bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2853: Sunitinib induces anti-angiogenic splice-variant VEGF165b through induction of CLK1 and CLK4 kinases in prostate cell line DU-145
Authors: Monks Anne , Hose Curtis D. , Doroshow James H.
Published: June 2012

Sunitinib is an oral anti-tumor agent active in renal carcinoma and GIST, which inhibits specific receptor tyrosine kinases that includes targeting vascular endothelial growth factor receptors (VEGFR). To understand mechanisms which may drive this agent's activity, transcriptional profiling (GeneChip® HT Human Genome U133 Array Plate Set) of the NCI-60 human tumor cell lines treated with 200 and 2000 nM sunitinib was completed. Examination of the data indicated the CDC2-like genes, CLK1 and CLK4, were upregulated in multiple cell lines after 2 h drug incubation, with a decreasing level of expression over 6 and 24 h. These kinases are implicated in regulation of alternative splicing of mRNA and may favor selection of the distal splice site of exon 8 in the VEGFA gene. These splice variants of VEGFA are designated VEGFAxxxb and have been shown to have anti-angiogenic properties; regulation of this form of alternative splicing has been identified as a potential novel strategy for anti-angiogenic therapeutics. To confirm whether sunitinib-induced CLK1 and CLK4 expression was indicative of a change in the splice variants of VEGFA, specific primers were used in TaqMan® Q-RT/PCR to distinguish the exon 8 distal splice variant VEGFA165b (anti-angiogenic) from the proximal splice variant VEGFA165a (pro-angiogenic) of the dominant mRNA isoform, VEGFA165. VEGFA165b variant gene expression was induced 2-4 fold over 24h, compared to < 2-fold change in the VEGFA165a expression in 2 cell lines (DU-145 and NCI-H322M) with robust sunitinib-induced CLK1 and 4 expression compared to no change in VEGFA165b expression in a cell line with no increase in CLK1 and 4 after sunitinib treatment. Using DU-145 cells, we also demonstrated a sunitinib-induced, 2-fold increase in VEGFA165b protein expression over 24 h. Inhibition of CLK 1 and 4 kinases with a specific CLK inhibitor, TG003, decreased constitutive gene expression of VEGFA165b in DU-145, indicating the importance of the CLK genes in regulating distal splice variants of the VEGFA165 mRNA isoform. In combination with sunitinib, CLK inhibition by TG003, prevented sunitinib-induced upregulation of the VEGFA165b splice variant. Thus we have uncovered a novel mechanism underlying the activity of the VEGFR inhibitor, sunitinib, where induction of CLK kinases change the balance of VEGFA splice variants towards expression of anti-angiogenic species through which modulation of angiogenesis may occur. Funded by NCI Contract No. HHSN261200800001E.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2853. doi:10.1158/1538-7445.AM2011-2853
Abstract 2854: Delineation of grade-specific molecular drivers in inducible preclinical mouse models of astrocytoma
Authors: Song Yurong , Zhang Qian , Bash Ryan , Yin Chaoying , Yang Chunyu , Gilbert Debra , Wang Sophie , Bullitt Elizabeth , Kafri Tal , McCarthy Ken D. , Louis David N. , Miller C. Ryan , Van Dyke Terry
Published: June 2012

Glioblastoma (GBM) is a uniformly lethal neoplasm that accounts for 20% of all primary brain tumors. Despite advances in multimodality therapy, the median survival for patients with this disease remains approximately 12 months and has changed little over the past 40 years. An increased understanding of the molecular genetic basis of gliomagenesis is urgently required to develop more effective therapies for this devastating disease. Recently, The Cancer Genome Atlas (TCGA) project reported the most extensive genetic analysis of GBMs enhancing our ability to query altered signaling pathways potentially involved. Yet, an understanding of disease etiology requires extensive modeling in mammalian genetic systems coupled with analyses of biological contributions of disease. Here we genetically perturbed the primary molecular pathways (pRb and Ras) in genetically engineered mouse models (GEM). Alterations were induced specifically in adult GEM astrocytes via a tamoxifen-inducible, human GFAP promoter-driven CreERTM allele. GEM with astrocyte RB pathway inactivation alone (T) developed low-grade astrocytomas (grade II). Constitutive KRAS activation alone (R) did not produce detectable brain pathology. GEM harboring both inactivated RB and constitutively active KRAS (TR) in astrocytes developed high-grade astrocytomas (grade III) with low frequency of GBM development (grade IV). Deletion of Pten further progressed the tumor to grade IV with high frequency. Mechanistically, pRb inactivation led to activation of MAPK pathway via Ras. Mutant Kras did not further activate MAPK. Instead, it activated PI3K pathway. Deletion of Pten further activated PI3K-AKT pathway, leading to higher GBM rate. These highly penetrant, inducible GEM models provide a unique system for delineating the downstream pathways and resulting biological abnormalities critical to astrocytoma pathogenesis and for preclinical drug and biomarker development. Initial studies have begun to define the relative contribution to disease of each disrupted pathway, including potential targets for therapeutic intervention.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2854. doi:10.1158/1538-7445.AM2011-2854
Abstract 2859: Identification of driver mutations in colon cancer using Sleeping Beauty mutagenesis
Authors: Starr Timothy K. , Allaei Raha , Sarver Aaron , Silverstein Kevin , O'Sullivan M. Gerard , Cormier Robert , Largaespada David
Published: June 2012

