bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4863: Streamlining NGS workflows using cancer samples by the application of the DNA Integrity Number (DIN) from the Genomic DNA ScreenTape assay
Authors: Schmidt Eva , Pechtl Isabell , McHoull Barry , Liu Melissa
Published: August 2015

The results of numerous molecular screening and assay methods often rely on the overall quality of the genomic DNA (gDNA) input material. However extraction of genetic material can be challenging and often results in low amounts or variable quality of gDNA samples, which are further subjected to time and cost intensive downstream applications. For example, array comparative genome hybridization (aCGH) and Next Generation Sequencing (NGS) can require intact, high quality gDNA to ensure high quality, unambiguous results. It is therefore widely recommended to perform an initial quality control (QC) of the input material. Especially as only the final step of these workflows reveals if meaningful results have been achieved. In order to provide an objective and automated measure to standardize the gDNA integrity assessment, a software algorithm has been developed. This functionality of the 2200 TapeStation system provides a numerical determination of the gDNA integrity and is referred to as the DNA Integrity Number (DIN). This study demonstrates how DIN obtained by the upfront QC of gDNA on the Agilent Genomic DNA ScreenTape assay has allowed for significant saving of sequencing and sample preparation overhead using cancer samples in NGS workflows.

Citation Format: Eva Schmidt, Isabell Pechtl, Barry McHoull, Melissa Liu. Streamlining NGS workflows using cancer samples by the application of the DNA Integrity Number (DIN) from the Genomic DNA ScreenTape assay. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4863. doi:10.1158/1538-7445.AM2015-4863
Abstract 4870: GenePool: A cloud-based technology for rapidly data mining large-scale, patient-derived cancer genomic cohorts including The Cancer Genome Atlas
Authors: Sanga Sandeep , Vladimirova Antoaneta , Goold Richard D. , Klingler Tod M.
Published: August 2015

GenePool is a cloud-based system for the secure storage, management, analysis, visualization and interpretation of large-scale human genomics data. The GenePool platform enables rapid interrogation of massive amounts of data generated by current laboratory methods and was designed to meet the needs of scientists engaged in cancer biomarker discovery and characterization. The software solution provides users with an intuitive interface, an easy way to store and select genomics datasets according to sample-associated metadata for analysis, and an automated system for performing routine, well-characterized genomics workflows. Results are presented with multiple options for annotation, sorting and data export, with a visualization tool that facilitates browsing of genomic data for biomarker identification. We will demonstrate how GenePool removes the data download, management, and computing burdens faced by researchers interested in working with large amounts of patient-derived cancer genomics exemplified by The Cancer Genome Atlas. To date, TCGA has completed molecular characterization of more than 9,000 patient samples from a target of 17,000, and will have released an estimated 2.5 petabytes of data at project completion.

We will present case studies using the >25 cancer cohorts worth of RNA-Seq, miRNA-Seq, Exome Sequencing, Protein Expression, Copy Number, and DNA Methylation data (provided to the community through the TCGA Data Portal) and made readily available for sample selection and analysis within GenePool. As validation of the GenePool platform, we will present results obtained within minutes that validate knowledge reported in the literature over the course of decades. We will then present novel findings for less understood cancer indications.

Note: This abstract was not presented at the meeting.

Citation Format: Sandeep Sanga, Antoaneta Vladimirova, Richard D. Goold, Tod M. Klingler. GenePool: A cloud-based technology for rapidly data mining large-scale, patient-derived cancer genomic cohorts including The Cancer Genome Atlas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4870. doi:10.1158/1538-7445.AM2015-4870
Abstract 4871: A new multiple feature approach for rapid and highly accurate somatic structural variation discovery from whole cancer genome sequencing
Authors: Xia Li C. , Bell John , Chen Jiamin , Zhang Nancy R. , Ji Hanlee P.
Published: August 2015

We present SWAN, a statistical framework with implemented software package for accurate detection of genomic structural variants in next-generation sequencing (NGS) data from cancer. SWAN is designed to sensitively detect submicroscopic and lesser size genome structural variants that could possibly present at low allele frequency in the sample as may be the case in clonal subpopulations of a primary tumor. The likelihood ratio scan statistics module of SWAN collectively quantifies evidence from read-pair, hang-reads based on Marked Poisson Process models while the softclipping module additionally integrates the evidence from remapped soft-clipped reads. Using a spike-in dataset, we compared SWAN against established structural variant callers in recovering simulated homozygous and heterogeneous variants of various sizes and types. SWAN maintained high accuracy, high precision and low false positives across this broad spectrum and outperformed all the other callers. In additional benchmark with golden standard real data from sample NA12878, SWAN again showed the best-balanced accuracy among all callers and was 30% more sensitivity in detecting validated deletions than the next runner-up caller LUMPY in 50x coverage. Even at 5x coverage, SWAN was able to find 40% of the validated deletions while LUMPY did less than 20%. As an application to whole cancer genome sequencing, we analyzed a matched sequenced colorectal adenocarcinoma sample and found 39 somatic deletions, insertions and duplications, some of which could potentially drive the cancer progression. SWAN is quite efficient in memory utilization and speed, and can be used for large-scale genome-wide structural variant genotyping and discovery based on NGS data.

