bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 454: The regulation of tumorigenesis by crosstalk between hypoxia and PPARdelta
Authors: Jeong Eunshil , Kim So Young , Lee Joo Young
Published: February 2011

Hypoxia, insufficient state of oxygen, is a feature of the tumor environment. Upon tumor growing, inside of tumor mass is hypoxic state. Hypoxia promotes tumor progression, expression of angiogenic mediators and tumor resistance to cancer therapy. Peroxisome proliferator-activated receptors (PPARs), ligand-activated transcription factors, also play an important role in the regulation of cancer cell growth and angiogenesis. However, it has not been entirely elucidated if hypoxia can regulate PPARdelta activation during tumorigenesis. Therefore, we investigated the relationship between hypoxic stress and PPARdelta activation in tumor cells and the contribution of the crosstalk between these factors to tumor cell growth. Hypoxia (<1% O2) and a hypoxia-mimetic (deferoxamine) increased mRNA levels and protein expression of tumor-promoting mediators such as IL-8, VEGF, and COX-2 in colon cancer cells (HCT116), but not in PPARdelta-knockout HCT116 cells. A synthetic ligand of PPARdelta(GW501516) induced the expression of IL-8, VEGF, and COX-2 in wild-type colon cancer cells but not in PPARdelta-knockout cells. Hypoxia increased both transcriptional activity and mRNA level of PPARdelta in HCT116 cells. In addition, overexpression of HIF-1alpha enhanced transcriptional activity of PPARdelta suggesting that HIF-1alpha regulates the activation of PPARdelta. The proliferation and migration rate of macrophages were enhanced by conditioned media derived from wild-type colon cancer cells but not PPARdelta-knockout cells. These suggest that the expression of IL-8, VEGF, COX-2 induced by hypoxic stress is dependent on PPARdelta activity and that HIF-1alpha and PPARdelta cooperate to promote tumor cell growth. [Supported by a grant from Research Center for Biomolecular Nanotechnology, GIST]

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 454.
Abstract 4542: The design, synthesis and in vitro evaluation of selective FR α and β and PCFT substrates with potent GARFTase inhibitory activity as antitumor agents
Authors: Gangjee Aleem , Wang Lei , Cherian Christina , Desmoulin Sita Kugel , Hou Zhanjun , Kisliuk Roy L. , Matherly Larry H.
Published: February 2011

Three folate transporters, the reduced folate carrier (RFC), folate receptors (FR) and the proton-coupled folate transporter (PCFT) account for most of the folate influx activity in mammalian cells. Lack of selectivity for folate transporters is one of the major causes of chemotherapy failure with antifolate drugs. We recently reported the potent glycinamideribonucleotide formyltransferase (GARFTase) inhibitory activity of a series of classical 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidines with a thienoyl for benzoyl-substituted side chain with FR and PCFT specificity over RFC. In an attempt to optimize for selective transport by FR and PCFT, and GARFTase inhibitory activity, a furan for benzoyl-substituted side chain analog with a 4-carbon bridge was synthesized. The target compound was tested against KB human tumor cells that express FR alpha, human RFC, and human PCFT using a fluorescence-based cytotoxicity screen, and showed remarkably potent subnanomolar inhibitory activity (IC50 of 0.29 nM). The furan analog was further tested in isogenic Chinese hamster ovary sublines, engineered to express human FR alpha (RT-16), FR beta (D4), hRFC (pC43-10), or hPCFT (R2/hPCFT4). The furan side chain compound again showed a subnanomolar IC50s for both FR alpha and FR beta expressing cells (IC50 of 0.16 nM for RT16, and 0.92 nM for D4). Thus, in comparison with the lead thienoyl side chain compound (IC50 of 2 nM for RT16, and 0.75 nM for D4), the furan analog exhibited similar activities toward FR beta, but a 12.5-fold increased activity toward FR alpha. For PCFT-expressing R2/hPCFT4 cells, the furan side chain compound (IC50 of 439 nM) was about 10-fold less potent than the thienoyl analog (IC50 of 43.4 nM). The furan analog was inactive against human dihydrofolate reductase (hDHFR) or human thymidylate synthase (hTS) (IC50 >10−5 M), suggesting that neither hDHFR nor hTS is a principal target. Metabolite protection from in vitro cytotoxicity in KB cells showed that thymidine (10 μM) did not affect the growth inhibitory effects of the furan analog whereas adenosine (60 μM) and 5-amino-4-imidazolecarboxamide (320 μM) were completely protective, identifying GARFTase as the likely intracellular target. Our results indicate that the side chain heterocyclic ring may control the transport selectivity for both FR and PCFT of the pyrrolo[2,3-d]pyrimidines.

