bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2389: A microfluidic system for the selection of circulating tumor cells that utilizes both affinity and size capture technologies
Authors: Smirnov Denis , Merdek Keith , Sprott Kam , Skelley Alison , Huang Richard , Tenney Don , Jiang Chunsheng , Milutinovic Aladin , Tims David , Dong Yi , Cain Jason , Wolfe Michele , Pierceall Bill , Carney Walter
Published: August 2012

Cancer is a disease of uncontrolled cell growth and dissemination. As tumors increase in size and vascularity, populations of cells break off from the primary tumor, enter into the circulation, and are transported to distant parts of the host. These circulating tumor cells (CTCs) may constitute a seed population that is responsible for subsequent growth of additional tumors (metastasis) in different tissues. Therefore capture, and subsequent characterization of CTCs from patient's blood, offers an opportunity to both study and monitor progression of the disease. One of the most common methodologies used for CTC capture is affinity based, where specific antibodies bind to cell surface antigens and allow cell capture. A second technique employs size filtration. CTCs are generally larger than normal leukocytes and this size differential can be exploited to enrich CTCs. Here we describe a CTC dual capture platform developed at On-Q-ity Inc. that combines both of these isolation techniques to offer more efficient CTC capture than either technology alone. Performance of On-Q-ity platform will be compared to Veridex's CellSearch. Utility of On-Q-ity platform for sophisticated molecular and cellular characterization of captured cells will also be described.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2389. doi:1538-7445.AM2012-2389
Abstract 2392: A novel approach for detecting viable and tissue-specific circulating tumor cells
Authors: Wu Ping , Oh Daniel , Chowdhury Wasim H. , Kudrolli Tarana A. , Liu Minzhi M. , Sokoll Lori , Rodriguez Ronald , Lupold Shawn E.
Published: August 2012

Circulating tumor cells (CTCs) offer a new strategy to access cancer tissue through a simple blood sample. CTCs levels can be predictive of overall survival in patients with metastatic cancers and changes in CTC levels following therapy can be indicative of therapeutic response. Moreover, CTCs may provide a means to select patients for specific therapeutic approaches by identifying active or susceptible pathways. However, most of the current methods for detecting CTCs have limitations including the need for specialized equipment, the reliance on cell capture through cell surface markers such as EpCAM, limited sensitivity, the inability to distinguish cancer from normal, tissue type, active pathways, or cell viability. Here we present a novel CTC detection approach which applies tissue-selective replicating adenoviruses and highly sensitive secreted reporters. These Circulating Tumor Cell Reporter Viruses (CTC-RVs) were generated by replacing the early viral E1A gene promoter with the prostate specific probasin promoter and prostate specific antigen enhancer (PSE-PBN). The secreted reporter gene, humanized metridia luciferase (hMLuc), was incorporated into the viral major late transcriptional unit, at the fiber gene locus, to limit reporter expression to only those cells in which the virus has replicated. Cells from total blood, including mononuclear cells and CTCs, are placed in tissue culture media and infected with the CTC-RV. After three days, viral replication dramatically amplifies reporter levels within infected prostate cancer cells with active Androgen Receptor pathways leading to the secretion of the bioluminescent reporter. Non-prostatic cells will not amplify the virus or express the reporter gene. LNCaP cellular dilution study results show that as few as five CTCs per million lymphocytes cells could be readily detected without any cell isolation. LNCaP cells diluted in healthy volunteer blood samples identified the optimal time and dosing conditions. Initial studies in clinical samples support the detection of viable CTCs with this assay. This new approach offers a means to quantify live CTC levels in a tissue-specific manner, without the need for cell capture, through the use of a secreted signal which can be detected separate from the large blood cell population.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2392. doi:1538-7445.AM2012-2392
Abstract 2399: Syntenin-1 promotes cell invasion though regulating Slug-dependent epithelial-mensenchymal transition in non-small cell lung cancer
Authors: Wang Lu-Kai , Chen Yuh-Ling , Hong Tse-Ming , Yang Pan-Chyr
Published: August 2012

