bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2023: The role of RASSF1A in FasL expression and function
Authors: Perera Janaki , Burger Jordyn , Aznavoorian-Cheshire Sadie , Pratt Joanne C.
Published: August 2012

RASSF1A is a tumor suppressor protein that is epigenetically silenced in a variety of tumor cells, including those of lymphocytic origin. Several previous studies have identified a role for RASSF1A in death receptor-dependent apoptosis in abnormal cells of epithelial origin downstream of tumor necrosis factor receptor 1 (TNF-R1) engagement; however relatively little is known about a possible role for Rassf1A in lymphocytic cells. Interaction of Fas ligand (FasL) on activated T lymphocytes with the Fas receptor on other immune cells plays a major role in down-modulating immune responses through activation-induced cell death (AICD). Apoptosis of T cells that have undergone clonal expansion in response to antigenic stimulation through the TCR is a key regulatory feature of the adaptive immune response that prevents the accumulation of excessive lymphocytes once the antigen has been cleared. We have examined the effect of RASSF1A overexpression on FasL transcriptional activation and function in the Jurkat human T cell line. We have demonstrated that over-expression of RASSF1A induced a significant increase in FasL promoter (FasLp) activity. Similar-fold increases in transcriptional activity were observed when the distal NFAT binding site (3X NFAT plus minimal promoter) within FasLp was used in co-transfection reporter assays; however, other transcription reporters (AP1, NFkB) were unaffected. Experiments with mutant Jurkat cell lines indicate that early signaling mediators, including tyrosine kinases and PLCγ, are critical in the RASSF1A-induced enhancement of FasL production. Poly (ADP-ribose) polymerase (PARP) cleavage is mediated by caspases, and is indicative of apoptosis. Supernatants from anti-CD3 stimulated Jurkat cells that stably overexpress RASSF1A show an increased ability to induce cleavage of PARP in apoptosis-sensitive J16 target cells when compared to anti-CD3 stimulated vector-transfected cells. Taken together, these results indicate that RASSF1A plays a role in expression of soluble FasL leading to increased apoptosis-inducing activity by these lymphocytic cells. The loss of this activity could contribute to outgrowth of malignant clones of lymphocytic origin in which RASSF1A is silenced.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2023. doi:1538-7445.AM2012-2023
Abstract 203: Hotair, a long non-coding RNA, exhibits pro-oncogenic activity in pancreatic cancer
Authors: Kim Kyounghyun , Safe Stephen
Published: August 2012

HOTAIR is a long intervening non-coding RNA (lincRNA) that represses specific gene transcription by interaction with the Polycomb Repressive Complex 2 (PRC2) that trimethylates histone H3 lysine 27, leading to reprograming of chromatin states. HOTAIR is a poor prognostic indicator for survival of breast, colon and liver cancer patients and is highly associated with cancer invasion and metastasis. In this study, we show that HOTAIR is more highly expressed in pancreatic tumors than normal tissue and is strongly correlated not only with cell invasiveness but also cell proliferation and death. Overexpression of HOTAIR increased cell invasion in pancreatic cancer cells expressing low levels of HOTAIR and this was previously observed in other cancer cell lines. However, unlike previous reports, HOTAIR knockdown in Panc1 and L3.6pL pancreatic cancer cells that overexpress this lincRNA decreased cell proliferation, altered cell cycle progression, and induced apoptosis, demonstrating an expanded function for HOTAIR in pancreatic cancer cells compared to other cancer cell lines. Results of gene profiling studies after HOTAIR knockdown showed that there was minimal overlap between HOTAIR-regulated genes in pancreatic vs. breast cancer cells and HOTAIR uniquely suppressed several interferon-related genes and genes related to cell cycle progression in pancreatic cancer cells. Consistently, Gene Set Enrichment Analysis (GSEA) shows that HOTAIR regulates gene sets relevant to cell proliferation using HOTAIR Knockdown gene profiling data and pancreatic patient gene expression data. Analysis of selected genes suppressed by HOTAIR in Panc1 and L3.6 pL cells showed by knockdown of EZH2 and chromatin immunoprecipitation assays that HOTAIR-mediated gene repression was both PRC-2-dependent and -independent. HOTAIR knockdown in L3.6pL cells also inhibited tumor growth in mouse xenograft model, further demonstrating the pro-oncogenic function of HOTAIR in pancreatic cancer. These results indicated that HOTAIR is a potential molecular target for inhibiting pancreatic cancer cell invasion, metastasis and proliferation for inducing cell death.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 203. doi:1538-7445.AM2012-203
Abstract 2031: Effect of 2-Deoxyglucose on colorectal cancer cell lines
Authors: Muley Pratik U. , Olinger Alex , Tummala Hemachand
Published: August 2012