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 4542.
Abstract 4544: Novel indolequinone inhibitors of thioredoxin reductase are active against human pancreatic cancer
Authors: Yan Chao , Shieh Biehuoy , Regan Phillip , Zhang Zhiyong , Colucci Marie , Chilloux Aurélie , Newsome Jeffery , Siegel David , Chan Dan , Moody Christopher J. , Ross David
Published: February 2011

A series of indolequinones was previously developed in our lab as potential antitumor agents against pancreatic cancer. Three general classes of indolequinone consisting of 2-methyl, 2-hydroxymethyl and 2-unsubstituted derivatives with varying leaving groups at the indole 3- position exhibited marked activity against human pancreatic cancer both in vitro and in vivo. Pancreatic cancer cell lines PANC-1, MIA PaCa-2, and BxPC-3 were used to ensure effects were not cell line specific and growth inhibitory effects were measured using both the MTT and clonogenic assays. Indolequinones displayed growth inhibitory activity against pancreatic tumor cells in vitro and two specific classes of indolequinone, the 2-unsubstituted and 2-hydroxymethyl classes, were particularly active agents with IC50 values in the 20-200nM range. Indolequinones were also found to be efficient inducers of apoptosis in pancreatic cancer cell lines at concentrations which induced growth inhibition. Selected indolequinones were also screened against the NCI-60 cell line panel to reveal the pattern of activity across various tumor types and were particularly effective against colon, renal, and melanoma cancer cells. The spectrum of activity of indolequinones in the NCI-60 was similar to established inhibitors of thioredoxin reductase. Indolequinones of both the 2-hydroxymethyl and 2-unsubstituted classes were found to inhibit thioredoxin reductase in MIA PaCa-2 cellspancreatic cancer at concentrations equivalent to those inducing growth inhibitory effects. The mechanism of inhibition of thioredoxin reductase by the indolequinones was studied in detail in cell-free systems using purified enzyme and the C-terminal selenocysteine of thioredoxin reductase was found to be the primary adduction site of the indolequinones using LC-MS analysis. Inhibition of thioredoxin reductase by indolequinones in cells resulted in a change of thioredoxin redox state and activation of the P38/JNK signaling pathway which may result in downstream apoptosis. Our results demonstrate that thioredoxin reductase is a potential target of the indolequinones in pancreatic cancer cells. (Supported by CA11441).

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 4544.
Abstract 4545: Anticancer activity of elesclomol correlates with low LDH levels and active mitochondrial respiration
Authors: Nagai Masazumi , Blackman Ronald K. , Rao Patricia E. , Wada Yumiko , Koya Keizo
Published: February 2011

Elesclomol is a first-in-class investigational drug that exerts anticancer activity through the elevation of reactive oxygen species (ROS). Our previous studies revealed that elesclomol selectively chelates copper and generates ROS via reduction of Cu(II) to Cu(I). In a Phase 3 trial in metastatic melanoma, the level of baseline lactate dehydrogenase (LDH) in patients emerged as an important prognostic factor for treatment outcomes with elesclomol. Additional investigations were undertaken to evaluate the relation between elesclomol activity and LDH levels. Because the copper redox reactions necessary for elesclomol activity can be influenced by changes in cellular metabolic properties, we investigated whether elesclomol activity varies between normoxic conditions (active mitochondrial respiration, low LDH) and hypoxic conditions (decreased mitochondrial respiration, high LDH). To assess the influence of hypoxic conditions, we evaluated elesclomol activity under several situations in which cells express high levels of Hypoxia Inducible Factor-1α (HIF1α), a transcription factor that induces a complete set of glycolytic enzymes, including LDH, and switches off glycolytic carbon flow to mitochondria. First, an increase in the level of HIF1α protein was observed in M14 melanoma cells at low oxygen level or high cell density. Elesclomol showed decreased cytotoxicity under both of these conditions. Second, we evaluated lines within the same cancer phenotype but showing distinct levels of HIF1α, and found that the high-HIF1α-expressing Caki-2 renal cancer line was resistant to elesclomol, while lower-HIF1α-expressing renal cancer cell lines were sensitive to elesclomol. Third, we assessed HIF1α levels and elesclomol activity in cells treated with CoCl2, a chemical mimetic of hypoxia. High levels of HIF1α induction were present in M14 cells treated with CoCl2, and elesclomol was significantly less active in these cells. The addition of oxamate, which selectively inhibits LDHA and activates pyruvate entry into mitochondria, restored the activity of elesclomol in the CoCl2-treated cells. These results support the hypothesis that elesclomol is more active under normoxic conditions (low LDH) and less active under hypoxic conditions (high LDH), consistent with the clinical findings. The results may be important for identifying patient populations for future clinical development of elesclomol.

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 4545.
Abstract 4557: In vivo near-infrared fluorescent quantification of therapy-induced apoptosis using Annexin-Vivo 750
Authors: Zhang Jun , Cuneo Gary , Narayanan Nara , Delaney Jeannine , Kossodo Sylvie , Bao Bagna , Rajopadhye Milind , Peterson Jeffrey D. , Madden Karen , Gao David Z. , Moonen Peter , Reutelingsperger Chris
Published: February 2011

