bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2901: An integrated matrix factorization algorithm for DNA methylation and gene expression identi es HNSCC clinical subtypes and GLI1 signaling speci c to HPV-negative HNSCC.
Authors: Fertig Elana J. , Markovic Ana , Danilova Ludmila V. , Gaykalova Daria A. , Cope Leslie , Chung Christine H. , Califano Joseph A. , Ochs Michael F.
Published: August 2014

Global methylation changes occur heterogeneously in primary head and neck squamous cell carcinoma (HNSCC). To infer methylation-driven changes in expression in subtypes of HNSCC, we integrated global gene expression and DNA methylation measurements in 44 HNSCC and 25 normal samples using a matrix factorization algorithm. This algorithm incorporated novel models to transform methylation and expression measurements to a common scale. Although unsupervised, this integrated matrix factorization inferred patterns common to gene expression and methylation data that differentiate clinical subtypes within the 44 HNSCC samples, including notably HPV status and tumor site. Informatic analysis of the genes associated with these patterns identified specific pathway activity related to HPV status. This algorithm uniquely linked DNA methylation changes and reactivation of genes in the Hedgehog pathway predominantly in HPV-negative samples. We further examined the pathway by determining expression levels of GLI1, one of the downstream target genes of the Hedgehog pathway, in two independent cohorts. We confirmed that GLI1 expression was higher in tumors compared to normal samples and HPV-negative tumors had higher GLI1 expression compared to HPV-positive tumors, suggesting that increased expression of GLI1 plays a role in HPV-negative HNSCC.

Citation Format: Elana J. Fertig, Ana Markovic, Ludmila V. Danilova, Daria A. Gaykalova, Leslie Cope, Christine H. Chung, Joseph A. Califano, Michael F. Ochs. An integrated matrix factorization algorithm for DNA methylation and gene expression identi es HNSCC clinical subtypes and GLI1 signaling speci c to HPV-negative HNSCC. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2901. doi:10.1158/1538-7445.AM2013-2901
Abstract 2902: Global bioinformatic analysis for transcription factor genomic binding sites from ChIP-sequencing.
Authors: Fu Junjiang
Published: August 2014

The technique for Chromatin Immunoprecipitation (ChIP) combining with high-throughput DNA sequencing (ChIP-seq), is better than the ChIP-on-chip, which might reveal interactions between proteins and DNAs by the new generation sequencing technology. ChIP-seq can accurately reveal all possible binding sites in the whole cellular genome of the transcription factors or interesting proteins. TWIST, an important transcription factor which has a highly conserved, basic helix - loop - helix (bHLH) domain, can bind with DNA motif CANNTG (E-box). However, ChIP-seq will generate millions of data and the exactly E-boxes of TWIST in the whole genome is unknown. In this study, we performed the global bioinformatic analysis for transcription factor TWIST genomic binding sites from ChIP-seq result in cell lines. MACS software was used to identify TWIST binding sites from ChIP-seq data. MEME, a motif discovery tool, was used to search for statistically over-represented motifs in and near TWIST-occupied regions. There are 26,229 binding sites and 90,659 E-boxes in whole genome, and 42,390 E-boxes within the peak of 100bp in the binding sites. The majority of ChIP-seq signals could be parsimoniously explained by a CABVTG motif within ±200bp of ChIP summit and, of these, CAGCTG is most prevalent. Specifically, the characteristics of E-box mainly are CACATG, CATATG, CAGATG, CAGCTG and CACCTG. The CAGCTG is most prevalent E-box with 9% in total, and most E-boxes located within ±200bp of ChIP summit with more than 70% in total. Additionally, further analysis in chromosome 6 revealed 1936 binding sites and 6,437 E-boxes. We defined 88 genes in which binding sites located at promoter region in chromosome 6. Through gene function prediction, they are divided into three clusters. Some genes from the clusters function as tumor development and necrosis, which will provide us the clues for studying TWIST net work regulation and signal pathways in cancer biology. Therefore, our results showed that bioinformatics analysis from high resolution ChIP-seq data may be used to drive effective discovery and dissect all of binding sites for transcript factors, including TWIST, in silico.

This work was supported by NSFC (30371493, 81172049). We thank Drs. R Chen, J Xu and Ms. J Cheng.

Citation Format: Junjiang Fu. Global bioinformatic analysis for transcription factor genomic binding sites from ChIP-sequencing. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2902. doi:10.1158/1538-7445.AM2013-2902

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 2914: To study DNA repair function of two closely related human lung adenocarcinoma cell lines, CL1-0 and CL1-5.
Authors: Wu Feng- Chang , Lin Yu-Fen , Lai Liang-Chuan , Chuang Eric Y , Tsai Mong-Hsun
Published: August 2014