Citation Format: Li C. Xia, John Bell, Jiamin Chen, Nancy R. Zhang, Hanlee P. Ji. A new multiple feature approach for rapid and highly accurate somatic structural variation discovery from whole cancer genome sequencing. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4871. doi:10.1158/1538-7445.AM2015-4871
Abstract 4873: Epicopy: Measuring DNA copy number variation using high-density methylation microarrays
Authors: Cho Soonweng , Kim Hyun-seok , Cope Leslie M. , Umbricht Christopher B.
Published: August 2015

Background:

Cancers exploit changes in both genomic copy number and DNA methylation to promote growth and escape tumor-suppressor pathways. Under the 2-hit hypothesis, a single gene is likely to be altered by multiple mechanisms at the same time and integrated analysis can sharpen the focus on the most likely drivers. Requiring larger amounts of input material and imposing additional costs may limit multiplatform analysis, especially in studies using archival tissues with long clinical follow-up information where the nucleic acids are degraded and yields are generally lower than are obtained from fresh tissues. Taking advantage of similarities between methylation arrays and SNP arrays, we developed Epicopy, a robust computational method to identify DNA copy number variation (CNV) using high-density Illumina Human Methylation 450K methylation microarrays, thereby delivering two complementary genetic and epigenetic profiles from a single chip.

Methods:

Epicopy was developed using data from thyroid carcinoma samples arrayed by The Cancer Genome Atlas (TCGA) and subsequently validated on breast and lung small cell carcinoma TCGA datasets. Using Epicopy, we identified circumstances where CNV information can be reliably measured by methylation microarrays.

Results:

Using TCGA SNP microarrays as the gold standard to assess the performance of methylation derived CNV data from the thyroid, breast, and lung small cell carcinoma datasets, we showed that Epicopy is able to detect CNVs identified by SNP arrays at a sensitivity of 0.69 and specificity of 0.90. Frequently occurring CNVs identified using Genomic Identification of Significant Targets in Cancer (GISTIC), were identified with even higher accuracy.

Conclusion:

Epicopy provides a robust method to obtain both copy number and methylation information from a single methylation microarray experiment and will add value to methylation microarrays at no additional cost to the user. Tools to highlight regions of high sensitivity and specificity will also be provided to help users decide on the feasibility of using Epicopy to identify CNVs in regions of interest. Epicopy is implemented in the R statistical language and will be made available as a freestanding package as part of the Bioconductor bioinformatics software project.

Citation Format: Soonweng Cho, Hyun-seok Kim, Leslie M. Cope, Christopher B. Umbricht. Epicopy: Measuring DNA copy number variation using high-density methylation microarrays. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4873. doi:10.1158/1538-7445.AM2015-4873
Abstract 4879: Reducing amplification artifacts in highly multiplex amplicon sequencing by using molecular barcodes
Authors: Peng Quan , Vijaya Satya Ravi , Lewis Marcus , Randad Pranay , DiCarlo John , Wang Yexun
Published: August 2015

Background

PCR amplicon sequencing has been widely used as a targeted approach for both DNA and RNA sequence analysis. Highly multiplex PCR has further enabled the enrichment of hundreds of amplicons in one simple reaction. At the same time, the performance of PCR amplicon sequencing can be negatively affected by issues such as high duplicate reads, polymerase artifacts and PCR amplification bias. Recently, people have made good progress at addressing those shortcomings by incorporating molecular barcodes into PCR primer design. So far, most work has been demonstrated using one to a few pairs of primers, which limits the size of the region one can analyze at one time.

Results

We described a simple protocol, which enables the use of molecular barcodes in highly multiplex PCR with hundreds of amplicons. Using this protocol and reference materials, we studied how molecular barcodes can increase the accuracy of variant calling at very low allelic frequency and reduce PCR amplification bias. We also evaluated its utility in profiling FFPE samples.

Conclusions

We demonstrated the successful implementation of molecular barcodes in highly multiplex PCR, at a scale many folds beyond earlier work. We showed that the new protocol combined the benefits of both highly multiplex PCR and molecular barcodes, i.e. the analysis of a very large region, low DNA input requirement, very good reproducibility and the ability to detect as low as 1% mutation with minimal false positives.

Citation Format: Quan Peng, Ravi Vijaya Satya, Marcus Lewis, Pranay Randad, John DiCarlo, Yexun Wang. Reducing amplification artifacts in highly multiplex amplicon sequencing by using molecular barcodes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4879. doi:10.1158/1538-7445.AM2015-4879
Abstract 4883: A novel target capture approach for next generation sequencing and its application to oncogenic mutation detection
Authors: Bei Yanxia , Galvin Brendan , Richard Cynthia
Published: August 2015

Due to the complexity and heterogeneity of cancer genetics, next generation sequencing has played an increasingly important role in cancer diagnosis, prognosis and therapeutics. An effective target enrichment strategy further simplifies data analysis associated with NGS and further reduces costs.

