bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4758: A new syngeneic MYCN-overexpressing neuroblastoma mouse model and MYCN-DNA vaccine
Authors: Stermann Alexander , Huebener Nicole , Seidel Diana , Shibina Anastasia , Fest Stefan , Gaedicke Gerhard , Lode Holger N.
Published: February 2011

Neuroblastoma (NB) is the most common solid extracranial tumor in childhood. High-level expression of MycN caused by amplification of the gene plays an important role in maintaining the malignant phenotype of NB and is the most significant marker for poor prognosis overall. Recent studies suggested that MYCN is a suitable target for immunotherapy, but up to now, a syngeneic NB mouse model over expressing MYCN and is not available to examine immunotherapeutic strategies in vivo.

Here, we report the development of a tetracycline inducible MYCN expressing murine NB cell line syngeneic to A/J mice and a new MYCN-DNA vaccine. The murine NB cell line C1300, syngeneic to A/J mice, was stably transfected with a tetracycline inducible MYCN expression vector system T-RExTM (Invitrogen). Stable transfection was verified by real-time PCR and Western-Blot, revealing significant high expression levels of MYCN RNA and protein, respectively. Characterization of the murine MYCN expressing cells in vitro and in vivo will be reported.

Furthermore, a new MYCN-DNA vaccine, based on epitopes encoding for three peptides from the murine MYCN protein sequence with high affinity to the A/J mouse MHC class I allele H2-Kk, was designed and tested in vivo for its ability to induce an antigen-specific immune response. Lymphocytes isolated from A/J mice vaccinated with the MYCN minigen vaccine effectively lysed wild type C1300 cells in cytotoxicity assays in contrast to lymphocytes from control mice. Additionally, lymphocytes from minigene vaccinated mice produced significantly higher amounts of IFN-γ after stimulation with irradiated C1300 cells, than lymphocytes from control mice, indicating the induction of cytotoxic T lymphocytes by the vaccine that actively kill even parental C1300 tumor cells with relatively low MYCN expression. We suspect that this effect will be enhanced when the newly established high MYCN-expressing C1300 cells are employed in a similar cytotoxicity assay. Results to support this hypothesis will be reported at the meeting.

In summary, we report the development of a new MYCN-DNA vaccine and a murine tetracycline inducible MYCN NB cell line providing an important new syngeneic mouse model for in vivo evaluation of new immunotherapeutic strategies.

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 4758.
Abstract 4760: A novel immune potentiating strategy for cancer vaccine
Authors: Qin Hong , Cha Soung-chul , Qin Xiao-feng , Liu Yong-Jun , Kwak Larry W.
Published: February 2011

A major obstacle for translational research of cancer vaccine is the weak immunogenicity of tumor antigens. Thus, the development of new, more potent strategy that aims at breaking such an immune tolerance is a prerequisite of vaccine therapy. Here we show that the unexpected effect of inducing sterile inflammation at vaccination sites by myotoxins potentiated induction of tumor-specific immunity by genetic vaccines and converted a non-immunogenic lymphoma idiotype antigen into a tumor rejection vaccine. This potent immunostimulatory effect was immune-mediated, requiring recruitment of dendritic cells (DC). Sterile inflammation induced by myotoxins was associated with upregulation of chemokines, proinflammatory cytokines, Toll-like receptors and their endogenous ligands, and activation of innate immunity. Mechanistic experiments in vivo also elucidated the requirement for genes triggering DC maturation including TLR signaling and CD40. These studies suggest that inducing sterile inflammation at vaccination sites generates a favorable microenvironment that promotes adaptive immunity. This novel mechanism of immune potentiation may be exploited for development of adjuvants for vaccines against cancer.

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 4760.
Abstract 4777: Human suppressor of cytokine signaling 1 controls immunostimulatory activity of monocyte-derived dendritic cells
Authors: Hong Bangxing , Ren Wenhong , Song Xiao-Tong , Evel-Kabler Kevin , Chen Si-Yi , Huang Xue F.
Published: February 2011

Dendritic cell (DC)-based tumor vaccines have only achieved limited clinical efficacy, underscoring the limitation of stimulatory strategies to elicit effective cytotoxic T lymphocyte (CTL) responses against self-tumor-associated antigens. Here, we investigate the role of human suppressor of cytokine signaling 1 (SOCS1), a feedback inhibitor of the Janus-activated kinase/signal transducer and activator of transcription signaling pathway, in regulating antigen presentation by human DCs (hDC). We find that human SOCS1 (hSOCS1)-silenced DCs have an enhanced stimulatory ability to prime self-antigen-specific CTLs in vitro and in a severe combined immunodeficient-hu mouse model. Human CTLs activated by SOCS1-silenced DCs, but not wild-type DCs, have an active lytic activity to natural antigen-expressing tumor cells. We further find that the capacity of hDCs to prime CTLs is likely controlled by SOCS1-restricted production and signaling of proinflammatory cytokines, such as interleukin-12. These results indicate a critical role of hSOCS1 in negatively regulating the immunostimulatory capacity of DCs and imply a translational potential of this alternative SOCS1 silencing strategy to develop effective DC vaccines.

