bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4984: Phosporylation of eIF4E confer resistance to cellular stress
Authors: Cajal Santiago Ramon y , Aasen Trond , Martínez Alba
Published: August 2012

Eukaryotic initiation factor (eIF) 4E mediates eIF4F association with the mRNA 5′ cap structure and is an essential factor for canonical protein translation. eIF4E can act as an oncogene when overexpressed in cell lines and high levels of eIF4E and phosporylated peIF4E have been described in a large number of human tumors. eIF4E has also been described as a component of cytoplasmic stress granules and processing bodies. Importantly, non-phosphorylated 4E-BP1 (often in response to stress) binds eIF4E to suppress protein translation. We demonstrated previously that high levels of peIF4E were associated with resistance to this suppressor effect in carcinoma cell lines. Because the role of peIF4E is not yet well established, we evaluated the role of eIF4E phosphorylation on in vitro stress resistance. Breast carcinoma cell lines (MDA-MB 231, MBA- MB 468 and HeLa cells) and an immortalized human keratinocyte cell line (HaCaT) were infected with retrovirus expressing wild type, hyperphosporylated (S209D) or hypophosporylated (S209D) mutants of eIF4E. Cells were subjected to different types of stress including oxidative (Arsenite), nutrient starving and cisplatin treatment. Immediate and long term response to stress was evaluated using colony formation, MTT cell proliferation assays, co-inmunoprecipitation and immunofluorescence microscopy. A significantly greater recovery after all forms of stress was observed in all the cell lines expressing hyperphosporylated eIF4E-S209D. After treatment with arsenite there was an increase of peIF4E, 4E-T and Hur and importantly higher increase of levels of Hur, CyclinD1, Mcl-1 and eIF4G in eIF4E-S209D expressing cells. Under normal condition we observed a unique colocalitzation of 4E-T and eIF4E-S209D in cytoplasmic bodies. This complex colocalize with Ago2 but not with markers of the stress granules (TIA-1) and processing bodies (DCP1A). By co-immunopreciptation, we observed that peIF4E binds to its carrier protein 4E-T, Ago2 and Hur. Conclusions: Phosphorylated eIF4E confer resistance to several stress conditions, including oxidative stress, DNA damage and nutrient deprivation. The fact that there was a relevant and significant increase of proteins such as Cyclin D1, Mcl-1 and Hur suggests that phosporylated eIF4E has an important role in selectively stimulating RNA translation under stress conditions, possibly via the formation of a unique type of 4E-T/peIF4E cytoplasmic bodies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4984. doi:1538-7445.AM2012-4984
Abstract 4987: Non-targeting siRNA induces NPGPx expression to cooperate with exoribonuclease XRN2 for releasing the stress
Authors: Wei Pei-Chi
Published: August 2012

Short interfering RNAs (siRNAs) target specific mRNAs for their degradation mediated by RNA-induced silencing complex (RISC). Persistent activation of siRNA-RISC frequently leads to non-targeting toxicity. However, how cells mediate this stress remains elusive. In this communication, we found that the presence of non-targeting siRNA selectively induced the expression of an ER-resident protein, non-selenocysteine containing phospholipid hydroperoxide glutathione peroxidase (NPGPx), but not other ER-stress proteins including GRP78, Calnexin, and XBP1. Cells suffering from constant non-targeting siRNA stress grew slower and prolonged G1 phase, while NPGPx-depleted cells accumulated mature non-targeting siRNA and underwent apoptosis. Upon the stress, NPGPx covalently bound to exoribonuclease XRN2, facilitating XRN2 to remove accumulated non-targeting siRNA. These results suggest that NPGPx serves as a novel responder to non-targeting siRNA-induced stress in facilitating XRN2 to release the non-targeting siRNA accumulation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4987. doi:1538-7445.AM2012-4987
Abstract 4990: PP2A signaling is overridden by MEK/ERK activity leading to suppression of Bim and protection of melanoma cells from ER stress-induced apoptosis
Authors: Tay Kwang Hong , Tseng Hsin-Yi , Jing Lei , Jiang Chen Chen , Ye Yan , Thorne Rick , Liu Tao , Hersey Peter , Zhang Xu Dong
Published: August 2012

Endoplasmic reticulum (ER) stress triggers apoptosis by activating Bim in diverse types of cells. This involves dephosphorylation of Bim by protein phosphatase 2A (PP2A). However, melanoma cells are largely resistant to ER stress-induced apoptosis, indicating that Bim activation is suppressed in melanoma cells undergoing ER stress. We show here that this is due to increased activation of MEK/ERK signaling that overrides the PP2A activity leading to enhanced Bim phosphorylation and subsequent proteosomal degradation. Upon pharmacological ER stress, the Bim protein expression was rapidly downregulated after an initial increase, despite its sustained upregulation at the transcriptional level. Although PP2A exerted the dephosphorylating effect on BimEL, its activity appeared to be superseded by increased activation of MEK/ERK signaling that led to enchanced phosphorylation and accelerated turnover of the protein. Moreover, in contrast to its upregulation by ER stress in MCF-7 and HEK293 cells, PP2A was downregulated in melanoma cells undergoing ER stress. Pharmacological activation or enforced expression of PP2A decreased ERK and Bim phosphorylation, up-regulated Bim expression, and rendered melanoma cells sensitive to ER stress-induced apoptosis. Notably, instead of direct interaction with Bim, PP2A reduced Bim phosphorylation indirectly by dephosphorylating ERK in melanoma cells. Together, these results identify a key resistance mechanism of melanoma cells against apoptosis induced by ER stress.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4990. doi:1538-7445.AM2012-4990
Abstract 4992: SPARC overexpression induces ER stress leading to autophagy-mediated apoptosis in neuroblastoma cells
Authors: Sailaja G. S. , Bhoopathi Praveen , Gorantla Bharathi , Tsung Andrew J. , Rao Jasti S.
Published: August 2012

