kinase 4 inhibitor CFI-400945 suppresses liver cancer through cell cycle perturbation and eliciting antitumor immunity HEPATOLOGY Chan, C., Yuen, V., Chiu, D., Goh, C., Thu, K. L., Cescon, D. W., Soria-Bretones, I., Law, C., Cheu, J., Lee, D., Tse, A., Tan, K., Zhang, M., Wong, B., Wong, C., Khong, P., Ng, I., Bray, M. R., Mak, T., Yau, T., Wong, C. 2022


    Prognosis of HCC remains poor due to lack of effective therapies. Immune checkpoint inhibitors (ICIs) have delayed response and are only effective in a subset of patients. Treatments that could effectively shrink the tumors within a short period of time are idealistic to be employed together with ICIs for durable tumor suppressive effects. HCC acquires increased tolerance to aneuploidy. The rapid division of HCC cells relies on centrosome duplication. In this study, we found that polo-like kinase 4 (PLK4), a centrosome duplication regulator, represents a therapeutic vulnerability in HCC.An orally available PLK4 inhibitor, CFI-400945, potently suppressed proliferating HCC cells by perturbing centrosome duplication. CFI-400945 induced endoreplication without stopping DNA replication, causing severe aneuploidy, DNA damage, micronuclei formation, cytosolic DNA accumulation, and senescence. The cytosolic DNA accumulation elicited the DEAD box helicase 41-stimulator of interferon genes-interferon regulatory factor 3/7-NF-κβ cytosolic DNA sensing pathway, thereby driving the transcription of senescence-associated secretory phenotypes, which recruit immune cells. CFI-400945 was evaluated in liver-specific p53/phosphatase and tensin homolog knockout mouse HCC models established by hydrodynamic tail vein injection. Tumor-infiltrated immune cells were analyzed. CFI-400945 significantly impeded HCC growth and increased infiltration of cluster of differentiation 4-positive (CD4+ ), CD8+ T cells, macrophages, and natural killer cells. Combination therapy of CFI-400945 with anti-programmed death-1 showed a tendency to improve HCC survival.We show that by targeting a centrosome regulator, PLK4, to activate the cytosolic DNA sensing-mediated immune response, CFI-400945 effectively restrained tumor progression through cell cycle inhibition and inducing antitumor immunity to achieve a durable suppressive effect even in late-stage mouse HCC.

    View details for DOI 10.1002/hep.32461

    View details for Web of Science ID 000778846300001

    View details for PubMedID 35302667

  • Hypoxia-induced macropinocytosis represents a metabolic route for liver cancer NATURE COMMUNICATIONS Zhang, M., Cui, J., Lee, D., Yuen, V., Chiu, D., Goh, C., Cheu, J., Tse, A., Bao, M., Wong, B., Chen, C., Wong, C., Ng, I., Wong, C. 2022; 13 (1): 954


    Hepatocellular carcinoma (HCC) invariably exhibits inadequate O2 (hypoxia) and nutrient supply. Hypoxia-inducible factor (HIF) mediates cascades of molecular events that enable cancer cells to adapt and propagate. Macropinocytosis is an endocytic process initiated by membrane ruffling, causing the engulfment of extracellular fluids (proteins), protein digestion and subsequent incorporation into the biomass. We show that macropinocytosis occurs universally in HCC under hypoxia. HIF-1 activates the transcription of a membrane ruffling protein, EH domain-containing protein 2 (EHD2), to initiate macropinocytosis. Knockout of HIF-1 or EHD2 represses hypoxia-induced macropinocytosis and prevents hypoxic HCC cells from scavenging protein that support cell growth. Germline or somatic deletion of Ehd2 suppresses macropinocytosis and HCC development in mice. Intriguingly, EHD2 is overexpressed in HCC. Consistently, HIF-1 or macropinocytosis inhibitor suppresses macropinocytosis and HCC development. Thus, we show that hypoxia induces macropinocytosis through the HIF/EHD2 pathway in HCC cells, harnessing extracellular protein as a nutrient to survive.