We have developed a novel target capture technique that offers advantages over traditional in-solution hybridization and multiplex PCR protocols. With this method, genomic DNA is first enzymatically nicked to generate single stranded DNA of desirable length, in a sequence independent manner. The DNA fragments are then rapidly hybridized to short probes that can be captured by beads. The off target sequence of the captured DNA, defined by the probes, is subsequently removed via enzymatic digestion. The resulting targeted sequence are ligated with NGS platform specific adaptors and amplified by PCR. With our method, genomic DNA can be converted into libraries ready for sequencing within 6 hours; and the procedure is easily scalable for automation.

To demonstrate the efficacy of this method, we have designed a cancer panel to interrogate 50 genes that are commonly mutated in cancer, and tested it on various DNA samples including FFPE. The results demonstrate that our approach captures targeted sequence with high specificity and uniformity, and detects variants with great accuracy and sensitivity.

Citation Format: Yanxia Bei, Brendan Galvin, Cynthia Richard. A novel target capture approach for next generation sequencing and its application to oncogenic mutation detection. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4883. doi:10.1158/1538-7445.AM2015-4883
Abstract 4885: TOMA OS-Seq: A clinically oriented targeted resequencing method for detecting all mutation classes in DNA isolated from FFPE, FNAs and plasma
Authors: Bouhlal Yosr , McKenzie Alexander , So Austin P.
Published: August 2015

Critical genetic errors, namely specific mutations, are required for the development and maintenance of cancer. Molecular diagnostics that leverage the breadth of next-generation sequencing (NGS) to identify this mutational landscape are therefore becoming an important clinical tool in the precision treatment of cancer. However, the widespread clinical adoption of NGS technologies is hampered not only by the challenges of assaying from various clinical matrices, but also by the complexity of the workflows utilized to create, analyze, and annotate an NGS library.

To address these challenges, an adaptation of oligo-selective sequencing (OS-Seq) was developed with a workflow designed specifically for the requirements of the clinical laboratory. Termed TOMA OS-Seq)a panel of 96 genes - each with diagnostic or prognostic value - can be sequenced end-to-end from as little as 1 ng of input material, enabling targeted library construction from diverse sources such as fine-needle aspirates (FNAs), plasma as well as formalin-fixed paraffin embedded (FFPE) tissue. The minimized number of high-efficiency steps reduces the requirement for PCR, allowing the identification of copy number alterations (CNAs), re-arrangements, insertions and deletions (indels), as well as single nucleotide variants (SNVs) from these matrices.

The performance of TOMA OS-Seq) was assessed using a panel of cell lines harboring known variants in all classes of mutations. Specifically, comparison with a 12-gene droplet digital PCR (ddPCR) based test (the Amplinome Test) demonstrated a high correlation (R2 > 0.95) of CNA measurements via TOMA OS-Seq) with ddPCR, when further challenged with FNAs. The application of TOMA OS-Seq) in assaying all mutation classes in circulating tumor DNA from plasma demonstrates the robustness and flexibility of this platform as clinically oriented targeted resequencing method.

Citation Format: Yosr Bouhlal, Alexander McKenzie, Austin P. So. TOMA OS-Seq: A clinically oriented targeted resequencing method for detecting all mutation classes in DNA isolated from FFPE, FNAs and plasma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4885. doi:10.1158/1538-7445.AM2015-4885
Abstract 4901: Comparison of gene expression platforms: RNA-Seq, Fluidigm, and Nanostring
Authors: Schleifman Erica B. , Motlhabi Maipelo , Cummings Craig , Nakamura Rin , Bosch Linda , Patel Rajesh , Do An , Watson Andrew , Sandmann Thomas , Darbonne Walter , McCaffery Ian , Peters Eric , Raja Rajiv
Published: August 2015

Accurately measuring the expression of genes in formalin-fixed paraffin embedded (FFPE) tumor tissues has long been a struggle due to the inherent degradation of RNA isolated from these materials. Accurate quantification of gene expression levels in FFPE samples can enable the testing of biomarker hypotheses in the clinic and can potentially be used for patient stratification or selection in clinical trials. Platforms such as Fluidigm, Nanostring and microarrays are currently the high-throughput technologies utilized. Each of these platforms has different drawbacks and challenges such as lack of sensitivity, reproducibility or dynamic range when working with degraded FFPE samples. Currently, whole transcriptome RNA sequencing (RNA-Seq) is the only platform that offers a truly high-throughput, sensitive, and reproducible method to accurately quantify mRNA levels in RNA derived from FFPE samples. RNA-Seq, however has not yet been widely evaluated and adapted for use with degraded FFPE samples. Here we report the direct comparison of whole transcriptome RNA-Seq with two platforms that are currently compatible with FFPE derived RNA, Fluidigm and Nanostring, to determine the accuracy and feasibility of using this technology on degraded RNA. Utilizing a collection of matched fresh frozen (FF) and FFPE samples we analyzed gene expression using all three platforms to allow for a direct evaluation of each technology. By comparing the results to the matching FF sample, we were able to determine the accuracy and sensitivity of each platform when using degraded FFPE derived RNA. By titrating down the input of FFPE RNA into the RNA-Seq library prep we were also able to define the optimal input of FFPE RNA needed to accurately and reproducibly quantify gene expression. This work allows for a systematic comparison of different gene expression platforms for their use with degraded FFPE RNA.

