bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4881: Polycomb protein EZH2 activates Wnt/β-catenin signaling to promote hepatocellular carcinoma development
Authors: Cheng Alfred S. , Lau Suki , Chen Yangchao , Kondo Yutaka , Li May , Feng Hai , Ching Arthur , Cheung Paul , Wong Kenneth , Jin Hongchuan , Choy Richard , Yu Jun , To Ka F. , Wong Nathalie , Huang Tim , Sung Joseph J.
Published: February 2011

Aberrant activation of the canonical Wnt pathway due to accumulation of

β-catenin occurs in approximately 70% of hepatocellular carcinomas (HCCs) and contributes to their initiation, development and progression. Genetic mutations in the components of this pathway account for only a subset of HCCs with β-catenin accumulation, suggesting that an alternative mechanism for Wnt/β-catenin activation is prevailing. Enhancer of zeste homolog 2 (EZH2) is known to promote tumorigenesis by down-regulating tumor-suppressor genes; however, whether EZH2 regulates oncogenic pathways is unclear. Using chromatin immunoprecipitation microarray, we uncovered 12 Wnt/β-catenin signal antagonists whose promoters were concordantly occupied by EZH2 and repressive histone modifications in hepatocellular HCC cells. EZH2 over-expressed in 42% (75/179) of human HCCs and significantly associated with β-catenin accumulation. Concomitant inhibition of EZH2 and histone deacetylase transcriptionally activated Wnt/β-catenin signal antagonists, suppressed T-cell factor-dependent transcriptional activity and down-regulated β-catenin transcriptional targets in HCC cells. Conversely, ectopic EZH2 over-expression in nontumorigenic hepatocytes increased cellular proliferation in a β-catenin-dependent manner and promoted tumorigenicity. This study uncovers an EZH2-mediated epigenetic mechanism that leads to Wnt/β-catenin signaling dyregulation in human HCC and suggests that therapeutic interventions targeting EZH2 may confer a clinical benefit in β-catenin-driven malignancies.

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 4881.
Abstract 4883: Differences in epigenetic patterns of HOX genes in normal lung and Non-small cell lung cancer
Authors: Lee Borum , Chun Sung-Min , Jin Min-Young , Jang Se Jin
Published: February 2011

HOX genes have determinative roles in cell fate during embryogenesis and adult cellular differentiation. Their dysregulation has been implicated in many human cancers. Loss of or de novo expression of the genes by methylation abnormality are also reported in non-small cell lung cancer (NSCLC). In this study, we screened the expression profiles of the HOX D genes in normal lung tissues, a bronchial epithelial cell line, 9 NSCLC cell lines and 10 normal-tumor pairs of NSCLC tissues and compared epigenetic patterns of the genes by Quantitative Real-Time PCR, Dual-Specific Bisulfite PCR (DSBP) and ChIP-on-chip assay. We showed frequent cancer specific de novo expression of HOX D9, D11, and D13 genes in NSCLC tissues and cells in contrast to normal lung tissues. In methylation assay, unexpectedly, the genes are frequently hypermethylated in cancerous samples but show low level of methylation in normal tissues. In ChIP-on-chip assay, we observed that normal tissues show repressive or bivalent histone modification pattern characterized by high level of H3K27me3 and low level of H4K20me3 and H4K16ac at the up-stream region of the genes. In contrast, cancer tissues showed permissive or active histone modification patterns at the region characterized by low level of H3K27me3 and high level of H4K20me3 and H4K16ac. The results suggest that normal epigenetic state of the genes in lung tissue could be defined as bivalent histone modification pattern, no DNA methylation and no gene expression. In lung cancer, epigenetic state of the genes are dysregulated to permissive histone modification pattern, DNA hypermethylation and de novo expression of the genes. (This study was supported by a grant of the Korea Healthcare technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea. (A084921), This study was supported by a grant of the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea. (A062254))

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 4883.
Abstract 4884: Polyamine analogues in combination with DFMO inhibits lysine-specific demethylase 1 and induces re-expression of aberrantly silenced genes
Authors: Wu Yu , Marton Laurence J. , Woster Patrick M. , Casero Robert A.
Published: February 2011

