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// Chia-Jung Liao 1 , Hsiang-Cheng Chi 1 , Chung-Ying Tsai 1 , Chi-De Chen 2 , Sheng-Ming Wu 1 , Yi-Hsin Tseng 1 , Yang-Hsiang Lin 1 , I-Hsiao Chung 1 , Ching-Ying Chen 1 , Syuan-Ling Lin 1 , Shiu-Feng Huang 3 , Ya-Hui Huang 4 , Kwang-Huei Lin 1 1 Department of Biochemistry, Chang-Gung University, Taoyuan, Taiwan 333, Republic of China 2 Chang Gung Molecular Medicine Research Center, Chang-Gung University, Taoyuan, Taiwan 333, Republic of China 3 Institute of Molecular and Genomic Medicine, National Health Research Institutes, Zhunan, Miaoli, Taiwan 350, Republic of China 4 Medical Research Center, Chang Gung Memorial Hospital, Taoyuan, Taiwan 333, Republic of China Correspondence to: Kwang-Huei Lin, e-mail: khlin@mail.cgu.edu.tw Keywords: Hepatoma, prognosis, sNEDD4, metastasis, apoptosis Received: October 21, 2014 Accepted: February 09, 2015 Published: March 20, 2015 ABSTRACT Despite numerous investigations on metastasis, the determinants of metastatic processes remain unclear. We aimed to identify the metastasis-associated genes in hepatocellular carcinoma (HCC). Potent metastatic SK-hep-1 (SK) cells, designated ‘SKM’, were generated using Transwell assay followed by selection in a mouse model. Genes expressed differentially in SKM and SK cells were identified via microarray analyses. A small form of Neural precursor cell-expressed developmentally downregulated 4 (sNEDD4) was identified to be overexpressed in SKM cells, which was confirmed as a novel transcript using liquid chromatography-mass spectrometry. In clinical specimens, sNEDD4 was significantly overexpressed in tumors and serves as a poor prognostic factor for male patients with HCC ( P = 0.035). Upon subcutaneous introduction of sNEDD4-overexpressing SK cells into flanks of nude mice, tumors grew faster than those of the control group. Furthermore, sNEDD4-mediated promotion of tumor metastasis was demonstrated in the orthotopic mouse model. Overexpression of sNEDD4 increased the invasive ability of SK cells through upregulation of matrix metalloproteinase 9 and inhibited serum deprivation-induced apoptosis via upregulation of myeloid cell leukemia 1. In conclusion, sNEDD4 is a novel metastasis-associated gene, which prevents apoptosis under nutrient restriction conditions. The present findings clearly support the prognostic potential of sNEDD4 for HCC.

The thyroid hormone, 3,3',5-triiodo-l-thyronine (T3 ), mediates several physiological processes, including embryonic development, cellular differentiation, metabolism and regulation of cell proliferation. Thyroid hormone (T3 ) and its receptor (TR) are involved in metabolism and growth. In addition to their developmental and metabolic functions, TRs play a tumor suppressor role, and therefore, their aberrant expression can lead to tumor transformation. Aberrant epigenetic silencing of tumor suppressor genes promotes cancer progression. The epigenetic regulator, Ubiquitin-like with PHD and ring finger domains 1 (UHRF1), is overexpressed in various cancers. In our study, we demonstrated that T3 negatively regulates UHRF1 expression, both in vitro and in vivo. Our results further indicate that UHRF1 regulation by T3 is indirect and mediated by Sp1. Sp1-binding elements of UHRF1 were identified at positions -664/-505 of the promoter region using the luciferase and chromatin immunoprecipitation assays. Notably, UHRF1 and Sp1 levels were elevated in subgroups of hepatocellular carcinoma patients and inversely correlated with TRα1 expression. Knockdown of UHRF1 expression should therefore provide a means to inhibit hepatoma cell proliferation. Expression of UHRF1 was downregulated by TRs, in turn, relieving silencing of the UHRF1 target gene, p21. Based on the collective findings, we propose that T3 /TR signaling induces hepatoma cell growth inhibition via UHRF1 repression.