Exogenous agents and endogenous processes, such as reactive oxygen species (ROS) can induce base oxidation and DNA lesions. One of the most severe DNA lesions is the double-strand break (DSB). There are several repair systems to fix the resulting DNA lesions to prevent error copies of genetic information to be carried to daughter cells. Oxidative stress is an important factor to induce DNA damage during normal cellular metabolism. In this study, we wanted to use lung cancer cells, CL1-0 and CL1-5 cells, which have differential invasiveness ability to characterize DNA repair function. Hydrogen peroxide (H2O2) was treated in lung adenocarcinoma cells to induced DNA DSB. By using immunoflurecence microscope, we found that CL1-5 had more gamma-H2AX foci (DNA DSB marker) and ROS accumulation than CL1-0 post both 1 mM and 5 mM H2O2 treatment. Moreover, CL1-0 had a tight G2/M checkpoint and has a longer G2 arrest after H2O2 treatment, but not found in CL1-5. In addition, increasing sub-G1 population (apoptotic cells) was found in H2O2-treated CL1-5 but less in CL1-0. Furthermore, phosphorylated-Akt (S473) was decreased in CL1-5, In contrast, phosphorylated-ATM (S1981) and Chk2 were increased after 5 mM H2O2 treatment for 90 min. In addition, the level of GSH was persistently lower at the steady state and post H2O2 treatment in CL1-5 by LC-MS/MS analysis. Besides, the apoptotic cells were decreased but no differences in CL1-0 while supplement reduced-GSH before H2O2 treatment. We therefore hypothesize that there are different processes on dealing with ROS scavenging post oxidative stress between CL1-0 and CL1-5. Taken together, using hydrogen peroxide for treating two lung cancer cell lines seem to have different DNA repair function to regulate cellular function. In the following experiments, the activities of antioxidant enzymes, superoxide dismutase (SOD), catalase as well as glutathione peroxidase (GPx) post oxidative stress in CL1-0 and CL1-5 will be determined. Moreover, we will examine the level of GSH in different tumor cell lines to investigate whether GSH involved in tumor invasion ability.

Citation Format: Feng- Chang Wu, Yu-Fen Lin, Liang-Chuan Lai, Eric Y Chuang, Mong-Hsun Tsai. To study DNA repair function of two closely related human lung adenocarcinoma cell lines, CL1-0 and CL1-5. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2914. doi:10.1158/1538-7445.AM2013-2914

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 2916: NOXX: Friend or foe for cellular proliferation.
Authors: Dho So Hee , Kim Ji Young , Kim Chang-Jin , Nauseef William M. , Choi So-Young , Lee Kwang-Pyo , Kwon Ki-Sun
Published: August 2014

Reactive oxygen species (ROS) are signaling molecules that determine cellular fate. In tumor progression, ROS have been suggested to function as a dual cell controller. They could induce cellular proliferation in low levels while inhibiting cellular proliferation in high levels. ROS can be produced by NOXX, a member of NADPH oxidase family. Here, we present that an increase in NOXX levels by activation of STAT5 are critical in tumor progression by comparing the NOXX levels of breast and lung tumor tissues to those of normal tissues. Consistently, depletion of NOXX causes tumor suppression of lung and breast cancer cells, and expression of NOXX induces proliferation of normal lung cells. Notably, we found that cisplatin, an antineoplastic agent, increases NOXX activity and inhibits cellular proliferation. Specifically, cisplatin activates NOXX to induce a high level of ROS in multiple ways through (i) an increase in NOXX levels, (ii) an increase in levels of c-abl which is an activator of NOXX, and (iii) a release of Ca2+, the other activator of NOXX. Together, NOXX is a double-edged sword for tumorigenesis in lung and breast cancers by modulating ROS levels, and will be a promising target for cancer therapy.

Citation Format: So Hee Dho, Ji Young Kim, Chang-Jin Kim, William M. Nauseef, So-Young Choi, Kwang-Pyo Lee, Ki-Sun Kwon. NOXX: Friend or foe for cellular proliferation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2916. doi:10.1158/1538-7445.AM2013-2916
Abstract 2920: Effect of nitric oxide on invasiveness and metabolism of cancer cells.
Authors: Caneba Christine A. , Marini Juan , Nagrath Deepak
Published: August 2014

Nitric oxide synthase (NOS) expression has been shown to be upregulated in cancer cells compared to normal cells. The resulting increased nitric oxide (NO) production changes the metastatic ability of cancer cells. The link between NOS expression, NO production, apoptosis, and drug resistance in ovarian cancer is beginning to be studied. However, the effect of NO on metabolism and invasiveness of ovarian cancer cells is not known. Here, using gas chromatography - mass spectroscopy based isotope analysis we show that highly-invasive ovarian cancer cells convert arginine to citrulline via the nitric oxide pathway as opposed to less-invasive ovarian cancer cells. We hypothesize that NO is important for the proliferation, migration, invasion, and drug resistance in highly-invasive ovarian cancer cells as opposed to less-invasive ovarian cancer cells, and that these changes in metastatic behavior are linked to changes in metabolism. We show that L-NG-Nitroarginine Methyl Ester (L-NAME), an inhibitor of NOS, decreases NO production and thus decreases proliferation, migration, and invasion of ovarian cancer cells, as well as decreases drug resistance. Using metabolic assays, we show that NO affects the glucose consumption, lactate secretion, and pyruvate uptake, and provides us with clues regarding the effect of NO on glycolytic activity of highly-invasive and less-invasive ovarian cancer cells. Lastly, we show that NO affects oxygen consumption rate in these cells, and thus oxidative phosphorylation. Our findings are the first to link NO production and invasiveness with metabolism in ovarian cancer cells.

