bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3871: Per1 is phosphorylated by ATM/ATR and is involved in the G2/M checkpoint response
Authors: Gery Sigal , Tabasyhi Takayuki , Nowak Verena , Alvarez Rocio , Sohn Julia , Koeffler Phillip H.
Published: February 2011

Circadian clocks are found in most organisms and influence a large array of cellular, physiological and behavioral processes. Consequently, disruption of circadian function results in severe dysfunctions and has been associated with various pathologies including cancer. Recent findings revealed intriguing links between the circadian system and another highly conserved biological system, the DNA damage response (DDR). Upon DNA damage, eukaryotic cells activate a complex signaling network to regulate cell cycle checkpoint arrest and/or apoptosis. At the core of the DDR are the ATM, ATR and DNA-PK kinases, which phosphorylate numerous downstream targets on preferred SQ motifs. We previously showed that the key circadian factor, Per1, associates with ATM and sensitize cells to DNA-damage induced apoptosis. In a recent screen, human Per1 Ser1263 was identified as a potential substrate for ATM/ATR. Per1 Ser1263 is part of a SQ motif conserved among mammalian Per1 proteins. To confirm that Per1 is phosphorylated on this site, we mutated murine Per1 Ser1264 (corresponding to human Ser1263) to alanine. 293T and HCT116 cells were transfected with either wild-type (WT) or S1264A mutant (SA) myc-tagged Per1 and treated with DNA-damaging agents. Western blot analysis demonstrated that Per1WT was recognized by phospho-specific SQ antibodies, while Per1SA was not. Furthermore, Per1 phosphorylation was significantly stronger in the treated cells. Interestingly, Per1 remained phosphorylated in ATM deficient cells. In contrast, silencing of ATR by shRNA in 293T cells attenuated Per1 phosphorylation. Immunoprecipitations experiments showed that both overexpressed and endogenously expressed Per1 associate with ATR. To examine the functional significance of Per1 phosphorylation, we transfected SKOV3 cells with either myc-Per1WT or myc-Per1SA, and monitored their cell-cycle progression and apoptotic response after DNA damage. Compared to control SKOV3 treated cells, Per1WT expression resulted in significant increased numbers of G2/M arrested and apoptotic cells. This increase was diminished by Per1S1264A mutation. Our findings implicate Per1 as an important ATM/ATR substrate, and emphasize the significance of proper circadian regulation to cell survival upon exposure to genotoxic stress.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3871.
Abstract 3893: Overexpression of FOXQ1 is important factor in tumorigenicity and tumor growth
Authors: Kaneda Hiroyasu , Arao Tokuzo , Tanaka Kaoru , Tamura Daisuke , Aomatsu Keiichi , Kudo Kanae , Sakai Kazuko , De Velasco Marco Antonio , Matsumoto Kazuko , Yoshihiko Fujita , Yamada Yasuhide , Tsurutani Junji , Okamoto Isamu , Nakagawa Kazuhiko , Nishio Kazuto , Nagai Tomoyuki , Furuta Kazuyuki
Published: February 2011

Forkhead Box Q1 (FOXQ1) is a member of the forkhead transcription factor family, and it has recently been proposed to participate in gastric acid secretion and mucin gene expression in mice. However, the role of FOXQ1 in humans and especially in cancer cells remains unknown.

We found that FOXQ1 mRNA is overexpressed in clinical specimens of colorectal cancer (28-fold / colonic mucosa). A microarray analysis revealed that the knockdown of FOXQ1 using siRNA resulted in a decrease in p21CIP1/WAF1 expression, and a reporter assay and ChIP assay showed that p21CIP1/WAF1 was one of target genes of FOXQ1. Stable FOXQ1-overexpressing cells (H1299/FOXQ1) exhibited elevated levels of p21CIP1/WAF1 expression and inhibition of apoptosis induced by doxorubicin or camptothecin. Although cellular proliferation was decreased in H1299/FOXQ1 cells in vitro, H1299/FOXQ1 cells significantly increased tumorigenicity (EGFP: 2/15, FOXQ1: 7/15) and enhanced tumor growth (437 ± 301 vs. 1735 ± 769 mm3, p<0.001) in vivo. Meanwhile, stable p21CIP1/WAF1 knockdown of H1299/FOXQ1 cells increased tumor growth, suggesting that FOXQ1 promotes tumor growth independent of p21CIP1/WAF1. Microarray analysis of H1299/EGFP and H1299/FOXQ1 revealed that FOXQ1-overexpression upregulated several genes that have positive roles for tumor growth including VEGFA, WNT3A, RSPO2 and BCL11A. CD31 and TUNEL staining of the tumor specimens showed that FOXQ1-overexpression mediated the angiogenic and anti-apoptotic effect in vivo.

