bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3204: The mRNA decay factor tristetraprolin (TTP) induces senescence in human papillomavirus-transformed cervical cancer cells by targeting E6-AP ubiquitin ligase
Authors: Sanduja Sandhya , Kaza Vimala , Dixon Dan A.
Published: February 2011

Cervical cancer is the second most common cancer among women worldwide. A necessary factor in the development of nearly all cases of cervical cancer is the human papillomavirus (HPV) infection. High-risk HPV (HPV16 and 18) mediates cellular transformation and carcinogenesis through the concerted actions of viral oncoproteins E6 and E7 that disrupt the normal cell cycle regulation by targeting the p53 and Rb tumor suppressor pathways respectively. In the present study, we found that expression of the mRNA decay factor TTP is lost in HPV18-positive HeLa cells and introduction of TTP in cells lead to inhibition of cell proliferation and tumorigenesis by inducing cellular senescence. Consistent with this, TTP-expressing cells showed enhanced p53 protein stability and p53-dependent transcriptional activity. In cells expressing TTP, no changes were observed in the RNA levels of E6 and E7 indicating that these viral factors are not targets of TTP-mediated RNA decay. Whereas the E6-associated protein (E6-AP), the cellular ubiquitin ligase required for E6-mediated p53 degradation, was found to be down regulated both at mRNA and protein levels in presence of TTP. Furthermore, the association of E6 and E6-AP promotes transcriptional activation of hTERT, and TTP-dependent down regulation of E6-AP resulted in inhibition of hTERT expression and decreased cellular telomerase activity. Analysis of E6-AP mRNA revealed potential AU-rich element (ARE) binding sites of TTP present in the 3’ untranslated region (3′UTR) and RNA-binding studies demonstrate a physical interaction between TTP and E6-AP mRNA leading to rapid decay of E6-AP mRNA in a 3’UTR-dependent manner. Similar results were obtained with other high-risk HPV-positive cell lines that employ E6-AP pathway to control p53 and hTERT levels. Consistent with this, we found TTP expression to be lost or significantly reduced in squamous cell carcinomas as compared to normal cervical tissue. Finally, our studies indicate that loss of TTP expression in cervical cancer occurs through epigenetic mechanisms involving promoter methylation and histone deacetylation. These findings demonstrate the ability of TTP to act as a tumor suppressor through inhibition of the E6-AP pathway and indicate TTP loss to be a critical event during HPV-mediated 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 3204.
Abstract 3208: Semaphorin 3F suppresses telomerase activity
Authors: Drenberg Christina D. , Bermudez Yira , Nicosia Santo V. , Kruk Patricia A.
Published: February 2011

Objectives: Telomerase is a ribonucleoprotein that is active in more than 90% of human tumors, including ovarian cancers. We have previously demonstrated that vascular endothelial growth factor (VEGF) stimulates telomerase activity in ovarian cancer cell lines through cellular VEGF receptors and the ERK1/2 pathway targeting Sp1 sites within the hTERT promoter. Since semaphorin 3F (S3F), a proposed tumor suppressor, has been shown to antagonize VEGF, the purpose of this study was to determine whether S3F inhibits telomerase

Methods: Telomerase-positive ovarian cancer cell lines (OV2008, C-13, SW626) and telomerase-negative non-tumorigenic, SV-40 large-T antigen-transfected human ovarian surface epithelium (HOSE) cell lines were evaluated for endogenous RNA and protein expression of S3F, VEGF, and their shared receptors. Cultured cells were either treated with recombinant S3F protein or transiently transfected with cDNA to over-express S3F. Conversely, these cells were transiently transfected with siRNA targeting S3F ± specific receptor inhibitors. RT-PCR and western blot confirmed S3F over-expression, knock-down of S3F, or inhibition of receptors. Telomerase activity was measured by telomerase PCR-ELISA, while RT-PCR and western immunoblotting were utilized to detect S3F as well as to explore the signaling pathway involved in S3F-mediated telomerase regulation.

Results: Telomerase activity was suppressed in ovarian cancer cell lines following treatment with S3F recombinant protein or S3F over-expression by transient transfection. Furthermore, siRNAs targeting S3F and NP-2 or treatment with the VEGF receptor inhibitor, CBO-P11, abrogated S3F mediated telomerase inhibition. Over-expression of S3F resulted in up-regulation of phosphorylation of Erk1/2.

