bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1776: Lymphomas associated with aberrant DNA rearrangements are suppressed by Mre11 mutation.
Authors: Smith Cheryl J. , Xie Jeffrey , Ferguson David O. , Sekiguchi JoAnn M.
Published: August 2014

Many translocations associated with spontaneous human lymphoid malignancies are mediated by short regions of sequence homology (microhomology) at the breakpoints. The hallmark features of this error prone pathway are deletions of flanking sequence and short homologies of 1-25 bases at the junctions that serve to align the DNA ends prior to joining. In addition to its importance in the etiology of human cancers, microhomology-mediated joining or alternative NHEJ (aNHEJ) also appears to play a significant role in therapy-induced oncogenic translocations.

Studies have provided compelling evidence that the DNA end-binding complex composed of Mre11 (nuclease), Rad50 (tetherer) and Nbs1 (signaler)–MRN–functions in the context of the aNHEJ pathway. In addition, in vivo and cell based studies have identified that deficiencies in components of the MRN complex reduce aNHEJ activity. However, it is not known whether the MRN complex participates in the generation of aNHEJ catalyzed translocations that can predispose to cancer. Therefore, our studies seek to better understand the contribution of Mre11/MRN to the aNHEJ pathway and thus, in generation of translocations.

We have obtained mice harboring an Mre11 conditionally targeted null allele as well as an Mre11 knock-in mutation that abrogates nuclease activity. We have included mutant mice in this study that succumb to early onset Pro-B lymphomas associated with clonal translocations containing microhomologies of 2-8 nucleotides. Thus, the spontaneously arising tumors in this mutant background represent a valuable in vivo system to assess the involvement of Mre11/MRN in aNHEJ mediated translocations.

We have determined that Mre11/MRN inactivation in the B-cell lineage suppresses Pro-B lymphoma in this mutant background. In future experiments we are investigating the impact of Mre11/MRN inactivation on various cancer phenotypes. Together, these studies will provide insights into the mechanisms underlying human lymphoma and may ultimately provide insights into targets for improved treatment of lymphoid malignancies.

Citation Format: Cheryl J. Smith, Jeffrey Xie, David O. Ferguson, JoAnn M. Sekiguchi. Lymphomas associated with aberrant DNA rearrangements are suppressed by Mre11 mutation. [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 1776. doi:10.1158/1538-7445.AM2013-1776
Abstract 1782: Regulation of ATM-dependent DNA damage signaling in human breast cancer cells by the RhoGEF Net1A .
Authors: Oh Wonkyung , Frost Jeffrey A.
Published: August 2014

The Neuroepithelioma transforming gene 1 (Net1) is a RhoGEF that is frequently overexpressed in human cancer, including breast cancer. We have previously reported that DNA damage activates Net1 to control p38 MAPK signaling and protect cells from ionizing radiation (IR) induced apoptosis. However, others have shown that Net1 activation contributes to cell death by activating a RhoB-Bim signaling pathway. Thus, the role of Net1 in controlling IR responses and cell survival is controversial. Here we show that the Net1A isoform is uniquely important for DNA double-strand break (DSB)-induced signaling and DNA repair. SiRNA-mediated knockdown of Net1A in human breast cancer cells reduced IR-stimulated ATM activation and signaling to its substrates Chk2 and H2AX. In addition, suppression of Net1A expression adversely affected cell survival after IR. We also observed that Net1A overexpression significantly reduced γH2AX foci formation after IR, which required the unique N-terminal region of Net1A. Importantly, this effect did not require Rho GTPase activation by Net1A, and was not recapitulated by overexpression of RhoA or RhoB. Using comet assays, we found that Net1A depletion substantially slowed DNA DSB repair in response to IR. Intriguingly, knockdown of Net1A promoted non-homologous end joining (NHEJ) in the absence of DNA damage, and Net1A was found to co-immunoprecipitate with the DNA-PK complex in an IR-regulated manner. Taken together, these data suggest a model in which Net1A functions as a non-catalytic binding protein to control DNA damage response signaling and DNA repair to affect cell survival after IR.

Citation Format: Wonkyung Oh, Jeffrey A. Frost. Regulation of ATM-dependent DNA damage signaling in human breast cancer cells by the RhoGEF Net1A . [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 1782. doi:10.1158/1538-7445.AM2013-1782
Abstract 1790: Mutagenic effects of inorganic particulate matter on Raji and HepG2 cell lines exposed to ultraviolet radiation.
Authors: Xiao Michael , El-Naggar Atif , Helsing Albert V. , Lynch Philip M. , Alegre Melissa M. , Robison Richard A. , O'Neill Kim L.
Published: August 2014