Aerobic glycolysis (Warburg Effect) has long been regarded as a hallmark of many types of cancers. Colorectal cancer is known to exist in hypoxic conditions and hence, it is believed that most of the colorectal carcinoma tumors depend more on glycolysis than other tumors. A number of drugs have been developed in the past to target the up-regulated glycolytic pathway of the tumor cells, including 2-Deoxy-glucose (2DG). Being structurally similar to D-Glucose, it is utilized in the glycolytic pathway and is phosphorylated to 2DG-phosphate, which starts to accumulate in the cells. The accumulated 2DG-phosphate, inhibits key glycolysis enzymes - hexokinase and phospho-glucoisomerase leading to inhibition of glycolysis, and thus starving the cells of ATP. Although this has been regarded as the major mechanism of 2DG, various observations question this being the sole mechanism of action. To further investigate the molecular mechanism of 2DG, we studied its effect on colorectal carcinoma cell lines (GC3/Cl, SW620 and SW480) alone or in combination with 5-fluorouracil (5FU). We have observed that colorectal carcinoma cells are highly susceptible to 2DG compared to other cancer cells or non-cancerous cells and exert an additive effect in combination with 5FU. 2DG treatment arrested cell cycle at G1/G0 checkpoint and promoted apoptosis. The treatment of 2DG also resulted in a transient increase in the expression of p21. A reduced expression of other cell cycle proteins such as cyclin D1 and cyclin A was also observed following the 2DG treatment. Here, we provide evidence that in colorectal carcinoma cell lines, the 2DG causes a cell cycle arrest in addition to glycolysis inhibition and this additional activity may be responsible for an increased susceptibility of colorectal cancer cell lines to 2DG.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2031. doi:1538-7445.AM2012-2031
Abstract 2035: The effect of KLF5 on cell cycle progression depends upon p53 status
Authors: Yang Yizeng , Lai Dongming , Tetreault Marie-Pier , Katz Jonathan P
Published: August 2012

Background: p53 mutation is one of the most common genetic events in esophageal squamous cell cancer (ESCC), and loss of Krüppel-like factor 5 (KLF5) in the context of p53 mutation transforms primary esophageal squamous epithelial cells. Interestingly, while KLF5 promotes proliferation in non-transformed esophageal squamous epithelial cells, KLF5 is growth inhibitory in ESCC. The mechanisms of this context dependent effect of KLF5 on cell Hypothesis: We hypothesized that the effect of KLF5 on cell cycle progression is dependent on p53 status. Methods: Human primary esophageal keratinocytes with or without either wild type or mutant p53R175H were transduced with lentivirus expressing inducible KLF5 shRNA or control. We investigated cell proliferation by MTT assay and cell cycle progression by flow cytometry. The expression of the cyclins and cyclin-dependent kinases were assessed by quantitave PCR. p21waf/cip1 activity was determined using a luciferase reporter. Results: In the context of wild-type p53, KLF5 suppression decreased cell proliferation and produced growth arrest at G2/M, with decreases in cyclin A2 and cyclin E1expression. In contrast, KLF5 loss in human primary keratinocytes harboring the common human p53 mutation p53R175H accelerated the cell cycle with more cells in G1/S and increased cyclin D1 expression. Interestingly, KLF5 directly bound within the p53 response elements in the regulatory region of p21waf/cip1 and activated its promoter activity only in cell with mutant p53. In addition, KLF5 suppression diminished p21waf/cip1 responsiveness to UV irradiation and growth factor depletion only in p53 mutant cells. Conclusions: Here, we identify a novel mechanism for context dependent effects of KLF5 on cell cycle via p53. These findings, in combination with the previously-established effects of KLF5 and p53 loss on cellular transformation, suggest that KLF5 is an important brake on the cell cycle and may function as a tumor suppressor in esophageal squamous cell cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2035. doi:1538-7445.AM2012-2035
Abstract 2036: Forced activation of Cdk1 via Wee-1 inhibition impairs homologous recombination in breast cancer cells
Authors: Van Vugt Marcel A. , Krajewska Malgosia , Bisselink Yvette , Seinstra Renee I. , Sillje Herman H. , de Vries Elisabeth G.
Published: August 2012

In response to DNA breaks, the so-called ‘DNA damage response’ provokes a cell cycle arrest to facilitate DNA repair. Recent findings have indicated that cells can respond to DNA damage throughout the cell cycle, except during mitosis. Specifically, various mitotic kinases, including Cdk1, Aurora A and Plk1, were shown to inactivate key DNA damage checkpoint proteins, including 53BP1. As a consequence, aberrant expression of mitotic kinases during interphase might modulate cellular responses to DNA damage. In this study we analyzed how aberrant activation of Cdk1 affects the cellular responses to DNA damage in breast cancer cells. We used Wee1 inhibition to enforce Cdk1 activation, which did not cause cytotoxicity in non-transformed cells. In contrast, Wee1 inhibition accelerated mitotic entry and caused radio-sensitization in p53-defective, but not p53-proficient breast cancer cells. Interestingly, we could show that Wee1 inhibition leads to elevation of Cdk1 activity in interphase breast cancer cells. When we subsequently analyzed DNA damage responses in cells with forced Cdk1 activation, we observed a marked reduction of the repair protein 53BP1 at the sites of DNA damage. In addition, Wee1 inhibition resulted in an increase in the number of γ-H2AX foci after irradiation, indicative of defective DNA repair. Indeed, when DNA repair was analyzed using in vivo endonuclease-induced recombination assays, compromised DNA repair after Wee1 inhibition was confirmed. Taken together, our data show that Wee1 inhibition leads to forced Cdk1 activation in interphase cells and that this interferes with normal DNA damage responses.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2036. doi:1538-7445.AM2012-2036
Abstract 204: Molecular function of the RNA binding protein EWS in RNA processing
Authors: Nieves Bethsaida I. , Niu Shuang , Vaka Dedeepya , Salzman Julia , Brown Patrick , Sweet-Cordero Alejandro I.
Published: August 2012