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 4777.
Abstract 4778: Epigenetic modulation of colorectal cancer cells for cancer-testis antigen-targeted immunotherapy
Authors: Chou Jeffrey , Voong Lilien N. , Chen Xiaoji , Yee Cassian , Warren Edus H.
Published: February 2011

Cancer-testis (C-T) antigens represent attractive potential targets for cancer immunotherapy due to their limited expression in normal somatic tissues and their widespread expression in malignant cells of diverse histologies. The utility of C-T antigens for immunotherapy of colorectal cancer (CRC), however, is limited because expression of C-T genes is repressed in most CRC cells by hypermethylation. We investigated whether treatment of CRC lines with the hypomethylating agent 5-aza-2′-deoxycytidine (DAC) could induce C-T gene expression in CRC cells and thereby sensitize them to recognition by CD8+ cytotoxic T lymphocytes (CTL) specific for the highly immunogenic C-T antigen NY-ESO-1.

A panel of CRC and primary dermal fibroblast lines was cultured with DAC or medium alone for 48 hours followed by culture with exposure to interferon γ (IFNγ) or medium alone for an additional 48 hours. After the treatment period, cells were harvested for collection of RNA, DNA, and protein; studied with flow cytometry for analysis of MHC class I and II expression; and tested for recognition by HLA-A*0201-restricted CTL specific for NY-ESO-1157–165 in a 51Cr release-cytotoxicity assay.

Quantitative RT-PCR analysis of the expression of 20 C-T genes in untreated and DAC-treated cells using a custom PCR array showed that the majority of C-T genes were expressed at low levels in untreated CRC cells but were induced in most CRC cell lines by DAC exposure. Fibroblasts did not show any significant C-T gene expression with or without DAC treatment. Western blot analysis for NY-ESO-1 expression confirmed that DAC induction of NY-ESO-1 transcription was also associated with increased protein expression. DAC-treated HLA-A*0201+ CRC lines were also recognized by CD8+ HLA-A*0201-restricted NY-ESO-1-specific CTL. CTL recognition was enhanced when the cells were treated with IFNγ in combination with DAC, with the most significant effect seen in CRC cells with low baseline expression of MHC class I molecules.

These data suggest that epigenetic modulation with DAC or related agents could be used to selectively induce expression of C-T antigens in CRC cells and thereby make them susceptible to C-T antigen-directed immunotherapy.

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 4778.
Abstract 4781: EGFR peptide-specific T cells in head and neck squamous cell carcinoma
Authors: Schuler Patrick J. , Boekers Petra , Engers Rainer , Arsov Christian , Greve Jens , Lang Stephan , Bier Henning , Whiteside Theresa L. , Hoffmann Thomas
Published: February 2011

In head and neck squamous cell carcinoma (HNSCC) expression of epidermal growth factor receptor (EGFR) correlates with poor prognosis and decreased survival rates. The cellular immune response to EGFR expression in vivo remains unclear.

The frequency of T-cells specific for HLA-A2.1 restricted to EGFR derived YLN peptide (YLNTVQPTCV) and KLF peptide (KLFGTSGQKT) was determined by corresponding tetramer/pentamer analysis in 18 HLA-A2.1+ HNSCC patients and 18 healthy HLA-A2.1+ individuals (NC). Patients’ results were correlated to EGFR expression obtained by immunohistochemistry in corresponding tumor sections. Proliferation and anti-tumor activity of peptide-specific T-cells was demonstrated by in vitro stimulation (IVS) with dendritic cells (DC) pulsed with the HLA-A2.1-restricted peptides.

For both EGFR peptides the average frequency of specific CD8+ T-cells (0.017% and 0.014%) was not significantly increased in the circulation of HNSCC patients compared to NC (0.019% and 0.014%). A large variability in the frequency of peptide-specific CD8+ T-cells was found in HNSCC patients (0.002 - 0.076%) and NC (0.002-0.134%). All (18/18) tumors showed EGFR expression. IVS with peptide-pulsed DC resulted in the expansion of EGFR-specific CD8+ T-cells, which were reactive against the EGFR+ cell line UD-SCC 8 in IFN-γ ELISPOT. T-cell expansion was significantly greater for the KLF-peptide than the YLN-peptide.

While EGFR peptide-specific T-cells were present in the circulation of HNSCC patients, their mean frequency was not increased to that in NC. Patients showed variable frequencies of peptide-specific T-cells. These T-cells were responsive in vitro to stimulation with the relevant EGFR peptides (YLN and KLF) and recognized EGFR+ tumor cells. Strategies for expansion of EGFR peptide-specific CTL may be important for future immunotherapy of HNSCC patients.