Conclusions: These data suggest that telomerase is a novel molecular target of S3F. Since, angiogenic and angiostatic factors play an important role in ovarian cancer tumorigenesis, S3F-mediated inhibition of telomerase represents a potential novel target for 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 3208.
Abstract 3209: The WRN RecQ helicase acts as a tumor suppressor in pancreatic adenocarcinoma
Authors: Chun Stephen G. , Yee Nelson S. , Qi Fang , Allsopp Richard , Davy Philip M. , Fong Keith S. , Carbone Michele , Bryant-Greenwood Peter K.
Published: February 2011

The vast majority of pancreatic adenocarcinoma is diagnosed in the geriatric population, and genes involved in cellular senescence such as the WRN-encoded RecQ helicase/exonuclease may be involved in pancreatic oncogenesis. Accumulating evidence suggests that WRN is a tumor suppressor gene (TSG), as a variety of cancers including pancreatic adenocarcinoma are associated with its epigenetic silencing or loss-of-function mutations. We hypothesize that wild-type WRN protects pancreatic epithelia from malignant transformation by regulating cell cycle progression and cell growth. First, we characterized a pancreatic adenocarcinoma cell line established from a human cadaver with Werner's syndrome (WS-Panc). WS-Panc had two loss-of-function mutations of WRN, formed adherent colonies in vitro, and expressed the pancreatic ductal epithelial marker cytokeratin 7. No oncogenic mutations of the epidermal growth factor receptor (EGFR), K-Ras nor B-Raf were detected by polymerase chain reaction (PCR), and immunologic assays revealed over-expression of lysyl oxidase-like 2, p53, and ß-catenin in WS-Panc. Fluorescent in situ hybrdization showed that WS-Panc over-expresses EGFR. Cytogenetic analyses showed normal karyotype. Strong telomerase activity was detected in WS-Panc by the Telomere Repeat Amplification Protocol. To test the hypothesis that WRN is a TSG, we forced ectopic expression of wild-type WRN in WS-Panc and are analyzing the biological properties and molecular features of transfected cells. Consistent with an anti-proliferative effect, wild-type WRN mRNA levels are uniformly repressed in a panel of sporadic pancreatic cancer cell lines from the American Type Culture Collection as shown by quantitative real-time PCR. In summary, we provide evidence that WRN is a TSG in pancreatic epithelia using WS-Panc and other pancreatic cancer cell lines. WS-Panc is the first cancer cell line carrying WRN loss-of-function mutations that represents a unique translational resource to elucidate WRN in the neoplastic state. Ongoing investigation of WRN in primary pancreatic cancer tissue and tumor models in vivo are expected to elucidate mechanistic links between aging and pancreatic cancer for the purpose of developing efficacious interventional strategies.

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 3209.
Abstract 321: Chromosomal and molecular genomic analysis of spontaneously transformed murine colon epithelial cells
Authors: McNeil* Nicole E. , Padilla-Nash* Hesed M. , Nguyen Quang Tri , Ried Thomas
Published: February 2011

Introduction: Colorectal cancer (CRC) represents one of the most common malignancies in the United States. The pathological progression from adenoma to carcinoma is accompanied by distinct and reproducible genetic alterations. Here we present a novel mouse model system for the sequential steps of tumorigenesis. This system will be helpful for functional validation of novel therapeutic targets in CRC.

Purpose: The aim of this study is to delineate the stage-specific genomic alterations and transcriptional deregulation that are associated with the spontaneous progression of normal murine colon epithelial cells to immortalized, transformed, and eventually tumorigenic cell lines.

Methods: Normal epithelial colon cells were selectively isolated from the large intestine from eight different isogenic five-six week old C57BL/6 mice. Primary colon cells were grown with reduced serum concentration while a fraction of these normal cells were cryopreserved for further analysis. The cells were sequentially recovered from culture based on distinct morphological changes: first, when the cells were in the pre-immortal stage as cells were actively growing; second, as the cells bypassed crisis, formed colonies and became immortal; third, at early transformation, as the cells first formed foci; fourth, mid-transformed (higher proliferation rates, later passages); and finally, as cells became less adherent, and exhibiting multiple foci. The cells were then analyzed using molecular cytogenetic methods (SKY and arrayCGH), and using global gene expression profiling.

Results: Using Spectral Karyotyping (SKY) we identified clonal chromosomal aberrations and recurrent chromosome imbalances, specifically reported here for late transformed cells and in tumor cells. Array CGH (aCGH) demonstrated a specific pattern of whole chromosome copy number gains (i.e. MMU10 and MMU15 which contain Mdm2 and Myc, respectively) and losses (ex. MMU4 which contains tumor suppressor gene p16/Ink4a/Cdkn2a) at the various stages of cellular transformation. Gene expression microarray identified the deregulation of specific genes that defined genomic signatures at each stage of transformation. Two of the eight late transformed cell lines gave rise to tumors in a nude mouse assay.

Conclusion: We present a novel mouse model of CRC that recapitulates the sequential steps of human colorectal tumorigenesis which affords us the possibility to experimentally test potential rational intervention strategies.

