bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4115: PI3Kα/δ inhibition has greater efficacy compared to PI3Kδ-selective inhibition in NHL with activated NFkB pathway .
Authors: Liu Ningshu , Haike Katja , Paul Juliane , Genvresse Isabelle , Wengner Antje M. , Laurent Dirk , O'Connell Damian , Mumberg Dominik , Ziegelbauer Karl
Published: August 2014

Follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) are the two of the most common Non-Hodgkin Lymphoma (NHL) wordwide. Although the introduction of anti-CD20 monoclonal antibodies has improved the outcome of patients with follicular lymphoma, a curative treatment or an improved treatment strategy for relapsed FL are still to be developed. DLBCL is an incurable, aggressive subtype of NHL with a high unmet medical need for effective therapeutics. Activation of the PI3K-AKT pathway by B cell receptor signaling and its role in the pathogenesis of FL and DLBCL have been highlighted in a number of studies, however, the relative importance of PI3K isoforms and effective application of PI3K inhibitors for the treatment of FL and DLBCL have not been fully addressed. To answer this question, we selected and characterized a panel of cell lines representing major subtypes (e.g. ABC and GCB) and frequent mutations (e.g. CD79, Bcl2, MyD88, CARD11, or EZH2) in FL and DLBCL. Analyzing the expression of PI3K isoforms indicated that not only PI3Kδ, an isoform known to be enriched in lymphocytes, but also PI3Kα is highly expressed in DLBCL and a subset of FL cell lines. Sensitivity profiling of the PI3Kα/δ inhibitor BAY 80-6946 (biochemical IC50=0.5 nM and 0.7 nM against PI3Kα and PI3Kδ, respectively) and the PI3Kδ-selective inhibitor CAL-101 confirmed that inhibition PI3Kα/δ is more effective than inhibition of PI3Kδ, particularly PI3Kα/δ inhibition showed a broader anti-tumor spectrum in the cell line panel. For example, unlike CAL-101, BAY 80-6946 revealed potent anti-tumor activity in NFκB activating MyD88 or CARD11 mutated DLBCL cell lines both in vitro and in vivo. Further analysis of mutation status and pathway inhibition discovered an unknown PI3K isoform-dependent crosstalk to the NFκB pathway in DLBCL. The detailed molecular mechanism and the strategy of developing PI3Kα/δ inhibitor as a single agent and rationale-based combination in FL and DLBCL will be discussed. Taken together, these findings provide further insights into the mechanism of action of BAY 80-6946 and support ongoing Phase I studies in FL and DLBCL patients (Lotze et al., J Clin Oncol 30, 2012, suppl, abstr 3019, data to be updated in the presentation).

Citation Format: Ningshu Liu, Katja Haike, Juliane Paul, Isabelle Genvresse, Antje M. Wengner, Dirk Laurent, Damian O'Connell, Dominik Mumberg, Karl Ziegelbauer. PI3Kα/δ inhibition has greater efficacy compared to PI3Kδ-selective inhibition in NHL with activated NFkB pathway . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4115. doi:10.1158/1538-7445.AM2013-4115
Abstract 4121: Leukemic FLT3 signals via Grb10 and elevated Grb10 expression correlates with poor prognosis in acute myeloid leukemia.
Authors: Uddin Kazi J. , Rönnstrand Lars
Published: August 2014

The adaptor protein Grb10 plays important roles in mitogenic signaling. However, its roles in acute myeloid leukemia (AML) are predominantly unknown. Here we describe the role of Grb10 in FLT3-ITD-mediated AML. The type III receptor tyrosine kinase, FLT3 is widely expressed in AML and approximately 30% of AML patients carry an oncogenic FLT3-internal tandem duplication (ITD) mutation. We observed that Grb10 physically associates with normal and oncogenic FLT3 through FLT3 phospho-tyrosine 572 and 793 residues. Although Grb10 expression did not affect FLT3 activation or stability, its expression enhanced FLT3-mediated Akt and STAT5 phosphorylation without affecting Erk or p38 phosphorylation. Selective Grb10 depletion reduced Akt and STAT5 phosphorylation in Ba/F3 and OCI-AML-5 cells. Grb10 regulates cell cycle by accelerating G2-M transition followed by an increased cell population in S-phase. Expression of Grb10 furthermore resulted in an increased proliferation, survival and colony formation of Ba/F3-FLT3-ITD cells. Grb10 expression was significantly increased in AML patients compared to healthy controls and was also elevated in patients carrying FLT3-ITD mutants. The elevated Grb10 expression correlated to relapse as well as to poor prognosis. In addition, Grb10 expression led to resistance to chemotherapy. These results suggest that Grb10 binds to FLT3 and induces PI3K-Akt and STAT5 signaling pathways resulting in an enhanced proliferation, survival and colony formation of hematopoietic cells that furthermore initiates drug-resistance and poor prognosis in AML.