Epidemiological studies have correlated exposure to ultraviolet-irradiated particulate matter with cardiovascular, respiratory, and malignant lung diseases. This study investigated the DNA damage induced by two major inorganic particulate matter compounds found in diesel exhaust, ammonium nitrate and ammonium sulfate on Burkitt's lymphoma (Raji) and hepatocellular carcinoma (HepG2) cell lines. We found a dose-dependent positive correlation of accumulated DNA damage in concentrations of ammonium nitrate and ammonium sulfate (25 μg/ml, 50 μg/ml, 100 μg/ml, 200 μg/ml, 400 μg/ml) with ultraviolet exposure (250 J/m2, 400 J/m2, 600 J/m2, 850 J/m2), as measured by the Comet Assay in both cell lines. There was a significant difference between the treated ammonium nitrate samples and negative control samples in Raji and HepG2 (p < 0.001). Apoptosis was shown in Raji and HepG2 cells when exposed to high concentrations of ammonium nitrate (200 μg/ml, 400 μg/ml) for 1 hour in samples without ultraviolet exposure, as assessed by the Comet Assay. However, the quantity of apoptotic cells greatly diminished after ultraviolet exposure at these concentrations. To further investigate and clarify the underlying mechanisms, we used Annexin V to quantify apoptosis. Flow Cytometry confirmed the results shown by the Comet Assay; ultraviolet exposure diminished apoptosis in ammonium nitrate-induced DNA damage in both Raji and HepG2. Ammonium sulfate induced DNA damage with or without ultraviolet influence was minimal as assessed by the Comet Assay in both cell lines, with a significant difference (p < 0.05) between the treated and negative control samples. These results indicate that the inorganic particulate compound, ammonium nitrate, induced DNA strand breaks at all concentrations and apoptosis at high concentrations in Raji and HepG2 cells, with ultraviolet radiation preventing apoptosis at high concentrations. We hypothesize that ultraviolet radiation inhibits an essential cellular mechanism, possibly involving p53, thereby explaining this phenomenon. Further studies are necessary to characterize the roles of apoptosis inhibition in DNA damage caused by inorganic particulate matter.

Citation Format: Michael Xiao, Atif El-Naggar, Albert V. Helsing, Philip M. Lynch, Melissa M. Alegre, Richard A. Robison, Kim L. O'Neill. Mutagenic effects of inorganic particulate matter on Raji and HepG2 cell lines exposed to ultraviolet radiation. [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 1790. doi:10.1158/1538-7445.AM2013-1790
Abstract 1793: ADAR1 regulates ARHGAP26 expression by RNA editing through destruction of the miR-30b-3p and miR-573 target sites.
Authors: Wang Qiong , Guo Zhendong , Zhang Weina , Wang Li
Published: August 2014

Rho GTPase activating protein 26 (ARHGAP26) is a putative tumor suppressor gene. It was observed to negatively regulate the small GTP-binding protein RhoA and be down-regulated in myeloid malignancies and metastatic brain cancers. Herein, we demonstrated that ARHGAP26 transcript undergoes extensive A-to-I RNA editing catalyzed by ADAR1 in its 3’-UTR. Either knocking down of ADAR1 by silencing or overexpression by ADAR1 expression vector decreased or increased the expression level of ARHGAP26 gene. Furthermore, luciferase assay showed that miR-30b-3p and miR-573 targeted the ARHGAP26, but the RNA editing abolished the effect of miR-30b-3p or miR-573. In addition, we observed that the level of the A-to-I RNA editing on ARHGAP26 transcript decreased significantly in human brain cancer tissues comparing to their controls, which contributes to the down-regulation of the ARHGAP26 expression in human cancer tissues. These data suggested that ADAR1 regulated the expression of ARHGAP26 gene by A-to-I RNA editing through destruction of the existing miR-30b-3p and miR-573 target sites. It implied that down-regulation of tumor suppressors through RNA editing is one crucial way to explain how dyregulation of RNA editing involves in tumorigenesis.

Citation Format: Qiong Wang, Zhendong Guo, Weina Zhang, Li Wang. ADAR1 regulates ARHGAP26 expression by RNA editing through destruction of the miR-30b-3p and miR-573 target sites. [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 1793. doi:10.1158/1538-7445.AM2013-1793

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 1801: ERK-mediated regulation of NFAT3 enhances CXCR4 expression in HeyA8 ovarian cell line.
Authors: Huang Keven , Kiefer Christine , Kamal Adeela
Published: August 2014

The G-protein coupled chemokine (C-X-C motif) receptor CXCR4 is linked to cancer, HIV, and WHIM (Warts, Hypogammaglobulinemia, Infections, and Myelokathexis) syndrome. While CXCR4 is widely studied and characterized extensively to be over-expressed in multiple human tumor tissues, we have seen poor surface CXCR4 expression in many solid tumor cell lines in vitro. To better understand how CXCR4 is regulated, cells grown under 3-D spheroid conditions were compared to normal adherent culturing conditions. When cultured as 3D spheroids, HeyA8 cells showed a dramatic increase in surface CXCR4 protein levels as well as mRNA transcripts. Furthermore, HeyA8 3D spheroids showed a decrease in pERK levels when compared to adherent cells. Using an inhibitor of the MEK-ERK pathway, the treatment of adherent HeyA8 cells with U0126 resulted in a significant increase in surface CXCR4 expression similar to 3D spheroids. Additional investigation using PCR array showed a wide range of transcription factors being up-regulated but notably a > 20 fold increase in NFAT3 transcription factor. Using a calcineurin inhibitor, cyclosporin A diminished the U0126 treated up-regulation of CXCR4 surface expression. Finally, Chromatin Immunoprecipitation analysis showed direct binding of NFAT3 on the CXCR4 promoter to increase CXCR4 expression in HeyA8 ovarian cell line. Taken together, our results suggest that phospho-ERK levels regulate CXCR4 expression through the NFAT3 signaling pathway.

