bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4873: Functional interactions between lysine 20 methylation and lysine 16 acetylation on histone H4 in DNA damage responses
Authors: Hsiao Kuei-Yang , Mizzen Craig
Published: February 2011

Individual histone post-translational modifications have been implicated in regulating many cellular events. Modifications can occur at high densities in the N-terminal tail domains of core histones, enabling the possibility that combinations of modifications regulate chromatin processes differently than modifications occurring individually. However, surprisingly little is known about the nature and functions of multi-site histone modification. This study investigates functional interactions between acetylation of lysine 16 (K16ac) and methylation of lysine 20 (K20me), two modifications that frequently occur together on molecules of histone H4. Methylation state-specific binding of p53-binding protein 1 (53BP1) to dimethyl K20 H4 (K20me2) and the formation of 53BP1 nuclear foci near double strand breaks are early events in DNA damage responses (DDR) that provide a platform for the assembly of downstream effectors. Since global levels of K20me2 are relatively stable and are established independently of DNA damage, we propose that reversible changes in the levels of K16ac co-modification act like a dynamic switch to modulate DDR responses mediated by K20me2. Analyses of the interaction of the K20me2 binding domain of 53BP1 with H4 peptides in vitro reveal that K16ac co-modification attenuates specific recognition of K20me1/2 by 53BP1. The possibility that K16ac and K20me2 interact functionally in vivo is demonstrated by our finding that K16 deacetylation accompanies the induction of H2AX phosphorylation, a well-known marker for DDR, following DNA damage by either UV irradiation or bleomycin treatment. Moreover, the effects of expressing K16 mutants of H4 and inhibition of histone deacetylases on 53BP1 foci formation induced by DNA damage are consistent with our hypothesis that K16ac co-modification negatively regulates the formation of 53BP1 foci. DDR mechanisms are frequently deregulated in cancer cells. Further investigation of the role of H4 modifications in DDR will enhance our understanding of how genome integrity is maintained in normal cells and may identify novel targets for future therapies.

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 4873.
Abstract 4874: Epigenetic gene silencing is co-regulated by DNMT3B and polycomb-mediated histone modifications in human cancer
Authors: Jin Bilian , Robertson Keith D.
Published: February 2011

CpG-cytosine methylation, the only epigenetic modification in mammalian DNA, is established and maintained by three DNA methyltransferases (DNMT): DNMT1, DNMT3A, and DNMT3B. Although essential for development and differentiation, DNA methylation patterns are frequently disrupted in cancer and contribute directly to carcinogenesis. Recent studies linking polycomb group (PcG) repression complexes (PRC1 and PRC2) to the DNMTs have begun to shed light on how methylation is targeted. By comparing genes regulated by DNMT3B, which we identified in previous studies, with known polycomb group target genes, we chose a panel of genes co-regulated by DNMT3B and PRC1/PRC2 to test whether these criteria (DNMT & PcG co-regulation) could accurately identify novel targets of epigenetic silencing in human colon cancer. Using reverse transcription-PCR, bisulfite genomic sequencing, and pyrosequencing, we show that the majority of these genes are frequently silenced in colorectal cancer cell lines and primary tumors. We then analyzed the histone code, DNMT, and PRC1/PRC2 binding by chromatin immunoprecipitation at epigenetically silenced loci to reveal a novel link between DNMT3B and PRC1, which was further confirmed by siRNA knockdown assays. Specifically, loss of DNMT3B resulted in significant reductions in ubiquitinated histone H2A levels, a mark catalyzed by the RING2 subunit of PRC1. Taken together, these studies suggest that patterns of epigenetic modifiers and the histone code influence the propensity of a gene to become hypermethylated in cancer and that DNMT3B plays an important role in regulating PRC1 function.

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 4874.
Abstract 4882: Loss of p300 reduces miR-let-7c and enhances ER-positive breast cancer cell tumorigenicity
Authors: Yuan Huijun , Sil Sukanya , Sharma Salil , Bishopric Nanette
Published: February 2011