Citation Format: Christine A. Caneba, Juan Marini, Deepak Nagrath. Effect of nitric oxide on invasiveness and metabolism of cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2920. doi:10.1158/1538-7445.AM2013-2920
Abstract 2925: p90RSK2 coordinates pro-apoptotic and anti-apoptotic signaling pathways to protect cancer cells from anoikis.
Authors: Jin Lingtao , Li Dan , Lee Jongseok , Alesi Gina N. , Fan Jun , Kang Hee-Bum , Wang Dongsheng , Fu Haian , Taunton Jack , Boggon Titus J. , Tucker Meghan , Gu Ting-Lei , Chen Zhuo G. , Shin Dong M. , Khuri Fadlo R. , Kang Sumin
Published: August 2014

How invasive and metastatic tumor cells evade from anoikis induction remains unclear. We found that knockdown of RSK2 sensitizes diverse cancer cells to anoikis induction, which is mediated through phosphorylation targets, including apoptosis signal-regulating kinase 1 (ASK1) and cAMP response element-binding (CREB). We provide evidence to show that RSK2 inhibits ASK1 by phosphorylating S83, T1109 and T1326 through novel mechanism in which phospho-T1109/T1326 inhibits ATP binding to ASK1, while phospho-S83 attenuates ASK1 substrate MKK6 binding. Moreover, RSK2-CREB signaling pathway provides anti-anoikis protection by regulating gene expression of protein effectors that are involved in cell death regulation, including anti-apoptotic protein tyrosine kinase 6 (PTK6) and pro-apoptotic inhibitor of growth protein 3 (ING3). PTK6 overexpression or ING3 knockdown in addition to ASK1 knockdown further rescued the increased sensitivity to anoikis induction in RSK2 knockdown cells. These data together suggest that RSK2 functions as a signal integrator to provide anti-anoikis protection to cancer cells in both transcription-independent and -dependent manners, in part by signaling through ASK1 and CREB, and contributes to cancer cell invasion and tumor metastasis.

Citation Format: Lingtao Jin, Dan Li, Jongseok Lee, Gina N. Alesi, Jun Fan, Hee-Bum Kang, Dongsheng Wang, Haian Fu, Jack Taunton, Titus J. Boggon, Meghan Tucker, Ting-Lei Gu, Zhuo G. Chen, Dong M. Shin, Fadlo R. Khuri, Sumin Kang. p90RSK2 coordinates pro-apoptotic and anti-apoptotic signaling pathways to protect cancer cells from anoikis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2925. doi:10.1158/1538-7445.AM2013-2925
Abstract 2943: Lapatinib restores TRAIL-mediated apoptosis in TRAIL-resistant Triple Negative Breast Cancer (TNBC) through an off-target strategy that appears to be independent of increased death receptor expression.
Authors: Lim Bora , Dolloff Nathan G. , Allen Joshua E. , Dicker David T. , El-Deiry Wafik S.
Published: August 2014

Triple negative breast cancer (TNBC) comprises 15-20% of breast cancer, and carries a poor prognosis. TRAIL (Tumor Necrosis Factor-related apoptosis inducing ligand), a member of the TNF-alpha family of death receptor ligands, induces apoptosis by binding death receptors (DR4 and DR5), and can be a good therapeutic target in TNBC based on the work of Lipkowitz at the NCI. Lapatinib, a dual HER2 and EGFR inhibitor has been shown to sensitize colon cancer and GBM cells to TRAIL-induced apoptosis through an off-target effect that involves JNK. In colon cancer cell lines, this sensitization was found to be due to increased DR4 and DR5 protein levels, and this was not affected by HER2 and EGFR dual specific inhibition. The majority of breast cancer cell lines including HCC 1937, a BRCA1-deficient TNBC cell line derived from a patient with BRCA1 mutation, are known to have constitutive death receptor endocytosis resulting in resistance to TRAIL. We hypothesized that treatment of TNBC cells with lapatinib may induce sensitization to TRAIL-induced apoptosis in normally TRAIL-resistant cell lines, but possibly not by increased death receptor protein expression since these cells have defective cell surface expression. MDA-MB-231 and HCC 1937 cell lines are both well-validated TNBC cell lines. MDA-MB-231 is known to be sensitive to TRAIL, whereas HCC 1937 is resistant to TRAIL. These two cell lines were pre-treated with 0-10 μM lapatinib for 48 hrs. Cells were then treated with His-tagged recombinant TRAIL (50 ng/mL) for 24 hr subsequently. Cell Titer Glo assay, and sub-G1 DNA content analysis by flow cytometry showed increments of apoptosis after lapatinib pre-treatment. This change was more prominent in HCC 1937, which is normally resistant to TRAIL-induced apoptosis. However western blotting did not show increased DR4/DR5 by pre-treatment with lapatinib in either MDA-MB-231 or HCC 1937. This result suggests that we may have identified an off-target effect of lapatinib in TNBC that appears to not involve increased death receptor expression. We are further exploring the previously described role for JNK in the off-target effect to further elucidate this TRAIL death receptor- and Her2/EGFR-independent mechanism of TRAIL sensitization by lapatinib that has implication for TNBC therapy.