Citation Format: Keven Huang, Christine Kiefer, Adeela Kamal. ERK-mediated regulation of NFAT3 enhances CXCR4 expression in HeyA8 ovarian 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 1801. doi:10.1158/1538-7445.AM2013-1801

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 1803: Transcriptional regulation of nicotinic acetylcholine receptors (nAChRs) by E2F family transcription factors in non-small cell lung cancer.
Authors: Schaal Courtney M. , Pillai Smitha , Johnson Jackie L. , Chellappan Srikumar
Published: August 2014

Nicotine, the addictive component of cigarette smoke, has been shown to promote cell proliferation, migration, invasion, and angiogenesis in multiple cancer types. Earlier studies from our lab had shown that nicotine can promote the growth and metastasis of non-small cell lung carcinoma (NSCLC) in mouse models. While a broad range of nAChRs have been found to be expressed on NSCLC cell lines, nicotine-mediated proliferation, invasion, and migration are facilitated predominantly through the α7 subunit. Consistent with this, α7 nAChR levels are elevated in NSCLC in mice that were administered nicotine. Stimulation of α7 nAChR with nicotine has been reported to activate Src, resulting in inactivation of the retinoblastoma (Rb) tumor suppressor protein and enhancing E2F-mediated transcription. The Rb-E2F transcriptional regulation pathway is known to induce genes involved in cell cycle progression, angiogenesis, and metastasis implicating it in tumor survival and progression. Given this background, attempts were made to elucidate whether nAChR genes are regulated by the Rb-E2F pathway. Analysis of a 2000bp promoter region of the human α7 gene revealed the presence of multiple E2F binding sites. Transient transfection experiments showed α7 to be responsive to multiple E2Fs. E2F1 was found to associate with the α7 promoter via chromatin immunoprecipitation assays. Depletion of E2F genes via small interfering RNA demonstrated a differential regulation of α7 by E2F family members 1-5. These results raise the possibility that exposure to nicotine stimulates the α7 signaling cascade resulting in elevated E2F1-mediated activation of various proliferative promoters, including that of α7 itself in a positive feedback mechanism. In addition to the α7 nAChR, recent genome wide association studies (GWAS) have identified a susceptibility locus for human lung cancer at 15q25.1 which encodes for nAChR subunits α3 and α5. Promoter analysis has revealed that both α3 and α5 have multiple potential E2F binding sites, as well. Preliminary knock down of E2F family members via small interfering RNA has suggested these subunits may also be differentially regulated by the E2F family of transcription factors. Further studies are under way to elucidate the role of E2F in the regulation of nAChR α3 and α5, and how this impacts nicotine signaling as well as growth and progression of NSCLC.

Citation Format: Courtney M. Schaal, Smitha Pillai, Jackie L. Johnson, Srikumar Chellappan. Transcriptional regulation of nicotinic acetylcholine receptors (nAChRs) by E2F family transcription factors in non-small cell lung 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 1803. doi:10.1158/1538-7445.AM2013-1803
Abstract 1804: Integrative genomics and expression analysis reveals enhanced SENP2-mediated desumoylation and a crosstalk with Neddylation pathway in squamous cell carcinoma of the lung.
Authors: Qian Jun , Wang Jing , Hoeksema Megan D. , Zou Yong , Espinosa Allan V. , Rahman Jamshedur , Zhang Bing , Massion Pierre P.
Published: August 2014

Chromosome 3q26-29 amplification is a critical genomic alteration in squamous cell carcinomas (SCC) of the lung. There remains no consensus on what driver genes may carry this amplicon throughout tumor progression and their functional implications. We hypothesized that the amplification level of a predefined amplicon in a tumor sample can be inferred based on gene expression data. The hypothesis was tested using the TCGA dataset with paired copy number and gene expression data and achieved an accuracy of 90%. Based on the TCGA dataset and six other independent gene expression datasets (GSE4573, GSE8894,GSE2109,GSE3141 and GSE17710), we inferred candidate 3q26-29 drivers by searching for genes that were consistently over-expressed in SCCs with a high-level of 3q26-29 amplification in all seven datasets. The top 20 candidate driver genes includes PSMD2, DCUN1D1,SENP2, ZNF639, FXR1, SENP5,PRKCI, PIK3CA and UBXN7, et al. Using NetWalker we further identified a potential driver network consisting of SENP2, DCUN1D1, UBXN7 and DVL3, which are involved in SUMOlyation and Neddylation pathway. The amplification and overexpression of four genes were confirmed by performing genome-wide Agilent 244k CGH profiling and 44k gene expression profiling on twenty-four SCC tumors. Knocking down using siRNA against SENP2 resulted in greatest cell growth inhibition on 3q26-28 amplified SCC cell line H520. Immunoprecipitation assay showed that SENP2 binds to endogenous MDM2 in H520 cells. Loss of SENP2 leads to increased SUMO1 expression and elevated p53 expression associated with a decrease in MDM2 expression. Furthermore, knocking down SENP2 reduced the expression of DCUN1D1 and DVL3 but not UBXN7 in H520 cells. Together these data suggest that SENP2-mediated desumoylation and crosstalk with the Neddylation pathway contribute to lung SCC progression.