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 4781.
Abstract 4783: Identification of a novel TAA, SPARC, as a target for immunotherapy of various cancers
Authors: Inoue Mitsuhiro , Hirata Shinya , Senju Satoru , Ikuta Yoshiaki , Hayashida Yuki , Tsunoda Takuya , Furukawa Yoichi , Nakamura Yusuke , Nishimura Yasuharu , Baba HIdeo
Published: February 2011

It is important to identify Tumor-associated antigens (TAAs) to direct the immune system to attack cancer in order to establish efficient anti-cancer-immunotherapy. We analyzed the gene expression profiles of gastric cancers by using the genome wide cDNA microarray consisting of probes for 20,340 genes, and found that SPARC (Secreted Protein Acidic and Rich in Cysteine) gene was overexpressed in diffuse-type gastric cancer tissues at higher levels in comparison to adjacent normal gastric tissues. The levels of expression of SPARC in pancreatic cancer and colorectal cancer tissues were also higher than those of normal counterparts. On the other hand, the expression of SPARC among the normal tissue specimens was detected only in limited tissue specimens at very low levels. Therefore, we evaluated the possibility of SPARC as a target antigen for anticancer immunotherapy. This study attempted to identify HLA-A24 (A*2402)-restricted and SPARC derived CTL epitopes.

To identify the SPARC-derived and HLA-A24-restricted CTL epitopes, we selected a total of 4 different candidate 9- or 10- amino acid peptides that were expected to have a higher binding affinity to HLA-A24 (A*2402) by the HLA Peptide Binding Predictions in the BIMAS software program. The H-2 Kd-binding peptide motif is comparable to that of HLA-A24 binding peptide. Thus, we examined CTL response of BALB/c mice to these SPARC-derived epitopes. We found that two SPARC derived peptides elicited high magnitude of CTL response in BALB/c mice in an H-2Kd-restricted manner. Therefore, we attempted to generate SPARC-specific CTLs from the PBMCs of healthy donors and various cancer patients positive for HLA-A24 by stimulation of PBMCs with these two peptides. SPARC peptides-reactive CTLs were successfully induced from PBMCs by in vitro stimulation with these two peptides in HLA-A24 (A*2402) positive healthy donors and cancer patients. Moreover the SPARC-reactive CTLs established from PBMCs derived from cancer patients by stimulation with these two peptides, exhibited cytotoxicity to the cancer cells expressing both HLA-A24 and endogenous or transgene-derived SPARC. On the other hand, the CTLs did not kill the cells which were negative for either HLA-A24 or SPARC.

Furthermore, the adoptive transfer of the SPARC-specific CTLs could inhibit the tumor growth in NOD/SCID mice bearing human cancer cells expressing both HLA-A24 (A*2402) and SPARC. These findings suggest that SPARC is a potentially useful target candidate for cancer immunotherapy.

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 4783.
Abstract 4788: Modulation of T lymphocytes by tumor-released survivin
Authors: Slater Jessica M. , Khan Salma , Wall Nathan R.
Published: February 2011

Introduction: The role of tumor infiltrating lymphocytes (TILs) in the progression of cancer has become an important area of research. Recent studies have shown that T lymphocytes within the tumor microenvironment have decreased activation and proliferation. Our lab has shown survivin is released from tumor cells within exosomes, and is capable of altering the phenotype of malignant cells. We hypothesize that this exosomally-released survivin prevent TIL activation and proliferation in T cell subsets and that Th2 type cytokines will be released in higher levels than Th1 cytokines. Methods: HeLa-S-WT cells that produce Flag-HA tagged survivin were growth in a bioreactor and survivin isolated from the media using Flag-HA tandem affinity purification. T lymphocytes from normal donors were cultured in control or survivin-containing media and activated with anti-CD28-anti-CD3 microbeads (32 ul/ml). Flow cytometric analysis of activation marker CD69 and the proliferation marker Ki-67 were performed at 24h and 6 days, respectively. Cytokine release was evaluated using a Cytokine Bead Array (BD Bioscience) for IFN-gamma, IL-2, IL-4 and IL-10. Results: CD69 expression was decreased significantly in CD8+ T cells after incubation with survivin (Control = 72 MFU, Treated = 57 MFU; p<0.05) while no significant change was seen in CD4+ T cells (Control = 65 MFU, Treated = 68 MFU). CD8+ T cells showed a significant reduction in Ki-67 expression after incubation with survivin when compared to controls (Control = 218 MFU, Treated = 139 MFU; p < 0.001), while CD4+ T cells showed little difference between control and treated groups (Control = 74 MFU, Treated = 81 MFU). Cells treated with survivin in activating conditions released higher amounts of IL-4 (Control = 4.8 pg/ml, Treated = 28.3 pg/ml) and decreased amounts of IFN-gamma (Control = 32.4 pg/ml, Treated = 16.9 pg/ml) compared to control activating conditions. IL-2 and IL-10 showed little difference between treatment groups. Conclusions: This data suggests that survivin in the tumor microenvironment is capable of negatively affecting cytotoxic T cell activation and proliferation. While survivin treatment does not affect the activation or proliferation of CD4+ subsets, the Th1: Th2 cytokine balance is altered when survivin is present. Thus, survivin in the tumor microenvironment could play a role in the suppression of immune surveillance by inhibiting the cytotoxic T cell response and preventing tumor death by increasing levels of Th2 cytokines and decreasing levels of Th1 cytokines.