Background Cervical cancer continues to threaten women’s health worldwide, and the incidence of cervical adenocarcinoma (AD) is rising in the developed countries. Previously, we showed that glucose-regulated protein 58 (Grp58) served as an independent factor predictive of poor prognosis of patients with cervical AD. However, the molecular mechanism underlying the involvement of Grp58 in cervical carcinogenesis is currently unknown. Methods DNA microarray and enrichment analysis were used to identify the pathways disrupted by knockdown of Grp58 expression. Results Among the pathway identified, the WNT signaling pathway was one of those that were significantly associated with knockdown of Grp58 expression in HeLa cells. Our experiments showed that β-catenin, a critical effector of WNT signaling, was stabilized thereby accumulated in stable Grp58 knockdown cells. Membrane localization of β-catenin was observed in Grp58 knockdown, but not control cells. Using a transwell assay, we found that accumulated β-catenin induced by Grp58 knockdown or lithium chloride treatment inhibited the migration ability of HeLa cells. Furthermore, an inverse expression pattern of Grp58 and β-catenin was observed in cervical tissues. Conclusions Our results demonstrate that β-catenin stability is negatively regulated by Grp58 in HeLa cells. Overexpression of Grp58 may be responsible for the loss of or decrease in membranous β-catenin expression in cervical AD. Electronic supplementary material The online version of this article (doi:10.1186/1471-2407-14-555) contains supplementary material, which is available to authorized users.

Triiodothyronine (T3) is a potent form of thyroid hormone mediates several physiological processes including cellular growth, development, and differentiation via binding to the nuclear thyroid hormone receptor (TR). Recent studies have demonstrated critical roles of T3/TR in tumor progression. Moreover, long-term hypothyroidism appears to be associated with the incidence of human hepatocellular carcinoma (HCC), independent of other major HCC risk factors. Dickkopf (DKK) 4, a secreted protein that antagonizes the canonical Wnt signaling pathway, is induced by T3 at both mRNA and protein levels in HCC cell lines. However, the mechanism underlying T3-mediated regulation of DKK4 remains unknown. In the present study, the 5' promoter region of DKK4 was serially deleted, and the reporter assay performed to localize the T3 response element (TRE). Consequently, we identified an atypical direct repeat TRE between nucleotides -1645 and -1629 conferring T3 responsiveness to the DKK4 gene. This region was further validated using chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA). Stable DKK4 overexpression in SK-Hep-1 cells suppressed cell invasion and metastatic potential, both in vivo andin vitro, via reduction of matrix metalloproteinase-2 (MMP-2) expression. Our findings collectively suggest that DKK4 upregulated by T3/TR antagonizes the Wnt signal pathway to suppress tumor cell progression, thus providing new insights into the molecular mechanism underlying thyroid hormone activity in HCC.

MicroRNAs (miRNAs) are thought to control tumor metastasis through direct interactions with target genes. Thyroid hormone (T3) and its receptor (TR) are involved in cell growth and cancer progression. However, the issue of whether miRNAs participate in T3/TR-mediated tumor migration is yet to be established. In the current study, we demonstrated that T3/TR negatively regulates mature miR-17 transcript expression, both in vitro and in vivo. Luciferase reporter and chromatin immunoprecipitation (ChIP) assays localized the regions responding to TR-mediated repression to positions -2234/-2000 of the miR-17 promoter sequence. Overexpression of miR-17 markedly inhibited cell migration and invasion in vitro and in vivo, mediated via suppression of matrix metalloproteinases (MMP)-3. Moreover, p-AKT expression was increased in miR-17-knockdown cells that led to enhanced cell invasion, which was blocked by LY294002. Notably, low miR-17 expression was evident in highly metastatic cells. The cell migration ability was increased by T3, but partially reduced upon miR-17 overexpression. Notably, TRα1 was frequently upregulated in hepatocellular carcinoma (HCC) samples and associated with low overall survival (P=0.023). miR-17 expression was significantly negatively associated with TRα1 (P=0.033) and MMP3 (P=0.043) in HCC specimens. Data from our study suggest that T3/TR, miR-17, p-AKT and MMP3 activities are interlinked in the regulation of cancer cell metastasis.