Human colon tumors contain hundreds of genetic and epigenetic anomalies, of which only a handful are responsible for the growth and development of the tumor. We have developed a forward genetic screen in mice that can identify those mutations capable of causing gastrointestinal tract tumors. By comparing the mutations identified in our screen with those found in human colon tumors we can pinpoint the drivers of carcinogenesis. The forward genetic screen uses the Sleeping Beauty (SB) DNA transposon as a random mutagen capable of both activating and inactivating genes. Using Cre recombinase under the control of the Villin promoter we can selectively activate this mutagenesis in intestinal epithelial cells in the mouse. Tumors generated in these mice are analyzed for transposon insertion sites using linker mediated-PCR in combination with high-throughput sequencing. This analysis provides us with genomic sequence that can be mapped to the mouse genome. Candidate cancer driver genes are identified because they are located in areas of the genome that are recurrently mutated by the SB transposon in multiple tumors.

We conducted the screen in wild-type mice and identified 77 candidate cancer genes, of which six were previously known human colon cancer genes (APC, BMPR1A, SMAD4, PTEN, FBXW7, CDK8). More importantly, we identified many novel candidate drivers that can now serve as potential targets in colon cancer therapy. We have extended our studies by conducting three more screens using mice that already contain a known driver mutation. The purpose of these new screens is to gain a better understanding of the genetic changes that cooperate with common mutations found in colon cancer. The three screens used mice that have a mutation in Apc (ApcMin), carry a dominant negative acting Trp53 (p53R270H), or are null for the tumor suppressor gene p19ARF. We have completed the screen in ApcMin mice and found an additional 30 candidate drivers of colon cancer that may specifically cooperate with the Apc mutation. The two final screens are in the process of completion and should uncover genes that cooperate with mutations in Trp53 and p19ARF. The results of these studies will greatly expand the list of potential therapeutic targets and will enhance our understanding of the genetic etiology of colon cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2859. doi:10.1158/1538-7445.AM2011-2859
Abstract 2882: CREB inhibition attenuates MM tumor growth and drug resistance
Authors: Shukla Arti , Hillegass Jedd M. , Macpherson Maximilian B. , Beuschel Stacie L. , Steele Chad , Mossman Brooke T.
Published: June 2012

Malignant mesothelioma (MM) has a poor prognosis because of its difficult diagnosis and chemoresistance. We have shown previously that cyclic AMP response element binding protein (CREB) is constitutively activated (phosphorylated) in MM cells and tissue arrays as compared to mesothelial cells or benign lesions. Moreover, inhibition of CREB results in increased Doxorubicin (Dox)-induced apoptosis by attenuation of prosurvival genes, Bcl2 and BclxL. Using intraperitoneal and subcutaneous SCID xenograft mice models injected with shCREB (CREB inhibited) human MM cells (Hmeso), we show that CREB inhibition significantly attenuates tumor growth in both models. In addition, CREB inhibition also results in additional decreases in tumor size after treatment with Dox (0.5mg/kg, ip, 3 times a wk for 6 wks), suggesting a role of CREB in drug resistance. TUNEL staining on tumor sections showed increased cell death in CREB-inhibited tumors. Assessment of human cytokine levels by Bioplex analysis in peritoneal lavage fluid (PLF) of tumor-bearing mice showed that CREB inhibition significantly attenuated levels of IL-6, IL-8, VEGF, MCP1, IFN-gamma, IP-10 and RANTES. Dox treatment per se increased levels of several cytokines (IL-6, IL-8, G-CSF, IP-10, MIP-1B, RANTES), however, in the presence of shCREB, these levels were significantly decreased after Dox treatment. Other cytokine levels like bFGF, IFN-gamma, MCP1 and VEGF were reduced following Dox treatment, and further decreases were observed in CREB inhibited (shCREB) tumors. Microarray analysis performed on shCREB-Hmeso cells in vitro showed several fold decreases in various tumorigenesis- related genes including PAK7, Calbindin 1, TIE-2, MCAM, CD24, CCL-5, c-Met, v-kit, MMP2, IL-6, and snail homolog 2 as compared to shControl-Hmeso cells. In addition, increases in mRNA levels of phosphatases, Dachund homolog, and Caspase-1 were also observed following CREB inhibition. The patterns of gene expression and cytokine release by shCREB suggest that CREB affects tumor growth by regulating proliferation/survival, angiogenesis, migration and apoptosis. In addition, we also show that inhibition of CREB expression renders MM tumors more susceptible to Dox. This study is supported by National Cancer Institute grant PO1CA114047 and a grant from the Vermont Genetics Network (VGN).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2882. doi:10.1158/1538-7445.AM2011-2882
Abstract 2894: Co-regulatory relationship between estrogen receptor alpha and the mTOR/S6K1 signaling pathways
Authors: Holz Marina K. , Maruani D. Myriam , Seligman Fannie , Spiegel Tirtza , Harris Emily , Shachter Avielle , Beren Faygel
Published: June 2012