Citation Format: Erica B. Schleifman, Maipelo Motlhabi, Craig Cummings, Rin Nakamura, Linda Bosch, Rajesh Patel, An Do, Andrew Watson, Thomas Sandmann, Walter Darbonne, Ian McCaffery, Eric Peters, Rajiv Raja. Comparison of gene expression platforms: RNA-Seq, Fluidigm, and Nanostring. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4901. doi:10.1158/1538-7445.AM2015-4901
Abstract 4907: The Emerging technology of Yb2Ti2O7 electrolyte-insulator-semiconductor biosensors for rapid screening of targeted mutation
Authors: Cheng Han Jhou , Shin Hsun Kao , Sow Neng Pang , Tung Ming Pan , Wen Hui Weng
Published: August 2015

Genetic mutations play an important role in the development of most cancers; thus, they have become key targets of investigation in the discovery of new biomarkers to help with cancer diagnostic and prognostic prediction, as well as monitor the development of drug resistance in cancer cells. We have now developed a technique by designing several probes that contain with different types of mutations, including substitution, insertion and deletion mutations for 6 or 12 bps (Table 1). Following this, we then mapped a colorectal cancer wild type KRAS cell line Caco2. To screen for different types of gene alterations,an electrolyte-insulator-semiconductor (EIS) device, incorporating a high-κ Yb2Ti2O7 sensing membrane deposited on Si substrates through co-sputtering to screen the genetic mutations, was made. X-ray diffraction and X-ray photoelectron spectroscopy were used to explore the structural features of these films annealed at various temperatures. The Yb2Ti2O7 EIS device, which annealed at 900 °C, exhibited a higher sensitivity of 59.4 mV/pH (in the solutions from pH = 2 to pH = 12), a lower hysteresis voltage of 1.6 mV (in the pH = 7→4→7→10→7), and a smaller drift rate of 0.16 mV/h (in the pH = 7 buffer solution) than those of the other annealing temperatures. We successfully demonstrated that high-κYb2Ti2O7 EIS devices with DNA-immobilized alginate films were able to screen all the designed mutation probes in Caco2 cells, and there was clear distinction from wild type based lines. In addition, even the substitution mutations were presented high resolution. Current measurement system provides a simple and inexpensive method for rapid and accurate detection of genetic changes in target genes; therefore it provided the high potential to serve as a diagnosis tool in clinical practice in the future.

Table 1.Design oligonucleotide probes.Oligonucleotide probessequencesTest 1wild type 24 bp5′-taggcaagagtgccttgacgatac-3′deletion 12 bp5′-taggca…………cgatac-3′deletion 6 bp5′-taggcaaga……tgacgatac-3′Test 2wild type 24 bp5′-ataggtgattttggtctagctaca-3′substitution 1 bp5′-ataggtgatttAggtctagctaca-3′insertion 6 bp5′-ataggtgattttGGGAACggtctagctaca-3′

Citation Format: Jhou Cheng Han, Kao Shin Hsun, Pang Sow Neng, Pan Tung Ming, Weng Wen Hui. The Emerging technology of Yb2Ti2O7 electrolyte-insulator-semiconductor biosensors for rapid screening of targeted mutation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4907. doi:10.1158/1538-7445.AM2015-4907
Abstract 4919: Tumor heterogeneity represented in human cancer cell lines
Authors: Volpe Lysa-Anne , Foulke John , Jackson Michael , Chen Luping , Randle David H. , Gitschier Hannah J. , Gardner Kelly , Tian Fang
Published: August 2015

EGFR has been found frequently mutated in human cancers, and more than 10% lung cancer patients associated with EGFR mutation. In addition, several critical mutations within the EGFR have been shown as gain of function mutations or can cause drug resistance to the EGFR inhibitors. A panel of ATCC tumor cell lines has been characterized for EGFR mutation and EGFR signaling pathway. Particularly, lung cell line NCI-H1975, which contains both drug sensitive mutation EGFR p.L858R and drug resistant mutation p.T790M, has displayed a resistance to EGFR inhibitor.Overall, NCI-H1975 cell line can be used as a nice model to study drug resistance, as well as to screen novel EGFR inhibitors that overcome the p.T790M-mediated resistance. Moreover, we have used next generation sequencing to analyze the mutation allelic frequency of both mutations, and observed tumor heterogeneity within this cell line. Impact of cell passage numbers on the mutation allele frequency of the cell line was also examined. Furthermore, a novel single cell western blot assay has been used to analyze the EGFR mutation, EGFR protein expression and downstream signaling within individual cells of three lung cancer cell lines at the single cell level. Collectively, NCI-H1975 cell line represents the acquired drug resistance found in clinical cancer patients, and this study demonstrates the tumor heterogeneity represented in human cell lines.