Slug, a transcriptional repressor, is involved in the regulation of epithelial-mesenchymal transition (EMT) during development and can promote cancer cell invasion and metastasis. Through Yeast-two hybrid screening, we found that syntenin-1, a tandem PDZ protein, can associate with Slug. Syntenin-1 is known to involve in many biological functions such as protein traffics and cytoskeleton remodeling. Recently, it has been found that overexpression of syntenin-1 may promote cancer cell invasion by altering defined biochemical and signaling pathways, including c-Src/ focal adhesion kinase (FAK) and NF-kappaB. Conversely, knock-down the expression of syntenin-1 can also increase cell aggregation. All these findings indicate that syntenin-1 may play an important role in cell adhesion and movement. Whether syntenin-1 is involved in the process of EMT should be further clarified. In this study, we showed that syntenin-1 could enhance Slug-dependent cell invasion and regulate the EMT in lung cancer cells. Syntenin-1 can interact with Slug through its PDZ-1 domain and increase their nuclear localization in cells. Overexpression of syntenin-1 increases Slug-induced cell migration and invasion though regulating the repression activity of Slug. Knock down the expression of syntenin-1 may also inhibit the repression activity of Slug on promoter of E-cadherin in highly invasive lung cancer cell lines. In Sum, Syntenin-1 may promote cell Invasion though regulating Slug-dependent EMT process in Non-small cell lung cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2399. doi:1538-7445.AM2012-2399
Abstract 24: Role of S100A14 in proliferation and invasion of oral cancer-derived cells
Authors: Sapkota Dipak , Costea Daniela , Bruland Ove , Vasstrand Endre N. , Ibrahim Salah
Published: August 2012

Members of multifunctional S100 protein family have been reported to be differentially expressed in a variety of human cancers and are functionally linked with various aspects of carcinogenesis. S100A14 is a recently identified member of the S100 protein family. Despite reports of altered expression in different cancer types, functional significance of S100A14 in carcinogenesis is largely unknown. In this study, we aimed to investigate the functional role of S100A14 proliferation and invasion of oral squamous cell carcinoma (OSCC)-derived cell. S100A14 mRNA and protein levels were found to be down-regulated in OSCCs and OSCC-derived cells compared to the corresponding normal controls. In addition, membranous S100A14 immunoexpression was found to be gradually lost in the invasive fronts/islands compared to the central/superficial part of OSCCs. Over-expression of S100A14, by using retroviral expression construct, was found to be associated with the inhibition of cell proliferation, induction of G1-arrest and up-regulation of p21 in OSCC-derived cell lines (CaLH3 and OSCC1) with wild type p53. Similarly, over-expression of S100A14 was associated with inhibition of invasive potential of OSCC-derived cells in the in vitro Matrigel invasion assay. S100A14 mediated inhibition of cellular invasion was found to be associated with modulation of mRNA signature of genes involved in tumor invasion and metastasis, in particular MMP1, MMP9. Taken together, these data suggest that similar to other multifunctional S100 protein members, S100A14 might be involved in OSCC progression by regulating cell proliferation and invasion. Hence, further characterization of the molecular pathways/targets of S100A14 might lead to the better understanding of proliferation and invasion of OSCC and this process, in future, might be useful as a diagnostic or therapeutic approach in cancers.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 24. doi:1538-7445.AM2012-24
Abstract 2406: The effect of anoxia/reoxygenation on epithelial-mesenchymal transition in human colon cancer cell lines
Authors: Okajima Manabu , Kokura Satoshi , Ishikawa Takeshi , Matsuyama Tatsuzo , Tsutiya Reiko , Adachi Satoko , Sakamoto Naoyuki , Naito Yuji , Yamane Tetsuro
Published: August 2012

Background and Aim: Epithelial-mesenchymal transition (EMT) is considered as crucial events in cancer metastasis. Anoxia/reoxygenation (A/R) is known to occur in cancer tissues, due to the process of angiogenesis and the change of the pressure in tissue growth. In this study, we investigated whether A/R induces EMT in the human colon cancer cells. Materials and Methods: Colon cancer cells, HT29 cells, were exposed to anoxia (2 h) followed by reoxygenation (4-22 h). To evaluate EMT, morphological changes were assessed by light microscopy, immunofluorescence and Western blot analyses. Moreover, RT-PCR for the EMT-related transcription factors (Snail and ZEB1) was performed and the activation of nuclear factor kappa B (NF-κB) was analyzed in the nuclear extracts using an ELISA method. To determine whether NF-kB was involved in A/R-induced EMT, HT-29 cells were treated with proteasome inhibitors. Results: Colon cancer cells exposed to A/R showed phenotypic changes consistent with EMT: spindle-shape and intercellular separation. The expression of E-cadherin on the cell surface and the total amount of E-cadherin protein were reduced after A/R. The EMT-related transcriptional factors such as Snail and ZEB1 were induced after exposure to A/R. Furthermore, exposure to A/R caused an activation of NF-κB and pretreatment with proteasome inhibitors significantly attenuated the effects of exposure to A/R. Conclusions: These results indicate that A/R induces EMT in human colon cancer cells through partially NF-κB dependent transcriptional pathway.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2406. doi:1538-7445.AM2012-2406
Abstract 2408: CD44s is required for a tumor-initiating stem-like mesenchymal phenotype in hepatocellular carcinoma via ligand-independent c-Met activation
Authors: Dang Hien T. , Ding Wei , Rountree Carl Bart
Published: August 2012