ERα mediates the proliferative effects of estrogen and represents an important clinical target in treatment of breast cancer. Tamoxifen is an anti-estrogen that has become the standard agent for the treatment of ER-positive breast cancer, where it acts as an antagonist. However, resistance to tamoxifen, and other endocrine or anti-estrogen therapies develops in many cases. One mechanism by which resistance develops is through phosphorylation of ERα, allowing it to act in estrogen-independent manner. Phosphorylation of Ser118 and Ser167 is important for transcriptional activity of ERα. The regulation of these sites is complex, involving inputs from the mTOR and MAPK signaling pathways. Importantly, ERα phosphorylation correlates with resistance to tamoxifen, and is a prognostic marker for disease progression and survival. Thus, the mechanism of these phosphorylation events, as well as the identity of the kinases responsible may have important clinical consequences. In this study we demonstrate that the mTOR/S6K1 pathway controls activation of ERα by phopshorylating it on these two critical residues.

Chromosomal region 17q23 containing the RPS6KB1 gene is frequently amplified in breast cancer cells, and S6K1 is overexpressed in about 30% of cases. Overexpression of S6K1 correlates with increased rapamycin sensitivity. The role of S6K1 in disease development and progression is supported by the observation that S6K1 overexpression is associated with poor prognosis in breast cancer patients. However, the reason for high-level amplification of the RPS6KB1 gene specifically in breast cancer, and not other cancer types is not well understood. In this study, we demonstrate that in response to estrogen activation, ERα activates expression of S6K1 by upregulating transcription of the RPS6KB1 gene. Increased levels of S6K1 promote further phosphorylation and activation of ERα, creating a positive feed-forward loop. We hypothesize that this relationship promotes breast cancer cell growth, thus providing a selective advantage to S6K1- and ERα-overexpressing cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2894. doi:10.1158/1538-7445.AM2011-2894
Abstract 2896: MEK inhibition controls EGFR level in EGFR-dependent NSCLC cells via deregulated expression of EGF-like ligands
Authors: Milewska Marta , Balko Justin Micah , Black Esther Penni
Published: June 2012

Purpose: The aim of the present study is to investigate the molecular mechanism underlying MEK inhibitor-induced EGFR down-regulation in EGFR-dependent NSCLC cell lines.

Methods: EGFR protein level in A549 and H1975 cell lysates was explored by Western blot assay. A549 and H1975 cell lysates were prepared and separated on SDS-polyacrylamide gels and blotted on PVDF membranes with antibodies specific for total or phosphorylated forms of EGFR, AKT, and total or phosphorylated ERK1/2. Calnexin or α-tubulin was used as loading control. Analysis of EGFR, HB-EGF and TGFα gene expression was performed using quantitative real time PCR (qPCR). Total RNA was extracted from cells and used for cDNA synthesis. qPCR was performed using Taqman On-Demand assays for EGFR, TGFα, HB-EGF and GAPDH. Relative quantification was applied using normalization to GAPDH.

Results: In the present study we observed that activation of the EGFR signaling cascade is attenuated by inhibition of MEK, resulting in down-regulation of EGFR in EGFR-dependent H1975 cells but not in EGFR-independent A549 cells. Interestingly EGFR down-regulation in H1975 cells does not occur at the level of transcription. However, mRNA expression of HB-EGF and TGFα, ligands overexpressed in EGFR-dependent H1975 cell line, decreased when treated with MEK inhibitor (AZD6244, Astra Zeneca). In contrast, no decrease in expression of either of the ligands was observed in the EGFR-independent A549 cell line following AZD6244 treatment. Interrogation of EGFR turnover using both protein degradation inhibitors and neutralizing anti-EGFR and anti-HB-EGF antibodies suggested that MEK inhibition down-regulates EGFR expression by directing EGFR to lysosomal degradation via deregulated EGFR and ligands interactions.