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 321.
Abstract 3213: GRHL2 enhances hTERT transcription by inhibiting promoter DNA methylation at 5’ CpG island
Authors: Chen Wei , Dong Qinghua , Shin Ki-Hyuk , Kim Reuben H , Oh JE , Park No-Hee , Kang Mo Kwan
Published: February 2011

We recently identified Grainyhead-like 2 (GRHL2), a mammalian homologue of Grainyhead in Drosophila, to be a novel transcription factor that binds to and regulates the activity of the human telomerase reverse transcriptase (hTERT) gene promoter. In the current study, we investigated the biological function of GRHL2 in normal human keratinocytes (NHK) and elucidated the molecular mechanism underlying GRHL2 regulation of hTERT gene expression. Primary NHK were infected with retroviral vectors expressing GRHL2 or the empty vector. GRHL2 transduction led to significant extension of the replicative lifespan of NHK by evading the senescence block. However, GRHL2 failed to cause immortalization of NHK, alone or in combination with Bmi-1, a polycomb group protein that bestows extended replication capacity to NHK. Enhanced proliferation of cells transduced with GRHL2 was also observed in three dimensional organotypic raft culture. Telomerase activity was detected in replicating NHK while it was rapidly lost during senescence. GRHL2 transduction increased the expression of hTERT and maintained detectable telomerase activity for extended time period, suggesting that GRHL2 positively influences the hTERT expression and telomerase activity in NHK. Upon knockdown of endogenous GRHL2, hTERT mRNA expression and telomerase activity were notably reduced. We previously showed that hTERT expression in NHK is regulated in part by promoter methylation at the 5’ CpG island. Thus, we determined the effects of GRHL2 on the methylation status of hTERT promoter by methylation-specific PCR (MSP). Loss of hTERT expression in the control NHK occurred with hTERT promoter hypermethylation. On the contrary, GRHL2 transduction notably reduced the extent of promoter methylation in NHK. This effect was not linked with enhanced cell proliferation because Bmi-1 transduction in NHK, which allowed prolonged cell proliferation, showed no effect on the hTERT promoter methylation. These data indicate that GRHL2 inhibits the promoter DNA methylation, thereby delaying hTERT gene inactivation and senescence of NHK. This study was supported by the grants R01DE18295, K02DE18959 from NIDCR/NIH.

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 3213.
Abstract 3217: Regulation of senescence-associated p-Erk1/2 (SA-p-Erk1/2) by PEA-15 and PKC-α in human diploid fibroblasts
Authors: Lee Yun Yeong , Kim Hong Seok , Choi Yong won , Lim In-Kyoung
Published: February 2011

Cellular senescence plays a key role in complicated biological processes, including development, aging, and tumorigenesis. Hallmarks of cellular senescence are metabolically active but not responsive to mitogens, therefore, decreased cell growth, cell cycle arrest, and reached to the flat and large cell shapes. We have recently reported that high expression of senescence-associated p-extracellular signal-regulated protein kinase 1/2 (SA-pErk1/2) can induce p21WAF1 expression via the activation of a transcription factor, SP1. In the present work, we investigated a biochemical regulation of the cytoplasmic sequestration of SA-pErk1/2 and found that PEA-15 (phosphoprotein enriched in astrocytes-15) could regulate translocation of SA-pErk1/2 to nuclei along with activation of PKC-α in senescent cells. It was proved by treatment of senescent cells with 12-O-tetradecanoylphorbol-13-acetate (TPA), which phosphorylated PEA-15 upon Ser104 residue via activation of PKC-α, which lead to release of SA-pErk1/2 form PEA-15 and then accompanied with nuclear translocation of SA-pErk1/2 along with PKC-α. Here, we, report the mechanism of cytoplasmic sequestration of SA-pErk1/2 and the relationship between PEA-15 and PKC-α in senescent human diploid fibroblasts (HDFs).

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 3217.
Abstract 3221: Analysis of the relationship between autophagy and senescence in response to Adriamycin in breast tumor cells
Authors: Di Xu , Henderson Scott C. , Gewirtz David A.
Published: February 2011

Autophagy is a conservative biological process, which involves the degradation of intracellular components (proteins or organelles) via the fusion of autophagosomes with lysosomes. Autophagy has been reported to have both cytoprotective and cytotoxic actions in response to either chemotherapy or radiation in different tumor model systems. In previous studies (Elmore LW et al., J Biol Chem. 2002 Sep 20; 277(38): 35509-15.), Adriamycin (doxorubicin), one of the primary chemotherapeutic drugs utilized in the treatment of breast cancer, was shown to induce a pronounced senescence response within 72hrs in MCF-7 breast tumor cells (at a clinically relevant concentration of 1μM, with a 2 hr exposure). Interestingly, Adriamycin was also observed to cause a time-dependent increase of autophagy within 72hrs (by acridine orange, Monodansylcadaverine) staining and electron microscopy). The current study was designed to evaluate the relationship between Adriamycin-induced autophagy and senescence in MCF-7 breast tumor cells. The antioxidants, GSH (glutathione) and NAC (N-acetyl cysteine), were previously shown to effectively block Adriamycin-induced senescence (Di X. et al., Biochem Pharmacol. 2009 Apr 1; 77(7):1139-50.). We now demonstrate that both GSH and NAC also effectively abolish Adriamycin-induced autophagy in MCF-7cells (as confirmed by the disappearance of punctuated red fluorescence staining (RFP-LC3) induced by Adriamycin. Unexpectedly, however, the autophagy inhibitors, 3-MA or wortmannin, failed to block Adriamycin-induced senescence (based on β-Gal staining). Another autophagy inhibitor, chloroquine, showed a significant blockade to β-Gal staining, although typical senescence morphology phenotypes, such as cell enlargement and flattening were still observed by phase contrast microscopy. Silencing of the autophagic protein, ATG5, failed to completely block Adriamycin-induced senescence, although the MCF-7/ATG5 knockdown cells showed decreased and delayed β-Gal staining. Taken together, our studies suggest that that autophagy proceeds and may contribute to the development of Adriamycin-induced senescence. The fact that blockade of senescence (by reactive oxygen scavengers) abrogates Adriamycin-induced autophagy further suggests that senescence and autophagy may be collaterally regulated in response to Adriamycin in MCF-7 cells. Both autophagy and senescence may be related to tumor dormancy (Gewirtz DA, Autophagy. 2009 Nov 24; 5(8).). Further studies are in progress to delineate the roles (cytoprotective or cytotoxic) of autophagy and the relationship of autophagy to senescence, tumor dormancy and sensitivity to chemotherapy and radiation.