Epigenetic gene silencing is an important mechanism for the loss of gene expression in cancer. Abnormal DNA CpG island hypermethylation and altered patterns of histone modifications are involved in aberrant silencing of tumor suppressor gene. The flavin-dependent lysine-specific demethylase 1 (LSD1) is the first enzyme identified to specifically demethylate Lysine 4 of histone H3. Methylation of H3K4 is an important mark associated with active chromatin transcription. The FAD-binding amine oxidase domain of LSD1 has considerable sequence homology to two polyamine oxidases SMO (spermine oxidase) and APAO (acetylpolyamine oxidase). Our laboratory has identified long chain polyamine analogues, named oligoamines, act as inhibitors of LSD1. Here we report the synergistic effects of specific oligoamines in combination treatment with 2-difluoromethylornithine (DFMO) in human colorectal cancer cells. DFMO is an inhibitor of ornithine decarboxylase (ODC), which is a rate-limiting enzyme to generate putrescine. Reducing the level of putrescine leads to accumulation of decarboxylated S-adenosylmethionine (dcSAM) and could subsequently result in alteration of DNA methyltransferase activity and changes in DNA methylation. In addition, DFMO treatment is known to increases uptake of circulating polyamines. Exposure of colorectal tumor cells to oligoamines and DFMO results in a synergistic global increase of H3K4me2 and induction of re-expression of aberrantly silenced genes including the secreted frizzled-related protein 2 (SFRP2) gene, which encodes Wnt signaling pathway antagonist and plays an anti-tumorigenesis role in colorectal cancer. Chromatin immunoprecipitation analysis indicates that the re-expression of SFRP2 is associated with increased H3K4me2 active marks at the gene promoter. The combination of LSD1-inhibiting oligoamines and DFMO represents a highly valuable and novel approach to epigenetic therapy of 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 4884.
Abstract 4887: Genetic analyses of chromatin remodelers in cancer
Authors: Riudalbas Laia S. , Melo Sónia A. , Mestres Anna Portela , Esteller Manel
Published: February 2011

Genetic analyses of chromatin remodelers in cancer

Laia S. Riudalbas1, Sónia A. Melo, Anna Portela & Manel Esteller.

1Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), 08907 L'Hospitalet, Barcelona, Catalonia, Spain

Chromatin remodelers are ATP dependent proteins that enable dynamic access to packaged DNA and tailor nucleosome composition in chromosomal regions. These protein complexes have been shown to regulate a wide range of cellular processes, including transcription regulation, DNA-damage response and DNA replication. Being that some components of this remodeler machinery were already characterized as tumor suppressor genes and/or oncogenic, we can hypothesize that the dysregulation of any of these molecules in tumor cells with microsatellite instability (MSI+), that show a higher rate of insertions and deletions, can lead to neoplastic transformation. For that we decided to screen all the exonic mononucleotide repeats of some of the most important chromatin remodelers with ATPase activity in colorectal, gastric and endometrial cancer cell lines, on the search for frameshift mutations that could alter protein function. We have mainly analyzed the catalytic subunits of remodeling complexes because any alteration of these crucial proteins could drive into a tumorigenic process. Until now no alteration was found in the cancer cell lines studied. This could be underscoring the importance of these genes to normal cellular functioning being that their disruption could impair cell viability.

This work was supported by Grants SAF2007-00027-65134, FIS PI08-0517, Consolider CSD2006-49, and CANCERDIP FP7-200620. L.S.R. is a research fellow of FIS - Instituto de Salud Carlos III, FI09/00218.

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 4887.
Abstract 4888: The effect of chromatin structure in regulation of telomere length in telomerase negative ALT cells
Authors: Jung A Ra , Kim Hye Ryun , Oh Bong-Kyeong , Jeung Hei-Cheul , Chung Hyun Cheol , Rha Sun Young
Published: February 2011

Telomeres are DNA-protein complexes at the ends of linear eukaryotic chromosomes and composed of tandem repeats of a TTAGGG sequence ending in a 3′ single-stranded overhang, or the G-strand overhang. And telomeres confer chromosomal stability protecting chromosome ends. Most immortalized human cell lines maintain and extend their telomere by activating telomerase. A small number of cells maintain and extend their telomere lengths without telomerase by homologous recombination of telomeres and this mechanism is called as alternative lengthening of telomere (ALT). There are many factors involved in telomere regulation. Recently, it has been known that chromatin structure is closely related to regulation of telomere length. Maintaining telomere length is important process in tumorigenesis and it has been suggested that telomeres are important target for cancer therapy. However, in ALT cells the effect of chromatin structure on regulation of telomere length is not understood. In this study, we induced change of chromatin structure by treating Trichostatin A (TSA), Histone deacetylase inhibitor, in ALT cells. Then, we measured APBs (ALT associated PML bodies) patterns, T-SCE (Telomere Sister Chromatid Exchange) frequency and telomere lengths. APBs patterns were analyzed by FISH (Fluorescence in situ hybridization) combined IF (Immunofluorescence staining). We compared the number of APBs in ALT cells with or without TSA. In GM 847 (ALT cell), APBs positive cells were found to be more frequent in GM 847 with TSA (63/100) compared to GM 847 without TSA (30/100) (p<0.001). A similar result was found in other ALT cell line, WI38-VA13. APB positive cells were more frequent in WI38-VA13 with TSA (38/100) compare to WI38-VA13 without TSA (18/100), (p<0.001). T-SCE frequency was determined by CO-FISH (Chromosome Orientation-FISH). Increse of T-SCE frequency was found in GM 847 with TSA (262/1026, 25.5%) compared to GM 847 without TSA (215/1026, 20.9%), (p<0.001). Telomere length was determined by telomere-FISH. Decrease of telomere length was found in GM 847 with TSA (telomere intensity mean 25.11%) compared to GM 847 without TSA (telomere intensity mean 29.8%), (p<0.001). This study demonstrated a close association between chromatin structure and telomere recombination in ALT cells and this result might provide insight into the mechanism of regulation of telomere length in telomerase-negative 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 4888.
Abstract 4893: EGFR signaling controls the chromatin binding status of BMI1 through a CK2/PP1 phosphorylation/dephosphorylation pathway and affects astrocytoma proliferation and neural stem cell differentiation
Authors: Hawes Jessica , Tuskan Robert , Akagi Keiko , Reilly Karlyne
Published: February 2011

