bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1693: Assessment of Nrf2 luciferase reporter assay and heme oxygenase expression as models of oxidative stress in in vitro cultures of lung cells exposed to cigarette smoke condensate
Authors: Fields Wanda R. , Liedtke Wolfgang , Li Jinju , Bombick Betsy
Published: February 2011

The Nrf2 signaling pathway is an important regulator associated with oxidative stress, xenobiotic metabolism, DNA damage and anti-inflammatory response. A key responsive gene in the Nrf2 regulatory pathway is heme oxygense 1 (HO-1), a cytoprotective enzyme that serves in an antioxidant capacity. An imbalance in antioxidant capacity observed in animal models or individuals with specific Nrf2 and HO-1 polymorphisms has been associated with susceptibility to lung diseases. The objective of this study was to characterize the response of Nrf 2 promoter regulation and HO-1 expression following exposure to cigarette smoke in lung models for oxidative stress. Cigarette smoke condensate (CSC) was prepared from Kentucky 3R4F (K3R4F) reference cigarettes. Nrf2 promoter regulation and HO-1 expression were evaluated in lung cells exposed to K3R4F CSC (0 − 90μg total particulate matter/ml media). Nrf2 promoter regulation was evaluated in transiently-transfected Nrf2 luciferase reporter cells exposed to K3R4F CSC for 0.5, 1, 2, 6 or 24 hours followed by quantification of luciferase activity. HO-1 expression was assessed following exposure to the K3R4F CSC in normal human bronchial epithelial (NHBE) cells for 3, 6 or 24 hours using real-time quantitative RT/PCR and enzyme linked immunoassay (ELISA). Nrf2 reporter cells exhibited dose-dependent increases in promoter activation following CSC exposure. The maximum response was observed between 6 and 24 hours of exposure with 15-, 25-, and 40-fold increases across the exposure range. The specificity of the promoter response was confirmed with co- exposure of the inhibitor all-trans retinoic acid with CSC. The CSC-induced Nrf2 activation was reduced towards control levels in the presence of the inhibitor. CSC also induced dose- and time-dependent increases on HO-1 mRNA and protein levels in NHBE cells. The mRNA changes ranged from 10- to 70-fold with maximal responses observed between 3 and 6 hours. The dynamic range of HO-1 protein response was observed and reached a level of 11-fold increase by 60 μg/ml. Collectively, these responses are consistent with our previously reported study which identified oxidative stress responsive genes in NHBE cells exposed to repetitive applications of CSC. The data suggest that changes in the Nrf2 regulatory pathway may serve as an effective indicator of oxidative stress and the impact of tobacco products in in vitro lung cell culture models.

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 1693.
Abstract 1694: Selective formation of the γ isomer of 1,N2-propanodeoxyguanosine adducts of acrolein through interactions with amine-containing molecules
Authors: Chung Fung-Lung , Wu Mona Y. , Dyba Marcin D. , Nath Raghu G.
Published: February 2011

Acrolein (Acr) is a highly reactive α,β-unsaturated aldehyde found ubiquitously in the environment and also formed endogenously as a major product from oxidation of polyunsaturated fatty acids. Acr can bind to deoxyguanosine (dG) to form two regioisomers of 1,N2-propanodeoxyguanosine adducts (α- or γ-Acr-dG depending on the position of the OH group). However, in most cases γ-Acr-dG is the major adduct detected in rodent and human tissues and Acr-treated cells. Our LC-MS/MS analyses of seven human lung and liver tissues confirmed previous findings that γ-Acr-dG is the major isomer. The reasons for the apparent prevalence of γ-Acr-dG in vivo are unclear. To determine whether the reaction of Acr with cellular molecules alter isomer formation, the soluble fraction of BEAS-2B cell lysate was reacted with equimolar concentrations of Acr and dG in 100 mM sodium phosphate buffer (pH 7.4) at 37° C for 24 h. Acr-dG was detected and quantified via HPLC-UV. In reactions without cell lysate, α- and γ-Acr-dG were detected in approximately equivalent amounts; however, in the presence of cell lysate the amount of α-Acr-dG decreased compared to γ-Acr-dG in a concentration-dependent manner. Similar results were obtained in the reactions with amino acids, histones, and polyamines under the same conditions or with a Tris buffer instead of a phosphate buffer. Additionally, LC-MS/MS analysis of reactions of these compounds with calf thymus DNA rather than dG yielded similar results. These results suggest that amino compounds are needed for the selective formation of the γ isomer. To investigate the intermediate(s) involved, Acr was reacted with arginine in 100 mM sodium phosphate buffer (pH 7.4). Analyses by HPLC-UV show that an initial compound forms quickly, but decomposes over time to give rise to a stable compound. These compounds were collected separately and reacted with dG. While Acr-dG was not detected in reactions with the stable compound, the initial compound predominately gave rise to γ-Acr-dG. It is likely that a similar or related unstable intermediate formed between Acr and an amine-containing molecule is responsible for the formation of DNA adducts in vivo. (Supported by NCI grant CA043159.)