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 3221.
Abstract 3223: Severe dietary restriction enhances cancer treatment: Protecting normal cells and sensitizing malignant cells to chemotherapy by fasting
Authors: Lee Changhan , Safdie Fernando , Raffaghello Lizzia , Wei Min , Longo Valter
Published: February 2011

Chemotherapy is a standard treatment modality for many cancers. However, its full clinical potential is greatly limited by toxic side-effects manifested as myelosuppression, gastrointestinal damage, fatigue, and in some case even death. Therefore, strategies or interventions that can selectively protect normal cells but not cancer cells will increase the therapeutic index of chemo drugs. We have previously reported that fasting can selectively protect tumor-bearing mice but not the cancer cells from chemo toxicity by down-regulating growth factors including IGF-I and its downstream pathways involving AKT/TOR and Ras. Here, using dietary and genetic interventions in yeast, mammalian cell culture, and mice, we show evidence that fasting not only provides selective protection to normal cells but may also sensitize certain cancer cells to chemotherapy or the severe dietary restriction itself.

Using mouse models of cancer, fasting for 48-hours was sufficient to drastically suppress tumor progression. In a mouse model of breast cancer, fasting alone caused more than a 50% decrease in tumor growth. Furthermore, when combined with chemotherapy, fasting sensitized cancer cells to chemo drugs and was able to further reduce tumor growth up to 90% compared to controls. In parallel, in vitro studies using these breast cancer cells revealed similar results where glucose restriction was sufficient to reduce proliferation and also sensitize the cells to 2 different types of the standard drugs for breast cancer treatment, doxorubicin and cyclophosphamide. Comparable results were obtained using mouse models of glioma, neuroblastoma and melanoma. Furthermore, in a metastatic model of melanoma, fasting/chemotherapy not only extended survival time, but also resulted in fewer organs affected by metastasis. Since chemotherapy is generally administered as a combination of drugs, we tested the effect of fasting on a cocktail of drugs in neuroblastoma-bearing mice and demonstrated significantly enhanced survival accompanied by reducing both drug toxicity and metastasis. In addition, we provide evidence that this protection is due to reduced DNA damage to normal cells. In fact, in our yeast studies, the deletion of the AKT/S6K homolog Sch9 or RAS provides enhanced protection against multiple types of chemotherapy-induced DNA mutations.

Considering the global increase in elderly population and that aging is the major risk factor for cancer, differential protection of normal cells concurrently with selective sensitization of malignant cells to chemotherapy may prove to be beneficial in cancer treatment.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3223.
Abstract 3224: Preclinical PKPD modeling and human dose projection of PF-04691502, a PI3K/mTOR dual inhibitor
Authors: Luu Kenneth T. , Zhang Eric , Bagrodia Shubha , Yuan Jing
Published: February 2011

PF-04691502 is a potent dual inhibitor of PI3K and mTOR, which are involved in regulating cell growth, proliferation and survival. The main objective of this work was to develop a preclinical pharmacokinetic-pharmacodynamic (PKPD) model for the compound and project the human efficacious dose. The PK of PF-04691502 in mouse obtained following oral dosing was described by a linear 2-compartment model which was used to drive two separate PD models: 1) an exponential growth model describing the tumor growth inhibition (TGI) and 2) an indirect-response model describing changes in intratumoral pAKT-S473/AKT in the U87MG xenograft mouse model. Based on the PK model, the half-life calculated from the linear elimination constant (k = 0.306 hr−1) was 2.3 hr and the plasma-compartment volume of distribution was 8.72 L/kg. The inter-compartmental distribution rate constants were 0.282 hr−1 and 0.212 hr−1 for k12 and k21, respectively. The model estimated in-vivo potency (IC50) for pAKT-S473/AKT suppression was 38.3 nM which was within the range of the IC50 (23 nM) for TGI (as parameterized by the model). The Imax estimated from the model was 0.975 (or 97.5%), which indicated nearly complete suppression of pAKT-S473/AKT production rate by PF-04691502. Based on the PKPD model and the scaled human PK, a clinical dose of 10 mg QD was determined to be adequate for maintaining the level of pAKT-S473/AKT below 50% of baseline. At steady-state PK this dose yielded a range of 50-80% human pAKT-S473/AKT suppression over the 24 hr dosing interval. In conclusion, PF-04691502 was determined to have a robust PKPD relationship in the preclinical animal model and was projected to have a clinically achievable efficacious dose of approximately 10 mg QD.