Citation Format: Jun Qian, Jing Wang, Megan D. Hoeksema, Yong Zou, Allan V. Espinosa, Jamshedur Rahman, Bing Zhang, Pierre P. Massion. Integrative genomics and expression analysis reveals enhanced SENP2-mediated desumoylation and a crosstalk with Neddylation pathway in squamous cell carcinoma of the lung. [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 1804. doi:10.1158/1538-7445.AM2013-1804
Abstract 181: Hepatic retinoid signaling in mouse hepatocarcinogenesis models.
Authors: Shirakami Yohei , Blaner William S. , Shimizu Masahito , Moriwaki Hisataka , Seishima Mitsuru
Published: August 2014

Retinoids are known to possess anti-cancer effects and a clinical trial of a synthetic retinoid for hepatocellular carcinoma (HCC) is ongoing recently in Japan. Hepatic retinoid metabolism is usually impaired during development of liver diseases. Loss of retinoid-containing lipid droplets upon hepatic stellate cell (HSC) activation is one of the first events in the development of liver diseases and retinoid stores are progressively lost from HSCs leading to hepatic fibrosis, cirrhosis, and HCC. In the present study, we employed two types of genetically-modified mice for investigating hepatic retinoid metabolism and signaling in diethylnitrosamine (DEN)-induced mouse hepatocarcinogenesis models. One is lecithin:retinol acyltransferas (LRAT) knockout (KO) mouse which lacks HSC lipid droplets and has much less hepatic retinoid stores, and the other is a transgenic mouse which expresses dominant negative form of retinoic acid receptor (dnRAR) to block retinoid signaling in the liver. Male 15 day-old mice were given intraperitoneal injections of DEN (25 mg/kg body weight). Eight-month-old DEN-treated Lrat KO mice showed lower liver tumor incidence and dnRAR transgenic mice showed higher incidence than that of respective control mice. In the acute phase after DEN injection, dnRAR transgenic mice exhibited higher hepatic levels of inflammatory cytokines. Two days after DEN injection, lower serum levels of alanine aminotransferase and decreased hepatic levels of cyclin D1 were observed in Lrat KO mice. Lrat KO mice also exhibited increased levels of retinoic acid-responsive genes, including p21, lower levels of cytochrome P450 enzymes required for DEN-bioactivation, and higher levels of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT). Our results indicate that Lrat KO mice, instead of almost no hepatic retinoid storage, are less susceptible to DEN-induced hepatocarcinogenesis due to increased hepatic retinoid signaling which is accompanied by higher expression of p21 in the liver. In conclusion, retinoid signaling plays a key role in these hepatocarcinogenesis models.

Citation Format: Yohei Shirakami, William S. Blaner, Masahito Shimizu, Hisataka Moriwaki, Mitsuru Seishima. Hepatic retinoid signaling in mouse hepatocarcinogenesis models. [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 181. doi:10.1158/1538-7445.AM2013-181
Abstract 1815: Genome-wide analysis of the molecular crosstalk between mitogenic and proinflammatory signaling in breast cancer.
Authors: Franco Hector L. , Nagari Anusha , Kraus W. Lee
Published: August 2014

The interplay between mitogenic (e.g., estradiol) and proinflammatory (e.g., TNFα) signaling pathways play key roles in determining the phenotypes and clinical outcomes of breast cancers. Both pathways promote dramatic changes in the transcriptomes of breast cancer cells, as well as downstream cellular effects, but the interplay between these two pathways at the transcriptional level has not yet been analyzed in a comprehensive way across the genome. We have used global nuclear run-on coupled with deep sequencing (GRO-seq), a genomic method that maps the location and orientation of all actively engaged RNA polymerases, to characterize the immediate transcriptional responses of MCF-7 breast cancer cells treated for short durations (40 min.) with either estradiol (E2), TNFα, or both. In addition, we have integrated these data with chromatin immunoprecipitation coupled with deep sequencing (ChIP-seq) for both estrogen receptor alpha (ERα), which mediates E2 signaling, and the p65 subunit of the NF-κB transcription factor, which mediates TNFα signaling. Our results indicate extensive transcriptional interplay between these two signaling pathways, which is observed for a number of classical mitogenic and proinflammatory protein-coding genes. In addition, GRO-seq has allowed us to capture the transcriptional crosstalk at genomic locations encoding for long non-coding RNAs (lncRNAs), a poorly characterized class of RNAs which have been shown to play important roles in cancer outcomes. The synergistic and antagonistic interplay between E2 and TNFα also manifests itself at the level of ERα and p65 binding, which relocalize to new binding sites that are not occupied by either treatment alone. Taken together, our results identify rapid, signal-dependent transcriptional changes that occur during proliferative signaling versus inflammatory signaling and show that these effects are more extensive than previously anticipated. Uncovering the transcriptional landscape of these two important pathways outside of the protein coding regions of the genome may lead to new ways of diagnosing and treating breast cancer.