Studies of brain tumors demonstrate the importance of a rare tumor cell population with the capability of regenerating tumors upon transplantation. These cancer stem cells (CSC) are believed to be more resistant to therapy than the bulk of brain tumor cells. Studies in mouse brain tumor models demonstrate that neural stem cells (NSC) can be an important initiating cell type for brain tumors, but it is unclear what is the relationship between NSC and CSC. EGF is a critical growth factor for NSC maintenance in culture and EGFR signaling is upregulated in glioblastoma. Similarly, the chromatin modulator BMI1 is necessary for NSC maintenance, and has been implicated in glioblastoma. We present studies linking cell surface EGF signaling and nuclear BMI1 chromatin binding. We demonstrate that BMI1 is specifically phosphorylated by EGFR signaling using EGF and TGFα, but not by many other oncogenic signaling ligands tested. We have identified three amino acids encompassing the phosphorylation site in response to EGFR signaling, and show that CK2 and PP1 are necessary for control of BMI1 phosphorylation. This phosphorylation alters the chromatin binding state of BMI1 and transcriptional regulation in astrocytoma cells. Interestingly we find that BMI1 can alter transcriptional profiles in both EGF-dependent and independent ways, suggesting that different signaling pathways could have specific effects on changing the BMI1-dependent epigenetic state of subsets of genes. Furthermore, EGF-induced phosphorylation of BMI1 is important for astrocytoma growth in vitro and in vivo, as well as maintenance of NSC.

Our data link signals outside the cell to the nucleus important for NSC maintenance and astrocytoma proliferation. These data represent a common pathway in NSC and tumor cells that may explain the importance of EGF signaling for NSC maintenance and gliomagenesis, and suggest a novel downstream pathway (CK2/PP1) involved in these processes.

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 4893.
Abstract 4896: The detection and analysis of epigenetic and genetic changes in ovarian tumor tissue
Authors: Poulin Matthew , Xu Jessica , Yan Liying
Published: February 2011

Human ovarian cancer is the fifth leading cause of cancer-related deaths. Most ovarian cancers are ovarian epithelial carcinomas or malignant germ cell tumors. Similar to other cancers, the early diagnosis of ovarian cancer is critical for treatment of the disease and the better chance for recovery. If diagnosed and treated early when the cancer is confined to the ovary, the 5-year survival rate is over 90%. Many studies involving various types of human cancers proved and aberrant miRNA expression occurs in many cancers, even at the early stage of the diseases, indicating its causative role. We have analyzed 10 ovarian tumors and their associated normal tissue, and two ovarian cancer cell lines, with methylation assays for over 25 different cancer related genes (p16, ESR, BRCA1, Rassf1, RAR beta, ECAD, etc.) including several miRNA genes. Global methylation assays show the ovarian tumors to be hypomethylated compared to the corresponding normal tissue. Several of the miRNA genes we have analyzed in ovarian tumor tissue show statistically changes in methylation pattern within their promoters compared with adjacent normal tissue. ANOVA analysis shows a variance with a p value of 0.00001. Expression analysis will be performed to correlate with the methylation data. Additionally, an extensive SNP analysis will be conducted on these tissues in order to establish specific screening parameters that will aid in early detection of this disease.

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 4896.
Abstract 4907: Methylated cell-free DNA in plasma as an early biomarker of pancreatic cancer
Authors: Mangold Kathy A. , Amick Ashley , Wang Vivien , Regner MaryAnn , Krafft Catherine , Du Hongyan , Brand Randall E. , Kaul Karen L.
Published: February 2011

Background: Presently there is an urgent need for better biomarkers for early pancreatic cancer (PaCa) diagnosis. Hypermethylation of several genes is known to correlate with presence of neoplasia in pancreatic tissue, and tumor DNA is known to be present in bodily fluids. With this background, we wanted to develop and validate a panel of methylated gene markers that would provide clinically relevant information when applied to plasma from those undergoing diagnostic procedures for pancreatic cancer.

Methods: Cell-free DNA was extracted from plasma obtained from 11 PaCa patients, 15 pancreatitis patients and 15 unaffected age-matched controls using either the automated EZ-1 (Qiagen, Valencia, CA) or the manual Charge Switch (Invitrogen, Carlsbad, CA) methods. The DNA was bisulfite treated using EZ DNA Methylation-Gold (Zymo Research, Orange, CA) then analyzed using a panel of quantitative real-time PCR assays to discriminate the methylated C or T that exists after bisulfite conversion, and normalizing the signal to the amplification level of a housekeeping gene (Col2A) to assess methylation of status of 13 genes known to be differentially methylated in pancreatic tissues. Several different combinations of 6 methylated gene markers (3-OST-2, ARF/p14, FOXE1, CDKN2/p16, ppENK, and RASSF1A) showed good discrimination between the 3 groups of patients to warrant further study with an additional 53 plasma specimens (from 21 PaCa patients, 17 pancreatitis patients, and 15 unaffected controls). Receiver operator curves were generated to examine the predictive probability between marker methylation and disease, and reliability of the regression model was evaluated by using the Hosmer-Leme show goodness-of-fit test.