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 1694.
Abstract 1695: Mechanisms of cytotoxicity of diepoxybutane, epichlorohydrin, and (1-chloroethenyl) oxirane
Authors: Bartley Bethany L. , Silvestri Vanesa L. , Ng Christopher , Hirsch David C. , Millard Julie T.
Published: February 2011

The bifunctional alkylating agents are an important class of drugs for the treatment of cancer, psoriasis, and some anemias. These compounds are widely believed to exert their cytotoxic effects through reactions with biological nucleophiles, with DNA interstrand cross-links considered to be the most lethal lesions. Indeed, for many families of bifunctional alkylators, DNA interstrand cross-linking correlates with cytotoxicity. Moreover, apoptotic potential may play a role in antitumor chemotherapeutic utility. We have established previously that diepoxybutane (DEB) and the structurally related compounds epichlorohydrin (ECH) and 1-(chloroethenyl)oxirane (CEO) form DNA interstrand cross-links in vitro. With the goal of elucidating the molecular and cellular mechanisms by which this family of epoxides exerts its cytotoxic effects, we are determining the relationships between interstrand cross-linking, LD50 values, and apoptotic potentials for DEB, ECH, and CEO in chicken 6C2 and human HL-60 cells. Preliminary results in 6C2 cells suggest that cytotoxicity follows the order DEB > CEO > ECH. Furthermore, flow cytometry with Annexin V-FITC/PI dual staining has revealed that the apoptotic potentials follow the order DEB >> ECH > CEO. Reverse-transcriptase real-time PCR analysis of HL-60 cells treated with DEB under conditions known to induce apoptosis suggests up-regulation of several key genes involved in the mitochondrial apoptotic pathway, including BAK1, BAX, DIABLO, and APAF1. Further work is underway to characterize the pathways by which ECH and CEO exert their apoptotic effects.

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 1695.
Abstract 1701: Elevated DNA adducts detected in human breast tumors than normal tissues from mammoplasty
Authors: Mungala Radha , Vadhanam Manicka , Chagpar Anees , Fleming Muffin , Fink Betsy , Gridley Mary , Vasconez Henry , Gupta Ramesh
Published: February 2011

Breast cancer is the most common cancer among women worldwide and the second leading cause of cancer-related death in women in the United States. The etiology of the majority of human breast cancers is unknown. Elevated levels of estrogen and exposure to environmental carcinogens have been suggested to be involved in human breast cancer development. Several studies have also provided evidence of the potential role of oxidative stress and lipid peroxidation in breast cancer etiology. Identification of DNA adducts and monitoring change in DNA adduct profiles/burden in normal and cancer tissue may provide clues for risk of breast cancer. In this study, we detected and compared the levels of a wide range of polar to lipophilic adducts in tumor (n= 23) and adjacent normal tissue (n= 30) from breast cancer patients with normal tissue (n=124) from mammoplasty using a 32P-postlabeling adductomics. Adducts were classified into polar (P-1, P-2a and P2b or 8-oxodG) and lipophilic (L-1) in descending order of their polarities. Our results indicated significantly elevated total DNA adduct levels in tumor and adjacent normal tissue of breast cancer patients compared to normal tissues from mammoplasty. The levels of most polar adducts (P-1) were not significantly different among the tumor, adjacent and normal breast tissue. However, relatively lesser polar adducts, i.e., P-2a, P-2b (8-oxodG) and L-1 adducts were significantly higher in tumor and adjacent normal tissues of breast cancer patients compared with normal tissues from mammoplasty. Thus, there is a clear trend of adduct accumulation in the breast tissues of cancer patients that could have acted as a precursor event in carcinogenesis process. Therefore, elevated oxidative DNA adducts such as 8-oxodG can be a possible biomarker for early detection of breast cancer risk and subjects with high DNA adduct load may potentially be at higher risk for cancer development, and could be stratified for chemopreventive intervention. (Supported from CA-90892 and Agnes Brown Duggan Endowment)

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 1701.
Abstract 1705: 14-3-3ζ is an androgen-regulated gene that activates the androgen receptor signaling and facilitates cell survival in prostate cancer
Authors: Murata Taro , Takayama Ken-ichi , Urano Tomohiko , Fujimura Tetsuya , Kumagai Jinpei , Horie-Inoue Kuniko , Ikeda Kazuhiro , Takahashi Satoru , Ouchi Yasuyoshi , Homma Yukio , Inoue Satoshi
Published: February 2011

