bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1133: Inactivation of BRG1 in non-small cell lung cancer (NSCLC) cell lines
Authors: Zhang Wei , Valencia Ilse , Yan Jingsheng , Chari Raj , Girard Luc , Soh Junichi , Tam Kit , Xie Yang , Lam Wan , Minna John , Gazdar Adi
Published: February 2011

Purpose: To study the frequency and gene expression profile of BRG1 inactivation in NSCLC cell lines. Background: BRG1 is the catalytic subunit of SWI/SNF chromatin remodeling complex and provides ATPase activity necessary for transcriptional regulation of many genes. BRG1 is frequently inactivated in lung cancers and loss of BRG1 correlated with poor prognosis. Methods: Forty NSCLC cell lines containing various gene mutations were analyzed. BRG1 cDNA was sequenced and SNP analysis was used to confirm the large deletion mutants. Gene expression profile was determined by Illumina human WG-6 V3 beadchip (detecting ∼ 48,000 transcripts, ∼25,000 genes). BRG1 mRNA levels were determined by both RNA microarray and qPCR methods. BRG1 protein expression was analyzed by immuno-blotting. Results: Thirteen of forty (33%) NSCLC cell lines had no BRG1 protein expression (n =11) or expressed abnormal size of BRG1 (n =2). BRG1 cDNA was sequenced in these cell lines as well as control cell lines. While 10 control cell lines with normal BRG1 protein expression were not found to contain mutations, eleven cell lines without BRG1 protein expression and one containing abnormal size BRG1 protein were found to have mutations, including deletions, point mutations and insertion. The deletions and point mutations of BRG1 are homozygous. Mutant BRG1 expressed either no or lower than normal levels of BRG1 mRNA. BRG1 inactivation was found more frequent in smokers (50%) than non-smokers (15%), and coexisted with KRAS mutation, but was mutually exclusive to EGFR mutation. Furthermore, inactivation of BRG1 and LKB1, a tumor suppressor gene also located on chromosome 19p and adjacent to BRG1, showed 73% concordance (P=0.03). Finally, RNA microarray analysis showed nearly 800 transcripts (False Discovery Rate <5%) down or up regulated in NSCLC cell lines with BRG1 inactivation compared to cell lines with wild type BRG1. Conclusions: 1) BRG1 is frequently mutated in NSCLC cell lines by deletions or point mutations. 2) There is excellent concordance (98 %) between mutation status and loss of protein expression. 3) Inactivation of BRG1 shows concordance with smoking history and LKB1 mutation, and is negatively correlated with EGFR mutation. 4) Inactivation of BRG1 has a major impact on gene expression profile of NSCLC cell lines. These data indicate that BRG1 may play an important role in lung tumorigenesis.

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 1133.
Abstract 1134: Analysis of the tumor supressor role of BRG1 in lung cancer
Authors: Romero-Ferraro Octavio A. , Gimenez-Xavier Pol , Morris Stephanie , Jhon Sam , Hager Gordon L. , Sanchez-Cespedes Montserrat
Published: February 2011

The BRG1 protein features a bromodomain and helicase/ATPase activity and is a central component of the SWI/SNF chromatin remodeling complex. At present, the tumor suppressor role of the BRG1 gene, also called SMARCA4, is strongly supported by several genetic and functional observations. First of all, somatic and homozygous genetic inactivation of BRG1 has been observed in a variety of cancer cell lines and studies in lung cancer cells have estimated the frequency of BRG1 inactivation in about thirty per cent of this tumor type. Here we have cloned two BRG1 mutations, lacking the ATPase domain, found in lung cancer cell lines. One of these is a missense change in a highly conserved aminoacid within the ATPase domain (W764R) identified in the NCI-H2126 cell line while the other is an in-frame deletion (E668-Q758 del) identified in the NCI-H1703 cell line. We have studied the capability of these mutations to affect the biological function of BRG1 and compared the efficiency of the wild type BRG1 and that of the two mutants in reducing cell growth in cancer cells. To determine the effect of these mutations on the activity of BRG1 we took advantage of a transcription reporter assay in the human lung carcinoma cell line (NCI-H1299) that lacks wild type BRG1 due to a homozygous mutation. In this system, the simultaneous transfection of the BRG1 constructs and a construct carrying the MMTV-Luciferase reporter plasmid and a GR (glucocorticoid) expression plasmid allows the determination of the contribution of BRG1 to the hormone-dependent regulation of MMTV transcription. Transfection of H1299 cells with these constructs resulted in an eightfold increase of MMTV transcription in the presence of hormone when the cells were transfected with the wild type BRG1 as compared as to the cells transfected with either the mutants or the empty vectors. In addition, we observed that overexpression of wild type BRG1, but not the mutant proteins, in several lung cancer cell lines lead to cell growth suppression. Thus, our present data support an important role of BRG1 tumor suppressor gene in cancer development.