The Ewing Sarcoma (EWS) protein is a member of the FET family of RNA-binding proteins and is involved in chromosomal translocations that generate oncogenic fusion genes found in a variety of sarcomas. EWS has been recently reported to be involved in regulating transcription, pre-mRNA processing, translation and DNA repair. However, the precise physiological role of EWS in RNA processing remains largely unknown. Using cross-linking immunopercipitation coupled with high-throughput sequencing (CLIP-Seq), we identified the full complement of RNAs bound to EWS in Hela cells. Analysis of RNA binding by EWS revealed that the greatest increase in binding was to mRNAs coding for A-I editing enzymes ADAR1, ADARB1 and ADARB2. EWS also bound to non-coding RNAs (lncRNAs) NEAT1 and MALAT1. Interestingly, these lncRNAs localize to specific subnuclear structures and have been implicated in regulating RNA editing and RNA splicing respectively. We hypothesize that EWS could play a role in regulating the subnuclear localization of other proteins involved in editing and splicing by virtue of its association with NEAT1 and MALAT1. We also found that binding of EWS to pre-mRNAs influenced alternative splicing. Paired-end RNA-Seq of the transcriptome revealed that knock down of EWS resulted in the over expression of 116 genes and the down regulation of 68 genes, including EWS itself. Further analysis identified 16 alternatively spliced genes following EWS knock down across two independent RNA seq experiments. Of these genes, 6 were also shown to bind EWS by CLIP-seq. These genes include, CENPA, CSTF1, RBBP6, RAC1, FANC1 and ANAPC5. These studies highlight a potential role for EWS as a critical regulator of alternative splicing and A-I editing.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 204. doi:1538-7445.AM2012-204
Abstract 2040: The Mre11 nuclease is critical for sensitivity of cells to Chk1 inhibition
Authors: Thompson Ruth H. , Montano Ryan E. , Eastman Alan R.
Published: August 2012

The Chk1 kinase is required for the arrest of cell cycle progression when DNA is damaged, thereby providing time for cells to repair their DNA and recover. Chk1 also stabilizes stalled replication forks. As a consequence, many Chk1 inhibitors have been developed and tested for their potential to enhance DNA damage-induced tumor cell killing. However, inhibition of Chk1 alone, without any additional exogenous agent, can be cytotoxic. Using the novel Chk1 inhibitor MK-8776 (previously known as SCH900776), we screened a panel of cell lines and found that some are killed rapidly at low concentrations while other cell lines continue to grow in the presence of much higher concentrations. Understanding the underlying mechanisms of this sensitivity is critical for defining which patients might respond best to therapy with Chk1 inhibitors. We have investigated the mechanism of sensitivity in U2OS osteosarcoma cells. Upon incubation with MK-8776, single-stranded DNA regions (ssDNA) and double-strand breaks (DSB) begin to appear within 6 h. These DSB have been attributed to the structure-specific DNA endonuclease, Mus81. The Mre11/Rad50/Nbs1 (MRN) complex is known to be responsible for the resection of DSB to ssDNA. However, we show that inhibition of the Mre11 nuclease activity leads to a decrease not only in the amount of ssDNA following Chk1 inhibition, but also in the formation of DSB, suggesting that DSB arise as a consequence of ssDNA formation. These findings were corroborated by the discovery that Mre11-deficient ATLD1 cells are highly resistant to MK-8776 and form neither ssDNA nor DSB following treatment, but once complimented with exogenous Mre11, the cells accumulate both ssDNA and DSB when incubated with MK-8776. Inhibition of Chk1 also leads to aberrant activation of Cdc25A and Cdk2, and our findings suggest that Mre11 provides the link between Cdk2 activation and Mus81. We propose that the variable sensitivity of cell lines to Chk1 inhibition reflects defects in this pathway. These data highlight a novel role for Mre11 in the production of DSB and may help define which tumors are more sensitive to MK-8776 alone or in combination with DNA damaging agents.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2040. doi:1538-7445.AM2012-2040
Abstract 2041: Her2/Neu has an essential role for the gamma-irradiation-induced ERK1/2 signaling activation and G2/M checkpoint response
Authors: Yan Ying , Greer Patrick M. , Kolb Ryan H. , Cowan Kenneth H.
Published: August 2012

