Jung-Hee Kwon

Overexpression of High-Mobility Group Box 2 Is Associated with Tumor Aggressiveness and Prognosis of Hepatocellular Carcinoma

Purpose: We investigated the expression of high-mobility group box 2 (HMGB2) in patients with hepatocellular carcinoma (HCC) and its clinical effects with underlying mechanisms. Experimental Design: HMGB2 mRNA levels were measured in 334 HCC patients by real-time reverse transcription-PCR and HMGB2 protein levels in 173 HCC patients by immunohistochemical studies. The HMGB2 expression level was measured by Western blotting for three HCC cell lines. To clarify the precise role of HMGB2 on cell proliferation, we did in vitro analysis with expression vectors and small interfering RNAs. Results: HMGB2 mRNA and protein expression were significantly higher in HCC than in noncancerous surrounding tissues (P < 0.0001) and showed a positive correlation (ρ = 0.35, P < 0.001). HMGB2 overexpression was significantly correlated with shorter overall survival time, both at mRNA (P = 0.0054) and protein level (P = 0.023). Moreover, HMGB2 mRNA level was an independent prognostic factor for overall survival in a multivariate analysis (P = 0.0037). HMGB2 knockdown by small interfering RNAs decreased cell proliferation, and overexpression of HMGB2 by expression vectors diminished cisplatin- and etoposide-induced cell death. Conclusions: Our clinical and in vitro data suggest that HMGB2 plays a significant role in tumor development and prognosis of HCC. These results can partly be explained by altered cell proliferations by HMGB2 associated with the antiapoptotic pathway. Clin Cancer Res; 16(22); 5511–21. ©2010 AACR.

Comparative interactomes of SIRT6 and SIRT7: Implication of functional links to aging

Sirtuins are NAD+‐dependent deacetylases that regulate a range of cellular processes. Although diverse functions of sirtuins have been proposed, those functions of SIRT6 and SIRT7 that are mediated by their interacting proteins remain elusive. In the present study, we identified SIRT6‐ and SIRT7‐interacting proteins, and compared their interactomes to investigate functional links. Our interactomes revealed 136 interacting proteins for SIRT6 and 233 for SIRT7 while confirming seven and 111 proteins identified previously for SIRT6 and SIRT7, respectively. Comparison of SIRT6 and SIRT7 interactomes under the same experimental conditions disclosed 111 shared proteins, implying related functional links. The interaction networks of interactomes indicated biological processes associated with DNA repair, chromatin assembly, and aging. Interactions of two highly acetylated proteins, nucleophosmin (NPM1) and nucleolin, with SIRT6 and SIRT7 were confirmed by co‐immunoprecipitation. NPM1 was found to be deacetylated by both SIRT6 and SIRT7. In senescent cells, the acetylation level of NPM1 was increased in conjunction with decreased levels of SIRT6 and SIRT7, suggesting that the acetylation of NPM1 could be regulated by SIRT6 and SIRT7 in the aging process. Our comparative interactomic study of SIRT6 and SIRT7 implies important functional links to aging by their associations with interacting proteins. All MS data have been deposited in the ProteomeXchange with identifiers PXD000159 and PXD000850 (http://proteomecentral.proteomexchange.org/dataset/PXD000159, http://proteomecentral.proteomexchange.org/dataset/PXD000850).

HMGB2 stabilizes p53 by interfering with E6/E6AP-mediated p53 degradation in human papillomavirus-positive HeLa cells

We investigated the effect of HMGB2 on the stability of p53 protein in HeLa cells. Overexpression of HMGB2 led to accumulation of the p53 protein, whereas HMGB2 knockdown with siRNA resulted in a substantial decrease in the p53 protein level. The HMGB2-dependent increase of p53 stability was specific for HPV-positive HeLa cells as HCT116 and MCF7 cell lines did not demonstrate this response. Co-expression of HMGB2 and HPV E6 prevented HPV E6 protein-mediated ubiquitination and degradation of p53. FACS analysis exhibited that HeLa cells transfected with HMGB2 displayed decreased cell proliferation, with a concomitant increase of the p53 protein and arrest of the cell cycle, predominantly in G1 phase. Our findings collectively suggest that HMGB2 could stabilize p53 by interfering with E6/E6AP-mediated p53 degradation in HPV-positive HeLa cells.