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 1134.
Abstract 1135: Enhanced activity of the CREB co-activator Crtc1 in LKB1 null lung cancer
Authors: Park Yoonsoo , Coxon Amy , Komiya Takefumi , Chen Wei-Dong , Zajac-Kaye Maria , Meltzer Paul , Karpova Tatiana , Kaye Frederic
Published: February 2011

Activation of Crtc1 (also known as Mect1/Torc1) by a t(11;19) chromosomal rearrangement underlies the etiology of malignant salivary gland tumors. Since LKB1 is a target for mutational inactivation in lung cancer and was recently shown to regulate hepatic Crtc2/CREB transcriptional activity in mice, we now present evidence suggesting disruption of an LKB1/Crtc pathway in cancer. Although Crtc1 is preferentially expressed in adult brain tissues, we observed elevated levels of steady-state Crtc1 in thoracic tumors. In addition, we demonstrate that somatic loss of LKB1 is associated with underphosphorylation of endogenous Crtc1, enhanced Crtc1 nuclear localization, and enhanced expression of the Crtc prototypic target gene, NR4A2/Nurr1. Inhibition of NR4A2 was associated with growth suppression of LKB1-null tumors, but showed little effect on LKB1-wildtype cells. These data strengthen the role of dysregulated Crtc as a bona fide cancer gene, present a new element to the complex LKB1 tumorigenic axis, and suggest that Crtc genes may be aberrantly activated in a wider range of common adult malignancies.

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 1135.
Abstract 1146: Three-color array CGH differentiates constitutional from acquired CNVs in cancer genomes
Authors: Jewell Ursula R. , Moon So Young , Benjes Suzanne M. , Morris Christine M.
Published: February 2011

In recent years microarray-based comparative genomic hybridisation (CGH) has become the assay of choice for high-resolution screening of genetic aberrations in research and clinical diagnostics, and has revealed a plethora of small, constitutional copy number variations (CNVs) in healthy individuals. The appearance of these benign CNVs on the array profile affects the ability to identify CNVs that are acquired and pathogenic, in particular if these are also small in size. It is possible to eliminate constitutional CNVs by using a matched self-reference for each sample, but this compromises ability to compare array profiles between different individuals. In addition, the ability to link specific constitutional CNVs or combinations of these to disease susceptibility is lost.

We have developed a new 3-color microarray CGH assay which allows identification of constitutional CNVs while retaining the ability to compare between different samples by simultaneous application of two distinctly labelled reference samples. The new 3-color microarray CGH assay uses three spectrally distinct fluorescent dyes to label the test sample, matched self-reference, and generic reference, for simultaneous hybridization to the same array platform. We have applied this new protocol to the analysis of samples from healthy individuals, leukemia patients, and the leukemia-derived cell line K562, and performed pair-wise comparison of 26 array-CGH profiles from the new 3-color array with corresponding profiles from conventional 2-color arrays.

Our results demonstrate that introducing the third dye has no adverse effect on array assay noise, as measured by paired ttest of the standard deviation of the log2 ratio of all clones on the 2-color and 3-color array profile. Copy number calls from the new 3-color array assays show a consistency of up to 97% with the corresponding conventional 2-color array assays, equivalent to the level of consistency that was achieved when repeating array-CGH on the same sample using the same protocol. The prevalence-adjusted Cohen Kappa test indicates almost perfect agreement between the new 3-color assay and conventional 2-color assay. Application of the new 3-color microarray assay has enabled us to identify 44 constitutional CNVs in 57% of our leukemia samples. At least one third of these CNVs have been reported as sites of normal copy number variation and are listed in The Human Genome Variation Database.

Introducing a third sample into the microarray assay is a new and more economical approach for high resolution genome-wide comparison of DNA, and therefore potentially amenable to uptake in both research and diagnostics.

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 1146.
Abstract 1152: A novel, practical method for improving the sensitivity and reproducibility of immunodetection
Authors: Hofseth Lorne J. , Bailey Anne
Published: February 2011

The sedentary nature of standard immune-detection techniques on a slide (e.g. immunohistochemistry, immunocytochemistry, and/or immunofluorescence) leads to the potential for tissue drying, and antibody pooling, ultimately resulting in uneven and inconsistent staining which can yield false negative or under readings. We therefore hypothesized that if we could provide motion in the antibody fluid while submerging the tissue laden slide, we could prevent such disadvantages. Here, we present data indicating that rocking such slides in an antibody bath leads to extremely even staining, highly reproducible results and greatly enhanced sensitivity. We found that for both nuclear and anti-nuclear antibodies, reaction sensitivity was increased 1000X to 1,000,000X over best practice with standard immune detection techniques. The immunodetection scores for p-P53/Serine 15, Ki-67 and Hsp 70 as well as experiment photographs comparing the sedentary to dynamic methods results are presented. References are also given.

