Kee Ho Lee

Integrative analysis of multiple gene expression profiles applied to liver cancer study

A statistical method for combining multiple microarray studies has been previously developed by the authors. Here, we present the application of the method to our hepatocellular carcinoma (HCC) data and report new findings on gene expression changes accompanying HCC. From the cross‐verification result of our studies and that of published studies, we found that single microarray analysis might lead to false findings. To avoid those pitfalls of single‐set analyses, we employed our effect size method to integrate multiple datasets. Of 9982 genes analyzed, 477 significant genes were identified with a false discovery rate of 10%. Gene ontology (GO) terms associated with these genes were explored to validate our method in the biological context with respect to HCC. Furthermore, it was demonstrated that the data integration process increases the sensitivity of analysis and allows small but consistent expression changes to be detected. These integration‐driven discoveries contained meaningful and interesting genes not reported in previous expression profiling studies, such as growth hormone receptor, erythropoietin receptor, tissue factor pathway inhibitor‐2, etc. Our findings support the use of meta‐analysis for a variety of microarray data beyond the scope of this specific application.

High prevalence of hepatitis B virus infection in patients with B-cell non-Hodgkin's lymphoma in Korea.

This study assessed the association of hepatitis B virus (HBV) and hepatitis C virus (HCV) infection with non‐Hodgkins lymphoma in a highly HBV‐endemic area. The prevalence of either HBV or HCV infection in 235 patients with non‐Hodgkins lymphoma was compared with that of an age‐ and sex‐matched hospital control group of 235 patients. The prevalence of HBV infection was higher in B‐cell non‐Hodgkins lymphoma (15.5%) than control (8.1%), but the prevalence of HCV infection in the non‐Hodgkins lymphoma patients (2.1%) and control group (3%) was similar. HBV prevalence increased significantly with age in the B‐cell non‐Hodgkins lymphoma patients. The presence of HBV proteins and DNA in lymphoma tissues and peripheral blood mononuclear cells (PBMCs) from HBV‐infected non‐Hodgkins lymphoma patients was also investigated using immunohistochemistry and PCR. HBV DNA was frequently detected in PBMCs from HBV‐infected non‐Hodgkins lymphoma patients, but HBV antigens were not. Therefore, HBV infection, but not HCV infection, was associated with B‐cell non‐Hodgkins lymphoma in Korea, suggesting a possible role for HBV in the development of non‐Hodgkins lymphoma. J. Med. Virol. 80:960–966, 2008.

Overexpression of Romo1 promotes production of reactive oxygen species and invasiveness of hepatic tumor cells

BACKGROUND & AIMS Chronic oxidative stress from reactive oxygen species (ROS) produced by the mitochondria promotes hepatocarcinogenesis and tumor progression. However, the exact mechanism by which mitochondrial ROS contributes to tumor cell invasion is not known. We investigated the role of ROS modulator 1 (Romo1) in hepatocellular carcinoma (HCC) development and tumor cell invasiveness. METHODS We performed real-time, semi-quantitative, reverse transcriptase polymerase chain reaction; invasion and luciferase assays; and immunofluorescence and immunohistochemical analyses. The formation of pulmonary metastatic nodules after tumor cell injection was tested in severe combined immunodeficient mice. We analyzed Romo1 expression in HCC cell lines and tissues (n = 95). RESULTS Expression of Romo1 was increased in HCC cells, compared with normal human lung fibroblast cells. Exogenous expression of Romo1 in HCC cells increased their invasive activity, compared with control cells. Knockdown of Romo1 in Hep3B and Huh-7 HCC cells reduced their invasive activity in response to stimulation with 12-O-tetradecanoylphorbol-13-acetate. Levels of Romo1 were increased compared with normal liver tissues in 63 of 95 HCC samples from patients. In HCC samples from patients, there was an inverse correlation between Romo1 overexpression and patient survival times. Increased levels of Romo1 also correlated with vascular invasion by the tumors, reduced differentiation, and larger tumor size. CONCLUSIONS Romo1 is a biomarker of HCC progression that might be used in diagnosis. Reagents that inhibit activity of Romo1 and suppress mitochondrial ROS production, rather than eliminate up-regulated intracellular ROS, might be developed as cancer therapies.