The 14-3-3 proteins are a family of regulatory molecules that modulate the functions of their binding partners. They are involved in many processes including protein synthesis/degradation, cell cycle regulation, apoptosis, and tumor development. We have identified one of 14-3-3 protein isoforms 14-3-3zeta as a novel androgen target gene by chromatin immunoprecipitation (ChIP) coupled with microarray analysis. The expression of 14-3-3zeta was upregulated in LNCaP prostate cancer cells at both mRNA and protein levels by androgen stimulation. We generated LNCaP cells stably expressing 14-3-3zeta or control empty vector, and found that less etoposide-induced cell death was observed in 14-3-3zeta-expressing cells compared with control cells. Cell proliferation and motility were increased in 14-3-3zeta-expressing cells. Imunoprecipitation study showed that 14-3-3zeta binds to androgen receptor (AR) in these cells. Luciferase assay showed that androgen-dependent PSA promoter activity was significantly increased in 14-3-3ζ-expressing cells. Androgen-dependent PSA expression was also increased in 14-3-3ζ-expressing cells. siRNA-dependent 14-3-3zeta knockdown revealed that androgen-dependent AR transactivation was reduced in LNCaP cells. Taken together, the present study indicates that 14-3-3ζ is a bona-fide androgen-regulated gene that plays an important role in activating AR-dependent signaling, thereby promoting cell growth, motility and acquirement of apoptosis resistance in prostate 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 1705.
Abstract 1706: Impact of PARP1 on AR signaling and therapeutic response in prostate cancer
Authors: Schiewer Matthew J. , Goodwin Jonathan , Knudsen Karen E.
Published: February 2011

Poly (ADP-ribose) polymerase 1 (PARP1) modifies various nuclear proteins by poly (ADP-ribose)-ylation, and has a known role in a multitude of chromatin-associated functions, including: transcriptional coregulation, modification of chromatin structure, regulation of insulators, and well-established activities in DNA repair. With regard to nuclear receptors, PARP1 promotes recruitment of topoisomerase IIβ (TopoIIβ) to estrogen-regulated promoters, leading to promoter DNA cleavage, factor exchange, and transcriptional activation. However, PARP1 function is known to be context dependent. Here, the impact of PARP1 on AR signaling was assessed under conditions of prostate cancer relevance. As will be discussed, key preliminary data strongly suggest that PARP1 function is critical for the pro-tumorigenic functions of AR. First, pharmacological inhibition of PARP1 enzymatic activity potently suppressed ligand-dependent AR transcriptional activity on target genes of clinical relevance. Second, the mechanism of PARP1-mediated AR regulation appears to be manifest at the level of chromatin, as inhibition of PARP1 failed to alter AR levels. This posit was assessed by chromatin immunoprecipitation assays to delineate the impact on AR signaling. Third, it was shown that suppression of PARP1 specifically inhibited AR-dependent prostate cancer cell proliferation in both early stage and late stage (castrate-resistant) models of disease. Lastly, it was observed that PARP1 inhibition cooperates with currently used genotoxic therapeutic modalities (IR and Docetaxel) in reducing cell survival, dependent on treatment scheduling.

In sum, our data identify PARP1 as a critical effector of AR activity at the level of chromatin, and suggest that abrogation of PARP1 function may cooperate with existing therapeutics to improve the response to therapeutic intervention.

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 1706.
Abstract 1707: Androgen responsiveness of the NKX3.1 gene is regulated by the proto-oncogene ETS1
Authors: Thomas Marc A. , Preece Darren M. , Bentel Jacqueline M.
Published: February 2011

Expression of the prostatic homeodomain protein NKX3.1 is androgen-regulated in both the normal prostate and in prostate cancer cells. Androgen responsiveness of NKX3.1 is mediated in part by the 3’ untranslated region (3'UTR) of the NKX3.1 gene, within which we have isolated 3 discrete regions of androgen-induced transcriptional regulatory activity including 2 androgen response elements (AREs). The proximal ∼2kb of the 5’ promoter of the NKX3.1 gene is incompletely characterised, however it is not androgen-responsive using conventional luciferase reporter assays. Investigation of the NKX3.1 proximal promoter has identified regulation of promoter activity by the ETS transcription factor, ETS1, which is mediated by an ETS binding site (EBS) located at −1008 relative to the NKX3.1 transcription start site. Overexpression of ETS1 in the LNCaP prostate cancer cell line results in upregulation of NKX3.1 protein levels, supporting a functional role of ETS1 in the regulation of NKX3.1 expression. ETS1 has been reported previously to transactivate the androgen receptor (AR) in a ligand-dependent manner on a subset of androgen-responsive promoters, suggesting cooperativity or co-regulation of AR and ETS1 signalling in prostate cancer cells. Treatment of LNCaP cells with 10−8M 5α-dihydrotestosterone (DHT) results in upregulation of endogenous ETS1 protein levels, while overexpression of exogenous ETS1 causes a concomitant increase in AR expression. A cooperative interaction between ETS1 and the AR on the NKX3.1 promoter is evident in LNCaP cells whereby overexpression of ETS1 is able to induce androgen responsiveness of the initial ∼2kb of the NKX3.1 promoter sequence and in particular the 76bp region surrounding the EBS. Reversal of DHT effects by the antiandrogen bicalutamide indicates an AR-mediated mechanism. ETS1 is overexpressed in a high proportion of prostate cancers with its expression aberrantly localised in the cytoplasm of a subset of prostate tumour cells. Findings of the present study support a complex mechanism of AR-mediated regulation of NKX3.1 expression via multiple regions of the NKX3.1 gene and modified by additional transcription factors in prostate cancer cells. Abnormal expression and/or function of these factors may contribute to the transcriptional deregulation of NKX3.1 expression and the downregulation of NKX3.1 protein levels that is frequently observed in advanced prostate tumours. Similarly, it is also feasible that upregulation of ETS1 contributes to the deregulation of AR signalling in advanced prostate cancers.