Conclusions: Our results suggest that loss of active ERK1/2 does not directly impact EGFR gene transcription and that MEK inhibition eliminates the signal for EGFR recycling and promotes degradation of EGFR. We assert that the MAPK cascade regulates production of EGF-like ligands responsible for maintaining high levels of active EGFR on which the cells depend. The difference in level of HB-EGF and TGFα ligands observed in EGFR-dependent cells indicates that EGFR signaling in these cells is maintained by both overexpression of ligands and maintenance of active ligands for EGFR interaction. Finally, our findings indicate that targeting EGFR ligand expression via the MAPK pathway may provide therapeutic benefit in EGFR-dependent lung cancer harboring resistant mutation to TKIs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2896. doi:10.1158/1538-7445.AM2011-2896
Abstract 291: Evaluation of blood flow in hepatocellular carcinoma: Assessment with computed tomography perfusion parameters and circulating angiogenic factors
Authors: Chen Ya-Wen , Chou Chen-Pin
Published: June 2012

Purpose: To study the relationships between CT perfusion parameters and circulating angiogenic factors in patients with hepatocellular carcinoma (HCC)

Materials and Methods: This study was HIPAA compliant and was approved by the institutional review board. All participants provided informed consents. CT perfusion parameters of HCCs among 21 patients were assessed using 21-phase liver images at a 64-slice multidetector CT. We analyzed CT perfusion parameters including total blood flow, arterial blood flow, portal blood flow and arterial fraction (arterial blood flow /total blood flow) of HCCs, surrounding liver parenchyma and HCC-parenchyma ratio. Interleukin 6 (IL-6), interleukin 8 (IL-8), vascular endothelial growth factor (VEGF), and platelet derived growth factor (PDGF) in plasma were measured using Bio-Plex multiplex immunoassay in 21 HCC patients and 8 healthy controls.

Result: CT perfusion parameters of HCCs showed statistically different from that of surrounding liver parenchyma in total blood flow (p=0.002), portal blood flow (p<0.001) and arterial fraction (p<0.001). Circulating IL-6, IL-8 and VEGF showed significantly higher levels in HCC patients than controls (p<0.05), and predicted HCC significantly better than chance (p<0.001). None of circulating angiogenic factors had significant correlation with any of CT perfusion parameters in HCC patients. Only HCC-parenchyma ratio of arterial blood flow showed significant positive correlation with circulating IL-8 (p<0.05).

Conclusion: CT perfusion parameters of HCC and circulating angiogenic factors are useful tools for HCC diagnosis. Only circulating IL-8 and HCC-parenchyma ratio of arterial blood flow showed significant correlation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 291. doi:10.1158/1538-7445.AM2011-291
Abstract 2920: Estrogen, tamoxifen, and Akt modulate expression of putative housekeeping genes in breast cancer cells
Authors: Shah Khyati Niral , Faridi Jesika S.
Published: June 2012

For quantitative reverse transcription-polymerase chain reaction (RT-qPCR), accurate data normalization requires the use of a stable reference gene. In estrogen-dependent cells, the identification of an appropriate housekeeping gene that is unaltered regardless of experimental conditions has been difficult. Additionally since cross-talk between the estrogen receptor and Akt pathways has been reported, the goal of this study was to identify the most stable reference gene for RT-qPCR (from a panel of nine putative housekeeping genes) that remains unaltered despite estrogen or tamoxifen treatment or stable overexpression of active Akt. Gene expression of nine candidate genes (GAPDH, ACTB, RPL13A, PGK1, TFRC, PPIA, HPRT1, B2M, and GUSB) was determined in parental and Akt overexpressing MCF-7 breast cancer cells treated with estrogen, tamoxifen, or vehicle, and gene stability was analyzed using two different statistical models. We observed that GAPDH, HPRT1, PGK1, and B2M were highly estrogen-regulated in parental MCF-7 cells. We also observed that seven out of the nine candidate genes were significantly altered in at least one of the Akt overexpressing MCF-7 cells. Based on our results, we suggest RPL13A as the most suitable internal reference gene that is both stable and remains unaltered in MCF-7 cells regardless of estrogen or tamoxifen treatment or Akt overexpression. In addition, our work emphasizes the importance of a preliminary study to validate each reference gene that will used for RT-qPCR.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2920. doi:10.1158/1538-7445.AM2011-2920
Abstract 2934: Novel approach for treating neural crest derived tumors: Selective inhibition of ganglioside biosynthesis with small molecules
Authors: Bai Xiaomei , Nguyen Tram , Brown Jillian , Glass Charles A. , Duron Sergio , Bhagwat Shripad S. , Crawford Brett E.
Published: June 2012

