bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3082: TGF-β synergistically stimulates malignant mesothelioma growth with defects in the Hippo pathway by inducing CTGF expression
Authors: Fujii Makiko , Toyoda Takeshi , Nakanishi Hayao , Yatabe Yasushi , Sato Ayuko , Matsudaira Yasue , Ito Hidemi , Murakami Hideki , Kondo Yutaka , Kondo Eisaku , Tsujimura Tohru , Hida Toyoaki , Osada Hirotaka , Sekido Yoshitaka
Published: August 2012

Malignant mesothelioma (MM) arises from the serosal cells of the pleural, peritoneal and pericardial cavities with poor prognosis because of its frequent diagnosis in advanced stages. The primary cause of this disease has often been linked to asbestos exposure, and the number of patients worldwide is expected to peak in the next two decades. There is no known curative therapy for MM and further understanding in molecular mechanism is needed to identify new candidates for targeted treatment. We found that the TGF-β pathway is associated with oncogenic mutations and loss of the tumor suppressor NF2, and induces pro-oncogenic biological activities. Loss of NF2 is one of the genetic lesions which have been related to the growth of human MM. The NF2 gene is responsible for neurofibromatosis type II syndrome and encodes Merlin which negatively regulates YAP function. Nearly 75% MM cases have inactivating mutations in the NF2 gene or in downstream signaling molecules of Hippo signaling cascade. We examined a functional interaction between the Hippo and TGF-β pathways and found that endothelin-1 and connective tissue growth factor (CTGF) are the only proteins affected by these two pathways in MM cells. The CTGF promoter contains both a YAP/TEAD-binding site and a consensus Smad-binding site adjacent to each other. The expression of CTGF induced by TGF-β in MM cell lines was greatly affected by the existence of YAP, by its binding with Smad3, forming a complex on CTGF promoter lesion. Knocking down CTGF expression in MM cells prolonged the survival of mice, and a significant association was seen between CTGF expression and extracellular matrix deposition in MM xenografts and in patient tissue specimens. This study elucidates a novel molecular mechanism induced by the TGF-β pathway, which may be applicable to malignancies with defective NF2 and of mesenchymal origin.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3082. doi:1538-7445.AM2012-3082
Abstract 3088: Essential roles of ATM in GM-CSF-induced bone marrow differentiation to dendritic cells
Authors: So Eui Young , Ouchi Toru
Published: August 2012

The gene mutated in ataxia telangiectasia (ATM) encodes 370-kD protein, belonging to a family of PI-3 protein kinases (PIKKs). ATM has been implicated in cellular responses under conditions of DNA damage that causes DNA lesions, playing crucial roles in activating DNA repair machinery and cell-cycle checkpoints. Recent studies have also illustrated that ATM regulates cellular growth, development, and stem cell renewal. Although mice model has demonstrated that ATM deficiency results in both multiple defects in DNA repair in response to ionizing radiation and severe developmental abnormality in T/B cells development as well as weight-loss, it remains unclear whether ATM is involved in development of other cell lineage. We found that levels of ATM are significantly low in primary bone marrow cells (BMs), and that they are increased when BMs’ are differentiated to dendritic cells (DCs) when stimulated with granulocyte macrophage colony-stimulating factor, GM-CSF. BMs derived from ATM mice (ATM-/- BMs) demonstrate decreased cell proliferation, and their differentiation to DCs is markedly impaired, suggesting that ATM is essential for development of DCs. ATM-deficiency is also associated with a decreased capability of BMs to activate T cells. We also found that an ATM specific inhibitor, KU-55933, abrogated GM-CSF-induced DCs development in BMs cell culture. ATM-deficiency in BMs also resulted in reduced expression of Akt compared with control cells, and inhibited Ser493 phosphorylation of Akt by GM-CSF. Levels of expression and phosphorylation of STAT5 were differentially regulated in ATM-/- BMs, and ectopic expression of STAT5 restored DCs differentiation of ATM-/- BMs. Together, our findings reveal that ATM plays a physiological role in BMs’ differentiation to DCs by regulating Akt/STAT5 pathways.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3088. doi:1538-7445.AM2012-3088
Abstract 3092: DSG3 facilitates cancer cell growth and invasion through DSG3-plakoglobin-TCF signaling pathway
Authors: Cheng Ann Joy , Chen Yin Ju
Published: August 2012