The Expression of Estrogen Receptors in Hepatocellular Carcinoma in Korean Patients

Expression of estrogen receptors (ER)-α and -β, as well as androgen receptor (AR), in hepatocellular carcinoma (HCC) is thought to be correlated with prognosis, survival, and male prevalence of HCC. These hypotheses are based on investigations of European patients; however the expression patterns of these receptors in Asian patients are largely unknown. In this study, we collected liver carcinoma and peritumor tissues from 32 patients (9 females and 23 males) in South Korea. The expression of ERs and ARs was studied using RT-PCR. Wild-type ER-α and AR were expressed in all of the samples investigated, and their expression was independent of the causal virus or patient sex. Expression of the ER-α variant was independent of sex (100% female vs. 91.3% male) and HCV and HBV status (91.3% vs. 100%). Wild-type ER-β was expressed more often in HCV patients than in HBV patients (95.7% vs. 44.4%; p < 0.05). In conclusion, the stronger ER-α variant expression in HCC tissues implies that this variant has an important role in HCC development. However, at least in Korean patients, expression of the ER-α variant (vER-α) is not related to male HCC prevalence. In addition, the predominant expression of ER-β in HCV patients suggests that it plays an important role in HCV-induced liver disease.

SIRT1 interacts with and protects glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from nuclear translocation: Implications for cell survival after irradiation

Upon apoptotic stimulation, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a cytosolic enzyme normally active in glycolysis, translocates into the nucleus and activates an apoptotic cascade therein. In the present work, we show that SIRT1 prevents nuclear translocation of GAPDH via interaction with GAPDH. SIRT1 depletion triggered nuclear translocation of cytosolic GAPDH even in the absence of apoptotic stress. Such translocation was not, however, observed when SIRT1 enzymatic activity was inhibited, indicating that SIRT1 protein per se, rather than the deacetylase activity of the protein, is required to inhibit GAPDH translocation. Upon irradiation, SIRT1 prevented irradiation-induced nuclear translocation of GAPDH, accompanied by interaction of SIRT1 and GAPDH. Thus, SIRT1 functions to retain GAPDH in the cytosol, protecting the enzyme from nuclear translocation via interaction with these two proteins. This serves as a mechanism whereby SIRT1 regulates cell survival upon induction of apoptotic stress by means that include irradiation.

SIRT1 deacetylates and stabilizes hypoxia-inducible factor-1α (HIF-1α) via direct interactions during hypoxia.

Upon shift to a hypoxic environment, cellular HIF-1α protein is stabilized, with a rapid decline in oxygen-sensitive hydroxylation. Several additional post-translational modifications of HIF-1α are critical in controlling protein stability during hypoxia. In the present study, we showed that SIRT1 stabilizes HIF-1α via direct binding and deacetylation during hypoxia. SIRT1 depletion or inactivation led to reduced hypoxic HIF-1α accumulation, accompanied by an increase in HIF-1α acetylation. Impaired HIF-1α accumulation was recovered upon inhibition of 26S proteasome activity, indicating that SIRT1 is essential for HIF-1α stabilization during hypoxia. Consistently, HIF-1α accumulation was enhanced upon overexpression of wild-type SIRT1, but not its dominant-negative form. SIRT1-mediated accumulation of HIF-1α protein led to increased expression of HIF-1α target genes, including VEGF, GLUT1 and MMP2, and ultimate promotion of cancer cell invasion. These findings collectively imply that hypoxic HIF-1α stabilization requires SIRT1 activation. Furthermore, SIRT1 protection of HIF-1α from acetylation may be a prerequisite for stabilization and consequent enhancement of cell invasion.