Histone deacetylase (HDAC) inhibitors have been extensively studied as potential anticancer treatments and several are currently in clinical trials. p300 is a nuclear histone acetyltransferase that is an important coordinator of multiple transcription-dependent cellular processes. HDAC inhibitors may directly and indirectly increase the activity of p300. The role of p300 is dependent on cell context but most or all of its biological activity stems from its ability to acetylate histones or non-histone nucleoproteins including p53, HSP) which regulate cell proliferation, differentiation and apoptosis. Whether p300 acts as a tumor suppressor or promoter is controversial, and its role in estrogen positive breast cancer remains to be defined, however, structural studies indicate that it has significant potential as a target for small molecule therapy. To address this issue, we have used a lentiviral approach to achieve 60-70% stable knockdown (KD) of p300 levels in estrogen receptor-positive breast cancer lines MCF-7 and BT 474 respectively. Growth curve and FACS analysis showed that loss of p300 shortened doubling times and increased G2 phase cells in both lines. Xenograft tumors formed from p300 KD cells were on average about three times larger at six weeks post implantation than control tumors (p<0.05). H&E and CD31 staining showed that p300 KD xenograft tumors have a more aggressive phenotype with increased vascularity. In searching for targets of p300 that might regulate its anti-tumor effect, we observed that the microRNA miR-let-7c was downregulated in p300-depleted MCF-7 and BT474 cells. Consistent with this, the miR-let-7c target N-Ras was up-regulated in both p300 KD cell lines and in p300 KD xenograft tumors, and adenovirus-mediated over-expression of p300 caused downregulation of N-RAS. Finally, gene expression analysis showed that loss of p300 resulted in upregulation of E-cadherin, laminin, collagen, IL-6, IFN, TGF-β and components of the Wnt signaling pathway, indicating a shift from epithelial to mesenchymal phenotype. Our data show that p300 loss or inactivation results in altered transcription patterns and suppression of anti-tumor microRNAs, including let-7, and may induce a more invasive and potentially metastatic phenotype. We conclude that p300 likely acts as a tumor suppressor in breast cancer and that activation of p300 may contribute to the anti-tumor effects of TSA.

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 4882.
Abstract 4885: Changes of H3K9 modification by increased expression of HDAC2 and SUV39H1 modulates chromosome condensation in A549 lung cancer cells
Authors: Park Jin-Ah , Kim Ae-Jin , Lee Joo-Kyung , Ham Myoung-Sun , Kim Keun-Cheol
Published: February 2011

Genetic information is condensed from interphasic chromatin to mitotic chromosome for correct segregation into two daughter cells. Phosphorylation of H3S10 is well-known as modification marker for chromosome condensation, but further modification at several histone residues could be required for correct mitosis. In this study, we show that H3K9me3 was dramatically increased at mitotic cells, whereas H3K9ac was decreased. Chromosome spreading analysis is shown that H3K9me3 was distinctly increased at a longitudinal section of chromosome at prophase and prometaphase. These combinational modification of H3K9 was resulted from the increased expression of HDAC2 and SUV39H1, suggesting that these proteins are involved in chromosome condensation. Interestingly, FACS analysis is shown that metaphasic progression was delayed in HDAC inhibition by TSA treatment. Moreover, HDAC2 expression was increased from late S phase to M phase, and SUV39H1 expression was sequentially followed from G2 to M phase, implicating that activities of HDAC2 and SUV39H1 are modulated during chromosome condensation. Therefore, we suggest that increased expression of HDAC2 and SUV39H1 facilitate chromosome condensation targeting combinational modification of H3K9.

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 4885.
Abstract 4900: MAGE-A11 expression in castration-recurrent prostate cancer: association with a global epigenetic defect
Authors: James Smitha R. , Forti Lena D. , Mohler James L. , Wilson Elizabeth M. , Karpf Adam R.
Published: February 2011

Despite the initial clinical response to androgen deprivation therapy, most prostate cancer patients subsequently develop castration-recurrent disease. Elucidation of the mechanisms driving this process is expected to lead to improved strategies to prevent and treat advanced prostate cancer. Epigenetic alterations, including DNA hypomethylation, play a key role in human cancer development. DNA hypomethylation is thought to promote oncogenesis in part by driving oncogene expression, but its potential role in castration-recurrent prostate cancer is unknown. Melanoma Antigen A-11 (MAGE-A11) is a cancer germline (CG) antigen that stabilizes the androgen receptor (AR), facilitates AR association with co-activators, and promotes AR transcriptional activity; thus it appears to play an oncogenic role in castration-recurrent disease. We have recently reported that MAGE-A11 is over-expressed in prostate cancer cell lines, prostate cancer xenografts, and human prostate cancer tissues; in each setting MAGE-A11 overexpression is associated with DNA hypomethylation at the MAGE-A11 promoter CpG island (Karpf et al., Molecular Cancer Research 7; 523-535, 2009). Critically, MAGE-A11 expression and promoter hypomethylation correlate with castration-recurrent cancer growth, both in the CWR-22 xenograft model and in primary human prostate cancer tissues. In the current study we examined whether MAGE-A11 expression and hypomethylation in prostate cancer is an isolated molecular event, or alternatively whether it coincides with a global DNA hypomethylation defect. To test this, we analyzed the global DNA methylation status of prostate cancer cell lines, CWR-22 cancer xenografts, and primary prostate cancer, in which MAGE-A11 expression and methylation was also characterized. We assessed global DNA methylation by measuring total genomic 5-methyl-2′-deoxycytidine (5mdC), LINE-1 repetitive DNA element methylation, and the expression and methylation of several other CG antigen genes, including MAGE-A1, NY-ESO-1, XAGE-1, and BORIS/CTCFL. Taken together, our data provide evidence for a global DNA hypomethylation phenotype in castration-recurrent prostate cancers that express MAGE-A11. Ongoing studies are using HpaII tiny fragment enrichment by ligation-mediated PCR (HELP assay) to define the epigenome of castration-recurrent prostate cancer displaying MAGE-A11 expression. In summary, our data provide evidence that global DNA hypomethylation contributes to castration-recurrent prostate cancer, at least in part by promoting MAGE-A11 expression.