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 1707.
Abstract 1708: Telomerase as an important target of androgen-signaling blockade for prostate cancer treatment
Authors: Liu Shuang , Qi Yanfeng , Ge Yubin , Duplessis Tamika , Rowan Brian G. , Cheng Helen , Rennie Paul S. , Zhang Haitao , Dong Yan
Published: February 2011

As the mainstay treatment for advanced prostate cancer, androgen-deprivation therapy (ADT) targets the action of androgen receptor (AR) by reducing androgen level and/or by the administration of anti-androgen that competes with androgens for binding to AR. Albeit effective in extending survival, ADT is associated with dose-limiting toxicity and the development of castration-resistant prostate cancer (CRPC) after prolonged use. Since CRPC is generally lethal and incurable, developing effective strategies to enhance the efficacy of ADT and circumvent resistance becomes an urgent task. Continuous AR signaling constitutes one major mechanism underlying the development of CRPC. The present study showed that methylseleninic acid (MSA), an agent that effectively reduces AR abundance, could enhance the cancer-killing efficacy of the anti-androgen bicalutamide in both androgen-dependent and castration-resistant prostate cancer cells. We found that combination of MSA and bicalutamide produced a robust downregulation of prostate-specific antigen and a recently identified AR target, telomerase and its catalytic subunit, telomere reverse transcriptase (hTERT). The downregulation of hTERT occurs mainly at the transcriptional level, through reducing AR occupancy of the hTERT promoter. Furthermore, apoptosis induction by the two agents is significantly mitigated by restoration of hTERT. Our findings thus indicate that MSA in combination with anti-androgen could represent a viable approach to improve the therapeutic outcome of ADT. Given the critical role of hTERT/telomerase downregulation in mediating the combination effect and the fact that hTERT/telomerase could be measured in blood and urine, hTERT/telomerase could serve as an ideal tumor-specific biomarker to monitor the efficacy of the combination therapy non-invasively. (Supported by the Department of Defense Prostate Cancer Training grant No. W81XWH-08-1-0291 (SL); the National Cancer Institute grant No. K01 CA114252 (YD); the American Cancer Society grant No. RSG-07-218-01-TBE (YD))

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 1708.
Abstract 1709: Activation of androgen signaling axis by NF-kappaB2/p52
Authors: Nadiminty Nagalakshmi , Lou Wei , Gao Allen C.
Published: February 2011

INTRODUCTION: Castration-resistant progression of prostate cancer occurs in >80% of patients after failure of hormonal therapy but the underlying mechanisms are incompletely understood. Emerging evidence implies that ligand-independent activation of the androgen receptor (AR) is of critical importance during this process. Our previous studies show that the levels of NF-κB2/p52 are elevated in prostate cancer cells and that active NF-κB2/p52 promotes prostate cancer cell growth in vitro and in vivo. In this study, we examined the role of NF-κB2/p52 in the aberrant activation of the AR during castration resistant progression, in contrast to the interaction between the AR and RelA/p65.

METHODS: The effects of NF-κB2/p52 on cell survival, growth and androgen responsiveness were examined in prostate cancer cells. Modulation of LNCaP tumor growth by p52 was examined in intact and castrated male nude mice. Effect of p52 or p65 on AR activation was examined using luciferase assays, EMSA, ChIP assays and ELISA.

RESULTS: Expression of NF-κB2/p52 enhanced androgen-sensitive LNCaP human CaP cell growth and clonogenic ability in androgen-deprived conditions in vitro. LNCaP cells expressing p52 exhibited protection from apoptotic cell death and cell cycle arrest induced by androgen-deprivation. Adenoviral mediated p52 expression in LNCaP cells induced tumor growth in castrated male nude mice. NF-κB/p52 induced the activation of the AR resulting in increased transactivation of AR-responsive genes such as PSA and NKX3.1 in a ligand-independent manner. NF-κB2/p52 enhanced nuclear translocation and transactivation of AR by interacting with its N-terminal domain and enhanced the recruitment of co-activators like p300 to the promoters of AR-dependent genes. These results were confirmed in 3 different prostate cancer cell lines: LAPC-4 (wild type AR), LNCaP (mutant AR) and C4-2 (androgen-insensitive). Transfection of p52 into LAPC-4 and LNCaP cells (which express low levels of p52) showed increased activation of the endogenous AR. Downregulation of endogenous p52 in C4-2 cells resulted in abrogation of AR constitutive activation. In contrast, we observed that p65 exhibited transcriptional repression of the AR, and did not induce expression of AR-responsive genes, PSA and NKX3.1.

CONCLUSIONS: These findings demonstrate that overexpression of NF-κB2/p52 induces castration-resistant growth and aberrant activation of the AR in LNCaP cells. NF-κB2/p52 may promote castration resistant progression by activating the AR under conditions of androgen deprivation.