DSDSG3 has been reported to act as an oncogene in head and neck cancers (HNCs), but the underlying mechanism of its oncogenic activity is unclear. In this study, we aimed to validate the proposed oncogenic function of DSG3 and investigate its downstream regulatory mechanisms. When we stably transfected HNC cells with a short hairpin DSG3-RNAi (shDSG3) to suppress DSG3 expression, reductions in cell growth and colony formation were observed, in addition to cell cycle arrest at the G0/G1 phase. Both cell migration and invasion abilities were also decreased. BecauseDSG3 is associated with plakoglobin which has been reported as a tumor suppressorin desmosomes, we examined whether or not the phenotypic alterations that occur following DSG3 silencing were mediated through the plakoglobin signaling pathway. Immunoprecipitation and immunofluorescent staining analyses revealed that DSG3 silencing disrupted the interaction of DSG3 with plakoglobin and induced the translocation of plakoglobin from the cytoplasm to the nucleus. Using a luciferase reporter assay and western blot analysis, we found that knockdown plakoglobin increasedTCF transcriptional activity butDSG3 silencing significantly reduced TCF transcriptional activity and subsequently suppressed the expression of the downstream target genes of the TCF transcription factors, including c-myc, cyclin D1, and MMP7, which are involved in the regulation of cell proliferation and invasion. Xenograft mice administered withDSG3 silencing significantly inhibited tumor growth in nude mice and also reduced c-myc, cyclin D1 and MMP7 expression. In conclusion, we found that DSG3 has an oncogenic function in HNC which is through DSG3-plakoglobin-TCF signaling pathway and facilitates cancer growth and invasion. Key words: DSG3, plakoglobin, TCF/LEF promoter, cell growth, cancer invasion

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3092. doi:1538-7445.AM2012-3092
Abstract 3097: Genomic instability of the breast tumor microenvironment: Stromal alterations or technological artifact
Authors: Rummel Seth K. , Valente Allyson L. , Kane Jennifer L. , Ellsworth Darrell L. , Shriver Craig D. , Ellsworth Rachel E.
Published: August 2012

Introduction: The breast tumor microenvironment plays an active role in tumorigenesis. Molecular alterations, including epigenetic modifications to DNA, and changes in RNA and protein expression have been identified in tumor-associated stroma; however, there is considerable debate as to whether the stroma is characterized by genomic instability or whether detection of chromosomal alterations in the breast stroma is a reflection of technological artifact rather than the true genomic content of the tumor microenvironment. Methods: Surgically-removed breast stroma specimens from 112 women undergoing reductive mammoplasty (n=7), prophylactic mastectomy (N=6) or mastectomy for a diagnosis of breast disease (n=99) were frozen in optimal cutting temperature medium. Allelic imbalance analysis was performed in 484 stromal specimens from 98 women using a panel of 52 microsatellite markers; SNP data was generated from a subset of 86 stromal specimens using 250K SNP arrays (Affymetrix). Copy number alterations were identified using Partek Genomics Suite. Results: AI was not detected in 93% (444/484) of stroma specimens. When compared to AI generated from 77 FFPE stroma specimens, 32 (42%) of which harbored at least one detectable AI event, the frequency of AI in the FFPE specimens (4.62%) was significantly higher (P<0.0001) than that found in frozen specimens (0.45%). Of the stroma specimens assayed using SNP arrays 95% (82/86) had no detectable alterations and the 11 copy number changes were small and not shared between specimens. Conclusions: The data presented here support a model in which the tumor microenvironment is genetically stable. The direct comparison of copy number alterations between FFPE and frozen research-grade specimens using identical methodologies suggests that past reports of significant AI/LOH in breast stroma, both adjacent to and distant from the tumor, reflects artifact in the archival specimens caused by formalin-fixation, paraffin-embedding and tissue storage.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3097. doi:1538-7445.AM2012-3097
Abstract 3099: Gastroesophageal junction adenocarcinoma displays abnormalities in homology directed repair
Authors: Dewalt Robin I. , Kesler Kenneth A. , Jalal Shadia I.
Published: August 2012

The incidence of adenocarcinoma of the gastroesophageal (GE) junction has risen dramatically over the last three decades. Outcomes continue to be poor with 5 year survivals estimated at 15%. These poor outcomes are related to the advanced presentation in the majority of patients in addition to the resistance esophageal adenocarcinoma displays to cisplatin based chemotherapy or radiation especially when each is administered alone. Only one-third of patients will have significant benefit and achieve complete responses when treated with the combination of cisplatin-based chemotherapy and radiation followed by surgical resection. Therefore, the need persists for overcoming resistance to cisplatin-based chemotherapy and radiation. Previous studies have highlighted the role of DNA repair pathways in resistance to cisplatin or radiation in a number of solid organ malignancies. The nucleotide repair pathway (NER) repairs the majority of cisplatin-induced DNA lesions while DNA double-strand breaks (DSBs) formed in response to radiation are repaired by homologous recombination (HR) and the non-homologous end-joining (NHEJ) pathways. We hypothesized that cisplatin and radiation resistance in esophageal adenocarcinoma is mediated through abnormalities in these DNA repair pathways. Using paired GE junction adenocarcinoma tumor samples with their matched normal tissue, we analyzed the expression of DNA repair genes in NER, HR and NHEJ pathways using quantitative RT-PCR. We identified overexpression of genes involved in HR in esophageal tumor samples in comparison to their normal counterparts. Eme1, an endonuclease present in complex with Mus81, showed consistent overexpression in GE junction adenocarcinoma samples. Mus81-Eme1 has been shown to play an important role in rescuing stalled replication forks and has been implicated in the repair of interstrand crosslinks, such as those formed in response to cisplatin therapy. Eme1 overexpression in esophageal adenocarcinoma could play a role in resistance to cisplatin through its impact on the repair of cisplatin induced DNA lesions. In addition, Eme1 overexpression could lead to genomic instability contributing to pathogenesis, metastases and therapeutic resistance. Further understanding the implications of Eme1 overexpression in these processes in GE junction adenocarcinoma will be of significant value.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3099. doi:1538-7445.AM2012-3099
Abstract 3101: Transition from extrachromosomal to intrachromosomal amplification in glioblastoma cells
Authors: Vogt Nicolas , Gibaud Anne , Debatisse Michelle , Bernard Malfoy
Published: August 2012