Citation Format: Lysa-Anne Volpe, John Foulke, Michael Jackson, Luping Chen, David H. Randle, Hannah J. Gitschier, Kelly Gardner, Fang Tian. Tumor heterogeneity represented in human cancer cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4919. doi:10.1158/1538-7445.AM2015-4919
Abstract 492: Epigenetic suppression of miRNA-203 promotes Ewing sarcoma malignancy
Authors: Hensel Tim , Heid Esther , Plehm Stephanie , Burdach Stefan , Richter Günther H.
Published: August 2015

Ewing sarcoma (ES), a malignancy of bone and soft tissue that affects children and young adults, is characterized by early metastasis to lung and bone and by oncogenic ews/ets translocations. We previously observed the histone methyltransferase Enhancer of Zeste, Drosophila, Homolog 2 (EZH2) to be regulated by EWS-FLI1 and highly increased in ES. We hypothesized non-coding RNAs to contribute to epigenetic processes and malignancy of ES. Here we show by ChIP on chip analysis that EZH2 occupies promoter regions of putative tumor suppressor miRNAs, oncogenic miRNAs and differentiation-associated miRNAs in ES and investigated their functional relevance by miRNA mimic, RNA interference, and functional assays. We show that hsa-miR-203a-3p (miR-203), a promoter of epidermal differentiation and trigger to exit cell cycle, was found epigenetic silenced in ES. We not only observed miR-203 up-regulation in different ES lines after EZH2 knock down or treatment with the EZH2 inhibitors GSK126 but also after histone de-acetylase inhibition (HDACi) via Trichostatin A (TSA) as well as MS-275. Similarly, treatment of ES lines with the DNA-methylation inhibitor 5′-azacytidine resulted in a strong up-regulation of miR-203 in ES. On the other hand, de-regulated expression of miR-203 after transient transfection with a miR-203-mimic identified a feed back loop resulting in a down-regulation of EWS-FLI1 and EZH2 in ES cell lines as compared to controls. In functional assays we observed that an increased expression of miR-203 suppressed contact dependent as well as independent growth in ES. In addition, in vitro invasiveness was strongly impaired after over-expression of miR-203. Taken together, we demonstrate the essential need for epigenetic suppression of miR-203 expression in ES that otherwise would suppress critical features of ES malignancy such as contact independent growth and invasiveness of ES. These results further provide a rationale for epigenetic combination therapy directed to reverse miR-203 suppression.

Citation Format: Tim Hensel, Esther Heid, Stephanie Plehm, Stefan Burdach, Günther H. Richter. Epigenetic suppression of miRNA-203 promotes Ewing sarcoma malignancy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 492. doi:10.1158/1538-7445.AM2015-492
Abstract 4942: Activation of Phosphatase toward the Retinoblastoma protein in breast and colorectal cancer cell spheroids
Authors: Egger Jacklynn , Antonucci Lisa , Lane Maria , Krucher Nancy A.
Published: August 2015

Excessive phosphorylation of the Retinoblastoma protein is found in most cancer tumor types. Several cyclin dependent kinase (cdk) inhibitors are in development or in clinical trials at the present time. Our previous studies have focused on the regulation of Rb phosphorylation by the phosphatase, PP1. Specificity toward substrates is imparted onto PP1 by many different interacting proteins. In proliferating cells, PP1 is associated with a regulatory protein called PNUTS (Phosphatase Nuclear Targeting Subunit). Our previous experiments have shown that PNUTS inhibits PP1 activity toward specific Rb phosphorylation sites. Further we have demonstrated that siRNA mediated knockdown of PNUTS in several cancer cell types (colon, breast, ovarian) leads to an increase in Rb specific PP1 activity, Rb dephosphorylation on several sites, and a dramatic induction of apoptosis. Furthermore, the ability of PNUTS siRNA to induce apoptosis was shown to be dependent on Rb expression. Interestingly, further studies showed that PNUTS knockdown had no effect on non-transformed breast and colorectal cells, in which Rb is not hyperphosphorylated. Thus we have developed a method to activate the Rb phosphatase in cells by knockdown of the PP1 binding protein, PNUTS. The current study was undertaken to analyze the effect of PNUTS knockdown in cancer cells grown in three-dimensional spheroids grown by incubation of cells in hanging drop plates. The effect of PNUTS knockdown on Rb phosphorylation, cell proliferation and cell viability using this model system will be presented.