c-Met, a high affinity receptor for hepatocyte growth factor (HGF), plays a critical role in cancer growth, invasion and metastasis. Hepatocellular carcinoma (HCC) patients with active HGF/c-Met signaling have a significantly worse prognosis. CD44, the receptor for hyaluronic acid, is utilized as a surface marker to identify mesenchymal tumor-initiating stem-like cells (TISC) in several cancers. CD44v6 (spice variant 6) has been shown to promote HGF activation of c-Met. In this work, we examine the role of CD44s as a deregulator and activator of c-Met signaling in HCC. In a transcriptome profile analysis of 149 human HCC cases, we identified a significant positive correlation (R2=0.8993) between up-regulated CD44 and c-Met expression. The human HCC cell lines MHCC97-H and Huh7 were further utilized to investigate the direct relationship between c-Met and CD44s. Metastatic MHCC97-H cells are 95% CD44+ and demonstrate ligand-independent c-Met activation. CD44+/c-Met+ MHCC97-H cells demonstrate a mesenchymal phenotype with low expression of E-cadherin, high level of tumorsphere formation, increased expression of fibronectin and Zeb2 compared to Huh7 cells, which have are CD44neg/c-Metneg. In MHCC97-H cells, a direct interaction between CD44s and c-Met was demonstrated by immunoprecipitation/immuno-blot analysis. Transient and stable inhibition of c-Met in MHCC97-H cells results in increased E-cadherin, decreased CD44s, and reduced tumor-initiating characteristics and loss of tumorsphere formation. Likewise, inhibition of CD44s demonstrates similar phenotype with increased E-cadherin, decreased tumorsphere formation, and decreased c-Met activation and loss of downstream Akt and Erk phosphorylation. CD44s knockdown recovered c-Met to a regulated HGF-dependent state. Selective inhibition of either PI3K (Ly294002) or MAPK (PD98059) pathways in MHCC97-H cells demonstrate that PI3K/Akt activation signals drive CD44s expression in a feed forward fashion. Conversely, the over-expression of CD44s in Huh7 cells demonstrates a mesenchymal phenotype with fibroblastic-like cell morphology, decreased E-cadherin, increased Zeb1, increased PI3K/Akt signaling, and increased tumorsphere formation compared to control Huh7 cells. Selective inhibition of PI3K/Akt signaling in CD44+ Huh7 cells resulted in reduced CD44s expression and decreased tumorsphere formation. In conclusion, CD44s induces a mesenchymal phenotype and tumor initiating characteristics in HCC through the deregulation of c-Met and activation of a positive feedback loop through PI3K/Akt signaling.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2408. doi:1538-7445.AM2012-2408
Abstract 2459: Membrane type 1 matrix metalloproteinase and integrin-linked kinase: A characterization of potential partners in ovarian cancer metastasis
Authors: Bruney Lana , Stack Sharon
Published: August 2012

Epithelial ovarian cancer (EOC) is the fifth leading cause of overall cancer death among American women. In 2010, 15,520 deaths were directly attributed to EOC; and 21,650 additional cases were diagnosed. When EOC is diagnosed prior to metastatic dissemination, the overall 5 year survival rate is 93%; however, over 75% of women with EOC are diagnosed with metastasis already present, dropping the survival rate to less than 20%. The process of EOC metastasis is unique in that epithelial cells detach from the primary tumor and shed into the peritoneal cavity as single cells and multicellular aggregates (MCA), which adhere intraperitoneally and invade the interstitial collagen-rich submesothelial matrix, where they proliferate to anchor secondary lesions. Proteolytic activity promotes the migration and collagen invasion crucial to metastatic success. Also key to cell migratory activity are focal adhesions (FA), integrin- and protease-rich cell adhesion sites that physically link the actin cytoskeleton to the extracellular matrix (ECM) and transduce signals into the intracellular compartment. Sites of adhesive contacts with collagen fibers have been associated with beta-1 integrin clustering and various matrix metalloproteinases. Membrane type 1 matrix metalloproteinase (MT1-MMP), an ECM degrading protease localized to the cell surface, has been shown to promote a collagen-invasive phenotype in ovarian carcinomas. Additionally, collagen-activated integrin-linked kinase (ILK), a beta-1 integrin cytoplasmic domain interactor, has been shown to regulate several biological processes that promote invasion and suppress anoikis. As initial steps to assess the contributions and potential inter-relationships of tumor cell invasion at FAs, presented here is a characterization of MT1-MMP and ILK expression in select ovarian cancer cell lines and in human EOC tumor tissue microarrays.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2459. doi:1538-7445.AM2012-2459
Abstract 2469: Increased cathepsin B expression in cultured tuberous sclerosis skin tumor cells and patient tumor tissues
Authors: Li Shaowei , Liu Ying , Wang Ji-an , Moss Joel , Darling Thomas N.
Published: August 2012