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 4788.
Abstract 479: p53 mutations and Ink4a/Arf deletion cooperate to induce metastatic skin carcinomas
Authors: Li Zhongyou , Mejia Olga , Caulin Carlos
Published: February 2011

Mutations in the p53 tumor suppressor gene are found in about half of the squamous cell carcinomas (SCC) that arise in the skin and oral cavity. Most of these mutations are missense mutations that result in the expression of altered forms of p53, some of which acquire oncogenic properties. We previously showed that the p53 gain-of-function mutation p53R172H cooperates with Ras activation during skin cancer initiation, progression to carcinoma and metastasis. However, it is unclear whether p53 gain- and loss-of-function mutations predispose differently to SCC formation when activated in the absence of additional oncogenic mutations. To address this question we generated mice in which deletion of p53 (loss-of-function) or activation of the p53R172H mutation (gain-of-function) were induced in mouse stratified epithelia. We found that over 70% of the mice developed skin or oral SCCs, with similar kinetics for mice with p53 gain- or loss-of-function mutations, suggesting that in the absence of Ras mutations the oncogenic role of p53R172H in SCC initiation is minimized. To identify mechanisms leading to SCC development in these mice we analyzed the status of pathways that are frequently involved in human SCCs. We observed that Erk1/2 activation was blocked in most of the SCCs. Consistently, oncogenic mutations in Ras genes were only found in <5% of the SCC analyzed. Notably, we found overexpression of cyclin D1 and activation of Akt in over 50% of the SCCs, suggesting an important contribution for these oncogenic alterations in SCC development in these mice. Importantly, p16INK4a and p19ARF were overexpressed in most of the SCCs, suggesting that in the absence of p53, alternative tumor suppressor mechanisms are activated to prevent tumor progression. To determine whether p53 mutations and p16/p19 cooperate in SCC formation we generated mice in which p53 mutations and deletion of the Ink4a/Arf locus were induced in the epidermis. These mice developed skin carcinomas significantly faster than mice with mutations in p53 only. In addition, in the presence of the Ink4a/Arf deletion, the p53R172H mutation induced accelerated SCC formation compared to deletion of p53, again suggesting that the oncogenic properties of mutant p53 are exposed in the presence of additional genetic alterations. The tumors that developed in these mice were classified as spindle cell carcinomas (SPCC) and generated metastasis in over 60% of the mice. Ongoing experiments are underway to identify molecular mechanisms involved in the generation of SPCCs and metastasis in this model.

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 479.
Abstract 4791: Inhibition of Tumor Formation in Mice following Vaccination with DNA Encoding a Novel Xenogeneic Tumor Associated Auto-Antigen
Authors: Bright Robert K. , Bright Jennifer D. , Schultz Heather N.
Published: February 2011

Tumor protein D52 (TPD52) is a novel auto-antigen involved in cellular transformation, proliferation and metastasis. Its over-expression has been demonstrated in several human malignancies including prostate, breast, and ovarian carcinomas. Murine TPD52 (mD52) has been shown to induce anchorage independent growth in vitro and spontaneous metastasis in vivo. Importantly, mD52 mirrors the function and normal tissue expression patterns of the human orthologue of TPD52 (hD52) and shares 86% identity at the amino acid level. Thus, TPD52 represents a self, non-mutated tumor associated auto-antigen that is important for maintaining a tumorigenic phenotype. The Transgenic Adeno-carcinoma of the Mouse Prostate (TRAMP) model was employed to study TPD52 as a vaccine antigen. Naïve mice were immunized with either mD52 or hD52 DNA. Following immunization, mice were challenged with a subcutaneous, tumorigenic dose of mD52 positive, autochthonous TRAMP-C1 tumor cells. Greater than 70% of mice were tumor free more than 10 months post tumor challenge when immunized with hD52 as a xenogenic DNA vaccine. Survivors of the initial tumor challenge rejected a second tumor challenge with TRAMP-C2 tumor cells given in the opposite flank approximately 138 days after the first challenge with TRAMP-C1, and remained tumor free for more than 6 months. The T cell cytokine secretion patterns from tumor challenge survivors indicated that a cellular immune response was involved in tumor rejection. These data suggest that hD52 xenogeneic DNA vaccination induced a memory, cellular immune response that resulted in superior protection from challenge with murine prostate-derived tumors that naturally over-express mD52 protein.