In conclusion, FOXQ1 is overexpressed in colorectal cancer and enhances tumorigenicity and tumor growth presumably through its angiogenic and anti-apoptotic effects. Our findings demonstrate that FOXQ1 is a new member of the cancer-related FOX family.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3893.
Abstract 3894: Regulation of VDUP1 expression in breast cancer cells by cyclopamine
Authors: Voigt Jeffrey M. , Bajaria Arjun , Dearborn Richard E.
Published: February 2011

VDUP1 (TXNIP) is a tumor suppressor protein that contributes to the regulation of cell proliferation through its role as an inhibitor of thioredoxin (TRX). VDUP1 expression is decreased in a variety of tumor cells relative to non-tumor cells and enforced expression of VDUP1 slows tumor cell growth. 1,25-Dihydroxyvitamin D3 (VD3) has long been known to increase VDUP1 expression in tumor cells, although the full extent of VDUP1 regulation is not known. Preliminary experiments with Drosophila mutants lacking Hedgehog (Hh) secretion suggested that VDUP1 expression could be regulated by the Hh signaling pathway. Experiments were conducted to determine the ability of cyclopamine (CYCLO), an inhibitor of Hh signaling, to regulate VDUP1 expression in breast cancer cells. MDA-231 and MCF-7 cells, grown in RPMI 1640 medium containing 10% fetal calf serum, were treated with CYCLO (5-20 μM) or vehicle (methanol) for 24-72 hrs. Using an anti-VDUP1 antibody, VDUP1 localization was determined by immunofluorescence staining of cells grown on gelatin-coated coverslips following fixation in 4% paraformaldehyde. VDUP1 expression in methanol-treated MDA-231 cells showed a greater expression of VDUP1 in nuclei, indicated by overlapping expression with the nuclear marker histone deacetylase, as compared to cytoplasm. Treatment with 20 μM CYCLO for 72 hours resulted in a decrease in nuclear VDUP1 expression, coupled with a dramatic increase in cytoplasmic VDUP1. This redistribution of VDUP1 from the nucleus to the cytoplasm was detected as early as 24 hours after initiation of treatment with 20 μM CYCLO and was also detected following treatment with 10 μM CYCLO for 72 hours. The CYCLO-induced decrease in nuclear VDUP1 was confirmed with Western blotting. In contrast, no such changes were detected in CYCLO-treated MCF-7 cells. To investigate the direct effect of CYCLO treatment on vdup1 promoter activity, a 1.7 kb fragment of the human vdup1 promoter was inserted upstream of a luciferase gene to generate a vdup1/pLuc plasmid. vdup1/pLuc was co-transfected into CYCLO-treated cells (20 μM), along with a β-galactosidase plasmid and luciferase activity was determined. CYCLO treatment of MDA-231 cells resulted in a significant decrease in luciferase activity, as compared to methanol-treated cells. No changes in luciferase activity were seen following similar experiments conducted with MCF-7 cells. These results demonstrate the novel finding that inhibition of Hh signaling alters both the level of VDUP1 expression and the intracellular distribution of VDUP1 in MDA-231 cells. Future experiments will be directed at determining the mechanisms responsible for the change in the subcellular distribution of VDUP1 seen following CYCLO treatment.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3894.
Abstract 3899: A coactivator role of CARM1 for the dysregulated β-catenin activity in colorectal cancer cell growth and gene expression
Authors: Ou Chen-Yin , Manegold Philipp , Kahn Michael , Stallcup Michael
Published: February 2011

Aberrant activation of Wnt/β-catenin signaling is recognized as a critical factor in the etiology of colorectal cancer. Evidence has suggested that dysregulated β-catenin activity is associated with the majority of colon cancers via activation of the expression of Wnt regulated oncogenes. In the nucleus, β-catenin regulates transcription by recruiting additional secondary coactivators. These secondary coactivators all have distinct and unique functions on Wnt/β-catenin target gene activation. For instance, CCAR1 (Cell Cycle and Apoptosis Regulator 1) has been shown to be important for the stable occupancy of β-catenin at the promoter of a Wnt target gene, and depletion of CCAR1 inhibits expression of several Wnt/β-catenin target genes and suppresses anchorage-independent growth of colon cancer cells (Ou CY et al. Journal of Biological Chemistry 2009;284(31);20629-20637).

Here, we show that CARM1 (coactivator-associated-protein-arginine-methyltransferase 1) interacts with β-catenin and positively modulates β-catenin-mediated gene expression. In colorectal cancer cells, which have constitutively high Wnt/β-catenin activity, depletion of CARM1 inhibits the expression of Wnt/β-catenin-mediated oncogenes and suppresses anchorage-independent growth. In colorectal cancer cells that do not have constitutively high Wnt/β-catenin activity, we found that activation of Wnt/β-catenin signaling by treatment with Wnt3a increased the expression of a subset of Wnt target genes. Utilizing these cells, we demonstrated that CARM1 is specifically required for the expression of these Wnt3a-induced targets. Upon Wnt3a stimulation, CARM1-dependent dimethylation of histone H3 arginine (Arg) 17, at a Wnt-regulated promoter, is associated with the recruitment of both β-catenin and CARM1. Arg17 dimethylation by Wnt3a ligands is compromised by CARM1 knockdown, whereas Wnt3a-induced β-catenin recruitment is not affected. Finally, in a nude mouse xenograft model, compared with CARM1 knockdown cells, wild type HT29 tumor xenografts grow significantly faster. We conclude that CARM1 is an important modulator of Wnt/β-catenin transcription and may represent a novel target for therapeutic intervention in cancers involving aberrantly activated Wnt/β-catenin signaling.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3899.
Abstract 3911: Dysegulation of Sox9-dependent gene expression leads to enhancement of angiogenesis and metastases in pancreatic cancer
Authors: Camaj Peter , Ischenko Ivan , Seeliger Hendrik , Krebs Stefan , Renner Andrea , Jauch Karl W. , Bruns Christiane J.
Published: February 2011