Neural crest derived tumors express high levels of a unique class of lipid linked glycan known as gangliosides. Gangliosides are involved in growth factor signaling by regulating complexes in lipid rafts. Genetic studies show that through aberrant expression of gangliosides, these tumors acquire aggressive growth properties. Prior to this research, the only ganglioside inhibitors identified were non-specific and broadly blocked virtually all glycolipid classes. These non-specific glycolipid inhibitors demonstrated anti-cancer activity in animal models of neural crest tumors. However, due to substantial off-target dose-limiting toxicity from lack of specificity for the ganglioside sub-class, they effectively cannot be used in humans for cancer treatment. Selective inhibition of gangliosides without affecting other glycan classes could potentially avoid these problems and provide an effective treatment for neural crest and other ganglioside-dependent tumors. To identify the first known selective inhibitors of gangliosides, we developed a novel molecular screening strategy for identifying selective small-molecule ganglioside inhibitors. This platform identified the first drug-like selective inhibitors of gangliosides. ZP10395, a lead compound, selectively and dose-dependently reduces gangliosides in multiple tumor cell lines and is 10-15 fold more potent than the existing non-specific inhibitors. Importantly, it does not inhibit other glycolipid classes associated with dose-limiting toxicity. Administering ZP10395 to a mouse xenograph melanoma model significantly reduced ganglioside production and slowed tumor growth in the presence of a reduced T-cell response. These results demonstrate the potential utility of specific ganglioside inhibitors for treating ganglioside dependent tumors.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2934. doi:10.1158/1538-7445.AM2011-2934
Abstract 2938: Inhibition of PI3K/AKT and MEK/ERK pathways act independently on p27 upregulation induced by downregulation of cathepsin B and uPAR in glioma
Authors: Gopinath Sreelatha , Alapati Kiranmai , Malla Rama Rao , Gondi Christopher S. , Mohanam Sanjeeva , Dinh Dzung H. , Rao Jasti S.
Published: June 2012

Cathepsin B and urokinase plasminogen activator receptor (uPAR) are overexpressed in gliomas. Deregulation of the G1 phase cell cycle machinery is a common feature of cancers. The abundance of cyclin-dependent kinase inhibitor p27Kip1 (p27) during the cell cycle determines whether cells will proliferate or become quiescent. In the present study, we carried out transfections using shRNA against cathepsin B and uPAR. We assessed cell cycle arrest using FACS analysis and used Western blot analysis to detect expression of ERK, AKT, p27, cyclin E, cyclin D, CDK4, CDK2, EGFR, β1 integrins, and the pocket proteins (p-Rb, p107 and p130). Specific inhibitors like Wortmanin (10 µM) and U0126 (10 µM) were used to further determine the roles of the AKT and ERK pathways. We used immunoprecipitation analysis to detect the respective interactions between EGFR and β1, p27 and CDK4, and p27 and CDK2. CHIP analysis was used to check for the recruitment of transcription factors FOXO3a, E2F1 and AP-1 to the p27 promoter. The results of the present study show that shRNA treatment efficiently downregulated expression of cathepsin B and uPAR and induced G0/G1 arrest through upregulation of p27. Downregulation of both p-ERK and p-AKT downstream of EGFR and β1 are involved, but independently, in p27 upregulation. Multiple transcription factors (namely, AP-1, E2F1 and FOXO3a) were involved in upregulation of p27 as determined by luciferase expression under the influence of promoter regions to which these transcription factors bind individually. Also, cathepsin B and uPAR downregulation reduced tumor growth and increased p27 nuclear expression in vivo. In summary, the reduction of p-ERK and AP-1, the increased expression of E2F1 and FOXO3a, and the reduced association of cyclin D-CDK4 and cyclin E-CDK2 complexes contributed to p27 upregulation as well as G0/G1 arrest induced by downregulation of cathepsin B and uPAR.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2938. doi:10.1158/1538-7445.AM2011-2938
Abstract 2941: Defining novel pathways that arrest genetically unstable tetraploid cells
Authors: Ganem Neil J. , O'Rourke Kevin P. , Pellman David
Published: June 2012

Tetraploid cells, which are a common byproduct of cell division failures, are genetically unstable and have the capacity to facilitate tumorigenesis. Consequently, p53-dependent tumor suppression mechanisms exist to limit the continued proliferation of these cells by promoting G1 cell cycle arrest. However, unlike other common cellular insults that activate p53 and promote G1 arrest, such as the DNA damage response, the mechanisms governing G1 arrest in response to tetraploidy remain largely unexplored. To gain insight into these mechanisms, we have developed a novel genome-wide RNAi screening assay to comprehensively identify proteins that are necessary to activate or maintain G1 cell cycle arrest after the induction of tetraploidy. Results from this screen, in combination with complementary bioinformatic approaches, have begun to illuminate the nature of the stresses associated with tetraploidy. More importantly, this work is shedding light on the adaptive mechanisms that tetraploid and near-tetraploid tumors may have evolved in order to overcome these stresses. These findings have the potential to lead to the identification of new pathways that can be targeted by chemotherapeutics to specifically reinforce G1 arrest in grossly aneuploid cancer cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2941. doi:10.1158/1538-7445.AM2011-2941
Abstract 2960: A combined bioinformatics and proteomics approach identifies DNA repair factors regulated by the APC/C
Authors: van Vugt Marcel A. , Fehrmann Rudolf S.N. , Ong Shao-En
Published: June 2012