Neuroblastoma is the most common extracranial solid tumor affecting children. Long-term survival for children (older than 18 months of age) with advanced disease is poor despite aggressive multimodal therapy. Normal cellular apoptosis is affected in multiple ways in a variety of diseases, including cancer. Our earlier studies showed that SPARC overexpression induced autophagy-mediated apoptosis in PNET cells. In the present study, we attempt to determine the signaling cascade associated with initiation of autophagy and apoptosis as a result of SPARC overexpression in combination with radiation. As demonstrated by TUNEL assay, neuroblastoma cells showed increased apoptosis with the combination treatment as compared to treatment with either SPARC or radiation alone. In corroboration, we also observed activation of caspase 3, and cleavage of PARP and Bid as determined by Western blotting. The combination treatment triggered endoplasimic reticulum (ER) stress and thereby an unfolded protein (UPR) response. This was apparent with the activation of stress receptors, pancreatic ER kinase (PERK) and inositol-requiring enzyme (IRE 1), as shown by western blotting. ER stress led to mitochondrial dysfunction through activation of eukaryotic initiation factor 2α (eIF2 α; another ER resident protein). In addition, we demonstrate that overexpression of SPARC induced IRE-JNK activation, leading to induction of the transcription factor CHOP. Inhibition of ER stress (or JNK activation) led to inhibition of autophagy-mediated apoptosis as demonstrated by decreased levels of LC3-II, and cleavage of caspase 3, PARP and Bid. Taken together, these results demonstrate the involvement of ER stress in regulating autophagy-mediated apoptosis in neuroblastoma cells treated with radiation and SPARC overexpression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4992. doi:1538-7445.AM2012-4992
Abstract 4993: IRE1-XBP1 branch of UPR is involved in tumor angiogenesis
Authors: Wang Yugang , Polverini Peter , Alam Goleeta , Ning Yu , Nör Jacques
Published: August 2012

The Unfolded Protein Response (UPR) is activated when the endoplasmic reticulum (ER) in tumor cells is subjected to environmental stress. We and others have previously shown that the eIF2α- PERK-ATF4 pathway, one of three of UPR pathways is involved in tumor angiogenesis by transcriptionally regulating the production of angiogenic mediators such as VEGF. However, role of other UPR pathways in tumor angiogenesis still remains to be elucidated. In this study we examined the role of the IRE1-XBP1 UPR pathway in promoting production of tumor-derived proangiogenic mediators. Upon ER stress IRE1 splices XBP1 into an active transcription factor that induces expression of several target genes. We used the gene-silencing approach in the oral squamous carcinoma cell line UM-SCC-81B to determine the effects of XBP1 knockdown on key angiogenic mediators. We were able to achieve over 80% knockdown of XBP1. mRNA levels of both EDEM1 and Grp78, downstream targets of XBP1 were also significantly inhibited confirming an efficient knockdown. UM-SCC-81B control cells as well as UM-SCC-81B-XBP1 knockdown cells were initially treated with low glucose (5mM) for 24 h to induce UPR. Next, mRNA and protein levels of VEGF were determined using Real-Time PCR and ELISA respectively. We observed a 45% decrease in VEGF mRNA levels and around 36% decrease in VEGF protein levels of UM-SCC-81B-XBP1 knockdown cells upon ER stress. Additionally, we detected a significant decrease in the transcription of several other key angiogenic mediators such as FGF2, IL6, IL8 and CTGF (p<0 .05) in UM-SCC-81B-XBP1 knockdown cells compared to control cells. These results suggest that the IRE1-XBP1 pathway of the UPR promotes tumor angiogenesis through modulating expression of proangiogenic mediators.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4993. doi:1538-7445.AM2012-4993
Abstract 4994: An integrated stress response activator causes cellular senescence and inhibits growth in multiple tumor cell types
Authors: Sayers Carly M. , Koong Albert C. , Koumenis Constantinos
Published: August 2012