Results: Statistical analysis of the 6 methylated gene markers for all 94 plasma specimens yielded an AUC of 0.638 (95% CI: 0.50-0.78) to differentiate PaCa from pancreatitis, and the goodness-of-fit for the model was good (p=0.697).

Conclusions: Cell-free DNA extracted from plasma is an appropriate sample for the study of gene methylation in PaCa. We utilized real-time assays to detect methylation in 6 target genes and found a good correlation between the methylation status of genes and the presence of PaCa. Further studies are needed to define the clinical utility of this approach. Funding was provided by a grant from the American Cancer Society - Illinois Division (#07-06) and by funds from the Department of Pathology and Laboratory Medicine at NorthShore.

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 4907.
Abstract 4908: Detection of bladder tumor by quantitative methylation specific real time PCR from urine sediment DNA
Authors: Chung Woonbok , Bondaruk Jolanta , Kim Daniel D. , Czerniak Bogdan , Issa Jean-Pierre J.
Published: February 2011

Bladder cancer is the fifth most frequent human cancer and causes approximately 3% of all cancer-related deaths. DNA hypermethylation is a common epigenetic abnormality in cancer and may serve as a useful marker to clone new cancer-related genes as well as a marker of the disease activity. Recently, we developed a high throughput genome wide methylation assay, Methylated CpG Island Amplification Microarray (MCAM). Using MCAM, we investigated methylation changes in about 8,000 genes in bladder tumors. Through this screening method and validation by bisulfite pyrosequencing, we have developed novel biomarkers for diagnosis and prognosis of bladder tumor. We characterized a group of genes that was found to be methylated in 80-90% of the tested bladder tumor samples. These candidate genes (DBC1, MYO3A, SOX11, SLC16A12, NPTX2, NKX6-2, A2BP1, PENK and CA10) were used to screen for bladder cancer in urine samples by quantitative methylation specific real time PCR, thus representing a non-invasive method for early detection. Based on these data, we established a panel of 6 biomarkers (MYO3A, SOX11, NKX6-2, PENK, CA10 and quantity of DNA in test urine) that can be used for bladder tumor screening in urine samples. The method showed 89.1 % sensitivity and 91.4 % specificity for detection of bladder tumor urines (n=128) compared to normal control urine (n=105) by 3 or more than 3 positive out of 6 biomarkers. Analyzing by stage of bladder tumors, 5 out of 5 (100 %) in Tis, 21 out of 29 (72 %) in Ta, 18 out of 19 (95 %) in T1, 57 out of 60 (95 %) in T2, 6 out of 6 (100 %) in T3 and 2 out of 2 (100 %) in T4 showed 3 or more than 3 positive out of 6 biomarkers. Thus, 83% of Tis-Ta-T1 stages and 95.6 % of T2-T3-T4 stages were detected in analyzed bladder tumor urines. This DNA methylation biomarkers panel will help the early detection and monitoring of bladder tumor 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 4908.
Abstract 4909: The role of CpG island methylator phenotype 2 (CIMP2) in early onset colorectal cancer patients
Authors: Li Carmen , Yuen Siu Tsan , Anthony  , Chan Kin Wang , Tsui Wendy Wai Yin , Chan Annie Suk Yee , Leung Suet Yi , Chan Tsun Leung
Published: February 2011

CpG Island Methylator Phenotype (CIMP), characterized by methylation at various promoter sites, has been reported as a subgroup in colorectal cancer. CIMP1 is often associated with microsatellite instability (MSI) as well as BRAF mutation while CIMP negative enriched with P53 mutant. Besides, a CIMP2 classification has also been established and indicated to be associated with KRAS mutation. Most of these experiments related to CIMP were carried out on tumor samples from late onset patients with a mean or median age of at least 60. Thus it was of interest to observe any deviation of such pattern among early onset colorectal cancer population. Bisulfite conversion of the extracted DNA from 73 tumor samples with MSS status were examined via pyrosequence for their methylation status among loci MINT1, MINT2 and MINT27, the most frequently methylated sites reported in CIMP 2 phenomenon. These samples were free from MLH1 methylation and BRAF mutation to exclude conditions of CIMP1. The age group ranged from 29 to 68 years old with a mean age of 50.7 years old. Sequencing of its genomic DNA for KRas mutation was also screened as per se to previous studies. It was found that CIMP2 appeared more frequently in patients over the age of 50 (p=0.04) when compared with patients below 50 years old. Furthermore, methylation of the CIMP 2 loci examined (MINT1, MINT2 and MINT27) were most likely accompanied with the mutation of KRas in patients over 50 years old (p=0.05) while there was no observed correlation of CIMP 2 loci and KRas mutation in patient samples under 50 years of age. Since the CIMP2 phenotype is not seen in early-onset CRC samples, our data suggests that an alternative pathway, but not CIMP, may be responsible for the tumorigenesis of early-onset MSS CRC. Moreover, current studies proposed chromosomal instability may be a distinct mechanism in the pathogenesis of CRC, thus investigation would be carried on this aspect.

