bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4895: A one-tube assay for simultaneous detection of gene mutation, fusion, copy number alteration and expression
Authors: Zheng Zongli , Shetty Ranjit , Liebers Matthew , Cambry Jessica L. , Bernardo Lindsay A. , Borger Darrell , Le Long P. , Iafrate John A.
Published: August 2015

There are growing demands for tumor genotyping in the clinic, with an ever-growing number of mutation/fusion targets and expression profiles needed for tumor diagnostics. We sought to develop a comprehensive yet efficient tumor genetic approach using next-generation sequencing (NGS) that would target the genomic DNA and mRNA components of a sample simultaneously, to decrease costs and labor in the lab, and take advantage of small/limited quantity tumor specimens.

We describe here a novel method termed one-tube anchored multiplex PCR (OneAMP) for combined DNA-and-RNA targeted sequencing, allowing simultaneous detection of gene expression, rearrangements, single nucleotide variations (SNVs), insertion and deletions (Indels) and copy number variation (CNV). In this approach, total nucleic acid is used as input for the assay, starting with cDNA synthesis, shearing, adenylation, and ligation with a half-functional molecule-indexed Y adaptor. The molecular index is a random oligomer and serves as a tag for tracing and counting sample templates from ligation through sequencing. Target enrichment is then achieved by two rounds of hemi-nested PCR using gene specific primers and adaptor primers. Gene specific primers for priming genomic DNA (intronic primer extension to exons for SNVs, Indels and CNV) and for priming cDNA (exonic primer extension for gene fusion and expression) have been optimized so they can be combined in one tube for PCR. A distinctive feature of OneAMP is that mRNA expression is normalized to genomic DNA copy number for the same gene, eliminating the need for normalization to housekeeping genes, which can eliminate significant bias across tissue types and individuals.

We developed a 210 gene OneAMP cancer panel that consists of important cancer genes that are mutated, fused, or with altered expression or copy number. We validated CNV calls with aCGH/FISH of the same samples and validated mRNA expression by comparing to array and qPCR. Finally SNVs, Indels, and fusions were validated with a large panel of samples with known mutation status.

We demonstrate the potential of OneAMP for the comprehensive detection of genetic abnormalities, preserving precious tumor samples, and reducing reagent usage and time.

Citation Format: Zongli Zheng, Ranjit Shetty, Matthew Liebers, Jessica L. Cambry, Lindsay A. Bernardo, Darrell Borger, Long P. Le, John A. Iafrate. A one-tube assay for simultaneous detection of gene mutation, fusion, copy number alteration and expression. [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 4895. doi:10.1158/1538-7445.AM2015-4895
Abstract 49: Interaction of the protein kinase C-related kinases with the TPα and TPβ isoforms of the T prostanoid receptor: role in thromboxane-induced neoplastic responses
Authors: Mulvaney Eamon P. , O'Sullivan Aine G. , Kinsella B Therese
Published: August 2015

Compelling epidemiological evidence concurs that long-term use of aspirin lowers the risk of many prevalent cancers. While such studies do not specify which cyclooxygenase-1/2 metabolite(s) are lowered by aspirin accounting for its prophylactic benefits, many reports show that some of its anti-cancer effects are due to its ability to inhibit thromboxane (TX)A2 generation, a substance more typically known for its role in hemostasis/thrombosis. In humans, TXA2 signals through the TPα and TPβ isoforms of the T prostanoid receptor (TP). There is increasing evidence supporting roles for the TXA2 axis in several neoplasms including prostate cancer, potentially due to the ability of TPα/TPβ to regulate key RhoA- and ERK-mediated signalling cascades that contribute to tumor growth & metastasis. Mechanistically, we recently identified protein kinase C-related kinase (PRK) 1 as a direct interacting partner of TPα/TPβ, a functional interaction essential for TXA2-induced prostate cancer (PCa) cell migration. Moreover, agonist-activation of TPα/TPβ induced phosphorylation of histone H3 at Thr11 (H3Thr11), a PRK1-catalysed marker of androgen-induced chromatin remodelling and transcriptional activation. This was the first demonstration that the TXA2/TP axis can lead to epigenetic changes previously only associated with androgens. As PRK1/PKN1 is a member of a wider subfamily that also includes PRK2/PKNγ and PRK3/PKNβ which, like PRK1, are widely implicated in cancers, we sought to investigate if PRK2 and PRK3 may interact with TPα/TPβ. Both PRK2 and PRK3 were found to interact with TPα and TPβ, interactions dynamically regulated following TP-agonist activation. Activation of the TPs also led to phosphorylation and catalytic activation of all three kinases. Furthermore, PRK1 and PRK2, but not PRK3, interacted with TPα and TPβ in PC-3 cells, while targeted disruption of PRK1 and PRK2, but not PRK3, impaired TP-mediated H3Thr11 phosphorylation, cell proliferation and migration of PC-3 cells. Considering the critical roles of TPα/TPβ and of the PRKs in cancer progression, the discovery of these distinct, yet regulated, interactions is functionally significant, not least in prostate cancer but also in other pathophysiologies where aberrant PRK1-3 &/or TPα/TPβ expression and function are implicated.

Funded by the Irish Cancer Society, Health Research Board Ireland and the Movember Foundation (# PCI12KIN, MRCG/2012/3).