Our genome is constantly challenged by endogenous and exogenous factors that induce a variety of DNA lesions. DNA double strand breaks are considered to most toxic type of DNA damage, and cells have evolved powerful mechanisms that can detect and repair such lesions. Two main mechanisms responsible for repair of DNA double strand breaks are Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR). The choice between these two types of DNA double strand break repair is strongly influenced by the cell cycle. Only cells in late S and G2 phase of the cell cycle can employ HR and this differential activity is underscored by a strict requirement for Cdk activity for HR. Mechanisms that enable HR during S-phase have been previously identified, but how exactly HR DNA repair is inactivated during the mitosis-to-G1 transition is poorly understood. Based on the observed strict cell-cycle regulation of HR repair, a role for the mitotic E3 ligase APC/C was suggested.

Here we present a combined proteomics and bioinformatics approach to identify APC/C targets and reveal components of DNA double strand break repair that are potential targets of the APC/C. Firstly, proteins were identified that contain evolutionary conserved and sterically accessibly APC/C targeting domains (KEN and D-boxes) using bioinformatics. Within this group of proteins with a known role in DNA repair were highlighted. Subsequently, a quantitative mass-spec approach was used to identify those proteins, whose abundance was readily lost upon mitotic exit. Reassuringly, we could identify many previously identified APC/C targets, that all showed loss of abundance during mitotic exit. We subsequently searched for DNA repair-associated factors that were down-regulated during mitotic exit and among those, we identified multiple topoisomerase subunits as likely APC/C substrates. We could subsequently confirm a role for the APC/C in regulating protein levels of these novel substrates using RNAi-mediated depletion of Cdh1 and live cell microscopy. In conclusion, we here present a novel method to identify APC/C targets and used this approach to uncover novel regulatory aspects of cell cycle-regulated DNA repair.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2960. doi:10.1158/1538-7445.AM2011-2960
Abstract 2967: Nucleolin phosphorylation by CK2 modulates its role in cell cycle checkpoint activation
Authors: Caglar Elif , Das Dibash , Borowiec James , Saxena Anjana
Published: June 2012

Nucleolin is a multifunctional abundant nucleolar protein and its gene expression is transactivated by the proto-oncogene c-myc during exponential cellular growth. This non-ribosomal protein is a major nucleolar phosphoprotein which is involved in ribosomal RNA processing, RNA polymerase I-catalyzed transcription, and the regulation of apoptosis, demonstrating both positive and negative effects on cell proliferation. During an unperturbed cell-cycle, interphase (CK2) and mitotic (Cdc2) kinases regulate nucleolin phosphorylation, modulating its function and nucleolar localization. Following cellular stress, nucleolin has been shown to inhibit chromosomal replication and to relay nucleolar stress signals mediated by its re-localization within the nucleus. Nucleolin also interacts with prominent components of the ARF (Alternate Reading Frame)/p53 checkpoint pathway that controls growth upon oncogenic stress signals. We have previously demonstrated that nucleolin binds to the p53-antagonist Hdm2, inhibits Hdm2-mediated degradation of p53 and causes p53-mediated apoptosis in an ARF-independent manner. Here we further dissect role/s of nucleolin phosphorylation in regulating the ARF/p53 checkpoint. We have generated a novel system by engineering human osteocarcinoma (NARF6) cells to induce expression of nucleolin-wt (wild-type) or -S6A (a phospho-mutant with six consensus site serines mutated to alanine, impairing phosphorylation by CK2). This novel system can control expression of both nucleolin and ARF by different promoters, allowing us to dissect the downstream events that are solely nucleolin- or ARF- dependent, or regulated by both. We found that increased levels of nucleolin-S6A significantly decreased ARF protein levels as compared to nucleolin-wt. Nucleolin-S6A was significantly more nucleoplasmic and, interestingly, cells that express S6A lacked nucleolar ARF expression. Although a dramatic loss in both ARF and p21 protein levels is evident with nucleolin-S6A expression, there was a net increase in p53 levels. Significantly, nucleolin-S6A expression caused an increase in the fraction of G1 cells in an ARF-independent manner. Thus, reduction of nucleolin phosphorylation by CK2 acts as a molecular switch causing the sub-nuclear re-localization of both nucleolin and ARF. These events lead to p53 activation, and the control of growth in both normal and stressed cells. These results also provide validation of our unique cellular system that allows us to manipulate nucleolin wt and mutant levels, facilitating the study of its physiological functions.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2967. doi:10.1158/1538-7445.AM2011-2967
Abstract 2996: DNA-damaging agents disrupt telomere structure
Authors: Samassekou Oumar
Published: June 2012