Skin hamartomas in tuberous sclerosis complex (TSC) are disfiguring red tumors that contain fibroblast-like cells, vessels, fibrous tissue, and elongated hair follicles. Our recent studies show that TSC skin tumor cells induce angiogenesis and hair follicle neogenesis in a xenograft mouse model. These morphogenic changes are likely caused by altered expression of proteins involved in tissue remodeling. To identify such factors, we carried out 2-D gel analysis of cell extracts of TSC2-null fibroblasts derived from a TSC periungual fibroma (PF) compared to fibroblasts from patient normal-appearing skin (NL). PF cells had greater levels of all three forms of cathepsin B than TSC normal fibroblasts. Cathepsin B, a member of the lysosomal cysteine proteases family that has been implicated in angiogenesis and extracellular matrix remodeling, exists in pro-, single chain and double chain forms, and these were increased in PF cells by 1.6-, 2.3- and 8.3-fold, respectively. Western blot analysis of TSC skin tumor fibroblasts from 5 patients showed dramatically greater levels of active cathepsin B than in paired patient NL fibroblasts. Tumor cells from PFs showed greater intracellular cathepsin B enzyme activity than NL controls. Western blot and ELISA analysis showed that media conditioned by TSC skin tumor cells from 5 different patients contained 4.1- to 36-fold more procathepsin B than media conditioned by NL fibroblasts. Immunohistochemical staining of TSC patient angiofibromas and PFs showed greater numbers of cathepsin B-positive cells than patient normal-appearing skin. These results suggest that TSC skin tumor cells may induce hamartoma morphogenesis through expressing and releasing high level of cathepsin B.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2469. doi:1538-7445.AM2012-2469
Abstract 2475: The abundance of SLC7a11 transporter in different tissues
Authors: Kondapalli Lavanya , Huang Ying , Lambros Maria P.
Published: August 2012

The purpose of this study is to investigate the abundance of the SLC7A11 transporter in different tissues. The SLC7A11 is a plasma membrane transporter which mediates the transport of one cystine into the cell in exchange for one intracellular glutamate. We performed RT-PCR and immunohistochemistry to demonstrate the abundance of SLC7A11 transporter in different tissues. Tissue array slides were purchased from US Biomax Inc. The tissues were stained with or without SLC7A11 primary antibody. The images were captured using the software NIS elements connected to Nikon Eclipse series microscope. The images were then analyzed for staining intensity compared to their negative controls by using the software, Imagej. Panc-1, BxPC-3 and MIAPaCa-2 are human pancreatic cancer cell lines which were purchased from ATCC. The cells were cultured at 370C in humidified atmosphere of 5% CO2 using MEM or RPMI medium with low cystine levels. RNA was extracted from all the cell lines and converted into cDNA for Real Time PCR analysis. PCR reactions were prepared using SYBR green (which acts as a nucleic acid stain), forward and reverse SLC7A11 or GAPDH primers, and diluted cDNA. The reactions were added into 96-well PCR strips and sealed to prevent evaporation of the samples. The ABI 7300 machine was run after configuring the software and finally analyzed using 7300 system SDS software. The immunohistochemistry images demonstrate that most cancers especially pancreatic cancers have a high abundance of SLC7A11 transporter compared to normal tissues. The RT-PCR results showed that PANC-1 has significantly higher SLC7A11 expression compared to BxPC-3 and MIAPaCa-2 cell lines. The results indicate that SLC7A11 transporter is highly expressed in cancer tissues.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2475. doi:1538-7445.AM2012-2475
Abstract 2478: Jagged 2 signalling is active in Langerhans cell histiocytosis and confers pathognomonic features on dendritic cells
Authors: Hutter Caroline , Kauer Max , Jug Gunhild , Simonitsch Ingrid , Kovar Heinrich , Gadner Helmut
Published: August 2012