Romo1 is a negative-feedback regulator of Myc

Degradation of Myc protein is mediated by E3 ubiquitin ligases, including SCFFbw7 and SCFSkp2, but much remains unknown about the mechanism of S-phase kinase-associated protein (Skp2)-mediated Myc degradation. In the present study, we show that upregulated Myc protein, which triggers the G1–S phase progression in response to growth-stimulatory signals, induces reactive oxygen species modulator 1 (Romo1) expression. Romo1 subsequently triggers Skp2-mediated ubiquitylation and degradation of Myc by a mechanism not previously reported in normal lung fibroblasts. We also show that reactive oxygen species (ROS) derived from steady-state Romo1 expression are necessary for cell cycle entry of quiescent cells. From this study, we suggest that the generation of ROS mediated by pre-existing Romo1 protein is required for Myc induction. Meanwhile, Romo1 expression induced by Myc during G1 phase stimulates Skp2-mediated Myc degradation in a negative-feedback mechanism.

Expression of the RERG Gene is Gender-Dependent in Hepatocellular Carcinoma and Regulated by Histone Deacetyltransferases

Ras-related, estrogen-regulated, and growth-inhibitory gene (RERG) is a novel gene that was first reported in breast cancer. However, the functions of RERG are largely unknown in other tumor types. In this study, RERG expression was analyzed in hepatocellular carcinomas of human patients using reverse transcriptase PCR analysis. In addition, the possible regulation of RERG expression by histone deacetyltransferases (HDACs) was studied in several cell lines. Interestingly, the expression of RERG gene was increased in hepatocellular carcinoma (HCC) of male patients (57.9%) but decreased in HCC of females (87.5%) comparison with paired peri-tumoral tissues. Moreover, RERG gene expression was increased in murine hepatoma Hepa1-6 cells, human breast tumor MDA-MB-231 cells, and mouse normal fibroblast NIH3T3 cells after treated by HDAC inhibitor, trichostatin A. Our results suggest that RERG may function in a gender-dependent manner in hepatic tumorigenesis and that the expression of this gene may be regulated by an HDAC-related signaling pathway.

Methylation of eukaryotic elongation factor 2 induced by basic fibroblast growth factor via mitogen-activated protein kinase

Protein arginine methylation is important for a variety of cellular processes including transcriptional regulation, mRNA splicing, DNA repair, nuclear/cytoplasmic shuttling and various signal transduction pathways. However, the role of arginine methylation in protein biosynthesis and the extracellular signals that control arginine methylation are not fully understood. Basic fibroblast growth factor (bFGF) has been identified as a potent stimulator of myofibroblast dedifferentiation into fibroblasts. We demonstrated that symmetric arginine dimethylation of eukaryotic elongation factor 2 (eEF2) is induced by bFGF without the change in the expression level of eEF2 in mouse embryo fibroblast NIH3T3 cells. The eEF2 methylation is preceded by ras-raf-mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK1/2)-p21Cip/WAF1 activation, and suppressed by the mitogen-activated protein kinase (MAPK) inhibitor PD98059 and p21Cip/WAF1 short interfering RNA (siRNA). We determined that protein arginine methyltransferase 7 (PRMT7) is responsible for the methylation, and that PRMT5 acts as a coordinator. Collectively, we demonstrated that eEF2, a key factor involved in protein translational elongation is symmetrically arginine-methylated in a reversible manner, being regulated by bFGF through MAPK signaling pathway.

Paclitaxel stimulates chromosomal fusion and instability in cells with dysfunctional telomeres: Implication in multinucleation and chemosensitization

The anticancer effect of paclitaxel is attributable principally to irreversible promotion of microtubule stabilization and is hampered upon development of chemoresistance by tumor cells. Telomere shortening, and eventual telomere erosion, evoke chromosomal instability, resulting in particular cellular responses. Using telomerase-deficient cells derived from mTREC-/-p53-/- mice, here we show that, upon telomere erosion, paclitaxel propagates chromosomal instability by stimulating chromosomal end-to-end fusions and delaying the development of multinucleation. The end-to-end fusions involve both the p- and q-arms in cells in which telomeres are dysfunctional. Paclitaxel-induced chromosomal fusions were accompanied by prolonged G2/M cell cycle arrest, delayed multinucleation, and apoptosis. Telomere dysfunctional cells with mutlinucleation eventually underwent apoptosis. Thus, as telomere erosion proceeds, paclitaxel stimulates chromosomal fusion and instability, and both apoptosis and chemosensitization eventually develop.