Telomeres are found at chromosome ends. When these ends are not protected by telomeric capping, the telomeres are recognized as double-strand breaks and the consequent recruitment of DNA repair machinery may affect cellular fate. Furthermore, telomeric capping proteins have been shown to interact with some of the proteins involved in DNA repair. A direct link thus exists between DNA repair and telomeres. However, few data are available on the changes in telomere structure associated with the different modes of DNA repair. To study the effects of three DNA-damaging agents on telomere structure, we exposed normal human primary fibroblasts to H202, gamma radiation, or UVB rays. The cells were then harvested at different times over a period of 24 hours. First the telomere nuclear architecture was studied by quantitative fluorescence in situ hybridization with a telomere-specific peptide nucleic acid probe followed by 3D telomere imaging. H202 treatment induced a significant reduction of telomere signals and a disruption of the nuclear volume that became normal after 24 hours of recovery. Also, telomere positioning was positively correlated to nuclear volume that disrupted over time. For gamma irradiation, the nuclear volume was positively correlated to the number of telomere signals and negatively correlated to telomere average intensity for each time point. UVB lead to a significant formation of telomeric aggregates and disruption of telomere nuclear shape by forming ellipsoid instead of circular shape.

Next, we studied the effects of these different DNA damage agents on the expression profile of telomere shelterin genes. The most affected genes were TRF1, POT1, TIN2 and Rap1. For instance, while the three agents showed a high induction of TIN2, gamma and H202 treatments presented high induction for POT1 and only H202 treatment induced significant increase of TRF1.

In conclusion, certain DNA-damaging events alter telomere structure by acting on nuclear telomere architecture and the expression profile of some telomere shelterin genes. The disruption of telomere structure due to acute insults might have an influence on cell homeostasis and the pathways involved can shed light for a better understanding of some pathological and therapeutic processes.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2996. doi:10.1158/1538-7445.AM2011-2996
Abstract 3: Induction of mitochondrial autophagy in leukemia by the Bcl-2 quadruplex-forming sequence
Authors: Sedoris Kara C. , Clarkson Cortney R. , Thomas Shelia D. , Miller Donald M.
Published: June 2012

Bcl-2 (B-cell CLL/lymphoma 2), a mitochondrial membrane oncoprotein functioning as an inhibitor of apoptosis, is commonly overexpressed in a variety of hematological malignancies promoting apoptotic resistance. Inhibition of Bcl-2 may result in sustained regression of leukemia/lymphoma. The promoters of several cancer-related oncogenes, including Bcl-2, contain sequences within nuclease hypersensitivity regions capable of forming quadruplex (four-stranded) DNA. The Bcl-2 quadruplex-forming sequence (Bcl-2 q, 23 bp) is located upstream of the P1 promoter of the Bcl-2 gene and is implicated in negative regulation of Bcl-2 transcription, however, the biological role of this sequence remains unclear. We hypothesize that treatment of leukemia cells with an oligonucleotide encoding Bcl-2 q induces cell death by inhibiting Bcl-2 gene expression. To determine the biological role of the Bcl-2 q on leukemic cell proliferation, U937 cells were treated with Bcl-2 q or the corresponding mutant sequence (MutBcl-2), which lacks runs of two or more guanines. Our results demonstrate that Bcl-2 q formed a stable parallel quadruplex structure and showed remarkable serum and intracellular stability. Treatment of leukemia cells with Bcl-2 q caused a significant dose and time-dependent decrease in cell proliferation after 3 and 6 days (IC50<5µM), which was not a result of cell cycle arrest. No change in growth of non-transformed stromal cells occurred in response to Bcl-2 q indicating the effect is specific for malignant cells. Confocal and flow cytometry analysis of cells treated with FITC-labeled Bcl-2 q or MutBcl-2 showed prominent uptake and nuclear localization of Bcl-2 q in contrast to MutBcl-2. Inhibition of cell proliferation corresponded with decreased Bcl-2 protein expression and increased expression of pro-apoptotic proteins Bax and Bak. This resulted in decreased mitochondrial transmembrane potential and significant autophagic degradation of mitochondria, characteristic of mitophagy. Induction of mitophagy was associated with increased Beclin-1 and LC3B expression and disruption of Bcl-2/Beclin-1 interaction. These results demonstrate striking growth inhibition in response to Bcl-2 q related to inhibition of Bcl-2 expression and induction of mitochondrial autophagy and shows considerable therapeutic potential.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3. doi:10.1158/1538-7445.AM2011-3
Abstract 30: Identification of genomic and epigenetic features of tumor suppressors and oncogenes in ovarian cancer
Authors: Wrzeszczynski Kazimierz O. , Varadan Vinay , Kamalakaran Sitharthan , Levine Douglas A. , Dimitrova Nevenka , Zhang Michael Q. , Lucito Robert
Published: June 2012