    View details for DOI 10.1038/s41467-022-28618-9

    View details for Web of Science ID 000757851300048

    View details for PubMedID 35177645

    View details for PubMedCentralID PMC8854584

  • The Amot/integrin protein complex transmits mechanical forces required for vascular expansion CELL REPORTS Zhang, Y., Zhang, Y., Kameishi, S., Barutello, G., Zheng, Y., Tobin, N. P., Nicosia, J., Hennig, K., Chiu, D., Balland, M., Barker, T. H., Cavallo, F., Holmgren, L. 2021; 36 (8): 109616


    Vascular development is a complex multistep process involving the coordination of cellular functions such as migration, proliferation, and differentiation. How mechanical forces generated by cells and transmission of these physical forces control vascular development is poorly understood. Using an endothelial-specific genetic model in mice, we show that deletion of the scaffold protein Angiomotin (Amot) inhibits migration and expansion of the physiological and pathological vascular network. We further show that Amot is required for tip cell migration and the extension of cellular filopodia. Exploiting in vivo and in vitro molecular approaches, we show that Amot binds Talin and is essential for relaying forces between fibronectin and the cytoskeleton. Finally, we provide evidence that Amot is an important component of the endothelial integrin adhesome and propose that Amot integrates spatial cues from the extracellular matrix to form a functional vascular network.

    View details for DOI 10.1016/j.celrep.2021.109616

    View details for Web of Science ID 000688508300051

    View details for PubMedID 34433061

  • Adaptive and Constitutive Activations of Malic Enzymes Confer Liver Cancer Multilayered Protection Against Reactive Oxygen Species HEPATOLOGY Lee, D., Zhang, M., Tsang, F., Bao, M., Xu, I., Lai, R., Chiu, D., Tse, A., Law, C., Chan, C., Yuen, V., Chui, N., Ng, I., Wong, C., Wong, C. 2021; 74 (2): 776-796


    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.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.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.

    View details for DOI 10.1002/hep.31761

    View details for Web of Science ID 000688494200017

    View details for PubMedID 33619771

  • Inhibition of CMTM4 Sensitizes Cholangiocarcinoma and Hepatocellular Carcinoma to T Cell-Mediated Antitumor Immunity Through PD-L1 HEPATOLOGY COMMUNICATIONS Chui, N., Cheu, J., Yuen, V., Chiu, D., Goh, C., Lee, D., Zhang, M., Ng, I., Wong, C. 2022; 6 (1): 178-193


    Liver cancers consist primarily of hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). Immune checkpoint inhibitors have emerged as promising therapeutic agents against liver cancers. Programmed cell death protein 1 (PD-1) is an immunoinhibitory receptor present on T cells that interacts with its ligand programmed death-ligand 1 (PD-L1) found on cancer cells. Blocking PD-1/PD-L1 binding improves T-cell survival, proliferation and cytotoxicity, which enhances their antitumor activity. Better understanding of the molecular mechanisms governing PD-1/PD-L1 response is essential to the development of predictive markers and therapeutic combinations that could improve the efficiency of anti-PD-1/PD-L1 treatment. Chemokine-like factor (CKLF)-like MARVEL transmembrane domain-containing 6 (CMTM6) has been recently identified as a major regulator of PD-L1. Another member in the CMTM family, CKLF-like MARVEL transmembrane domain-containing 4 (CMTM4), has been shown to compensate for the effects of CMTM6 when CMTM6 is lost. Interestingly, we found that CMTM4 is the major regulator of PD-L1 in the context of liver cancer. Up-regulated CMTM4 in patients with HCC and ICC is associated with poor patient survival, potentially due to its function in stabilizing PD-L1 expression, hence facilitating escape from T cell-mediated cytotoxicity. We confirmed the role of CMTM4 as a positive regulator of PD-L1 in multiple HCC and ICC cell lines and demonstrated that CMTM4 stabilizes PD-L1 through posttranslational mechanisms. In vivo, suppression of Cmtm4 inhibited HCC growth and increased CD8+ T-cell infiltration in immunocompetent mice. Furthermore, we found that depletion of CMTM4 sensitized HCC tumor to anti-PD-L1 treatment compared with control. This suggests that CMTM4 expression level could be a predictive marker for patient response to anti-PD-L1 treatment, and CMTM4 depletion can potentially be used to enhance the clinical benefits of anti-PD-L1 immunotherapy in patients with liver cancer.