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 3224.
Abstract 3226: Targeting PI3K signaling in a xenograft model of nasopharyngeal carcinoma
Authors: Merritt David M. , Smith Pamela , Thorley-Lawson David A.
Published: February 2011

Nasopharyngeal carcinoma (NPC) is a cancer of the head and neck and is particularly common in Southern China and parts of the developing world. NPC may be a significant world health problem since it is likely to be underreported in parts of Southeast Asia and North Africa. The nonkeratinizing forms of nasopharyngeal carcinoma are divided into two subtypes based on histology (“differentiated” WHO type II and “undifferentiated” WHO type III.) Our analysis of gene expression data from an Affymetrix study of type II and type III NPC tumor biopsies using the Connectivity Map, a bioinformatics algorithm developed at the Broad Institute, suggests that the PI3K-Akt-mTOR pathway may be more highly activated in NPC type III. The array data may also reflect differential sensitivity to PI3K-Akt-mTOR pathway inhibitors and that the underlying genetic lesions that drive Akt activity in NPC type II and NPC type III are different. In addition, analysis using an algorithm that analyzes sets of functionally related genes, Gene Set Enrichment Analysis (GSEA), revealed that in NPC type III tumors, there is an increased expression of genes associated with epithelial-mesenchymal transition (EMT); a developmental program characterized by loss of cell adhesion, repression of E-cadherin expression and increased cell mobility. EMT has been reported to be a feature of several metastatic cancers. We performed biochemical assays in vitro on NPC cells that supported these findings, and then developed a xenograft tumor model that replicates invasive and metastatic NPC in immunodeficient mice. This model allows us to track tumor development by bioluminescence non-invasively over time. Blocking the PI3K-Akt-mTOR signaling pathway with Rapamycin, an mTOR inhibitor, had a significant effect on the growth of metastases.

Taken together, this suggests that the activation of the PI3K-Akt-mTOR pathway and EMT in NPC type III tumors may provide a molecular basis for the distinction between NPC type II and NPC type III, and that the type III phenotype is associated with metastatic progression in nasopharyngeal carcinoma. It may also provide prognostic and therapeutic implications for more efficacious treatment specifically targeting metastasis in NPC.

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 3226.
Abstract 3231: Chronic nicotine stimulates lung adenocarcinoma in vivo and in vitro via modulation of nicotinic acetylcholine receptor regulated excitatory and inhibitory neurotransmission characteristic of nicotine addiction
Authors: Al-Wadei Hussein A. , Al-Wadei Mohammed H. , Schuller Hildegard M.
Published: February 2011

Nicotine addiction is characterized by changes of nicotinic acetylcholine receptors (nAChRs) in the brain to increase the production of excitatory neurotransmitters noradrenaline/adrenaline while reducing inhibitory neurotransmitter γ-aminobutyric acid (GABA). We tested the hypothesis that nAChR-regulated neurotransmitters regulate lung adenocarcinoma (AC) and that nAChR changes in AC cells analogous to nicotine addiction in the brain contribute to the development and progression of this cancer.

Mice carrying xenografts of the human AC cell line NCI-H322 received no treatment or were given nicotine in the drinking water for 30 days with and without intraperitoneal injections of GABA. Xenograft sizes were measured. Noradrenaline and adrenaline in serum and cAMP in blood cells and tumor cells were measured by immunoassays. Protein expression of α7nAChR, α4β2nAChR, p-CREB, p-ERK1/2, glutamate decarboxylase 65 (GAD65) and GABA in xenografts were analyzed by Western blotting. Nicotine increased xenograft sizes and upregulated receptor protein of both nAChRs in xenografts while increasing noradrenaline, adrenaline and cAMP in the blood. Xenograft expression of GAD65 and GABA were suppressed by nicotine while p-CREB, p-ERK and cAMP were induced. GABA reversed all these effects of nicotine. In vitro exposure of AC cells for 7 days to nicotine upregulated and sensitized the α7nAChR, resulting in a significant increase in noradrenaline/adrenaline production in response to agonist and enhancement of its cAMP-dependent signaling responses. At the same time, the α4nAChR was desensitized by chronic nicotine causing a significant reduction in GABA production.

Nicotine-induced predominance of stimulatory noradrenaline and deficiency in inhibitory GABA in AC cells analogues to the nicotine addicted brain may therefore contribute to the development and progression of AC in smokers and GABA may be useful for the marker-guided prevention and adjuvant therapy of AC.