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 1152.
Abstract 1156: Applying targeted Next-Gen Sequencing and miRNA expression profiling for cancer biomarker discovery
Authors: Leonard Jack T.
Published: February 2011

The Biomarker Discovery Center in Heidelberg, Germany develops new approaches to cancer biomarker identification and validation using high throughput targeted next-generation sequencing and miRNA assays to characterize patient samples from the German Cancer Research Center (DKFZ) and other leading research institutions. Using HybSelect™, febit's product for sequence capture, we demonstrated high throughput processing of patient samples upstream of targeted Next-Gen Sequencing. Overlapping probes were designed to capture the genomic regions of interest (i.e., targets), and then the Geniom Biochips were synthesized for the HybSelect procedure. DNA libraries were prepared from clinical specimens using bar-coded sequencing adaptors from Life Technologies, and the targets were enriched using the HybSelect Sequence Capture Protocol. Bar-coded targets from hundreds of patient samples were sequenced using a SOLiD System with a febit Geniom RT Analyzer. The variants were compared to the reference sequences from healthy individuals. In a related study, changes in miRNA expression profiles were examined in certain cancers. The relative stability of miRNA and the relatively accessible nature of bodily fluids such as blood make miRNA a promising molecule for cancer biomarker discovery. For evaluation of the miRNA expression profiles of cancer patients, we utilized a Microfluidic Primer Extension Assay (MPEA), which starts with the hybridization of unlabeled total RNA samples to Geniom miRNA Biochips. After hybridization, selective enzymatic elongation of the correctly hybridized miRNAs was carried out with biotinylated nucleotides, and then detected with SAPE (streptavidin-conjugated phycoerythrin). The content included on Geniom miRNA Biochips was based on the latest Sanger miRBase update, thus guaranteeing that all human miRNAs, including the most recently validated miRNAs were addressed. With only 130 ng of total RNA required, this method dramatically reduced the amount of sample needed to reliably measure miRNAs and to develop predictive miRNA signatures. Analyses of complex miRNA expression patterns in blood of cancer patients compared to those of healthy donors allowed for distinct classification of the samples.

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 1156.
Abstract 1158: Single step multiplex amplification and barcoding of FFPE samples for Next-Gen sequencing
Authors: Isaksson Magnus , Johansson Henrik , Roos Fredrik , Falk Elin , Moens Lotte , Ericsson Olle , Nilsson Mats
Published: February 2011

We present data outlining application of the Selector Technology to successfully amplify and sequence sets of exons in both fresh frozen and formalin fixed paraffin embedded tumor samples. We also show preliminary data where the Selector Technology has been adapted to incorporate sequencing primers and barcodes during amplification thereby replacing the laborious sample preparation with a single PCR step which open up for streamlined large scale parallel sample preparation of patient cohorts. The technology has been scaled to amplify as many as 17,000 amplicons in one single reaction tube. The Selector Technology is suitable for convenient and scalable single tube amplification of large sets of genes. The technology provides unbiased amplification of exons for resequencing providing unparalleled on target alignment of >95% of reads mapping to amplified regions and >90% of exon base bp at 20-30% of average amplicon sequencing depth providing cost effective high coverage resequencing. Concordance with HapMap genotypes is 99% and the reproducibility (R2) between replicates is 0.98 at the single nucleotide level. High reproducibility provides the possibility to score copy number variations in tumor samples as well as calling copy number neutral loss of heterozygosity. These features are demonstrated using matched tumor-normal breast cancer samples. Conclusively, the Selector Technology is a convenient, high performance technology for large scale sample preparation of cancer samples.

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 1158.
Abstract 1164: Parallel preparation of targeted resequencing libraries from 480 genomic regions using multiplex PCR on the Access Array system
Authors: Kaper Fiona , Wang Jun , Anderson Megan J. , Chen Peilin , Lin Min , Pieprzyk Martin , Jones Robert C. , May Andrew P.
Published: February 2011

Next generation sequencing platforms have dramatically reduced sequencing costs. However, it currently remains too expensive to routinely resequence entire human genomes in order to discover genetic variants or somatic mutations underlying tumorigenesis. Therefore, a need exists for multiplexed, targeted amplification methods that allow for the analysis of multiple genomic regions in large cohorts. Available targeted enrichment technologies are either aimed at the capture of regions of interest from a single sample, exhibit uneven representation or require significant amounts of input material. The novel microfluidic platform, the Access Array system, combines 48 samples with 48 primer sets resulting in 2,304 simultaneously occurring PCR amplifications requiring as little as 50ng DNA per sample. PCR products generated on the Access Array system can be used for sequencing on all next-generation sequencing platforms, including 454 GS FLX and Illumina GAII. To increase coverage and throughput, PCR reactions can be multiplexed within Access Array chips generating up to 480 amplicons per sample.