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 4900.
Abstract 4903: Oxidative DNA damage and its relationship with GSTP1 methylation in prostate cancer
Authors: Fnu Rajnee , Sharma Haripaul , Gupta Sanjay
Published: February 2011

Prostate cancer is the most common cancer diagnosed in men and is the second leading cause of cancer-related death in the United States. The risk factor for prostate cancer includes both genetic and epigenetic modifications. Growing evidence suggests that epigenetic mechanisms such as DNA methylation affect gene expression in tissue-specific manner. The most frequent epigenetic alteration observed in human prostate cancer is hypermethylation of gluthathione-S-transferase-pi class (GSTP1) in the promoter region which causes loss or its decrease expression. Inactivation of GSTP1 renders prostate epithelial cells vulnerable to oxidative DNA damage from both oxidants and electrophile carcinogens to acquire relevant genetic alteration and neoplastic transformation. Extensive observations suggest that reactive oxygen species may cause accumulated DNA damage which might contribute to increased incidence of prostate cancer. However, the role of GSTP1 and its correlation with DNA damage has not been studied. Based on the assumption that DNA damage is prevalent in cancer cells compared to nontumorous tissue, we took a panel of 24 human prostate specimens obtained after surgery which comprised of matched primary tumor and adjacent nontumorous (benign) prostate tissues. Oxidative DNA damage was measured by 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels through highly sensitive and quantitative ELISA assay. GSTP1 methylation was determined by methyl-specific PCR and 5-methylcytosine estimation was performed by HPLC analysis. The 8-OHdG levels were higher in cancer specimen compared to their benign counterparts. The 8-OHdG levels in DNA of prostate cancer tissues ranged from 0.151 to 0.616ng/μg DNA, whereas the levels in benign tissue range from 0.149 to 0.318ng/μg DNA, respectively. The increase in 8-OHdG levels correlate with GSTP1 methylation which was higher in cancer specimens compared to the benign counterparts. Furthermore, levels of 5-methylcytosine were higher in tumor specimens compared to benign tissue. Taken together, our studies suggest that there is a link between GSTP1 methylation and DNA damage which correlates to epigenetic modification in prostate cancer. Our results are in agreement with the prediction that positive relationship occur between GSTP1 inactivation and DNA damage in prostate cancer.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

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 4903.
Abstract 4913: The Bmi-1 polycomb protein antagonizes the (−)-Epigallocatechin-3-gallate dependent suppression of skin cancer cell survival
Authors: Balasubramanian Sivaprakasam , Adhikary Gautam , Eckert Richard L.
Published: February 2011

The Polycomb Group (PcG) proteins are epigenetic suppressors of gene expression that play a key role in promoting cell survival. This suppression is achieved via the action of two multiprotein PcG complexes - PRC2 (eed) and PRC1 (Bmi-1). These complexes act to modulate gene expression by increasing histone methylation and reducing acetylation - ultimately leading to a closed chromatin conformation and gene silencing. Activity of these proteins is associated with increased cell proliferation and survival. We show increased expression of key PcG proteins in immortalized keratinocytes and skin cancer cell lines. We examine the role of two key PcG proteins, Bmi-1 and Ezh2, and the impact of the active agent in green tea polyphenol, (−)-epigallocatechin-3-gallate (EGCG), on the function of these regulators. EGCG treatment of SCC-13 cells reduces Bmi-1 and Ezh2 level and this is associated with reduced cell survival. The reduction in survival is associated with a global reduction in histone H3-K27-trimethylation (H3 K27-3M), a hallmark of PRC2 complex action. This change in PcG protein expression is associated with reduced expression of key proteins that enhance progression through the cell cycle (cdk1, cdk2, cdk4, cyclin D1, cyclin E, cyclin A, and cyclin B1) and increased expression of proteins that inhibit cell cycle progression (p21 and p27). Apoptosis is also enhanced, as evidenced by increased caspase 9, 8 and 3 cleavage and increased PARP cleavage. EGCG treatment also increases Bax and suppresses Bcl-xL expression. Vector-mediated enhanced Bmi-1 expression reverses these EGCG-dependent changes. These findings suggest that green tea polyphenols reduce skin tumor cell survival by influencing PcG-mediated epigenetic regulatory mechanisms.