Gene amplification is one of the genomic alterations found in cancer cells by which oncogenes may be over-expressed. Amplified sequences may be found within homogeneously staining regions of the chromosomes (hsr) or on autonomously replicating circular and acentric extrachromosomal DNA molecules, named double minutes (dmins). Integration of dmins is a recognized pathway for hsr formation, but the molecular mechanisms involved in this process are still poorly understood. We analyzed three xenografted glioblastomas with dmins at early passages in mice that evolved in hsr at late passages. Using various approaches (FISH, PCR, quantitative-PCR, chromosome walking, DNA sequencing, genome-wide SNP analysis), we characterized the events leading to dmin formation, evolution and reintegration, and determined the structure of the integration sites. In all three glioblastomas, dmins constituted by the fusion of several syntenic or non-syntenic DNA fragments formed upon chromosome fragmentation were present at early passages. The fragments were fused by a non-homologous end-joining (NHEJ) mechanism. Later, a new type of dmin was formed by the fusion of fragments from both the initial dmin and other chromosomes, suggesting a second round of extensive DNA fragmentation. The new type of dmin then gave rise to an hsr. It is not known whether the new dmin was amplified extrachromosomally before reintegration or if one molecule was integrated, then amplified in the chromosome by a mechanism that did not involved breakage-fusion-bridge cycles since the target chromosome was not rearranged outside of the integration site. The integration sites were characterized at the nucleotide level. No preferential integration sequence was found. No significant homologies existed between extrachromosomal and intrachromosomal sequences at the integration sites. The structure of the integration site may be simple, with a single small deletion, or very complex, exhibiting deletions, duplication and insertion of fragments coming from other regions of the genome. Fusions between fragments were mainly driven by NHEJ. Current data allow us to propose a mechanism of hsr formation through integration of extrachromosomal DNA molecules. At the best of our knowledge, it is the first reported molecular characterization of hsr insertion sites.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3101. doi:1538-7445.AM2012-3101
Abstract 3179: Targeted re-sequencing of cancer-related genes from matched FFPE and fresh-frozen tumor samples using the Illumina sequencing platform
Authors: Bibikova Marina , Chien Jeremy , Ho Vincent , April Craig , Munchel Sarah , Jaeger Erich , Cooper Samantha , Grocock Russell , Nielsen Fiona , Tarabishy Yaman , Visscher Daniel , Manion Megan , Liu Jonathan , Wieben Eric , Hartmann Lynn , Kalli Kimberly , Shridhar Viji , Fan Jian-Bing
Published: August 2012

High throughput sequencing technologies open up a new dimension in cancer genomics by enabling the characterization of cancer genomes at base-pair resolution. To date, the majority of large-scale genomics projects rely on fresh-frozen biospecimens in their studies, which are difficult to obtain and often lack long-term clinical data. Unlike fresh-frozen samples, archived formalin-fixed paraffin-embedded (FFPE) tissues are more readily accessible, and are often associated with known clinical outcomes and more complete clinical annotations. However, sequencing library preparation methods need to be further optimized with regard to applicability to FFPE samples. To test whether next-generation sequencing technologies could overcome previously reported artifacts associated with formalin fixation and report accurate sequencing results, we compared whole-genome and targeted enrichment DNA sequencing data obtained from five FFPE tumor samples for which matching frozen tissues were available. We successfully generated sequencing libraries using 1 µg of genomic DNA as input material and a modified TruSeq™ sample preparation protocol. We designed a custom enrichment pool targeting exons from 215 cancer-related genes and utilized this pool to pull-down and sequence approximately 1.3 Mb region of the genome with an average 400x coverage depth to test if deep sequencing would improve validation of tumor-specific somatic mutations. We used the Illumina sequence analysis pipeline and the Windows-based second generation DNA sequencing software NextGENe® to analyze the data and identify sequence variations that are different from the human reference genome. CNV detection was overall higher among FFPE samples compared with fresh-frozen samples, demonstrating that tissue processing impacts sequencing data quality. We obtained good concordance in variant calls between matched FFPE and fresh-frozen samples. Discordant variant calls were mainly due to low depth of coverage in the regions where variant calls were made. Improvements to DNA sequencing methods for archived samples will significantly enhance cancer research and will result in more reliable prediction of individual cancer therapies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3179. doi:1538-7445.AM2012-3179
Abstract 3183: Breast Cancer MethylSeq: Analysis of bisulfite converted breast cancer genomes using microdroplet-based targeted sequencing
Authors: Samuels Michael L. , Wu Chia-Chin , Brayer James , Warner Jason , Link Darren , Harris Alan , Oesterreichs Steffi , Lee Adrian , Milosavljevic Aleksandar
Published: August 2012