As proof-of-principal experiments, we have carried out multiplexed amplifications of a set of commonly mutated cancer gene exons across 48 genomic DNA samples. In initial experiments, 580 primer pairs were validated in individual PCR reactions in 96-well plates. Each primer pair was designed to include 5’ sequences that allow for the incorporation of 454 and Illumina adapters necessary for subsequent emPCR and cluster generation, respectively. 480 primer pairs that produced a single band of the correct size, as determined on a Caliper LabChip system, were selected for multiplex PCR experiments. Primer pairs yielding amplicons with a similar size were combined in groups of 10 sets, resulting in 48 primer pools of 10 primer pairs each. Multiplex PCR was carried out on Access Array chips, followed by harvesting of the 48 amplicon pools. Each pool was diluted and then subjected to a second round of PCR in standard 96-well plates with barcoded universal primers corresponding to the 454 and Illumina sequences. The resulting products are 48 uniquely barcoded amplicon pools, each comprising 480 amplicons derived from one sample, that are ready for sequencing. We will present sequencing data generated on both 454 and Illumina systems.

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 1164.
Abstract 1165: Image-guided Raman endoscopy for in vivo clinical detection of cancer in the stomach
Authors: Zheng Wei , Teh Seng Khoon , Ho Khek Yu , Teh Ming , Yeoh Khay Guan , So Jimmy Bok Yan , Zhu Feng , Huang Zhiwei
Published: February 2011

Objective: Raman spectroscopy is a unique optical vibrational spectroscopic technique that is capable of noninvasively probing biomolecular changes associated with cancerous transformation. The aim of this study was to investigate the feasibility of a novel imaged-guided Raman endoscopy system developed for distinguishing cancer lesions from the non-cancerous tissues in the human stomach in vivo.

Materials and Methods: A fiber-optic Raman endoscopy system was utilized for tissue Raman spectroscopic measurements at 785 nm laser excitation. A total of 20 in vivo gastric Raman measurement sites were used in this study, in which 15 sites were from 10 patients who underwent gastroscopic inspection, and 5 were from the suspicious lesion sites of 5 patients who underwent endoscopic ultrasound procedure. The histopathogical examinations on the biopsied tissues from the Raman measurement sites showed that 15 were non-cancerous and 5 were cancers. Multivariate statistical algorithms including principal component analysis (PCA) and linear discriminant analysis (LDA) were employed to determine the diagnostic sensitivity and specificity.

Results: High-quality in vivo gastric Raman spectra in the range of 800-1800 cm−1 can be collected in less than 1 second, together with the simultaneous utilization of white-light reflectance imaging modality for guiding the Raman endoscopic probe to the suspicious tissue sites. Raman spectral shapes were significantly different between non-cancerous and cancers, particularly in the spectral ranges of 850-900 cm−1, 1000-1090 cm−1, 1200-1305 cm−1 and 1600-1800 cm−1 which contain signals related to hydroxyproline for collagen, phenylalanine, amide III, amide I and C=C stretching of lipids. The Raman technique together with PCA-LDA-based analytic algorithm yielded diagnostic sensitivity of 96.1% and specificity of 100% for detection of cancer from the non-cancerous gastric tissues in vivo.

Conclusion: This study illustrates for the first time that the image-guided Raman endoscopy technique has promising clinical potential for rapid in vivo diagnosis of cancer tissues in the stomach.

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 1165.
Abstract 1174: Mechanisms of Fn14 overexpression in invasive breast tumors
Authors: Niska Joshua R. , Willis Amanda L. , Ramos Pilar , Mancini Catherine M. , Getz Julie E. , Cunliffe Heather E.
Published: February 2011

The fibroblast growth factor-inducible 14 (Fn14) signaling axis plays an important role in the regulation of breast cancer cell invasion. Breast tumors express Fn14 whereas normal mammary epithelium does not, and Fn14 expression correlates with poor prognostic indicators. The molecular mechanisms driving aberrant Fn14 expression in breast cancer have not yet been determined. This investigation aims to elucidate the mechanisms underlying Fn14 over-expression. Soluble tumor necrosis factor-like weak inducer of apoptosis (TWEAK), the cognate ligand for Fn14, was found to induce Fn14 expression in some breast cancer cell lines via a proposed autoregulatory mechanism. We also provide evidence to support the likelihood that membrane-bound TWEAK can stimulate Fn14 expression through adjacent tumor-cell interaction. EGF and a variety of other receptor tyrosine kinase ligands known to play a role in breast tumor progression stimulated de novo Fn14 expression in a panel of breast cancer cell lines. We therefore hypothesized that Fn14 expression may be induced by aberrant activation of the MAPK signaling pathway. Using an MCF7 model of TPA-mediated MAPK pathway activation and in vitro invasion using Matrigel-coated Transwell Boyden chambers, we observed TPA-mediated expression of Fn14 concomitant with a dramatic increase in cell invasion. Fn14 expression and invasion in this model were abrogated in the presence of U0126, a specific inhibitor of Mek1/2, implicating a role for MAPK signaling in regulation of Fn14 expression. From previously published microarray-based surveys of genomic aberrations in breast cancer, we also hypothesized that constitutive Fn14 expression may be due to Fn14 gene amplification. We performed fluorescence in situ hybridization analysis of 53 breast tumors and identified 15/53 (28%) tumors with additional copies of Fn14 relative to centromere 16. Fn14 was not amplified in normal breast epithelium or in Fn14-negative cell lines. We conclude that deregulation of Fn14 expression can occur through diverse molecular mechanisms associated with breast tumor progression. A more complete definition of the spectrum of molecular aberrations leading to induction of Fn14 expression in breast cancer requires further studies.