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 4791.
Abstract 4799: Epigenetic antagonism between Polycomb and SWI/SNF complexes during oncogenic transformation
Authors: Wilson Boris G. , Shen Xiaohua , McKenna Elizabeth S. , Wang Xi , Cho Yoon-Jae , Koellhoffer Edward C. , Nguyen Phuong T.L. , Pomeroy Scott L. , Orkin Stuart H. , Roberts Charles W.M.
Published: February 2011

Epigenetic alterations serve critical roles in cell fate decisions and have increasingly been implicated in cancer formation. The Polycomb Group (PcG) protein EZH2 mediates epigenetic gene silencing, displays pro-oncogenic properties in cell lines and is frequently overexpressed in cancer. Conversely, loss of function mutations in the tumor suppressor gene SNF5, a core subunit of the SWI/SNF chromatin remodeling complex, occur in several types of aggressive human cancers. Intriguingly, analysis of homeotic mutants in Drosophila suggests that PcG and SWI/SNF can serve antagonistic roles in the epigenetic control of gene expression. Here we have investigated functional relationships between these epigenetic regulators in oncogenic transformation by examining the role of EZH2 in tumors driven by SNF5 loss. Notably, SNF5-mutant tumors, despite being highly aggressive, are diploid and genomically stable, making them an ideal model with which to elucidate epigenetic drivers of oncogenesis. We show that targeted inactivation of Snf5 leads to increased expression of Ezh2 and that EZH2 is upregulated in SNF5- deficient cancers. A key mechanism by which SNF5 loss promotes oncogenesis is via downregulation of the p16INK4a tumor suppressor. We show that inactivation of Ezh2 restores normal levels of p16INK4a to Snf5-deficient fibroblasts. We further show that reducing the levels of EZH2 in a human SNF5-deficient tumor cell line leads to increased levels of p16INK4a accompanied by slowed proliferation and cellular senescence. Most importantly, using conditional mouse models, we show that inactivation of EZH2 completely blocks tumor formation driven by SNF5 loss. Our results demonstrate an essential role for Ezh2 in oncogenic transformation in vivo and show that perturbing the epigenetic balance of gene expression mediated by EZH2 silencing and SWI/SNF activation can have a profound effect on tumor formation. Further, we establish that targeted inhibition of EZH2 in vivo can have potent anti-cancer 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 4799.
Abstract 4803: A novel mitochondrial isoform of mammalian DNA methyltransferase 1
Authors: Shock Lisa M. , Thakkar Prashant V. , Peterson Erica J. , Moran Richard G. , Taylor Shirley M.
Published: February 2011

Cancer cells, in contrast to normal cells and tissues, fulfill their energy requirements by increased glycolysis, with a distinct downregulation of oxidative phosphorylation in the mitochondria. Regulation of enzymes of the glycolytic pathway under the anoxic conditions prevalent in human solid tumors by stabilization of HIF1α have been intensely investigated; however, the mechanisms regulating transcription of the mitochondrial genome, and its coordinate regulation with the nuclear genome are less well understood. Eukaryotic mitochondrial DNA carries a low but significant level of cytosine methylation, but molecular mechanisms responsible for the generation of this modification and its role in mitochondrial function has not been studied. The mitochondrial genome encodes 13 polypeptides involved in oxidative phosphorylation, as well as the tRNAs and rRNAs required for mitochondrial translation. All other proteins required for mitochondrial function are encoded by the nuclear genome and translocated to the mitochondria using mitochondrial targeting sequences frequently located at the N-terminus. We have identified a novel isoform of mammalian DNA methyltransferase 1 (DNMT1) that translocates to the mitochondrion, using a conserved N-terminal mitochondrial targeting sequence; the mRNA encoding this isoform is expressed from an upstream transcription start site. De novo methyltransferases DNMT3a and DNMT3b are not found in mammalian mitochondria. Expression of the mitochondrial DNMT1 isoform (mtDNMT1) is regulated by nuclear respiratory factor 1 (NRF1) and coactivator PGC1α, which together upregulate several nuclear-encoded mitochondrial genes involved in the electron transport chain, following hypoxic stress. The mitochondrial isoform (mtDNMT1) is also preferentially upregulated following loss of p53 function, leading to specific effects on mitochondrial gene expression and potentially affecting mitochondrial function. We therefore suggest that mitochondrial DNA methylation, established and maintained by mtDNMT1, plays a role in coordination of mitochondrial gene expression and that the expression of the mtDNMT1 isoform is under the control of p53.