Citation Format: Eamon P. Mulvaney, Aine G. O'Sullivan, B Therese Kinsella. Interaction of the protein kinase C-related kinases with the TPα and TPβ isoforms of the T prostanoid receptor: role in thromboxane-induced neoplastic responses. [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 49. doi:10.1158/1538-7445.AM2015-49
Abstract 4903: Microarray gene expression analysis of human prostate cancer PC-3 cells targeted by Salmonella typhimurium A1-R
Authors: Zhao Ming , Wu Wen , Lu Chongyi , Zhang Yong , Hoffman Robert M. , Qi Hui , Yang Meng , Gu Yanqin , Lu Yiyu , Su Shibing , Zhang Hui , Zhou Qianmei , Chen Qilong
Published: August 2015

The transcriptional profile of PC-3 human prostate cancer cells infected with tumor-targeting Salmonella typhimurium A1-R (A1-R) was analyzed with by microarray. PC-3 is highly sensitive to A1-R. Numerous genes involved in diverse cellular processes, including apoptosis, necrotic cell death, neutrophil chemotaxis and innate immunity were significantly up-regulated by A1-R bacterial infection. Such up-regulated genes include TNF-a, Fos and JUN which are involved in cell death and IL-1A and CXCL which are involved in neutrophil chemotaxes. Knowledge of the up-regulated gene profile will be exploited for enhancing prostate tumor targeting by A1-R.

Keywords: Salmonella typhimurium A1-R, human prostate cancer, microarray, apoptosis, necrotic cell death, neutrophil chemotaxis, tumor-targeting.

Citation Format: Ming Zhao, Wen Wu, Chongyi Lu, Yong Zhang, Robert M. Hoffman, Hui Qi, Meng Yang, Yanqin Gu, Yiyu Lu, Shibing Su, Hui Zhang, Qianmei Zhou, Qilong Chen. Microarray gene expression analysis of human prostate cancer PC-3 cells targeted by Salmonella typhimurium A1-R. [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 4903. doi:10.1158/1538-7445.AM2015-4903
Abstract 4909: Increased IR-A/IR-B ratio in non-small cell lung cancers associates with lower epithelial-mesenchymal transition signature and longer survival in squamous cell lung carcinoma
Authors: Huang Jiaqi , Zhu Wei , Jiang Liyan , Streicher Katie , Morehouse Chris , Brohawn Philip , Ge XiaoXiao , Dong Zhengwei , Yin Xiaolu , Zhu Guanshan , Gu Yi , Mahadevia Parthiv , Higgs Brandon W. , Yao Yihong
Published: August 2015

To evaluate the insulin receptor isoform mRNA expression status in NSCLC patients RNA-seq data from 614 NSCLC [355 adenocarcinomas(LUAD) and 259 squamous cell carcinomas (LUSC)] and 92 normal lung specimens were obtained from The Cancer Genome Atlas (TCGA) to evaluate the mRNAexpression of insulin receptor isoform A (IR-A) and insulin receptor isoform B(IR-B). The differential expression status of the insulin receptor isoforms in NSCLC patients was confirmed using qRT-PCR assays with lung cancer cDNA arrays and primary tumor samples. The mRNA expression levels of IR-B were significantly lower in some NSCLC samples compared to normal lung specimens,including both LUAD and LUSC. Notably, no IR-B transcripts were detected - only the IR-A isoform was expressed in 11% of NSCLC patients. This decrease in IR-B expression contributed to an elevated IR-A/IR-B ratio, which was also associated with lower epithelial-mesenchymal transition gene signatures in NSCLC and longer patient survival under standard of care in LUSC. In addition to NSCLC, RNA-seq data from TCGA revealed a similar increase in IR-A/IR-B ratio in many other cancer types, with high prevalence in acute myeloid leukemia,glioblastoma multiforme, and brain lower grade glioma. Our results indicate a common reduction of the mRNA expression level of IR-B and an increased IR-A/IR-B mRNA ratio in NSCLC and other tumor types. The relationship of altered IR-A/IR-B ratios with cancer progression and patient survival should be prospectively explored in future studies.

Citation Format: Jiaqi Huang, Wei Zhu, Liyan Jiang, Katie Streicher, Chris Morehouse, Philip Brohawn, XiaoXiao Ge, Zhengwei Dong, Xiaolu Yin, Guanshan Zhu, Yi Gu, Parthiv Mahadevia, Brandon W. Higgs, Yihong Yao. Increased IR-A/IR-B ratio in non-small cell lung cancers associates with lower epithelial-mesenchymal transition signature and longer survival in squamous cell lung carcinoma. [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 4909. doi:10.1158/1538-7445.AM2015-4909
Abstract 4911: Mutually exclusive FGFR2, HER2, and KRAS gene amplifications in gastric cancer revealed by multicolor FISH
Authors: Das Kakoli , Gunasegaran Bavani , Tan Iain Beehuat , Deng Niantao , Lim Kiat Hon , Tan Patrick
Published: August 2015