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 1709.
Abstract 1716: BRCA1 expression modulates apoptosis and survival in response to retinoic acid in breast cancer cells
Authors: Pickholtz Itay , Papa Moshe Z. , Yarden Ronit I.
Published: February 2011

The vitamin A metabolite retinoic acid (RA) plays key roles in development, cell proliferation and differentiation. RA derivatives also display anti-cancinogenic activities. The different physiological effects of RA are mediated by retinoic acid receptors (RARs) and retinoid-X receptos (RXRs) that are transcription factors and members of the nuclear hormone receptor superfamily. Transcriptional activation by RA trigger cell cycle arrest, differentiation and apoptosis in certain tissues and cell types but in other tissues RA appear to promote rather than inhibit cell survival.

Recently, RA derivatives were shown to bind and activate the orphan nuclear hormone receptor, peroxisome proliferator-activated β/δ (PPARβ/δ) when it hetrodimerizes with RXR. The partitioning of RA between the RAR and PPARβ/δ receptors is regulated by the balance between two intracellular lipid binding proteins: cellular retinoic acid binding protein II (CRABPII) and fatty acid binding protein 5 (FABP5). While CRABP-II delivers RA to RAR-RXR and facilitates anti-proliferative effects, FABP5 shuttles RA to PPARβ/δ-RXR and promotes pro-survival functions.

Interestingly, the breast and ovarian tumor suppressor, BRCA1 and RA share the ability to direct cell-cycle arrest and apoptosis, and both BRCA1 and RA regulate transcription and chromatin structure. We hypothesize that BRCA1 and RA pathways work in concert to inhibit cell growth in breast cancer cells. We found that the inhibitory effects of all-trans RA (ATRA) and 9-cisRA correlate with BRCA1 expression. Both RA derivatives inhibited BRCA1-proficient MCF7 breast cancer cells growth by inducing apoptosis. ATRA and 9-cisRA did not inhibit the growth of BRCA1-null or BRCA1-silenced breast cancer cells despite similar levels of RA receptors (RAR and RXR) expression. We show here that in BRCA1-proficient cells, the FABP5/CRABP-II ratio is constant facilitating apoptotic cell death, while in BRCA1-deficient cells FABP5/CRABP-II ratio is elevated, and coincides with increased phosphorylation of the pro-survival factor, AKT, supporting RA-mediated cell survival. Additional apoptotic and cell cycle genes were differentially regulated between BRCA1-deficient and proficient cells in response to RA. However, RA-induction of the metabolic enzyme CYP26 gene expression was not altered between BRCA1-deficient and proficient cells, suggesting the cooperation between RA and BRCA1 is affecting cell proliferation but not necessarily all spectrum of RA 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 1716.
Abstract 172: A role for the demethylation of microRNA-496 in MBD2-mediated repression
Authors: Alvarado Sebastian G. , Andrews Stephen D. , Chik Flora , Szyf Moshe
Published: February 2011

BACKGROUND: MicroRNAs are small RNA molecules that have the ability to repress target mRNAs through binding in their 3’ UTR. Prior work has shown that the expression of methyl binding domain protein 2 (MBD2) induces a variety of prometastatic genes through direct demethylation.

HYPOTHESIS: MBD2 turns on microRNA expression through promoter demethylation that in turn suppress other genes.

RESULTS: Methylated DNA Immunoprecipitation (mDIP) and MBD2 ChIP arrays identified a subset of microRNAs that change with the overexpression and transient knockdown of MBD2 in MCF10A and MCF7 cell lines, respectively. Methylation of these microRNAs with bisulfite sequencing and validation of MBD2 binding with quantitative PCR (qPCR) revealed the possible induction of hsa-mir-496 by MBD2. Expression of mir-496 was shown to be induced in MBD2 transfected MCF10A cells and repressed in MCF7 and MDA-231 cells with qPCR. Luciferase promoter assays revealed hsa-mir-496 promoter to be regulated by methylation and driven forward by co-transfection with MBD2. In silico scans of possible targets of hsa-mir-496 identified CPEB3 as a putative target. Expression of CPEB3 was shown to be inversely proportional to MBD2 in MBD2 transfected MCF10A cell lines and transient knockdowns of MBD2 in MCF7 and MDA-231 cell lines with qPCR.

CONCLUSIONS: The discovery of microRNAs has shed a whole new light on transcriptional regulation and with it has raised several questions about specific cell states. Considering that MBD2 causes complete transformation of a cell and plays a large role in tumorigenicity it is essential to characterize the roles of microRNA action with regards to MBD2 and its respective network of action. More importantly to show MBD2 as a key regulator of microRNA expression can further reinforce its role as a DNA demethylase and a key regulator of cancer.

FUTURE DIRECTIONS: Future work will involve testing mir-496 action in the cell with its putative target CPEB3 using 2-O-methyl primer assays in conjunction with luciferase reporter assays in order to probe the functionality of the CPEB3 3′UTR. The aforementioned results and directions will give us a refined picture of our model as well as attempt to define a system of MBD2-microRNA action.