Supported by a grant from the National Lung Cancer Partnership and LUNGevity Foundation.

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 3231.
Abstract 3232: The proteome analysis of mice lung tumors
Authors: Yamano Shotaro , Kakehashi Anna , Tago Yoshiyuki , Tei Keikou , Wei Min , Wanibuchi Hideki
Published: February 2011

We have previously shown that lung squamous cell carcinoma (SCC) is induced by 20 weeks treatment with N-nitroso-tris-chloroethylurea (NTCU) The aim of the present study was to investigate the expression patterns of proteins in lung adenocarcinoma (AdCa) and SCC Seven mice were treated with single i p injection of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 2 mg/0 1 ml saline/mouse) on the first experimental day and sacrificed after 55 weeks (AdCa-induced model) Twenty six mice were treated topically with NTCU (0 01 M 75 μl/mouse) twice a week for eighteen weeks and sacrificed after 22 weeks (SCC-induced model) SCC and AdCa samples were collected from each group and proteome analysis was carried out by using QSTAR Elite LC-MS/MS 187 proteins were identified with 95% confidence or higher and quantified with ProteinPilot 2 0 Software Significant overexpression of 52 proteins (e g SP-C, TIM) were detected in lung AdCa compared to normal-appearing tissues and 46 proteins (e g cytokeratins 6A, 5, 19) were detected in SCC Expression patterns of proteins largely differed between AdCa and SCC Furthermore AdCa and SCC are suggested to be derived from type II pneumocytes and clara cell, respectively

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 3232.
Abstract 3234: AlgiMatrix™ 3-D cell culture system as an in vitro tumor model for H460 non-small cell lung cancer cell line
Authors: Desai Utkarsh S. , Chougule Mahavir B. , Sachdeva Mandip Singh
Published: February 2011

Objective: In cancer research, three dimensional (3D) cell culture models are rapidly gaining importance as they create a pragmatic microenvironment and mimic an in vivo system, which helps to understand cell-cell interactions. AlgiMatrix sponge (Invitrogen Corporation, Carlsbad, CA, USA) is a non toxic and biodegradable material made from alginate, which seems to construct a cell culture model that resembles normal cell characteristics and morphology. The main objective of the current study was to appraise the AlgiMatrix 3D cell culture system as a 3D invitro tumor model and to ascertain the anticancer activity of docetaxel. Methods: Cell titration was done to optimize seeding density (0.05, 0.10, 0.15, 0.25, 0.50, 0.75, 1.0, 1.25, 1.50, 3 & 6 million H460 cells/well using 6-well plate) and to determine the optimal incubation time for the formation of spheroids. On day 4, 9 & 13, the number and average size of the spheroids were determined using inverted phase contrast microscopy and imaging software. Further, optimized cell density (0.15 million cells/well) was used to determine the IC50 & IC90 values of docetaxel. The alamarBlue® assay was performed to determine the number of viable cells in control & treatment wells. Results: Optimized seeding density and the optimal incubation time was found to be 0.15 & 0.25 million H460 cells/well and 14 days respectively. Average size and the total number of the spheroids for 0.15 and 0.25 million H460 seeded cells was determined as shown in the table.     Time PointsAverage size of spheroids (µm)Total Number of spheroids 0.15 million0.25 million0.15 million0.25 million4th day177.88 ± 61.79220.69 ± 61.2748 ± 477 ± 89th day205.35 ± 78.03232.90 ± 82.8790 ± 8189 ± 1513th day232.27 ± 89.07263.94 ± 86.40153 ± 18228 ± 17

Total number of cells obtained by alamarBlue® assay on 14th day was 38.64 ± 4.94 & 42.92 ± 3.29 million cells/well for 0.15 & 0.25 million seeded cells respectively. The IC50 & IC90 concentration of the docetaxel was found to be 193.19 µM & 290.47 µM when ascertained with 0.15 million cells/well. Conclusion: These results strongly support that the AlgiMatrix 3D Cell Culture System may be used as an in-vitro tumor model and to evaluate cytotoxicity of anticancer drugs.

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 3234.
Abstract 3245: SRC-3: A prostate cancer oncogene with the same modus operandi in different niches
Authors: Tien Jean , Liu Zhaoliang , Liao Lan , Wang Fen , Xu Jianming
Published: February 2011

Prostatic adenocarcinoma will affect one in six American men and represents the second leading cause of cancer death in this population. Tumors that are locally invasive/ metastatic at diagnosis cause the majority of prostate cancer deaths. SRC3 is a p160 family coactivator overexpressed in invasive prostate cancers. A recent study in our laboratory demonstrated it is also highly expressed in tumors of the TRAMP mouse, a cancer model in which an androgen-responsive probasin promoter drives expression of the SV40 viral oncogene in several prostate cell types. Global deletion of SRC3 in TRAMP mice arrested primary prostate tumors at the well-differentiated stage and eliminated metastasis.