Targeted sequencing using microdroplet-based PCR provides a method to selectively and uniformly amplify thousands of genomic regions of interest (ROIs) for efficient next generation sequencing (NGS). Recently, this approach has been adapted for the analysis of bisulfite-treated DNA, enabling strand-specific determination of cytosine methylation status with single-base pair resolution. This droplet PCR-based targeted approach significantly reduces cost and improves sequencing depth compared to whole-genome sequencing, allowing researchers to power larger studies to discover and validate how methylation and genomic aberrations affect cancer development. Here we report results from our ongoing work using a breast cancer targeted panel (Breast Cancer MethylSeq Panel) for sequence specific analysis of both epigenomic methylation and genomic mutation in breast tumor samples. The panel includes PCR primer pairs designed to target ∼2,700 ROIs, leaving room for an additional 1,300 target ROIs of interest to be added by individual researchers. Use of this single panel will provide information about CpG methylation and genomic sequence enabling analysis of multiple content types including: A) cell subtype; B) genomic mutation; C) surrogate gene expression subtypes using promoter CpG islands or other methylation markers; D) copy-number variation (CNV); E) Loss of Heterozygosity (LOH); F) methylation-mediated gene silencing and loss of imprinting. The Breast Cancer MethylSeq microdroplet library, comprising individual primer pairs for PCR in droplets, will be tested and validated using a set of 64 bisulfite-converted samples including 8 breast cancer cell lines, a range of ER positive and negative tumors and TCGA samples, and a set of 8 metastatic tumors from a single individual. We will report overall sequencing success metrics, as well as concordance with metadata on the various ROI classes (A-F above). The deep and comprehensive breast tumor sequencing enabled by this targeting panel will allow researchers to identify epigenomic alterations and genomic mutations across a range of assay types using just one assay. The goal for development and validation of this panel is to provide the breast cancer research community a unique tool for identifying and correlating cancer subtypes with clinical outcomes.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3183. doi:1538-7445.AM2012-3183
Abstract 3190: Capture and molecular characterization of ovarian cancer-derived exosomes
Authors: Ruf Ingrid K. , Briggs Hilary , Tewari Muneesh
Published: August 2012

Exosomes are secreted, membrane-bound vesicles of 50-100 nm known to carry cellular messenger RNAs (mRNA) and microRNAs (miRNA). Several reports have indicated that tumor-derived exosomes are released into the circulation of patients with a variety of cancer types, including epithelial ovarian cancer. The goals of this study are (i) to develop methods for specific capture and molecular profiling of ovarian cancer tumor-derived exosomes from blood plasma and (ii) determine whether information carried in tumor-derived exosomes can serve as a reliable surrogate for tumor tissue for measurement of key miRNA and mRNA biomarkers. In our work to date, we have used exosomes derived from conditioned media of ovarian cancer cell lines to develop and optimize methods for the isolation and physical characterization of exosomes. Using Nanoparticle Tracking Analysis (NTA) technology (NanoSight, Inc.) to directly count and accurately size exosome populations, our initial data indicates that traditional ultracentrifugation protocols may fail to recover a major fraction of exosomes present in conditioned media. The basis for this finding is currently under investigation and the latest results will be presented. In parallel, we are developing affinity capture approaches to isolate specific subpopulations of exosomes using antibodies directed against epithelial cell adhesion molecule (EpCAM) and other exosomal surface proteins. Initial results indicate that a subpopulation of ovarian cancer-derived exosomes can be purified in this manner; molecular characterization of exosomal contents is in progress. Finally, we are analyzing clinical plasma specimens from patients and controls to: (i) test whether bulk properties of exosomes (e.g., size and number) differ between cases and controls, and (ii) perform specific capture of tumor-derived plasma exosomes and compare exosomal mRNA/miRNA profiles to matched tumor tissue to determine the degree of concordance between exosomal and tissue RNA profiles. Latest results from these studies will be presented. Ultimately, we anticipate that this work will lead to methods for molecular phenotyping of epithelial ovarian cancers in individual patients using a blood-based approach. This work was supported in part by a Stand Up To Cancer/AACR Innovative Research Grant to M. Tewari.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3190. doi:1538-7445.AM2012-3190
Abstract 3193: A new approach for colocalization of proteins using HRP and AP enzyme detection in paraffin embedded tissues
Authors: Gonzalez Rachel M. , Ding Li , Xiao Ming , Zhang Xiaoping
Published: August 2012