Background: The understanding of invasion, angiogenesis and metastasis is essential for the development of new targeted molecular therapy against pancreatic cancer. The aim of this study is to analyse the regulation of differentially expressed genes in highly proangiogenic and prometastatic compared to low metastatic and low angiogenic pancreatic cancer cells

Materials and methods: The transcriptome of the low metastatic pancreatic cancer cell line and its highly angiogenic and metastatic derivate L3.6pl have been compared under the normoxic and hypoxic conditions using Affymetrix HGU133plus 2.0 arrays. The hierarchic cluster analysis has been performed using SOTA algorithm. Promoters of the differentially expressed genes have been analysed using oPOSSUM 2 program. Affymetrix data have been validated via qRT-PCR and via ELISA. Chromosomal DNA containing promoters of the relevant genes has been amplified via PCR and sequenced.

Results: The Affymetrix analysis revealed clusters of differentially regulated genes with regard to the cell line and the oxygen level. While VEGF expression was upregulated in FG cells under hypoxic condition, it was constitutively upregulated already under normoxic conditions in L3.6pl and no further stimulation was observed under hypoxic conditions. This regulation pattern was similar in a cluster of genes that share a promoter sequence coding for a binding site for the transcription factor Sox9. Sox9 itself shows a similar regulation, leading to the assumption that expression changes of Sox9 are involved in the different hypoxic gene expressions. Promoter analysis of Sox9 in FG and L3.6pl cells showed mutations in the promoter region in L3.6pl, while the VEGF promoter was unchanged. Further analysis has shown that this mutation is not relevant for biological function. Refined analysis revealed differential Sox9 promoter methylation as reason for observed phenomenon.

Conclusions: Proangiogenic genes such as VEGF are upregulated in the more aggressive cell line L3.6pl, compared to FG. We identified a gene cluster that is constitutively upregulated in L3.6pl, independent of hypoxia. These genes are controlled by the transcription factor Sox9. A Sox9 promoter differential methylation may be the reason for the escape from hypoxic regulation in L3.6pl cells and thus may contribute to the higher angiogenic and metastatic potential. Taken together, Sox9 may be an interesting new diagnostic and therapeutic target in pancreatic cancer progression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3911.
Abstract 393: The SHrN™ scid hairless NOD mouse model: Development and characterization
Authors: Williams Shanice V. , Wildt Sheryl J. , Brooks Andrew I. , Cooper Dale M.
Published: February 2011

The SHrN™ scid hairless NOD mouse was developed to provide a hairless triple-immunodeficient mouse model to the research community. The non-obese diabetic (NOD) mouse carrying the Prkdcscid mutation (NOD scid mouse) is an established xenograft model for humanization, oncology, and immunology research. It has proven to be particularly useful in elucidating mechanisms of spontaneous lymphoma and thymic tumors, prostate cancer research, and the engraftment of hematopoietic stem cells. As scid mice are haired, it is often necessary to shave these mice to facilitate tumor observation and evaluation. However, shaving animals does not prevent autofluorescence of hair follicles while imaging scid mice. We have transferred the hairless mutation (Hrhr) from the outbred HsdOla:MF1-Hrhr to the NOD.CB17-Prkdcscid/NCrHsd mouse to generate the inbred hairless NOD.Cg-Prkdcscid Hrhr/NCrHsd mouse, or commonly named scid hairless NOD (SHrN™). Herein we describe the breeding program utilized to generate the SHrN™ model using marker assisted backcrossing methodology. A combination of single nucleotide polymorphism (SNP) allelic discrimination and quantitative PCR (QPCR) analysis was employed to genotype offspring for the Prkdcscid and Hrhr mutations. SNP genotypes were determined using the Fluidigm Biomark™ High Throughput SNP Genotyping Platform. In each backcross generation, a total of 560 SNPs was measured on all offspring homozygous for Prkdcscid and heterozygous for Hrhr to select the most optimal breeders with the highest percentage of NOD genetic background. Littermates identified to have a genetic background 99% concordant to the NOD mouse were sibling-mated to produce F1 breeders homozygous for both Prkdcscid and Hrhr mutations. The immunologic characteristics of this model are also described, which include B and T cell deficiency, a functional deficit of NK cells, and decreased macrophage and complement function. As the least leaky, most immunocompromised hairless model available, the SHrN™ model will become a valuable enhancement to oncology and immunology research, with particular benefit to imaging technology studies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 393.
Abstract 3936: Expression of DNA Repair Factor DDB2 predicts chemoresistance in various cancer cell lines
Authors: Takimoto Rishu , Murase Kazuyuki , Onuma Yoshiyuki , Hirakawa Masahiro , Yoshida Makoto , Sato Tsutomu , Sato Yasushi , Miyanishi Koji , Kobune Masayoshi , Kato Junji
Published: February 2011