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 172.
Abstract 1722: c-Met overexpression contributes to the acquired apoptotic resistance of non-adherent ovarian cancer cells through a cross-talk mediated by phosphatidylinositol 3-kinase and extracellular signal-regulated kinase 1/2
Authors: Tang Maggie K. S. , Zhou Hong Y. , Yam Judy W. P. , Wong Alice S. T.
Published: February 2011

Ovarian cancer is the most lethal gynecological cancer mainly due to widespread peritoneal dissemination and malignant ascites. Key to this is the capacity of tumor cells to escape suspension-induced apoptosis (anoikis), which also underlies their resistance to chemotherapy. Here we utilized a non-adherent cell culture model to investigate molecular mechanisms of apoptotic resistance of ovarian cancer cells that may mimic the chemoresistance found in solid tumors. We found that ovarian cancer cells acquired a remarkable resistance to anoikis and apoptosis induced by exposure to clinically relevant doses of two front-line chemotherapeutic drugs cisplatin and paclitaxel when grown in three-dimensional (3D) than monolayer cultures. Inhibition of the hepatocyte growth factor (HGF) receptor c-Met, which is frequently overexpressed in ovarian cancer, by a specific inhibitor or small interfering RNA blocked the acquired anoikis resistance and restored chemosensitivity in 3D, not in 2D cultures. These effects were found to be dependent on both phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular-signal regulated kinase (ERK) 1/2 signaling pathways. Inhibitors of PI3K/Akt abrogated ERK1/2 activation and its associated anoikis resistance in response to HGF, suggesting a signaling relay between these two pathways. Furthermore, we identified, as a mechanism of this cross-talk, a central role of Ras. Interestingly, Ras did not lie upstream of PI3K/Akt, whereas PI3K/Akt signaling to ERK1/2 involved Ras. These findings shed new light on the apoptotic resistance mechanism of non-adherent ovarian cancer ascites cells (This study was supported by Hong Kong Research Grants Council 7599/05M and Committee on Research and Conference Grant HKU200807176203 to A.S.T.W.).

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 1722.
Abstract 1728: Membrane androgen receptor ligands: a novel therapeutic approach for the treatment of cancer
Authors: Alevizopoulos Konstantinos , Bacopoulos Nicholas G. , Stournaras Christos
Published: February 2011

Membrane Androgen Receptor (mAR) is a novel, yet of unknown identity, membrane receptor frequently over-expressed in aggressive prostate cancer. mAR is not related to the classical intracellular androgen receptor (AR), a well known therapeutic target for prostate cancer treatment. Consequently, mAR induces tumour cell death by apoptosis in both AR-positive and AR-negative cell lines or in the presence of anti-androgens (e.g. flutamide). Early experimental results with membrane-targeted steroids (androgen-albumin conjugates) have proved the validity of mAR as a novel target of cancer therapy, as based on the anti-cancer action observed in prostate xenograft models (reviewed in IUBMB Life. 2009 Jan;61(1):56-61).

In this study, we describe novel small molecule steroidal mAR ligands that exhibit in vitro anti-cancer activity in the nanomolar range in prostate cancer cell lines. Furthermore, and as based on the identification of functional membrane androgen receptors in non-prostate cancer cells by immuno-fluorescence analysis, we show that selective novel mAR ligands exert broad anti-cancer action in additional cell line panels including colon, non-small cell lung cancer, central nervous system, breast or multi drug resistant cells. Notably, our compounds are selective for mAR binding since control displacement assays failed to identify significant interactions with recombinant Androgen Receptors.

To assess the anti-cancer of our compounds in vivo, we measured the ability of a prototype mAR ligand, MDX-146, to inhibit tumour growth of human PC-3 prostate cancer xenografts. Our results show clear tumour growth inhibition effects of MDX-146 in animals and propose a potential therapeutic role of specific mAR ligands for the treatment of prostate and other cancers.

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 1728.
Abstract 173: Frequent concomitant epigenetic silencing of the SFRPs and SOX1, Wnt/β-catenin regulators, in human hepatocellular carcinoma
Authors: Lin Ya-Wen , Hsieh Chung-Bao , Shih Yu-Lueng , Hsieh Tsai-Yuan , Tsao Chun-Ming , Yan Ming-De
Published: February 2011