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 4913.
Abstract 4914: Methylation profiling of CpG islands in multiple drug resistant cell line
Authors: Hung Tsai-Hsien , Shen Chih-Jie , Chen Chuan-Mu , Chong Kowit Yu
Published: February 2011

Multiple drug resistance (MDR) is a major issue to attenuate the effectiveness of chemotherapy to many human malignancies. Despite extensively investigation, MDR inhibitors have been discovered serve side effect. Recently, scientists have reported that proteasome inhibitor, Bertezomib (PS-341), induced apoptosis in a variety of cancer cells and recognized as a novel anticancer therapy approach. Previous study revealed that bertezomib inhibits DNA methyltransferase and led to DNA hypomethylation in myeloid leukemia. Despite its success, some patients are resistant to ongoing proteasome inhibitor treatment. These reports suggested that chemoresistant cell line may develop a novel mechanism to against proteasome inhibitor. To verify the hypothesis, we used human CpG island microarray to indentify epigenetic change and genes expression profile in parental human uterus sarcoma cell line (MES-SA) and MES-SA variant drug resistant cell line (Dx5-C5) Our data has shown that the global genes were hypomethylated in both PS-341 treated MES-SA and Dx5-C5 cell line. Furthermore, the Wnt signalling pathway and cell cycle related gene have significant altered in DNA methylation pattern of Dx5-C5. In summary, our study demonstrated that the cell cycle related gene(s) and upstream gene(s) of Wnt pathway altered its DNA methylation pattern and mRNA expression level in MDR cell line. These data may help us to develop a new strategy for treatment of multiple drug resistance cancer cells.

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 4914.
Abstract 4918: Quality assessment of DNA derived from up to 30 year old archival tissues from lung cancer patients for PCR-based methylation analysis using SMART-MSP and MS-HRM
Authors: Kristensen Lasse S. , Wojdacz Tomasz K. , Thestrup Britta B. , Hager Henrik , Hansen Lise Lotte
Published: February 2011

Background: The High-Resolution Melting (HRM) technology has recently been introduced as a rapid and robust analysis tool for the detection of DNA methylation. The methylation status of multiple tumor suppressor genes may serve as biomarkers for early cancer diagnostics, for prediction of prognosis and for prediction of response to treatment. Therefore, it is important that methodologies for detection of DNA methylation continue to evolve. Sensitive Melting Analysis after Real Time - Methylation Specific PCR (SMART-MSP) and Methylation Sensitive - High Resolution Melting (MS-HRM) are two methods for single locus DNA methylation detection based on HRM.

Methods: Here, we have assessed the quality of DNA extracted from 30-years-old Formalin Fixed Paraffin Embedded (FFPE) tissue for DNA methylation analysis using SMART-MSP and MS-HRM. The quality assessment was performed on DNA extracted from 54 Non-Small Cell Lung Cancer (NSCLC) samples derived from FFPE tissue, collected over 30 years and grouped into five-years-intervals. For each sample, the methylation level of the CDKN2A (p16) and RARB promoters were estimated using SMART-MSP and MS-HRM assays designed to assess the methylation status of the same CpG positions. This allowed for a direct comparison of the methylation level estimated by the two methods for each sample.

Results: The methylation level of the CDKN2A promoter was successfully determined by SMART-MSP and MS-HRM in all of the 54 samples. The same methylation estimates were obtained by the two methods in 46 of samples. The methylation level of the RARB promoter was successfully determined by SMART-MSP in all of samples. When using MS-HRM to assess RARB methylation five samples failed to amplify and 15 samples showed a melting profile characteristic for heterogeneous methylation. Out of the remainder 34 samples, for which the methylation level could be estimated, 27 gave the same result as observed when using SMART-MSP.

Conclusion: MS-HRM and SMART-MSP can be successfully used for single locus methylation studies of DNA derived from up to 30-years-old FFPE tissue samples. Furthermore, it can be expected that MS-HRM and SMART-MSP will provide similar methylation estimates when assays are designed to analyze the same CpG positions.

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 4918.
Abstract 4925: Promoter methylation of RASSF1A Tumor Suppressor Gene Modulates the Effect of Microtubule-Targeting Agent, docetaxl, in Breast Cancer
Authors: Eun-Young Gil , Jo Uk Hyun , Jung Hae Yun , Woo Ok Hee , Seo Jae Hong , Kim Aeree , Lee Eun-Sook , Cho Kyu-Ran , Kim Yeul Hong , Kim Jun Suk , Kim In Sun , Park Kyong Hwa
Published: February 2011

RASSF1A has been previously demonstrated to be inactivated by CpG island methylation in breast cancer cell lines and primary tumor tissues. There have been evidences suggesting that methylation of this gene have a significant impact on biological characteristics of breast tumors and have a relationship with prognosis in breast cancer. The tumor suppressive effect of RASSF1A has been reported to be mediated by its 2 important roles in other cells; induction of cell cycle arrest and apoptosis. Therefore, it is possible that expression status of RASSF1A by promoter methylation may alter patient's sensitivity to chemotherapeutic agents affecting cell cycle. However, there is few data regarding the relationship between the methylation status of RASSF1A gene and clinical response to chemotherapeutic agents in breast cancer patients. In this current study, we show that RASSF1A gene expression was suppressed in the human breast cancer cell lines (MCF-7, MDA-MB-231, and ZR75-1) by the mechanism of promoter methylation using methylation -specific PCR. It was confirmed by the results that treatment with the DNA demethylating agent, 5-aza-2′-deoxycytidine, reactivated the expression of RASSF1A at the level of mRNA and protein. Re-expression of RASSF1A gene in breast cancer cell lines could decrease the cell growth and proliferation by both decreasing cyclin D1 expression and inducing cyclin A and B1 accumulation, then resulting in cell cycle arrest at G2/M phase. In addition, re-introduction of RASSF1A further increased docetaxel-induced cell death in breast cancer cells. We next questioned whether the methylation status of RASSF1A gene is associated with clinical response to taxane-based chemotherapy in breast cancer patients. Fresh frozen primary breast cancer tissues were ananlyzed for methylation status of RASSF1A gene using MS-PCR. Thirty three out of 39 patients (84.6%) showed RASSF1A methylation and 6 had un-methylated gene. Patients who had unmethylated or partially methylated RASSF1A gene showed some mRNA in RT-PCR analysis. And, the clinical response to taxane-based chemotherapy was significantly better in the un-, or partially-methylated patient group (100% vs 62.5%). These results suggest that hypermethylation of the CpG island promoter of RASSF1A may play an important role in breast cancer pathogenesis and is a modulating factor for docetaxel activity in breast cancer patients.