Citation Format: Hector L. Franco, Anusha Nagari, W. Lee Kraus. Genome-wide analysis of the molecular crosstalk between mitogenic and proinflammatory signaling in breast 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 1815. doi:10.1158/1538-7445.AM2013-1815
Abstract 1816: SULT1A1 gene expression level is correlated with gene copy number in different human epithelial and breast cancer cell lines.
Authors: Yao-Borengasser Aiwei , Rogers Lora J. , Penny Rosalind B. , Edvana Vineetha K. , Kadlubar Susan A.
Published: August 2014

Sulfotransferase isoform 1A1 (SULT1A1) is a member of a subfamily of cytosolic enzymes which catalyze the transfer of a sulfonate group from 3’-phosphoadenosine 5’ -phosphosulfate (PAPS) to a range of xenobiotic and endogenous compounds such as drugs, carcinogens, and steroid hormones. SULT1A1 plays a role in detoxification of tamoxifen (TAM), a drug for estrogen receptor (ER) positive breast cancer treatment and chemoprevention. Besides its involvement in drug metabolism, SULT1A1 also contributes to increased cancer risk. SULT1A1 activity varies several-fold among individuals. Earlier studies have shown that individuals can possess one to five copies of the SULT1A1 gene. SULT1A1 gene expression is higher in individuals with more than one copy number, although there is no significant difference among individuals who have more than 3 copy numbers. We determined the copy number of SULT1A1 in cell lines ZR75, MCF7, T47D, MDA-MB-231, and MCF10A using a pre-designed Custom Plus TaqMan® Copy Number kit from Life Technology. Similar to the human population, the human cell lines tested also contain one to five copies of SULT1A1. Genomic DNA of MCF7, MDA-MB-231, and MCF10A cells contains only one copy of SULT1A1, while ZR75 DNA contains 3 copies and T47D DNA contains 5 copies of SULT1A1. To determine if copy number correlates with gene expression level, we compared the amount of SULT1A1 mRNA by RT-PCR in each cell line with corresponding copy numbers. Cell lines with one copy of SULT1A1 (MCF7 and MCF10A) had lower gene expression; while cell lines with more than one copy number (ZR75 and T47D) had higher gene expression. ZR75 cells expressed significantly more SULT1A1 mRNA than that of T47D, even though ZR75 DNA has two less copies than T47D cells. Although MDA-MB-231 DNA contains one copy of SULT1A1, no SULT1A1 gene expression was detected, indicating that other mechanisms exist in regulating SULT1A1 gene expression. In summary, the current data suggest that differential SULT1A1 gene expression levels in human breast cancer cells could be partially regulated by its copy numbers. Furthermore, the cell lines tested here could be used as in vitro models to study the mechanisms of SULT1A1 gene expression regulations since the copy number differences in those cell lines represented the copy number differences in human population.

Citation Format: Aiwei Yao-Borengasser, Lora J. Rogers, Rosalind B. Penny, Vineetha K. Edvana, Susan A. Kadlubar. SULT1A1 gene expression level is correlated with gene copy number in different human epithelial and breast cancer cell lines. [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 1816. doi:10.1158/1538-7445.AM2013-1816
Abstract 1819: Over-expression of PIM1 kinase contributes to CYP24A1 gene up-regulation in prostate cancer.
Authors: Luo Wei , Chernov Mikhail , Ma Yingyu , Johnson Candace , Trump Donald
Published: August 2014

The traditional role of vitamin D is in the control of calcium homeostasis and bone mineralization. Over the past 2 decades, studies have demonstrated that vitamin D is useful for the prevention and treatment of various cancers. 24-hydroxylase, encoded by CYP24A1 gene, is the key enzyme which inactivates vitamin D. Increased expression of CYP24A1 has been found in several human tumors. CYP24A1 is regulated directly by 1,25(OH)2D3 (1,25D3) through genomic signaling. Studies have revealed that protein kinase signaling pathways may also be involved in the regulation of CYP24A1 expression. By screening a chemical compound library, we discovered that small chemical compounds with inhibitory activity toward protein kinases including PIM1 and HIPK2 inhibited CYP24A1 expression. Furthermore, siRNA-mediated PIM1 knockdown reduced 1,25D3-induced CYP24A1 mRNA and protein expression in human prostate cancer PC3 cells. In contrast, HIPK2 knockdown had no effect. The serine/threonine kinase PIM1 has been implicated as an oncogene in various human cancers including lymphomas, gastric, colorectal and prostate carcinomas. We analyzed mRNA expression of CYP24A1 and PIM1 in 26 case-matched pairs of human benign and malignant prostate by qRT-PCR. CYP24A1 expression was increased in 6 out of 26 malignant prostate samples compared to benign lesions (> 1.5 fold). PIM1 expression was increased in 10 out of 26 malignant prostate samples compared to benign lesions (> 1.5 fold). Five of the 6 samples with increased CYP24A1 in malignant lesions also had increased PIM1 expression. Fisher's exact test shows that increased CYP24A1 expression is significantly associated with increased PIM1 expression in malignant prostate (P = 0.0184). These data indicate that PIM1 may be involved in increased CYP24A1 expression in prostate cancer.

This study was supported by NIH/NCI grants 5R01CA067267 and 5R01CA095045.

Citation Format: Wei Luo, Mikhail Chernov, Yingyu Ma, Candace Johnson, Donald Trump. Over-expression of PIM1 kinase contributes to CYP24A1 gene up-regulation in 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 1819. doi:10.1158/1538-7445.AM2013-1819
Abstract 1820: TP63 regulation of epithelial to mesenchymal transition in an experimental prostate cell model.
Authors: Olsen Jan Roger , Øyan Anne Margrete , Rostad Kari , Hellem Margrete R. , Liu Jie , Li Lisha , Micklem David , Haugen Hallvard , Lorens James B. , Ke Xisong , Rotter Varda , Lin Biaoyang , Kalland Karl-Henning
Published: August 2014

Background: p63 is a transcription factor that is central for epithelial homeostasis and development. In our cell culture model of epithelial to mesenchymal transition (EMT) in a human prostate cells, p63 was one of the most down-regulated transcription factors during EMT. This led us to investigate the role of p63 in EMT by a gain and loss of function approach.