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 4909.
Abstract 4910: Identification of putative epigenetic markers for head and neck squamous cell carinoma recurrence
Authors: Rettori Marianna M. , de Carvalho Ana Carolina , Maia Danielle C. C. , Carvalho Andre L. , Kowalski Luiz Paulo , Vettore Andre L.
Published: February 2011

Head and neck squamous cell carcinoma (HNSCC) is one of the most frequent types of cancer but the lack of early detection and high incidence of local-regional recurrence lead to high mortality. Therefore, it is important to develop new molecular markers to be used as diagnostic and prognostic indicators. An important mechanism for gene transcriptional inactivation is hypermethylation at the CpG islands within the promoter regions. The presence of hypermethylation might be useful as a molecular marker for tumor cell detection which could be useful for early detection of tumor recurrence. The aim of this study was to evaluate promoter hypermethylation of suppressor genes via quantitative methylation-specific PCR (Q-MSP) in tumor and body fluid samples (saliva) as a means to detect epigenetic alterations specific to HNSCC. Tumor samples and salivary rinse were collected before and after the last curative treatment from 180 consecutive HNSCC cases and every 6 months during the patient follow-up. The pattern of aberrant methylation of 24 genes was analyzed and allowed the selection of a panel of 5 genes that have a specificity of 95% and a sensitivity of 38% in the detection of hypermethylated cells in saliva samples from recently diagnosed HNSCC patients. The efficacy of this panel may be useful in early diagnosis of HNSCC recurrence is being evaluated.

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 4910.
Abstract 4916: MAL, a frequently methylated gene marker for early detection of breast cancer
Authors: Hillocks Yolanda M.N. , Lo Pang-Kuo , Teo Wei Wen , Fackler Mary Jo , Sukumar Saraswati
Published: February 2011

Hypermethylation of CpG islands in the promoter region of tumor suppressor genes is a common event in cancer, and often results in transcriptional silencing. Computational searches of existing large databases for genes underexpressed in multiple cancers, combined with CpG island analysis of those genes, identified a novel methylation marker gene in breast cancer called MAL (Maturation-Associated protein for T-Lymphocyte). MAL encodes a 17kD lipid raft protein that is found in mature T-cells and participates in T-cell differentiation and T-cell receptor signaling. Two previous studies have reported gene promoter hypermethylation of MAL in primary breast tumors and breast cancer cell lines. Furthermore, Horne et al found MAL inactivation is predictive of benefit from adjuvant chemotherapy. We propose that alteration of MAL expression is functionally important in breast cancer. In addition, we propose that MAL has the potential to serve as a serum methylation marker for early detection of breast cancer. MAL gene methylation as assessed in breast cancer cell lines and primary tumors by quantitative multiplex methylation specific PCR (QM-MSP) showed that it is frequently methylated in cell lines (40/48; 83%), in primary tumors (11/20; 55%) and in distant metastasis (18/35; 51%), but not in normal breast tissues. Hypermethylation of MAL resulted in its decreased expression in the majority of the samples tested. Treatment of breast cancer cell lines with demethylating agents and histone deacetylase inhibitors restored MAL expression, thus establishing epigenetic alterations as the major mechanism of silencing. The function of MAL is being deciphered at both the biological and biochemical level by performing overexpression and knockdown studies in breast cells using already constructed expression vectors. The characterization and validation of MAL, a lipid raft protein, as a potential DNA methylation marker in breast cancer, particularly in serum, could provide a valuable early detection and prediction tool for 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 4916.
Abstract 492: Esophageal administration of manganese superoxide dismutase plasmid liposomes (MnSOD-PL) reverses thoracic irradiation sensitivity of nitric oxide synthase one homologous recombinant negative (NOS1 -/-) mouse
Authors: Epperly Michael W. , Rajagopalan Malolan S. , Rwigema Jean-Claude , Stone Brandon , Goff Julie , Dixon Tracy M. , Franicola Darcy , Greenberger Joel S.
Published: February 2011