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 4925.
Abstract 4928: 14-3-3ζ-mediated hypomethylation of c-myc oncogene by downregulation of Dnmts contributes to initiation and progression of breast cancer
Authors: Xu Jia , Yu Dihua
Published: February 2011

The development of breast cancer is a multistep process and the molecular mechanisms underlying this process are still elusive. Previously, we discovered that 14-3-3ζ overexpression not only contribute to breast cancer progression but also play an important role in breast cancer initiation, including an early stage of breast disease-atypical ductal hyperplasia (ADH). However, the underlying mechanisms remain unclear. Recently, 14-3-3ζ has been shown to bind to Histone H3, Methylation Binding Proteins (MBPs) and HDAC family members, suggesting 14-3-3ζ may play a role in epigenetic regulation. As an initial effort to investigate whether 14-3-3ζ has a role in epigenetic regulation and whether epigenetic regulation by 14-3-3ζ plays a role in breast cancer initiation, we established 14-3-3ζ stable overexpressing lines (10A.14-3-3ζ) in non-tumorigenic MCF10A human mammary epithelial cells (HMEC) and analyzed molecular alterations by cDNA microarray in the 10A.14-3-3ζ cells versus the 10A.vec control cells in a 3D culture system. Interestingly, we found that c-myc, a well known oncogene which is overexpressed in many types of cancer, was upregulated at the mRNA level in the 10A.14-3-3ζ cells versus the 10A.vec cells. We further confirmed c-Myc upregulation at both mRNA level by RT-PCR and protein level by Western Blotting. Additionally, we performed Reverse Phase Protein Array (RPPA) analysis with a series of validated antibodies, and we found that Dnmt1, the key DNA methyltransferase in mammals, was downregulated in 10A.14-3-3ζ cells compared to 10A.vec cells. We have confirmed Dnmt1 downregulation by western blotting. Since c-myc oncogene are well known to be upregulated by hypomethylation as a consequence of Dnmt1 downregulation, these data suggest that 14-3-3ζ may contribute to breast cancer initiation and progression by upregulation of oncogene c-myc via promoter hypomethylation. We will continue this study to provide a better understanding on how 14-3-3ζ contributes to breast cancer initiation and progression via epigenetic deregulation (demethylation) of c-myc.

This study will bring insight into how 14-3-3ζ overexpression elicits the phenotypic changes during the development of breast carcinomas through global deregulation of transcriptome as well as specific gene expressions. Our studies could provide a better understanding of the role of 14-3-3ζ in breast cancer initiation and progression.

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 4928.
Abstract 4933: Expression of the N-myc downstream-regulated gene 2 (NDRG2) is frequently suppressed by promoter hypermethylation in human gastrointestinal and pancreatic cancers
Authors: Yamamura Akihiro , Miura Koh , Karasawa Hideaki , Abe Keiko , Mizuguchi Yasuhiko , Jin Guo , Gu Zhaodi , Fukushige Shinichi , Kaneko Naoyuki , Kinouchi Makoto , Ando Toshinori , Yazaki Nobuki , Tanaka Naoki , Sase Tomohiko , Shibata Chikashi , Sasaki Iwao , Horii Akira
Published: February 2011

It is well known that inactivation of tumor suppressor gene (TSG) by promoter hypermethylation plays an important role during the development and progression of cancer. NDRG2 (N-myc downstream regulated gene 2) is considered to be one of the candidate TSGs. Significant down-regulation in various human cancers were observed, but the molecular mechanisms of reduced expression and the ways to carcinogenesis are largely not well understood. To elucidate the mechanisms in expressional suppression of NDRG2, we studied the methylation status of the putative promoter region in this gene. NDRG2 has two promoters; we first found that the mRNA transcription predominantly starts from the promoter containing exon 1b in organs of the digestive system. Then the expression level of NDRG2 was analyzed by quantitative real-time RT-PCR using 20 colorectal, 6 gastric, and 5 pancreatic cancer cell lines; many cell lines showed reduced expression. Bisulfite modification followed by nucleotide sequencing analyses in the putative promoter region revealed that the methylation status correlated with the suppressed expression. We extended our analysis to the primary tumors by immunohistochemical examination to elucidate clinical significance of suppressed expression of NDRG2 in tumorigenesis; matched normal and cancerous tissues from cancer patients with digestive system were analyzed and observed suppressed expression of NDRG2 in 19 of 20 colorectal, 8 of 10 gastric, and 85 of 86 pancreatic cancer specimens. We then designed a set of primer pairs for methylation specific PCR and examined the relationship between expression level of NDRG2 and promoter hypermethylation in resected primary tumor specimens. Association between methylation status and clinicopathologic features are now studied and the newly obtained results will be presented.