Oncogenic activation of the Wnt/β-catenin signaling pathway is common in hepatocellular carcinoma (HCC). The secreted frizzled-related proteins (SFRPs) function as negative regulators of Wnt signaling and have important implications for carcinogenesis. Recently, we have shown that SFRPs are frequently downregulated through promoter hypermethylation in HCC cell lines and clinical HCC tissues. Furthermore, we have demonstrated that restoration of SFRPs could attenuate Wnt signaling in HCC cells with β-catenin point mutation, decrease abnormal accumulation of free β-catenin in the nucleus, and then suppress cell growth. Sox1 encodes a transcription factor involved in the regulation of embryonic development and cell fate determination. Structurally related to TCF/LEFs, several members of the Sox family, including Sox17, Sox3, Sox7, and Sox9, have also been implicated in repressing β-catenin activity by a mechanism that is not well understood. In this study, we found that the Sox1 was severely down-regulated by quantitative reverse transcription polymerase chain reaction (QRT-PCR). Next, we employed bisulfite modification and methylation-specific PCR (MS-PCR) to analyze the feature of Sox1 promoter methylation in the HCC cell lines and found that the majority of the tested cell lines exhibited hypermethylation. Meanwhile, we also examined sixty primary HCC surgical samples and adjacent nonturmorous liver tissues from Taiwan Liver Cancer Network (TLCN). These results showed that aberrant promoter methylation of SOX1 were found in 32 of 60 HCCs (53.3%), in 13 of 50 cirrhotic livers (26%), and in one of 30 normal control tissues (3.3%). Concomitantly, SFRPs was epigenetically inactivated in HCC cell lines and most of all the tumors with methylated SOX1. A significant correlation between the methylation of both genes was found (p >0.05). Furthermore, overexpression of SOX-1 in HCC cells could suppress TCF-dependent transcriptional activity and suppress colony formation. Taken together, our data suggest that SOX1 is a negative modulator of Wnt/β-catenin signaling, and that SOX1 silencing due to promoter hypermethylation is an early event during tumorigenesis and may contribute to aberrant activation of Wnt signaling in HCC.

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 173.
Abstract 1732: Elevated expression of YKL-40, a cancer biomarker, is associated with tumor angiogenesis
Authors: Yan Wei , Cao Jackie Qing , Arenas Richard B. , Bentley Brook , Shao Rong
Published: February 2011

Growing evidence from clinical investigations has shown that increased serum levels of YKL-40 are correlated with short survival and metastasis in a broad type of human cancers. These data suggest that YKL-40 in the blood may present a powerful tool for cancer diagnosis and prognosis. However, pathological activity of YKL-40 in cancer development is poorly understood. Here, we explored a functional role for YKL-40 in vasculature development of human cancer. In a survey of twenty cases of breast ductal carcinoma in situ (DCIS) and thirty-eight cases of infiltrating ductal carcinoma (IDC), we found that expression levels of YKL-40 in specimens from DCIS were barely detectable, but different levels of YKL-40 were present in IDC. Nine out of thirty-eight IDC cases showed high levels of YKL-40 and nine cases displayed medium levels of YKL-40, whereas the rest twenty cases were low or negative. Interestingly, in an analysis of vasculature formation in these IDC samples, we found that different blood vessel densities significantly correlated with different degrees of YKL-40 expression. To test this possible angiogenic activity of YKL-40 in tumor development, we utilized angiogenic xenograft models by subcutaneous injection of SCID/Beige mice with a brain tumor line U87 cells which express strong endogenous YKL-40 and U87 cells with siRNA knockdown of YKL-40. siRNA knockdown of YKL-40 notably suppressed tumor angiogenesis and tumor development relative to high levels of YKL-40 expressed in control U87 cells. Further, to dissect cellular and molecular mechanisms underlying YKL-40-induced tumor angiogenesis, we treated human microvascular endothelial cells with YKL-40 and found that YKL-40 was capable of binding to heparan sulfate on ectodomain of membrane receptor syndecan-1 (S1). The activated S1 prompted its interaction with adjacent receptor integrin αvβ3, leading to activation of intracellular cascades including phosphorylation of focal adhesion kinase and MAP kinase. A series of individual signaling inhibitors blocked YKL-40-induced angiogenesis in vitro. Collectively, these data strongly suggest that YKL-40 expressed in cancer activates vascular endothelial cell angiogenesis to promote tumor development. These findings may hold promise for developing novel therapeutic agents targeting YKL-40, a cancer biomarker that is overexpressed in a wide range of human cancers.

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 1732.
Abstract 1735: Expression of circadian genes correlate with liver metastasis and prognosis in colorectal cancer
Authors: Oshima Takashi , Kunisaki Chikara , Sato Tsutomu , Yamada Roppei , Fujii Shoich , Yamamoto Naoto , Shiozawa Manabu , Akaike Makoto , Rino Yasushi , Masuda Munetaka , Imada Toshio
Published: February 2011