Drug-induced ex vivo apoptotic tests are widely used pre-clinically to monitor chemotherapeutic efficacy. Real-time non-invasive in vivo imaging and quantification of therapy-induced tumor apoptosis would impact drug discovery significantly. As a key apoptotic marker, phosphatidylserine (PS) translocated to the outer leaflet of the plasma membrane can be detected by fluorescent-labeled Annexin A5 specifically with high affinity. We synthesized an in vivo imaging contrast agent Annexin-Vivo 750 by conjugating a human recombinant Annexin A5 to a near-infrared fluorophore with em 770 nm. Fluorescence microscopy showed 4h anti-FAS Ab treatment significantly increased the number of Annexin-Vivo 750 positive Jurkat cells, confirming that the agent binds to PS on apoptotic cells. Similarly, flow cytometry showed anti-FAS Ab increased Annexin-Vivo 750 positive cells from 15% to 55%. TUNEL positive cells were increased from 0% to 65.14 ± 3.93% (p=0.0004) by anti-FAS antibody. The TUNEL data correlate very well with flow cytometry data and are consistent with the fluorescence microscopy data. In vivo imaging of Annexin-Vivo 750 was performed in nude mice implanted with human colorectal carcinoma HT-29 cells. Once tumors reached a volume of 142.36 + 8.3 mm3 mice were randomized and treated intraperitoneally with cyclophosphamide (170 mg/kg) or vehicle at time 0h. The agent was injected at 24h with a dose of 2.4 nmol/animal and the tumors were imaged at 26h with an FMT2500 quantitative tomography imager for in vivo real-time 3D Annexin-Vivo 750 quantification. Tumor fluorescence signal was 2.55 fold higher with than without cyclophosphamide (p=0.0002), while tumor volumes were the same (p=0.35). Fluorescent signal decreased significantly by 24h and returned to baseline at 48h post agent injection. Likewise, intratumoral injection of TNFα (2.5 μg) plus IFNγ subcutaneous injection (2500 units) also significantly increased HT29 tumor Annexin-Vivo 750 fluorescence (3 fold compared to control, p=0.0099, 24h after treatment, 2h post-agent injection). TUNEL positive HT-29 cells were increased from 11.16 ± 7.05 % to 41.77 ± 8.78 % (p=0.0004) by cyclophosphomide, indicating apoptosis. These results demonstrate that Annexin-Vivo 750 in vivo imaging can be used for detecting, quantifying and monitoring apoptosis following cancer treatments, underscoring its value in drug discovery and development in cancer and potentially in other clinically important areas, such as cardiovascular-, autoimmune-, inflammatory- and neurodegenerative diseases.

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 4557.
Abstract 457: Hypoxia-mediated upregulation of Pim-1 contributes to tumor survival
Authors: Chen Jian , Kobayashi Masanobu , Darmanin Stephanie , Qiao Yi , Gully Christopher , Zhao Ruiying , Kondo Satoshi , Wang Hua , Wang Huamin , Yeung Sai-Ching Jim , Lee Mong-Hong
Published: February 2011

Introduction: The proto-oncogene pim-1 regulates apoptosis and the cell cycle by phosphorylating target proteins. Overexpression of Pim-1 can cause genomic instability and contribute to lymphomagenesis. Tumor hypoxia directly promotes genomic instability and changes the responses of cancer cells to many chemotherapy agents, resulting in chemoresistance. It is not clear whether Pim-1 is involved in hypoxia-mediated tumor survival and drug resistance.

Result: (1) Hypoxia stabilize Pim-1 by preventing its ubiquitin-mediated proteasomal degradation and cause Pim-1 translocation from the cytoplasm to the nucleus. (2) Overexpression of Pim-1 increases NIH3T3 cell transformation exclusively under hypoxia, suggesting that Pim-1 expression under hypoxia may be implicated in the transformation process of solid tumors. (3) Introduction of dominant negative Pim-1 resensitizes pancreatic cancer cells to apoptosis induced by glucose-deprivation under hypoxia. Also, administration of siRNAs for Pim-1 resensitizes cancer cells to glucose deprivation under hypoxic conditions, while over-expression of Pim-1 causes solid tumor cells to become resistant to glucose deprivation. (4) Dominant negative Pim-1 reduces tumorigenicity and sensitizes tumors to chemotherapy in pancreatic cancer cells and HeLa xenograft mouse models.

Conclusions: Our studies indicate that Pim-1 plays a distinct role in solid tumor formation and chemoresistance, implying that Pim-1 may be a novel target for cancer therapy.

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 457.
Abstract 4573: Discovery of candidate biomarkers for colorectal cancer using mass spectrometry-based proteomic analysis of cell line conditioned media
Authors: Karagiannis George S. , Diamandis Eleftherios P.
Published: February 2011

Colorectal cancer (CRC) represents the third leading cause of cancer-related deaths with approximately 655,000 deaths annually, worldwide. Specific biomarkers for early detection of CRC are needed, mainly because the currently applied serum biomarkers (CEA, CA 19.9) lack adequate sensitivity and specificity for screening purposes, while colonoscopy is invasive, with considerable cost, risks and inconvenience. In the present study, we utilized a mass spectrometry-based platform to analyze the proteome of the conditioned media from 12 CRC cell lines with diverse genetic profiles, to capture the disease heterogeneity. Proteins were trypsin-digested and fractionated by strong cation-exchange and reverse-phase liquid chromatography prior to mass spectrometric analysis with an LTQ Orbitrap instrument. The samples were run in triplicate and results were searched against the IPI human forward and reverse database, using MASCOT and X!Tandem search engines. We were able to identify approximately 2,000 unique proteins for each cell line, with reproducibility ranging from 71 to 82% among the triplicates. The efficiency of this approach was signified by the large number of known biomarkers, such as CEA, successfully identified in our list of candidates, as well as by the identification of internal control proteins, such as kallikreins 6, 7, 10, and 11, which were also quantified by ELISA in cancer cell supernatants. The acquired protein datasets were further subjected to bioinformatic analysis using Gene Ontology. The proteins were assigned a subcellular localization and 35-40% were classified as extracellular or plasma membrane proteins. Further mining of this vast database by using bioinformatics tools and experimental approaches (such as multiple reaction monitoring and ELISA) could lead to shortlists of proteins that may have utility as circulating CRC biomarkers.