Transformation of prostatic luminal epithelial cells (LECs) into invasive cancers is a key feature of the TRAMP phenotype. Therefore, we hypothesized selective deletion of SRC3 in these cells would inhibit primary tumor growth and metastasis. To test the hypothesis, we crossed floxed SRC3/TRAMP mice with mice harboring a Cre recombinase gene driven by the LEC-specific probasin promoter. Efficacy of SRC3 knockout in SRC3c−/−/TRAMP mice was confirmed via immunohistochemistry and Western blotting.

We compared prostate tumor size, composition and metastases in SRC3c−/−/TRAMP vs. WT/TRAMP mice at 12 (n=3/5), 18 (n=7/7), 24 (n=9/9) and 30 (n=6/6) weeks of age. We observed no statistically significant differences in prostate or GU tract weight at any time point, though a trend toward decreased tumor size in the knockout was evident at 30 weeks. Tumor composition differed between SRC3c−/−/TRAMP and WT/TRAMP mice at 24 and 30 weeks. While synaptophysin(+) (neuroendocrine) cells were virtually absent in dedifferentiated regions of WT/TRAMP prostate, large synaptophysin(+) cell clusters were visible in the majority of sections taken from prostates of in SRC3c−/−/TRAMP mice. Neuroendocrine cell clusters were positive for SRC3 and negative for AR. Interestingly, 24 and 30 week-old SRC3c−/−/TRAMP mice also had tumors of the liver, lung and kidney: common sites of neuroendocrine metastasis in the TRAMP model.

Given that SRC3c−/−/TRAMP mice have primary tumors of equivalent size to WT/TRAMP but skewed toward neuroendocrine composition, SRC3 deletion in luminal epithelial cells appears to inhibit cancer growth in a cell autonomous fashion thus enabling neuroendocrine proliferation and metastasis. Since global SRC3−/−/TRAMP mice have no neuroendocrine tumors, our findings indicate SRC3 may also act as an oncogene in neuroendocrine 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 3245.
Abstract 3246: Important differences between exosome biosignatures from prostate cancer patient plasma samples and prostate cancer cell lines
Authors: Tinder Teresa , Pawlowski Traci , Spetzler David , Conrad Amber , Ellis Phil , Kennedy Patrick , Tyrell Annemarie , Jariwala Unnati , Kuslich Christine
Published: February 2011

Exosomes are endosome-derived vesicles secreted by many cell types, including tumor cells. The vesicles are formed intracellularly by invagination followed by fusion with the multivesicular body (MVB). The MVB ultimately merges with the plasma membrane, leading to exocytosis of the exosomes, which consequently have a similar membrane protein composition as their cell of origin; this provides the exosomes with unique biosignatures. Furthermore, these circulating exosomes participate in cellular communication by transporting mRNAs, microRNAs and proteins to target cells where they can elicit biological responses. This study compared the biosignatures identified from prostate cancer (PCa) cell line-derived exosomes to those found in exosomes isolated from the plasma of PCa patient samples. To develop an exosome-specific protein and RNA signature for PCa, we studied RNA and surface membrane protein profiles by flow cytometry (FACS) of exosomes derived from four prostate cancer cell lines, VCaP, 22Rv1, LNCaP and DU145. The exosome biosignatures identified from the cell lines were then measured in plasma exosomes isolated from PCa patients. We found that there was variability between the four cell line exosome-specific mRNA expression levels and exosome surface protein content. The mRNA exosome biosignatures identified in the four cell lines were not found in the exosomes from plasma samples of patients with PCa. Using a combination of antibodies for B7H3, PSMA and CD63 by FACS, we identified a protein signature from all prostate cancer cell line exosome populations that defined a specific subpopulation of exosomes containing all 3 proteins on their surface. Interestingly, this same exosome subpopulation was not found in exosomes derived from patients with PCa. Additionally, exosome-specific mRNA expression of two mRNA transcripts often found to be overexpressed in PCa, STEAP1 and SPINK1, were consistently identified in exosomes derived from prostate cancer cell lines VCaP and 22Rv1, but were only found in the plasma derived exosomes from one out of eight patients with PCa. Our results show that prostate cancer cell lines are not appropriate models for the development of exosome-specific disease profiles in prostate cancer; this will be investigated in additional cell lines. We find that patient samples are essential to identify disease-specific exosome biosignatures. These findings support the development of a versatile diagnostic platform based on exosome-specific biosignatures found in the plasma of patient samples for prostate and other 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 3246.
Abstract 3250: Bioluminescence imaging of prostate cancer progression and response to therapy in PTEN-deficient mice
Authors: Svensson Robert U. , Henry Michael D.
Published: February 2011

Prostate specific deletion of PTEN function in mice has been reported to recapitulate histological progression of human prostate cancers. Here we describe an improvement on this previously characterized model of prostate cancer by incorporation of a luciferase reporter allele. Specifically, the cancer initiating event is coupled to activation of firefly luciferase from the endogenous ROSA26 promoter in the same prostate epithelial cells. Additionally, we have extensively backcrossed our model onto the albino C57BL/6J background which enhances BLI sensitivity. Consistent with previous studies, we show that progression of prostate cancer from 3-12 weeks is rapid and is correlated with an increase in bioluminescence. However, longitudinal BLI from 12-40 weeks reveals a plateau in bioluminescence signal that is correlated with a halt in cellular proliferation as evidenced by Ki67 staining at necropsy. This may be related to activation of a cellular senescence pathway as reported previously in PTEN deficient mice. We further demonstrate the utility of this model to evaluate the effects of diet induced obesity on disease progression and to monitor response to therapy. Our model may offer advantages in sensitivity and specificity of cancer detection compared to other similar approaches.