NOS1 -/- mice are more sensitive to 29 Gy thoracic or 9.5 Gy total body irradiation (TBI) than control C57BL/6NHsd, or NOS2 -/- or NOS3 -/- mice (p < 0.0001 and p = 0.0006). Histological examination of the esophagus, lungs, heart and intestine revealed no explanation of the rapid death of NOS1 -/- mice. Blood and bone marrow isolates six days after TBI (1 day before the NOS1 -/- mouse death) showed similar decrease in blood counts for all the mouse strains. NOS1 -/- mouse marrow irradiated to doses ranging from 0 to 8 Gy showed CFU-GM, BFU-E and CFU-GEMM radiosensitivity similar to that from control mice. There were also no differences in serum electrolytes, liver or renal function tests in irradiated NOS1 -/- compared to C57BL/6NHsd mice. RNA extracted from esophagus, stomach or intestine at day 0, 1 or 6 following 9.5 total body irradiation showed by qualitative RT-PCR analysis of TNF-σ, NF-κβ, and INF-γ similar cytokine expression between NOS1 -/- and C57BL/6NHsd mice. NOS1 -/- and C57BL/6NHsd mice were irradiated to 9.5 Gy total body or 20 Gy to the thoracic cavity and observed by video camera. NOS1 -/- mice had seizures before death following both TBI or thoracic irradiation, while no seizures were detected in dying C57BL/6NHsd mice. NOS1 -/- mice irradiated to 9.5 Gy to the brain (with the remainder of the body shielded) did not produce death from seizures. NOS1 -/- were intraesophageally administered MnSOD-PL (100 µg plasmid DNA) and irradiated to 20 Gy to the upper body 24 hr later. NOS1 -/- mice administered MnSOD-PL had normalized survival compared to control irradiated NOS1 -/- mice: 50% survival at 12 days for irradiated control NOS1 -/- mice compared to 128 days for MnSOD-PL treated NOS1 -/- mice (p = 0.0311). The data indicate that esophageal neuronal abnormalities which cause pyloric stenosis in the strain may be responsible for both the rapid thoracic irradiation sensitivity leading to seizure and its reversal by intraesophageal MnSOD-PL gene therapy.

Supported by NIAID grant U19AI068021, and NIH grant T32AG21885

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 492.
Abstract 4920: Effects of PMEPA1 in prostate tumor cell growth and tumor formation
Authors: Li Hua , Li Hongyun , Raymundo Eliza , Thangapazham Rajesh , Sreenath Taduru , Dobi Albert , Srivastava Shiv
Published: February 2011

Introduction: PMEPA1 is an androgen-induced gene. We have shown that androgen receptor transcriptionally activates PMEPA1 and PMEPA1 negatively regulates androgen receptor by recruiting the E3 ubiquitinatin ligase NEDD4 to AR, therefore promoting proteasome-dependent AR degradation, thus, forming a remarkably tight feedback loop between AR and PMEPA1. Moreover, PMEPA1 mRNA was found in decreased amounts in prostate carcinoma. Previous data in cell culture models of prostate cancer have shown that DNA Methylation of the PMEPA1 gene may play a role in decreased PMEPA1 expression in human prostate cancer cells.

Experimental procedures: We generated stable PMEPA1 shRNA expressing LNCaP cells and analyzed this cell line by cancer biological assays including cell counting, soft agar and cell invasive assays. To evaluate the Methylation status of PMEPA1 promoter genomic DNA from primary human prostate tumors was examined by Methylation sensitive PCR method.

Results: PMEPA1 is successfully knocked down in the PMEPA1 shRNA expressing LNCaP cell line as detected at the mRNA and protein levels. The PMEPA1 knockdown cell line expressed AR protein at elevated levels and consistent elevation of AR signaling was evident in the study. Interestingly, the size of PMEPA1 knockdown cell showed altered morphology when compared to stale transfectant non-targeted shRNA LNCaP cells. In the PMEPA1 knockdown cell line we found higher number of cells in the S phase of the cell cycle compared with the control. In agreement with this observation the cellular proliferations were also enhanced under both androgen dependent and independent conditions. Moreover, the PMEPA1 knockdown cells have stronger invasive activity compared to control cells. The promoter methylation study of human prostate tumor derived genomic DNA revealed DNA Methylation within the PMEPA1 promoter downstream region in 20% of the examined samples.

Conclusions: PMEPA1 knockdown may enhance proliferation and invasiveness of prostate cancer cell by the elevation of AR levels. Thus PMEPA1 gene may act as a tumor suppressor gene in human prostate cancer cells. DNA methylation may play a role in silencing PMEPA1 gene expression in a subset of patients with lost or decreased PMEPA1 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 4920.
Abstract 4922: KRAS and BRAF mutations influence correlation structure of locus-specific CpG island methylation and CIMP in colorectal cancer
Authors: Ogino Shuji , Tanaka Noriko , Nosho Katsuhiko , Huttenhower Curtis , Quackenbush John , Haigis Kevin , Fuchs Charles
Published: February 2011

Background: The CpG island methylator phenotype (CIMP-high or CIMP1) is a distinct phenotype associated with microsatellite instability (MSI) and BRAF mutation in colon cancer. Recent investigations suggested that a separate KRAS mutation-associated CIMP subtype (CIMP-low or CIMP2) exists. However, no study has deciphered the interrelationship between KRAS, BRAF, CIMP and locus-specific CpG island methylation, utilizing causal modeling by structure equation model (SEM) analysis.