Citation Format: Kazi J. Uddin, Lars Rönnstrand. Leukemic FLT3 signals via Grb10 and elevated Grb10 expression correlates with poor prognosis in acute myeloid leukemia. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4121. doi:10.1158/1538-7445.AM2013-4121
Abstract 4125: The retinoid-induced G0S2 protein undergoes ISG15ylation and destabilization.
Authors: Lu Yun , Mustachio Lisa Maria , Chinyengetere Fadzai , Yang Wei , Ma Tian , Dong Jessica , Sekula David J. , Freemantle Sarah J. , Dmitrovsky Ethan
Published: August 2014

The interferon stimulated protein ISG15 was one of the first ubiquitin-like modifiers discovered. Similar to ubiquitin, ISG15 is conjugated to cellular proteins through a three-step enzymatic cascade involving an activating enzyme E1 (UBE1L), a conjugating enzyme E2 and a ligase E3. Conjugated ISG15 is removed from substrate proteins by the UBE1L-ISG15 deconjugase Usp18 (UBP43). To date, few direct target proteins of the ISG15 system were found and the biologic implications of their ISG15ylation are actively under study. We previously reported that the G0/G1 switch gene 2 (G0S2) was a direct target of all-trans-retinoic acid (RA) treatment in acute promyelocytic leukemia (APL). Prior work by others found that G0S2 was able to regulate lipolysis by inhibiting adipose triglyceride lipase (ATGL). The current study extends prior work by reporting that G0S2 was induced in APL cells by RA-treatment coordinately with UBE1L, ISG15 and the deconjugase Usp18. G0S2 was also shown to be a previously unrecognized target of ISG15ylation. G0S2 species were co-immunoprecipitated with ISG15. The functional consequence of this modification was confirmed by finding that G0S2 protein stability was directly affected by the ISG15 deconjugase Usp18. Loss of Usp18 expression by transfection of individual siRNAs into studied cells led to G0S2 protein destabilization. In contrast, engineered gain of Usp18 expression in the same cells stabilized G0S2 protein. Agents that can inhibit specific protein degradation pathways were also examined. Findings revealed that G0S2 protein was stabilized by treatment with proteasome or caspase inhibitors, but was not affected by lysosome or aggresome inhibitors. Thus, these findings indicate that G0S2 is a novel ISG15 substrate and that this modification substantially reduces G0S2 protein stability. Taken together, these results provide a mechanistic basis for cross-talk between interferon and retinoid signaling at the level of the G0S2 protein that undergoes ISG15ylation. This provides evidence for a role of lipolysis regulation in both interferon and retinoid signaling. The functional consequences of this are under study in mouse models.

Citation Format: Yun Lu, Lisa Maria Mustachio, Fadzai Chinyengetere, Wei Yang, Tian Ma, Jessica Dong, David J. Sekula, Sarah J. Freemantle, Ethan Dmitrovsky. The retinoid-induced G0S2 protein undergoes ISG15ylation and destabilization. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4125. doi:10.1158/1538-7445.AM2013-4125
Abstract 4127: Expression profiling of paired formalin-fixed, paraffin-embedded (FFPE) and fresh-frozen tissue samples on Ion Torrent PGMTM.
Authors: Gu Jian
Published: August 2014

Archived formalin-fixed paraffin-embedded (FFPE) specimens represent excellent resources for biomarker discovery, but it has been a major challenge to study gene expression in these samples due to mRNA degradation and modification during fixation and processing. We have developed methods for small RNA and whole transcriptome analysis of FFPE samples on the Ion Torrent PGM™ System using Ion total RNA-Seq Kit v2 and RiboMinus Eukaryote System v2.

Paired FFPE and fresh-frozen RNA samples from lung adenocarcinoma tissue were used in this study. Small RNA libraries were prepared using the Ion Total RNA-Seq kit v2 with modified protocol, followed by sequencing on PGM™ system. Results show that small RNA extracted from FFPE sample was successfully converted to small RNA library. Although percentage of reads mapped to miRBase was slightly lower when compared to fresh-frozen sample, the overall miRBase coverage and detection sensitivity were comparable. For whole transcriptome analysis, RiboMinus Eukaryote System v2 kit was used to deplete rRNA from FFPE samples, followed by library construction using the Ion Total RNA-Seq kit v2 with a modified protocol. Significant reduction in rRNA reads was observed after sequencing. High RefSeq correlations were observed between fresh-frozen and FFPE samples.

In conclusion, a full spectrum of gene expression data, from both small RNA and whole transcriptome, were generated from FFPE samples using Ion Total RNA-Seq kit v2 with modified protocols. High correlations were observed between paired fresh-frozen and FFPE samples, demonstrating the reliability of the modified workflow for gene expression profiling on FFPE samples. This study provides feasibility for systematic gene expression analysis on FFPE samples from cancer and other diseases.

Citation Format: Jian Gu. Expression profiling of paired formalin-fixed, paraffin-embedded (FFPE) and fresh-frozen tissue samples on Ion Torrent PGMTM. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4127. doi:10.1158/1538-7445.AM2013-4127
Abstract 4132: Rapid microfluidic-based isolation of tumor cells from pleural effusions for cytodiagnostics.
Authors: Che James , Mach Albert , Go Derek , Talati Ish , Ying Yong , Rao Jianyu , Di Carlo Dino , Kulkarni Rajan
Published: August 2014