Accumulating evidence suggests that DNA repair enzyme activity may predict sensitivity to chemotherapeutic agents in cancer cells. The status of a DNA repair enzyme, such as XPF/ERCC4 or XPD/ERCC2, has been reported to serve as a prognostic indicator as well as an indicator of chemosensitivity. Another such candidate is the protein product of the DDB2 gene, p48, that interacts with the p127 protein to form the UV-damaged DNA binding complex (DDB). DDB2, a DNA repair factor induced by tumor suppressor p53, plays an important role in the nucleotide excision repair (NER) of UV-damaged DNA. Mice lacking DDB2 developed spontaneous tumors at a high rate, suggesting a tumor suppression function for DDB2. In this study, we hypothesized that DDB2 expression correlated with sensitivity to chemotherapeutic agents. To this end, we compared the cytotoxicity levels of DDB2-expressing and non-expressing cell lines upon exposure to chemotherapeutic agents. As a result, downregulation of DDB2 using siRNA restored chemosensitivity. DDB2 may be a molecular marker for prediction of chemosensitivity and a new target molecule for cancer chemotherapy.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3936.
Abstract 3943: Oxidatively induced DNA base damage in non-small cell lung cancer
Authors: Senol Ozlem , Resmi Halil , Sevinc Can , Sanli Aydin , Onen Ahmet , Taylan Ebru , Dizdaroglu Miral , Kirkali Guldal
Published: February 2011

Non-small cell lung cancer (NSCLC) accounts for 80-85% of all cases of lung cancer and is known to be caused by smoking and other carcinogenic agents. Oxidatively induced DNA damage is involved in multiple modifications and mutations in DNA, thereby in pathogenesis of cancer as well as other diseases. In this study, we compared the tumor tissues to the surrounding healthy tissues obtained from 29 NSCLC patients who had undergone surgical resection. The levels of oxidatively induced DNA lesions such as 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua), 8-hydroxyguanine (8-OH-Gua) and (5′S)-8,5′-cyclo-2′-deoxyadenosine (S-cdA) were measured by gas chromatography/isotope-dilution mass spectrometry and liquid chromatography/isotope-dilution mass spectrometry. We found that the level of S-cdA was statistically significantly greater in cancer tissues than that in control tissues (p=0.0063), whereas the levels of FapyGua and 8-OH-Gua showed no significant difference between the two groups. There was no significant correlation between DNA damage and age, smoking or pathological stage. These results provide evidence for significant accumulation of S-cdA in tumor tissues compared to surrounding healthy tissues in NSCLC. The evaluation of the alterations in the levels of DNA lesions may contribute to the elucidation of the DNA repair defects in NSCLC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3943.
Abstract 3982: Super-resolution imaging of drug delivery to live cells using real-time structured illumination microscopy
Authors: Lefman Jonathan , Cheuk Tai , Metaferia Belhu , Tsang Patricia , Scott Keana , Stranick Stephan , Khan Javed
Published: February 2011

Imaging the effects of drug delivery in live cells is most often performed by confocal microscopy, which has a resolution limited by the diffraction of light. Here, we report the development of a novel method for imaging drug delivery to live cells using real-time super-resolution microscopy in wide-field mode. Structured illumination microscopy (SIM) is one of several super-resolving microscopy techniques and is capable of imaging beyond the resolution of conventional microscopy to less than 100 nm. In SIM, each super-resolution (SR) image is computationally constructed from a dataset of numerous raw images. Due to the computational time required for SR image construction, datasets are usually post-processed to avoid slowdowns during the acquisition process. Although post-processing is convenient, this approach has limited use since SR images are not available during image acquisition and thus cannot be utilized for real-time evaluation of the sample. Utilizing SR images in real-time is particularly important when objects of interest are smaller than the resolution limit of conventional microscopy. Applications such as determining optimal focus and finding desirable regions of interest are depending on real-time feedback to user. When performing real-time SIM imaging live cells, image acquisition, processing, and the display of SR images must occur in rapid succession thereby reducing the effects of molecular movement. To achieve the data processing speed necessary for real-time imaging of live cells, we utilized a graphics processing unit (GPU) to perform high-speed SR image construction. Each function for processing SIM data was optimized for parallel operation using GPU kernels. Excluding data transfer overhead, the actual GPU processing time for synthesizing the SR image was 45 milliseconds. Compared with parallel processing on a multi-core computer (CPU), using the GPU achieved a 45-fold increase in processing speed. Overall, data throughput using the GPU attained an 8-fold speed increase over CPU processing, thus allowing real-time high resolution imaging. We applied this SIM platform to record the action of nocodazole on microtubule depolymerization in HeLa cells. We are currently developing this real-time imaging method to investigate the delivery and release mechanism of therapeutic cargoes contained within nanoparticles.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3982.
Abstract 4000: Cellular MRI of human breast cancer cells labeled with gadofluorine M
Authors: Lizardo Michael M. , Ronald John A. , Chen Yuanxin , Misselwitz Bernd , Rutt Brian K. , Chambers Ann F.
Published: February 2011