Gastric cancer (GC) is a major cause of global cancer mortality. Previous genomic studies have reported that several RTK-RAS pathway components are amplified in GC, with individual tumours often amplifying one component and not others (“mutual exclusivity”). Here, we sought to validate these findings for three RTK/RAS components (FGFR2, HER2, KRAS) using fluorescence in situ hybridization (FISH) on a series of gastric tumours, cell lines and patient-derived xenografts. Applying dual-colour FISH on 137 gastric tumours (89 FFPE surgical resections and 48 diagnostic biopsies), we observed FGFR2 amplification in 7.3% and HER2 amplification in 2.2% of GCs. GCs exhibiting FGFR2 amplification were associated with high tumour grade (p = 0.034). In FISH positive tumours, striking differences in copy number levels between cancer cells in the same tumour were observed, suggesting intra-tumour heterogeneity. Using a multicolour FISH assay allowing simultaneous detection of FGFR2, HER2, and KRAS amplifications, we confirmed that these components exhibited a mutually exclusive pattern of gene amplification across patients. The FISH data was also strongly correlated with Q-PCR levels and at the protein level by immunohistochemistry. Our data confirms that RTK/RAS components are mutually exclusively amplified in GC, and demonstrates the feasibility of identifying multiple aneuploidies using a single FISH assay. Application of this assay to GC samples, particularly diagnostic biopsies, may facilitate enrollment of GC patients into clinical trials evaluating RTK/RAS directed therapies. However, the presence of intra-tumour heterogeneity may require multiple biopsy samples to be obtained per patient, before a definitive diagnosis can be attained.

Citation Format: Kakoli Das, Bavani Gunasegaran, Iain Beehuat Tan, Niantao Deng, Kiat Hon Lim, Patrick Tan. Mutually exclusive FGFR2, HER2, and KRAS gene amplifications in gastric cancer revealed by multicolor FISH. [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 4911. doi:10.1158/1538-7445.AM2015-4911
Abstract 4912: Screening of multiplex gene mutations using cell-free DNA in lung cancer patients
Authors: Kwon Nak-Jung , Sung Jae Sook , Seong Ahreum , Kim Hyunmi , Lee Woo Chung , Kim Yeul Hong , Yang Kap-Seok , Seo Jeong-Sun
Published: August 2015

Cell free DNA present in the blood stream shows much potential as a useful cancer maker for early diagnosis and cancer progression monitoring. Especially, analyzing the cell free DNA with Next Generation Sequencing (NGS) technology allows high through put examination of various genes concurrently at a low cost. On average, healthy person's serum contains only about 5ng/ml of cell free DNA, whereas a cancer patient's serum contains up to 1,500ng/ml of cell free DNA. For these reasons, cell free DNA could be a useful cancer marker to further our understanding of the patient's conditions. However, sequencing cell free DNA using NGS platform requires various quality control steps to ensure the quality and the quantity of the cell free DNA extracted and the libraries produced from them.

In this study, we examined the viability of using cell free DNA on NGS platforms. First, DNA was extracted and sequencing libraries were prepared using currently available commercial kits. Next, the quality of the libraries prepared was evaluated through whole genome sequencing on MiSeq. Lastly, lung cancer driver mutation was analyzed using TruSight Cancer Content Set and Ion AmpliSeq Cancer hotspot Panel. Two panels were chosen in order to study the viability of capture hybridization method and amplicon method.

Citation Format: Nak-Jung Kwon, Jae Sook Sung, Ahreum Seong, Hyunmi Kim, Woo Chung Lee, Yeul Hong Kim, Kap-Seok Yang, Jeong-Sun Seo. Screening of multiplex gene mutations using cell-free DNA in lung cancer patients. [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 4912. doi:10.1158/1538-7445.AM2015-4912
Abstract 4922: Analysis of copy number changes in HNSCC by MLPA
Authors: Dalay Nejat , Gurbuz Orkun , Baltaci Elif , Karaman Emin , Buyru Nur
Published: August 2015

Gene amplification is a common finding in a broad spectrum of tumor types, often leading to increased levels of gene expression, a mechanism by which genetic changes contribute to the initiation or progression of cancer. HNSCC is among the neoplasias that frequently show amplification of specific genomic regions. Previous cytogenetic and molecular genetic studies by Fluorescence in situ Hybridization (FISH) and comperative genome hybridization (CGH) and other molecular methods showed that frequently amplified chromosomal regions in primary HNSCC tumors are 3q, 8q, 8p, 11q, 9q. Generally, proto-oncogenes reside in these amplified regions and amplification of these often leads to increased levels of gene expression. Therefore, in this study we investigated amplification of 22 different oncogenes mapping to these regions. The analyses were performed by MLPA on an ABI 3100 genetic analyzer and relative peak areas of the amplification products were evaluated using the Coffalyser MLPA analysis software.

Analysis of 50 HNSCC tumor tissue samples showed that the most frequently amplified genes were from the regions 17q12 (MED1), 11q13.3 (CCND1), 8q (MYC and MTDH) and 8p (ZNF703) with frequencies of 24%, 24%, 20%, 20% and 18%, respectively.

In total, Copy Number Alterations of the CCND1 and MED1 genes were observed in 12 of 50 tumor samples. For the CCND1 gene 6 of these alterations were heterozygous duplications and the remaining 6 were triplications. On the other hand, for the MED1 gene duplications were observed in 8 samples and trriplications were detected in 4 samples. Triplication of the MTDH gene was not observed in any of the amplified samples. However, one third of the ZNF703 amplifications were triplications.