Pleural effusions accumulate in patients with cancer or other illnesses and are often drained to provide symptomatic relief. Cells within this fluid are concentrated and analyzed to determine the cause of effusion. In patients with cancer, tumor cells may be present in the effusions and provide a source of cells for further diagnostics and mutational analysis. However, blood cell components and other non-cellular materials can create a significant background and can obscure the search for tumor cells. Furthermore, blood cell impurities can adversely impact the search for genomic or proteomic changes in the tumor cells by creating a large wild type background. A purity of at least 20% of target cells is necessary to ensure that background signal is suppressed. We have developed a microfluidic based “Centrifuge Chip” for the rapid isolation and purification of cancer cells. The Centrifuge Chip utilizes microvortices to trap cells based on size without the need for affinity-based capture. Isolated cells can subsequently be released on-demand and are viable to grow or can be analyzed for molecular changes. We utilized this chip for the isolation of tumor cells and mesothelial cells at high purity from pleural effusions and similar complex fluids such as blood. We then stained the cells with CK/DAPI for tumor cells and CD45 for white blood cells. Samples with high numbers of CK positive cells were deemed suspicious for malignancy; all our results were cross-checked to the standard Cytospin results. We routinely achieved improved purities in all samples, with greater than 70-fold increase in purity for samples positive for malignancy, and are developing a quantitative PCR (QPCR) based system to confirm the presence of activating EGFR or KRAS mutations in lung cancer cells isolated from patients with malignant pleural effusions secondary to lung cancer. The Centrifuge Chip can achieve rapid isolation of tumor cells from complex biofluids and allows for easier access to such cells for molecular analysis that will provide clinicians with critical information required for targeted cancer therapies.

Citation Format: James Che, Albert Mach, Derek Go, Ish Talati, Yong Ying, Jianyu Rao, Dino Di Carlo, Rajan Kulkarni. Rapid microfluidic-based isolation of tumor cells from pleural effusions for cytodiagnostics. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4132. doi:10.1158/1538-7445.AM2013-4132
Abstract 4137: An improved assay system for cell migration and invasion.
Authors: Partridge Jeff , Rimsa Stephen , Flaherty Paula
Published: August 2014

In vitro cell migration and invasion assays are frequently used as model systems for quantifying the directed movement of cells towards a chemoattractant stimulus, or to measure how a particular drug, antibody or extra cellular matrix (ECM) coating affects that movement. Analysis of this movement is often accomplished through the use of visual methods that are either time-consuming, labor-intensive, or subjective. To address these issues, an improved fluorescence blocking version of the Boyden chamber was developed. As cells migrate or invade through a unique fluorescence blocking microporous membrane, they can be detected using a bottom-reading spectrofluorometer or fluorescence microscope. Cells remaining in the upper chamber of the insert are shielded from this detection, allowing for quantitation of cell numbers in a homogeneous assay system.

The technology blocks ≥ 98% transmission of light from 360 - 690 nm as measured by transmission spectrophotometry, and improvements have been made over previous embodiments such that >50% more light in the range of 360 - 490 nm is blocked. These enhancements now permit the use of the blue fluorescent Hoechst dyes and DAPI for direct cell enumeration and multiplexing. Functional applications of this technology were demonstrated in several well-established assays: endpoint staining in a model tumor invasion system yielded a Z’-factor >0.5; real-time chemotaxis of monocytes demonstrated a peak signal between 25-35 minutes; and migration of human endothelial cells to VEGF was demonstrated with high reproducibility (CV < 15%). Additionally, the measured IC50 values of chemotherapeutic compounds including paclitaxel and doxorubicin remained consistent with previously reported results. FluoroBlok™ cell culture inserts can efficiently be used for high-throughput cell migration studies, are automation friendly and yield highly reproducible results.

Citation Format: Jeff Partridge, Stephen Rimsa, Paula Flaherty. An improved assay system for cell migration and invasion. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4137. doi:10.1158/1538-7445.AM2013-4137
Abstract 4141: Tumor cell enrichment is critical for assessing cell signaling pathways in glioblastoma multiforme.
Authors: Mueller Claudius , deCarvalho Ana C. , Mikkelsen Tom , Calvert Valerie , Espina Virginia , Liotta Lance A. , Petricoin Emanuel F.
Published: August 2014

Introduction & Methods: Glioblastoma tumor cells are generally assumed to be mixed with few distinct non-tumorigenic cell types and few cells of the microenvironment in general, owing to their location in the brain and the high tumor cell density compared to normal brain parenchyma. However, even small cell populations can have a significant impact on the abundance of phosphorylated proteins. We have analyzed the level and/or phosphorylation of 133 proteins in laser capture microdissected glioblastoma primary tumors (n=39) and compared the results to non-tumor cell enriched, whole tissue lysates.

Results: A large portion (44 percent) of endpoints were significantly different between matched tumor cell enriched and non-enriched samples - even when limiting the investigation to tumor samples with at least 90 percent tumor content. EGFR protein levels, which is highly expressed in tumor cells compared to normal brain parenchyma, increased in enriched and non-enriched samples following EGFR mutation, while EGFR phosphorylation was only significantly increased in tumor cell enriched samples. In contrast, PTEN, which has a higher expression in normal brain compared to tumor cells, was only found to be reduced in tumor cell enriched samples from patients with either PTEN mutation or loss in PTEN copy number, with no difference seen in non-enriched samples. Patient stratification by either phosphorylated EGFR, Akt, mTOR, Stat1, VEGFR2 or Bcl-2 based on non-tumor cell enriched samples was dramatically different from tumor cell enriched samples, showing significant misclassification for up to 40 percent of patients even in high tumor content samples.

Conclusions: This study demonstrates the critical necessity for selected tumor cell population procurement when investigating protein levels and phosphorylation in glioblastoma. Only tumor cell enriched samples correctly reflected expected tumor biology in the face of EGFR and PTEN mutations. Moreover, tumor cell enrichment had a dramatic effect on the stratification of clinical patient samples that would form the basis for targeted therapy selection.