Gamma-irradiation (IR)-induced double strand DNA breaks frequently results in G2/M cell cycle arrest, allowing time for DNA repair prior to re-entering cell cycle. Previous studies from our laboratory have shown that the G2/M checkpoint activation following exposure of MCF-7 breast cancer cells to IR is dependent on the activation of extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) signaling. Studies presented in this report indicate that IR exposure of several breast cancer cell lines (MCF-7, MDA-MB-231, SKBR3 and T47D) is associated with a marked increase in HER2/neu phosphorylation and that the inhibition of HER2/neu using specific inhibitors abrogates the IR-induced activation of both MEK1/2 and ERK1/2 in these cells. Furthermore, the HER2/neu inhibition also results in attenuation of IR-induced G2/M arrest and concomitant diminution of IR-induced activation of ATM, ATR, Chk1 and Chk2 kinases. In contrast, inhibition of HER1/EGFR using specific inhibitors has no detectable effect on IR-induced increase of ERK1/2 phosphorylation as well as the induction of G2/M arrest following IR exposure. Collectively, results in this report suggest that HER2/neu plays an essential role in the activation of IR-induced of MEK/ERK signaling and subsequent G2/M checkpoint response.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2041. doi:1538-7445.AM2012-2041
Abstract 2042: The RAX/PACT-PKR stress-signaling pathway promotes p53 sumoylation and stability to induce G1 arrest
Authors: Bennett Richard L. , Pan Yu , Hui Teng , May W. Stratford
Published: August 2012

Cellular stresses including IL-3 withdrawal from factor-dependent hematopoietic cells, inflammatory cytokine treatment or viral infection promote RAX/PACT-dependent activation of the interferon-induced, double-stranded RNA activated protein kinase PKR. Previously, we and others demonstrated that activated PKR phosphorylates the alpha subunit of eIF2 to inhibit protein synthesis and initiate apoptosis. In addition, recent reports indicate that PKR may regulate the transcription factors p53, STAT1 and NF-κB. Now we find that RAX/PACT interacts with the SUMO E2 ligase Ubc9 to stimulate p53-Ubc9 association and reversible p53 sumoylation on lysine 386. In addition, stable or transient expression of RAX/PACT in a variety of cell lines promotes p53 stability and activity to increase p53 target gene expression. Significantly, while expression of RAX/PACT, PKR or p53 alone has little effect on the cell cycle of p53-null H1299 cells, co-expression of p53 with either RAX/PACT or PKR promotes a 25 - 35% increase of cells in G1. In contrast, co-expression of RAX/PACT with the sumoylation-deficient p53 (K386R) mutant or with p53 and the desumoylase SENP1 fails to induce such a G1 arrest. Furthermore, RAX/PACT-induced, p53-dependent G1 growth arrest is inhibited by expressing the dominant-negative PKR (K296R) mutant, and expression of RAX/PACT in pkr+/+ but not pkr-/- MEF cells promotes p53 and p21 expression following gamma irradiation. Significantly, expression of exogenous RAX/PACT promotes phosphorylation of wild-type but not p53 (K386R) on serine 392, and p53 stability is decreased in cells with reduced RAX/PACT or PKR following doxorubicin treatment. Collectively, results indicate that in response to stress the RAX/PACT-PKR signaling pathway may promote p53 translational activation leading to G1 cell cycle arrest by a mechanism dependent on p53 sumoylation and phosphorylation that regulates protein turnover. Importantly, these novel findings may indicate a wider role for the RAX/PACT-PKR signaling pathway to direct sumoylation-dependent subcellular localization, protein stability or protein-protein interactions of downstream targets in response to cellular stress.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2042. doi:1538-7445.AM2012-2042
Abstract 2045: BubR1 is modified by sumoylation during mitotic progression
Authors: Yang Feikun , Hu Liyan , Yao Yixin , Yu Jianxiu , Khodjakov Alexey , Pagano Michele , Dai Wei
Published: August 2012

BubR1 functions as a crucial component that monitors proper chromosome congression and mitotic timing during cell division. We investigated molecular regulation of BubR1 and found that BubR1 was modified by an unknown post-translation mechanism during the cell cycle, resulting in a significant mobility shift on denaturing gels. We termed it BubR1-M as the nature of modification was not characterized. Extended (>24 h) treatment of HeLa cells with a microtubule disrupting agent including nocodazole and taxol or release of mitotic shake-off cells into fresh medium induced BubR1-M. BubR1-M was derived from neither phosphorylation nor acetylation. Ectopic expression coupled with pulling down analyses showed that BubR1-M was derived from SUMO modification. Mutation analysis revealed that lysine 250 was a crucial site for sumoylation. Significantly, compared with the wild-type control, ectopic expression of a sumoylation-resistant mutant of BubR1 induced chromosomal missegregation and mitotic delay. Combined, our study identifies a new type of post-translational modification that is essential for BubR1 function during mitosis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2045. doi:1538-7445.AM2012-2045
Abstract 2056: Molecular delineation of TIP150 function underlying kinetochore microtubule dynamics during mitotic chromosome segregation
Authors: Ward Tarsha L.
Published: August 2012