The ability to non-invasively locate and quantify tumor cell number at a metastatic site would be of considerable interest in pre-clinical and clinical metastasis research. To this end, our objective is to develop positive-contrast cellular magnetic resonance imaging (MRI) for use in quantifying tumor cell number at a metastatic site in vivo. The goals of the current study were to characterize the in vitro labeling of MDA-MB-231-luc-D3H2LN human breast cancer cells with a novel positive contrast MR agent Gadofluorine M (GdF), and to demonstrate the detectability of GdF-labeled cells in vitro at clinical strength MRI (1.5 and 3Tesla) scanners. Briefly, cells (1.5 × 107cells) were loaded by simple incubation for 24h with GdF at concentrations ranging from 25μM to 10mM. GdF-loading from 25μM to 1000μM did not result in significant changes in metabolic activity. Higher GdF-loading concentrations, 2500μM and 10mM, resulted in a 16% and 21% reduction in metabolic activity, respectively. The lowest GdF-loading concentration used (25μM) resulted in a significant (9-fold) increase in MR signal enhancement which then plateaued from 100μM to 1000μM. T1-relaxometry of cell pellets demonstrated a similar statistical trend in relaxation rates of cell pellets for all loading conditions, and at both field strengths. Inductively-coupled plasma atomic emission spectroscopy was performed on the cell pellet extracts to assess the average amount of GdF that had accumulated per cell at each incubating concentration. Since the relaxivity of an MR contrast agent can be affected by its intracellular location, we assessed whether internalized GdF-cc (GdF analog where the mannose moiety was replaced with carbocyanine) was cytosolic or compartmentalized into endosomes, via immunostaining for mannose-6-phosphate receptor. Using confocal fluorescence microscopy, we found that GdF is largely cytosolic and did not co-localize to late endosomes, an optimal situation for achieving maximum positive-contrast by T1-shortening since endosomal compartmentalization can lessen the enhancement seen in MR images. From these results, we have determined that the GdF loading concentration of 1000μM permits the highest payload for detection by cellular MRI in clinical field strength scanners. Information from this proof-of-principle study will support future work to develop positive-contrast cellular MRI as a new approach to quantitative imaging of metastatic cells in vivo.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4000.
Abstract 4025: SHIP-1 is involved in resistance to alkyl-lysophospholipid induced apoptosis in S49 T-cell lymphoma cells
Authors: Alderliesten Maaike , Klarenbeek Jeffrey , van der Luit Arnold , van Lummel Menno , van Blitterswijk Wim , Verheij Marcel
Published: February 2011

Edelfosine (ALP, Et-18-OCH3) is a prototype alkyl-lysophospholipid that has effective antitumoral activity. It inserts in the membrane and accumulates in lipid rafts leading to apoptosis in S49 mouse T cell lymphoma cells. A variant cell line, made resistant to ALP (S49AR) shows in impaired uptake of ALP. S49AR cells are not only resistant to a variety of ALP analogues, but also to DNA damage and Fas/CD95 death receptor induced apoptosis, suggesting another cause for resistance in addition to impaired ALP uptake. Here we studied the ALP resistance of S49AR. We observe an upregulation of phosho-PKB/Akt and phospho-ERK1/2 which are key proteins in the canonical survival signaling pathways. However inhibition of ERK and/or PKB/Akt using pharmacological inhibitors has no effect on the resistance of the S49AR cells to apoptotic stimuli. PKB/Akt and ERK1/2 activation is regulated by PI(3,4,5)P3 formation. The levels of phosphoinositides in the various cell lines were measured with HPLC. The cell lines resistant to apoptotic inducing agents show decreased levels of the diverse phosphopoinositides. SHIP-1 is a SH2 domain containing inositol phosphatase that removes the 5′ phosphate of PI(3,4,5)P3 and is mainly expressed in hemotopoietic cells. It decreases the pool of PI(3,4,5)P3 thereby negatively regulating the activation of PKB/Akt. We see down regulation of SHIP-1 both on gene expression and protein level in the S49AR compared to S49 cells. Other components of the phosphoinositide metabolism, like PI3Kinase and SHIP2, did not show any changes. Resensitization of the S49AR cells to ALP leads to restoration of SHIP-1 expression. Knocking down SHIP-1 using siRNA causes resistance to various ALPs, DNA damage and Fas/CD95 death receptor induced apoptosis. Microarray analysis show that the S49AR and S49siSHIP cells show remarkably similar expression profile which are distinct from the expression profiles shown by S49 and S49mock cells.