Amplification of the CCND1 and MYC genes are frequently observed in different types of cancers including HNSCC. More recently overexpression of MTDH has been associated with lymph node metastasis and poor-survival in HNSCC. However, to our knowledge there is no data in the literature investigating the ZNF703 gene in HNSCC. Our data indicate that ZNF703 may represent a candidate gene the role of which warrants more detailed analysis in HNSCC.

Citation Format: Nejat Dalay, Orkun Gurbuz, Elif Baltaci, Emin Karaman, Nur Buyru. Analysis of copy number changes in HNSCC by MLPA. [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 4922. doi:10.1158/1538-7445.AM2015-4922
Abstract 4929: An ARMS PCR assay for detection of PI3KCA mutations in FFPE tumor samples
Authors: Yu Dehua Derek
Published: August 2015

The activating mutations of phosphatidylinositol-3-kinase subunit pl10a (PIK3CA) occur in a variety of cancer types and serve as the predictive markers for response to inhibitors targeting a number of receptor tyrosine kinases (RTKs) and PI3K/AKT pathway. Therefore detection of PI3KCA mutations will guide patient selection for effective personalized cancer therapy. Here we reported a new method for the rapid and reliable detection of PI3KCA mutations by using modified mutation-specific ARMS primers.

In this real-time PCR system, the mutant gene is selectively amplified by the modified ARMS primers and the amplicon extending signals are collected by the taqman-probe at the same time.

The ARMS assay could detect 5 mutations on PI3KCA gene with a sensitivity of 0.5% under the background of a total of 10ng wild-type genomic

DNA. By using this assay, we analyzed the PI3KCA mutations in 113 Chinese breast cancer FFPE samples in comparison with Sanger sequencing. Our results indicated that the positive rates were 27.4

%, while 24.8% when measured by Sanger sequencing. The coincidence rates of the two methods were 89.3

% for the positive samples and 96.5% for the negative samples. The total coincidence rate was 97.3%. In conclusion, our study provides a basis for the detection of somatic mutations in PI3KCA in a routine clinical setting. The

ARMS PCR assay allows the detection of the PI3KCA mutations in FFPE samples with better sensitivity and feasibility than the current assays used in clinic.

Note: This abstract was not presented at the meeting.

Citation Format: Dehua Derek Yu. An ARMS PCR assay for detection of PI3KCA mutations in FFPE tumor samples. [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 4929. doi:10.1158/1538-7445.AM2015-4929
Abstract 493: Drug response profiling to inform individualized treatment approaches in high risk leukemia
Authors: Frismantas Viktoras , Rinaldi Anna , Dobay Maria Pamela , Higi Salome , Eugster Sabrina , Marovca Blerim , Horvath Peter , Delorenzi Mauzro , Kunz Joachim , Bandapalli Obul R. , Cario Gunnar , Stanulla Martin , Kulozik Andreas E. , Muckenthaler Martina , Eckert Cornelia , Radimerski Thomas , Bourquin Jean-Pierre , Bornhauser Beat C.
Published: August 2015

Novel treatment approaches are needed for patients with acute lymphoblastic leukemia (ALL) who respond poorly to current therapies. The genotypic diversity identified recently by next generation sequencing technologies within ALL calls for individualized novel strategies. However, functional correlations of oncogenic lesions with drug response profiles are ill defined for ALL. We have established an imaging-based cell viability analysis platform to generate drug response profiles for primary patient-derived ALL samples co-cultured with mesenchymal stroma cells, expecting to derive functional information directly from individual patient samples. Such response profiles may mirror perturbations in relevant cellular programs that could be exploited therapeutically. Our pipeline integrates high-content screening, newly developed bioinformatics tools and biochemical approaches. We screened a library of 65 compounds for activity in 37 precursor B-ALL and 23 T-ALL samples including refractory cases. Cross-sample comparisons revealed that cells from relapsed refractory patients showed a more resistant phenotype for most of the drugs. While only a few agents including genotoxic drugs showed activity across all samples, we detected selective activity of given drugs that distinguish patient sample groups. MLL-rearranged and TCF3-HLF-positive ALL samples as well as a subgroup of T-ALL cases were highly sensitive to the BCL-2 specific BH3-mimetic ABT-199 suggesting BCL-2 dependency for these cases. Multiparametric analyses of in vitro responses predicted in vivo activity of ABT-199 in xenografted mice. Moreover, we could identify synergistic activity of ABT-199 with clinical and preclinical compounds, such as topotecan or epigenetic modifiers. Our drug response profiling pipeline contributes to the identification of distinct vulnerabilities in leukemia and may facilitate the selection of candidate drugs for further development into clinical application.

Citation Format: Viktoras Frismantas, Anna Rinaldi, Maria Pamela Dobay, Salome Higi, Sabrina Eugster, Blerim Marovca, Peter Horvath, Mauzro Delorenzi, Joachim Kunz, Obul R. Bandapalli, Gunnar Cario, Martin Stanulla, Andreas E. Kulozik, Martina Muckenthaler, Cornelia Eckert, Thomas Radimerski, Jean-Pierre Bourquin, Beat C. Bornhauser. Drug response profiling to inform individualized treatment approaches in high risk leukemia. [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 493. doi:10.1158/1538-7445.AM2015-493
Abstract 4944: Identification of B-cell lymphoma 6 as a novel therapeutic target in glioblastoma
Authors: Chen Ye , Xu Liang , Hazawa Masaharu , Thippeswamy Anand M. , Yang Henry , Müschen Markus , Lin De-Chen , Koeffler Phillip
Published: August 2015

Background

Glioblastoma multiforme (GBM) is one of the most aggressive cancers in humans. Identification and characterization of the GBM-promoting factors is imperative for the development of novel and effective clinical interventions. We recently discovered that the expression of B-cell lymphoma 6 (BCL6), a gene involved in the development of B cell lymphoma, is up-regulated in GBM. This study aims to explore the functional implications of BCL6 in this disease.