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 4933.
Abstract 4935: Hyperemethylation of retinoid X receptors genes in non-small cell lung cancers and its clinical implication
Authors: Lee Su M. , Kim Mi J. , Lee Ji Y. , Park Jae Y. , Kim Dong S.
Published: February 2011

Retinoid X receptors (RXRs) are nuclear receptors for retinoids that play a critical role in the regulation of growth and differentiation in normal and tumor cells. Deregulation of RXR expression has been reported in non-small cell lung cancer (NSCLC); however, the mechanism underlying the impaired expression of RXRs in lung cancer is not known. Aberrant methylation of promoter CpG islands is known to be a major mechanism for inactivation of tumor suppressor genes. We investigated the methylation status of the RXR genes in 139 surgically-resected NSCLCs and correlated the results with the clinicopathologic characteristics of the patients. Methylation in the tumors was detected at RXRα (5.7%), RXRβ (4.3%), and RXRγ (23.7%). RT-PCR analysis showed that RXRγ methylation correlates with mRNA expression. Methylation of the RXRγ gene was not significantly associated with the prognosis of patients; however, when the patients are categorized by smoking status, the effect of RXRγ methylation on prognosis was significantly different between never- and ever-smokers (P = 0.003 [test for homogeneity]); specifically, RXRγ methylation was associated with a significantly worse survival in never-smokers, whereas it exhibited a better survival outcome in ever-smokers, although not statistically significant. This finding suggests that methylation-associated down-regulation of the RXRγ gene may play a differential role in the carcinogenesis of NSCLCs according to smoking status, but further studies are needed to confirm this.

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 4935.
Abstract 4948: Silencing of Hint1, a novel tumor suppressor gene, by promoter hypermethylation in human breast cancer
Authors: Zhang Yu-jing , Li Haiyang , Joe Andrew K. , Hibshoosh Hanina H. , Weinstein Bernard , Santella Regina M.
Published: February 2011

The HINT1 protein, a member of the histidine triad (HIT) family, is highly conserved in diverse species and ubiquitously expressed in mammalian tissues. Hint1 has been proven a haploinsufficient suppressor gene. In previous studies, it was demonstrated that promoter hypermethylation of Hint1 may play a role in the development of colon cancer and hepatocellular carcinoma (HCC). In the present study, we investigated the aberrant methylation of the Hint1 promoter and explored possible relationships between aberrant methylation and clinicopathological features and other parameters in breast cancer. Hypermethylation of Hint1 was evaluated by methylation specific PCR (MSP) in 81 breast cancer samples, of which 17 cases (21%) displayed Hint1 promoter hypermethylation. A statistically significant inverse correlation was found between Hint1 promoter hypermethylation and p27Cip1/Kip1 expression. This is consistent with our previous finding that HINT1 up-regulates cellular levels of p27Cip1/Kip1. No significant correlation was found between the Hint1 promoter methylation status and the expression of p53, p21, estrogen receptor (ER), progesterone receptor (PR) and HER2/neu. Taken together, these results suggest that promoter hypermethylation of Hint1 may contribute to carcinogenesis of breast cancer.

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 4948.
Abstract 495: Inhibition of the prolyl isomerase Pin1 reduces CD133 and Sox2 expression and enhances radiosensitivity in glioblastoma cells
Authors: Chen Edmund I. T. , Huang Yu-Wen , Lai Li-Chuan
Published: February 2011

Pin1, a prolyl isomerase, specifically recognizes the phosphorylated Ser/Thr-Pro motifs and induces cis/trans isomerization of its target proteins. Such conformational changes have profound effects on the function of Pin1 substrates. Pin1 has been shown to be overexpressed in many human cancers, and Pin1 over-expression is often associated with up-regulation of cyclin D1 and β-catenin. Here we report that, in cell culture studies, enhanced Pin expression occurs in radiation-resistant glioblastoma cells (GBM-R) or CD133-expressing glioblastma cells (S1) compared with controls. Ectopic expression of Pin1 but not mutated Pin1(K63A) increased the viability of GBM cells to IR.CD133-expressing glioma cells are known to play a key role in tumor recovery after treatment and are resistance to radiotherapy. Here we showed that Pin1 knock-down in GBM-R or GBM-CD133 (+) cells enhanced their sensitivities to radiation or anti-GBM drug TMZ. Moreover, Pin1 knockdown increased apoptopic population in GBM-CD133 (+) cells. It appeared that Pin1 knockdown reduced the ability of S1 cells to repair double-stranded breaks. Interestingly, Pin1 knockdown decreases the expression of stemness genes including CD133, Sox-2, Bmi1 and c-Myc etc., and silencing of Pin1 can interfere with sphere formation in S1 cells. Our results suggest that Pin1 is an important regulator of the resistance of GBM-R or GBM-CD133 (+) to radiotherapy.