Citation Format: Claudius Mueller, Ana C. deCarvalho, Tom Mikkelsen, Valerie Calvert, Virginia Espina, Lance A. Liotta, Emanuel F. Petricoin III. Tumor cell enrichment is critical for assessing cell signaling pathways in glioblastoma multiforme. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4141. doi:10.1158/1538-7445.AM2013-4141
Abstract 4151: A novel, fast, and efficient technology for the preparation of high quality NGS libraries.
Authors: Kurihara Laurie , Beaubien Ron , Perry Jeff , Kelchner Vanessa , McUsic Drew , Mishra Anjali , Laliberte Julie , Chupreta Sergey , Makarov Vladimir
Published: August 2014

Today's next-generation sequencing (NGS) investigators and clinicians must extract and analyze complex sequence data from a wide range of sample types. Preparing fragment libraries from these samples for whole genome, exome or targeted gene panel sequencing by NGS is a laborious process, characterized by long prep times and sample compatibility constraints due to DNA input quantity, genomic complexity, and molecular integrity. The workflows for most commercially-available kits are variations on traditional modifications for the repair, adaptation, and selection of DNA fragments, requiring 2-4 hours of preparation time before sequencing. As a consequence, NGS users cite sample preparation as the second most time-consuming step and bottleneck in their NGS workflows (following data analysis and interpretation; Bioinformatics, Oct. 2011). We present a novel technology that enables faster and more efficient NGS fragment library preparation, which increases yield and is compatible with many sample types, including damaged, cross-linked samples derived from FFPE or ChIP or samples with limiting DNA quantity. This method conveys these advantages because it does not require intact, double-stranded DNA for adapter ligation, and the streamlined workflow results in significant time savings relative to the workflows of commercial products. Side-by-side comparisons of sequencing data from libraries prepared using this new technology versus those from existing commercial products demonstrates equivalent or improved genome coverage, and suitability for damaged or cross-linked samples.

Citation Format: Laurie Kurihara, Ron Beaubien, Jeff Perry, Vanessa Kelchner, Drew McUsic, Anjali Mishra, Julie Laliberte, Sergey Chupreta, Vladimir Makarov. A novel, fast, and efficient technology for the preparation of high quality NGS libraries. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4151. doi:10.1158/1538-7445.AM2013-4151
Abstract 4152: New methods for the isolation and analysis of biomarkers from exosomes in cell culture media and bio-fluids.
Authors: Gutierrez Sara , Strug Ivona , Cappione Amedeo , Smith Janet L. , Mabuchi Masaharu , Nadler Timothy
Published: August 2014

Microvesicles, such as exosomes, are small particles secreted by many types of cells under both normal and pathological conditions such as cancer. These microparticles contain RNA and protein (both membrane-bound and cytosolic) from their cells of origin and thus represent a burgeoning resource for biomarker identification. While plasma and urine offer convenient, non-invasive access to sufficient sample volumes for screening purposes, major obstacles to this effort include the problematic issues of exosome isolation methods, the presence of over-abundant proteins or interfering cell debris, and the wide dynamic range of protein expression. It is clearly recognized in many fields that the quality of sample preparation ultimately impacts their performance in downstream analyses; this is particularly true where the target in question is a low abundant protein. Here we present an ultrafiltration-based method for the isolation of microvesicles from cell culture media as well as body fluids. To assist in process optimization, we used a novel infrared (IR) based biomolecule detection system to evaluate lysis and extraction conditions and analyze protein and lipid content. This IR system is less influenced by reducing agents and detergents than either BCA or Bradford assays. Following isolation, samples were analyzed using a rapid immunodetection technique, permitting detection of biomarkers more quickly and efficiently than ever before.

Citation Format: Sara Gutierrez, Ivona Strug, Amedeo Cappione, Janet L. Smith, Masaharu Mabuchi, Timothy Nadler. New methods for the isolation and analysis of biomarkers from exosomes in cell culture media and bio-fluids. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4152. doi:10.1158/1538-7445.AM2013-4152
Abstract 4159: Expression of miRNAs associated with DNA repair capacity in women with breast cancer.
Authors: Matta Jaime L. , Morales Luisa , Ortiz Carmen , Zayas Celestino , Suarez Erick
Published: August 2014

MicroRNAs (miRNA) are endogenous, short (19-24 nucleotides) non-protein-coding RNAs that regulate gene expression at the post-transcriptional level via binding to 3′-untranslated regions of protein-coding transcripts. A low DNA repair capacity (DRC) is an important risk factor for breast cancer (BC). We are currently investigating whether miRNAs expression is associated with dysregulation of DRC in women with BC. To that effect, we obtained plasma from 30 BC cases and 30 controls selected based on their DRC levels. DRC was measured in lymphocytes using a host cell reactivation assay. Samples were analyzed for miRNA expression utilizing protocols from Applied Biosystems (Life Technologies). After QA/QC, 27 BC cases and 29 controls whose DRC data was available from our cohort study were selected and miRNAs isolated from plasma. The miRNA expression profiling was performed utilizing the RT-PCR TaqMan Array Human MicroRNA A Cards v 2.0 (Applied Biosystems). Single-stranded cDNA was synthesized from 200 ng of total RNA in 8 Multiplex RT primer pool reactions containing stem-looped RT primers that were specific to mature miRNAs. U6 snRNA-001973 was selected for normalization based on our own experimental validations. To quantify the association of the miRNA expression the fold change () was estimated for every detector with the p-values based on the t-test. Significant candidate miRNAs were determined with the false discovery rate of 4%. The results showed that twelve miRNAs are underexpressed in patients with BC relative to controls. These candidate miRNAs were associated with the expression of twenty seven DNA repair genes. Two of these genes (RPA3, MMS19L) are part of the NER pathway which has recently been linked to breast carcinogenesis. Our pilot study data suggests that differential expression of some miRNAs might be associated with dysregulation of DRC in women with BC.