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 1174.
Abstract 118: Analysis of biological pathways associated with epirubicin and/or cyclophosphamide response by pathway enrichment analysis in human breast cancers
Authors: Shen Kui , Rice Shara , Wang Dakun , Gingrich Dave , Brower Stacey , Ervin Paul , Gabrin Michael , Song Nan
Published: February 2011

Recently, a number of different approaches for improving cancer treatment have been developed. One scientific approach is to understand the biochemical pathways and coding genes involved in cancer causation, while other approaches look to distinguish the biochemical pathways involved in drug response. Many chemotherapeutic drugs have been developed targeting different cellular properties, and even more targeted therapies impacting specific oncogenic pathways are on the horizon. Pathways implicated in drug response have been studied to determine why some tumors might respond to a given chemotherapy while others do not. Understanding these pathways may provide potential for improving therapeutic efficacy. Pathway analysis methods have been proposed to discover pathways of interests using gene expression profiling.

In this study, the biological pathways associated with response to Epirubicin (E), Cyclophosphamide (C) or the combination of the two drugs (combo EC) were evaluated in both breast cancer cell lines and patients. Thirty immortalized breast cancer cell lines were evaluated for response to these drugs using ChemoFx®. Area under the dose-response curve (AUC) was calculated to measure the chemosensitivity. We applied pathway enrichment to detect specific biochemical pathways associated with these drugs based on biological pathway databases (Kyoto Encyclopedia of Genes and Biocarta), breast cancer cell lines’ drug responses and public microarray data. Comparison of pathways associated with single drug E, C, and combo EC revealed that there are pathways associated with drug combination but not significantly associated with single drugs. These drug response-associated pathways offer great potential for new biomarker identification and drug targeting discovery.

In addition, EC associated pathways in breast cancer patients were identified using public microarray data and the associated patient clinical outcomes. Of importance, four biological pathways related to adherens junction, leukocyte transendothelial migration, regulation of actin cytoskeleton and ribosomal protein were identified as EC response-associated pathways in both breast cancer cell lines and patients. These common EC response-associated pathways indicate the potential possibility to use cell line in vitro drug responses to predict patient chemotherapy outcomes.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 118.
Abstract 1186: PTEN expression predicts biochemical recurrence in prostate cancer
Authors: Stone Steven , Sangale Zaina , Veneroni Silvia , Reid Julia , Colecchia Maurizio , Carlson Amy , Valdagni Riccardo , Gutin Alexander , Daidone Maria G. , Zaffaroni Nadia
Published: February 2011

Prostate cancer is the most commonly diagnosed malignancy in men, but many patients have indolent disease that is unlikely to cause significant morbidity or mortality. Biomarkers that accurately predict disease severity would enable physicians to more appropriately manage patient care. Data from previous studies indicated that the molecular status of PTEN (phosphatase and tensin homolog) might be prognostic in prostate cancer patients after radical prostatectomy (RP). PTEN expression was evaluated on surgical specimens by IHC using anti-PTEN antibody 138G6 (Cell Signaling Technology) in 132 men treated with RP. Approximately half the patients also had neoadjuvant hormonal blockade therapy (BAT). PTEN IHC scores were derived from the weighted average of percent positive cells x intensity (0, 1 or 2). PTEN status (positive or negative) was determined using a predefined IHC score of ≤ 20 for negatives. Using this scoring system, 24% of the tumors were PTEN negative. Association with biochemical recurrence was evaluated using Cox PH models and likelihood ratio tests. Since we were testing a prior hypothesis about the prognostic utility of PTEN, the corresponding p-values are one-sided. In a univariate analysis on the overall series, PTEN status was associated with biochemical recurrence (p-value = 0.0046, HR = 2.3; 95% CI: 1.27, 4.33). Most of the clinical parameters typically associated with recurrence were affected by neoadjuvant therapy and rendered uninterpretable. Nevertheless, pathological tumor stage (p-value = 4.3 × 10−6, HR = 6.0; 95% CI: 2.35, 15.22) was still associated with outcome. In multivariate analyses, PTEN status was a significant predictor of outcome after adjusting for pathological stage (p-value = 0.009), neoadjuvant therapy (p-value = 0.014), or both (p-value = 0.04). These data support the hypothesis that PTEN status predicts outcome after RP, and suggest further, that PTEN may be a clinically important molecular marker for defining aggressive prostate cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1186.
Abstract 1187: The role of PI3K as a biomarker
Authors: Amatulli Michael , Brennan Laura A. , Deevi Dhanvanthri S. , Prewett Marie , Tonra James R. , Paz Keren
Published: February 2011

