Felice Ho-Ching Tsang

Enhancer of zeste homolog 2 epigenetically silences multiple tumor suppressor microRNAs to promote liver cancer metastasis

Epigenetic alterations and microRNA (miRNA) deregulation are common in hepatocellular carcinoma (HCC). The histone H3 lysine 27 (H3K27) tri‐methylating enzyme, enhancer of zeste homolog 2 (EZH2) mediates epigenetic silencing of gene expression and is frequently up‐regulated in human cancers. In this study we aimed to delineate the implications of EZH2 up‐regulation in miRNA deregulation and HCC metastasis. Expressions of a total of 90 epigenetic regulators were first determined in 38 pairs of primary HCCs and their corresponding nontumorous livers. We identified EZH2 and its associated polycomb repressive complex 2 (PRC2) as one of the most significantly deregulated epigenetic regulators in primary HCC samples. Up‐regulation of EZH2 was next confirmed in 69.5% (41/59) of primary HCCs. Clinicopathologically, EZH2 up‐regulation was associated with HCC progression and multiple HCC metastatic features, including venous invasion (P = 0.043), direct liver invasion (P = 0.014), and absence of tumor encapsulation (P = 0.043). We further demonstrated that knockdown of EZH2 in HCC cell lines reduced the global levels of tri‐methylated H3K27, and suppressed HCC motility in vitro and pulmonary metastasis in a nude mouse model. By interrogating the miRNA expression profile in EZH2‐knockdown cell lines and primary HCC samples, we identified a subset of miRNA that was epigenetically suppressed by EZH2 in human HCC. These included well‐characterized tumor‐suppressor miRNAs, such as miR‐139‐5p, miR‐125b, miR‐101, let‐7c, and miR‐200b. Pathway enrichment analysis revealed a common regulatory role of these EZH2‐silenced miRNAs in modulating cell motility and metastasis‐related pathways. Our findings suggest that EZH2 exerts its prometastatic function by way of epigenetic silencing of multiple tumor suppressor miRNAs. Conclusion: Our study demonstrated that EZH2 epigenetically silenced multiple miRNAs that negatively regulate HCC metastasis. (HEPATOLOGY 2012)

Long non-coding RNA HOTTIP is frequently up-regulated in hepatocellular carcinoma and is targeted by tumour suppressive miR-125b

Hepatocellular carcinoma (HCC) is one of the most common human cancers. Recently, emerging evidence has suggested the role of long non‐coding RNAs (lncRNAs) in human carcinogenesis. In this study, we aimed to investigate the expression and functional implications of lncRNAs in human HCC.

Prognostic significance and therapeutic potential of eukaryotic translation initiation factor 5A (eIF5A) in hepatocellular carcinoma

Using comparative proteomic and genomic approaches, the authors identified eukaryotic translation initiation factor 5A (eIF5A) as an oncofetal molecule highly abundant in mouse embryonic livers and human hepatocellular carcinoma (HCC) cell lines. To evaluate the oncogenic role and prognostic significance of eIF5A in HCC, we investigate the expression patterns of the two isoforms (eIF5A1 and eIF5A2) in a cohort of 258 HCC cases by cDNA microarray. Both eIF5A isoforms were expressed in the tumors, and clinically correlated eIF5A1 with more numbers of tumor nodules and eIF5A2 with tumor venous infiltration in HCC. In a separate cohort of 50 HCCs, high level of eIF5A2, but not eIF5A1, was associated with elevated levels of deoxyhypusine synthase and deoxyhypusine hydroxylase that catalyze post‐translational hypusination of eIF5A protein. Interestingly, N1‐guanyl‐1,7‐diaminoheptane (GC7), which is an inhibitor for the first step of eIF5A hypusination, was shown to significantly impair the cell proliferation and invasion of primary HCC cells (HepG2 and Hep3B). To further demonstrate the tumorigenic role associated with eIF5A, a drastic reduction of cell proliferation was associated with suppression of eIF5A2 by transfecting Hep3B, H2‐P and H2‐M HCC cells expressing high level of this isoform using small interfering RNA (siRNA) against eIF5A2. For these assays, a milder response was usually observed in normal hepatocyte cell line. Therefore, these findings suggest that eIF5A plays an important role in HCC tumorigenesis and metastasis, and targeting eIF5A hypusination by GC7 inhibitor or eIF5A2 by RNA interference (RNAi) may offer new therapeutic alternatives to HCC patients.