Supported by grants S06 GM008239-20, MBRS-RISE GM082406 [NIH-NIGMS] and 1SCA157250-2 from the NCI Center to Reduce Health Disparities, and NIH-MBRS Program (NIGMS).

Citation Format: Jaime L. Matta, Luisa Morales, Carmen Ortiz, Celestino Zayas, Erick Suarez. Expression of miRNAs associated with DNA repair capacity in women with breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4159. doi:10.1158/1538-7445.AM2013-4159
Abstract 4160: miR-34a as a radio sensitizer of triple negative breast cancer.
Authors: Bhardwaj Anjana , Ganesan Nivetha , Molkentine David , Raju Uma , Bedrosian Isabelle
Published: August 2014

Introduction: Triple negative breast cancer (TNBC) is known to be relatively resistant to radiation therapy resulting in a comparatively higher rates of locoregional recurrence compared with non-TNBC patients. The mechanisms behind this relative radio resistance remain unknown. The goal of this study is to investigate the role of three miRNAs namely, miR-34a, miR-21, and miR-210, which are implicated in DNA damage-repair process, in the response of TNBC cell lines to radiation therapy. Methods: Seven TNBC breast cancer cell lines were used for the study. The levels of miR-34a, miR-21 and miR-210 were evaluated by taqman- based quantitative PCR assay. These cells were exposed to 2 Gy radiation dose and colonogenic survival was assessed after about 2 weeks. Survival fraction after 2 Gy (SF2), which is the ratio of number of cell colonies formed after 2 Gy radiation vs. the number of cell colonies with out radiation, was determined. The SF2 factor is a reliable index to measure radio sensitivity, greater the SF2 factor more radio resistant the cells are. Results: The SF2 value of the TNBC cells varied from 0.31 to 0.74 with MDA-MB-468, HCC-70 being the most radio sensitive cell lines and MDA-MB-436, BT-20 being the most radio resistant cell lines. MDA-MB- 231, BT-549 and Hs 578T were found to be moderately radio resistant breast cancer lines. The oncomiR -21 expression levels in these 7 TNBC cell lines varied from 0.85 to 4.18 fold with a very poor correlation with SF2 (r2=0.08). The oncomiR 210 levels varied from 0.49 to 9.19 fold and also did not exhibit any correlation with radio resistance in TNBC cells (r2= 0.141). Whereas the tumor suppressor miR, miR-34a expression showed an inverse correlation with radio resistance (r2=0.8) in TNBC cells. The radio resistant cell line (e.g., MDA-MB-436, SF2 = 0.74) expressed significantly low levels of miR-34a (0.016 fold) relative to miR -34a levels (set as 1) in HCC 70, which was found to be a radiosensitive cell line with SF2 of 0.309. Conclusions: Our preliminary data indicates that miR 34a may play a role in the response of TNBC cells to radiotherapy.

Citation Format: Anjana Bhardwaj, Nivetha Ganesan, David Molkentine, Uma Raju, Isabelle Bedrosian. miR-34a as a radio sensitizer of triple negative breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4160. doi:10.1158/1538-7445.AM2013-4160
Abstract 4166: MicroRNA-449a enhances radiosensitivity in lung adenocarcinoma cells.
Authors: Liu Yi-Jyun , Lin Yu-Fen , Chen Yi-Fan , Luo En-Ching , Sher Yuh-Ping , Tsai Mong-Hsun , Chuang Eric Y. , Lai Liang-Chuan
Published: August 2014

Lung cancer is the leading cause of cancer-related mortality worldwide. Radiotherapy is often applied for treating lung cancer, but it often fails because of the relative non-susceptibility of lung cancer cells to radiation. MicroRNAs (miRNAs) have been reported to modulate the radiosensitivity of lung cancer cells and have the potential to improve the efficacy of radiotherapy. The purpose of this study was to identify a miRNA that can adjust radiosensitivity in lung adenocarcinoma cells. Two lung adenocarcinoma cell lines (CL1-0 and CL1-5) with different metastatic ability and radiosensitivity were used. In order to understand the regulatory mechanisms of differential radiosensitivity in these isogenic tumor cells, both CL1-0 and CL1-5 were treated with 10 Gy radiation, and were harvested respectively at 0, 1, 4, and 24 h after radiation exposure. The changes in expression of miRNA upon irradiation were examined using Illumina Human microRNA BeadChips. Twenty-five miRNAs were identified as having differential expression post-irradiation in CL1-0 or CL1-5 cells. Among these miRNAs, miR-449a, which was down-regulated in CL1-0 cells at 24 h after irradiation, was chosen for further investigation. Overexpression of miR-449a in CL1-0 cells effectively increased irradiation-induced DNA damage and apoptosis, altered the cell cycle distribution and eventually led to sensitization of CL1-0 to irradiation.