Co-localization of two proteins is a powerful tool in immunofluorescence, however the stability of the fluorescence limits the ability to screen large sample sets. Double and triple staining with the ability to visualize co-localization of proteins using HRP and AP enzymes in paraffin embedded tissue may prove to be a useful tool for discovery and functional analysis of gene and protein expression. Here we present a novel chromogen mix by GBILabs.com of Emerald and Red that yields a third color (Dark Blue/Purple) when proteins are co-localized using HRP and AP enzymes for detection. Initial tests evaluated combinations of ER, Her2, CEA, Keratin, PR, p53, PCNA, and Ki67 in 10 breast and 10 colon cancer cases to screen the percentage of co-localized proteins in these cases. In addition, a 70 case breast cancer tissue array containing duplicate samples and known diagnosis of ER, PR, and Her2 for each case were screened with ER, PR, Her2, p53, PCNA, or Ki67and keratin antibodies. The core tumors sections showed co-localization of PCNA, Ki67 and p53 proteins. This new double and triple staining protocols in combination with tissue arrays, will allow researchers to rapidly determine the association between expression of two or more proteins in the tissue and if proteins co-localize within the cell.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3193. doi:1538-7445.AM2012-3193
Abstract 3200: The AKT inhibitor AZD5363 is selectively active in PIK3CA mutant gastric cancer cell lines, and sensitizes a gastric cancer explant model to docetaxel
Authors: Li Jing , Davies Barry R. , Zhou Minhua , Zhang Jingchuan , Xu Yan , Tang Lili , Wang Huiying , Liu Charles , Yin Xiaolu , Ji Qunsheng , Yu Derek Dehua
Published: August 2012

Activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway is a common phenomenon in cancer due to multiple mechanisms, including mutation of PI3KCA, loss or mutation of PTEN, or over-expression of receptor tyrosine kinases. We recently developed a novel AKT kinase inhibitor, AZD5363. To explore the activity of AZD5363 and elucidate the correlation between AZD5363 response and genetic alterations in gastric cancer, we investigated the effects of pharmacological inhibition of AKT on a panel of 20 gastric cancer cell lines. We demonstrated that gastric cancer cells with PIK3CA mutations were selectively sensitive to AZD5363. Interestingly, we found that HGC27, a cell line harboring both PI3KCA mutation and PTEN loss, displayed the greatest sensitivity to this AKT inhibitor, suggesting possible synergy between these two genetic alterations. To further dissect the role of PI3KCA mutation and PTEN loss in response to AKT inhibition, we tested, using in vivo xenografts, the antitumor activity of AZD5363 in HGC27 and two other primary gastric cancer models containing either PI3KCA mutation or PTEN loss. Our data indicated that AZD5363 monotherapy treatment led to complete tumor regression in the HGC27 xenograft model, correlating with the induction of cell apoptosis and inhibition of PI3K/AKT pathway. On the other hand, the same treatment evoked only a moderate response (∼60% TGI) in the PI3KCA mutant primary xenograft model. Whilst monotherapy AZD5363 or docetaxel were not active in the PTEN negative, PIK3CA wild-type primary xenograft model, significant anti-tumor activity was seen when AZD5363 was combined with docetaxel. These results suggest that PI3KCA mutation is an important determinant of response to AKT inhibition in gastric cancer. Moreover, combination with AZD5363 can overcome innate resistance to docetaxel in a gastric cancer explant model.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3200. doi:1538-7445.AM2012-3200
Abstract 3203: High quality DNA obtained with an automated DNA Extraction method with 15 to 40 year old formalin fixed paraffin embedded (FFPE) blocks from normal and cancer tissues
Authors: Niland Erin , McGuire Audrey D. , Cox Mary L. , Sandusky George E.
Published: August 2012