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 4573.
Abstract 4575: Covalent incorporation of a new class of dye-based affinity baits in sieving hydrogel nanoparticles overcome roadblocks to cancer biomarker discovery
Authors: Luchini Alessandra , Tamburro Davide , Douglas Temple , Espina Benjamin H. , Lepene Benjamin S. , Zhou Weidong , Espina Virginia , Petricoin Emanuel F. , Liotta Lance A.
Published: February 2011

Cancer biomarker discovery and clinical application has been seriously hampered by low abundance, instability and complex formation with high abundance proteins (e.g. albumin). N-isopropylacrylamide (NIPA) based hydrogel particles, consisting of a bait-containing core and a protective shell, overcome these major issues associated to biomarker discovery and measurement. Particles perform in one step, in solution, a) total sequestration and concentration, b) size sieving and c) complete protection form degradation of LMW protein biomarkers in blood and urine. In this study, novel dye molecules were selected as affinity ligands for broad classes of biomarkers; dyes were chosen for their ability to bind proteins, glycoproteins and nucleic acid for tissue histology, to bind textile proteins for fabric staining or for their known property to bind allergens. Dyes containing primary amine group (e.g. Acid Black 48, Basic Red 9) were coupled to NIPA/acrylic acid (AAc) particles by condensation of the amine group the carboxylic group. The ability of particles to capture and concentrate analytes was tested against a panel of low abundance, labile tumor relevant biomarker that include angiogenic growth factors (bFGF, VEGF, PDGF), IGF-1 and IGF binding proteins, interleukins (IL-6, IL-8), chemokines (CXCL12, CCL24), pro-apoptotic Bcl-2 proteins (Bak, Bax), and nucleic acids. Technologies used to screen dye functionalized particles include 1 D gel electrophoresis, western blotting, immunoassay, and mass spectrometry.

Results showed that dye based baits have extremely high affinity for the target analytes so that particles capture all the analyte present in solution and that the affinity of biomarkers to the bait is unique and specific to the chemical structure of the bait molecule.

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 4575.
Abstract 4595: ERK/MAPK regulation of ERRγ in Tamoxifen resistant breast cancer
Authors: Mazzotta Mary M. , Liu Minetta C. , Clarke Robert , Riggins Rebecca B.
Published: February 2011

Breast cancer is a serious public health problem that is the second-most common cause of cancer-related death in women. Endocrine therapy, like Tamoxifen (TAM), has made significant contributions to the successful management of ER+ breast cancer but unfortunately 30-50% of these tumors may acquire endocrine resistance. However, not all ER+ breast cancers are the same. Two of the major classifications of breast cancer are invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC). In comparison to IDC, a significantly greater percentage of ILCs are estrogen receptor alpha-positive (ER+) and progesterone receptor-positive (PR+). Therefore, women diagnosed with this tumor subtype should be ideal candidates for endocrine or antiestrogen therapy. However, clinical studies differ as to whether ILC patients experience better or worse survival following antiestrogen treatment when compared to ER+ IDC patients.

In a model of TAM-resistance in ILC, we have shown that the orphan nuclear receptor estrogen related receptor gamma (ERRγ) plays a functional role in the resistant phenotype of LCCTam cells, ERRγ overexpression is sufficient to induce TAM resistance, and we show for the first time that ERRγ is serine-phosphorylated in TAM-resistant breast cancer cells in association with increased expression and activity of extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK; ERK). We have also recently found an IDC-derived model of TAM-resistance to overexpress ERRγ. Therefore, we hypothesize that ERK regulates ERRγ transcriptional activity and is a critical determinant of ERRγ-induced TAM resistance in breast cancer.

To test this hypothesis, we used two antiestrogen-sensitive and -resistant breast cancer cell line pairs: IDC-derived RR cells and their sensitive controls MCF7, and the ILC-derived LCCTam cells and their sensitive controls SUM44. In both sensitive controls we found TAM alone to cause cell death. When we added the ERK inhibitor, U0126, to the TAM treatments, cell death increased. The resistant cell lines showed no effect with the addition of TAM alone, but the TAM treatment coupled with the inhibitor restored sensitivity and caused cell death. Additionally, we tested the transcriptional activity of ERRγ in the presence or absence of the ERK inhibitor. We found each of the four cell lines to show decreased ERRγ transcriptional activity in the presence of U0126.

Future studies include the identification of the serine residues on ERRγ being phosphorylated by ERK, and whether ERRγ overexpression is associated with increased ERK expression and activity, poor overall survival, and poor response to TAM in breast cancer patients treated with endocrine therapy.