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 4803.
Abstract 4805: A quantitative high-throughput screen identifies novel potential epigenetic modulators of gene transcription
Authors: Wichterman Jennifer L. , Huang Ruili , Huang Wenwei , Austin Christopher , Inglese James , Martinez Elizabeth , Johnson Ronald
Published: February 2011

Epigenetic regulation of gene expression is important in many biological processes including tumor formation. While some key modulators of epigenetic control like histone deacetylases and DNA methyltransferases are the subject of intensive research, little else is known of other targets or modifications that regulate this process. We used an image-based cell assay that measures induction of a silenced GFP-estrogen receptor (ER) reporter to identify novel classes of compounds involved in epigenetic regulation. This Locus Derepression (LDR) assay was screened against 282,000 compounds using quantitative high-throughput screening, a method that assays compounds at multiple concentrations. Structure-activity relationships of the 680 actives recovered from the screen indicated 127 clusters and 110 singletons. Over 200 compounds were retested in the LDR cells and were counter-screened against the parental line to identify fluorescent artifacts. Of the 72 compounds that retested as active and nonfluorescent, 57 were tested as independent samples to confirm activity. Confirmation studies indicated 23 compounds showed consistent activity in LDR cells and were not fluorescent. Confirmed actives are being characterized further to identify the targets and pathways these molecules modulate. These compounds represent important tool compounds for understanding the molecular mechanisms of epigenetic control of transcription.

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 4805.
Abstract 4811: Compression-induced cell distension stimulates coordinated migration of mammary carcinoma cells
Authors: Tse Janet M. , Cheng Gang , Tyrrell James A. , Wilcox-Adelman Sarah A. , Jain Rakesh K. , Munn Lance L.
Published: February 2011

Uncontrolled cell proliferation of a solid tumor in a confined space not only creates oxidative stress (hypoxia), but also generates mechanical compressive stress, which can influence the tumor cells and modify their interactions with neighboring cells and the extracellular matrix. Little is known about how such compressive stress affects cancer progression. Here we show, for the first time, that externally-applied compressive stress results in faster migration of mammary carcinoma cells. Compression induces migration of mammary carcinoma cells in a coordinated sheet, initiated by “leader cells” - single cells at the leading edge of the sheet, extending long filopodia. The formation of leader cells is dependent on the geometry of association with neighboring cells, but the frequency of leader-cell formation - and sheet migration speed - is increased by applying compression perpendicular to the sheet. Accompanied by redistribution of fibronectin deposition, applied compression enhances cell-matrix adhesion and stabilizes cell distension independent of actomyosin contractile machinery. Our results suggest that mechanical stress accumulated during tumor growth is sufficient to enhance cell-substrate adhesion and stimulate cancer cell migration. This mechanism opens the door to a new class of targets for blocking mechanical stress pathways.

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 4811.
Abstract 4812: Collagen regulation of Snail expression in pancreatic cancer involves TGF-β1 signaling
Authors: Shields Mario A. , Dangi-Garimella Surabhi , Krantz Seth , Bentrem David J. , Munshi Hidayatullah G.
Published: February 2011

Pancreatic cancer, the fourth leading cause of cancer death in the U.S., is associated with pronounced desmoplastic reaction; however, the role of this collagen-rich fibrotic reaction in pancreatic cancer progression is poorly understood. Previously, we had shown that the collagen increased expression of the key proteinase membrane type 1-matrix metalloproteinase (MT1-MMP). Now we show that collagen can also increase expression of the zinc-finger transcription factor Snail to enhance pancreatic cancer invasion. Relative to cells grown on tissue culture plastic, PDAC cells grown in 3D collagen gels induced Snail. As we had previously published that TGF-β1 increases Snail levels, we examined the contribution of TGF-β1 signaling to collagen-induced Snail expression. Inhibiting TGF-β1 signaling with siRNA against TGF-β1 receptor type I (TbRI) or with a highly specific inhibitor of TbRI kinase activity attenuated the effect of collagen on Snail levels. To further examine the role of Snail in PDAC progression, we generated Snail-inducible PDAC cells lines. Expression of Snail in PDAC cells grown in 3D collagen gels enhanced invasion, which was blocked in the presence of the MMP inhibitor GM6001. Overexpression of Snail in PDAC cells increased MT1-MMP, while siRNA against Snail decreased MT1-MMP and also attenuated collagen-induced MT1-MMP expression. Snail expression of MT1-MMP requires ERK1/2 signaling and blocking ERK1/2 phosphorylation inhibited Snail-induced invasion in 3D collagen gels. To provide in vivo support for our findings that Snail can regulate MT1-MMP, we examined the expression of Snail and MT1-MMP in human PDAC tumors and found a statistically significant positive correlation between MT1-MMP and Snail in these tumors. Overall, our data demonstrate that collagen-rich milieu can increase the expression of Snail, and suggests that the desmoplastic reaction actively contributes to PDAC 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 4812.
Abstract 4824: Little evidence of human papillomavirus in lung tumor tissue
Authors: Koshiol Jill , Rotunno Melissa , Gillison Maura , van Doorn Leen-Jan , Tarantini Letizia , Song Hebin , Quint Wim , Struijk Linda , Goldstein Alisa , Chaturvedi Anil , Hildesheim Allan , Taylor Philip , Wacholder Sholom , Bertazzi Pier Alberto , Landi Maria Teresa , Caporaso Neil
Published: February 2011