Citation Format: Bora Lim, Nathan G. Dolloff, Joshua E. Allen, David T. Dicker, Wafik S. El-Deiry. Lapatinib restores TRAIL-mediated apoptosis in TRAIL-resistant Triple Negative Breast Cancer (TNBC) through an off-target strategy that appears to be independent of increased death receptor expression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2943. doi:10.1158/1538-7445.AM2013-2943
Abstract 2948: c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK) are involved in local anesthetics-induced apoptosis in human thyroid cancer cells.
Authors: Cheng Shih-Ping , Chang Yuan-Ching , Hsu Yi-Chiung , Huang Shih-Yuan , Hu Meng-Chun , Chi Chin-Wen , Yu Sung-Liang
Published: August 2014

Local anesthetics are known to induce apoptosis in clinically relevant concentrations. However, it is unknown whether the effects are also present in thyroid cancer cells and the mechanisms are not well uncharacterized. We sought to explore the effects of lidocaine and bupivacaine on thyroid cancer cells in terms of cell growth, colony formation, cell cycle, induction of apoptosis, and mitochondrial injury. At 24 hour, lidocaine had an IC50 value of 6.8 mM to 7.3 mM, whereas bupivacaine had an IC50 value of 1.3 mM to 3.1 mM. In search for a molecular mechanism, based on microarray and pathway analysis, we determined that apoptotic activity is mediated by the c-jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK). Our data suggest that induction of apoptosis by local anesthetics has the potential to improve the regional control of thyroid cancer.

Citation Format: Shih-Ping Cheng, Yuan-Ching Chang, Yi-Chiung Hsu, Shih-Yuan Huang, Meng-Chun Hu, Chin-Wen Chi, Sung-Liang Yu. c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK) are involved in local anesthetics-induced apoptosis in human thyroid cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2948. doi:10.1158/1538-7445.AM2013-2948
Abstract 295: PPARγ-induced KLF4 in regulation of cell growth and migration in human hepatoma cells.
Authors: Hsu Hui-Tzu , Sung Ming-Ta , Chi Chin-Wen
Published: August 2014

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Recently, a zinc finger transcription factor Krüppel-like factor 4 (KLF4) was found to be reduced in tumors of HCC patients. Additionally, results from our laboratory showed that overexpressed KLF4 repressed epithelial-mesenchymal transition (EMT) process and cell motility by induction of E-cadherin expression and reduction of EMT markers in hepatoma cells. Interestingly, it has been found that PPARγ agonists upregulated KLF4 in colorectal cancer cells. However, the mechanism of PPARγ-induced KLF4 in the EMT process of HCC remains unclear. In this study, Hep3B, PLC/PRF/5, and Mahlavu human hepatoma cells were treated with PPARγ agonists rosiglitazone, troglitazone, and PPARγ antagonist GW9662, respectively. After rosiglitazone or troglitazone treatment for various time periods, growth inhibition was observed in both Hep3B and PLC/PRF/5 cells at 48 hours by surforhodamine B assay. Mahlavu cells showed less response to rosiglitazone and troglitazone treatment as compared to the other two cell lines. However, pretreatment of GW9662, a PPARγ antagonist, prior to PPARγ agonists treatment did not block the PPARγ agonists-induced cell growth inhibition. Moreover, cotreatment of PPARγ agonist and antagonist showed additive growth inhibition effect. In addition, rosiglitazone and troglitazone inhibited the migration of Hep3B, PLC/PRF/5 and Mahlavu cells differentially in transwell assay. Western blotting analysis showed upregulation of KLF4 and E-cadherin in Hep3B and PLC/PRF/5 cells after rosiglitazone and troglitazone treatment. Interestingly, decreased KLF4 and E-cadherin were observed in Hep3B, PLC/PRF/5, and Mahlavu cells after GW9662 treatment, indicating that the regulation of KLF4 and E-cadherin were PPARγ-dependent. Moreover, GW9662 also inhibited cell migration of Hep3B, PLC/PRF/5, and Mahlavu cells, with a concomitant decrease of vimentin. These results together suggested that PPARγ agonists and antagonist not only inhibited the growth but also decreased migration of hepatoma cells. Modulation of the PPARγ agonists induced expression of KLF4 and E-cadherin played important role in migration of hepatoma cells.

Citation Format: Hui-Tzu Hsu, Ming-Ta Sung, Chin-Wen Chi. PPARγ-induced KLF4 in regulation of cell growth and migration in human hepatoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 295. doi:10.1158/1538-7445.AM2013-295
Abstract 2961: The N-terminal projection domain of the microtubule associated protein tau inhibits TNFα signaling.
Authors: Debnath Shawon , Hannon Peter , Toropova Kristina , Rodriguez Nelson Nunez , Alonso Alejandra , Fata Jimmie Eugene
Published: August 2014