The microtubule cytoskeleton orchestrates the cellular dynamics and plasticity that underlies cellular morphogenesis. Microtubule binding proteins known as “TIP” proteins regulate the dynamic properties of microtubules during chromosomal segregation and cellular division. Our recent study revealed that TIP150 is a novel plus end tracking protein interacting with EB1 and MCAK in vitro and in vivo (EMBO Reports. 2009. 10:857-65). However, it is unclear whether and how this interaction regulates chromosome segregation in mitosis. We hypothesize that Tip150 is essential for chromosome segregation, and governs proper kinetochore-microtubule attachment at the microtubule plus end. To delineate the molecular function of TIP150 and its regulation in chromosome segregation and the mitotic cell cycle, a combination of biophotonic analyses with RNA interference was employed. Immunofluorescence reveals that TIP150 localizes to the outer most part of the kinetochore and to the ends of growing microtubules by its C-terminal domain. Suppression of TIP150 resulted in an increase in mitotic defects, reduced kinetochore-microtubule attachment, and perturbed chromosome oscillation dynamics. Furthermore, the use of total internal reflection microscopy reveals a novel role for TIP150-EB1 elicited tracking to the microtubule ends. We reason that TIP150 is essential for kinetochore-microtubule attachment in mitosis and microtubule dynamics.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2056. doi:1538-7445.AM2012-2056
Abstract 2064: Atm is required for homeostasis within the lactating mammary gland
Authors: Dyer Lisa M. , Brown Kevin D.
Published: August 2012

ATM is a protein kinase activated in response to DNA damage and oxidative stress. Limited evidence suggests that ATM functions in mammary gland development and tumor suppression. However, we now document that mice with germ line loss of Atm exhibit severely blunted mammary gland development. To examine a role for Atm within the mammary gland we generated a novel mouse line with conditional deletion of Atm (AtmWAP-cre) in the mammary epithelium. Characterization of the AtmWAP-cre mouse line indicated that reduced Atm expression results in a progressive lactation defect as judged by a reduction in pup growth rate, aberrant lobulo-alveolar structure, and significantly diminished expression of several milk protein genes in mid-lactation dams. We also document that lactating AtmWAP-cre dams exhibit both morphological and molecular features consistent with premature entry into involution. These aberrant events in Atm-deficient glands occur independently of established Jak/Stat signaling mechanisms that normally control mammary gland development. We found that RNA harvested from lactating Atm-deficient mammary glands contain heightened levels of oxidized guanine (8-oxoGua) indicating unchecked response to oxidative damage. Finally, we knocked down ATM expression in human and mouse mammary epithelium-derived lines and measured increased sensitivity to oxidative stress. This study supplies evidence for a novel biological function for Atm within the mammary gland and provides new insight into the critical importance of oxidative stress response during the high metabolic burden associated with the process of lactation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2064. doi:1538-7445.AM2012-2064
Abstract 2102: High resolution fluorescence imaging of cellular DNA and RNA enabled by the synthesis of complex oligonucleotide libraries
Authors: Ach Robert A. , Yamada Alice , Tsang Peter , Pekrun Katja , Scheffer-Wong Alicia , Bruhn Laurakay
Published: August 2012

Fluorescence in situ Hybridization (FISH) is a powerful technique for studying the structure, organization, and localization of nucleic acids within individual cells. However, the use of FISH has often been dependent upon access to cloned template DNA for the generation of probes. Clones for specific regions may be unavailable, or the genomic region of interest may contain repetitive and other non-informative sequences which can be problematic for FISH analysis. We have leveraged our ability to chemically synthesize DNA in massively parallel reactions to produce libraries of oligonucleotides up to 200 bases in length that can be used to generate FISH probes. The sequences of the oligonucleotides in these libraries are selected in silico using empirically determined criteria so as to avoid repetitive elements or regions homologous to other non-targeted loci. Using oligonucleotide library-derived FISH probes on DNA, human genomic regions as small as 1.8 kb and as large as whole chromosomes can be visualized in both metaphase and interphase cells using the same simple assay protocol. Because of the inherent flexibility in our probe design methods, we readily visualized regions rich in repeats and/or GC content. Using oligonucleotide library-derived FISH probes on RNA, we have been able to detect the localization of a variety of both coding and non-coding RNAs in fixed cells, using both conventional fluorescence and structured illumination microscopy. Simultaneous hybridization of FISH probes labeled with different fluorophores enables visualization of multiple sequences at once. Using probes designed specifically to transcribed vs. non-transcribed regions, we have been able to simultaneously detect DNA and RNA from the same locus in the same FISH assay. Our oligo FISH methods are readily compatible with the co-detection of cellular proteins by immunocytochemistry. The ability to generate high performance FISH probes using chemically synthesized oligo libraries that can work flexibly with co-detection of other molecules yields a valuable tool for studies of how localization of specific nucleic acids impacts biological function.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2102. doi:1538-7445.AM2012-2102
Abstract 2110: An attempt to replicate the results of genome-wide association studies on lung cancer susceptibility loci in a Korean population
Authors: Kang Hyo-Gyoung , Bae Eun Young , Lee Shin Yup , Jeon Hyo-Sung , Park Jae Yong
Published: August 2012