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 4595.
Abstract 4606: An in vitro and in vivo evaluation of novel anticancer agents in a triple negative breast cancer model
Authors: Hawthorne Tamorah , Gallien Joseph , Gibbs Lee , Jones Janeque , Muir Shannon , Rhodes Lyndsay V. , Driver Jennifer , Khalili Hafez , Burow Matthew , Parker-Johnson KiTani A.
Published: February 2011

Patients diagnosed with triple negative breast cancer, which is the absence of the estrogen (ER), progesterone (PR), and Her2/neu receptor have a poor prognosis and there is a critical need to develop novel antineoplastic agents for this breast cancer sub-type. Therefore, the focus of this project is to identify novel anticancer agents with in vitro and in vivo antiproliferative activity in a triple negative breast cancer model. Previously, our laboratory synthesized a group of novel isochalcones called DJ compounds that demonstrated significant in vitro antiproliferative effects. Cell proliferation of MDA-MB-231 cells was measured using the alamar blue dye method and produced IC50 values of DJ52, DJ56, and DJ82 at 10−6M, 10−5M, and 10−5M, respectively. Binding studies have indicated that these agents do not bind the ER but appear to inhibit a phosphorylation step in the epidermal growth factor (EGFR) receptor pathway. In vivo studies were also conducted by implanting tumors derived from MDA-MB231 cells into SCID mice to determine tumor regression was measured over 20 days. DJ52 at 50mg/kg caused significant decrease (p value <.05) decreased tumor volume by nearly fifty percent compared to the control with vehicle alone. Therefore, these data suggests that DJ52 has merit for further evaluation for potential intervention in patients diagnosed with triple negative breast cancer. Our laboratory continues our efforts to understand the mechanism of action of these agents to potentially provide alternative pharmacological options for these patients for better health outcomes.

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 4606.
Abstract 461: Identification of COX-2 as a major actor of the transcriptomic adaptation of endothelial and tumor cells to cyclic hypoxia: impact on angiogenesis and metastases
Authors: Daneau Géraldine , Boidot Romain , Martinive Philippe , Feron Olivier
Published: February 2011

Purpose: Cyclic hypoxia in tumors originates from heterogeneities in red blood cell flux and influences tumor cells but also endothelial cells lining tumor blood vessels. Whether pO2 fluctuations, in particular transient reoxygenation periods, alter the well-known hypoxia-inducible factor (HIF)-dependent gene program is largely unknown. Experimental design: We compared the transcriptomic profiles of endothelial and tumor cells exposed to cyclic hypoxia vs continuous hypoxia to uncover a possible differential impact on angiogenesis and metastases. Results: Microarray analyses identified early genes that were selectively induced by cyclic hypoxia in endothelial cells. Among them, we focused on PTGS2 since the observed increase in mRNA expression led to a significant increase in the expression and the activity of COX-2 (the protein product of PTGS2). HIF-1α was shown to be stabilized by cyclic hypoxia (despite reoxygenation periods) and to favor COX-2 induction, as validated by the use of echinomycin and HIF-1α targeting siRNA. Using a specific COX-2 inhibitor and a dedicated COX-2-targeting siRNA, we documented that COX-2 accounted for the higher endothelial cell survival and angiogenic potential conferred by cyclic hypoxia. Cyclic hypoxia also led to a preferential COX-2 induction in tumor cells and, contrary to continuous hypoxia, fostered a higher metastatic take of pre-challenged tumor cells. Conclusions: Our study documents that PTGS2/COX-2 is part of a cyclic hypoxia gene signature and largely accounts for the unique phenotype of endothelial and tumor cells exposed to fluctuations in pO2, thereby offering new perspectives for the clustering of tumors expressing COX-2 together with other cyclic hypoxia-responsive genes.

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 461.
Abstract 4613: Targeting ER-KDM1 axis to sensitize therapy resistant breast cancer cells to hormonal therapy
Authors: Cortez Valerie , Nair Sujit S. , Tekmal Seshidar Rao , Vadlamudi Ratna K.
Published: February 2011

Estrogen-induced breast carcinogenesis is characterized by global changes in histone modifications. KDM1 (also known as LSD1) a histone demethylase enzyme, plays a key role in establishing specific histone methylation marks at ERα target gene promoters. Emerging evidence suggests histone methylation imposes ligand dependency; and deregulation of this epigenetic pathway potentially contributes to hormonal independence and adaptive resistance in breast cancer. Our study examined the therapeutic efficacy of pargyline, a KDM1 inhibiting drug, and evaluated the therapeutic benefit to curb growth of therapy resistant breast cancer cells. We used several model cell lines shown to exhibit resistance to hormonal therapy including MCF7-HER2, MCF7-Tam, MCF7-Ca-LTLT, MCF7-PELP1 and parental MCF7 cells as controls. Reporter gene assays showed KDM1 enhanced ERα- mediated transcription in therapy resistant cells. KDM1 functionally interacts with ERα coregulator PELP1 and is co-recruited with PELP1 to ERα target genes. Pargyline treatment substantially inhibited ERα transactivation functions. ChIP analysis revealed distinct activating histone methylation modifications at growth regulatory ERα target genes in aggressively growing and therapy resistant breast cancer cells. Breast cancer models treated with pargyline facilitated reversal of the observed specific modifications and thereby inhibited the growth of breast cancer cells in vitro and in vivo nude mice models. Pargyline also showed significant effect in blocking local estrogen synthesis via alterations in histone methylation modifications at the aromatase promoter. Combinational therapy using three agents that: (a) block ERα's nuclear actions (tamoxifen or letrozole), (b) ERα's extranuclear actions (dasatinib) and (c) ERα's epigenetic modifications (pargyline) showed most promising therapeutic effect compared to single agent therapy on the growth of therapy resistant cells. Our results suggest that histone methylation modifications play a role in therapy resistance and validate the therapeutic potential of pargyline in combinational therapies. Collectively, our results suggests targeting KDM1 axis with pargyline in combination with current endocrine therapies will have better therapeutic effect and may inhibit or delay development of hormonal resistance. This study is funded by Komen grant KG090447.