Proteomic expression signature distinguishes cancerous and nonmalignant tissues in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is an aggressive liver cancer but clinically validated biomarkers that can predict natural history of malignant progression are lacking. The present study explored the proteome-wide patterns of HCC to identify biomarker signature that could distinguish cancerous and nonmalignant liver tissues. A retrospective cohort of 80 HBV-associated HCC was included and both the tumor and adjacent nontumor tissues were subjected to proteome-wide expression profiling by 2-DE method. The subjects were randomly divided into the training (n = 55) and validation (n = 25) subsets, and the data analyzed by classification-and-regression tree algorithm. Protein markers were characterized by MALDI-ToF/MS and confirmed by immunohistochemistry, Western blotting and qPCR assays. Proteomic expression signature composed of six biomarkers (haptoglobin, cytochrome b5, progesterone receptor membrane component 1, heat shock 27 kDa protein 1, lysosomal proteinase cathepsin B, keratin I) was developed as a classifier model for predicting HCC. We further evaluated the model using both leave-one-out procedure and independent validation, and the overall sensitivity and specificity for HCC both are 92.5%, respectively. Clinical correlation analysis revealed that these biomarkers were significantly associated with serum AFP, total protein levels and the Ishaks score. The described model using biomarker signatures could accurately distinguish HCC from nonmalignant tissues, which may also provide hints on how normal hepatocytes are transformed to malignant state during tumor progression.

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

Epigenetic deregulation plays an important role in liver carcinogenesis. Using transcriptome sequencing, we examined the expression of 591 epigenetic regulators in hepatitis B‐associated human hepatocellular carcinoma (HCC). We found that aberrant expression of epigenetic regulators was a common event in HCC. We further identified SETDB1 (SET domain, bifurcated 1), an H3K9‐specific histone methyltransferase, as the most significantly up‐regulated epigenetic regulator in human HCCs. Up‐regulation of SETDB1 was significantly associated with HCC disease progression, cancer aggressiveness, and poorer prognosis of HCC patients. Functionally, we showed that knockdown of SETDB1 reduced HCC cell proliferation in vitro and suppressed orthotopic tumorigenicity in vivo. Inactivation of SETDB1 also impeded HCC cell migration and abolished lung metastasis in nude mice. Interestingly, SETDB1 protein was consistently up‐regulated in all metastatic foci found in different organs, suggesting that SETDB1 was essential for HCC metastatic progression. Mechanistically, we showed that the frequent up‐regulation of SETDB1 in human HCC was attributed to the recurrent SETDB1 gene copy gain at chromosome 1q21. In addition, hyperactivation of specificity protein 1 transcription factor in HCC enhanced SETDB1 expression at the transcriptional level. Furthermore, we identified miR‐29 as a negative regulator of SETDB1. Down‐regulation of miR‐29 expression in human HCC contributed to SETDB1 up‐regulation by relieving its post‐transcriptional regulation. Conclusion: SETDB1 is an oncogene that is frequently up‐regulated in human HCCs; the multiplicity of SETDB1 activating mechanisms at the chromosomal, transcriptional, and posttranscriptional levels together facilitates SETDB1 up‐regulation in human HCC. (Hepatology 2016;63:474–487)

RNA N6‐methyladenosine methyltransferase‐like 3 promotes liver cancer progression through YTHDF2‐dependent posttranscriptional silencing of SOCS2

Epigenetic alterations have contributed greatly to human carcinogenesis. Conventional epigenetic studies have predominantly focused on DNA methylation, histone modifications, and chromatin remodeling. Recently, diverse and reversible chemical modifications of RNAs have emerged as a new layer of epigenetic regulation. N6‐methyladenosine (m6A) is the most abundant chemical modification of eukaryotic messenger RNA (mRNA) and is important for the regulation of mRNA stability, splicing, and translation. Using transcriptome sequencing, we discovered that methyltransferase‐like 3 (METTL3), a major RNA N6‐adenosine methyltransferase, was significantly up‐regulated in human hepatocellular carcinoma (HCC) and multiple solid tumors. Clinically, overexpression of METTL3 is associated with poor prognosis of patients with HCC. Functionally, we proved that knockdown of METTL3 drastically reduced HCC cell proliferation, migration, and colony formation in vitro. Knockout of METTL3 remarkably suppressed HCC tumorigenicity and lung metastasis in vivo. On the other hand, using the CRISPR/dCas9‐VP64 activation system, we demonstrated that overexpression of METTL3 significantly promoted HCC growth both in vitro and in vivo. Through transcriptome sequencing, m6A sequencing, and m6A methylated RNA immuno‐precipitation quantitative reverse‐transcription polymerase chain reaction, we identified suppressor of cytokine signaling 2 (SOCS2) as a target of METTL3‐mediated m6A modification. Knockdown of METTL3 substantially abolished SOCS2 mRNA m6A modification and augmented SOCS2 mRNA expression. We also showed that m6A‐mediated SOCS2 mRNA degradation relied on the m6A reader protein YTHDF2‐dependent pathway. Conclusion: METTL3 is frequently up‐regulated in human HCC and contributes to HCC progression. METTL3 represses SOCS2 expression in HCC through an m6A‐YTHDF2‐dependent mechanism. Our findings suggest an important mechanism of epigenetic alteration in liver carcinogenesis. (Hepatology 2018;67:2254‐2270).

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.

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

BACKGROUND & AIMS 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. METHODS 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. RESULTS 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. CONCLUSION 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.