The Integrated Stress Response (ISR) is a program elicited by mammalian cells to attenuate global translation in response to a variety of stressors. There are four branches of the ISR, including those mediated by PERK, which responds to endoplasmic reticulum stress, and GCN2, which is activated by amino acid deprivation. Activation of these kinases ultimately leads to translational upregulation of the transcription factor ATF4, which modulates the expression of genes involved in amino acid transport and synthesis, oxidative stress, and differentiation. Complete abrogation of the ISR or its overactivation by pharmacological agents in the presence of hypoxic or nutrient stress has been shown to lead to cell death. Here, we employed a small molecule screen to identify modulators of ATF4 expression via a reporter construct with the 5′-UTR of ATF4 fused to the luciferase gene. From this screen, a compound with ATF4-activating properties was identified. Multiple assays have been used to characterize the effects of this drug, referred to as E235, on both transformed and normal cells. Specifically, we have shown that E235 induces the ISR in a dose-dependent manner in tumor cell lines (HT1080 fibrosarcoma, B16F10 melanoma) but not in normal human diploid cells. The drug also reduces cell viability and proliferation at low μM concentrations, which does not appear to be dependent on apoptosis. By performing live cell microscopy, we observed that after prolonged (>24h) treatment with 1μM E235, the majority of cells displayed senescence-like morphology. This was confirmed by additional experiments, such as staining for senescence-associated β-galactosidase activity and immunoblotting for the upregulation of the cdk inhibitor p21. Future experiments will focus on establishing the roles of ATF4, p21 and p53 in E235-mediated senescence.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4994. doi:1538-7445.AM2012-4994
Abstract 4995: Glucose-regulated protein 78 regulates crosstalk between apoptosis and autophagy to determine antiestrogen responsiveness
Authors: Cook Katherine L. , Shajahan Ayesha N. , Jin Lu , Warri Anni , Hilakivi-Clarke Leena A. , Clarke Robert
Published: August 2012

While over 70% of breast cancers express estrogen receptor α (ER+), drugs targeting these receptors such as Tamoxifen (TAM) or Faslodex (fulvestrant, ICI) often fail to cure these patients. Many ER+ patients fail to respond (de novo resistance) or lose drug responsiveness (acquired resistance), making endocrine resistance a major clinically relevant problem. The molecular mechanisms of endocrine therapy resistance still remain unclear. We show that antiestrogen resistant cells with an acquired resistant phenotype overexpress glucose-regulated protein 78 (GRP78), which is not observed in de novo resistance. Moreover, we show increased expression of GRP78 in MCF7/LCC9 (estrogen independent, ICI resistant, TAM cross-resistant) when compared to the MCF7/LCC1 (estrogen independent, TAM and ICI sensitive) breast cancer cells and tumors. These data reflect the response of aromatase inhibitor resistant breast cancers to second line antiestrogen therapy, and further support the hypothesis that antiestrogen resistance and estrogen independence are separate phenotypes. Targeting GRP78 with RNAi restored antiestrogen sensitivity in endocrine therapy resistant cells (MCF7-RR and MCF7/LCC9), whereas expressing GRP78 cDNA conferred resistance on antiestrogen sensitive breast cancer cells (MCF7 and MCF7/LCC1). We show that GRP78 integrates cellular signaling to inhibit apoptosis (as determined by cleaved caspase-7, cleaved PARP, and annexin V staining) and stimulate prosurvival autophagy (as determined by LC3-II, p62, and GFP-LC3 punctate). Moreover, GRP78-stimulated autophagy is dependent on TSC2/AMPK-mediated mTOR inhibition. Endocrine resistance induced by GRP78 overexpression is prevented through an inhibition of autophagy by ATG5 knockdown, while caspase inhibition prevented GRP78 RNAi-mediated therapy resensitization. Furthermore, knockdown of GRP78 and beclin 1 (BECN1) synergistically restores antiestrogen sensitivity in resistant cells. The use of GRP78 to integrate the cellular functions of apoptosis and autophagy to control cell fate raises the provocative question that this signaling may be widely applicable and represent a major stress response that explains how many different cell types could respond to stressors and acquire resistance to therapeutic interventions.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4995. doi:1538-7445.AM2012-4995
Abstract 4996: Genome-wide DNA methylation profiles in hepatocellular carcinoma
Authors: Shen Jing , Wang Shuang , Zhang Yujing , Kappil Maya , Wu Hui-Chen , Kibrya Muhammad G. , Wang Qiao , Jasmine Farzana , Ahsan Habib , Lee Po-Huang , Yu Ming-Whei , Chen Chien-Jen , Santella Regina M.
Published: August 2012

Alterations in DNA methylation frequently occur in hepatocellular cancer (HCC). We have previously demonstrated that hypermethylation in candidate genes can be detected in plasma DNA prior to HCC diagnosis. To identify with a genome-wide approach additional genes hypermethylated in HCC that could be used for more accurate analysis of plasma DNA for early diagnosis, we analyzed tumor and adjacent non-tumor tissues from 62 Taiwanese HCC cases using Illumina methylation arrays that screen 26,486 autosomal CpG sites. After Bonferroni adjustment, a total of 2,324 CpG sites significantly differed in methylation level, with 684 CpG sites significantly hypermethylated and 1,640 hypomethylated in tumor compared to non-tumor tissues. Array data were validated with pyrosequencing in a subset of 5 of these genes; correlation coefficients ranged from 0.92 to 0.97. Analysis of plasma DNA from 38 cases demonstrated that 37% to 63% of cases had detectable hypermethylated DNA (≥5% methylation) for these 5 genes individually. At least one of these genes was hypermethylated in 87% of cases, suggesting that measurement of DNA methylation in plasma samples is feasible. The panel of methylated genes indentified in the current study will be further tested in large cohort of prospectively collected samples to determine their utility as early biomarkers of hepatocellular carcinoma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4996. doi:1538-7445.AM2012-4996
Abstract 4999: Genome-wide methylation array of human prostate tissues using illumina infunium 450K bead chip reveals distinct DNA methylation signatures with potential as clinical predictors
Authors: Kwabi-Addo Bernard , Wang Songping , Lee Norman H. , Ittmann Michael , Suer Funda , Devaney Joseph
Published: August 2012