Results: Over-expression of the predominant epithelial isoform ΔNp63α in mesenchymal EPT1 and EPT2 cells led to gain of several epithelial characteristics without resulting in a complete mesenchymal to epithelial transition (MET). When p63 was knocked down in epithelial EP156T cells complementary results were observed. Global gene expression analyses found that ΔNp63α induced gene modules involving cell adhesion genes in mesenchymal like cells. ChIP-seq analyses confirmed the enriched binding of p63 to regulatory areas of genes associated with cell adhesion in prostate epithelial cells.

CDH1 and ZEB1 are two elemental factors in the control of EMT. Over-expression and knockdown of these factors, respectively, were not sufficient alone or in combination with ΔNp63α for full reversal of the mesenchymal phenotype in EPT cells.

Conclusion: The partial reversion of epithelial to mesenchymal transition might reflect the ability of ΔNp63α, as a key co-ordinator of several epithelial gene expression modules, to reduce epithelial to mesenchymal plasticity (EMP). The potential of reduced EMP in order to counteract metastasis and the utility of ΔNp63α expression in this regard may provide a novel strategy to counteract the metastatic process.

Citation Format: Jan Roger Olsen, Anne Margrete Øyan, Kari Rostad, Margrete R. Hellem, Jie Liu, Lisha Li, David Micklem, Hallvard Haugen, James B. Lorens, Xisong Ke, Varda Rotter, Biaoyang Lin, Karl-Henning Kalland. TP63 regulation of epithelial to mesenchymal transition in an experimental prostate cell model. [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 1820. doi:10.1158/1538-7445.AM2013-1820
Abstract 1822: An integrated approach to identify clinically relevant long non-coding RNAs (lncRNAs) in cancer.
Authors: Liu X. Shirley
Published: August 2014

Despite growing appreciation of the number and importance of long non-coding RNAs (lncRNAs) in normal physiology and diseases, the knowledge on cancer-related lncRNAs is still limited. We utilized probes from array-based expression profiles that are uniquely mapped to annotated lncRNAs to investigate the expression of 10,207 lncRNAs in about 1,300 tumor samples over four different cancer types. By integrating the expression profiles of these lncRNAs with clinical outcome and somatic copy number alterations (SCNA), we identified lncRNAs that are associated with specific cancer subtypes and clinical prognosis, and predicted those that are potential drivers of cancer progression. We further validated our computational predictions by experimentally confirming the oncogenic functions of two novel lncRNAs in prostate cancer. Our results demonstrate that public genomic data contain a wealth of information on disease-relevant non-coding RNAs, and integrating orthogonal datasets and clinical information could facilitate the discovery of clinically relevant lncRNAs and help prioritize their functional studies.

Citation Format: X. Shirley Liu. An integrated approach to identify clinically relevant long non-coding RNAs (lncRNAs) in 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 1822. doi:10.1158/1538-7445.AM2013-1822

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 1825: The inhibitory effect of long non-coding RNA MALAT1 on Smads signaling pathway.
Authors: Zhang Jinqiang , Han Chang , Wu Tong
Published: August 2014

Long non-coding RNA (lncRNA) is arbitrarily defined as RNA longer than 200 bases that does not encode mRNA, rRNA or tRNA. LncRNAs are involved in almost every aspect of cellular and molecular biology. In most cases, lncRNAs assemble with proteins as RNA-protein complexes to execute their functions. We hypothesize some important molecular in critical signaling pathway, such as Smad2 and Smad3 in TGF-β/Activin pathway, might have their lncRAN partner/partners to fine regulate their function. Based on the hypothesis, we carried on little scale screen and finally identified lncRAN MALAT1 can exclusively bind to pSmad2 and pSmad3 in hepatocellular carcinoma cell lines Hep3B, SK-Hep1 and cholangiocarcinoma cell line CCLP1 by methods of RNA Immunoprecipitation (RIP). After quantifying by qRT-PCR, we found that MALAT1 enriched by anti-pSmad2/3 antbodies is >15 times more than that by IgG control. Co-localization of MALAT1 and pSmad2 and 3 in nuclear speckles of Hep3B cells were also proved by methods of combining FISH and immunofluorescence staining. Further bioinformatics analysis and immunoprecipitation reveal that pSmad2/3 binding to MALAT1 through SET domain containing 2 (SETD2) indirectly. Accordingly, if SETD2 was knockdown by siRNA, the binding between pSmad2/3 with MALAT1 was proved decreasing dramatically by methods of RIP. We also found that MALAT1 knockdown by shRNA or antisense oligonucleotide will increase the Smad2/3 signaling activity significantly, which was proved by Smad2/3 luciferase reporter constructs, DNA pull-down using synthesized oligonucleotide containing consensus sequence of Smads binding elements (SBE) and enhanced inhibition effect of TGF-β on the proliferation of Hep3B and CCLP1 cells. Next, Protein Phosphatase 1A (PPMA1), which act as a Smad phosphatase to terminate pSmad2/3 activity, was proved to bind to MALAT1 and MALAT1 knockdown decreased the binding of pSmad2/3 to PPM1A. Therefore, MALAT1 (together with SETD2) might act as scaffold to facility the interaction between pSmad2/3 and PPM1A, which lead to the dephosphorylation of pSmad2/3. This kinetically decrease of pSmad2/3 dephosphorylation might be the reason of increased Smads signaling activity in MALAT1 knockdown cells.