Concluding, SHIP-1 is downregulated in ALP resistant T cell lymphoma cells concurrent with a general downregulation of phosphoinositide levels in the cells which is associated with the resistance to ALP induced apoptosis. How ALP resistance is caused is yet unknown and is the scope of current research.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4025.
Abstract 4027: The lectin concanavalin-A induces cyclooxygenase-2 expression through an IKKgamma/NFkB-dependent pathway in U87 glioblastoma cells
Authors: Sina Asmaa , Proulx-Bonneau Sébastien , Annabi Borhane
Published: February 2011

The lectin from Canavalia ensiformis (Concanavalin-A, ConA), one of the most abundant lectins known, enables one to mimic biological lectin/carbohydrate interactions that regulate extracellular matrix protein recognition. As such, ConA is known to induce membrane type-1 matrix metalloproteinase (MT1-MMP) and is, therefore, routinely used to assess MT1-MMP-mediated functions. Given that gliomas with increasing histological grade demonstrated MT1-MMP expression that correlated with high expression of cyclooxygenase (COX)-2, we specifically assessed the early proinflammatory cellular signaling processes triggered by ConA, a lectin known to induce MT1-MMP expression, in the regulation of COX-2. We found that treatment with ConA or direct overexpression of a recombinant MT1-MMP resulted in the induction of COX-2 expression. This increase in COX-2 was correlated with a concomitant decrease in phosphorylated AKT suggestive of cell death induction, and was independent of MT1-MMP's catalytic function. ConA and MT1-MMP-mediated intracellular signaling and induction of COX-2 was also validated in both wild-type and Nuclear Factor-kappaB (NF-κB) p65−/− mutant mouse embryonic fibroblasts (MEF), but was abrogated in NF-κB1 (p50)−/− and in I kappaB kinase (IKK) γ−/− mutant MEF cells. Collectively, our results highlight an IKK/NF-κB-dependent pathway linking MT1-MMP-mediated intracellular signaling to the induction of COX-2 by ConA. That signaling pathway could account for the inflammatory balance responsible for the therapy resistance phenotype of glioblastoma cells, and prompts for the design of new therapeutic strategies that target cell surface carbohydrate structures and MT1-MMP-mediated signaling.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4027.
Abstract 4046: Androgen-mediated phenotypic change in prostate carcinoma cells and the microRNA-ome
Authors: Segal Corrinne , Powell Chris J. , Gooderham Nigel J.
Published: February 2011

Activation of the androgen receptor (AR) results in phenotypic change in androgen-responsive cells; for example, in the human prostate carcinoma cell line (LNCaP) androgens mediate cell proliferation. Factors controlling the range of phenotypic responses mediated by androgens are only partially understood, but include both transcriptional and post-transcriptional mechanisms. One such mechanism that appears to offer wide ranging, yet highly selective control, is the expression of the miRNA-ome. In the present study, we assessed the influence of endocrine-active compounds that act via the AR pathway on the microRNA (miRNA) profile. As negative regulators of gene expression, miRNA are short non-coding RNA that are able to suppress translation or induce degradation of their target mRNA transcripts (post-transcriptional) leading to decreased protein expression.

The effect of exposure of LNCaP cells to the AR agonists mibolerone and dihydrotestosterone, and the antagonist bicalutamide, was determined at various time points up to 120 hours. Phenotypic response to exposure was assessed as an alteration of proliferative ability, expression of prostate-specific antigen (PSA) and alteration of the miRNA-ome. As expected, each of the agonists significantly induced proliferation, compared to vehicle control, and the antagonist decreased proliferation. Concurrent with changes in proliferation, were an up-regulation of PSA secretion for mibolerone and DHT. The temporal effects of exposure to androgenic compounds on the expression profile of 850 miRNAs was assessed using Agilent's miRNA microarray version 3. The ability of androgens to alter the miRNA-ome was confirmed and found to be temporally-dependent. Bioinformatic analysis of the phenotypic changes induced by the androgens is reflected in the changes in the miRNA-ome.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4046.
Abstract 4048: Increased expression of PACS-1 in cervical cancer is associated with loss of microRNA449a
Authors: Veena Mysore S. , Raychaudhuri Santanu , You Huihong , Basak Saroj K. , Zainabadi Kayvan , Thomas Gary , Srivatsan Eri
Published: February 2011