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 4613.
Abstract 4617: Novel antiestrogens, SS1020 and SS5020, for breast cancer therapy and prevention
Authors: Shibutani Shinya , Suzuki Naomi , Liu Xiaoping , Kim Hyo Jeong , Laxmi Y. R. Santosh , Okamoto Yoshinori
Published: February 2011

Tamoxifen (TAM) is widely used as a first-line endocrine therapy for breast cancer patients and as a prophylactic agent for women at high risk of developing this disease. Long-term administration of TAM has serious side effects, including endometrial cancer. These side-effects are due to the DNA damaging and/or estrogenic action of the drug. Raloxifene (RAL) approved as a chemopreventive agent for postmenopausal women at high risk for invasive breast cancer retains an estrogenic action. The poor bioavailability of RAL requires high doses to obtain an efficacy equivalent to that achieved with TAM. Several other antiestrogens developed were dropped out from the clinical trials due to their undesirable effects on the uterus. Therefore, development of new antiestrogen alternatives, but free of genotoxic and estrogenic potential, having significant antitumor potential beyond TAM and RAL is urgently required. SS1020 and SS5020 were developed as a new triphenylethylene antiestrogen and benzopyran antiestrogen, respectively, in our laboratory. Although TAM is a hepatic carcinogen and produces a high level of DNA damage in rats, both SS1020 and SS5020 did not produce such DNA damage. Unlike TAM and RAL, SS1020 and SS5020 had no detectable uterotrophic activity in OVX-rats treated even with a high dose (10 mg/kg). These indicate that SS1020 and SS5020 are free of genotoxic and estrogenic activities. The antitumor potential of SS1020 or SS5020 against 7,12-dimethyl-benz(a)anthracene-induced mammary carcinoma in rats and human MCF-7 breast cancer xenograft in athymic nude mice was superior to that of TAM and RAL. SS1020 has a much higher bioavailability than RAL, endowing it with higher antitumor potential than RAL. In addition, the development of estrogen-induced mammary tumor in rats was completed inhibited by dietary intake of SS1020, indicating that this compound has preventive potential. Taken together, SS1020 and SS5020 lacking genotoxic and estrogenic actions have anti-breast cancer activity superior to that of TAM and RAL. Both SS1020 and SS5020 are safer and more effective antiestrogen candidates for breast cancer therapy and prevention.

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 4617.
Abstract 4639: Prognostic significance and molecular associations of CDKN2A (p16) promoter methylation and loss of expression in colorectal cancer
Authors: Shima Kaori , Nosho Katsuhiko , Baba Yoshifumi , Cantor Mami , Giovannucci Edward L. , Fuchs Charles S. , Ogino Shuji
Published: February 2011

Background: p16 (CDKN2A/Ink4a) is a cyclin-dependent kinase inhibitor and functions as a tumor suppressor. CDKN2A promoter methylation and gene silencing are associated with the CpG island methylator phenotype (CIMP) in colorectal cancer. However, prognostic significance of CDKN2A methylation or loss of p16 expression independent of CIMP status has not been studied.

Patients and Methods: Utilizing 940 colorectal cancers (stage I-IV) in two independent cohort studies (the Nurses’ Health Study and the Health Professionals Follow-up Study), we quantified DNA methylation in the CDKN2A promoter real-time PCR (MethyLight) and detected hypermethylation in 269 tumors (29%). By immunohistochemistry, we detected loss of p16 (CDKN2A) expression in 25% (200/804) of tumors. We also analyzed DNA methylation in LINE-1 and 7 other CpG islands (CACNA1G, CRABP1, IGF2, MLH1, NEUROG1, RUNX3 and SOCS1); microsatellite instability (MSI); KRAS, BRAF and PIK3CA mutations; and expression of β-catenin, p53, p21, p27, cyclin D1, fatty acid synthase (FASN), and cyclooxygenase-2 (PTGS2, COX-2).

Results: In multivariate logistic regression analysis, CDKN2A methylation was independently associated with CIMP-high [adjusted odds ratio (OR) 41.6; 95% CI, 21.6-80.0; vs. CIMP-0], CIMP-low (adjusted OR 5.66; 95% CI, 3.75-8.53; vs. CIMP-0), and COX-2 negativity (adjusted OR 0.46; 95% CI, 0.29-0.74; COX-2 -positive vs. negative). Both CDKN2A promoter methylation and loss of p16 were significantly associated with high overall mortality in univariate Cox regression analysis [hazard ratio (HR), 1.31; 95% CI, 1.07-1.60; p=0.009 (CDKN2A methylation), HR, 1.30; 95% CI, 1.03-1.63; p=0.026 (loss of p16)] but not in multivariate analysis [HR, 0.79; 95% CI, 0.57-1.09; p=0.14 (CDKN2A methylation), HR, 1.00; 95% CI, 0.79-1.27; p=0.98].