The phosphatidylinositol 3-kinase (PI3K) signaling pathway has been implicated in tumorigenesis. Evidence over the past years suggests a pivotal role for the catalytic subunit, PIK3CA, in human cancers. Receptor tyrosine kinases (RTKs) on the cell surface are key regulators of PI3K activity, and RTK antagonists have demonstrated significant anticancer effects in animal models and in patients. However, among tumor harboring activating mutations of PIK3CA, the therapeutic value of RTK antagonists may be compromised. The mutation status of PIK3CA may, therefore, serve as a potential biomarker for both prognosis and response to RTK inhibitors.

Somatic mutations of the pik3ca gene occur primarily in three hot spots, E542K, E545K (exon 9) and H1047R (exon 20). These mutations are detected in a broad spectrum of cancer indications including breast, colorectal and lung. This study was designed to explore the nature of PIK3CA mutations among numerous human cancer cell lines, utilizing mostly cells with the capacity to form tumors in mice. Primers corresponding to exon 9 and 20 of human pik3ca gene were designed. Genomic DNA was harvested from over 200 lines, derived from different human cancer indications. The pik3ca locus was sequenced and its WT or mutation status was determined.

Of these cells, 8% presented mutations equally distributed between exon 9 and 20. The frequency of PIK3CA mutations was particularly high among colon cancer lines, with fewer incidences among lung, breast and ovarian cancer lines. Interestingly, 50% of the PIK3CA mutated lines presented a concurrent mutation of either KRAS or BRAF. The capacity of PIK3CA mutation-bearing cell lines to respond to RTK blockers was then evaluated, and the mechanism contributing to such phenomenon was further discussed.

These results stress PI3K diagnosis value and its potential role as a valid biomarker for patient susceptibility.

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 1187.
Abstract 1191: Flotillin-2 overexpression is associated with an invasive urothelial cell carcinoma phenotype
Authors: Veerakumarasivam Abhimanyu , Saeb-Parsy Kasra , Scott Helen E. , Neal David E. , Kelly John D.
Published: February 2011

Invasive urothelial cell carcinoma (UCC) is associated with poor clinical outcome and is characterized by a genotype that is distinct from superficial disease. Exploiting a multi-component mining strategy on a set of high-throughput genome-wide experimental data, we probed for genes associated with an invasive phenotype. Fresh frozen UCC tissues were profiled using global expression microarrays and array-based comparative genomic hybridization (aCGH). Differentially expressed genes were functionally annotated by gene ontology and compared to available SAGE libraries and expression array datasets. We identified overexpression of a lipid raft associated protein, Flotillin 2 (FLOT2) in invasive UCC. The FLOT2 locus (17q11-q12) was associated with copy number gains in 15% of the tumors. We further characterized Flot2 protein expression in an independent tissue microarray series by immunohistochemistry. Flot2 protein expression increased with clinical risk. Interestingly, carcinoma in situ (CIS) tumors demonstrated highest level of expression. In vitro, FLOT2 expression increased with the degree of invasiveness of the UCC cell lines. Knockdown of FLOT2 lead to a significant reduction in the invasive phenotype; whilst overexpression of wild-type FLOT2 enhanced invasion in non-invasive cells. The precise mode of action of Flot2 remains to be elucidated but it is predicted to play an important role in transmembrane signal transduction, cell adhesion and endocytosis. FLOT2 overexpression has also been shown to enhance the spreading of cells, formation of filopodia as well as melanoma progression and metastasis. Thus, the functional targeting experiments and gene-dosage dependent FLOT2 overexpression in invasive UCC confirm a link between FLOT2 and pro-invasive cancer phenotype.

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 1191.
Abstract 1193: Prospective validation of multiple biomarkers for improved clinical decision-making in patients with urothelial carcinoma of the bladder
Authors: Lotan Yair , Youssef Ramy , Shariat Shahrokh F. , Alhalbi Feras , Ashfaq Raheela , Sagalowsky Arthur I.
Published: February 2011

Objectives: To prospectively assess the predictive value of a panel of biomarkers (cyclin E1, p53, p21, p27 and pRB/Ki-67) for staging of patients with urothelial carcinoma of the bladder (UCB).

Methods: 174 consecutive patients treated with transurethral resection (TUR) and/or radical cystectomy (RC) for UCB were prospectively included in the study starting in January 2007. Standardized, automated immunohistochemical staining and scoring was performed. A prognostic score (PS; ≤2 altered biomarkers=Favorable; >2 altered biomarkers=Unfavorable) was defined and correlated with clinical and pathological data.