    View details for DOI 10.1002/hep4.1682

    View details for Web of Science ID 000675131100001

    View details for PubMedID 34558800

    View details for PubMedCentralID PMC8710793

  • Genome-wide CRISPR-Cas9 knockout library screening identified PTPMT1 in cardiolipin synthesis is crucial to survival in hypoxia in liver cancer CELL REPORTS Bao, M., Yang, C., Tse, A., Wei, L., Lee, D., Zhang, M., Goh, C., Chiu, D., Yuen, V., Law, C., Chin, W., Chui, N., Wong, B., Chan, C., Ng, I., Chung, C., Wong, C., Wong, C. 2021; 34 (4): 108676


    Hypoxia, low oxygen (O2), is a key feature of all solid cancers, including hepatocellular carcinoma (HCC). Genome-wide CRISPR-Cas9 knockout library screening is used to identify reliable therapeutic targets responsible for hypoxic survival in HCC. We find that protein-tyrosine phosphatase mitochondrial 1 (PTPMT1), an important enzyme for cardiolipin (CL) synthesis, is the most significant gene and ranks just after hypoxia-inducible factor (HIF)-1α and HIF-1β as crucial to hypoxic survival. CL constitutes the mitochondrial membrane and ensures the proper assembly of electron transport chain (ETC) complexes for efficient electron transfer in respiration. ETC becomes highly unstable during hypoxia. Knockout of PTPMT1 stops the maturation of CL and impairs the assembly of ETC complexes, leading to further electron leakage and ROS accumulation at ETC in hypoxia. Excitingly, HCC cells, especially under hypoxic conditions, show great sensitivity toward PTPMT1 inhibitor alexidine dihydrochloride (AD). This study unravels the protective roles of PTPMT1 in hypoxic survival and cancer development.

    View details for DOI 10.1016/j.celrep.2020.108676

    View details for Web of Science ID 000613631500018

    View details for PubMedID 33503428

  • Adipose-derived stem cells and cancer cells fuse to generate cancer stem cell-like cells with increased tumorigenicity JOURNAL OF CELLULAR PHYSIOLOGY Chan, Y., So, C., Yau, K., Chiu, K., Wang, X., Chan, F., Tsang, S. 2020; 235 (10): 6794-6807


    Adipose-derived stem cells (ADSCs) are a type of mesenchymal stem cells isolated from adipose tissue and have the ability to differentiate into adipogenic, osteogenic, and chondrogenic lineages. Despite their great therapeutic potentials, previous studies showed that ADSCs could enhance the proliferation and metastatic potential of breast cancer cells (BCCs). In this study, we found that ADSCs fused with BCCs spontaneously, while breast cancer stem cell (CSC) markers CD44+ CD24-/low EpCAM+ were enriched in this fusion population. We further assessed the fusion hybrid by multicolor DNA FISH and mouse xenograft assays. Only single nucleus was observed in the fusion hybrid, confirming that it was a synkaryon. In vivo mouse xenograft assay indicated that the tumorigenic potential of the fusion hybrid was significantly higher than that of the parent tumorigenic triple-negative BCC line MDA-MB-231. We had compared the fusion efficiency between two BCC lines, the CD44-rich MDA-MB-231 and the CD44-poor MCF-7, with ADSCs. Interestingly, we found that the fusion efficiency was much higher between MDA-MB-231 and ADSCs, suggesting that a potential mechanism of cell fusion may lie in the dissimilarity between these two cell lines. The cell fusion efficiency was hampered by knocking down the CD44. Altogether, our findings suggest that CD44-mediated cell fusion could be a potential mechanism for generating CSCs.