Langerhans cell histiocytosis (LCH) is an enigmatic disease defined by the accumulation of eponymous Langerhans-cell-like cells of unknown origin. It is a rare disease that may affect any age group, although its most severe clinical course affects predominantly young children. The clinical manifestations range from single lesions that can resolve spontaneously to systemic disease that requires intensive chemotherapy and bone marrow transplantation. Here we demonstrate that LCH cells exhibit a unique transcription profile that clearly separates them from indigenous dendritic cells (DCs), i.e. Langerhans cells, myeloid dendritic cells, and plasmacytoid dendritic cells, indicating a distinct DC entity that occurs under pathologic conditions. Molecular analysis revealed that LCH cells selectively expressed NOTCH ligand Jagged 2 (JAG2) and were the only DCs that coexpressed both NOTCH ligand and receptor. In line, activated NOTCH was detected in LCH but not control biopsies. Furthermore, we show that JAG2 signalling induces key LCH markers CD1a and Langerin on MoDCs, suggesting a role of NOTCH signalling in LCH ontogenesis. Interestingly, JAG2 also strongly induced matrix-metalloproteinase (MMP) 1 in MoDCs, which is highly expressed in LCH lesions and mediates massive tissue destruction and remodelling. The MMP induction by JAG2 was selective for DCs and not recapitulated in monocytes. Together these findings strongly suggest that Jagged2 mediated NOTCH activation confers phenotypic and functional aspects of LCH to DCs. Thus, interference with NOTCH signalling may prove an attractive target to combat this disease.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2478. doi:1538-7445.AM2012-2478
Abstract 248: JNK-FoxO3a dependent apoptosis in quercetin treated MDA-MB-231 human breast cancer cells
Authors: Kim Haesung , Choi Byung-Soo , Lee Sang-Soo , Kim Lee-Su , Ryu Byung-Yoon , Nam Ju-Suk
Published: August 2012

FoxO transcription factors are well known as key tumor suppressors in mammalian cells. The suppression of FoxOs in cancer cells is thought to be mainly due to activation of multiple onco-kinases, such as Akt and SGK, by a phosphorylation-ubiquitylation-mediated cascade. On the other hand, phosphorlyation of FoxO by JNK relocalizes it from the cytoplasm to the nucleus allowing transcription of FoxO target genes. In this study, we applied human breast cancer cells to determine whether the activity of FoxO3a is regulated during the quercetin mediated apoptosis. After treatment with the indicated amount of quercetin (5-80uM) for 5days, the growth of human breast cancer cell lines including MDA-MB-231, MDA-MB-486 and MCF-7 cells was reduced in dose- and time-dependent manner. FACS analysis with AnnexinV/PI double staining showed that the proportion of early apoptotic cancer cells were significantly increased in quercetin treated cells. It represents the potent apoptotic effect of quercetin in breast cancer cells. Furthermore, a marked elevation of FoxO3a transcriptional activity was detected at 20μM of quercetin using FHRE-reporter assay, as well as increased protein level of FoxO3a was detected by western blotting. And also it was determined that the activation of FoxO3a lead to the upregulation of FasL expression during the apoptotic process. To identify the signaling mechanism of FoxO3a regulation, we examined the activation of several well known upstream kinases such as Akt, Erk, IKKβ, AMPK and JNK. Notably, a significant increase of JNK phosphorylation was observed in quercetin treated cells. In addition, the inhibition study with pharmacological inhibitor (SP600125) showed that the involvement of JNK for the regulation of FoxO3a activity is critical in quercetin treated breast cancer cells. These results demonstrate that quercetin induces FoxO3a-related apoptosis by activation of JNK in MDA-MB-231 cells, and suggest the possibility that JNK-FoxO3a signaling pathway contributes mainly to the apoptotic process of breast cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 248. doi:1538-7445.AM2012-248
Abstract 2482: Carnitine palmitoyltransferase 1A (CPT1A): a transcriptional target of PAX3-FKHR and mediates PAX3-FKHR-dependent motility in alveolar rhabdomyosarcoma cells
Authors: Liu Lingling , Chen Taosheng
Published: August 2012