Citation Format: Jacklynn Egger, Lisa Antonucci, Maria Lane, Nancy A. Krucher. Activation of Phosphatase toward the Retinoblastoma protein in breast and colorectal cancer cell spheroids. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4942. doi:10.1158/1538-7445.AM2015-4942
Abstract 4949: HAS2 is a critical effector for AGL mediated regulation of tumor growth
Authors: Guin Sunny , Ru Yuanbin , Lew Carolyn R. , Agarwal Neeraj , Owens Charles , Theodorescu Dan
Published: August 2015

In bladder cancer, reduced levels of Amylo-alpha-1-6-glucosidase-4-alpha-glucanotransferase (AGL), an enzyme involved in glycogenolysis and mutated in glycogen storage disease type III, enhances proliferation in vitro and tumor growth in vivo. To identify how reduced levels of AGL promote bladder cancer growth, we gene expression profiled two bladder cancer cell lines with and without siRNA mediated AGL depletion. This identified that hyaluronic acid synthase 2 (HAS2), an enzyme responsible for hyaluronic acid (HA) synthesis, is upregulated with AGL depletion. We validated this finding in several additional bladder cancer cell lines and also found that HA levels were 2-fold higher bladder cancer cells with low AGL compared to control. Interestingly, siRNA induced knockdown of HAS2 preferentially reduced monolayer, anchorage independent and xenograft growth in bladder cancer cells with low AGL. 4-Methylumbelliferone (4-MU), an inhibitor of HA synthesis, had similar effects. Analysis of human bladder cancer tissues showed that AGL and HAS2 mRNA expression are negatively correlated in 5/8 patient datasets (N = 725). Bladder cancer patients with high HAS2 and low AGL expression had worse survival than patients with the reciprocal relationship between these two genes suggesting that HAS2 is a driver of bladder tumor growth with AGL loss establish the HAS2/HA axis as a major driver and target of therapy in bladder tumors with low AGL.

Citation Format: Sunny Guin, Yuanbin Ru, Carolyn R. Lew, Neeraj Agarwal, Charles Owens, Dan Theodorescu. HAS2 is a critical effector for AGL mediated regulation of tumor growth. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4949. doi:10.1158/1538-7445.AM2015-4949
Abstract 4962: Inhibition of TGF-β-RUNX3 signaling by activated Shh is correlated with the development of TGF-β resistance in gastric cancer cells
Authors: Park Seong Hye , Kim Jung Lim , Kim Bo Ram , Na Yoo Jin , Oh Sang Cheul
Published: August 2015

Three members of the Runt-related (RUNX) family of genes, RUNX1, RUNX2, and RUNX3 transcription factors, are known as developmental regulators important in the inception and progression of a variety of human cancers. RUNX3 is a tumor suppressor in gastric cancer, and its expression is frequently inactivated by DNA methylation or its protein mislocalized in many cancer types, including gastric and breast cancer. In this study hypothesized that the relationship between Shh signaling and RUNX3 may be involved in the development of TGF-β resistance.

Here, we was investigated a role and mechanism for RUNX3 reported a functionally important component in transforming growth factor-beta (TGF-beta) mediated signaling pathway. Cell proliferation, measured by an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay that treat TGF-b in SHH overexpressed gastric cancer cell. The result showed that inhibit the proliferation by TGF-beta treatment in the control cells in a dose-dependent pattern but did not, SHH overexpressing gastric cancer cells. RT-PCR analysis showed that the expression of Runx3 in SHH overexpressing cells with TGF-beta. RUNX3 mRNA level not affected in control and SHH overexpressing cellls. When Shh overexpressing cells were treated with the proteasome inhiitors, MG132, level of Runx3 was decrease. RUNX3 are known to be primarily localized to the nucleus. Immunofluorescence indicate translocation of Runx3 from the nuleus to the cytoplasm in SHH overexpressing cells. On the other hand, treatment with SHH blocking antibody inhibited the moved from the nucleus to the cytoplasm in SHH overexpressing cells. These results indicate that suppression of TGF-β-RUNX3 signaling by activated Shh is correlated with the development of TGF-β resistance in gastric cancer.

Citation Format: Seong Hye Park, Jung Lim Kim, Bo Ram Kim, Yoo Jin Na, Sang Cheul Oh. Inhibition of TGF-β-RUNX3 signaling by activated Shh is correlated with the development of TGF-β resistance in gastric cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4962. doi:10.1158/1538-7445.AM2015-4962
Abstract 4963: Prognostic significance of the expression of nuclear EIF5A2 in human melanoma
Authors: Khosravi Shahram , Martinka Magdalena , Ong Christopher J
Published: August 2015

Eukaryotic translation initiation factor 5A2 (EIF5A2) is an oncogene that is upregulated in several different cancers. We previously showed that cytoplasmic EIF5A2 expression increases with melanoma progression and inversely correlates with melanoma patient survival. In this study, we used immunohistochemistry and tissue microarray (TMA) using a large number of melanocytic lesions (n = 459) to examine the expression profile of nuclear EIF5A2 in melanoma progression in addition to the correlation between nuclear EIF5A2 expression and melanoma patient survival. We found that nuclear EIF5A2 expression was significantly upregulated in primary melanomas compared with normal nevi and dysplastic nevi, as well as in metastatic melanomas compared with primary melanomas, normal nevi and dysplastic nevi. Nuclear EIF5A2 expression had a significant inverse correlation with overall and disease-specific 5-year survival of all and primary melanoma patients. Nuclear EIF5A2 expression was also significantly correlated with melanoma thickness and AJCC stages, suggesting the possible role of nuclear EIF5A2 in melanoma cell invasion. We then investigated the correlation between the expression of nuclear EIF5A2 and MMP-2 which is one of the important factors for promoting cancer cell invasion. Nuclear EIF5A2 expression and strong MMP-2 expression were directly correlated as well, and their concurrent expression was significantly associated with worse overall and disease-specific 5-year survival of all and primary melanoma patients. We also studied the correlation between nuclear and cytoplasmic EIF5A2 expression and found that they were significantly correlated, and simultaneous expression of both was significantly associated with poor overall and diseases-specific 5-year survival of all and primary melanoma patients. Multivariate Cox regression analysis revealed that nuclear expression of EIF5A2 was an adverse independent prognostic factor for melanoma patients. In conclusion, we for the first time demonstrated the nuclear expression of EIF5A2 as an independent prognostic marker in melanoma and its role in melanoma progression and patient survival. As a result, nuclear EIF5A2 may have the potential to serve as a therapeutic marker for melanoma.