Methods

We analysed the transcriptional level of BCL6 in primary glioma samples, and screened the protein expression in a panel of human GBM cell lines as well as primary GBM explants. cDNA microarray analysis was conducted to identify the potential downstream targets of BCL6 in GBM. Small-interfering RNA (siRNA), small hairpin RNA (shRNA), and CRISPR-mediated gene silencing strategies were applied to examine the biological function of BCL6. Finally, inhibitors targeting BCL6 were tested in glioma cells.

Results

In silico analysis revealed elevated BCL6 mRNA levels in both primary GBM samples and cell lines when compared with neural stem cell or non-tumor brain tissues. Western blot confirmed the prevalent expression of BCL6 in GBM cell lines as well as primary GBM explants. Functional studies showed that BCL6 played an important role in promoting cell growth. Depletion of endogenous BCL6 through siRNA, shRNA or CRISPR in GBM cells led to marked inhibition of cell proliferation both in vitro and in vivo.

Transcriptome analysis showed that BCL6 is an important regulator of TP53 signaling pathway. Knockdown of BCL6 increased the expression of p53, p21CIP and p27KIP. CHIP-qPCR further confirmed that BCL6 interacted directly to the TP53 promoter and suppressed its expression.

Moreover, BCL6 negatively regulated MEK-ERK activation in GBM cells. BCL6 depletion reduced the phosphorylation levels of both MEK1/2 and ERK1/2, but had little effect on AKT phosphorylation. Our further investigations uncovered that BCL6 modulated the expression of several key regulators of MEK-ERK pathway, such as SPRY1, SPRY2 and AXL. Finally, BCL6 inhibitor showed marked anti-proliferation activity in GBM cells, suggesting that targeting BCL6 may serve as a new strategy to treat GBM.

Conclusion

Our data suggest that BCL6 is involved in glioma tumorigenesis through regulating both the TP53 and MEK-ERK pathways, and that BCL6 is a potential therapeutic target for glioma treatment.

Citation Format: Ye Chen, Liang Xu, Masaharu Hazawa, Anand M. Thippeswamy, Henry Yang, Markus Müschen, De-Chen Lin, Phillip Koeffler. Identification of B-cell lymphoma 6 as a novel therapeutic target in glioblastoma. [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 4944. doi:10.1158/1538-7445.AM2015-4944
Abstract 4945: A novel role for drebrin in regulating progranulin bioactivity in bladder cancer
Authors: Xu Shi-Qiong , Buraschi Simone , Morcavallo Alaide , Genua Marco , Shirao Tomoaki , Peiper Stephen C. , Gomella Leonard G. , Belfiore Antonino , Iozzo Renato V. , Morrione Andrea
Published: August 2015

Bladder cancer (BC) is one of the most common cancers in the United States with 56,390 estimated new cases and 11,170 estimated deaths in 2014. The majority of deaths is due to metastatic spread. Understanding the molecular mechanisms regulating bladder tumor cell invasion and progression toward metastases is essential for developing better therapies to treat bladder cancer patients.

The growth factor progranulin plays an important role in cell proliferation, wound healing and transformation. We have recently established a critical role for progranulin in bladder cancer insofar as progranulin promotes urothelial cancer cell motility and contributes, as an autocrine growth factor, to the transformed phenotype by modulating invasion and anchorage-independent growth. In addition, progranulin expression is upregulated in invasive bladder cancer tissues compared to normal controls. However, the molecular mechanisms of progranulin action in bladder cancer have not been fully elucidated.

In this study, we searched for novel progranulin-interacting proteins using pull-down assays with recombinant progranulin and proteomics. We discovered that drebrin, an F-actin binding protein, bound progranulin in urothelial cancer cells. We characterized drebrin function in various urothelial cancer cell lines and showed that drebrin is critical for progranulin-dependent activation of the Akt and MAPK pathways and modulates F-actin remodeling, motility, invasion and anchorage-independent growth. In addition, drebrin regulates tumor formation in vivo and its expression is upregulated in bladder cancer tissues compared to normal tissue controls.

Our data are translationally relevant as indicate that drebrin exerts an essential functional role in the regulation of progranulin action and may constitute a novel target for therapeutic intervention in bladder tumors. In addition, drebrin may serve as novel biomarker for bladder cancer.

This work was supported by the Benjamin Perkins Bladder Cancer Fund, National Institutes of Health Grants RO1 CA164462 (A.M., R.V.I.), and RO1 CA39481 and RO1 CA47282 (R.V.I.), Associazione Italiana per la Ricerca sul Cancro (AIRC) (grant n. 10625/12), AIRC project Calabria 2014 and Fondazione Cassa di Risparmio di Calabria e Lucania PON01_01078 (A.B.). Alaide Morcavallo was supported in part by Fondazione Diabete Ricerca.