Alveolar rhabdomyosarcoma (ARMS) has a high propensity to metastasize, leading to its aggressiveness and a poor survival rate among those with the disease. More than 80% of aggressive ARMSs harbor a PAX3-FKHR fusion transcription factor, which regulates cell migration and promotes metastasis, most likely by regulating the fusion protein's transcriptional targets. Therefore, identifying druggable transcription targets of PAX3-FKHR that are also downstream effectors of PAX3-FKHR-mediated cell migration and metastasis may lead to novel therapeutic approaches for treating ARMS. To identify genes whose expression is directly affected by the level of PAX3-FKHR in an ARMS cellular-context, we first developed an ARMS cell line in which PAX3-FKHR is stably down-regulated, and showed that stably downregulating PAX3-FKHR in ARMS cells significantly decreased the cells’ motility. We also found that when PAX3-FKHR was downregulated, the expression level of carnitine palmitoyltransferase 1A (CPT1A) decreased. Analysis of the CPT1A promoter identified 4 putative PAX3-FKHR binding sites. Results of mutational analysis, promoter reporter assays, and electrophoretic mobility shift assays showed that PAX3-FKHR binds directly and specifically to a paired-domain binding-site in the CPT1A promoter region, indicating that CPT1A is a novel direct transcriptional target of PAX3-FKHR. Downregulating CPT1A, either by gene knockdown or by using a pharmacologic modulator of CPT1A activity, decreased cell motility in ARMS cells. Taken together, we have identified CPT1A as a novel direct transcriptional target of PAX3-FKHR and revealed the novel function of CPT1A in promoting cell motility. CPT1A may represent a novel therapeutic target for the treatment of ARMS.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2482. doi:1538-7445.AM2012-2482
Abstract 2484: Whole-exome sequencing of a rare case of familial childhood acute lymphoblastic leukemia
Authors: Healy Jasmine , Saillour Virginie , Spinella Jean-François , Vidal Ramon , Bareke Eric , Richer Chantal , Larivière Mathieu , Busche Stephan , Ge Bing , Montpetit Alexandre , Pastinen Tomi , Sinnett Daniel
Published: August 2012

Acute lymphoblastic leukemia (ALL) is the most common cancer in children, accounting for approximately 25% of all pediatric cancer cases. However, familial childhood ALL is extremely rare. Few families with multiple non-twinned siblings diagnosed with childhood ALL have been reported, and to date no highly penetrant leukemia susceptibility gene(s) has been identified to explain this uncommon occurrence. We postulated that pure (nonsyndromic) familial childhood ALL could result from the accumulation of disadvantaging rare DNA variants in predisposing genes or biological pathways. To address this hypothesis, we used next-generation sequencing technologies to capture and re-sequence the whole-exomes of a family comprising the mother, father and two male non-twinned affected siblings (sibling A and sibling B). Both brothers were diagnosed with the identical ALL subtype, pre-B hyperdiploid childhood ALL, three years apart. The similar clinical and molecular characteristics of the siblings suggest shared etiologic factors. Using the Agilent SureSelect All Human Exon 38 Mb Kit and the SOLiD 3 Plus system, we captured and sequenced a total of 17.5 Gb of sequence for the entire family, with a mean coverage of 47X. For each individual, approximately 96% or 36.4 Mb of the targeted bases were covered α1X and 70% of the targeted bases or 26.4 Mb passed our thresholds for variant calling. We identified 52,038 positions at which the called allele(s) differed from the reference genome in at least one of the four family members. In total, we identified 19,096 germline variants in sibling A and 28,061 in sibling B, of which 2,355 (12.3%) and 2,125 (7.6%), respectively, were previously undiscovered in dbSNP. We investigated non-synonymous homozygous variant and compound heterozygous positions shared between the siblings, as well as genes/pathways with increased burden of rare non-synonymous variants. Based on several criteria (PolyPhen annotation, known allele frequency, etc.), we identified variants that are strong functional candidates to explain this case of pure familial childhood ALL. In parallel, high-density genotyping was also performed (Illumina Omni 2.5M) for quality control and structural variant detection, allowing the identification of putatively shared copy number variants that may also be involved in leukemogenesis. Though independent validation and functional assessment is required, this is the first study to identify genetic factors involved in pure familial childhood ALL.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2484. doi:1538-7445.AM2012-2484
Abstract 2487: CONSERTING: an accurate method for detecting focal and gross somatic copy number alterations in cancer genome by next generation sequencing
Authors: Chen Xiang , Wang Jianmin , Roberts Kathryn , Pounds Stanley , Dyer Michael , Mullighan Charles , Downing James , Zhang Jinghui
Published: August 2012