Results: The study comprised of 102 RC patients and 72 TURBT cases. Unfavorable PS were noted in 36.3 % of RC cases and 16.6 % of TURBT cases matching the more advanced pathological stages of RC patients. In RC group; an unfavorable PS was significantly associated with advanced tumor stage (p=0.02), the presence of LVI (p=0.017), recurrence (p=0.02) and survival (p=0.048). In the subgroup of patients who had biomarker evaluation at TUR and then underwent RC (n=32), 75 % and 50% of the cases with unfavorable PS showed upstaging and LN involvement at RC versus 25 % and 12.5 % in cases with favorable PS. Unfavorable PS at TUR was associated with the presence of LVI (p=0.001) and LN metastasis (p=0.026). Conclusions: The preliminary analysis of our ongoing prospective trial strongly suggests that a panel of five biomarkers improves the identification of patients at risk of upstaging at RC. An unfavorable PS may identify patients who are most likely to benefit from neo-adjuvant and adjuvant chemotherapy combined with RC.

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 1193.
Abstract 120: Cellular import of the inhibitor of apoptosis (IAP) protein survivin
Authors: Khan Salma , Slater Jessica M. , Neidigh Jonathan W. , Wall Nathan R.
Published: February 2011

The inhibitor of apoptosis (IAP) protein survivin is found overexpressed in virtually all human cancers and has been shown to correlate with poor prognosis. Recent work performed by our laboratory and that of others has demonstrated that survivin, when found or expressed extracellularly, is associated with a more aggressive disease form. In a variety of cancer cell lines grown in conditioned medium, rich with a flag/HA-tagged form of survivin, intracellular accumulation was readily detected. We found that survivin was internalized constitutively at a rate that was not affected by chelation of the conditioned medium in HeLa S cervical cancer cells. Instead, survivin internalization was modestly affected by lipid membrane stasis, induced by cooling the cells and the conditioned medium prior to and during incubation leading us to investigate if survivin's intracellular accumulation was dependent upon clathrin-coated pit-dependent endocytosis. Using dual-affinity protein purification and mass-spectrometry, survivin was found associating directly with clathrin, TNFR1, and the transferrin receptor (TfR), indicating that the process of survivin uptake may be through endocytosis. In addition, short-inhibitory RNA (siRNA) knockdown of TNFR1 and TfR as well as antibody blocking of these receptors significantly reduced survivin's intracellular accumulation. Further characterization of this novel survivin pool and its intracellular accumulation may reveal new strategies for the downregulation of survivin in cancer and ultimately better treatment strategies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 120.
Abstract 1202: MDM2 and its interaction with MYCN in neuroblastoma survival
Authors: He Jing , Gu Lubing , Li Jiansha , Zhang Hailong , Zhou Muxiang
Published: February 2011

The multifunctional oncoprotein MDM2 plays both p53-dependent and p53-independent roles in cancer initiation, progression and the development of resistance to therapy. Using a siRNA strategy to silence MDM2, which is highly expressed in neuroblastoma, we discovered that MDM2 regulates tumor cell growth and survival through distinct mechanisms in MYCN-amplified and non-MYCN-amplified neuroblastomas. MDM2 inhibition in a MDM2-overexpressing/non-MYCN-amplifying neuroblastoma cell line, SH-EP1, resulted in p53 activation, growth inhibition and apoptosis. In contrast, MDM2 inhibition in the MDM2-overexpressing/MYCN-amplifying neuroblastoma cell lines NB-1691 and IMR32 did not activate p53, despite expression of wild-type p53 in these lines. However, the siRNA silencing of MDM2 in NB-1691 and IMR32 cells did lead to complete inhibition of cell proliferation. In examining MYCN expression in these MYCN-amplified cells, we found that inhibition of MDM2 significantly downregulated MYCN expression at both the mRNA and protein levels. In a p53-null/MYCN-amplifying neuroblastoma cell line, LA1-55N, inhibition of MDM2 also decreased MYCN expression, inhibiting cell growth and apoptosis. These results suggest that MDM2 plays a p53-independent role in MYCN-amplified neuroblastomas. A critical factor for disease progression, we believe deregulation of MYCN expression may contribute significantly to the oncogenic properties of MDM2 in neuroblastoma.