Tumor Necrosis Factor-alpha (TNFα) is a multifunctional pro-inflammatory cytokine implicated in inducing both tumor promotion and tumor inhibition. This paradoxical effect can be partly explained by the fact that TNFα acts as a regulator of both proliferation and apoptosis; however the mechanisms and factors associated with how or whether a tumor cell responds to a microenvironment rich in TNFα have not been fully resolved. Here we provide evidence that the microtubule associate protein Tau inhibits TNFα signaling through its N-terminal domain and implicate it as a novel regulator of the cellular response to this key cytokine. Our findings indicate that over-expression of Tau in the human breast cancer cell lines MCF7, SkBr3 and in the pheochromocytoma cell line PC12 suppresses TNFα induced apoptosis evident in the control cells. TNFα induced cell apoptosis in control cells is triggered as early as 6 hrs post-treatment with hallmark features of cell shrinkage, membrane blebbing, disruption of tubulin architecture and caspase-3 activation. While over-expression of wild type full length Tau isoform in these cell lines, completely abrogates all TNFα associated apoptotic phenotypes. Aside from inhibiting TNFα induced apoptosis, Tau over-expression also interferes with TNFα induced NFκB activation. This is evident by a significant decrease in NFκB nuclear translocation in Tau over-expressed cells compared to controls. Suppression of TNFα signaling by Tau is not associated with its tubulin interaction since a truncated N-terminal projection domain lacking all microtubule binding domains still provides resistance to TNFα induced apoptosis and NFκB activation. These findings lead us to conclude that the functional domain of Tau that regulates TNFα response lies within its N-terminal region and current mechanistic studies indicate this region inhibits TNFα receptor clustering, which is needed for signaling. Our data provide direct evidence that the N-terminal domain of the microtubule associated protein Tau can inhibit TNFα signaling in tumor cells. These findings indicate a novel function for Tau and add to our knowledge of how a tumor cell responds to TNFα signaling.

Citation Format: Shawon Debnath, Peter Hannon, Kristina Toropova, Nelson Nunez Rodriguez, Alejandra Alonso, Jimmie Eugene Fata. The N-terminal projection domain of the microtubule associated protein tau inhibits TNFα signaling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2961. doi:10.1158/1538-7445.AM2013-2961

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 2978: DNA methylation changes during epithelial-to-mesenchymal transition in ovarian cancer cells.
Authors: Cardenas Horacio , Goswami Chirayu , Nephew Kenneth P. , Matei Daniela
Published: August 2014

Epithelial-to-mesenchymal transition (EMT) is a required step in the process of metastasis and has been linked to cancer cell stemness. We hypothesized that aside from defined genetic changes associated with EMT, epigenetic mechanisms are also involved. We determined DNA methylation changes in SKOV3 ovarian cancer (OC) cells undergoing TGFβ-induced EMT by using the Infinium HumanMethylation450 array. Methylation of sites was expressed as ß-values ranging from 0 (completely unmethylated) to 1 (completely methylated). Methylation of 468 and 390 sites changed significantly (P<.01, ANOVA and FDR<0.05) after 48h and 120h of TGFβ stimulation, of which 31 and 72 sites changed >1.25-fold. The majority of changes (∼66%) reflected TGFβ-induced demethylation. Methylation of 160 sites changed similarly at 48 and 120h indicating that approximately 40% of changes were sustained. PCA analysis demonstrated that the removal of TGFβ was associated with reversal of DNA methylation changes at most sites and restitution of the baseline methylome. Pathway analysis identified DNA methylated genes involved in functional networks relevant to EMT and cancer progression, including: 1) cell morphology and development (39 genes), 2) cell growth and proliferation, and cell death and survival (34 genes), 3) cellular assembly and organization (22 genes), 4) DNA replication, recombination and repair (17 genes), and 5) gene expression, cell signaling, and cellular movement (15 genes). Validation of the relevant genes is ongoing. Treatment of OC cells with the DNA methyl transferase inhibitor SGI-110 prevented TGFβ-induced EMT and reduced by two-fold the number of cells with stem cell characteristics (ALDH1+). These results demonstrate that DNA methylation is a dynamic process involved in regulation of genes implicated in EMT and metastasis.

Citation Format: Horacio Cardenas, Chirayu Goswami, Kenneth P. Nephew, Daniela Matei. DNA methylation changes during epithelial-to-mesenchymal transition in ovarian cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2978. doi:10.1158/1538-7445.AM2013-2978
Abstract 2985: The histone demethylase JMJD1A induces neuroblastoma cell migration and invasion.
Authors: Ling Dora , Tee Andrew , Nelson Charlotte , Xu Ning , Atmadibrata Bernard , Liu Pei Y. , Pasquier Eddy , Marshall Glenn M. , Suzuki Takayoshi , Zhang Yi , Liu Tao
Published: August 2014

Patients with neuroblastoma due to N-Myc oncogene-amplification die of tumour metastasis. However, little is known about how N-Myc induces cell migration, invasion and metastasis. The histone demethylase JMJD1A activates target gene transcription by demethylating lysine

9 residues of histone H3 (H3K9). Here we demonstrated that N-Myc up-regulated the expression of JMJD1A in N-Myc oncogene-amplified human neuroblastoma cells by directly binding to JMJD1A gene promoter. Affymetrix gene array studies revealed that the gene most significantly up-regulated by JMJD1A was the long intergenic noncoding RNA Metastasis-associated Lung Adenocarcinoma Transcript 1 (MALAT1).

Consistent with this finding, chromatin immunoprecipitation showed that JMJD1A bound to MALAT1 gene promoter. Importantly, JMJD1A siRNAs, MALAT1 siRNAs and small molecule JMJD1A inhibitors impeded neuroblastoma cell migration and invasion. Taken together, our data identify a novel pathway through which N-Myc causes neuroblastoma cell migration, invasion and potentially metastasis.