Citation Format: Shi-Qiong Xu, Simone Buraschi, Alaide Morcavallo, Marco Genua, Tomoaki Shirao, Stephen C. Peiper, Leonard G. Gomella, Antonino Belfiore, Renato V. Iozzo, Andrea Morrione. A novel role for drebrin in regulating progranulin bioactivity in bladder cancer. [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 4945. doi:10.1158/1538-7445.AM2015-4945
Abstract 4948: The cytosolic domain of a disintergrin and metalloprotease (ADAM) 15 promotes non-small cell lung cancer (NSCLC) proliferation
Authors: Hou Hsin-Han
Published: August 2015

Emerging evidence has indicated that proteins of a disintergrin and metalloprotease (ADAM) family contribute to cancer progression and metastasis. One member of this family, ADAM15, was reported to over-express in multiple cancers, including gastric, lung, breast, and prostate cancers. It has been shown that the enzymatic activities of its extracellular metalloprotease domain mediate ErbB signaling pathway and promote breast cancer proliferation and migration. Nevertheless, in non-small cell lung cancer (NSCLC), the role of ADAM15 and its correlation to patient prognosis is still not well understood. Moreover, unlike in breast cancer, the enzymatic activities of the ADAM15 extracellular domain appeared to be independent of ErbB signaling and lung cancer proliferation, indicating an alternative mechanism of ADAM15 in promoting lung cancer progression. Here, we demonstrated that ADAM15 was over-expressed in primary NSCLC tissues compared to the adjacent normal tissues. Patients with ADAM15 high-expressing lung tumors had shorter survival times. Furthermore, we examined different isoforms of ADAM15 and discovered that, isoform 6 (i6), which contains the most cytoplasmic Src homology 3 (SH3) binding motifs, was significantly upregulated in primary lung cancer tissues. Comparison of i6 and isoform 1 (i1), which has no cytoplasmic SH3 binding motifs, revealed that the cytoplastmic domain is essential for lung cancer proliferation, as well as aggressiveness in an in vitro lung cancer migration model. We also showed that ADAM15 i6, but not i1, mediated ErbB signaling pathway through physical interaction with growth factor receptor-bound protein 2 (Grb2), and activation of Src homolog 2 domain containing (Shc). Most importantly, overexpression of ADAM15 i6 promoted NSCLC cell growth in a xenograft mouse model while knockdown of i6 resulted in tumor shrinkage. Thus, we identified a novel mechanism of the ADAM15 cytoplasmic domain in NSCLC tumor progression, which will not only shed light on the molecular mechanisms of ADAM proteins, but also facilitate development of novel therapy in NSCLC.

Citation Format: Hsin-Han Hou. The cytosolic domain of a disintergrin and metalloprotease (ADAM) 15 promotes non-small cell lung cancer (NSCLC) proliferation. [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 4948. doi:10.1158/1538-7445.AM2015-4948
Abstract 4952: WWOX and LOT1 genes are down-regulated in non-small cell lung cancer (NSCLC)
Authors: Baykara Onur , Ekizoglu Eratak Seda , Kaynak Kamil , Koseoglu Hikmet , Buyru Nur
Published: August 2015

Lung cancer is a result of uncontrolled cell growth in lung tissue and is the most common type of cancer leading to death in men and second after breast cancer in women. Even though smoking is the main reason of lung cancer, genetic and environmental factors play an important role in the onset and prognosis of the disease. The WW domain-containing oxidoreductase (WWOX) gene is a putative tumor suppressor gene located on chromosome 16q23.3-24.1 and is composed of 9 exons. Several studies have reported the role of WWOX gene in different types of carcinomas including lung, breast, ovarian, stomach and nasopharyngeal cancer. The 46kDa WWOX protein has two WW (tryptophan) domains which can recognize and bind the proline-rich region (PPXY) of other proteins. Its main function is to inhibit tumor cell growth. WWOX also plays an important role in wnt/beta-catenin signaling pathway.

The lost-on-transformation 1 (LOT1/ZAC1/PLAGL1) gene is another putative tumor suppressor gene located on chromosome 6q24-25, a common site known for loss of heterozygosity in many tumors. Deletions of the 6q region are observed in different types of cancers including breast, prostate, small cell lung cancer and mesotheliomas. The LOT1 gene codes for a 55 kDa protein with seven C2H2 type zinc finger motifs located in amino terminal half of the protein. This zinc-finger nuclear transcription factor interacts with other proteins and transcription factors exerting antiproliferative effects in cancer cells.

The aim of this study was to investigate the expression levels of the WWOX and LOT1 genes by RT-PCR in tumor tissue samples from 39 patients with non small cell lung cancer (NSCLC). We observed down-regulation of WWOX and LOT1 expression in 82.1% and 48.7% of the patients, respectively. Expression levels of both genes were down-regulated simultaneously in 18 (46.1%) of the patients. When we analyzed the association between gene expression and age, sex, smoking, histology and stage, we found that WWOX expression was decreased in 28 of 31 (90.3%) patients older than 50 years (p = 0.02). Lower expression levels of both genes supports the hypothesis that WWOX and LOT1 act as tumor suppressors in NSCLC and that both genes may have a common effect on tumor progression which warrants further studies to analyze a possible cooperation between the two proteins.