Citation Format: Yi-Jyun Liu, Yu-Fen Lin, Yi-Fan Chen, En-Ching Luo, Yuh-Ping Sher, Mong-Hsun Tsai, Eric Y. Chuang, Liang-Chuan Lai. MicroRNA-449a enhances radiosensitivity in lung adenocarcinoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4166. doi:10.1158/1538-7445.AM2013-4166
Abstract 417: SMAD3 upregulation is a novel biomarker of poor prognosis and epithelial-mesenchymal transition in pancreatic cancer.
Authors: Yamazaki Ken , Masugi Yohei , Effendi Kathryn , Sakamoto Michiie
Published: August 2014

Pancreatic cancer has one of the worst survival rates among all cancers. Features like epithelial-mesenchymal transition (EMT) are often seen in pancreatic tumors and correlate with poor prognosis. The purpose of this study was to identify factors involved in EMT in pancreatic cancer that may serve as prognostic indicators. Gene-expression profiling identified overexpression of SMAD3 in pancreatic cancer with high-grade solitary cell infiltration, one of the EMT-like features. SMAD3 expression was then evaluated through immunohistochemical analysis using clinical specimens. SMAD3 was accumulated in the nuclei of tumor cells (positive rate ranging from 0% to 75%), but was not detected in most of the epithelial cells in the pancreatic duct. Upregulated SMAD3 (≥15% positive in nuclei of tumor cells) correlated with malignant characteristics, such as higher tumor grade (P = 0.001) and lymph node metastasis (P <0.001), as well as with EMT-like features, such as reduced E-cadherin (P = 0.013) and elevated vimentin (P = 0.032). SMAD3 knockdown in pancreatic cancer cells resulted in increased E-cadherin expression, decreased vimentin expression and decreased cell motility. The suppressed EMT-like features by SMAD3 knockdown were observed even in SMAD4-inactivated pancreatic cancer cells, suggesting that SMAD3 is involved in EMT induction, regardless of SMAD4 status. In clinical cases, SMAD3 upregulation and EMT-like features correlated with shorter survival time. Moreover, SMAD3 and E-cadherin were identified as independent prognostic factors by multivariate analysis, and the group of patients with both upregulated SMAD3 and reduced E-cadherin had the least favorable prognosis compared with other groups. Taken together, SMAD3 seems to play an important role in malignant progression of pancreatic cancer, and may provide a potential biomarker for poor prognosis of pancreatic cancer.

Citation Format: Ken Yamazaki, Yohei Masugi, Kathryn Effendi, Michiie Sakamoto. SMAD3 upregulation is a novel biomarker of poor prognosis and epithelial-mesenchymal transition in pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 417. doi:10.1158/1538-7445.AM2013-417
Abstract 4172: MiR-215 expression in tumor tissue and in vitro effects of its replacement in colorectal cancer.
Authors: Faltejskova Petra , Svoboda Marek , Mlcochova Jitka , Srutova Klara , Fabian Pavel , Vyzula Rostislav , Slaby Ondrej
Published: August 2014

Background: MicroRNAs (miRNAs) constitute a robust regulatory network with post-transcriptional regulatory efficiency for almost one half of human coding genes, including oncogenes and tumor suppressors.

Methods: We determined the expression profile of 667 miRNAs in colorectal cancer (CRC) tissues and paired non-tumoral tissues and identified 42 differentially expressed miRNAs. One of the most significantly altered miRNAs was miR-215, significantly down-regulated in tumor tissue. We chose miR-215 for further validation on an independent cohort of 125 clinically characterized CRC patients and for in vitro functional studies on DLD1, HCT116 and HT29 cell lines.

Results: MiR-215 was proved to be significantly decreased in CRC tumor tissues (p<0.0001) and to be negatively correlated with clinical stage (p<0.0001) and tumor grade (p=0.04). In vitro analyses showed that ectopic expression of miR 215 decreases viability in DLD1 (p=0.05) and HCT116 (p=0,05) cells, increases apoptosis in HCT116 (p=0.005), promotes cell cycle arrest (inhibited G1/S transition) in HCT-116 colon cancer cells (p=0.01) and decreases migration in DLD1 (p=0.05) and HT-29 cells (p=0.05). We further confirmed transcription factor HOXB9 as direct target of miR-215 on the mRNA and protein level.

Conclusions: These findings indicate that miR 215 play an important role in CRC as tumor suppressor and contributes to CRC pathogenesis.

Supported by Internal Grant Agency of Czech Ministry of Health, grant numbers: NT13549-4/2012 a NT13860 4/2012.

Citation Format: Petra Faltejskova, Marek Svoboda, Jitka Mlcochova, Klara Srutova, Pavel Fabian, Rostislav Vyzula, Ondrej Slaby. MiR-215 expression in tumor tissue and in vitro effects of its replacement in colorectal cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4172. doi:10.1158/1538-7445.AM2013-4172
Abstract 4173: MicroRNA expression of non-terminal respiratory unit type lung adenocarcinoma.
Authors: Kim Mi Hyun , Lee Min Ki , Shin Dong Hoon , Lee Chang Hun
Published: August 2014

Background: Lung adenocarcinomas can be classified into two major subtypes, terminal respiratory unit (TRU) type and non-TRU type. We previously reported that non-TRU type adenocarcinoma has unique clinical and morphological features compared with TRU type adenocarcinoma. In brief, among 36 cases of non-terminal respiratory unit type adenocarcinoma, 24 cases revealed mucous columnar cell changes that were continuous with bronchial ciliated columnar cells. The mucous columnar cells became dysplastic showing loss of cilia, disorientation, and enlarged nuclei. Adenocarcinoma arose from these dysplastic mucous columnar cells and, characteristically, this type of adenocarcinoma showed acute inflammation, and honeycombing changes in the background. TTF1 immunostaining was consistently negative. In a case study with 14 males and 10 females, including 12 smokers or ex-smokers, EGFR and KRAS mutations were detected in 3 and 6 patients, respectively. We think that this kind of adenocarcinoma arising through mucous columnar cell change belongs to non-terminal respiratory unit type adenocarcinoma, and mucous columnar cell change is a precursor lesion of pulmonary adenocarcinoma.