Conclusion: Both CDKN2A promoter methylation and loss of p16 expression in colorectal cancer are not independently associated with patient survival. However, CDKN2A promoter methylation is independently associated with CIMP and COX-2 negativity.

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 4639.
Abstract 4660: Double combination array analysis detected A kinase anchor protein 12 (AKAP12) gene as a new tumor suppressor gene of hepatocellular carcinoma
Authors: Hayashi Masamichi , Nomoto Syuji , Kanda Mitsuro , Okamura Yukiyasu , Fujii Tsutomu , Sugimoto Hiroyuki , Takeda Shin , Nakao Akimasa
Published: February 2011

Background Hepatocellular carcinoma (HCC) is the fifth most common malignancy and is caused environmental and genetic factors. However, little is known about the genetic characteristics of HCC cells. In this study, we investigated a novel tumor suppressor gene in HCC by double combination array method, consisting of both expression array analysis and karyotyping analysis of single-nucleotide polymorphism (SNP) array, using the same surgical specimen. Methods We performed double combination array analysis of the HCC sample from a 68-year-old woman with chronic hepatitis type C. The Affymetrix HGU133A and HGU133B was used for expression profiling and the Affymetrix GeneChip Mapping 500K array was used for SNP array. We verified the identified gene by using HCC cell lines and 48 primary HCC tissues of patients who had undergone hepatectomy at our hospital. Results AKAP12 gene expression was decreased in tumor tissue by the expression array analysis. This gene is said to produce a structurally diverse protein associated with protein kinase A, which blocks tumor cell growth and invasion. The copy number at locus 6q24-q25 did not show chromosomal deletion by the SNP array analysis. This gene was also revealed to have a CpG rich promoter region. We could recognize promoter hypermethylation by sequence analysis of the HCC cell lines. Furthermore, AKAP12 gene expression was reactivated after 5-aza-2′-deoxycytidine (5-aza-dC) treatment. As for the 48 HCC samples, 41 cases (85%) showed promoter hypermethylation by methylation specific PCR. In these cases, the values of AKAP12 gene expression in tumor tissues, which were examined by quantitative real-time PCR, were significantly lower (P = 0.0001) than those in adjacent non-cancerous tissues. Finally, we evaluated the downregulated status of AKAP12 and the clinico-pathological findings. The downregulated cases were correlated with poor overall survival rate, although the diffenrence was not significant (P = 0.115). Conclusion Our double combination array method successfully detected a tumor suppressor gene of HCC which has yet to be identified. The downregulation of AKAP12 gene occurred by promoter hypermethylation and correlated with poor prognosis.

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 4660.
Abstract 4679: Integrin and FAK signaling regulate ARHI-induced autophagy
Authors: Lu Zhen , Liao Warren S. , Suh Grace K. , Bast Robert C.
Published: February 2011

Autophagy selectively degrades cellular proteins, protein aggregates and organelles, providing energy in a nutrient deprived environment. We have reported that re-expression of ARHI, a maternally imprinted Ras-related tumor suppressor gene that is down regulated in more than 60% of ovarian cancers, induces autophagy. Persistence for ARHI-induced autophagy for several days is lethal for ovarian cancer cells in culture. In xenografts, however, ARHI-induced autophagy does not kill ovarian cancer cells, but rather is required to maintain a dormant state in the absence of a vascular supply. When ARHI is downregulated in vivo, previously dormant xenografts grow promptly and outgrowth of cancers is inhibited by treatment of dormant cancer cells with chloroquine. Growth factors (IGF-1, IL-8, VEGF) and ECM components (fibronectin) in the cancer microenvironment can rescue ovarian cancer cells from ARHI-induced autophagic death in cell culture. Rescue was associated with partial reversal of ARHI-mediated inhibition of PI3K signaling and AKT and mTOR activity. Consequently, we have hypothesized that both autophagy and survival factors (IGF-1, fibronectin) are required to maintain dormancy, where autophagy provides energy for avascular xenografts and survival factors prevent autophagic death by partially reversing inhibition of PI3K signaling. Here, we show that re-expression of ARHI in SKOv3 ovarian cancer cells inhibited integrin β1 expression and decreased FAK activation, stimulated by fibronectin and growth factors (IGF-1). Consistent with the importance of ECM, addition of fibronectin, a receptor for αvβ1 integrin, induced FAK activity and diminished ARHI-induced autophagy; whereas addition of neutralizing antibody against integrin β1 decreased FAK and Akt activities, enhancing autophagy. Autophagy induced by ARHI is dramatically reduced in cells that overexpress FAK and knockdown of FAK by RNA interference enhances ARHI-induced autophagy in ovarian cancer cells cultured on fibronectin. Our results suggest that the ECM and growth factors in the tumor microenvironment function to coordinate the activities of integrin β1, FAK and Akt to regulate ARHI-mediated autophagy, contributing to the survival of dormant ovarian 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 4679.
Abstract 4683: Coexistence incidence and clinicopathologic characteristics of gastrointestinal stromal tumors with gastrointestinal carcinoma
Authors: Wen Li , Ren Xu-gong , Huang Yan-li
Published: February 2011