Prostate cancer (PCa) harbors a myriad of aberrant genomic and epigenetic alterations. Epigenetic inactivation of genes in PCa is largely based on transcriptional silencing by aberrant CpG methylation of CpG rich promoter regions. Candidate gene-based studies have identified a handful of aberrant CpG DNA methylation events in PCa. To better understand the role of aberrant epigenetic alterations and identify biological pathways likely to be affected by methylation mediated alterations in gene expression in PCa, we have examined the methylation status of 450,000 (450K) cytosine microarray (illumina). The 450K microarray includes CpG islands/shores/shelves/open sea, non-coding RNA and sites surrounding the transcription start sites for coding genes, but also for the corresponding gene bodies and the 3′-UTR. We demonstrate that the 450k DNA methylation array can significantly detect CpG methylation changes in radical prostatectomy prostate tissue samples. Cluster analysis revealed distinct DNA methylation profiles for prostate cancer tissues in comparison with non-malignant tissues. A total of 225 CpG loci (p > 0.00049) showed differential methylation in the cancer tissues in comparison with non-malignant tissues. Each of the 225 CpG locus was validated in silico using an independent, publicly available methylome dataset from the Cancer Genome Atlas. The majority of these loci were informative particularly in the prostate cancer. Overall, cluster analysis showed gene set enriched in pathways including transcriptional modifications, apoptosis/autophagy, chromatin assembly, growth factor signaling, cell cycle regulation and oxidative-redox reaction. This study shows that CpG locus methylation arrays could reveal important biological differences in the epigenome between prostate cancer and non-malignant tissues and provide potential markers for disease detection and/or progression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4999. doi:1538-7445.AM2012-4999
Abstract 500: DMP1 activates osteolytic cycle in a tumor environment
Authors: Eapen Asha S. , George Anne
Published: August 2012

Bone metastasis is one of the skeletal malignancies that could be caused by breast cancer. Dentin matrix protein 1 (DMP1) is an acidic noncollagenous protein localized specifically in the mineralized matrix of bone and dentin. It is a multifunctional protein, involved in gene regulation (within the cell) and HAP nucleation (in the ECM). DMP1 is endocytosed by preosteoblasts, triggering the calcium dependent and calcium independent signaling pathways resulting in a series of downstream events leading to osteoblast differentiation. These signaling events facilitate the activation of a calcium dependent stress-induced p38 MAP kinase and a calcium independent integrin mediated ERK1/2 MAP kinase pathway resulting in the expression of downstream target genes such as Runx2 and osteocalcin. Results from this study have shown that the osteoblast cells upon differentiation secrete receptor activator of nuclear factor kappa B ligand (RANKL) that stimulates the differentiation of precursor monocytes to osteoclasts. The study also focuses on identifying the role of DMP1 in breast cancer cells that could enhance osteoclast formation thereby accelerating bone resportion. Thus, the study reveals a new mechanism by which DMP1 can activate the vicious cycle leading to the aggressive growth and behavior of the cancer cells. To address the mechanism by which DMP1 might contribute to the osteolytic process, DMP1 was overexpressed in the metastatic cancer cell line MDA MB231. Upregulation of markers like Runx2, MMP2, MMP9, RANKL, OPN, BSP, VEGF and the activation of Smad2/3 and MAP kinase pathways (p38 and ERK1/2 pathways) were observed in overexpressed cells. Results from this study have shown that DMP1 secreted by the breast cancer cells into the extracellular environment could stimulate the differentiation of osteoblasts, leading to the secretion of factors that signal monocytes to differentiate to osteoclast thus resulting in bone metastasis. Overall, the results obtained from this study identify a new role for DMP1 in the differentiation of breast cancer tumor cells that may be directly related to their metastatic potential. Supported by NIH DE 115657 and the Brodie Endowment Fund

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 500. doi:1538-7445.AM2012-500
Abstract 5004: CpG island methylator phenotype in bladder cancer
Authors: Chung Woonbok , Bondaruk Jolanta , Zhang Nianxiang , Jelinek Jaroslav , Estecio Marcos , Liang Shoudan , Czerniak Bogdan , Issa Jean-Pierre J.
Published: August 2012