Citation Format: Dora Ling, Andrew Tee, Charlotte Nelson, Ning Xu, Bernard Atmadibrata, Pei Y. Liu, Eddy Pasquier, Glenn M. Marshall, Takayoshi Suzuki, Yi Zhang, Tao Liu. The histone demethylase JMJD1A induces neuroblastoma cell migration and invasion. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2985. doi:10.1158/1538-7445.AM2013-2985
Abstract 2989: Loss of methylation in a regulatory region of BCL2 in MDS/AML.
Authors: Lee Sangmin , Galili Naomi , Nimer Stephen , Minden Mark D. , Murty Vundavalli V. , Jhanwar Suresh , Raza Azra , Tycko Benjamin
Published: August 2014

Background:

Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic disorders characterized by ineffective hematopoiesis and increased risk of progression to acute myeloid leukemia (AML). A hallmark of MDS is increased apoptosis, while AML undergoes decreased apoptosis and increased proliferation. BCL2, an anti-apoptotic protein, is overexpressed in high grade MDS and AML and may confer poor prognosis and increased chemotherapy resistance. In this study we examined the DNA methylation status of BCL2 in MDS and AML.

Methods:

We conducted DNA methylation profiling on genomic DNA extracted from bone marrows of 22 refractory anemia with ringed sideroblasts (RARS), 8 refractory anemia with excess blasts (RAEB), 21 de novo AML, 21 therapy related AML, and 7 normal volunteers. We used both Illumina 27K BeadChip and 450K BeadChip methylation arrays to assess CpG methylation. Standard student T-test was used to screen differentially methylated MDS and AML CpG sites compared to normal samples. We validated the microarray data by combined bisulfite restriction analysis (COBRA) and bisulfite sequencing.

Results:

We identified a region in the BCL2 gene body that was among the most differentially methylated in MDS and AML versus normal bone marrow. Within that amplicon, BCL2 in both RARS, a low grade MDS, and RAEB, a high grade MDS, were hypermethylated compared to normal bone marrow (43% vs 18%, 48% vs 18%, respectively, p<0.05). BCL2 in de novo AML and therapy related AML were hypomethylated compared to normal bone marrow (12% vs 35%, 13% vs 35%, respectively, p<0.05). We validated these results by COBRA and bisulfite sequencing. This region coincided with H3K27ac and H3K4m1 enhancer type marks in ENCODE data in normal cells.

Conclusions:

Our study identifies a region of BCL2 that is hypermethylated in both low and high risk MDS while hypomethylated in AML compared to normal bone marrows. We hypothesize aberrant epigenetic modification of BCL2 plays a key role in apoptosis and transformation from MDS to AML, and our data support the idea of combining demethylating drugs with BCL2 antagonists. We are currently investigating whether BCL2 protein expression correlates with methylation, and plan to map the histone marks with CHIP-seq to correlate chromatin accessibility and DNA methylation in BCL2.

Citation Format: Sangmin Lee, Naomi Galili, Stephen Nimer, Mark D. Minden, Vundavalli V. Murty, Suresh Jhanwar, Azra Raza, Benjamin Tycko. Loss of methylation in a regulatory region of BCL2 in MDS/AML. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2989. doi:10.1158/1538-7445.AM2013-2989
Abstract 3: Molecular markers of prostate cancer: Investigation of microRNA profiles as a marker in patients with high-risk prostate cancer.
Authors: Stegmaier Petra , Kirste Simon , Mo Xiaokui M. , Zynger Debra L. , Chakravarti Arnab , Hlavin Bell Erica
Published: August 2014

Purpose:

The aim of this study was to investigate the microRNA (miRNA) profile in prostate carcinoma as biomarkers to identify high-risk prostate cancer patients who may benefit from post-prostatectomy radiation therapy using global miRNA profiling.

Method:

Tumor from Formalin-Fixed Paraffin-Embedded (FFPE) tissues were obtained from 9 radical prostatectomy specimens resected for high-risk prostate cancer, to perform a pilot study using the Nanostring miRNA platform from isolated RNA. An optimized protocol utilizing the Ambion RecoverAll digestion buffer and the Qiagen miRNeasy FFPE kit was used to isolate total RNA. Each prostatectomy specimen was marked by a genitourinary pathologist and 1 mm cores of enriched areas of tumor or normal epithelial or stromal tissue were obtained. To analyze global miRNA expression, the Nanostring nCounter human miRNA Expression Assay Kit was used. The data was further analyzed in collaboration with The Ohio State University Center for Biostatistics.

Results:

The expression of miRNA in prostate tumor samples was compared to normal epithelium. It showed that seven miRNAs (miR-375, miR-18a, miR-25, miR-183, miR-641, miR-15b, miR-548a-3p) that were >1.5-fold upregulated and 4 miRNAs (miR-660, miR-1915, miR-200b, miR-205) <1.5-fold downregulated. Three (miR-375, miR-183, miR-205) were in accordance with previous studies published on prostate cancer. Expression of six up-regulated miRNAs (miR-205, miR-877, miR-K12-4-5p, miR-548b-3p, miR-34c-5p, ebv-miR-BART10) correlated with high risk (T>3 and/or Gleason score ≥ 8) and five miRNAs (up-regulated: miR-10b, miR-205; down-regulated: miR-574-5p, miR-30c, miR-499-5p) with pathological tumor stage 2 and 3.