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 1202.
Abstract 1205: Frequent detection of MDMX isoforms in human ovarian carcinomas
Authors: Loebel Claudia , Wolf Anja , Richter Julia , Thomssen Christoph , Hauptmann Steffen , Bartel Frank
Published: February 2011

The p53 tumor suppressor pathway is inactivated in many human tumors either by p53 mutations or through overexpression of negative regulators, such as MDM2 and MDMX. In analogy to MDM2, besides the full-length mRNA several transcript variants, alternatively, as well as, aberrantly spliced transcripts of MDMX have been identified in different tumor cell lines. However, so far there are no studies regarding the expression of MDMX transcript variants in ovarian cancer tissue samples. We therefore screened 34 snap frozen ovarian tumor samples, using a high-temperature First-Strand cDNA synthesis kit to avoid reverse transcriptase artefacts. We identified in addition to MDMX-S numerous new MDMX transcripts in 16 of the 34 tumor samples that were either spliced at genuine exon/intron boundaries or at cryptic splice sites within exons. Furthermore, we could detect the transcript variants MDMX-A and MDMX-XALT2 firstly in ovarian cancer tissue. The MDMX splice variants can be classified into two groups: some isoforms have only retained the p53 binding domain, such as MDMX-S; in contrast, other splice forms consist of an intact C-terminus including the RING-finger domain, such as MDMX-XALT2. In addition, we have analyzed the expression of MDMX splice forms in ovarian cancer cell lines after treatment with chemotherapeutic agent. We observed a significant increase of the MDMX-S splice form while the expression levels of full-length MDMX decreased. This suggests that MDMX splice forms may play a role in mediating chemoresistance by attenuating the p53-pathway. In ongoing studies, we are investigating the role of frequently detected MDMX splice forms regarding their impact on chemoresistance in ovarian cancer in more detail.

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 1205.
Abstract 1209: Par-4 as therapy for colon cancer
Authors: Irby Rosalyn B. , Kline Christina L. , Sharma Arun K.
Published: February 2011

Par-4 (Prostate apoptosis response-4) is a tumor suppressor that induces apoptosis in tumor cells in response to apoptotic stimuli. It has been reported to be reduced in a number of human cancers. We examined the expression levels of Par-4 in human colon cancers and explored the possibility of using Par-4 as a treatment for colon cancer. We found that expression of Par-4 is reduced in human colon cancers 2.65-fold compared to paired normal colon. To assess the role of Par-4 in colon cancer growth, the human colon cancer cell line, HT29, with low native Par-4 expression, was transfected to overexpress Par-4. It has been shown that apoptosis was higher in the transfected cells than in the mock transfected cells. It was also shown that tumors growing in nude mice were smaller when the cells were transfected to overexpress Par-4. Importantly for therapeutic considerations, it has been reported that Par-4 expressing cells can have a bystander effect on tumor cells that are not transfected, also inducing apoptosis in the untransfected cells. We hypothesized that HT29 colon cancer cells producing Par-4 in vivo would secrete Par-4 into the animal system and result in lower tumor volume of tumors created with wild type cells growing distally.

Methods: To show proof of concept, nude mice were injected subcutaneously with wild type HT29 cells. Half of the mice were also injected with HT29 cells overexpressing Par-4 in the opposite flank. Mice were treated with 5-fluorouracil to test the sensitivity of tumors to the chemotherapy.

Results: At the end of the first week, the wild type tumors in the mice with Par-4 tumors also were 1.8-fold smaller (p=0) than those tumors in mice who received only wild type tumor cell injections. Over the course of three weeks, the wild type tumors growing in mice without Par-4 tumors increased in volume 12-fold more than those wild type tumors growing in mice with Par-4 tumors. In addition, in the event that Par-4 tumors resolved completely (5% of the cases) the wild type tumors in those mice showed increased growth rates. This data suggests that overexpression of Par-4 in tumor cells in vivo not only sensitized those cells to chemotherapy, but had a bystander effect on tumor cells growing distally to the Par-4 overexpressing cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1209.
Abstract 1216: The mechanism related to anti-apoptotic property of SOX2 in prostate cancer
Authors: Jia Xianpei , Li Xuefei , Mou Wenjun , Zhang Shu , Tan Xiaoyue , Liu Cheng-Hu , Xiang Rong , Li Na
Published: February 2011

SOX2 is one of the key regulatory genes to maintain the pluripotency and unlimited cell growth properties in embryonic stem cells. Recently, more and more researches on cancer stem cell (CSC) support the hypothesis that CSC is the progenitor of cancer cells and contributes to the chemoresistant properties of cancer cells. Although previous study showed SOX2 contributed to the tumorigenic properties in breast and gastric tumor, its function in prostate cancer and apoptosis are still largely unknown. In this study, we used immunohistochemistry method to analyze the expression of SOX2 in prostate cancer tissues and found that the degree of SOX2 staining increased when the Gleason histological grade went higher. We further investigate SOX2's function in cell growth and apoptosis process by using human prostate cancer cells-DU145 with SOX2 overexpression or down-regulation by shRNA. We found that SOX2 increased cell growth and the percentage of cells in S phase in cell cycle assay, it also increased the apoptosis resistant properties of DU145 with reduced mRNA expression of Orai1, STIM1 and decreased store-operated Ca2+ entry (SOCE) activity and cytosolic Ca2+ concentration. This study first investigated SOX2's function in apoptosis process of prostate cancer and elucidated SOX2's regulatory effect on SOCE activity. Our study supports the concept that targeting SOX2 to increase the SOCE activity may be a potential strategy in prostate cancer therapy.

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 1216.