DNA hypermethylation is a common epigenetic abnormality in cancer, and some cancers are characterized by the presence of intense hypermethylation at multiple loci, termed CpG island methylation phenotype (CIMP). CIMP has not been fully characterized in bladder cancer. In a pilot study, we examined the methylation status of 38 loci in a panel of 26 bladder cancer and identified 14 genes whose methylation was concordant, supporting the presence of CIMP. In an expanded series of 108 cancers, we found that a distinct subset displays concerted hypermethylation at a large number of genes, confirming the existence of CIMP in bladder cancer. Unsupervised hierarchical K-means clustering identified three distinct DNA methylation groups of bladder cancers, CIMP-N (negative), CIMP-L (low), and CIMP-H (high) representing 39%, 33% and 28% of cancer overall. Compared to CIMP-N cases, CIMP-H cases were more frequently current smokers (p<0.05), had more H-RAS and K-RAS mutation (15% vs. 0 %, P< 0.05), had less FGFR3 mutations (8% vs. 32 %, P<0.05) and more RB1 loss (35% vs. 10%, P<0.05). Genome wide analysis of 85 bladder cancers by MCAM (methylated CpG island amplification microarray) showed an average of 12.4 % of genes hypermethylated in CIMP-H, 6.5 % in CIMP-L and 4.4 % in CIMP-N (CIMP-N vs. CIMP-L, p=0.0006 and CIMP-L vs. CIMP-H, p<0.0001). Affected genes were enriched for PcG targets, and gene ontology analysis showed clustering at Homeobox and voltage-gated cation channel based on gene expression of CIMP targets. Our study suggests that CIMP is an important molecular variable in bladder cancer heterogeneity.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5004. doi:1538-7445.AM2012-5004
Abstract 5005: Large-scale DNA methylation profiling in gastrointestinal stromal tumors (GIST) reveals epigenetic regulation of SPP1 as an independent prognostic factor
Authors: Haller Florian , Zhang Jitao David , Riazalhosseini Yasser , Ward Aoife , Balwierz Aleksandra , Korn Bernhard , Schaefer Inga-Marie , Cameron Silke , Ghadimi B. Michael , Agaimy Abbas , Fletcher Jonathan A. , Hoheisel Jörg , Wiemann Stefan , Sahin Özgür
Published: August 2012

Aims Gastrointestinal stromal tumors (GISTs) have distinct gene expression patterns according to anatomical localisation and genotype, with potential impact on biological aggressiveness. DNA methylation of CpG dinucleotides in the promoter region of genes is an important mechanism for regulation of gene expression. We performed a large-scale analysis of DNA methylation in GISTs to identify methylation patterns associated with clinico-pathological parameters and prognosis. Methods Seventy-five GISTs with either KIT or PDGFRA mutations were analysed for DNA methylation of 1.505 CpG sites in the promoter regions of 807 cancer-related genes using a large-scale approach (Cancer Methylation Panel I, Illumina). Epigenetic regulation of Secreted Phosphoprotein 1 (SPP1) expression was analysed at mRNA and protein levels in GIST882 and GIST48b cells after treatment with a demethylating agent. Impacts on tumorigenesis-related signalling pathways were analysed by Western Blot after stimulation of GIST cell lines with SPP1. Prognostic impact of SPP1 at methylation and protein levels were investigated in 75 and 121 GIST tumor samples, respectively, and compared to classical prognostic parameters tumor size, mitotic counts and tumor localisation. Results Principal component analysis revealed distinct DNA methylation patterns according to anatomical localisation, genotype and mitotic counts. Hypomethylation of a CpG dinucleotide 647 base pairs upstream of the transcription start site of SPP1 was significantly correlated to SPP1 mRNA and protein level, and also to tumor progression (p=0.003). In vitro analyses confirmed epigenetic regulation of SPP1 expression, and demonstrated potential impact on proliferation and invasion. High protein level of SPP1 was significantly correlated to shorter disease-free survival in univariate (p=2x10−7) and multivariate (p<1x10−6) analyses. Conclusion The expression of SPP1 is regulated epigenetically in GISTs, and high expression of SPP1 is a novel and independent prognostic parameter in GISTs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5005. doi:1538-7445.AM2012-5005
Abstract 5006: High-throughput TR-FRET cellular assays for interrogating histone H3 methylation
Authors: Bi Kun , Dudek Jeanne , Hermanson Spencer
Published: August 2012

Post-translational modifications such as phosphorylation, acetylation and methylation play important roles in regulating the structures and functions of histones, which in turn regulate gene expression and DNA repair and replication. Histone modifying enzymes, such as deacetylases, methyltransferases and demethylases, have been pursued as therapeutic targets for various diseases. However, detection of the activities of these enzymes in high-throughput cell-based formats has remained challenging. We have developed high-throughput LanthaScreen® cellular assays for histone H3 site-specific modifications. These assays utilize cells expressing green fluorescence protein (GFP) tagged-histone H3 transiently delivered via BacMam and terbium labeled-anti-histone H3 modification-specific antibodies. Robust time-resolved Foerster resonance energy transfer signals were detected for H3 lysine-9 di-methylation (K9me2), lysine-4 di- (K4me2) and tri-methylation (K4me3) and lysine-27 tri-methylation (K27me3). Consistent with previous reports, hypoxic stress increased K4 methylation levels and methyltransferase G9a inhibitor UNC-0638 decreased K9me2 levels significantly with little effects on other modifications. Further validation of these assays using methyltransferases-specific RNAi oligos demonstrated that EZH2-specific RNAi reduced the level of K27me3 with little effect on the other three modifications, whereas G9a RNAi and SMYD3 RNAi reduced K9me2 and K4me2/3, respectively. The use of BacMam gene delivery system enables the measurement of these histone methylations in a variety of cell backgrounds including primary cells. In conclusion, we have developed homogenous cellular assays that can enable the high-throughput screening for modulators of histone H3 methylation at various lysine sites.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5006. doi:1538-7445.AM2012-5006
Abstract 501: The carboxypeptidase inhibitor TIG1 interacts with cathepsin L2 and inhibits cathepsin L2-stimulated cellular invasion
Authors: Jiang Shun-Yuan , Tsai Fu-Ming , Shyu Rong-Yaun , Wu Chang-Chieh , Tsai Tzung-Chieh , Wang Chun-Hua
Published: August 2012