Objectives: We investigated whether microRNA (miRNA) expression profiles can differentiate between non-TRU type and TRU type lung adenocarcinoma.

Methods: MiRNA expression in lung adenocarcinoma with non-TRU and TRU type was analyzed by microarray analysis with 1205 human and 144 human viral miRNAs.

Results: Eight pairs of lung adenocarcinomas and corresponding noncancerous lung tissues (4 for non-TRU type and 4 for TRU type) were analyzed by microarray analysis. Initially, 44 miRNAs showed statistical differences in expression between lung cancer tissues and corresponding noncancerous tissues. Comparison analyses between non-TRU type and TRU type revealed 30 miRNAs with statistically different expression. Particularly, hsa-mir-494 and ebv-mir-BART19 were upregulated with more than 5-fold difference as compared with TRU type adenocarcinoma. Similarly, hsa-mir-551b was downregulated at more than 5-fold as compared with TRU type adenocarcinoma. Only 2 miRNAs (hsa-mir-1 and hsa-mir-133b) were shared in both non-TRU and TRU type of adenocarcinoma.

Conclusion: We demonstrated that non-TRU type adenocarcinoma showed different miRNAs expression patterns compared with TRU type adenocarcinoma, suggesting different carcinogenic pathways for these tumors. Further studies are required for validation the results from microarray analysis.

Citation Format: Mi Hyun Kim, Min Ki Lee, Dong Hoon Shin, Chang Hun Lee. MicroRNA expression of non-terminal respiratory unit type lung adenocarcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4173. doi:10.1158/1538-7445.AM2013-4173
Abstract 4174: miR-17-92 as a therapeutic target in gastric cancer.
Authors: park Dong M. , Lee Seok C. , Kim Hark K.
Published: August 2014

We previously reported that 82 gastric cancer patients overexpress miR-17-5p, compared with healthy volunteers [median tumor/normal ratio, 1.6; false discovery rate<0.001]. Although 13q31.3 amplification has been reported in gastric cancer, the exact frequency of MIR17HG gene amplification in gastric cancer has not been systematically investigated. Using combined CGH and expression array analyses, we identified genomic abnormalities associated with miR-17-5p expression. No copy number alteration of MIR17HG was identified among the 32 study patients. Median or higher levels of miR-17-5p expression in gastric cancer patients were associated with copy number gain and overexpression of known oncogenes such as MYC and FGFR2. Based on mRNA expression microarray, genes differentially expressed between the high miR-17-expressing tumors and healthy volunteers were significantly enriched in MYC target genes (LS P value, 0.007). MYC knockdown in SNU-16 gastric cancer cells significantly decreased miR-17-92 expression. Although transcriptional activation, rather than gene amplification, was identified as the primary mechanism underlying miR-17-92 overexpression in gastric cancer, miR-17-5p inhibition led to suppressed monolayer and anchorage-independent growth and increased G1/S phase ratio in KATO-III cells which harbor FGFR2 gene amplification. Hence, miR-17-92 may serve as a therapeutic target in gastric cancer (supported by the Proteogenomic Research Program through the National Research Foundation of Korea funded by the Korean Ministry of Education, Science and Technology; and by Korean National Cancer Center Grant 121005).

Citation Format: Dong M. park, Seok C. Lee, Hark K. Kim. miR-17-92 as a therapeutic target in gastric cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4174. doi:10.1158/1538-7445.AM2013-4174
Abstract 4175: Characterization of hsa-miR-135b in human neuroblastoma cell line SK-N-BE(2).
Authors: Castillo Chelsea A. , Chang Judy C. , Hung Jaclyn Y.
Published: August 2014

Background: MicroRNAs (miRs) are an abundant class of small (∼22 nucleotides), non-coding, single-strand RNAs (ncRNA) that negatively regulate gene expression at a post-transcriptional level. miRs are thought to act by inhibiting the translation of messenger RNAs (mRNAs) into protein in addition to promoting mRNA degradation. These regulatory ncRNAs play an important role in the control of many biological processes and their deregulation has been implicated in a variety of pathological conditions, including many cancers. Emerging evidence supports the concept that in many cancer types a pool of cancer cells responsible for cancer maintenance and long-term remission failure exhibit stem-like properties. Using a high throughput miR microfluidic card assay qRT-PCR approach, we identified 26 miRs (23 up-regulated and 3 down regulated) as stem-like associated miRs in six pediatric cancer cell lines, including neuroblastoma cell line SK-N-BE(2). hsa-miR-135b was one of the miRs that was up-regulated. The purpose of this study was to characterize the functional consequences of hsa-miR-135b silencing in the SK-N-BE(2) cell line.

Methods: hsa-miR-135b expression was knocked down (KD) using Lipofectamine® transfection reagent and a stable cell line was generated in the presence of puromycin. Cell cycle distribution as well as the effect of inhibition on the ability to form neurospheres (read-out for self-renewal) was determined. To investigate the effect on anchorage-independent growth, the ability of the KD cells to form colonies in soft agar was measured. Cell capacity for invasion was tested using basement membrane matrix invasion culture system. Western blot analysis was performed on the components of the Wnt/****B-catenin signaling pathways.