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 495.
Abstract 496: Enhance efficacy of photodynamic therapy in combination with selenium in TC-1 animal model
Authors: Oh Eun Kyung , Wen Lanying , Bae Sumi , Lee Sung Ha , Cho Young Lae , Ahn Woong Shick
Published: February 2011

Photodynamic therapy (PDT) is a promising cancer treatment modality that involves the interaction of the photosensitizer, molecular oxygen and light of specific wavelength to destroy tumor cells. To improve efficacy of PDT, the efforts are underway to develop combination protocols. Selenium is an essential trace element and has anticarcinogenic properties on a various human cancer model. The aim of this study was to investigate the synergetic anti-tumor effects of PDT plus selenium in vitro and in vivo.

To compare the anti-proliferative effects by PDT, Selenium, or PDT plus selenium, MTT assay, microscopic observation and FACS analysis were performed. Apoptosis signaling pathways were analyzed using mouse signal transduction pathwayRT profiler PCR array (Superarray). For the vivo study, when tumor size was approximately 8 to 10 mm, Radachlorin (10mg/kg b.w.) was injected in to the tail vein at 3 hrs before irradiation with 100 J/cm2 of light and then selenium (1ug/kg b.w) was administrated daily during 20 days.

Our results demonstrate that combination of selenium with PDT strongly inhibits growth of TC-1 cell and tumor in TC-1 cell implanted mice when compared to the other groups. Moreover, PDT and selenium treatment in TC-1 cells had apoptosis features in microscope and FACS analysis. Increased apoptosis was associated with tumor inhibition in the combination therapy group. In signal transduction pathway Superarray, genes closely relate to NFkB, p53, and phopholipase C pathway such as vcam1, mdm2, and fos were very significantly down-regulated at least 10 fold in TC-1 cell following co-treatment of PDT and selenium. Furthermore, anti-apoptosis related genes and tumor-promoting genes, birc2, birc3, hk2, ccl2, ptgs2, wisp1, mmp10 and wisp1 were also significantly down-regulated. Up-regulated genes by co-treatment of PDT and selenium had relatively low ranges of fold change than fold range of down-regulated genes and were related with cell survival and proliferation mechanism.

These data indicate that combination therapy of selenium and PDT could more effectively induce tumor suppression response compared to PDT or Selenium alone. We suggest that combination therapy using PDT and selenium can be a useful to for inducing synergistic anticancer effect.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

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 496.
Abstract 4965: The epithelial-specific ETS transcription factor ESE1 links inflammation with prostate cancer transformation and progression
Authors: Longoni Nicole , Ortelli Erica , Kunderfranco Paolo , Genini Davide , Grand Maurizia Mello , Chiorino Giovanna , Malek Anastasia , Catapano Carlo V. , Carbone Giuseppina M.
Published: February 2011

Altered expression of ETS transcription factors by chromosomal translocation is a frequent event in prostate cancers. However, deregulated expression of other oncogenic ETS factors can be important for the pathogenesis of this disease. By analyzing the expression of multiple ETS genes in prostate cancer and normal prostate by genomic profiling and quantitative real-time PCR we found that the epithelial-specific ETS factor ESE1 was highly expressed (>4 fold) in 44% of prostate tumors compared to normal prostate. ESE1 has been previously shown to be induced by pro-inflammatory stimuli and to regulate the expression of genes involved in inflammation. Treatment of immortalized prostate epithelial cells (LHS) and prostate cancer cells 22RV1 with the pro-inflammatory cytokine IL-1beta induced ESE1 at RNA and protein levels. Concomitantly we observed that cells acquired ability to grow in poly-hema coated plates and an EMT phenotype. To define the role of ESE1 in prostate cancer progression we established 22RV1 prostate cancer cells stably expressing ESE1 (22RV1-ESE1). ESE1 expressing cells were capable to grow in poly-hema coated plates and when injected in nude mice formed larger subcutaneous tumors compared to control cells. Global gene expression profiles indicate robust changes with many genes differentially expressed in 22RV1-ESE1 cells compared to control cells, including key genes involved in cell invasion and metastasis such as COX2 and MMP-10. Consistently, 22RV1-ESE1 cells had greater ability to migrate compared to parental cells in Boyden Chamber and wound-healing assays. Furthermore, when implanted in mice via tail vein injection, 22RV1-ESE1 cells displayed greater ability to form lung metastasis compared to control cells. These results suggest that ESE1 contribute to the acquisition of transforming properties such as the ability to invade and metastasize. Induction of ESE1 during chronic inflammation could be an essential step for prostate cancer initiation and may co-operate with other ETS alterations for prostate cancer progression.