The tazarotene-induced gene 1 (TIG1), also known as retinoic acid receptor responder 1 (RARRES1), is a putative tumor suppressor gene. The gene encodes a carboxypeptidase inhibitor for ATP/GTP binding protein-like 2 (AGBL2). This study identified TIG1 binding proteins and characterized the activities of TIG1 on cellular invasion of HCT116 colon cancer cells stimulated by the cathepsin L2 (CTSL2). The cytoplasmic portion of the TIG1A isoform was used for identification of TIG1 binding proteins using the yeast two-hybrid screening, and six genes, CTSL2, DNAJC8, SPATA22, SPINK2, VAC14, and C6orf225, were selected. CTSL2, a cysteine protease with an increased expression shown in breast and colon carcinomas, was selected for analysis. Immunoprecipitation analysis using transiently transfected cytosols confirmed the binding between both TIG1 isoforms and CTSL2. Both TIG1A and CTSL2 were secreted proteins; however, the interaction of both proteins in culture supernatants was not detected. Further immunoprecipitation analysis using purified recombinant proteins demonstrated direct binding between both TIG1 isoforms and CTSL2. Cellular invasion of HCT116 cells was increased by 52 to 71% in CTSL2 transiently transfected cells. Both TIG1 isoforms had no effects on cellular invasion of HCT116 cells. However, TIG1A and TIG1B significantly inhibited CTSL2-stimulated cellular invasion by 65.1 and 62.9%, respectively. In conclusion, both TIG1 isoforms interact with CTSL2 and inhibited CTSL2-stimulated cellular invasion of HCT116 cells. The results suggest that TIG1 may suppress CTSL2-mediated cellular invasion through inhibiting the activation of CTSL2 protease, and merit for further investigation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 501. doi:1538-7445.AM2012-501
Abstract 5014: Differences in DNA methylation pattern of primary bladder tumours correlate with their metastatic potential
Authors: Stubendorff Beatrice , Dudziec Ewa , Catto James , Sanjmyatav Jimsgene , Gaida Mieczyslaw , Wunderlich Heiko , Grimm Marc-Oliver , Junker Kerstin
Published: August 2012

Introduction: The prognosis for patients with metastatic bladder cancer is poor with a 5-year survival rate of only 6%. Early prediction of the metastatic risk in primary tumours can lead to improvements in prognosis and therapy. Currently, there are no parameters available that enable an individual risk assessment for patients with metastatic bladder cancer. Therefore, identification of new reliable prognostic markers for early prediction of tumour spread is required. The aim of this project is to determine whether changes in DNA methylation correlate with the metastasis risk and to identify a specific DNA methylation pattern that provides a reliable tool for prognosis assessment. Materials & methods: Genomic DNA was isolated from 23 invasive bladder tumour tissues with and without lymph node metastases. For enrichment of methylated fragments genomic DNA was incubated with 5-Methylcytosin antibody. Input and IP were labelled with Cy3 and Cy5. Labelled DNA was hybridizes onto CpG island microarrays. Cluster analysis enabled selection of differentially methylated candidate genes. The actual methylation state was confirmed by cleavage of genomic DNA with methylation dependent restriction enzyme (McrBc) followed by quantitative PCR. Results: In total, 201 promoter CpG islands show highly significant differences in DNA methylation between metastasised and non-metastasised primary bladder tumours (p<0,01). In metastasised tumours 74 CpG islands are hypermethylated and 127 CpG islands are hypomethylated. Data base research for identification of candidate genes resulted in 61 hypermethylated and 58 hypomethylated genes that can clearly assigned to the identified promoter associated CpG islands. Validation of methylation level of candidate gene RASSF1 resulted in higher methylation in metastatic tumours (mean methylation: 38%) than in non-metastatic tumours (mean methylation: 26%). RASSF1 methylation shows strong correlation to T stage (r=0,65). Methylation of candidate gene KISS1R shows significant differences between patient groups (p=0,001). Mean methylation in metastatic tumours is 48% and in non-metastatic tumours 22%. Conclusion: Primary bladder tumours show significant differences in their DNA methylation pattern in correlation to their metastatic potential. KISS1R methylation is strongly associated with the metastatic risk of primary tumours. Further analysis will demonstrate the correlation between changes in DNA methyaltion and expression of corresponding genes. The impact of DNA methylation on tumour behaviour shall be determined in functional analysis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5014. doi:1538-7445.AM2012-5014
Abstract 5015: Regulation of histone modification and chromatin structure by p53-PADI4 pathway
Authors: Tanikawa Chizu , Nakamura Yusuke , Matsuda Koichi
Published: August 2012