Citation Format: Onur Baykara, Seda Ekizoglu Eratak, Kamil Kaynak, Hikmet Koseoglu, Nur Buyru. WWOX and LOT1 genes are down-regulated in non-small cell lung cancer (NSCLC). [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 4952. doi:10.1158/1538-7445.AM2015-4952
Abstract 4955: Somatic alteration and depleted nuclear expression of BAP1 in human esophageal squamous cell carcinomas
Authors: Mori Takahiro , Sumii Makiko , Fujishima Fumiyoshi , Ueno Kazuko , Nagasaki Masao , Ishioka Chikashi , Chiba Natsuko
Published: August 2015

We have found a case of somatic non-synonymous mutation of BAP1, substituting phenylalanine to isoleucine at codon 170 (F170I) out of 49 patients with esophageal squamous cell carcinomas (ESCs) examined by Sanger's direct sequencing. By in vivo Ubiquitination assay, deubiquitininase activity was suppressed in F170I, comparing with wild BAP1. BAP1 localization assay revealed that F170I mutant preferred to localize within cytoplasm although wild BAP1 mostly localizes within nucleus. Microarray analysis revealed that F170I has drastically altered gene expression profile. The following gene-ontology analyses indicated that expression of genes involved in oncogenic pathways has drastically altered by F170I. We have also found that BAP1 expression was depressed around 10% of ESCs examined by immunohistochemistry. It is interesting that nuclear BAP1-depleted cases have strong cytosolic expression of BAP1, instead. As nuclear localization of BAP1 is important in tumor suppressor function, BAP1 may be functionally inactivated around 10% of ESCs.

Citation Format: Takahiro Mori, Makiko Sumii, Fumiyoshi Fujishima, Kazuko Ueno, Masao Nagasaki, Chikashi Ishioka, Natsuko Chiba. Somatic alteration and depleted nuclear expression of BAP1 in human esophageal squamous cell carcinomas. [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 4955. doi:10.1158/1538-7445.AM2015-4955
Abstract 4966: Identification of deubiquitinating enzyme USP19 as a regulator of EWS/FLI1 protein turnover in Ewing sarcoma
Authors: Gierisch Maria E. , Lopez-Garcia Laura A. , Pfistner Franziska , Niggli Felix K. , Schaefer Beat W.
Published: August 2015

Ewing sarcoma belongs to the family of pediatric tumors which arise most commonly in bone. The majority of Ewing sarcoma is characterized by a balanced translocation between chromosomes 11 and 22 which encodes for the uniquely expressed fusion protein EWS/FLI1. Tumor cells are crucially dependent on expression of the fusion protein. Protein degradation is an important and highly regulated process in all cells and novel insights are beginning to be applied for cancer therapy. We aim to investigate the mechanism of turnover with the goal to diminish EWS/FLI1 protein and thereby identify novel targets for Ewing sarcoma treatment.

Here, we show first that EWS/FLI1 protein stabilizes upon proteasome inhibition. Co-immunoprecipitation of EWS/FLI1 with ubiquitin shows a characteristic ubiquitination smear indicating that EWS/FLI1 is ubiquitinated and a substrate of the ubiquitin-proteasome system. We characterized the turnover itself as rather slow. Interestingly, the half-life time of the fusion protein is increased by four-fold compared to the full length protein FLI1. This increased stability possibly contributes to the oncogenic and resistant features of the fusion protein.

To identify a deubiquitinating enzyme capable of regulating EWS/FLI1 turnover, we selected 20 deubiquitinating enzymes based on their expression in Ewing cell lines and tumors in-silico. Then, we established a screening approach directly measuring EWS/FLI1 protein levels as a read-out by an ELISA-like assay, allowing us to monitor the amount of 3x flag-tagged EWS/FLI1 protein. After transient knocked down of the 20 candidates, the screening revealed ubiquitin specific protease 19 (USP19) as a promising candidate. Depletion of USP19 via siRNA or shRNA was capable to diminish endogenous EWS/FLI1 protein level which led to modulation of its target gene levels. Vice versa, USP19 overexpression stabilized EWS/FLI1 protein level by twofold. Most importantly, stable knockdown of USP19 in Ewing cell lines led to a 40% reduction of cell growth and proliferation. Currently, we further characterize the influence of USP19 on EWS/FLI1 turnover and Ewing cell viability in vivo to identify novel possibilities for therapeutic interventions.

Citation Format: Maria E. Gierisch, Laura A. Lopez-Garcia, Franziska Pfistner, Felix K. Niggli, Beat W. Schaefer. Identification of deubiquitinating enzyme USP19 as a regulator of EWS/FLI1 protein turnover in Ewing sarcoma. [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 4966. doi:10.1158/1538-7445.AM2015-4966
Abstract 4970: USP15 regulates SMURF2 kinetics through C-lobe mediated deubiquitination
Authors: Eichhorn Pieter J.A. , Iyengar Prasanna , Jaynes Patrick , Verma Chandra
Published: August 2015