Design: Utilizing the database of 861 colorectal cancers, DNA methylation at 16 CpG islands [CACNA1G, CDKN2A (p16), CHFR, CRABP1, HIC1, IGF2, IGFBP3, MGMT, MINT-1, MINT-31, MLH1, NEUROG1, p14 (CDKN2A/ARF), RUNX3, SOCS1, and WRN] was quantified by real-time PCR (MethyLight). We also examined MSI status and DNMT3B expression. To evaluate the effect of KRAS and BRAF mutation on the correlation structure of the CpG island markers and MSI, we categorized tumors into 4 groups; Group 1 (N=440) with wild-type KRAS and BRAF; Group 2 (N=308) with mutant KRAS and wild-type BRAF; Group 3 (N=107) with wild-type KRAS and mutant BRAF; and Group 4 (N=6) with mutant KRAS and BRAF, which was excluded from further analyses. We analyzed data using clustering analysis, principal component analysis (PCA) and SEM.

Results: In unsupervised disjoint clustering analysis of the methylation markers, only 4 markers (CACNA1G, IGF2, RUNX3, SOCS1), were clustered with CIMP-high regardless of KRAS or BRAF mutation. In Groups 1 and 2, two latent CIMP classes, CIMP-low and CIMP-high, were assumed. However, in Group 3, CIMP-high status and CIMP-low status were clustered together so that only one latent class, “CIMP”, was assumed. PCA yielded similar findings to clustering. Based on the results of the cluster analysis and PCA and the literature data, we applied causal modeling by SEM analysis. In SEM analyses, the correlation structures between CIMP, locus-specific CpG island methylation and MSI differed according to KRAS and BRAF status, which was in agreement with results by PCA analyses. Differences between Group 1, Group 2 and Group 3 were highly significant p<0.0001).

Conclusions: KRAS and BRAF mutations influence correlation structures between locus-specific CpG island methylation, and CIMP status in colorectal cancer. Our data suggest the role of KRAS and BRAF mutations in modifying propensity of CpG island methylation in a locus-specific manner.

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 4922.
Abstract 4927: MGMT promoter methylation is enriched in glioblastoma derived spheres as compared to respective original tumor tissue and is associated with loss of expression
Authors: Sciuscio Davide , Diserens Annie-Claire , Martinet Danielle , Vlassenbroeck Ilse , Hamou Marie-France , Janzer Robert-Charles , Pollo Claudio , Stupp Roger , Levivier Marc , Hegi Monika E.
Published: February 2011

Epigenetic silencing of the O6-methylaguanine DNA methyl transferase (MGMT) gene by promoter hypermethylation blunts repair of O6-methyl guanine and has been shown to be a predictive factor for benefit from alkylating agent therapy in glioblastoma. Quantitative methylation specific PCR (Q-MSP) suggests that not all cells in a glioblastoma exhibiting MGMT methylation carry a methylated MGMT allele, even after adjustment for tumor cell content. This raises the question of the clinically relevant methylation threshold for predicting response to therapy for treatment stratification. There are several potential reasons: heterogeneity of glioblastoma; contaminating normal tissue; a heterogenous methylation pattern not correctly detectable by the assay, or presence of MGMT methylation in distinct subpopulations of cells, namely tumor initiating cells. In order to address the question we investigated the degree and pattern of MGMT promoter methylation in 10 paired samples of glioblastoma and spheres derived thereof using Q-MSP and methylation specific clone sequencing. We observed that in MGMT methylated glioblastoma, the percentage of methylated alleles ranged from 10 to 90%. In contrast, in most of the respective glioblastoma derived spheres, cultured under stem cell conditions, methylated alleles were highly enriched (10/10 clones), even if both MGMT-alleles were retained. The methylation pattern was conserved in the spheres, even when passing them in nude mice. Most importantly, this hypermethylation was associated with complete loss of MGMT expression. For one case we observed only 50% methylated clones in the sphere fraction, associated with low expression of MGMT.

Taken together, our data suggest that in MGMT methylated glioblastoma, cells with MGMT promoter hypermethylation are enriched in the sphere fraction which supposedly comprises cancer initiating cells. Thus, even a low percentage of MGMT methylation in a glioblastoma could be predictive marker for a benefit from alkylating agent therapy.

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 4927.
Abstract 4937: Hypermethylation of semaphorin 3B promoter in malignant pleural mesothelioma
Authors: Koyama Nobuyuki , Zhang Jialing , qun Hu , Goto Yoshiya , Ishikawa Yuichi , Shimizu Yoshihiko , Sakaguchi Kozo , Kobayashi Kunihiko , Hagiwara Koichi
Published: February 2011

Semaphorin 3B (SEMA3B) is a putative tumor suppressor gene that is inactivated by promoter hypermethylation and allelic loss on chromosome 3p21.3 in lung cancer. While malignant pleural mesothelioma (MPM) is associated with poor outcome, its pathogenic mechanism remains unclear. The purpose of this study is to elucidate a novel molecular pathogenesis of MPM from the investigation of the methylation status of SEMA3B promotor. After informed consent, we used surgically resected specimens of 28 MPM cases. The methylation was detected by methylation-specific PCR for genomic DNA extracted from individual specimens, and then correlation between the methylation status and clinicopashological features was analyzed. While 17 of 28 MPM cases harbored SEMA3B methylation (60.7%), there was no significant difference in any factors including survival time. SEMA3B promotor is frequently hypermethylated in MPM compared to lung cancer, and might be associated with the pathogengesis of MPM.