The identification of genetic and epigenetic alterations from primary tumor cells has become a common method to identify genes critical to the development and progression of cancer. We provide a bioinformatic analysis of copy number variation and DNA methylation covering the genetic landscape of ovarian cancer tumor cells. We individually examined the copy number variation and DNA methylation for 42 primary ovarian cancer samples using our MOMA-ROMA technology and 379 tumor samples analyzed by The Cancer Genome Atlas. We have identified 346 genes with significant deletions or amplifications among the tumor samples. Utilizing associated gene expression data we predict 156 genes with significantly altered copy number and correlated changes in expression. Among these genes CCNE1, POP4, UQCRB, PHF20L1 and C19orf2 were identified within both datasets. We were specifically interested in copy number variation as our base genomic property in the prediction of tumor suppressors and oncogenes in the altered ovarian tumor. We therefore identify changes in DNA methylation and expression for all amplified and deleted genes. We predicted 615 potential oncogene and tumor suppressor candidates by integrating these multiple genomic and epigenetic data types. Genes with a strong correlation for methylation dependent expression exhibited at varying copy number aberrations include CDCA8, ATAD2, CDKN2A, RAB25, AURKA, BOP1 and EIF2C3. Moreover, well characterized tumor suppressors and oncogenes exhibit varying CNV, methylation and expression features within cancers. We therefore compared significant copy number variation and methylation effects on expression for many known tumor suppressors, oncogenes and cancer related genes in order to identify altered gene function properties specific to primary ovarian tumors. Here, we present the analysis of multiple genomic modalities from two primary ovarian tumor datasets for the identification of candidate genes. We predict genes with epigenetic and genomic tumor suppressor and oncogenic properties having both analyzed and identified known validated ovarian cancer genes and identifying new candidates.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 30. doi:10.1158/1538-7445.AM2011-30
Abstract 3002: Multiplexing ChIP-Seq and rapid chromatin preparation from solid mammalian tissues for low cell ChIP assays
Authors: Tobler Andreas R. , Pickle Loni , Marnellos George , Chen Tom Z.
Published: June 2012

Currently, the chromatin immunoprecipitation assay (ChIP) is the most powerful method utilized to selectively enrich for DNA sequences bound by a particular protein in living cells. We developed a SOLiD™ ChIP-seq kit, which offers an optimized ChIP workflow and an efficient SOLiD™ ChIP-seq library construction from relatively low cell number samples. ChIP procedure is usually laborious, time consuming, and typically requires large starting cell numbers. We use MAGnify™ Chromatin Immunoprecipitation System, which is suitable for fast enrichment of chromatin complexes and DNA recovery. It is able to use lower starting cell numbers for ChIP and allows faster throughput to investigate different chromatin and transcription time-course events as well as enable antibody screening to determine ChIP compatibility. In addition, we develop a sensitive ChIP-seq library construction procedure which enables users to construct a complex library using as low as 1 ng ChIP DNA. Combining with SOLiD™'s ultra high sequencing throughput, SOLiD™ ChIP-seq offers a highly sensitive, hypothesis-neutral approach to accurately characterize protein-DNA interactions at genome-wide scale. Here we paired the SOLiD™ Fragment Library Barcoding Kit, which enables users to pool fragment libraries prior to emulsion PCR and then conduct multiplexed sequencing analysis, with the SOLiD™ ChIP-Seq Kit to characterize Estrogen Receptor alpha binding sites in breast cancer cells utilizing the SOLiD™ platform. In addition, we demonstrate a novel, rapid, and low input per ChIP tissue collection protocol for researchers utilizing solid mammalian tissue to study protein-DNA interaction via the MAGnify™ ChIP System. The 50 cent assay is reduced to less than 1 cent per reaction and it reduces reported homebrew amount of tissue from approximately 30mg to less than 1mg per ChIP reaction when paired with the powerful MAGnify™ ChIP system.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3002. doi:10.1158/1538-7445.AM2011-3002
Abstract 3010: Evaluation of genome wide analysis platforms for chromatin immunoprecipitation
Authors: Ma Lin
Published: June 2012

Chromatin states can influence transcription directly by altering the packaging of DNA to allow or prevent access to DNA-binding proteins, or they can modify the nucleosome surface to enhance or impede recruitment of effector protein complexes. Genome-wide mapping of protein-DNA interaction and epigenetic marks helps to better understand the transcriptional regulation. Prior to the development of next gen sequencing technologies, chip on chip or location analysis was the method of choice for exploring genome wide protein:DNA occupancy patterns. ChIP-seq is increasing in popularity due to the data density and lack of interrogation bias that this platform provides. While this application is becoming more popular, few studies have been performed which explore differences in binding site profiles obtained from both methods.

Here we present data which compares the performance of ChIP antibodies by ChIP-chip as well as by ChIP-seq. For theses studies, chIP-chip using Agilent microarrays and ChIP-seq using the Illumina Genome Analyzer platform were performed. These studies may serve as a basis to allow the development of specifications for acceptable performance on these genome wide platforms to facilitate the comparison of data sets in shared databases and in published research.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3010. doi:10.1158/1538-7445.AM2011-3010