Background: Lung cancer kills over one million people worldwide each year. Human papillomavirus (HPV) has been proposed as a potential cause. Over 50 studies have evaluated HPV in lung tumor tissue over the last 20 years with mixed findings, but most were severely underpowered or had other methodological limitations. While several HPV-associated cancers have been identified (e.g., cervical, tonsillar), the role of HPV in lung carcinogenesis remains controversial.

Methods: We tested 450 lung cancer cases in the Environment And Genetics in Lung cancer Etiology (EAGLE) Italian population-based, case control study for HPV DNA. DNA was extracted from ethanol or formalin-fixed, paraffin-embedded tumor tissue under strict PCR clean conditions and was evaluated for HPV16 and 18 by type-specific, real-time PCR targeted to the E6 or E7 gene. The number of cell equivalents evaluable in these reactions was estimated by real-time PCR targeted to the human ERV3 gene. DNA purity was assessed by standard spectrophotometry (260/280 ratio).

Results: 79.6% (n=358) of the 450 cases were male, 48.9% (n=220) were current smokers, 44.0% (n=198) were former smokers, 6.7% (n=30) were never smokers, and 0.4% (n=2) had unknown smoking status. 399 of 450 cases (88.67%) met quality indices for DNA quantity (at least 100 evaluable cell equivalents) and quality (260/280 ratio of 1.5 to 2.2). Of these, 398 (99.75%, 95% confidence interval: 98.61%-99.99%) were negative for HPV16 and 18. One case, a female former smoker with adenocarcinoma, was positive for HPV16 at low copy number (0.4 per 1000 cell equivalents). We are currently evaluating a subset of cases (n=100) for a broad spectrum of HPV types by PCR with SPF10 primers and a cocktail of conservative probes recognizing at least 54 mucosal HPV genotypes in a microtiter plate format (DEIA). To date no HPV has been detected in the 30 cases tested.

Conclusions: Using extensive laboratory efforts to avoid DNA contamination and sensitive HPV DNA detection assays, we found no evidence to support a role for HPV in lung cancer in a representative Western population. Our results provide the strongest evidence to date to rule out a role for HPV in lung 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 4824.
Abstract 4827: A novel FGFR2 haplotype is associated with increased risk of breast cancer in African American women
Authors: Shetty Priya B. , Barnholtz-Sloan Jill S. , Guan Xiaowei , Nyante Sarah J. , Millikan Robert C.
Published: February 2011

Objectives: The objective of this study was to identify new and validate known SNPs and haplotypes in FGFR2 associated with risk of breast cancer in African American women.

Methods: Study participants were from the Carolina Breast Cancer Study, a population-based case-control study conducted in North Carolina. Cases and controls were frequency-matched on age and race. 1536 SNPs and 158 AIMs (ancestry informative markers) were genotyped with the Illumina GoldenGate assay. 89% of the SNP panel and 91% of the AIMs passed quality control. Associations between single SNPs and breast cancer were examined using logistic regression in SAS 9.2, and linkage disequilibrium (LD) between SNPs was tested with SAS/Genetics. Haplotypes were constructed and evaluated for association with breast cancer using generalized linear models as implemented in Haplo. stats. Statistical analyses were conducted on the whole dataset and stratified by race and age at diagnosis. 10 single SNPs in FGFR2 were analyzed. Of these 10 SNPs, rs11200014, rs2981579, rs1219648 and rs2420946 were further analyzed in haplotype analysis in order to compare our results with previous studies. As African Americans were over-sampled, all analyses were adjusted to account for study design.

Results: There were 3748 participants in the study; 742 cases (37.6% of cases) and 658 controls (37.0% of controls) were African American, and 1230 cases and 1118 controls were non-African American. 9 SNPs in all women, 9 SNPs in older women (> 50 years at diagnosis), 7 SNPs in younger women (< 50 years at diagnosis), 8 SNPs in African Americans and 9 SNPs in non-African Americans were associated with increased risk of breast cancer after adjusting for European ancestry and/or race and age. In all subjects, the ATGT, GTGT and GTAT haplotypes were associated with increased odds of breast cancer after adjusting for age, race and ancestry. In African American women, the GTGT haplotype was associated with significant increased risk of breast cancer while the ATGT haplotype was associated with increased risk in non-African American women. Interestingly, the GTGT haplotype was also found to be associated with increased risk of breast cancer among women aged <50 years old at diagnosis.