This work was supported by National Institutes of Health grants CA102325, CA106280, CA134568, DK077776 (to T.W.) and CA137729 (to C.H.)

Citation Format: Jinqiang Zhang, Chang Han, Tong Wu. The inhibitory effect of long non-coding RNA MALAT1 on Smads signaling pathway. [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 1825. doi:10.1158/1538-7445.AM2013-1825
Abstract 1827: HnRNP I interacts with lncRNA UCA1 and regulates its stability.
Authors: Huang Jianguo , Mo Yin-Yuan
Published: August 2014

Background: The human genome also makes a large number of non-coding RNAs including long non-coding RNAs (lncRNAs) and microRNAs in addition to protein-coding genes. While microRNAs are well characterized, little is known about lncRNAs. Emerging evidence has implicated lncRNAs in gene regulation; and they are often aberrantly expressed in a variety of human diseases, in particular cancer. The heterogeneous nuclear ribonucleoprotein I (hnRNP I) is a member of the large hnRNP family and has been implicated in mRNA splicing, stability and translational regulation of certain RNA transcripts. During screening for lncRNA-interacted proteins, we identified hnRNP I as a binding partner for UCA1. However, it remains unclear how hnRNP I interacts with UCA1 and whether such an interaction impacts UCA1-mediated cell growth and responsiveness to anti-cancer drugs.

Methods: To determine cell growth and response to doxorubicin, we performed MTT assays as well as cell counting assays. Protein levels from treated cells were analyzed by western blot. Real-time RT-PCR was used to determine relative expression of genes. Interaction between hnRNP I and UCA1 was confirmed by RNA co-immunoprecipitation and RNA pulldown. RNA stability was determined by treating MCF-7 cells with actinomycin D, followed by real-time RT-PCR. hnRNP I was knock down by transient transfection of hnRNP I siRNAs.

Results: We found that UCA1 induced proliferation and increased resistance to doxorubicin in MCF-7 cells. RNA pulldown assays revealed that UCA1 RNA directly interacted with hnRNP I. This interaction was further confirmed by RNA immunoprecipitation by hnRNP I antibody. Real-time RT-PCR revealed that knockdown of hnRNP I by RNAi reduced the level of UCA1 in MCF-7 cells and it also repressed the induction of UCA1 by doxorubicin. Further experiments suggested that hnRNP I impacts the UCA1 stability. We showed that half-life of UCA1 was about 12 h but was decreased to about 6 h in the presence of hnRNP I siRNAs. We further showed that the phosphorylated form of hnRNA I, predominantly in the cytoplasm, was a major form that interacts with UCA1.

Conclusion: Taken together, these results suggest that hnRNP I plays a role in regulation of the stability of UCA1. Since previous work implicates the involvement of UCA1 in tumor cell growth and chemoresistance, a better understanding of the underlying mechanism will provide new insight of targeting UCA1 in cancer therapy.

Citation Format: Jianguo Huang, Yin-Yuan Mo. HnRNP I interacts with lncRNA UCA1 and regulates its stability. [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 1827. doi:10.1158/1538-7445.AM2013-1827
Abstract 1831: Functional analysis of a novel long ncRNA involved in the tumorigenicity of colorectal cancer.
Authors: Taniue Kenzui , Kurimoto Akiko , Sugimasa Hironobu , Nasu Emiko , Nagashima Takeshi , Okada-Hatakeyama Mariko , Akiyama Tetsu
Published: August 2014

In recent years, the advent of high-throughput sequencing technologies has revealed that in addition to protein-coding genes, there are an even larger number of RNA transcripts that do not encode proteins and are instead defined as noncoding RNAs (ncRNAs). Numerous ncRNAs are misregulated in various diseases, especially cancer. We therefore attempted to identify and characterize novel long ncRNAs that regulate the tumorigenicity of colorectal cancer. Here we show that a novel long ncRNA, termed NR-X, is required for proliferation and tumorigenicity of colorectal cancer cells. We further show that NR-X is upregulated under hypoxic conditions and induces expression of hypoxia-responsive genes. Moreover, we succeeded to identify a chromatin-related protein that is associated with NR-X. In this presentation, we would like to present our data on the molecular functions of NR-X and discuss its significance in tumorigenesis.

Citation Format: Kenzui Taniue, Akiko Kurimoto, Hironobu Sugimasa, Emiko Nasu, Takeshi Nagashima, Mariko Okada-Hatakeyama, Tetsu Akiyama. Functional analysis of a novel long ncRNA involved in the tumorigenicity of colorectal 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 1831. doi:10.1158/1538-7445.AM2013-1831
Abstract 1837: Exosomal non-coding RNAs as mediators for cell-cell communications .
Authors: Singh Ramesh , Mo Yin-Yuan
Published: August 2014

Introduction: Exosomes are 30-100nm membrane vesicles of endocytic origin containing proteins, mRNAs, microRNAs and lincRNAs. MicroRNAs are small and lincRNAs are large intergenic non-coding RNAs. They are emerging as key regulators of diverse cellular processes including cancer. In this study, we investigated the exosome-mediated transfer of microRNAs and lincRNAs, regulation and functional significance in breast cancer.