Transforming growth factor-β (TGFβ) is a multifunctional cytokine that acts as a tumour suppressor in normal cells and as an oncogene in advanced tumour cells. In the later scenario, TGFβ signaling enhances pro-invasive and pro-metastatic properties of the cancer cell. Ubiquitin modification of the TGF-β pathway components is emerging as a key mechanism of TGF-β pathway regulation. To limit TGF-β responses, TGF-β signaling is regulated through a negative feedback loop whereby the E3 ligase SMURF2 targets the TGF-β receptor (TβR) complex for ubiquitin-mediated degradation. Counteracting this process, a number of deubiquitinating (DUBs) enzymes have recently been identified that deubiquitinate and stabilize the TβR. However the precise mechanism by which these DUBs act on TβR function remains poorly defined. Here, we demonstrate that apart from targeting the TBR complex directly USP15 also deubiquitinates SMURF2 resulting in enhanced TβR stability and downstream pathway activation. Through proteomic analysis, we show that USP15 modulates the ubiquitination of Lys734, a residue required for SMURF2 catalytic activity. Our results show that SMURF2 is a critical target of USP15 in the TGF-β pathway and may also explain how USP15 and SMURF2 target multiple complementary protein complexes in other pathways.

Citation Format: Pieter J.A. Eichhorn, Prasanna Iyengar, Patrick Jaynes, Chandra Verma. USP15 regulates SMURF2 kinetics through C-lobe mediated deubiquitination. [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 4970. doi:10.1158/1538-7445.AM2015-4970
Abstract 4971: Detection of receptor and non receptor tyrosine kinases in the culture filtrates of astrocytes and cancer cells
Authors: Gogineni Venkateswara Rao
Published: August 2015

As demonstrated in our previous work, meningioma cells shed their membrane receptors and these receptors function in a cell-free environment. The purpose of this study was to determine if receptor and non-receptor kinases were similarly being released in different cancers. Using antibody arrays, cell culture filtrates from glioma and prostate cancer cell lines were assessed to determine if solubilized receptors and kinases were present. Supernatants from cells treated with Brefeldin-A were analyzed similarly. Further, culture filtrates concentrated by ultrafiltration were subjected to luminescence based kinase assays and western blotting to assess the active forms. Cell viability was measured with Trypan blue staining in all the experiments. Our results demonstrate that an array of receptors are solubilized into culture filtrates of brain and prostate cancer cell lines within the first 24 hours. Simultaneously, soluble receptors in the culture filtrates of astrocytes within 48 hours of culture were also detected. Compared to astrocytes, cancer cells showed elevated levels of receptors and kinases released at 48 hours. As many as 15 receptor or non-receptor tyrosine kinases including Src family of kinases were found in the culture filtrates of the brain and prostate cancer cell lines. Treatment with Brefeldin-A inhibited the release of receptors and kinases partially, but markedly, within first 24 hours suggesting a secretory pathway for their release. Additionally, phosphorylation of tyrosine kinases was detected in the western blots within 24 hours. Kinase assays further confirmed active nature of the extracellular kinases, particularly, when checked for the active form of Lyn kinase in the culture filtrates, phosphorylation on Tyrosine-507 was detected along with the total forms. Cell viability assays on the cultured cells revealed more than 98% viability, thus eliminating the possibility of the release of detected kinases from dead cells. In light of this study, we propose a new niche for solubilized receptor and tyrosine kinase functioning that might have a role in intercellular signaling in cell-free conditions both in disease and physiological statuses.

Note: This abstract was not presented at the meeting.

Citation Format: Venkateswara Rao Gogineni. Detection of receptor and non receptor tyrosine kinases in the culture filtrates of astrocytes and 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 4971. doi:10.1158/1538-7445.AM2015-4971
Abstract 4985: Notch1 as a key mediator in promoting advanced castration-resistant prostate cancer
Authors: Stoyanova Tanya , Faltermeier Claire , Smith Bryan , Goldstein Andrew , Zhang Xi , Drake Justin , Lee John , Orellana Sandra , Blum Steven , Cheng Donghui , Pienta Kenneth , Huang Jiaoti , Witte Owen
Published: August 2015

The first line of treatment for men with advanced prostate cancers is androgen deprivation therapy. However, the disease commonly relapses in its lethal metastatic form referred to as castration-resistant prostate cancer (CRPC). CRPC is the primary cause of prostate cancer specific mortality in men. Current therapies including chemotherapeutic agents improve median overall survival by only few months. The mechanisms that distinguish clinically localized indolent tumors from lethal CRPC are unclear. Here we demonstrate that ectopic expression of Notch1 promotes progression to poorly differentiated carcinoma when combined with pathways that are altered in advanced disease but are insufficient to drive aggressive prostate cancer alone. Notch1 driven tumors are resistant to androgen deprivation. Transcriptional profiling reveals that these tumors display features of epithelial to mesenchymal transition, a morphological change associated with tumor aggressiveness and metastasis. Our study provides the first functional evidence that Notch1 signaling axis is a key mediator in promoting advanced prostate cancer and may represent a new therapeutic target for the advanced disease.

Note: This abstract was not presented at the meeting.

Citation Format: Tanya Stoyanova, Claire Faltermeier, Bryan Smith, Andrew Goldstein, Xi Zhang, Justin Drake, John Lee, Sandra Orellana, Steven Blum, Donghui Cheng, Kenneth Pienta, Jiaoti Huang, Owen Witte. Notch1 as a key mediator in promoting advanced castration-resistant prostate cancer. [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 4985. doi:10.1158/1538-7445.AM2015-4985