Conclusions: The GTGT haplotype in FGFR2 is a newly discovered haplotype that is associated with increased risk of breast cancer in African American and younger women. Previous studies have primarily focused on post-menopausal, European American women, and these results highlight the need to conduct genetic epidemiology studies in a variety of racial/ethnic populations.

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 4827.
Abstract 4829: Plasma coenzyme Q10 levels and postmenopausal breast cancer risk: The Multiethnic Cohort Study
Authors: Chai Weiwen , Caberto Christian P. , Shvetsov Yurii B. , Wilkens Lynne R. , Franke Adrian A. , Cooney Robert V. , Marchand Loic Le , Henderson Brian E. , Kolonel Laurence N. , Goodman Marc T.
Published: February 2011

Background: Coenzyme Q10 (CoQ10) is a component of the mitochondrial electron transport chain and is considered an important cellular antioxidant. Lower circulating CoQ10 levels are reported in patients with breast cancer, but epidemiologic evidence from large prospective studies is lacking. We examined the association of plasma CoQ10 levels with postmenopausal breast cancer risk in prospectively collected blood samples.

Methods: Plasma levels of total CoQ10 were measured by HPLC with UV detection after precolumn coulometric oxidation among 160 incident postmenopausal breast cancer cases and 289 controls in the Multiethnic Cohort Study. Cases and controls were individually-matched on age, sex, ethnicity, study location (Hawaii or California), hormone replacement therapy use, date/time of collection and hours of fasting. Conditional logistic regression was used to compute odds ratios (ORs) and 95% confidence intervals (CIs).

Results: Plasma CoQ10 levels were positively associated with breast cancer risk among postmenopausal women in the current study. The OR for the highest versus the lowest tertile was 1.6 (95% CI: 1.0 - 2.6; P = 0.05). The strongest association was observed for postmenopausal women under age 60 (OR for the highest versus the lowest tertile: 3.9; 95% CI: 1.1 - 14.0). Results were similar after adjusting for breast cancer risk factors.

Conclusions: Higher CoQ10 levels in postmenopausal women are associated with increased breast cancer risk in this multiethnic population. Future studies are needed to define the origin of circulating CoQ10, its physiological meaning, and potential role in cancer etiology and progression.

This work was supported by NIH Grants CA132149, CA33619. One of the authors (WC) was supported by NIH Training Grant CA90956.

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 4829.
Abstract 483: Genome-wide analysis identify molecular regulators of metastasis
Authors: Ryu Seongho , Mittal Vivek
Published: February 2011

Metastasis of primary tumors to distant target organs such as the lungs is a major factor contributing to cancer-related mortality. To identify key miRNAs of metastasis we have performed high throughput Solexa sequencing on tumors harvested form variety of metastatic tumor models. In this study, we will present the identification of novel candidates of microRNAs and their target genes. We also validate their biological properties both in vitro and in vivo models of breast and prostate cancer metastasis. Since relatively little is known about regulation system of miRNAs that governs metastasis, the identification and validation of upstream factors will be of great benefit in understanding metastasis regulation. We performed a meta-analysis combines miRNA expression profiles and genome-wide epigenetic modification signatures obtained by using ChIP-SEQ technique. We will also discuss potential clinical significance of key candidates identified in this screen.

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 483.
Abstract 484: Treatment strategy to reverse osteolytic breast cancer metastasis using osteoblasts in a murine xenograft model
Authors: Bodenstine Thomas M. , Beck Benjamin H. , Welch Danny R.
Published: February 2011

The most common site of breast cancer metastasis is bone. The majority of these metastases are osteolytic, characterized by an increase in bone degradation via an upregulation of osteoclast activity. These effects lead to complications including fracture, contractile pain and hypercalcemia, significantly reducing the quality of life in these patients. The role of bone forming osteoblasts at sites of osteolytic lesions is less understood. We have recently shown that breast cancer cells are capable of inducing apoptosis of osteoblasts in vitro as well as in vivo using murine xenograft models. This effect would further exacerbate osteolytic disease, and may explain why treatment strategies capable of inhibiting osteolysis (bisphosphonates) are not capable of repairing lost bone. We hypothesize that modulating the number of osteoblasts at sites of osteolytic metastases, may aid in the repair of resorbed bone. To provide proof-of-principle evidence, we have engineered the murine pre-osteoblast cell line, MC3T3-E1, to overexpress Bcl2, conferring resistance to apoptosis. These cells will be used with an in vivo model of osteolysis using GFP-labeled breast cancer cells previously reported by our laboratory. Apoptosis resistant MC3T3-E1 will be injected into bone both before, and after induction of osteolysis and rates of bone resorption and repair will be measured. The results of this project will provide the first in vivo evidence that restoration of osteoblasts at sites of osteolytic metastases can aid in the repair of lost bone and will test for the first time, experimental manipulation of osteoblast number and function in osteolytic breast cancer metastasis.

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 484.