Conclusion:

Our results suggest great potential for the use of miRNA profiling as a novel diagnostic and prognostic biomarker in defining the signatures of prostate cancer at different stages. We are currently completing the RNA isolation and downstream analyses in correlation with clinical data for 80 additional high-risk prostate cancer cases from the University hospital of Freiburg, Germany.

Citation Format: Petra Stegmaier, Simon Kirste, Xiaokui M. Mo, Debra L. Zynger, Arnab Chakravarti, Erica Hlavin Bell. Molecular markers of prostate cancer: Investigation of microRNA profiles as a marker in patients with high-risk prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3. doi:10.1158/1538-7445.AM2013-3
Abstract 3005: Cdca7l is a sex-specific modifier of astrocytoma and glioblastoma.
Authors: Reilly Karlyne M. , Lee Min-Hyung , Schaick Jessica Amlin-Van
Published: August 2014

Astrocytoma and glioblastoma-multiforme (GBM) are the most common primary brain tumors and are currently incurable. Both astrocytoma and GBM show male-predominance, with a male-to-female ratio of 1.3:1, respectively. Previously, our lab has demonstrated that astrocytoma/GBM-tumorigenesis in the Nf1-/+;Trp53-/+cis (NPcis) mouse model shows gender-bias in certain genetic contexts. Using linkage-analysis in the NPcis mouse, we have identified a male-specific genetic modifier locus on distal chromosome 12, named Arlm1 for Astrocytoma-resistance-locus-in-males1. To identify candidate genes for the Arlm1 modifier of glioma risk, we used combinational bioinformatics approaches and cross-species comparisons, identifying Cell-division-cycle-associated-7-like (CDCA7L) as a strong candidate for a male-specific astrocytoma susceptibility gene among 503 candidate genes. To investigate the role of CDCA7L in astrocytoma/GBM, we analyzed CDCA7L in both human and mouse astrocytoma cell lines in both genders. CDCA7L expression was upregulated in astrocytoma cells compared to normal brain with male-predominance. shRNA-mediated CDCA7L-silencing in male-derived astrocytoma cells led to decrease of both cell growth and viability in both human and mouse. Further mechanistic study revealed that CDCA7L-depletion in male-derived astrocytoma cells was connected with the induction of Caspase3-activation and reduction of CyclinD1-expression, suggesting the oncogenic role of CDCA7L in astrocytoma/GBM. On the other hand, CDCA7L-overexpression in female-derived astrocytoma cells showed less degree of cell proliferation and viability increases. Strikingly, CDCA7L-depletion in human female-derived astrocytoma cell line, U87MG caused increase of both cell growth and viability. These data highlight the sex-specificity of CDCA7L in astrocytoma/GBM tumorigenesis and provide evidence for the first modifier of male-specific susceptibility to brain tumors.

Citation Format: Karlyne M. Reilly, Min-Hyung Lee, Jessica Amlin-Van Schaick. Cdca7l is a sex-specific modifier of astrocytoma and glioblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3005. doi:10.1158/1538-7445.AM2013-3005
Abstract 3007: Down-regulation of HOXC6 in serous ovarian cancer.
Authors: Tait David L. , Bahrani-Mostafavi Zahra , Vestal Carol , Richardson Christine , Mostafavi M. Taghi
Published: August 2014

Objective: The Homeobox (HOX) family of genes consists of 39 genes encoding transcription factors important to morphogenesis and cell differentiation. Usually inactive in normal differentiated tissues, dysregulation of HOX genes has been reported in several malignancies. HOX genes have been linked to genes important in malignant transformation including vascular endothelial growth factor, Ras, interleukin-8, and fibroblast growth factor. We have previously reported up-regulation of HOXA2, HOXA7 and HOXB7 in ovarian cancer. Little is known about HOXC6 gene expression in ovarian cancer. In this study we evaluated HOXC6 expression in normal and malignant human ovarian tissue to characterize expression of HOXC6 in serous ovarian cancer.

Methods: Microarray analysis of mRNA from LCM captured cells from human ovarian tissues was performed on 11 samples of normal, and malignant ovarian tissues. These samples were analyzed using the Affymetrix Human EXON 1.0 ST microarray to distinguish the differential pattern of mRNA expression between malignant and normal samples. Real-time reverse transcription PCR was utilized to confirm down-regulation of HOXC6 genes as determined by microarray analysis. HOXC6 protein in tissue was evaluated by immunohistochemistry.

Results: Microarray analysis demonstrated significant down-regulation of the HOXC6 gene in malignant serous ovarian tumor samples compared to normal ovarian epithelial cells of control samples. RT-PCR and immunohistochemistry confirmed HOXC6 down-regulation in malignant samples. A 115-fold difference in malignant and normal primary tissue samples and cell lines was seen in the RT-PCR analysis. By immunohistochemistry nuclear staining of HOXC6 protein was seen in the nuclei of normal ovarian surface epithelial tissues but significantly reduced or absent in malignant ovarian samples.