    View details for DOI 10.1002/jcp.29574

    View details for Web of Science ID 000509771700001

    View details for PubMedID 31994190

  • Induction of Oxidative Stress Through Inhibition of Thioredoxin Reductase 1 Is an Effective Therapeutic Approach for Hepatocellular Carcinoma HEPATOLOGY Lee, D., Xu, I., Chiu, D., Leibold, J., Tse, A., Bao, M., Yuen, V., Chan, C., Lai, R., Chin, D., Chan, D., Cheung, T., Chok, S., Wong, C., Lowe, S. W., Ng, I., Wong, C. 2019; 69 (4): 1768–86


    Hepatocellular carcinoma (HCC) is one of the most prevalent and lethal cancers worldwide which lacks effective treatment. Cancer cells experience high levels of oxidative stress due to increased generation of reactive oxygen species (ROS). Increased antioxidant-producing capacity is therefore found in cancer cells to counteract oxidative stress. The thioredoxin system is a ubiquitous mammalian antioxidant system which scavenges ROS, and we demonstrate that it is vital for HCC growth as it maintains intracellular reduction-oxidation (redox) homeostasis. Transcriptome sequencing in human HCC samples revealed significant overexpression of thioredoxin reductase 1 (TXNRD1), the cytosolic subunit and key enzyme of the thioredoxin system, with significant correlations to poorer clinicopathological features and patient survival. Driven by the transcriptional activation of nuclear factor (erythroid-derived 2)-like 2, the master protector against oxidative stress, TXNRD1 counteracts intracellular ROS produced in human HCC. Inhibition of TXNRD1 through genetic inhibition hindered the proliferation of HCC cells and induced apoptosis in vitro. Administration of the pharmacological TXNRD1 inhibitor auranofin (AUR) effectively suppressed the growth of HCC tumors induced using the hydrodynamic tail vein injection and orthotopic implantation models in vivo. Furthermore, AUR sensitized HCC cells toward the conventional therapeutic sorafenib. Conclusion: Our study highlights the reliance of HCC cells on antioxidants for redox homeostasis and growth advantage; targeting TXNRD1 resulted in dramatic accumulation of ROS, which was found to be an effective approach for the suppression of HCC tumor growth.

    View details for DOI 10.1002/hep.30467

    View details for Web of Science ID 000462612400032

    View details for PubMedID 30561826

  • Assessment of Stabilization and Activity of the HIFs Important for Hypoxia-Induced Signalling in Cancer Cells. Methods in molecular biology (Clifton, N.J.) Chiu, D. K., Zhang, M. S., Tse, A. P., Wong, C. C. 2019; 1928: 77-99


    Blood vessels in tumors contain chaotic branching structures and leaky vessel lumens, resulting in uneven supply of oxygen in the tumor microenvironment. High metabolic and proliferation rate of tumor cells further depletes the local oxygen supply. Therefore, hypoxia is a common phenomenon in multiple solid malignancies. Hypoxia-inducible factors (HIFs) regulate the transcription of a spectrum of genes, which are vitally important for tumor cell adaption under hypoxia, and shape the tumor microenvironment to become more favorable for progression. HIFs are involved in almost every step of cancer development through inducing angiogenesis, metabolic reprogramming, metastasis, cancer stemness maintenance, chemoresistance, and immune evasion. Here, we describe methods for the assessment of HIF activity, as well as identification of novel transcriptional targets of HIFs in vitro and in vivo.

    View details for DOI 10.1007/978-1-4939-9027-6_6

    View details for PubMedID 30725452

  • RNA N6-methyladenosine methyltransferase-like 3 promotes liver cancer progression through YTHDF2-dependent posttranscriptional silencing of SOCS2 HEPATOLOGY Chen, M., Wei, L., Law, C., Tsang, F., Shen, J., Cheng, C., Tsang, L., Ho, D., Chiu, D., Lee, J., Wong, C., Ng, I., Wong, C. 2018; 67 (6): 2254–70


    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.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).