The circadian rhythms are daily oscillations in various biological processes, and generated by the feedback loops of eight core circadian genes. These eight genes are, Clock, casein kinase I ε (CKI ε), cryptochrome1 (Cry1) and cryptochrome2 (Cry2), Period1 (Per1), Period2 (Per2) and Period3 (Per3), Bmal1. Recent studies have suggested that the circadian genes participate in the growth and development of various cancers. This study examined the relations of the circadian genes expression to clinicopathological factors and prognosis in patients with colorectal cancer. We studied surgical specimens of cancer tissue and adjacent normal mucosa obtained from 202 patients with untreated colorectal cancer. The relative expression levels of the circadian genes in the specimens were measured by quantitative real-time, reverse-transcription polymerase chain reaction. Expressions of Clock and CKI ε in cancer tissue were significantly higher than those in adjacent normal mucosa. Expressions of Per1 and Per3 in cancer tissue were significantly lower than those in adjacent normal mucosa. Analysis of the relations between clinicopathological features and expressions of the eight circadian genes showed that high expression of the Bmal1 and low expression of the Per1 correlated with liver metastasis. In the analysis of the relations between prognosis and expressions of the eight circadian genes, the prognosis of high expression of the Per2 was significantly better than that of low expression of the Per2. Our results suggest that over-expression of the Bmal1 and reduced expression of the Per1 might promote liver metastasis, and reduced expression of Per2 might shorten prognosis in colorectal cancer. Thus over-expression of the Bmal1 gene and reduced expression of the Per1 gene may be a useful predictor of liver metastasis. And reduced expression of Per2 may be useful as prognostic factor in patients with colorectal 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 1735.
Abstract 1737: Lipocalin-2/NGAL expression is associated with poor prognostic variables in human mammary, endometrial and ovarian carcinomas
Authors: Ross Jeffrey S. , Sheehan Christine E. , Kallakury Bhaskar V.S.
Published: February 2011

BACKGROUND: Recent literature has shown that lipocalin-2, a lipophilic ligand transporter member of the lipocalin family has a possible role as a biomarker and a modulator of human cancers. Elevated levels of lipocalin-2 have been demonstrated in several types of human cancer. However, its role as a prognostic biomarker has not been widely reported.

DESIGN: Formalin-fixed, paraffin-embedded tissue sections from 146 invasive mammary, 124 endometrial and 59 ovarian carcinomas were immunostained by automated method (Ventana Medical Systems Inc., Tucson, AZ) using rat monoclonal lipocalin-2/NGAL (R&D Systems, Minneapolis, MN). Cytoplasmic immunoreactivity was semiquantitatively scored based on staining intensity and distribution and the results were correlated with morphologic and prognostic variables.

RESULTS: Cytoplasmic lipocalin-2 overexpression was observed in 78/146 (53%) of invasive mammary carcinomas and correlated with PR positive status [63% PR positive versus 41% PR negative, p=0.012] and disease recurrence [69% recurrent versus 48% non-recurrent, p=0.021). On multivariate analysis, advanced stage, lipocalin-2 overexpression, ER negative status, metastatic disease at diagnosis and positive node status were independent predictors of disease recurrence; while advanced stage, metastatic disease at diagnosis and positive node status were independent predictors of shortened survival. In endometrial carcinomas, cytoplasmic lipocalin-2 overexpression was observed in 47/124 (38%) tumors, and correlated with tumor grade (52% grade 3 vs. 43% grade 2 vs. 21% grade 1, p=0.045) and with ER negative status [52% ER negative vs 28% ER positive, p=0.049]. There was no correlation with disease recurrence or overall survival. In invasive ovarian carcinomas, cytoplasmic lipocalin-2 overexpression correlated with tumor grade (0% grade 1 vs. 40% grade 2 vs. 67% grade 3, p=0.006) and advanced tumor stage (32% stage I vs. 65% stage III vs. 100% stage IV, p=0.018). Lipocalin-2 expression did not correlate with patient survival.

CONCLUSION: Lipocalin-2 overexpression correlates with varying markers of biologic aggressiveness in human mammary, endometrial and ovarian carcinomas warranting further study.

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 1737.
Abstract 1744: αB-crystallin as a novel marker of lymph node metastasis in breast cancer
Authors: Kim Haesung , Lee Younok , Kang Hee Joon , Kim Lee-Su
Published: February 2011

αB-crystallin, the member of small heat shock protein, is known as an anti-apoptotic protein and is associated with aggressive tumor behavior. In addition, recent study revealed that αB-crystallin was overexpressed in a metastatic variant of the GI101A human breast carcinoma cell line. The purpose of this study was to investigate whether expression of αB-crystallin was associated with lymph node metastasis. And also we evaluated the relationship of expression of αB-crystallin and other established prognostic factors. Eighty two patients who underwent breast cancer surgery and chemotherapy at Hallym Sacred Heart hospital were enrolled. Expression of αB-crystallin was determined by immunohistochemical staining using monoclonal antibody (SPA-222, Stressgen Biotech.) and graded as negative=0, weakly positive=1, moderately positive=2 and highly positive=3. Estrogen receptor, progesterone receptor, cERB-2, lymphatic tumor emboli, histologic grades and other tumor markers were compared with αB-crystallin expression. Expression of αB-crystallin in breast cancer tissues was associated with lymph node metastasis (p=0.01), lymphatic tumor emboli (p<0.01), high nuclear grade (p=0.02) and triple negative status (p=0.02). Five of 6 visceral metastasis cases showed highly expression of αB-crystallin. Results of this study revealed a strong relationship between αB-crystallin and lymph node metastasis of breast cancer. And also the expression of αB-crystallin significantly correlated with other breast tumor markers. We thought that αB-crystallin could be a novel marker of lymph node metastasis 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 1744.