Genome-wide association studies (GWASs) have identified three chromosomal regions at 5p15, 6p21, and 15q25 as being associated with lung cancer risk in European populations. This study was performed to confirm these associations in Korean patients with lung cancer. The genotypes of rs2736100, rs402710, rs401681, and rs31489 at 5p15, rs9295740 at 6p22, and rs2036534 and rs6495309 at 15q25 were determined in 1094 lung cancer patients and 1100 healthy controls frequency-matched for age and gender. The single nucleotide polymorphisms (SNPs) at 5p15 and 15q25 were significantly associated with lung cancer risk. The magnitude of effect was similar to that reported in previous studies and the association was in the same direction. The effect of SNPs at 5p15 region on the risk of lung cancer was significant only in adenocarcinoma. The two SNPs at 15q25 region were significantly associated with lung cancer risk in ever-smokers and squamous cell carcinoma. However, there was no association between the SNP at 6p22 and lung cancer risk. We confirmed the association between SNPs at the 5p15 and 15q25 regions and the risk of lung cancer in a Korean population.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2110. doi:1538-7445.AM2012-2110
Abstract 2115: REV1, DNA polymerase ≤ and the fanconi anemia proteins FANCD2 and FANCI function together during repair
Authors: Sharma Shilpy , Canman Christine E.
Published: August 2012

Fanconi anemia (FA) is a chromosomal instability disorder characterized by bone marrow failure, congenital abnormalities and increased incidence of malignancies. Cells derived from FA patients exhibit cellular hypersensitivity to interstrand crosslink (ICL)-inducing agents, including mitomycin C (MMC) and cisplatin. ICLs are sensed and repaired by a complex process which involves multiple DNA repair proteins including the FA factors as well as proteins that function in nucleotide excision repair, translesion DNA synthesis and homologous recombination (HR). REV1 (a Y family translesion DNA polymerase) and DNA Polymerase ≤ (Polα, comprised of the catalytic REV3 protein and REV7 accessory subunit) play important roles in performing DNA replication across blocking lesions. Previous studies have demonstrated an epistatic relationship between Polα and genes encoding proteins comprising the Fanconi anemia core complex with regards to resistance to ICLs. Additionally, REV1, Polα, and the FANCD2/FANCI heterodimer have each been shown individually to be involved in promoting HR repair, although the reasons for their involvement remain unclear. Here, we show that human REV1 and REV3 exhibit an epistatic relationship with FANCD2 and FANCI in homology-directed repair of a defined DNA double-stranded break and cellular resistance to ICLs created by MMC. We also demonstrate that MMC-induced chromosomal anomalies are increased in REV1 or REV3-depleted cells and FANCD2 depletion does not further exacerbate this phenotype. Additionally, we demonstrate for the first time that full length human REV1 and REV3 interact with FANCD2 and FANCI in vivo as detected by co-immunoprecipitation studies. We therefore propose a model where replication fork stalling during HR repair DNA synthesis is resolved through Polα-dependent translesion DNA synthesis which is regulated by the Fanconi anemia pathway.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2115. doi:1538-7445.AM2012-2115
Abstract 212: gp78/AMFR is involved in ubiquitination of PGI/AMF
Authors: Wang Ying , Xie Youming , Nangia-Makker Pratima , Balan Vitaly , Raz Avraham
Published: August 2012

Phosphoglucose isomerase/autocrine motility factor (PGI/AMF) is a multifunctional protein that intracellularly catalyzes the interconversion of glucose 6-phosphate and fructose 6-phosphate in both glycolytic and gluconeogenesis pathways and extracellularly acts as a cytokine associated with aggressive tumor phenotype. Secreted PGI/AMF promotes cancer cell invasion and metastasis by stimulating cell motility via an autocrine manner following binding to its seven-transmembrane glycoprotein receptor gp78/AMFR. Gp78/AMFR also demonstrates an additional function in the endoplasmic reticulum (ER) as ubiquitin ligase (E3). Thus, gp78/AMFR represents a potential link between ubiquitination, ER-associated degradation (ERAD) and metastasis. We questioned if gp78/AMFR is involved in the ubiquitination of PGI/AMF, and whether the ubiquitinated PGI/AMF can be related to cancer progression and metastasis. In this study, we found that indeed PGI/AMF can be ubiquitinated and gp78/AMFR is involved in this process. PGI/AMF was predominantly ubiquitinated via K29-linked ubiquitin chains. Monoubiquitinated PGI/AMF increases the secretion of PGI/AMF and this is the first report to show that monoubiquitinated PGI/AMF is secreted out of the cells. As secretion of PGI/AMF plays a significant role in tumor cell migration and invasion during metastasis, these studies assist in understanding the regulation of PGI/AMF and the mechanisms of its secretion, which may provide a new therapeutic target for tumor metastasis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 212. doi:1538-7445.AM2012-212
Abstract 2127: SSB1 is essential for embryogenesis and maintenance of genomic stability in mice
Authors: Khanna KumKum , Shi Wei , Bain Amanda
Published: August 2012

