Seung Oe Lim

Epigenetic Changes Induced by Reactive Oxygen Species in Hepatocellular Carcinoma: Methylation of the E-cadherin Promoter

BACKGROUND & AIMS In addition to genetic alterations, epigenetic changes underlie tumor progression and metastasis. Promoter methylation can silence tumor suppressor genes, and reactive oxygen species (ROS) promote DNA damage, although the relationship between ROS and epigenetic changes in cancer cells is not clear. We sought to determine whether ROS promote hypermethylation of the promoter region of E-cadherin, a regulator of the epithelial-to-mesenchymal transition, in hepatocellular carcinoma (HCC) cells. METHODS HCC cells were exposed to H(2)O(2) or stably transfected to express Snail, a transcription factor that down-regulates E-cadherin expression. E-cadherin and Snail expression levels were examined by real-time reverse-transcriptase polymerase chain reaction and immunoblot analyses. The methylation status of E-cadherin was examined by methyl-specific polymerase chain reaction, bisulfite sequencing, and chromatin immunoprecipitation. The interactions between Snail, histone deacetylase 1, and DNA methyltransferase 1 were assessed by immunoprecipitation/immunoblot and immunofluorescence analyses. ROS-induced stress, E-cadherin expression, Snail expression, and E-cadherin promoter methylation were confirmed in HCC tissues by immunoblot, immunohistochemistry, and methyl-specific polymerase chain reaction analyses. RESULTS We demonstrated that ROS induce hypermethylation of the E-cadherin promoter by increasing Snail expression. Snail induced DNA methylation of the E-cadherin promoter by recruiting histone deacetylase 1 and DNA methyltransferase 1. In human HCC tissues, we observed a correlation among ROS induction, E-cadherin down-regulation, Snail up-regulation, and E-cadherin promoter methylation. CONCLUSIONS These findings provide novel mechanistic insights into epigenetic modulations induced by ROS in the process of carcinogenesis. They are potentially relevant to understanding the activity of ROS in silencing various tumor suppressor genes and in subsequent tumor progression and metastasis.

Epithelial–Mesenchymal Transition Induced by TNF-α Requires NF-κB–Mediated Transcriptional Upregulation of Twist1

Proinflammatory cytokines produced in the tumor microenvironment facilitate tumor development and metastatic progression. In particular, TNF-α promotes cancer invasion and angiogenesis associated with epithelial-mesenchymal transition (EMT); however, the mechanisms underlying its induction of EMT in cancer cells remain unclear. Here we show that EMT and cancer stemness properties induced by chronic treatment with TNF-α are mediated by the upregulation of the transcriptional repressor Twist1. Exposure to TNF-α rapidly induced Twist1 mRNA and protein expression in normal breast epithelial and breast cancer cells. Both IKK-β and NF-κB p65 were required for TNF-α-induced expression of Twist1, suggesting the involvement of canonical NF-κB signaling. In support of this likelihood, we defined a functional NF-κB-binding site in the Twist1 promoter, and overexpression of p65 was sufficient to induce transcriptional upregulation of Twist1 along with EMT in mammary epithelial cells. Conversely, suppressing Twist1 expression abrogated p65-induced cell migration, invasion, EMT, and stemness properties, establishing that Twist1 is required for NF-κB to induce these aggressive phenotypes in breast cancer cells. Taken together, our results establish a signaling axis through which the tumor microenvironment elicits Twist1 expression to promote cancer metastasis. We suggest that targeting NF-κB-mediated Twist1 upregulation may offer an effective a therapeutic strategy for breast cancer treatment.

EGFR modulates microRNA maturation in response to hypoxia through phosphorylation of AGO2

MicroRNAs (miRNAs) are generated by two-step processing to yield small RNAs that negatively regulate target gene expression at the post-transcriptional level. Deregulation of miRNAs has been linked to diverse pathological processes, including cancer. Recent studies have also implicated miRNAs in the regulation of cellular response to a spectrum of stresses, such as hypoxia, which is frequently encountered in the poorly angiogenic core of a solid tumour. However, the upstream regulators of miRNA biogenesis machineries remain obscure, raising the question of how tumour cells efficiently coordinate and impose specificity on miRNA expression and function in response to stresses. Here we show that epidermal growth factor receptor (EGFR), which is the product of a well-characterized oncogene in human cancers, suppresses the maturation of specific tumour-suppressor-like miRNAs in response to hypoxic stress through phosphorylation of argonaute 2 (AGO2) at Tyr 393. The association between EGFR and AGO2 is enhanced by hypoxia, leading to elevated AGO2-Y393 phosphorylation, which in turn reduces the binding of Dicer to AGO2 and inhibits miRNA processing from precursor miRNAs to mature miRNAs. We also identify a long-loop structure in precursor miRNAs as a critical regulatory element in phospho-Y393-AGO2-mediated miRNA maturation. Furthermore, AGO2-Y393 phosphorylation mediates EGFR-enhanced cell survival and invasiveness under hypoxia, and correlates with poorer overall survival in breast cancer patients. Our study reveals a previously unrecognized function of EGFR in miRNA maturation and demonstrates how EGFR is likely to function as a regulator of AGO2 through novel post-translational modification. These findings suggest that modulation of miRNA biogenesis is important for stress response in tumour cells and has potential clinical implications.