Citation Format: Shahram Khosravi, Magdalena Martinka, Christopher J Ong. Prognostic significance of the expression of nuclear EIF5A2 in human melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4963. doi:10.1158/1538-7445.AM2015-4963
Abstract 4967: The BAP1 deubiquitinase promotes triple-negative breast cancer partially by stabilizing the KLF5 transcription factor
Authors: Chen Ceshi , Qin Junying , Zhou Zhongmei
Published: August 2015

The transcription factor KLF5 is highly expressed in basal triple-negative breast cancer (TNBC) and promotes breast cancer cell proliferation, survival and tumor growth. However, the mechanism by which KLF5 is regulated in breast cancer remains unclear. In this study, we performed a genome-wide siRNA library screening against deubiquitinases (DUBs) and identified BAP1 as a bona fide KLF5 DUB. BAP1 interacts with KLF5 directly and stabilizes KLF5 via deubiquitination. KLF5 is in the BAP1/HCF-1 complex, and this newly identified complex promotes cell cycle progression partially by inhibiting p27 gene expression. Furthermore, BAP1 knockdown inhibits tumorigenicity and lung metastasis, which can be partially rescued by ectopic KLF5 expression. Collectively, our findings not only identified BAP1 as the first DUB for KLF5 but also revealed a critical mechanism for KLF5 expression regulation in TNBC. BAP1 could be a potential therapeutic target for TNBC and other cancers.

Citation Format: Ceshi Chen, Junying Qin, Zhongmei Zhou. The BAP1 deubiquitinase promotes triple-negative breast cancer partially by stabilizing the KLF5 transcription factor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4967. doi:10.1158/1538-7445.AM2015-4967
Abstract 4968: microRNA-mediated silencing of COP1 and altered ubiquitination of key oncogenic transcription factors promote cancer stem cell (CSC) phenotype and prostate cancer progression
Authors: Dallavalle Cecilia , Albino Domenico , Civenni Gianluca , Curti Laura , Ostano Paola , Mello-Grand Maurizia , Garcia-Escudero Ramon , Chiorino Giovanna , Catapano Carlo V. , Carbone Giuseppina M. R.
Published: August 2015

Mutations and deletions of ubiquitin ligase complex proteins are emerging as important mechanisms driving tumorigenesis. In this study we report an alternative mechanism, depending on microRNA-mediated post-transcriptional silencing, leading to altered ubiquitination of key oncogenic proteins and induction of tumorigenic and CSC properties in prostate epithelial cells (PrECs). We found that increased expression of miR-424 promotes malignant transformation of normal PrECs by targeting the E3 ubiquitin ligase COP1. miR-424 was upregulated in primary prostate cancers and prevalently in ERG negative tumors. Bioinformatics and functional analysis demonstrated that miR-424 targeted COP1 mRNA in PrECs drastically reducing its protein level. Moreover, miR-424 and COP1 expression were inversely correlated in prostate tumors and associated with enrichment of CSC features. Functionally, over-expression of miR-424 or knockdown of COP1 in PrECs increased anchorage-independent growth, cell migration and prostatosphere formation under CSC selective conditions. Consistently, inhibition of miR-424 or over-expression of COP1 prevented these phenotypic changes in normal PrECs and prostate cancer cell lines. Moreover, COP1 knockdown reproduced closely the effects of miR-424 upregulation in PrECs. Mechanistically, we found that silencing of COP1 by miR-424 resulted in reduced turnover and increased level of several oncogenic transcription factors, including known COP1 substrates like c-JUN and ETV1. Furthermore, we identified STAT3 as a novel substrate of the miR-424/COP1 axis. Consistently, miR-424 upregulation and COP1 silencing increased STAT3 protein level and enhanced basal and cytokine induced STAT3 activity in PrECs and prostate cancer cells. These effects were blocked by miR-424 inhibition and COP1 overexpression. These results establish the miR-424/COP1 axis as a relevant oncogenic pathway acting through deregulation of key transcription factors and with important prognostic and therapeutic implications.