Single-stranded DNA binding proteins (SSBs) are essential for multiple DNA transactions, including regulation of DNA damage checkpoints and repair of DNA damage. Previously, we reported the functional characterization of these proteins in human cells using specific siRNA and provided the initial evidence that both SSBs are essential for efficient repair of DNA double strand breaks by the homologous recombinational repair pathway, and for efficient initiation of the ATM/ATR dependent signaling pathway (Richard et al., 2008). However cellular model systems have inherent limitations. To decipher the role of these newly identified proteins at the organismal level, we have generated conditional SSB1 and SSB2 knockout (KO) mouse models. The constitutive SSB1 KO mice show late embryonic lethality and die perinatally. The conditional inactivation of SSB1 in adult mice is associated with testicular atrophy, reduced fertility and increased genomic instability. We also find compensatory up-regulation of SSB2 protein levels in multiple tissues of conditional SSB1 KO mice. In addition, we report increased radiosensitiity of SSB1-/- mice in response to ionizing radiation, and increased genomic instability, which manifests as a broad spectrum of tumours at approximately 1 year of age. T cells isolated from these animals exhibited mild G1/S and G2/M checkpoint defects, and a persistence of DNA damage as seen by increased gamma-H2AX and 53BP1 foci in response to α-irradiation. Collectively, these results demonstrate an essential role of SSB1 in the maintenance of genomic stability in-vivo. Reference Richard, DJ., Bolderson, E., —et al. Khanna KK (2008). Single-stranded DNA binding protein is critical for genomic stability. Nature 453: 677-681

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2127. doi:1538-7445.AM2012-2127
Abstract 2143: Smg-GDS regulates cellular proliferation in squamous cell carcinomas of the head and neck
Authors: Bock Jonathan M. , Wrege Kay , Johnston Nikki , Mackinnon Alexander C. , Williams Carol L.
Published: August 2012

Squamous cell carcinoma of the head and neck (SCCHN) is a major worldwide source of morbidity and mortality, with over 26,000 estimated new cases and 6,000 deaths from pharyngeal and laryngeal cancer in 2011 in the the United States alone. SCCHN is an aggressive disease which is characterized by local invasion and lymphatic and distant metastasis. Ras and Rho family GTPases are known to regulate tumor cell proliferation in numerous human cancers including SCCHN. SmgGDS, an unusual guanine nucleotide exchange factor, has previously been shown in other systems to activate these small GTPases and directly regulate cellular proliferation through control of small GTPase prenylation and cellular localization. Little is known about the role of SmgGDS in the progression of SCCHN. These studies were begun to characterize the expression of SmgGDS in SCCHN and assess the role that SmgGDS plays in SCCHN proliferation. SCCHN tumor tissue microarray analysis demonstrated increased SmgGDS expression in tumor tissue from various head and neck sites compared to normal tissues, with evidence for higher SmgGDS expression at the pushing tumor boarders in invasive cancers. Significantly increased amounts of SmgGDS protein and RNA expression were also noted in head and neck tumor cell lines compared to cultured normal head and neck tissue cell lines. To assess the function of Smg-GDS expression in SCCHN, UM-SCC-1 cells were transfected with siRNA directed toward the 60.7 and 55.8 kD SmgGDS isoforms, with significant decreases in S-phase distribution for all treatments. Blocking both 60.7 and 55.8 kD SmgGDS isoforms with siRNA induced an 89% decrease in S-phase distribution as determined by Click-iT Edu cell cycle proliferation assay. These studies confirm an active role for SmgGDS protein function in the promotion of cancer cell proliferation in SCCHN, and provide the basis for further investigations into the role of small GTPases in head and neck neoplasia.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2143. doi:1538-7445.AM2012-2143
Abstract 2153: Active PAK6 induces aneuploidy in breast cancer cells
Authors: Lu Michael L.
Published: August 2012

The natural history of breast cancer remains largely undefined. More than 50% of the clinical specimen exhibits aneuploidy. Among these aneuploid cancers, 50% has chromosome copy number near tetraploid. The high incidence of aneupoidy in advanced breast cancer is thought to be a byproduct of chromosome mis-segregation resulting from mutations of checkpoint-regulating tumor suppressor genes associated with disease progression. However, new large-scale sequencing projects reveal that most tumors contain approximately 14-20 different mutant genes, suggesting that genomes and karyotypes of cancer cells are equally heterogeneous. This notion revives the old concept that aneuploidy resulting from chromosome mis-segregation or other similar mechanisms may play an active role leading to transformation and disease progression. A novel p21-activated kinase 6 (PAK6) was recently identified to be an androgen receptor (AR) and estrogen receptor (ER) interacting protein. An estrogen-stimulated PAK6 activation was observed in breast cancer BT474 cells. This activation is mediated by estrogen-stimulated activation of PKA. At the cellular level, active PAK6 promotes breast cancer MCF7 cell metastatic phenotypes including increased cell motility, matrigel invasion and soft agar growth. Further characterization of breast cancer MCF7 cells expressing PAK6 constitutive active mutant revealed an accumulation of cells with higher ploidy as compared to that of parental MCF7. Using a proteomic approach to determine the PAK6 downstream target, we identified that Cdt1, a DNA pre-replicative complex (pre-RC) subunit, is overexpressed in response to PAK6 activation. In eukaryotes, the expression and stability of Cdt1 are strictly regulated to ensure only one round of DNA replication in each cell cycle. Deregulated overexpression of Cdt1 has previously been demonstrated to induce aneuploidy and malignant transformation due to re-initiation of DNA replication within the same S-phase. The identification of Cdt1 overexpression in response to PAK6 activation provides a plausible mechanism for the observed accumulation of higher ploidy cells in MCF7 expressing active PAK6. These findings directly link the hormonal signals to the DNA replication process and maintenance of genomic integrity.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2153. doi:1538-7445.AM2012-2153
Abstract 2155: Inhibition of the ERK1/2 pathway by RanBPM
Authors: Atabakhsh Elnaz , Schild-Poulter Caroline
Published: August 2012