DNA and RNA have been used as markers of tissue quality and integrity in tissues the last decade. In this research study, kidney, liver, colon, lung, and breast genomic quality were analyzed in tissues from surgical cancer patients and post-mortem cases obtained with an IRB approved protocol and informed/or next of kin consent. DNA extraction was performed on formalin-fixed paraffin-embedded tissues (FFPE) from both groups of cases from the 1970's through the 2000's and formalin fixed / long term 70% ethanol storage(15 + years). To extract the DNA from the FFPE tissues, we soaked all tissues in xylene to remove the paraffin, digested the tissue with a proteinase K solution, and used an automated extraction machine to extract the DNA. A similar SOP was used for the formalin fixed/ 70% long term ethanol-stored tissue, except the tissue was first homogenized rather than soaked in xylene. DNA quality and yield were evaluated using a spectrophotometer. Gel electrophoresis was used to evaluate the length of the DNA in about 20% of the 200 samples. Samples that had optical density (OD) readings above 1.60 and below 2.10 with a DNA yield above 100 µg/mL were deemed to be high quality DNA. FFPE tissues yielded the highest DNA concentration and quality (OD reading of 1.87 and yield of 625 ug/ml) with greater than 95% of the samples meeting the above specifications, while the formalin fixed/long term 70% ethanol stored tissue yielded both the lowest DNA concentration and quality (yield of 52 ug/ml and OD reading of 1.48). In conclusion, the results show that high quality DNA can be extracted from FFPE tissue using an automated DNA extraction procedure with paraffin blocks from the early 1970's to present, and the DNA extracted is useful for clinical and experimental studies. The DNA from tissues stored in aqueous media (formalin fixed / long term 70% ethanol storage) may be useful in some research applications, but the DNA is partially degraded compared to the tissue stored in paraffin blocks.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3203. doi:1538-7445.AM2012-3203
Abstract 3263: Expression of Bim, Noxa, and Puma in non-small cell lung cancer
Authors: Sakakibara-Konishi Jun , Oizumi Satoshi , Kikuchi Junko , Kikuchi Eiki , Mizugaki Hidenori , Kinoshita Ichiro , Dosaka-Akita Hirotoshi , Nishimura Masaharu
Published: August 2012

Background: The BH3-only members of the Bcl-2 protein family have been proposed to play a key role in the control of apoptosis and in the initiation of the apoptotic pathways. In this study, we evaluated the expression of Bim, Noxa, and Puma in non-small cell lung cancer (NSCLC). Methods: A total of 135 surgically resected NSCLCs were immunohistochemically assessed for Bim, Noxa, and Puma expression. The immunoscores were determined, and then its correlation with either the clinicopathological variables or the survival outcomes were analyzed. Results: Immunohistochemical reactivity for Bim, Noxa, and Puma was detected in the cytoplasm of the tumor cells. Bim expression was associated with several clinicopathological factors, including sex (p<0.001), smoking habit (p=0.03), pathological histology (p=0.001), pathological T stage (p=0.03), pathological disease stage (p=0.02), and differentiation of tumor (p<0.001). Multivariate logistic regression analysis showed a significant correlation between low Bim expression and squamous cell carcinoma (p=0.04), in addition to a correlation between high Bim expression and well differentiated tumors (p=0.02). Analysis of cellular biological expression demonstrated a link between low Bim expression and high Ki67. While Noxa expression was also shown to be correlated with both smoking habit (p=0.02) and the pathological histology (p=0.03), there was no strong association observed between the expression and the clinical features when they were examined by a multivariate logistic regression analysis. No correlations were noted between Puma expression and any of the variables. Our analyses also indicated that the expression levels of the BH3-only proteins were not pertinent to the survival outcome. Conclusions: The current analyses demonstrated that Bim expression in the NSCLCs was associated with both squamous cell carcinoma histology and tumor proliferation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3263. doi:1538-7445.AM2012-3263
Abstract 33: Implementing CAD cells quantify the relationships between Ig-CAM-mediated adhesion, force transduction, and actin dynamics
Authors: Fuller Miles H. , Schaefer Andrew , Mejean Cecile , Forscher Paul
Published: August 2012

Several neuro-degenerative diseases and motor defects have been linked to malfunctions in the motility of neurons. This project is focused on the determining whether the roles of the transmembrane proteins NCAM and L1-CAM can be studied using the CAD cell line as a model. CAD cells are a cancer cell line that come from mice and can be stimulated to grow as either neurons or glial cells. In addition, this cell line has been shown to be an easily transfected line. In order to determine usefulness of this cell line several techniques were used. Western blots were implemented in order to determine if the NCAM and/or L1-CAM and proteins known to be involved in their signaling pathways were present in the cell line. Western blots were then confirmed by immunocytochemistry staining. Next, the CAD cell line was transfected with photoactivateable actin. Finally, immuno beads were attached to the membranes of these CAD cells and then holographic optical tweezers were used to determine whether or not thsee ligands coupled to the retrograde actin flow in the cells. The combination of these experiments should provide the evidence for whether or not the CAD cells would be a feasible model to study the nature of the NCAM and L1-CAM proteins and their relationship to the movement of actin in the cytoskeleton of neurons.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 33. doi:1538-7445.AM2012-33
Abstract 3328: Targeted inactivation of EZH2 by CDF inhibits pancreatic tumor growth mediated by re-expression of lost miRNAs in tumors
Authors: Bao Bin , Ali Shadan , Banerjee Sanjeev , Logna Farah , Azmi Asfar , Kong Dejuan , Ahmad Aamir , Li Yiwei , Padhye Subhash , Sarkar Hazlul H.
Published: August 2012