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 4965.
Abstract 4981: ICI182,780 induces P-cadherin upregulation in breast cancer cells through histone modifications at the promoter level: The role of C/EBPbeta in CDH3 gene activation
Authors: Albergaria André , Ribeiro Ana Sofia , Pinho Sandra , Milanezi Fernanda , Carneiro Vítor , Sousa Bárbara , Sousa Sónia , Oliveira Carla , Machado José Carlos , Seruca Raquel , Paredes Joana , Schmitt Fernando
Published: February 2011

P-cadherin is a classical cadherin which has been extensively studied concerning its functions and its prognostic value in breast cancer. Moderate to intense expression of P-cadherin has been identified in 30% - 50% of invasive ductal carcinomas of the breast, being reported to be strongly associated with proliferative and high histological grade, decreased patient survival and as a marker of the aggressive behaviour of basal-like and HER-2 tumour subtypes. In vitro, P-cadherin overexpression is also able to induce increased cell invasion and migration. However, only a few works have been published in order to decipher the regulatory mechanisms behind P-cadherin overexpression in breast cancer. P-cadherin is up-regulated by the antiestrogen ICI 182,780, suggesting that the lack of ERalpha signalling may be responsible for the aberrant P-cadherin expression in breast cancer, but the mechanisms underlying this ICI-induced aggressive behaviour are completely unknown. The aim of this study was to determine whether the induction of the CDH3 gene is a direct effect of ICI 182,780 at CDH3 promoter level or would require the prior induction of other genes/proteins or epigenetic mechanisms.

We found that ICI 182,780 increases CDH3 promoter activity as well as induces an epigenetic mechanism that lead to activating histone modifications at putative C/EBPbeta binding sites in at the CDH3 promoter. We also demonstrate that this transcription factor is able to directly activate P-cadherin transcription in breast cancer cells and that these two proteins are highly associated in human breast carcinomas, being both significantly related with high grade and proliferative invasive breast carcinomas. This study brings forward the characterization of putative intracellular pathways and epigenetic mechanisms that are able to induce P-cadherin overexpression in breast cancer cells and, in general, contribute to clarify the ability of ICI 182,780 to induce expression of signal transduction genes normally repressed by oestrogen/ER signalling. In addition, we showed for the first time that the transcription factor C/EBPbeta is able to regulate P-cadherin overexpression in breast cancer.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

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 4981.
Abstract 4985: Identification of PARP-1 as a novel KLF8 interacting and regulating protein
Authors: Lu Heng , Wang Xianhui , Urvalek Alison M. , Li Tianshu , Yu Lin , Zhu Jinhua , Lin Qishan , Zhao Jihe
Published: February 2011

KLF8 transcription factor regulates important genes including cyclin D1 and E-cadherin and plays a critical role in cell cycle progression, oncogenic transformation and EMT. Transcription factors need to interact with both DNA and other proteins in order to regulate transcription. However, little is known about KLF8-protein interaction. Using mass spectrometry and co-immunoprecipitation (co-IP), we identified PARP-1, a chromatin-associated enzyme that catalyzes protein poly(ADP-ribosyl)ation (PARylation), as one of novel proteins that interact with KLF8. Co-IP and western blotting indicated that KLF8 is a PARylation substrate. Mutation of the cystein residues in the first and second zinc-finger motifs of KLF8 abolishes its interation with PARP1. Luciferase assays showed KLF8 activation on the cyclin D1 promoter was significantly reduced when PARP1 was physically deleted or functionally inhibited or when KLF8-PARP-1 interation was disrupted. Surprisingly, immunofluorescent staining demonstrated that KLF8 was localized in the nuclei of the wild-type mouse embryonic fibroblasts (MEF), but was mislocalized in the perinuclear cytoplasm of the PARP-1−/− MEFs, which could be rescued by re-expression of PARP1. Interestingly, while the PARP1-interation deficient KLF8 mutant was also mislocalized in the cytoplasm of the wild-type MEFs, inhibition of PARP1 activity in the MEFs did not affect the nuclear localization of wild-type KLF8. Cychloheximade (CHX)-chase assay revealed the stability of KLF8 dramatically reduced by loss of PARP-1 or when KLF8-PARP-1 interaction was disrupted. Ubiquitination assay showed heavier ubiquitin chains in PARP-1−/− MEFs or when KLF8-PARP-1 interaction was disrupted. Furthermore, DNA damage assay under Doxorubin treatment indicated KLF8 involved in PARP-1-dependent DNA damage repair pathway. Those results suggest that PARP-1 work as a co-activator in KLF8's transcriptional activity on Cyclin D1 promoter, keep KLF8's nuclear localization, inhibit KLF8's ubiquitination and contribute to KLF8's stability. This work has identified PARP1 as a novel binding partner and post-translational modifier of KLF8 and a critical regulator of KLF8 localization, function and stability in the cells.

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 4985.