Histone proteins are modified in response to various external signals, however their mechanisms are still not fully understood. We here report in vivo and in vitro citrullination of arginine 3 residue of histone H4 (H4R3) in response to DNA damage through the p53-PADI4 pathway. We also observed DNA damage-induced citrullination of lamin C through the p53-PADI4 pathway. Citrullinated H4R3 (cit-H4R3) and citrullinated lamin C are located around fragmented nuclei in apoptotic cells. Ectopic expression of PADI4 led to chromatin decondensation and promoted DNA cleavage, while Padi4-/- mice exhibited resistance to radiation-induced apoptosis in the thymus. We also found loss of function mutations of the PADI4 gene in several cancer cell lines. Furthermore, the level of cit-H4R3 was negatively correlated with that of p53 protein in cancer cells and with tumor size in non-small cell lung cancer tissues. Our findings reveal that citrullination of H4R3 would be an “apoptotic histone code” to detect damaged cells and induce nuclear fragmentation, which plays a crucial role in carcinogenesis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5015. doi:1538-7445.AM2012-5015
Abstract 5022: Immune biology drives cancer specific methylation profiles in blood
Authors: Koestler Devin C. , Marsit Carmen J. , Christensen Brock C. , Accomando William , Langevin Scott M. , Houseman Eugene A. , Nelson Heather H. , Karagas Margaret R. , Wiencke John K. , Kelsey Karl T.
Published: August 2012

Blood leukocytes from patients with solid tumors exhibit complex and distinct cancer-associated patterns of DNA methylation. However, the biological mechanisms underlying these patterns remain poorly understood. Since epigenetic biomarkers offer significant clinical potential for cancer detection, we sought to address a mechanistic gap in recently published works, hypothesizing that this variation is in large part due to shifts in leukocyte populations. To discern differentially methylated regions (DMRs) among leukocyte subtypes, we assessed epigenome-wide DNA methylation in sorted, purified, healthy human peripheral blood leukocyte subtypes. Performing a targeted analysis of leukocyte DMRs from epigenome-wide blood methylation data in three independent case-control studies of different cancers revealed that leukocyte DMRs predict case status with a high degree of accuracy (area under the curve = 0.82, 0.83, and 0.67, forhead and neck squamous cell carcinoma (HNSCC), ovarian, and bladder cancer, respectively). These results demonstrate that blood-derived differences in DNA methylation patterns in patients with solid tumors are largely attributable to systematic differences in the DMRs that define specific, known leukocyte sub-populations, suggesting that the unique epigenetic signatures in blood from solid tumor patients arise as a result of immune responses represented by shifts in leukocyte populations.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5022. doi:1538-7445.AM2012-5022
Abstract 5023: CpG pethylator phenotype (CIMP) methylation pattern in Puerto Ricans Hispanics colorectal cancer patients
Authors: Lacourt Mercedes Y. , Reyes Jeyshka , Lozada María E. , Rodriguez Heriberto M. , Castillo Xiomara , Cardona Alberto , Bernabe Raúl D. , Cruz-Correa Marcia
Published: August 2012

Introduction: The CpG island methylator phenotype (CIMP), characterized by extensive promoter methylation, seems to be a distinct epigenotype of colorectal cancer (CRC). CIMP hypermethylation blocks transcriptional activation of several genes associated with tumor suppression, cell cycle, DNA repair, differentiation and apoptosis. Moreover, CIMP hypermethylation is an important epigenetic change associated with gene silencing, poor prognosis and lower response to chemotherapy in CRC patients. However, prevalence of CIMP status and specific clinicopathological features has not been examined in Puerto Rican Hispanic CRC patients. In this study, we determined the CIMP status in PR Hispanics with CRC and examined its association with phenotypical characteristics. Methods: Methylation-specific PCR (MSP) was used to evaluate DNA methylation in eight CIMP-specific promoters (CACNA1G, IGF2, NEUROG1, RUNX3, SOCS1, hMLH 1, CRABP1 and p16) gene in eighty-nine CRC cases. No-CIMP status was defined as the absence of methylation, CIMP-Low status as one to four methylated loci, and CIMP-High status as five or more methylated loci. Clinicopathological characteristics were correlated with CIMP status using Wilcoxon rank tests, Chi square and Fisher exact t-tests, as appropriate using STATA 10.0. Results: Eighty-nine CRC cases (mean age at diagnosis 60.5 ± 10.3 years; 49 males) were evaluated. Tumors were mostly located in the distal colon (71.0%). The 5.6 percent (5/89) of CRC cases were CIMP-High, 88% (87.6/89) were CIMP-Low, and 6.7% (6/89) were No-CIMP. CRABP1 (69.7%), IGF2 (48.3%) and p16 (43.8%) were the three most commonly methylated genes. CIMP-High/Low tumors were more common among women and among individuals ≥ 60 y/o (borderline statistical significance). There were no significant statistical associations between CIMP status and tumor staging, differentiation, tumor location or survival. Discussion: We report the CIMP status in Puerto Rican Hispanics with CRC. CIMP-hypermethylation may be less frequent among Hispanics than previously reported in other racial and/or ethnic groups, with a discrete clinicopathological phenotype and gene methylation pattern. A comprehensive understanding of the differences in epigenetic silencing of promoter regions by hypermethylation will shed light on the mechanisms resulting in colorectal tumorigenesis in Hispanic patients. Current efforts are underway to characterize a large number of Puerto Rican Hispanics CRC patients which may help tailor screening and therapeutic strategies for this population.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5023. doi:1538-7445.AM2012-5023
Abstract 5025: Regulation of miR-145 expression by Foxo3a, C/EBP-β and p53 in cancer cells
Authors: Zhou Nanjiang , Mo Yin-Yuan
Published: August 2012