EZH2 plays a critical role in the regulation of cell survival, proliferation and cancer stem cell (CSC) function, contributing to tumor aggressiveness. Increased expression of EZH2 has been found in human pancreatic cancer (PC); however, the mechanism(s) by which EZH2 mediates its biological effects is not known, and there is no avenue for inactivation of EZH2. In this study, we examined the effect of CDF, a novel Curcumin analogue, on tumor cell growth in vitro and in vivo and assessed whether the effect of CDF is due to inactivation of EZH2 and CSC function mediated by deregulation of microRNAs (miRNAs). We found that CDF decreased cell survival, clonogenicity, formation of pancreatospheres, cell migration and invasion, and attenuated CSC function in human PC cells, which was associated with inactivation of EZH2 expression and NF-κB activity, and increased expression of miRNAs such as let-7a, b, c, d, miR-26a, miR-101, miR-146a, and miR-200b, c. Further mechanistic studies showed that the re-expression of miR-101 led to decreased expression of EZH2 and EpCAM. Moreover, CDF inhibited tumor growth in a mouse orthotopic model of PC, which was associated with decreased expression of EZH2, Notch-1, CD44, EpCAM and Nanog, and increased expression of let-7, miR-26a and miR-101 in tumor remnant. From these results, we conclude that the inhibition of tumor growth and tumor cell aggressiveness by CDF is in part due to down-regulation of EZH2, which was mediated by up-regulation of specific miRNAs, suggesting that CDF could be useful for the prevention of tumor progression and/or treatment of PC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3328. doi:1538-7445.AM2012-3328
Abstract 3335: The potential role of lateral inhibition in the maintenance of glioblastoma cancer stem cells
Authors: Lim Kah Jing , Eberhart Charles G. , Bar Eli E.
Published: August 2012

Glioblastoma (GBM) is a malignant cancer of the central nervous system. Tumor cells are highly resistant to conventional therapies and tumors always recur. Cancer stem cells are thought to be critical for long-term growth and therapeutic resistance of GBM. It is therefore important to understand the signaling mechanisms maintaining GBM cancer stem cells. Notch is known to be an important signaling pathway during neural development, and can promote the maintenance of stem and progenitor cells via the process of lateral inhibition. Lateral inhibition is a contact-dependent signaling mechanism by which some cells within a field remain stem-like while others differentiate. In normal development, Notch ligands initiate this process by binding to the receptor on an adjacent cell, signaling to this neighbor to remain undifferentiated. Although Notch is known to be a critical player in the maintenance of stem-like cancer cells in various tumor types, including GBM, it is not known if the mechanism of lateral inhibition is also conserved in neural cancer stem cells. To test this hypothesis, we engineered a model system where isogenic cells expressing low and high levels of the Jag1 ligand are co-cultured followed by flow-based separation and determination of Notch pathway activity in each respective population. These experiments suggest that lateral inhibition is conserved in GBM. In the GBM neurosphere lines HSR-GBM1 and 040821, co-culture induced expression of Notch targets Hes5 and Hey1 in Jag1-low “signal receiving” cells and inhibited expression of these targets in Jag1-overexpressing “signal sending” isogenic cells. Preliminary experiments suggest that the ligand intracellular domain (ICD) is involved in this process. Our data suggest conservation of a developmental mechanism regulating Notch activation in cancer, and may affect our understanding of how to treat Notch-dependent tumors.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3335. doi:1538-7445.AM2012-3335
Abstract 334: Identification of novel regulatory genes controlling epithelial to mesenchymal transition in cultured breast cancer cells
Authors: Lehtinen Laura , Mäkelä Rami , Kallioniemi Olli , Iljin Kristiina
Published: August 2012