Results: Our results indicated that inhibition of hsa-miR-135b impaired sphere formation (read-out for self renewal) as well as growth of colonies on a semi-solid culture media. No significant difference in cell cycle distribution was observed between control and hsa-miR-135b KD cells.

Conclusion: Our findings suggest that in our model system, hsa-miR-135b likely plays a role in the maintenance of cancer stem cells and tumor progression. Therefore, it is a promising therapeutic target in neuroblastoma patients.

Citation Format: Chelsea A. Castillo, Judy C. Chang, Jaclyn Y. Hung. Characterization of hsa-miR-135b in human neuroblastoma cell line SK-N-BE(2). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4175. doi:10.1158/1538-7445.AM2013-4175
Abstract 4187: Regulation of NRSF/REST by miR-217; Implications during pancreatic acinar ductal trans-differentiation.
Authors: Azevedo-Pouly Ana Clara P. , Jiang Jinmai , Sutaria Dhruvitkumar , Schmittgen Thomas
Published: August 2014

Pancreatic ductal adenocarcinoma is believed to arise from exocrine acinar cells following trans-differentiation to cells with a more epithelial phenotype. A pancreas enriched cluster of miRNAs (miR-216a/b-217) is located on chromosomes 2 and 11 in humans and mice, respectively. These miRNAs are significantly reduced in patient's tumor specimens and during tumor progression in a transgenic mouse model of pancreatic ductal adenocarcinoma. To investigate a role for miR-216/-217 during acinar ductal trans-differentiation, we used a model by which primary mouse pancreatic acini trans-differentiate into epithelial-like cells when cultured on matrigel. Over a 4 day culture period, the acini develop an epithelial morphology and display reduced mRNA expression of the acinar marker amylase, increased expression of the epithelial marker cytokeratin-19 (KRT19) and reduced expression miR-216/-217. Forced expression of miR-217 using a lentiviral vector (LV) decreased KRT19 mRNA expression compared to the empty vector control treated cells on day 4 of the trans-differentiation. To implicate miR-217 targets in regulating acinar trans-differentiation, we considered REST/NRSF, a transcriptional repressor of neuronal genes in non-neuronal tissues. REST mRNA expression increased during the 4 day trans-differentiation implying regulation of REST by miR-217. Regulation of REST by miR-217 was validated using a luciferase assay and western blotting. Furthermore, two REST target genes were upregulated by the miR-217 LV on day 4 of the trans-differentiation. Studies are ongoing to determine if this increase in expression results from miR-217 suppressing REST protein levels. Additionally, miR-217 LV infection of 3 pancreas cancer cell lines reduced REST protein and caused significant cell death. Our current focus is to establish a link between miR-217 suppression of REST and cell death. We report a connection between reduced miR-217 in several pancreas model systems. We hypothesize a novel role for REST in pancreas homeostasis and cancer.

Citation Format: Ana Clara P. Azevedo-Pouly, Jinmai Jiang, Dhruvitkumar Sutaria, Thomas Schmittgen. Regulation of NRSF/REST by miR-217; Implications during pancreatic acinar ductal trans-differentiation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4187. doi:10.1158/1538-7445.AM2013-4187
Abstract 4194: The hereditary genetic polymorphism of mir-608 predicts the clinical outcome and expression of IL-6 in patients with esophageal squamous cell carcinoma .
Authors: Yang Pei-Wen , Hsieh Ching-Yueh , Huang Yu-Ting , Huang Ya-Chuan , Chiang Tzu-Hsuan , Lee Jang-Ming
Published: August 2014

PURPOSE: To investigate the effect of the single nucleotide polymorphisms (SNPs) in microRNA-related genes on the prognosis of patients with esophageal squamous cell carcinoma (ESCC).

PATIENTS AND METHODS: A total of 504 patients with a diagnosis of ESCC were enrolled in the study. The genotypes of miRNA-related SNPs were analyzed from the genomic DNA of peripheral leukocytes and were correlated with the clinical outcome of patients, which was first evaluated in a randomly assigned training set (n=129) and subsequently verified by a replication set (n= 375).

RESULTS: In training group, the heterozygous CG genotype of mir-608 rs4919510 was associated with reduced risk for adverse clinical outcomes (HR [95% CI] =0.47 [0.27-0.82] for death; HR [95% CI] =0.47 [0.29-0.77] for recurrence). In contrast, both homozygous CC and GG genotypes showed unfavorable for prognosis. The associations were confirmed in an independent replication group (GG+CC vs. CG, HR [95% CI] =1.36 [1.07-1.73], P=0.013 for death; HR [95% CI] = 1.26 [1.00-1.59], P=0.048 for recurrence). The prognostic effect was more pronounced among patients with advanced tumor stages and in those treated with concurrent neoadjuvant chemo- radiotherapy (CCRT). Functional relevance of rs4919510 was correlated with the serum IL-6 levels. GG variant was significantly associated with IL-6 over-expression in advanced ESCC patients.

CONCLUSION: The hereditary genetic variants in mir-608 can serve as an independent predictor for the clinical outcome of ESCC patients. The prognostic effect of rs4919510 may be partially mediated by impairing the ability in regulating IL-6 expression.

Citation Format: Pei-Wen Yang, Ching-Yueh Hsieh, Yu-Ting Huang, Ya-Chuan Huang, Tzu-Hsuan Chiang, Jang-Ming Lee. The hereditary genetic polymorphism of mir-608 predicts the clinical outcome and expression of IL-6 in patients with esophageal squamous cell carcinoma . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4194. doi:10.1158/1538-7445.AM2013-4194