Hypothyroidism has been associated with significantly elevated risk for hepatocellular carcinoma (HCC), although the precise underlying mechanisms remain unknown at present. Thyroid hormone (T3) and its receptor (TR) are involved in metabolism and growth. Endoglin is a T3/TR candidate target gene identified from our previous studies. Here, we demonstrated that T3 positively regulates endoglin mRNA and protein levels, both in vitro and in vivo. The thyroid hormone response elements of endoglin were identified at positions -2114/-2004 and -2032/-1973 of the promoter region using the electrophoretic mobility shift assay and chromatin immunoprecipitation assay. Endoglin was downregulated in the subgroups of HCC patients and significantly associated with histology grade (negative association, P=0.001), and this expression level was significantly associated with TRα1 in these HCC patients. Our results clearly indicate that p21 is involved in T3-mediated suppression of cell proliferation. Knock down of endoglin expression in HCC cells facilitated p21 polyubiquitination and promoted cell proliferation in the presence of T3. The data collectively suggest that T3/TR signaling suppresses cell proliferation by upregulating endoglin, in turn, affecting p21 stability. The results indicate that endoglin has a suppressor role to inhibit cell proliferation in HCC cell lines.

Gastric cancer is the sixth leading cause of cancer‐related death in Taiwan, and the identification of related factors is essential to increase patient survival. ADP‐ribosylation factor 1 (ARF1) was initially identified using 2‐D electrophoresis combined with MALDI–time‐of‐flight mass spectrometry. ADP‐ribosylation factor 1 belongs to the Ras superfamily or GTP‐binding protein family and has been shown to enhance cell proliferation. In the current study, we evaluated the potential of ARF1 as a biomarker for gastric cancer detection. ADP‐ribosylation factor 1 mRNA was upregulated in tumor tissues (compared with adjacent non‐tumor tissues, n = 55) in approximately 67.2% of gastric cancer patients. Expression of ARF1 protein was additionally observed using Western blot and immunohistochemistry (IHC) analyses. The clinicopathological correlations of ARF1 were further evaluated. Elevated ARF1 expression was strongly correlated with lymph node metastasis (P = 0.008), serosal invasion (P = 0.046), lymphatic invasion (P = 0.035), and pathological staging (P = 0.010). Moreover, the 5‐year survival rate for the lower ARF1 expression group (n = 50; IHC score

A second corresponding author, Chia-Siu Wang, was not signified. The author Chia-Siu Wang can be contacted at wangcs@adm.cgmh.org.tw Also, there is an error in Figure 3. The correct Figure 3 can be seen here: [^]

The thyroid hormone T(3) regulates differentiation, growth, and development. We demonstrated that methionine adenosyltransferase 1A (MAT1A) was positively regulated by T(3) identified by cDNA microarray previously. The expression of the MAT1A was upregulated by T(3) in hepatoma cell lines overexpressing thyroid hormone receptors (TRs). Additionally, these findings indicate that MAT1A may be regulated by CCAAT/enhancer binding protein (C/EBP). The critical role of the C/EBP binding sites was confirmed by the reporter or chromatin immuno-precipitation (ChIP) assay. In addition, C/EBP was upregulated in hepatoma cells after T(3) treatment and ectopic expression of MAT1A inhibited cell migration and invasion in J7 hepatoma cells. Conversely, knockdown of MAT1A expression increased cell migration. Together, these findings suggest that the expression of the MAT1A gene is mediated by C/EBP and is indirectly upregulated by T(3). Finally, TR was downregulated in a small subset of hepatocellular carcinoma cells concomitantly reduced the expression of C/EBPalpha and MAT1A.