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 4937.
Abstract 4938: Aberrant methylation and expression of p21 on lung cancer and malignant pleural mesothelioma
Authors: Ueno Tsuyoshi , Teramen Hiromu , Toyooka Shinichi , Kubo Takafumi , Soh Junichi , Asano Hiroaki , Tsukuda Kazunori , Yamane Masaomi , Oto Takahiro , Miyoshi Shinichiro
Published: February 2011

Background: The cell cycle progression of which alteration is well known in human cances involves sequential activation and inactivation of cyclin-dependent kinases. The p21 (CDKN1A) plays a key role in regulating the cell cycle. Loss of expression or function of p21 (CDKN1A) has been implicated in the genesis or progression of many human malignancies. DNA methylation in tumor suppressor gene has been established as an important mechanism for gene silencing in human malignancies. In this study, we examined the methylation and expression status of P21 gene in lung cancer and malignant pleural mesothelioma (MPM). Methods: We used 14 non-small-cell lung cancer (NSCLC) cell lines and 20 primary tumors, 12 small-cell lung cancer (SCLC) cell lines, 7 MPM cell lines and 10 primary MPMs. Methylation of P21 gene was examined with methylation-specific PCR assay. p21 expression was examined by RT-PCR and western blotting. Cell lines with methylation were treated with 5-aza-CdR to confirm restoration of p21 expression. Results: Aberrant methylation of P21 was found in 20.6% of NSCLCs (28.6% and 15% in cell lines and primary NSCLCs, respectively), 8.3% of SCLC cell lines, 5.9% of MPMs (0% and 10% in MPM cell lines and primary MPMs, respectively). In NSCLC tumors, P21 methylation was detected only in smokers and squamous cell carcinoma cases but not in never smokers and adenocarinomas with bronchioloalveolar carcinoma feature. Loss of P21 mRNA expression were observed in 4 NSCLC cell lines (50.0%), 4 SCLC cell lines (33.3%) and none of MM cell lines (0%) while loss of protein expression was present in 10 NSCLC cell lines (83.3%), 6 SCLC cell lines (58.3%) and 2 MM cell lines (33.3%). mRNA expression of P21 was restored with 5-Aza-CdR treatment, indicating that methylation was responsible for silencing. These results suggested that 1) P21 methylation was one of the mechanisms of loss of P21 mRNA expression, and 2) post-transcriptional modification is responsible for loss of p21 protein expression. Conclusion: Aberrant methylation of p21 is present in lung cancer, especially for NSCLC, and rare in MPM. p21 protein expression was frequently loss in NSCLC and SCLC, partially due to DNA methylation.

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 4938.
Abstract 494: Identification and characterization of novel kinases that regulate BRCA1 expression and function
Authors: Wang Wenjia , Harlan-Williams Lisa M. , Jensen Roy A.
Published: February 2011

Transcriptional and functional regulation of breast cancer susceptibility gene 1 (BRCA1) in the pathogenesis of sporadic breast cancers is poorly understood. About 90% of all breast cancers are considered sporadic, and 30-40% of these cases exhibit decreased expression of BRCA1 in the absence of mutations at its genetic locus. Loss of BRCA1 accelerates growth of tumor cells, while expression of BRCA1 leads to growth arrest and apoptosis. These observations support further examination of regulatory pathways of BRCA1 to restore its expression and function.

To identify potential regulators of BRCA1, we developed a functional assay based on the role of BRCA1 in DNA damage repair. BRCA1 acts as a central scaffold that assembles a complex of repair proteins to sites of double-strand breaks (DSBs) following exposure to ionizing radiation (IR). Immunostaining for BRCA1 reveals these repair sites as punctate ionizing radiation-induced foci (IRIF). The ability of BRCA1 to localize to DSBs depends on both its expression and phosphorylation status. We evaluated the effect of siRNAs against kinases on BRCA1 IRIF formation because 1) phosphorylation of BRCA1 regulates many of its functions, 2) bioinformatics provides BRCA1 phosphorylation sites where the kinase involved is unknown, and 3) kinases play key roles in signaling pathways to influence gene expression or phosphorylation status. A number of kinases were identified that affected the ability of BRCA1 to form IRIF. However, the mechanism of kinase influence is unclear. Secondary siRNA screening assays have validated the kinase hits and lead kinases have been selected for further characterization. We are currently determining whether the effects of the lead kinases on BRCA1 are at the transcriptional or post-translational level and how these changes affect the ability of BRCA1 to repair DSBs by homologous recombination, to regulate the G2/M cell cycle checkpoint, and to modulate expression of downstream genes. Additionally, we are elucidating the signaling pathways that explain how the lead kinases regulate BRCA1. Once completed, we will have identified and characterized the role of the lead kinases on BRCA1 expression and function. This knowledge can translate into a novel therapy for BRCA1-related sporadic breast cancers.

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 494.