The Identification of somatically acquired DNA copy number alterations (CNA) is extremely important for finding causal genes in cancer. Although next-generation sequencing (NGS) can assay a genome at base-pair resolution, accurate identification of CNA from NGS data has been hindered by sequencing artifacts, coverage bias and paralogous genomic duplication. As a result, existing algorithms tend to generate many CNA segments that are not corroborated by an alternative assay such as SNP array, suggesting high false discovery rate. To improve CNA analysis for NGS data, we developed Copy Number Segmentation by Regression Tree in Next Generation Sequencing (CONSERTING), a novel copy number detection method that integrates read depth analysis with structural variation (SV) detection and adjustment for sequencing artifacts. Analysis of simulated data shown that CONSERTING outperforms existing methods by achieving high sensitivity of focal copy number detection (CONSERTING: 0.80, CNV-Seq: 0.26, SegSeq: 0.19, FREEC: 0.02) at a low false discovery rate (CONSERTING: 0.09, CNV-Seq: 0.19, SegSeq: 0.46, FREEC: 0.07). When applied to matching tumor/normal samples of hematopoietic malignancies and solid tumors, CONSERTING showed a high concordance with SNP array, with a mean of 18 uncorroborated CNA segments compared with 104 by CNVSeq, 182 by SegSeq and 63 by FREEC. In addition, CONSERTING is able to discover bona fide focal CNAs in genes of biological importance that were not detectable by SNP array, such as a 36 kb homozygous deletion in SH2B3 and a 20 kb deletion in CDK6. Finally, CONSERTING is able to find CNAs and SVs present in tumor subclone that are missed by SNP array or SV analysis of NGS data. The novel CNAs and SVs reported here were experimentally validated, demonstrating that the high sensitivity and accuracy of CONSERTING enable discovery of key genetic lesions in cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2487. doi:1538-7445.AM2012-2487
Abstract 2492: Integrated genomics on molecular subgroups in medulloblastoma
Authors: Kool Marcel , Jones David T.W. , Remke Marc , Jaeger Nathalie , Korshunov Andrey , Schlanstein Maria , Northcott Paul A. , van Meeteren Netteke Schouten , van Vuurden Dannis , Clifford Steven C , Pietsch Torsten , Von Bueren Andre O. , Rutkowski Stefan , Cho Yoon-Jae , McCabe Martin , Collins Peter , Haberler Christine , Ellison David , Gilbertson Richard , Pomeroy Scott , Doz Francois , Delattre Olivier , Taylor Michael D. , Lichter Peter , Pfister Stefan M.
Published: August 2012

Medulloblastoma is the most common malignant brain tumor in childhood. Molecular studies from several groups around the world demonstrated that medulloblastoma is not one disease but comprises a collection of distinct molecular subgroups. However, all these studies reported on different numbers of subgroups. The current consensus is that there are only four major subgroups, which are now called WNT, SHH, Group 3 and Group 4. A better understanding of each of these molecular subtypes is urgently warranted to improve treatment strategies and the overall survival of patients and ultimately also the quality of life for those that survive medulloblastoma. We used the data of seven independent studies on medulloblastoma for further characterization of these molecular subtypes. All cases (n = 550) were analyzed by expression profiling and for most cases SNP or array-CGH data were available. Data are presented for all medulloblastomas together and for each subgroup separately. For validation purposes we compared the results of this meta-analysis with another large medulloblastoma cohort (n = 408) for which subgroup information was obtained by immunohistochemistry. Results from both cohorts are highly similar and show how distinct the molecular subtypes are with respect to their transcriptome, DNA copy number aberrations, demographics, and survival. Interestingly, the data also showed how different medulloblastomas are between infants, children and adults. In infants, for instance, almost all medulloblastomas are classified as SHH or Group 3, whereas in adults most medulloblastomas are of the SHH subtype and almost never of Group 3. Recent next generation sequencing data (whole genome and exome) generated in our laboratory for a large series of pediatric and adult medulloblastomas show that the spectrum of genetic mutations is also very different, not only between the molecular subtypes, but also between the different age categories. All these data clearly show that medulloblastoma is not one disease. Results from these molecular analyses will form the basis for prospective multi-center studies and will have an impact on how the different variants of medulloblastoma will be treated in the future.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2492. doi:1538-7445.AM2012-2492
Abstract 2494: Identification of mithramycin analogues with increased potency and more selective inhibition of EWS-FLI1 for the treatment of Ewing sarcoma
Authors: Gebregiorgis Meti , Woldemichael Girma , He Min , Kang Min H. , Núñez Luz E. , Morís Francisco , Smith Malcolm A. , Helman Lee J. , Grohar Patrick J.
Published: August 2012