Citation Format: Cecilia Dallavalle, Domenico Albino, Gianluca Civenni, Laura Curti, Paola Ostano, Maurizia Mello-Grand, Ramon Garcia-Escudero, Giovanna Chiorino, Carlo V. Catapano, Giuseppina M. R. Carbone. microRNA-mediated silencing of COP1 and altered ubiquitination of key oncogenic transcription factors promote cancer stem cell (CSC) phenotype and prostate cancer progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4968. doi:10.1158/1538-7445.AM2015-4968
Abstract 4974: IL-1 receptor-associated kinase-1 and -4 expression in Hodgkin lymphoma
Authors: Giffi Victoria S. , Younger Kenisha M. , Singh Zeba , Chen Qing , Davila Eduardo , Kimball Amy S.
Published: August 2015

BACKGROUND: The Toll-like receptor (TLR) mediates nuclear factor kappa B (NFkB) activation in lymphocytes using a family of intermediary proteins called IL-1 receptor associated kinase (IRAK). Mutations within and upregulation of this pathway have been described in the carcinogenesis of non-Hodgkin lymphoma (NHL), but it is not known whether IRAK proteins are present in Hodgkin lymphoma (HL). Furthermore, Epstein-Barr virus (EBV) has been shown to activate NFkB in HL but its association with proteins upstream of NFkB is not described.

METHODS: 1. We performed Western blots on two Hodgkin Reed-Sternberg (HRS) cell lines to detect total IRAK-1 and -4, and phospho-IRAK-1 and -4 (the activated forms). 2. Archived lymph node biopsy samples from Hodgkin lymphoma patients diagnosed at the University of Maryland were stained for IRAK-4.

RESULTS: 1. IRAK-1, IRAK-4, p-IRAK-1 and p-IRAK-4 were present in the malignant HRS cells in two HRS cell lines (HDLM-2 and KM-H2). 2. Seventeen lymph node biopsy samples showed marked positivity for IRAK-4 (mean of 2.3 on a 0-4 scale), as well as lesser positivity in surrounding reactive lymphocytes and histiocytes (mean of 1.6). The only sample to achieve a staining level of 4 was of mixed cellularity subtype, and was EBER-positive and HIV-negative.

CONCLUSIONS: The IRAK proteins are present in the malignant HRS cells of Hodgkin lymphoma. Further work will examine IRAK-1, and phospho-IRAK-1 and -4 in the lymph node samples. Future studies should also examine whether IRAK causes downstream NFkB activation in HL as it does in NHL, and whether HRS cells depend on IRAK's presence for survival. A possible association with EBV infection will also be investigated. IRAK is a targetable protein with inhibitors available, so this and future studies may reveal new therapeutic strategies in relapsed Hodgkin lymphoma.

Citation Format: Victoria S. Giffi, Kenisha M. Younger, Zeba Singh, Qing Chen, Eduardo Davila, Amy S. Kimball. IL-1 receptor-associated kinase-1 and -4 expression in Hodgkin lymphoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4974. doi:10.1158/1538-7445.AM2015-4974
Abstract 4976: Inhibition of eIF4B suppresses the growth and induces the apoptosis of Bcr-Abl positive cells
Authors: Chen Ke , Yang Jian-Ling , Guo Guijie , Chen Jilong
Published: August 2015

Alterations in translation are associated with various human malignancies, but the precise pathogenic processes and mechanistic underpinnings remain to be better understood. The oncogenic tyrosin kinase Bcr-Abl, which is believed to induce chronic myelogenous leukemia (CML), could cause the dysregulation of mRNA translation initiation by activating a series of signal pathways. Eukaryotic initiation factor 4B (eIF4B), a critical element in the assembly of translation initiation complex, has been shown to anticipate in Bcr-Abl-mediated tumorigenesis. eIF4B is activated through Ser422 phosphorylation by several pathways including JAK/STAT/Pim and PI3K/AKT/mTOR. Such redundant activation provides mechanistic bases for the less potent antitumor consequences under single-pathway targeted therapy. Interestingly, profound synergy was observed against cell survival and tumor growth and greater decrease of eIF4B Ser422 phosphorylation was detected upon the combination of Pim and mTOR inhibitors. Knockdown of eIF4B would increase the dependence of Bcr-Abl positive cells on Pim and mTOR signaling and then sensitize cells to the synergistical effect mediated by the combined inhibition. In addition, inhibiting mTOR pathway by shRNA targeting AKT or mTOR renders cells dependent on Pim pathway and knockdown Pim1 would sensitize cells to mTOR inhibitor. Thus, eIF4B acts as a prominent role in Bcr-Abl-driven pathogenesis by playing as the convergent point of these pathways which are active in Bcr-Abl positive tumors. Our results reveal a novel mechanism by which Bcr-Abl-induced cellular transformation can be blokced and provides a rationale for combination therapy targeting the dysregulation of translation for CML.

Note: This abstract was not presented at the meeting.

Citation Format: Ke Chen, Jian-Ling Yang, Guijie Guo, Jilong Chen. Inhibition of eIF4B suppresses the growth and induces the apoptosis of Bcr-Abl positive cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4976. doi:10.1158/1538-7445.AM2015-4976