Introduction: With unique geographic features, mountain climbing has been a popular outdoor activity in Taiwan. Related conferences on safety issues were held by Ministry of Education. It is found that mortality and morbidity of high mountain syndrome (HMS) is highly reverse related to timely diagnosis and management. Generally, it is not easy for mountain climbers to manage this condition without an expert on site. Several climbers died from HMS every year. In 2005, wireless internet service was provided in Yushan National Park (YSNP), the highest national parkin Northeast Asia. We therefore began this project - building an emergent HMS consulting system based on this service.

Gene amplification provides a means for overexpression of cell growth promoting genes driving tumor formation and progression. Indeed, cancer genomic analyses have indicated that gene amplification occurs somatically in certain regions of human cancer genome. Among them, amplification of the 11q13.3 is a frequent event in human cancers including head and neck squamous cell carcinoma. Our whole genome copy number alterations (CNAs) study also revealed amplification of 11q13.3 in oral cavity squamous cell carcinoma (OSCC) in Taiwan. This region contains at least 5 cancer-related genes including CCND1 and FADD. CCND1 amplification has been demonstrated as an oncogenic driver gene in breast, bladder, head and neck, liver and lung squamous cancer. FADD, an apoptotic effector, was previously identified as a novel cancer driver gene in laryngeal/pharyngeal cancer. It is warranting for further investigation to determine whether FADD amplification is also a cancer driver in other types of human cancer. Therefore, this study was designed to explore whether FADD is a cancer driver in Taiwanese OSCC based on CNAs, protein expression and their clinical implications. A total of 346 male OSCCs were examined for gene copy number using TaqMan CNV analysis and protein expression using immunohistochemistry. We found that the intensity of FADD protein expression was strongly correlated with gene copy number gain/amplification. Gene copy number gain/amplification and high protein expression was observed in 70 (20.23%) and 199 (57.51%) OSCCs, respectively. Both FADD gene copy number gain/amplification and high protein expression were significantly associated with lymph node metastasis (p Citation Format: Chia-Jung Liao, Huei-Tzu Chien, Shiang-Fu Huang, I-How Chen, Chun-Ta Liao, Hung-Ming Wang, Ling-Ling Hsieh. Clinical implications of FADD copy number gain/amplification and high protein expression in areca-quid-associated oral cavity squamous cell carcinomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3855. doi:10.1158/1538-7445.AM2015-3855

Cervical cancer is still a serious problem threatening to women health worldwide. The incidence of cervical adenocarcinoma (AD) is rising in more developed countries. Previously we have identified that glucose-regulated protein 58 (Grp58) serves as a poor independent prognostic factor for cervical AD. The molecular mechanism underlying Grp58 involved cervical AD carcinogenesis is unknown. The DNA microarray and enrichment analysis were used to identify pathway maps perturbed by knockdown of Grp58 expression. The WNT singling map is one of the significant enriched pathways. The β-catenin, vital effector of WNT signaling, was stably accumulated in Grp58 knockdown stable cells. A membrane fashion of β-catenin was observed in Grp58 knockdown but not control cells. By using transwell assay, we have demonstrated that accumulation of β-catenin, which was induced by Grp58 knockdown or lithium chloride treatment, inhibited the migration ability of HeLa cell. Furthermore, an exclusive expression pattern of Grp58 and β-catenin was observed in cervix tissues. In sum, we have demonstrated that β-catenin stability is negatively regulated by Grp58. Overpression of Grp58 might result in lost or decreased of membranous β-catenin in cervical cancer to promote cancer progression. Citation Format: Chia-Jung Liao, Syuan-ling Lin, Tzu-Hao Wang, Kwang-Huei Lin. Glucose-regulated protein 58 modulates β-catenin protein stability in cervical adenocarcinoma. [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 3290. doi:10.1158/1538-7445.AM2014-3290