Ran binding protein-M (RanBPM, also called RanBP9) is a nucleo-cytoplasmic protein that has been implicated in a multitude of cellular roles, including the regulation of transcription, protein modifications and stability, and extracellular receptor signaling pathways. However its function within the cell remains largely unknown. Previous studies in our lab have identified RanBPM as a pro-apoptotic protein that mediates its effects through modulation of Bcl-2 and Bcl-XL levels. Here we present evidence that RanBPM functions to regulate apoptotic activation through regulation of the ERK1/2 signaling pathway. First, we determined that siRNA-mediated knockdown of RanBPM expression led to elevated Bcl-2 and Bcl-XL mRNA expression, suggesting regulation by RanBPM at the transcriptional level. Additionally, expression of RanBPM affected ectopically expressed Bcl-2 protein levels. Together, these findings suggested that RanBPM regulated the expression of Bcl-2 and Bcl-XL at both the transcriptional and post-translational levels. The ERK1/2 signaling cascade has previously been shown to regulate the expression of Bcl-2 and Bcl-XL at both the transcriptional and post-translational levels. We found that that RanBPM-deficient cells exhibited enhanced ERK1/2 phosphorylation and activation, while re-expression of RanBPM led to a marked decrease in the levels of phosphorylated ERK1/2. Furthermore, enhanced ERK1/2 activation in RanBPM-deficient cells contributed to the elevated levels of Bcl-2 expression observed in these cells, as inhibition of ERK1/2 activation correlated with decreased Bcl-2 protein expression. We characterized regulation of ERK1/2 signaling by RanBPM, and identified that RanBPM targets this pathway downstream of Ras, through modulation of c-Raf stability. Our studies revealed that downregulation of RanBPM expression promotes phosphorylation and stabilization of c-Raf, leading to elevated c-Raf protein expression. Finally we observed that downregulation of RanBPM expression promotes cellular transformation, as RanBPM-deficient cells exhibit increased cell growth rates and enhanced cell migration. Together, these findings suggested that RanBPM acts as a novel inhibitor of ERK1/2 signaling, and that downregulation of RanBPM expression leads to oncogenic transformation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2155. doi:1538-7445.AM2012-2155
Abstract 2161: MLK3 signals to paxillin in chemokine-induced migration and invasion, and is necessary for breast cancer metastasis
Authors: Chen Jian , Gallo Kathleen A.
Published: August 2012

Breast cancer metastasis is the major cause for mortality of patients with invasive breast cancer. Metastasis involves multiple steps including cancer cell invasion through basement membrane, intravasation, survival in the circulation, extravasation and colonization of distant organs. Identification of the signaling pathways through which prometastatic factors promote cancer cell migration and invasion is key to develop effective therapeutics for breast cancer. Mixed lineage kinase 3 (MLK3) is a MAP3K involved in activating each of the three major MAPK signaling pathways. The MLK3-JNK-AP-1 signaling axis has been shown to be required in breast cancer cell migration and invasion. Herein, we demonstrate that the prometastatic factors, CXCL12 and HGF, signal through MLK3-JNK to induce phosphorylation of the focal adhesion scaffold, paxillin on Ser 178, which is required for migration and invasion of human breast cancer cells. Furthermore, we show that MLK3 regulates the phosphorylation of paxillin at Tyr 118 and promotes the interaction of FAK with paxillin, which can be blocked by inhibition of MLK3. To further understand the role of paxillin phosphorylation at S178, a nonphosphorylatable paxillin mutant S178 A was generated. Expression of S178A blocks Y118 phosphorylation of paxillin in breast cancer cells. Silencing of mlk3, inhibition of JNK, or expression of the nonphosphorylatable paxillin S178A increases focal adhesion numbers and increases Rho activity, suggesting that the MLK3-JNK-paxillin axis limits Rho activity in breast cancer cells. Furthermore, MLK3 silencing is sufficient to prevent formation of metastases in a mouse xenograft model of spontaneous breast cancer metastasis. Our findings have identified a novel role of MLK3 in regulation of focal adhesion dynamics and cytoskeletal remodeling in the process of breast cancer cell migration and suggest that MLK3 may be a potential therapeutic target for metastatic breast cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2161. doi:1538-7445.AM2012-2161