Breast cancer prognosis is tightly correlated with the degree of spread beyond the primary tumor. A prerequisite for breast cancer cell dissemination is activation of a process called “epithelial - mesenchymal transition” (EMT). During EMT, epithelial cells lose epithelial characteristics, such as E-cadherin expression and gain mesenchymal properties, such as vimentin expression. The reversal process to EMT is mesenchymal-epithelial transition (MET), which occurs during colonization of the disseminated cancer cells to distant sites. Thus, EMT should be considered as a transient and reversible process in cancer progression. Despite the clinical importance, EMT and MET have been largely studied from a focused signalling standpoint, which may provide a biased view of the biological processes involved. In this study, RNAi technology followed up by lysate microarray analysis (LMA) using E-cadherin and vimentin as endpoint markers was performed to identify novel EMT and MET regulatory genes in cultured breast cancer cells. The screening conditions were optimized for the estrogen receptor (ER) positive E-cadherin expressing luminal MCF-7 breast cancer cells and for ER negative vimentin expressing mesenchymal bone metastatic MDA-MB 231 cells. The results from the replicate screens with >1012 siRNAs targeting 599 genes (1-3 siRNAs / gene) amplified and /or over-expressed in breast cancer cells were found to correlate (r > 0.9), and the validated siRNAs targeting E-cadherin and vimentin were among the top repressing hits for the corresponding endpoints, indicating reliability of the protocol. Interestingly, several novel putative E-cadherin (E-cadherin reducing siRNAs, genes inducing loss of metastatic potential) and vimentin (vimentin reducing siRNAs, metastasis-promoting candidate genes) regulators were identified and validated using secondary assays, indicating that our knowledge on EMT and MET is still far from comprehensive. Functional characterization of the identified candidate regulators in cultured breast cancer cells is on-going. Since modulation of the differentiation state of breast cancer cells is a promising therapeutic strategy, better understanding of EMT and MET may provide novel therapeutic opportunities to block breast cancer cell dissemination.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 334. doi:1538-7445.AM2012-334
Abstract 3366: An inhibitor of the NF-kappaB pathway targets cancer stem cells and prevents tumor recurrence
Authors: Gomez-Cabrero Azucena , Liao Debbie , Nguyen Giang , Reisfeld Ralph
Published: August 2012

Multidrug resistance and tumor recurrence are two of the major challenges in cancer treatment. Cancer stem cells (CSCs), found in most solid tumor types, have been proposed as the root cause of cancer growth and recurrence. These CSCs share similarities with normal Stem Cells, such as “stemness” gene profile expression, overexpression of Sox2, Nanog and Oct4, as well as ability to grow in spheres and to repopulate tumors. Additionally, CSCs also have a high resistance to chemotherapeutic agents, probably due to their dormancy and overexpression of ABC transporters. Since CSCs markers are highly variable between cell lines and tumor samples, we used a functional assay to select a drug candidate targeting CSCs, based on their capability to efflux dyes such as Hoechst 33324 by a side population (SP). This drug decreased SP and spheres formation in vitro, and reduced protein expression of CSC genes such as Sox2, Nanog, Survivin and Oct4. Another serious limitation of cancer chemotherapy is its toxic side effect due to poor targeting to the tumor and its microenvironment (TME). To address this issue, we developed targeted nanoparticles (tNP) to deliver chemotherapeutic drugs specifically to tumor cells and their TME, resulting in substantially reduced systemic toxicity, as well as eradication of tumors and prevention of their recurrence in a preclinical setting.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3366. doi:1538-7445.AM2012-3366
Abstract 3368: Efficient delivery of prostate cancer gene therapy via adipose-derived mesenchymal stem cells in vivo
Authors: Zolochevska Olga , Figueiredo Marxa L.
Published: August 2012

It is critical to develop new therapeutic approaches for prostate cancer, since there is no effective treatment for patients in the advanced stages of this disease. And although many gene therapy approaches have been evaluated to date, clinical responses unfortunately remain poor. We have examined the potential of developing more effective cell-based gene therapies for preventing prostate cancer progression, using adipose-derived mesenchymal stem cells (ASC) as vehicles. The present study reports promising preliminary observations using this novel gene therapy modality. ASCs can be isolated using simpler methods as compared to those involving bone marrow mesenchymal cells, and are much more easily expanded at early passage numbers to therapeutic quantities. And since current techniques allow isolation of ASCs from an individual's own adipose tissue, this could help prevent immune reaction, suggesting that ASCs could be far more efficient as a gene delivery vehicle than viral vectors currently in clinical trials. In this study we have examined the potential of new antiangiogenesis and proapoptotic therapies delivered by ASCs and evaluated their ability to reduce prostate tumor growth rate in vitro and in vivo. We expressed in ASCs several secreted antitumor gene therapies (PEDF, Mda7, or IFNb), and all were found to be highly effective in almost complete prevention of tumor progression in vivo using prostate cancer xenograft models. Overall, ASC-delivered PEDF prevented HUVEC tube formation in vitro and induced strong apoptosis of cocultured cancer cells, suggesting a potential bystander effect that might be useful for therapeutic applications. Similar antitumor findings were observed when Mda7 or IFNb were delivered by ASCs, with induction of strong cell cycle arrest, and viability reduction by an increase in the apoptosis of cocultured tumor cells as assessed by Caspase 3/7 detection. The transgene expression was overall non-toxic and did not appear to interfere with the normal differentiation potential of ASCs (osteogenesis or adipogenesis). Therefore, this novel cellular delivery method for gene therapy appears to be effective and highly promising for translational applications.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3368. doi:1538-7445.AM2012-3368