    View details for DOI 10.1002/hep.29683

    View details for Web of Science ID 000434113900019

    View details for PubMedID 29171881

  • Hepatitis transactivator protein X promotes extracellular matrix modification through HIF/LOX pathway in liver cancer ONCOGENESIS Tse, A., Sze, K., Shea, Q., Chiu, E., Tsang, F., Chiu, D., Zhang, M., Lee, D., Xu, I., Chan, C., Koh, H., Wong, C., Zheng, Y., Ng, I., Wong, C. 2018; 7: 44


    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.

    View details for DOI 10.1038/s41389-018-0052-8

    View details for Web of Science ID 000433997800004

    View details for PubMedID 29799025

    View details for PubMedCentralID PMC5968027

  • Histone methyltransferase G9a promotes liver cancer development by epigenetic silencing of tumor suppressor gene RARRES3 JOURNAL OF HEPATOLOGY Wei, L., Chiu, D., Tsang, F., Law, C., Cheng, C., Au, S., Lee, J., Wong, C., Ng, I., Wong, C. 2017; 67 (4): 758–69


    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.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.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.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.

    View details for DOI 10.1016/j.jhep.2017.05.015

    View details for Web of Science ID 000410775900013

    View details for PubMedID 28532996

  • Folate cycle enzyme MTHFD1L confers metabolic advantages in hepatocellular carcinoma JOURNAL OF CLINICAL INVESTIGATION Lee, D., Xu, I., Chiu, D., Lai, R., Tse, A., Li, L., Law, C., Tsang, F., Wei, L., Chan, C., Wong, C., Ng, I., Wong, C. 2017; 127 (5): 1856–72


    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.

    View details for DOI 10.1172/JCI90253

    View details for Web of Science ID 000400381000026

    View details for PubMedID 28394261

    View details for PubMedCentralID PMC5409797

  • NDUFA4L2 Fine-tunes Oxidative Stress in Hepatocellular Carcinoma CLINICAL CANCER RESEARCH Lai, R., Xu, I., Chiu, D., Tse, A., Wei, L., Law, C., Lee, D., Wong, C., Wong, M., Ng, I., Wong, C. 2016; 22 (12): 3105–17


    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.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.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.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.

    View details for DOI 10.1158/1078-0432.CCR-15-1987

    View details for Web of Science ID 000377958800030

    View details for PubMedID 26819450

  • Transketolase counteracts oxidative stress to drive cancer development PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Xu, I., Lai, R., Lin, S., Tse, A., Chiu, D., Koh, H., Law, C., Wong, C., Cai, Z., Wong, C., Ng, I. 2016; 113 (6): E725–E734


    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.

    View details for DOI 10.1073/pnas.1508779113

    View details for Web of Science ID 000369571700009

    View details for PubMedID 26811478

    View details for PubMedCentralID PMC4760787

  • Switching of Pyruvate Kinase Isoform L to M2 Promotes Metabolic Reprogramming in Hepatocarcinogenesis PLOS ONE Wong, C., Au, S., Tse, A., Xu, I., Lai, R., Chiu, D., Wei, L., Fan, D., Tsang, F., Lo, R., Wong, C., Ng, I. 2014; 9 (12): e115036


    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.

    View details for DOI 10.1371/journal.pone.0115036

    View details for Web of Science ID 000347239900019

    View details for PubMedID 25541689

    View details for PubMedCentralID PMC4277479

  • Lysyl Oxidase-Like 2 Is Critical to Tumor Microenvironment and Metastatic Niche Formation in Hepatocellular Carcinoma HEPATOLOGY Wong, C., Tse, A., Huang, Y., Zhu, Y., Chiu, D., Lai, R., Au, S., Kai, A., Lee, J., Wei, L., Tsang, F., Lo, R., Shi, J., Zheng, Y., Wong, C., Ng, I. 2014; 60 (5): 1645–58


    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.These findings integrate the clinical relevance, molecular regulation, and functional implications of LOXL2 in HCC metastasis.

    View details for DOI 10.1002/hep.27320

    View details for Web of Science ID 000344352400023

    View details for PubMedID 25048396