Hepatitis B viral X protein interacts with tumor suppressor adenomatous polyposis coli to activate Wnt/β-catenin signaling

HBV X protein is a transactivator of several cellular signaling pathways including Wnt which contributes to HBV associated neoplasia. The Wnt/β-catenin pathway is associated with HCC-initiating cells. Here we perform a functional screen for host factors involved in the transactivational properties of HBx. We identify adenomatous polyposis coli (APC) as a binding partner of HBx and further determine that HBx competitively binds APC to displace β-catenin from its degradation complex. This results in β-catenin upregulation in the nucleus and the activation of Wnt signaling. We show that Wnt inhibitors curcumin and quercetin target downstream β-catenin activity and effectively repress HBx-mediated regulation of c-MYC and E-cadherin. Our results provide a pathological mechanism of HBx induced malignant transformation.

Downregulation of catalase by reactive oxygen species via hypermethylation of CpG island II on the catalase promoter.

Catalase, which decomposes reactive oxygen species (ROS), is reduced in hepatocellular carcinoma (HCC); however, the reasons are poorly defined. In this study, it is demonstrated that prolonged exposure to ROS induced methylation of CpG island II on the catalase promoter and downregulated catalase expression at the transcriptional level in HCC cell lines. In addition, hypermethylation of CpG island II was also observed in tumor tissues, together with a decrease in catalase mRNA and protein expression levels when compared to non‐tumor tissues. From these data, we suggest that ROS may downregulate catalase through the methylation of promoter during the development of HCC.

Glycosylation and stabilization of programmed death ligand-1 suppresses T-cell activity

Extracellular interaction between programmed death ligand-1 (PD-L1) and programmed cell death protein-1 (PD-1) leads to tumour-associated immune escape. Here we show that the immunosuppression activity of PD-L1 is stringently modulated by ubiquitination and N-glycosylation. We show that glycogen synthase kinase 3β (GSK3β) interacts with PD-L1 and induces phosphorylation-dependent proteasome degradation of PD-L1 by β-TrCP. In-depth analysis of PD-L1 N192, N200 and N219 glycosylation suggests that glycosylation antagonizes GSK3β binding. In this regard, only non-glycosylated PD-L1 forms a complex with GSK3β and β-TrCP. We also demonstrate that epidermal growth factor (EGF) stabilizes PD-L1 via GSK3β inactivation in basal-like breast cancer. Inhibition of EGF signalling by gefitinib destabilizes PD-L1, enhances antitumour T-cell immunity and therapeutic efficacy of PD-1 blockade in syngeneic mouse models. Together, our results link ubiquitination and glycosylation pathways to the stringent regulation of PD-L1, which could lead to potential therapeutic strategies to enhance cancer immune therapy efficacy.

p53 inhibits tumor cell invasion via the degradation of snail protein in hepatocellular carcinoma

MINT‐7718939: Snai1 (uniprotkb:O95863) physically interacts (MI:0915) with MDM2 (uniprotkb:Q00987) by anti tag coimmunoprecipitation (MI:0007)

Crystal structure of human nucleophosmin-core reveals plasticity of the pentamer-pentamer interface

Crystal structure of human nucleophosmin-core reveals plasticity of the pentamer–pentamer interface Hyung Ho Lee, Hyoun Sook Kim, Ji Yong Kang, Byung Il Lee, Jun Yong Ha, Hye Jin Yoon, Seung Oe Lim, Guhung Jung, and Se Won Suh* 1 Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul 151-747, Korea 2 Department of Biological Science, College of Natural Sciences, Seoul National University, Seoul 151-747, Korea

PARP inhibitor upregulates PD-L1 expression and enhances cancer-associated immunosuppression

Purpose: To explore whether a cross-talk exists between PARP inhibition and PD-L1/PD-1 immune checkpoint axis, and determine whether blockade of PD-L1/PD-1 potentiates PARP inhibitor (PARPi) in tumor suppression. Experimental Design: Breast cancer cell lines, xenograft tumors, and syngeneic tumors treated with PARPi were assessed for PD-L1 expression by immunoblotting, IHC, and FACS analyses. The phospho-kinase antibody array screen was used to explore the underlying mechanism of PARPi-induced PD-L1 upregulation. The therapeutic efficacy of PARPi alone, PD-L1 blockade alone, or their combination was tested in a syngeneic tumor model. The tumor-infiltrating lymphocytes and tumor cells isolated from syngeneic tumors were analyzed by CyTOF and FACS to evaluate the activity of antitumor immunity in the tumor microenvironment. Results: PARPi upregulated PD-L1 expression in breast cancer cell lines and animal models. Mechanistically, PARPi inactivated GSK3β, which in turn enhanced PARPi-mediated PD-L1 upregulation. PARPi attenuated anticancer immunity via upregulation of PD-L1, and blockade of PD-L1 resensitized PARPi-treated cancer cells to T-cell killing. The combination of PARPi and anti-PD-L1 therapy compared with each agent alone significantly increased the therapeutic efficacy in vivo. Conclusions: Our study demonstrates a cross-talk between PARPi and tumor-associated immunosuppression and provides evidence to support the combination of PARPi and PD-L1 or PD-1 immune checkpoint blockade as a potential therapeutic approach to treat breast cancer. Clin Cancer Res; 23(14); 3711–20. ©2017 AACR.

Reactive oxygen species downregulate catalase expression via methylation of a CpG island in the Oct-1 promoter.

Reactive oxygen species (ROS) caused oxidative stress plays a key role in carcinogenesis. The POU domain transcription factor Oct‐1 and catalase is closely associated with ROS. However, a correlation between these two key proteins has not been demonstrated before. In this report, we show that Oct‐1 acts as an activator of catalase, by binding to the catalase promoter in hepatocellular carcinoma (HCC) cell lines. In addition, we suggest that Oct‐1 is downregulated by ROS via CpG island methylation in its promoter. These findings contribute to a better understanding of the epigenetic changes induced by ROS in the process of carcinogenesis.