Approach: Exosomes were isolated by ultracentrifugation and stained with PKH26 dye for cellular uptake in different cells by confocal microscopy. This uptake was also shown by exosome isolated from stable 293T cell line overexpressing the pCDH-CD63-GFP fusion protein. To determine the effect of nSMase2 on exosome secretion, stable cell lines were established with smpd3 gene. Levels of microRNAs and lincRNAs secreted in media were determined by real time PCR and their function validated through western blot and cell invasion.

Results: We first showed that exosomes can be transferred among different cell lines through direct uptake. We found that miR-10b is highly expressed in metastatic breast cancer MDA-MB-231 cells as compared to non-metastatic breast cells. Also, nSMase2 promoted the exosome-mediated miR-10b secretion whereas ceramide inhibitor suppressed this secretion. Importantly, western blot revealed that upon uptake, miR-10b can still reduce the protein level of its target genes. We also found lincRNA 21A and BC200 to be secreted through exosome in cell type dependent manner with functional significance.

Conclusion: We found that the exosomes secreted from cancer cells containing microRNA and lincRNA can be taken up by other cells and have functional role in the recipient cells suggesting the importance of exosome-mediated RNA transfer in tumor microenvironment.

Citation Format: Ramesh Singh, Yin-Yuan Mo. Exosomal non-coding RNAs as mediators for cell-cell communications . [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 1837. doi:10.1158/1538-7445.AM2013-1837
Abstract 1839: Long non-coding RNA SS-001 regulates proliferation, cell cycle, and migration in esophageal adenocarcinoma cells.
Authors: Song Jee Hoon , Meltzer Stephen J.
Published: August 2014

Esophageal adenocarcinoma (EAC) is one of the most rapidly increasing cancers in Western countries, but its underlying molecular mechanisms have not yet been completely elucidated. Recently, the discovery of long non-coding RNAs (lncRNAs) has added a new layer of complexity to the molecular architecture, identifying these molecules as emerging key regulators of diverse biological pathways. Increasing evidence shows that lncRNA dysregulation can lead to diseases such as cancer, but their potential involvement in EAC is not yet well-understood.

In this study, we identified a set of lncRNAs that were differentially expressed in esophageal cancers vs. normal tissues by massively parallel RNA sequencing. Expression levels were validated using quantitative real-time PCR. One lncRNA, designated SS-001, was significantly overexpressed and hypomethylated in EAC cell lines and tissues vs. normal esophageal epithelial cells and adjacent non-tumor tissues, respectively. SS-001 is an intergenic lncRNA located on chromosome 21.

In vitro assays in EAC cell lines demonstrated that knockdown of SS-001 resulted in inhibition of cell proliferation, the cell cycle, and migration. Moreover, patients with high SS-001 expression levels had a relatively poor prognosis. Our findings suggest that SS-001 may play a significant role in the development or progression of EAC and has the potential to serve as a prognostic biomarker.

Citation Format: Jee Hoon Song, Stephen J. Meltzer. Long non-coding RNA SS-001 regulates proliferation, cell cycle, and migration in esophageal adenocarcinoma 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 1839. doi:10.1158/1538-7445.AM2013-1839
Abstract 1857: Dichloroacetate (DCA), pyruvate dehydrogenase kinase (PDK) inhibitor, enhances the cytotoxic effect induced by metformin .
Authors: Choi Yong Won , Lim In Kyoung
Published: August 2014

It has been recently reported that metformin, widely used as an antidiabetic drug, reduces risk of cancer development in human and animals based on its inhibitory effects observed in the several cancer cells and mouse tumor models. Metformin shows anti-tumor activity indirectly by enhancing insulin sensitivity and decreasing the level of insulin and IGF in serum. And it was also reported that metformin has direct anti-tumor activity through AMPK activation and mTOR inhibition. We also observed that metformin induces cytotoxicity in several different types of cancer cell lines. However when metformin was treated in high glucose condition, cytotoxic effect of metformin was significantly reduced and AMPK activation and mTOR inhibition were not observed. Metformin increased glycolysis by inhibiting mitochondrial respiratory complex I activity weakly and also inhibiting pyruvate dehydrogenase activity more significantly. So cytotoxic effect induced by metformin was dependent on glucose availability. Significant mitochondrial ROS production was observed in metformin treated cancer cells. NADPHs are produced by enhanced glycolysis through the pentose phosphate pathway. Since reducing power of NADPH is important for protection from ROS induced damage, we hypothesized that enhanced glycolysis by inhibition of PDH activity observed in metformin treated cancer cells is a protective role from metformin induced ROS injury. So when PDH activity was enhance by dichloroacetate (DCA), pyruvate dehydrogenase kinase (PDK) inhibitor, ROS production was more increased and resulted in significant cell death.

Citation Format: Yong Won Choi, In Kyoung Lim. Dichloroacetate (DCA), pyruvate dehydrogenase kinase (PDK) inhibitor, enhances the cytotoxic effect induced by metformin . [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 1857. doi:10.1158/1538-7445.AM2013-1857