Edgar Engleman, Postdoctoral Faculty Sponsor
Hypoxia regulates the mitochondrial activity of hepatocellular carcinoma cells through HIF/HEY1/PINK1 pathway
CELL DEATH & DISEASE
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
Induction of Oxidative Stress Through Inhibition of Thioredoxin Reductase 1 Is an Effective Therapeutic Approach for Hepatocellular Carcinoma
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.)
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
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
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
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
Hypoxia inducible factor HIF-1 promotes myeloid-derived suppressor cells accumulation through ENTPD2/CD39L1 in hepatocellular carcinoma
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
Folate cycle enzyme MTHFD1L confers metabolic advantages in hepatocellular carcinoma
JOURNAL OF CLINICAL INVESTIGATION
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
Hypoxia induces myeloid-derived suppressor cell recruitment to hepatocellular carcinoma through chemokine (C-C motif) ligand 26
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
NDUFA4L2 Fine-tunes Oxidative Stress in Hepatocellular Carcinoma
CLINICAL CANCER RESEARCH
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
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
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
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