Cervical cancer is the second most common cancer in women worldwide. Human papilloma virus infection is the primary cause of cervical cancer, although it cannot cause cancer by itself. Molecular markers involved in cervical carcinogenesis are needed. A two-dimensional polyacrylamide gel electrophoresis (2-DE) proteomic strategy was used to discover genes with exceptional expression from four paired cervical cancer tissues. Immunoblotting and immunohistochemical analysis with 52 paired specimens and 107 sections were used to confirm the results of 2-DE, and the clinical significance was estimated. The molecular functions of ER-60 were investigated in vitro and in vivo. We identified ER-60 was upregulated in 71.2% of cancerous tissues. ER-60 expression differed significantly between patients with different histological types and differentiation grades (P = 0.013 and 0.045, respectively). Patients with high ER-60 expression had a lower 5-year overall survival (OS) and recurrence-free survival (RFS) rate (P = 0.038 and 0.017, respectively). High ER-60 expression was an independent prognostic factor of poor RFS (P = 0.019). Knockdown of ER-60 expression in HeLa cell decreased cell motility. The lung metastasis was inhibited when introduced ER-60-knockdown cells into severe combined immunodeficiency mice. We observed accumulation of b-catenin and regulation of b-catenin downstream CD44 and S100A4 expression in ER-60-knockdown cell were responsible for this migration inhibition. Taken together, ER-60 overexpression is a potential prognostic factor of poor OS and an independent factor of poor RFS in cervical cancer. ER-60 may be involved in cervical cancer invasion by modulating b-catenin protein stability and signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3807. doi:10.1158/1538-7445.AM2011-3807

Introduction: The global yearly incidence and mortality of cervical cancer are 471,000 annual new cases and 233,000 deaths. Almost 80% cases occur in less developed countries, where it accounts for 15% of cancer in women. Cervical carcinoma ranks as the leading malignancy of genital tract malignancy in the developing countries. The recurrence indicate poor prognosis while the clinicopathological risk factors exist. In order to identify the potential molecular biomarker, proteomic analysis was performed and gesolin protein was selected. Materials and Methods: The plasma from normal healthy and cervical cancer, and tissue of cervical cancer plus adjacent normal tissue were collected for Western blotting assay, enzyme-linked immunosorbent assay (ELISA) or immunohistochemical stain (IHC). One hundred and twenty four patients with cervical cancers underwent surgical management were enrolled to evaluate IHC Histoscore of gesolin according to the multiply of immunoreactivity and involved percentage. The Transwell assay was conducted for cell migration analysis in HeLa cell line. The statistical analysis include independent t tests, Kaplan-Meier method using the log-rank test and the Cox proportional hazard model Receiver operating characteristics (ROC) curve analysis was used to determine cut-off points of the predictive parameters in univariate analysis. Results: The Western blot and ELISA assay both demonstrated the overexpression of plasma gelsolin in cases with cervical cancer rather than normal population, and with staged III/IV rather than stage I/II cases, In addition, gelsolin protein level also increased in conditional medium of cultured HeLa cell. Thus in vitro cell migration assay revealed decreased cell numbers while adding the gelsolin neutralizing antibody or small interference RNA compared with adding mock, non-specific antibody or Luciferase-shRNA. The other clinical parameters including stage, tumor size and lymph node status also put into consideration. The best cut-off point of gesolin Histosore to clarify the recurrence by ROC curve analysis is 115. The gelsolin Histosore (≤ 115 vs > 115, P= 0.018) and lymph node metastasis (no vs yes, P Conclusion: Overexpression of plasma and cytoplasmic gelsolin protein in cervical cancer promote cell migration by binding to actin cytoskeleton for rearrangement and cell motility. Among the clinicopathological parameters, the gelsolin overexpression and lymph node metastasis disclosed the recurrence-free survival impact by multivariate analysis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1740.