Conclusion: HOXC6 is significantly differentially expressed comparing normal ovarian tissue and serous malignant ovarian tissue based on both tissue mRNA and protein analysis. This finding of HOXC6 down-regulation in ovarian cancer is unique and opposite of the up-regulation of other HOX genes previously reported. Down-regulation of HOXC6 in serous ovarian cancer may be an important finding relative to oncogenic pathways and biomarker development. This suggests dysregulation of HOXC6 genes may be an early event in malignant transformation and additional studies to validate the role of the HOXC6 gene in ovarian cancer are warranted.

Citation Format: David L. Tait, Zahra Bahrani-Mostafavi, Carol Vestal, Christine Richardson, M. Taghi Mostafavi. Down-regulation of HOXC6 in serous ovarian cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3007. doi:10.1158/1538-7445.AM2013-3007
Abstract 3011: The role of peroxidasin in human prostate cancer.
Authors: Woods Keith
Published: August 2014

Peroxidasin is a peroxidase associated with kidney disease and its function is not totally known. Peroxidasin was identified first in the common fruit fly, or Drosophilia, where its function has not been identified, but is expressed in different stages of development. When peroxidasin was identified in the human system, it was found in colon cancer cell lines as the product of a p-53 response gene. It was originally called melanoma gene 50 because it was speculated to be a potent-melanoma associated antigen. In previous studies, we have found using real-time PCR array, that peroxidasin is up regulated 300-fold in ARCaP prostate cancer cell line overexpressing Snail transcription factor. Snail is the transcriptional factor that is involved in Epithelial-Mesenchymal Transition, or EMT, which is associated with increased migration and metastasis. Snail down-regulates cell-cell association genes like E-cadherin and up-regulates mesenchymal genes like vimentin. We hypothesized that the enzyme, peroxidasin, will be one of the causes for increased cellular migration and increased tumor progression in aggressive prostate cancer. By using Western Blot, immunofluorescence and immunohistochemical analyses, peroxidasin presence was examined in normal prostate epithelial tissue, prostate cancer patient tissue, non-metastatic and metastatic prostate cancer cell lines. We also utilized LNCaP and ARCaP prostate cancer cell lines that were modified to over-express Snail to better ascertain the regulation of peroxidasin by Snail in these cell lines. So far, we have found that peroxidasin is increased in prostate cancer patient tissue as compared to normal epithelial tissue. Peroxidasin is also expressed at varying levels in prostate cancer cell lines. LNCaP and ARCaP Snail-transfected cells also expressed more peroxidasin as compared to empty vector Neo control cells. Migration, invasion, and adhesion assays will be performed to examine the role of peroxidasin in these biological pathways. Based on the results, future studies will focus on the migration of prostate cancer cells towards nerve cells and if peroxidasin presence is significant to this process. Overall, we have shown that peroxidasin is highly expressed in prostate cancer and its expression is mediated by Snail transcription factor.

Supported by grants P20MD002285-01 & G12RR003062-22.

Citation Format: Keith Woods. The role of peroxidasin in human prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3011. doi:10.1158/1538-7445.AM2013-3011
Abstract 3018: Identification of genes up-and down-regulated by dermcidin in G-361 malignant melanoma cell line.
Authors: Belizario Jose E. , Perez Marcela
Published: August 2014

Dermcidin (DCD) is a human gene mapped to chromosome 12q13 region, only identified in primates and humans, and normally expressed in the eccrine glands of skin and brain. Several studies have confirmed DCD-derived peptides contribute to innate and immune surveillance and in the oncogenic processes of breast, prostate and skin cancers, as revealed by its role as a growth factor and cell survival. We have further explored DCD function and its tumorigenic potential on skin melanocytes by specifically knocking down its expression in G-361 malignant melanoma cells via expressing constitutively short hairpin RNA against DCD mRNA. Biological and biochemical assays showed that the "knockdown" in the expression of DCD in two cell clones decreased significantly the in vitro growth in cell culture and tumor formation in nude mice. Similar results were obtained treating nude mice bearing G-361 melanoma xenografts with rabbit polyclonal antibodies against DCD protein (1 mg/Kg, i.p., once week, and one month). Here, we present a summary of a DNA microarray-based study that identified the genes that are up- and down-regulated in a G-361 pLKO control clone and a clone expressing constitutively short hairpin RNA against DCD mRNA. A total of 213 genes were differentially and repeatedly expressed in the G-361-DCD-RNAi clone IBC-I (>3-fold change and P < 0.005). Relative to pLKO vector control data profiling, 99 genes were up-regulated and 120 genes were down-regulated. Next, we validated various genes by real time-PCR. Systematic functional analysis of the differentially expressed genes using DAVID platform revealed significant enrichment for U1-U5 spliceosomal complex genes (SNORs) required for post-transcriptional processing of rRNA, mRNA and tRNA as well as small nucleolar RNA and non-coding RNAs (ncRNAs). Further studies are on way to prove the functional connections among ncRNAs in DCD-induced the development and progression of melanoma malignancy.

Citation Format: Jose E. Belizario, Marcela Perez. Identification of genes up-and down-regulated by dermcidin in G-361 malignant melanoma cell line. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3018. doi:10.1158/1538-7445.AM2013-3018