Cervical cancer is one of the leading causes of female death world wide with approximately 50,000 deaths per year. We identified a 300 kb deletion of chromosome 11q13 in cervical cancer and mapped eleven genes to this region. Of these, phosphofurin acidic cluster sorting −1 (PACS-1) gene encodes a multi-functional sorting protein involved in secretory pathway membrane traffic. Expression studies showed the presence of an abnormal PACS-1 transcript in tumorigenic cell lines. Protein expression studies in paired normal and cervical tumors showed over-expression of PACS-1 protein in tumor specimens. RT-PCR indicated that protein over-expression was related to over-expression at the RNA level. These findings together with the absence of detectable mutations in PACS-1 exons in HeLa or SiHa cervical carcinoma cell lines raised the possibility that dysregulation of a microRNA (miRNA) may alter PACS-1 expression in cervical cancer. Consistent with this possibility, a UCSC genome database search showed the presence of binding sites for miRNA449a in the 3′ UTR (untranslated region) of the PACS-1 RNA transcript. Co-transfection of PACS-1 3′UTR luciferase reporter with miRNA449a resulted in reduced expression of luciferase. Expression of miR449a in HeLa and SiHa cells led to reduced expression of PACS-1 and increased expression of p53 and p21 proteins. FACS analysis of HeLa cells transfected with miR449a showed a reduction in S phase indicating the growth suppressive effect of miR449a. These results indicate that miR449a regulates PACS-1 expression and loss of miR449a expression leads to over-expression of PACS-1. This in turn seems to result in the destabilization of p53 in cervical cancer. Our findings suggest PACS-1 plays a role in cervical cancer development and its expression is regulated post-transcriptionally by miRNA449a.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4048.
Abstract 4049: MiR-187 is a marker of poor prognosis in estrogen receptor-positive breast cancer
Authors: Mulrane Laoighse , Madden Stephen , Brennan Donal J. , McGee Sharon F. , Jirström Karin , Veer Laura van't , Bernards Rene , Higgins Des , Gallagher William M. , O'Connor Darran P.
Published: February 2011

Resistance to endocrine-directed therapy represents a significant problem in the management of breast cancer. Here we describe an integrated bioinformatics and translational pathology approach to identifying novel microRNAs (miRNA) controlling this phenomenon.

Differential gene expression can, in part, be attributed to the activity of specific miRNAs. Given a database of miRNA binding site motifs and gene expression levels determined by transcriptomic profiling, correspondence analysis, between group analysis and co-inertia analysis can be combined to produce a ranked list of miRNAs associated with a specific gene signature and phenotype. We have applied this approach to examine gene expression data from 69 breast cancer patients treated with tamoxifen (1) and have identified miR-187 as the principle miRNA associated with poor response to anti-estrogen therapy.

Expression of miR-187 was subsequently evaluated in a cohort of breast cancer patients (n=111), by locked nucleic acid in situ hybridisation (LNA-ISH) on tissue microarrays, and quantified by automated image analysis. LNA-ISH signal was validated by miR-187-specific Taqman PCR in a subset of tumours. High miR-187 expression was significantly associated with poor outcome in ER+ breast cancer patients (p = 0.05) and correlated with increased tumour size (p=0.009), higher grade (p=0.005) and PR negativity (p=0.014). Additionally, the prognostic significance of the miR-187 target genes OCA2 and HIPK3 was examined in an independent dataset comprising 155 breast cancer patients treated with adjuvant tamoxifen (2). Low mRNA expression of both OCA2 (p=0.0001) and HIPK3 (p=0.016) was found to be significantly associated with poor outcome. These data support the hypothesis that miR-187 and its targets play an important role in anti-estrogen resistance and may be useful as biomarkers to aid treatment options and as potential drug targets to restore tamoxifen sensitivity in resistant patients.

1.

Kok M et al. Comparison of gene expression profiles predicting progression in breast cancer patients treated with tamoxifen. Breast Cancer Res Treat 2009;113(2):275

2.

Chanrion M et al., A gene expression signature that can predict the recurrence of tamoxifen-treated primary breast cancer. Clin Cancer Res 2008;14(6):1744-52.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4049.
Abstract 4057: Negative regulation of prostate specific membrane antigen by androgen is mediated by TMPRSS2-ERG gene fusion in VCaP prostate cancer cells
Authors: Yin Lihong , Rao Pravin , Wang Jianghua , Ittmann Michael , Heston Warren D.W.
Published: February 2011

Prostate specific membrane antigen (PSMA) is a type II transmembrane glycoprotein over-expressed in prostate carcinoma. Elevated PSMA expression has been correlated with aggressive disease and risk of early recurrence after radical prostatectomy. This molecule is also expressed in the neovasculature of multiple nonprostate solid tumors. Consequently, this unique cell marker has been targeted for diagnosis and therapy of prostate cancer (PCa) and other malignancies. Recently studies have shown that a significant fraction of PCa harbor a signature gene fusion between the androgen-responsive TMPRSS2 gene and transcription factors in the ETS family - most commonly ERG. The TMPRSS2-ERG fusion is likely the cause for over-expression of ERG in PCa. PSMA, unlike ERG, is negatively regulated by androgen. Elucidation of the relationship between TMPRSS2 gene fusions and PSMA expression could reveal mechanisms to alter PSMA expression and disease course. The purpose of this investigation was to determine whether PSMA gene expression could be regulated by the TMPRSS2-ERG fusion in prostate cancer cells.