Vaccinia-related kinase 1 promotes hepatocellular carcinoma by controlling the levels of cell cycle regulators associated with G1/S transition

We identified the specific role of vaccinia-related kinase 1 (VRK1) in the progression of hepatocellular carcinoma (HCC) and evaluated its therapeutic and prognostic potential. VRK1 levels were significantly higher in HCC cell lines than a normal hepatic cell line, and were higher in HCC than non-tumor tissue. VRK1 knockdown inhibited the proliferation of SK-Hep1, SH-J1 and Hep3B cells; moreover, depletion of VRK1 suppressed HCC tumor growth in vivo. We also showed that VRK1 knockdown increased the number of G1 arrested cells by decreasing cyclin D1 and p-Rb while upregulating p21 and p27, and that VRK1 depletion downregulated phosphorylation of CREB, a transcription factor regulating CCND1. Additionally, we found that luteolin, a VRK1 inhibitor, suppressed HCC growth in vitro and in vivo, and that the aberrant VRK1 expression correlated with poor prognostic features of HCC. High levels of VRK1 were associated with shorter overall and disease-free survival and higher recurrence rates. Taken together, our findings suggest VRK1 may act as a tumor promoter by controlling the level of cell cycle regulators associated with G1/S transition and could potentially serve as a therapeutic target and/or prognostic biomarker for HCC.

SIRT6 Depletion Suppresses Tumor Growth by Promoting Cellular Senescence Induced by DNA Damage in HCC

The role of Sirtuin 6 (SIRT6) as a tumor suppressor or oncogene in liver cancer remains controversial. Thus, we identified the specific role of SIRT6 in the progression of hepatocellular carcinoma (HCC). SIRT6 expression was significantly higher in HCC cell lines and HCC tissues from 138 patients than in an immortalized hepatocyte cell line, THLE-2 and non-tumor tissues, respectively. SIRT6 knockdown by shRNA suppressed the growth of HCC cells and inhibited HCC tumor growth in vivo. In addition, SIRT6 silencing significantly prevented the growth of HCC cell lines by inducing cellular senescence in the p16/Rb- and p53/p21-pathway independent manners. Microarray analysis revealed that the expression of genes involved in nucleosome assembly was apparently altered in SIRT6-depleted Hep3B cells. SIRT6 knockdown promoted G2/M phase arrest and downregulation of genes encoding histone variants associated with nucleosome assembly, which could be attributed to DNA damage. Taken together, our findings suggest that SIRT6 acts as a tumor promoter by preventing DNA damage and cellular senescence, indicating that SIRT6 represents a potential therapeutic target for the treatment of HCC.

AROS Is a Significant Biomarker for Tumor Aggressiveness in Non-cirrhotic Hepatocellular Carcinoma.

Despite a low risk of liver failure and preserved liver function, non-cirrhotic hepatocellular carcinoma (HCC) has a poor prognosis. In the current study, we evaluated an active regulator of SIRT1 (AROS) as a prognostic biomarker in non-cirrhotic HCC. mRNA levels of AROS were measured in tumor and non-tumor tissues obtained from 283 non-cirrhotic HCC patients. AROS expression was exclusively up-regulated in recurrent tissues from the non-cirrhotic HCC patients (P = 0.015) and also in tumor tissues irrespective of tumor stage (P < 0.001) or BCLC stage (P < 0.001). High mRNA levels of AROS were statistically significantly associated with tumor stage (P < 0.001), BCLC stage (P = 0.007), alpha fetoprotein (AFP) level (P = 0.013), microvascular invasion (P = 0.001), tumor size (P = 0.036), and portal vein invasion (P = 0.005). Kaplan-Meir curve analysis demonstrated that HCC patients with higher AROS levels had shorter disease-free survival (DFS) in both the short-term (P < 0.001) and long-term (P = 0.005) compared to those with low AROS. Cox regression analysis demonstrated that AROS is a significant predictor for DFS along with large tumor size, tumor multiplicity, vascular invasion, and poor tumor differentiation, which are the known prognostic factors. In conclusion, AROS is a significant biomarker for tumor aggressiveness in non-cirrhotic hepatocellular carcinoma.