This work is supported by NIH grant R21 CA137490

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 3250.
Abstract 3251: Zebrafish: Is it a viable model for prostate cancer
Authors: Jagadeeswaran Pudur , Hutson Angela , Radhakrishnan Uvaraj , Kim Seongcheol
Published: February 2011

Since its introduction as a vertebrate model organism for developmental studies, the Zebrafish model has continued to improve in its useful applications for studying human disease and more recently cancer. However, because there has been no evidence for the presence of prostate glands in fish, this model has not been exploited to study cancer of the prostate gland. In this abstract, we present evidence that fish carry prostate-like cells and, therefore, we postulate that zebrafish could be used as a model for prostate cancer research.

We hypothesized that even though a well-defined prostate gland is not present in fish, there may be a few cells similar to thyroid gland. As a first approach, we developed an experimental design for detection of Prostate Specific Antigen (PSA) gene and protein expression, as well as PSA gene mapping. PSA is a serine protease that binds in seminal plasma to protein C inhibitor, which is involved in the clotting cascade in humans. When we analyzed the zebrafish genome, we found a PSA gene that is syntenic to human PSA and at the protein level zebrafish PSA is 70% homologous to human PSA. We also found PSA mRNA by RT-PCR analysis of the total testes RNA. In situ hybridization of the 4 day old zebrafish larva revealed expression of PSA in gonad. Treatment of zebrafish larvae with the anti-androgen, hydroxyl-flutamide, showed a PSA response profile similar to that in mammalian models in which there is a reduction in PSA mRNA at low doses and an increase at high doses with a return to baseline levels. Based on these findings we contend zebrafish have cells which are functionally equivalent to the prostate gland. We further believe that this information supports the notion of using zebrafish as a model to generate prostate cancer -like symptoms and propose for future studies expression of SV40 large T antigen under the PSA promoter to be used to demonstrate proof of principle. Such a model would be useful to explore not only early detection of prostate cancer but also to provide the opportunity for large scale studies of chemical suppression of cancer progression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3251.
Abstract 3256: Investigating the intrinsic signaling differences in MET- and ERBB2-driven breast cancers
Authors: Graveel Carrie R. , DeGroot Jack D. , Dykema Karl , Sigler Robert E. , Furge Kyle A. , Vande Woude George F.
Published: February 2011

Understanding the signaling pathways that drive aggressive breast cancers is critical to the development of effective therapeutics. Over 20% of breast cancers overexpress ERBB2 and are associated with an aggressive tumor phenotype and poor outcome. Patients with ERBB2-positive tumors are often treated with the humanized monoclonal antibody trastuzumab, yet the majority of patients develop resistance. Recent studies show that the receptor tyrosine kinase MET is expressed in ERBB2 positive tumors and contributes to trastuzumab resistance in ERBB2-expressing cell lines. Other studies have shown that MET correlates with poor clinical outcome independent of ERBB2. We are utilizing mouse models to examine the intrinsic signaling differences between MET and ERBB2-mediated breast cancers.

We have developed a novel mouse model of mutationally activated MET (Metmut) that develops a high incidence of diverse mammary tumors with basal characteristics. Using this mouse model and human breast cancer tissue, we have examined the role of MET in aggressive breast cancers. By gene expression and tissue microarray analysis, we observe that high MET expression in human breast cancers significantly correlates with estrogen receptor negative/ERBB2 negative tumors and with basal breast cancers (Graveel et al., 2009). We performed gene expression analysis on mammary tumors from Metmut mice and activated ErbB2 (Neu-Ndl2-5) mice. Unsupervised hierarchical analysis revealed that solid Metmut tumors clustered separately from Metmut tumors with a mixed pathology and that solid Metmut tumors have expression patterns very similar to ERBB2 tumors. The similarity of solid Metmut tumors to ErbB2 tumors suggests that MET signaling may be influential in ERBB2-driven tumors. To investigate this hypothesis further, we performed a parametric gene set enrichment analysis (PGSEA) and observed that the Ras activation signature was significantly associated with the Metmut mixed tumors. Currently, we are also investigating expression patterns of MET and ERBB2 in human breast cancers by coimmunostaining. Collectively, our studies indicate that MET may play a critical role in the development of the most aggressive breast cancers and may be a rational therapeutic target for trastuzumab-resistant ERBB2 breast 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 3256.