We employed VCaP cells, which harbor TMPRSS2-ERG fusion, and LNCaP cells, which lack the fusion. Our data showed that after 24h androgen R1881 treatment, ERG and TMPRSS2-ERG mRNA level were increased in VCaP cells and unchanged in LNCaP cells. PSMA and androgen receptor (AR) mRNA level were dramatically decreased in VCaP cells and only modestly decreased in LNCaP cells. Treatment with the androgen antagonist flutamide partially restored PSMA and AR expression in androgen-treated VCaP cells. Knocking down ERG by siRNA in VCaP cells enhances PSMA expression both in the presence and absence of R1881. When LNCaP cells transfected with TMPRSS2-ERG fusion transcripts, PSMA was down-regulated both in the presence or absence of R1881. Using PSMA-based luciferase reporter assays, we found TMPRSS2-ERG fusion can inhibit PSMA activity at the transcriptional level. Our data indicated that negative regulation of PSMA by androgen is mediated by TMPRSS2-ERG gene fusion in VCaP prostate cancer cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4057.
Abstract 4061: Depleting the cellular level of glutathione and subsequently S-glutathionylation of keap1 by electrophilic agents activates keap1-nrf2 pathway
Authors: Zhang Xiaoyu , Ma Zhongjun , Sun Fang , Shen Liming
Published: February 2011

S-glutathionylation is a reversible post-translational modification of protein cysteine with critical roles in oxidative stress and signal transduction. Keap1-Nrf2 pathway has emerged as a master regulator for the endogenous antioxidant response, which is critical in defending cells against carcinogenesis. Herein, we demonstrated that after depleting the cellular level of glutathione (GSH) by electrophilic agent (the GSH level decreased to 23 percent of control after the agent treatment for 4 h), Keap1 was modified by S-glutathionylation and Nrf2 expression increased both in nucleus and total cellular homogenates. The modification could be blocked by the addition of exogenous GSH, suggesting depleting the cellular level of GSH and alteration of intracellular GSH/GSSG ratio might be essential for S-glutathionylation of Keap1 and activating Keap1-Nrf2 pathway. Flexidock program was employed to elucidate the influence of Keap1 S-glutathionylation on Keap1-Nrf2 complex. After enforcing a disulfide bond between GSH and Cys434 residue in Keap1 and energy minimization, the two carboxylate oxygen atoms of GSH participate in hydrogen bond interaction with Arg483 and Arg380 residues in Keap1, which had been previously verified bound to DxETGE motif of Nrf2. The distance between GSH O1 and Arg483 NH1, GSH O2 and Arg380 NH1 were 2.587 Å and 2.569 Å, respectively, which could form potent hydrogen bonds and diminish the interaction between Keap1 and Nrf2, resulting in the accumulation of Nrf2 in the nucleus. We concluded from our findings that the activation of Keap1-Nrf2 pathway was triggered by Keap1 S-glutathionylation and conjugation with intracellular GSH might be a key event involved in the Nrf2 inducing activities of electrophilic agents.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4061.
Abstract 4063: Estrogen receptor alpha functions as an RNA binding protein to regulate GATA3 protein levels: a novel role for ER
Authors: Joyce Karen , Hostetter Christine , Chamoun George , Keen Judith C.
Published: February 2011

The role of estrogen receptor alpha (ER) as a transcription factor has been well established. ER transcriptionally activates genes by binding to DNA directly within the gene promoter region or by binding to DNA indirectly through protein-protein dependent mechanisms. Many ER-responsive genes that are activated through ER-mediated transcriptional activation have been identified, including GATA3. While the role of GATA3 in breast cancer still remains largely unknown, numerous reports have shown that ER and GATA3 are coordinately expressed in breast cancer and that ER may play a role in regulating GATA3 expression. A recent report by Eeckhoute et al (2007) has shown that ER transcriptionally activated GATA3 expression in T47D breast cancer cells. Using MCF7 cells, we have recently identified a novel role for ER as an RNA binding protein that binds to GATA3 mRNA. Interestingly, silencing of ER using siRNA probes slightly increased GATA3 mRNA levels, however GATA3 protein levels decreased significantly. This decrease in protein was not due to improper export of GATA3 mRNA from the nucleus to the cytoplasm where protein translation occurs, as GATA3 mRNA expression was equivalent in both the nuclear and cytoplasmic compartments. This suggested that the decrease in protein expression was not due to impaired GATA3 mRNA transport but could be due to impaired protein translation. Since silencing of ER had dramatically different effects on RNA and protein levels, this data suggested that ER protein may function as an RNA binding protein that binds to the GATA3 mRNA to regulate its protein translation. Using biotin pulldown techniques, we found that ER does function as an RNA binding protein by binding directly to a site within a 125 nucleotide region of the GATA3 3‘UTR. This role of ER as an RNA binding protein is consistent with the data of Lanz et al (1999, 2000) that demonstrated that ER protein can bind to an RNA transcript, the steroid receptor co-activator, SRA. When bound to SRA, however, ER did not alter the RNA levels or protein expression of SRA but did result in increased activation of ER. Therefore, our data is the first to identify a novel role for ER as a RNA binding protein that can regulate the protein expression of the RNA transcript to which it is bound. Altering such ER-RNA interactions could potentially provide a new therapeutic avenue for the treatment of breast cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4063.