REST-dependent expression of TRF2 renders non-neuronal cancer cells resistant to DNA damage during oxidative stress.

REST is a neuronal gene silencing factor ubiquitously expressed in non‐neuronal tissues. REST is additionally believed to serve as a tumor suppressor in non‐neuronal cancers. Conversely, recent findings on REST‐dependent tumorigenesis in non‐neuronal cells consistently suggest a potential role of REST as a tumor promoter. Here, we have uncovered for the first time the mechanism by which REST contributes to cancer cell survival in non‐neuronal cancers. We observed abundant expression of REST in various types of non‐neuronal cancer cells compared to normal tissues. The delicate roles of REST were further evaluated in HCT116 and HeLa, non‐neuronal cancer cell lines expressing REST. REST silencing resulted in decreased cell survival and activation of the DNA damage response (DDR) through a decrease in the level of TRF2, a telomere‐binding protein. These responses were correlated with reduced colony formation ability and accelerated telomere shortening in cancer cells upon the stable knockdown of REST. Interestingly, REST was down‐regulated under oxidative stress conditions via ubiquitin proteasome system, suggesting that sustainability of REST expression is critical to determine cell survival during oxidative stress in a tumor microenvironment. Our results collectively indicate that REST‐dependent TRF2 expression renders cancer cells resistant to DNA damage during oxidative stress, and mechanisms to overcome oxidative stress, such as high levels of REST or the stress‐resistant REST mutants found in specific human cancers, may account for REST‐dependent tumorigenesis.

Abstract 351: Comparative studies on SIRT6 and SIRT7 interactomes: Implications of important roles in cancer via associating with interacting partners

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Sirtuins are NAD+-dependent deacetylases that regulate a range of cellular processes. Although anti-aging effects of sirtuins have been proposed, the proteins interacting with SIRT6 and SIRT7 remain elusive. In the current study, we identified SIRT6- and SIRT7-interacting proteins and compared their interactomes. Our data revealed 129 novel interacting proteins for SIRT6 and 122 for SIRT7 while confirming 7 and 111 previously identified proteins for SIRT6 and SIRT7, respectively. Comparison of the SIRT6 and SIRT7 interactomes under a same experimental condition disclosed 111 shared proteins indicated that functional associations. The functional networks of interactomes exhibited that the binding partners are associated with important biological processes in aging and age-related diseases including cancer. Two proteins, NPM1 and NCL localized in the nucleus and interacted with SIRT6 and SIRT7. NPM1 was identified as a novel substrate and its acetylation level decreased by both SIRT6 and SIRT7. Additionally, in senescent cells, the acetylation level of NPM1 was increased in conjunction with decreased level of SIRT6 and SIRT7, suggesting that acetylation of NPM1 is regulated by SIRT6 and SIRT7 in the aging process. Our findings provide insights on functional relationship between SIRT6 and SIRT7 as well as their roles in DNA repair, chromatin assembly and cancer cell proliferation, and aging. Citation Format: Namgyu Lee, Jung-Hee Kwon, Sung Jin Park, Kwan Yong Choi. Comparative studies on SIRT6 and SIRT7 interactomes: Implications of important roles in cancer via associating with interacting partners. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 351. doi:10.1158/1538-7445.AM2014-351