Background: MicroRNAs are master gene regulators that control gene expression through the posttranscriptional repression mechanism. Accumulating evidence indicates that microRNAs are often dysregulated in human cancers, and they may function as oncogenes or tumor suppressors. Our laboratory and other groups have previously shown that miR-145 is a tumor suppressor that is downregulated in a variety of tumors. However, it is not fully understood about the underlying mechanism of miR-145 regulation. In this study, we characterized miR-145 regulation involving transcription factors FoxO3a, C/EBP-β and p53 in cancer cells. Methods: We previously constructed the miR-145 promoter luciferase reporter in pGL3-basic, which was used in this study to determine the effect of FoxO3a, C/EBP-β or p53 on miR-145 promoter activity. p53 expression vector was constructed in pEGFP; C/EBP-β and FoxO3a were cloned into pCDH lentiviral vector carrying copGFP. Western blot and fluorescence microscope were used to determine expression of exogenous genes. Endogenous miR-145 levels were determined by TaqMan real time PCR after reverse transcription as well as by in situ hybridization. The expression vectors or luciferase reporter were introduced into cells by either transfection or infection. Results: We showed that miR-145 functions a downstream effector of Akt, and impacts tumor growth and invasion. For example, suppression of Akt by serum starvation or PI3K inhibitor LY29 significantly activates p53, which in turn binds to the miR-145 promoter and induces miR-145 expression. Furthermore, we showed that in addition to p53, FoxO3a also functions a positive regulator of miR-145. Of considerable interest, FoxO3a induces miR-145 expression in the mutant p53 background; however, in the wild type p53 background, p53 suppresses the ability of FoxO3a to induce miR-145. On the other hand, C/EBP-β serves as a negative regulator of miR-145 in both wild type p53 and mutant p53 backgrounds, which is likely in part through regulation of Akt activity. Experiments are underway to further dissect the regulation of miR-145. Conclusion: Together, these results suggest a sophisticated regulatory system controlling miR-145 expression in cancer cells. Thus, a better understanding of this regulatory network will aid in the identification of novel cancer biomarkers and therapeutic targets, and improve cancer diagnosis and treatment.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5025. doi:1538-7445.AM2012-5025
Abstract 5028: MicroRNA-212 targets multiple signaling pathways in prostate cancer
Authors: Ramalinga Malathi , Srivastava Anvesha , Dimtchev Alexander , Soldin Offie , Li James , Marian Catalin , Suy Simeng , Collins Sean P. , Kumar Deepak
Published: August 2012

Prostate cancer (PCa) is the most common type of cancer in men in the United States. While metastasis is the most common cause of death among PCa patients, no specific markers have been assigned to the severity of the disease. Identification of molecular factors could improve diagnosis and therapeutic intervention of PCa. MicroRNAs represent a promising new class of circulating biomarkers owing to their inherent stability and resilience. Studies in our lab have demonstrated that miR-212 is a differentially modulated circulatory microRNA in prostate cancer patients. Identification and validation of miRNA targets provides functional insights in mechanisms of prostate carcinogenesis and strategies for therapy. The present study was aimed to investigate and validate the targets for miR-212 to elucidate its role in prostate cancer and identify potential biomarkers and/or therapeutic targets for prostate cancer. Common mRNA targets of miR-212 were extracted from four web-based databases: TargetScan, miRanda, RNAhybrid, and PicTar. Some of the important common targets for miR-212 included DEDD, E2F5, GRM3, G2L1, SPRED1, SIRT1, SOS1, RASA1, PEA15, MYC, MAPK3, MAPK1 and HMGA2. Out of these SIRT1, SOS1, SPRED1, RASA1 were picked for further validation based on their documented role in carcinogenesis and tumor progression. Transfection studies with miR-212 mimics were performed in PC3 and LnCap prostate cancer cell lines. Western blotting was used to see the initial expression of various targets. 3′UTR-Luciferase reporter assay was performed for validating the target inhibition by miR-212. In addition, Cell proliferation assay and wound-healing assay were performed to evaluate the effects of miR-212 on PCa cell proliferation and migration. In summary the present study identifies and validates miR-212 targets and examines the functional significance of miR-212 in prostate cancer (PCa) cells lines. *Contributed equally.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5028. doi:1538-7445.AM2012-5028