Objective Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumors of the digestive tract. The coexistence with other gastrointestinal carcinoma is unique. The aim of this study was to investigate the coexistence of GIST with other primary gastrointestinal carcinoma and its clinicalpathologic features. Methods 113 patients had been diagnosed as GIST in three local hospitals from 2002 to 2008. The surgical specimens were analyzed with an immunohistochemical panel that included staining for CD117, CD34, SMA and Ki-67. Results 16.8 % of (19 of 113) patients with GIST were co-diagnosed as gastrointestinal carcinoma. The average age at diagnosis was 57 years (range, 43 to 66), two years older than that of the patients only with GIST (range, 43 to 71). The patients with the coexistence of GIST and gastrointestinal carcinoma included 11 males and 8 females. The male-to -female ratio was 1.38:1, which is higher than that of the patients of GIST without the carcinoma (1.24:1). 26.3% GIST with the carcinoma was found in the esophagus (5 of 19) and 73.7% in the stomach (14 of 19) and non in the intestine. In contrast, only 5.3% GIST without the carcinoma was found in the esophagus (5 of 94) which was less incidence in the esophagus than that of coexistence of GIST and the carcinoma (P < 0.05), 84.0% in the stomach and 10.6% in the intestine, which higher incidence in the intestine than that of coexistence of GIST and the carcinamo (P < 0.05). The mean diameter of 19 GISTs with the carcinoma was 1.91 cm (range, 0.6 to 3.8 cm) smaller than that of 94 GISTs without the carcinoma (5.42 ± 6.17 cm, p < 0.05). 15.8% of the patients with coexistences of GIST and the carcinoma showed lower dysplasia, whereas 41.5 % of GIST patients without the carcinoma had the dysplasia and 33.0 % was malignant (p < 0.05). The average mitotic count in GIST with the carcinoma was 0.74/high-power field (HPF) (range, 0-4/HPF), and much less than that (3.78/HPF) of GIST without the carcinoma (range, 0 to 53/HPF, P < 0.05). The average proliferative index (positive number of Ki-67/HPF) of GIST with the carcinoma was 7.16/HPF (range 0 to 22) and less than that (19) of GIST without the carcinoma (range, 0 to 107). Conclusion 16.8% of GIST was co-diagnosed with gastrointestinal carcinoma. More coexistence of GIST and the carcinoma occurred in the esophagus. The GIST with the carcinoma was less proliferative and aggressive than the GIST without the Carcinoma.

[Key words] gastrointestinal stromal tumor; pathology; carcinoma

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 4683.
Abstract 4688: A relationship between environmental tobacco smoke (ETS) and epidermal growth factor receptor (EGFR) mutations in never smoker patients with non-small cell lung cancer
Authors: Lee Young Joo , Cho Byoung Chul , Choi Hye Jin , Lee Jin Gu , Kim Joo-Hang
Published: February 2011

Purpose

The etiology of EGFR-mutant lung cancer remains unknown. The high incidence of EGFR mutations are observed in life-time never smokers. We investigated whether the risk factors of lung cancer in never smoker may be associated with a mutant-EGFR dependent carcinogenesis. Especially, we focused on the effect of ETS on EGFR-mutant lung cancer.

Patients and Methods

We enrolled 179 consecutive never smokers who were newly diagnosed with NSCLC. The history of ETS exposure was obtained with a standardized questionnaire that included exposure period, place, and duration. The nucleotide sequences of exons 18-21 on EGFR gene were determined using nested PCR amplification. Those patients with an exon 19 deletion and exon 21 point mutations were selected as a subgroup with activating mutations for further analysis.

Results

The median total smoker-year of patients with ETS exposure was higher than that of those without (25 years vs. 15 years; P = 0.002). The incidence of EGFR mutations was significantly lower in patients with ETS exposure than in those without (38.5% vs. 61.4%; P = 0.008). The proportion of exon 19 mutation was lower in patients with ETS exposure (59.6% vs. 81.5%; P = 0.050). In a logistic regression model that adjusted for gender and histology, an adjusted odds ratio (AOR) for the risk of EGFR mutations with exposure to ETS was 0.40 (95% confidential interval [CI], 0.20-0.81; P = 0.011). In quartile groups based on total smoker-year, the AORs for the lowest- to highest-quartile groups were 0.59 (95% CI, 0.23-1.49), 0.50 (0.17-1.50), 0.48 (0.20-1.18), and 0.22 (0.08-0.62) (Ptrend = 0.028). The period and place of ETS exposure had no different effect on the incidence of EGFR mutations. Patients with ETS exposure showed a lower response rate to EGFR tyrosine kinase inhibitors than did patients without ETS exposure (24.6% vs. 44.8%; P = 0.053).

Conclusions

Exposure to ETS is negatively associated with the incidence of EGFR mutations in lung cancer in never smokers. There was an inverse trend between the cumulative dose of ETS exposure and the frequency of EGFR mutations. A mutant-EGFR-dependent carcinogenesis may be different from a tobacco-smoke-associated carcinogenesis.

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 4688.