Background: Ewing Sarcoma Family Tumors (ESFT) is characterized by a chromosomal translocation, most commonly, between chromosomes 11 and 22, resulting in the fusion of the EWS and FLI1 genes. The fusion protein EWS-FLI1 is known to function as a constitutively activated transcription factor responsible for the oncogenic phenotype of ESFT. The identification of potent and specific inhibitors of EWS-FLI1 offers the hope of improved outcomes for ESFT patients. We have recently identified mithramycin, an antineoplastic antibiotic produced by Streptomyces plicatus, as a potent inhibitor of EWS-FLI1. In this report, we have investigated a series of mithramycin structural analogs in an effort to find compounds with better pharmacological properties. Methods: We evaluated the relative effects of the mithramycin analogs on ESFT viability using MTS, XTT and IncuCyte® real time cell confluence assays. The compounds were screened against a panel of pediatric cell lines within the Pediatric Preclinical Testing Program (PPTP) to evaluate selectivity. Western blot analysis and luciferase assays were used to determine the effects on EWS-FLI1 downstream target expression. Results: The mithramycin analogs 6 and 16 exhibit better potency and improved selectivity relative to mithramycin for both cell viability and downstream target inhibition. Furthermore, compound 6 is known to have a higher 200mg/kg maximum tolerated dose (MTD) relative to the 5 mg/kg MTD of mithramycin in xenograft models. Conclusions: In comparison to the drug mithramycin, we have shown that some of its structural analogs display better pharmacological properties such as increased potency and more specificity for ESFT cells. These results indicate that studies of analogs of DNA binding drugs are an important aspect of transcription factor drug development. We are working to further characterize the mechanism of these analogs and evaluate them in an in vivo model. The ultimate goal would be to translate these compounds to the clinic and improve therapeutic outcomes in patients with ESFT.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2494. doi:1538-7445.AM2012-2494
Abstract 2495: Sepiapterin reductase interacts with Ornithine Decarboxylase and regulates neuroblastoma proliferation
Authors: Lange Ingo , Koomoa Dana-Lynn T. , Bachmann Andre S.
Published: August 2012

Ornithine Decarboxylase (ODC) is a key, rate-limiting enzyme in mammalian polyamine biosynthesis that is upregulated in various types of cancer including neuroblastoma (NB). MYCN is a transcription factor and proto-oncogene that has been shown to be a key prognostic factor associated with advanced stage NB. ODC is a transcriptional target of MYCN, and has been found to play a critical role in NB progression. Previous studies have shown that inhibition of ODC through drugs such as alpha-difluoromethylornithine (DFMO) depletes intracellular polyamine pools and induces G1 cell cycle arrest. Our previous studies have shown that the anti-proliferative effect of DFMO in NB involves upregulation of the cyclin dependent kinase inhibitor p27 and downregulation of MYCN. In a search for novel regulators of ODC, we recently discovered that sepiapterin reductase (SPR), a key enzyme in the biosynthesis of tetrahydrobiopterin, binds to ODC and may regulate ODC activity through a novel mechanism. Co-immunoprecipitation experiments using NB cell lysates show that ODC interacts with SPR. In addition, immunofluorescence staining and laser-scanning confocal microscopy confirm the co-localization of ODC and SPR. Furthermore, downregulation of SPR expression using siRNA targeting SPR results in significant inhibition of NB proliferation, without affecting ODC protein levels. These results suggest that the interaction between SPR and ODC may be critically important to ODC activity and function and may constitute a novel mechanism for the regulation of NB proliferation. This interaction may play a critical role in fundamental physiological and pathophysiological processes including the development and progression of NB. Understanding the novel interaction between ODC and SPR might play a crucial role for the development of new drugs targeting SPR for the treatment of NB and other MYC/ODC-driven diseases.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2495. doi:1538-7445.AM2012-2495
Abstract 2502: Next-generation sequencing: Integrated exotome analysis in human multiple neuroblastoma
Authors: Hiyama Eiso , Hiyama Keiko
Published: August 2012

Background: Although multifocal neuroblastoma (NBL) is rare, its susceptibility is due to germline aberration and this predisposition gene may also be involved in sporadic neuroblastoma tumorigenesis. Sequence capture enrichment strategies and massively parallel next generation sequencing (NGS) were used for gene discovery because of their alterations. Methods: Out of approximately 500 NBL samples, 10 samples derived from 5 multifocal neuroblastoma cases were analyzed by a method for whole-exome sequencing coupling Agilent whole-exome capture to the Illumina DNA-sequencing platform. Using this technology, we investigated consistent point-mutations and insertions-deletions in these cases. Then, we analyzed the expression levels of these candidate genes in 298 sporadic NBL samples. Results: Among these 5 cases, one case showed ALK mutation in both of his multifocal tumors and germline mutation of ALK exon 23 (F1174L) was identified. The remaining 4 cases showed 57 candidate loci with consistent point-mutations and insertions-deletions including NBPF family genes and cell division cycle protein (CDC) genes. The expression levels of these candidate genes revealed that two genes were significantly decreased in these multifocal NBL tumors, except for one ALK mutated case. Then, we examined the expression levels of these two genes in sporadic NBL tumors. The expression levels of these candidate genes were significantly low in 34 and 59 NBL tumors, respectively but the low expression of both genes were detected in only two NBL tumors.Conclusions: Whole-exome sequencing using NGS in multifocal neuroblastoma revealed several candidate germline-mutated genes including ALK in such NBL-susceptibility cases. The expression levels of these genes also indicated that heterogeneous subtypes exist in sporadic NBLs. NGS can be used in research and diagnostic settings to screen for mutations and deletion/insertion in number of loci in genetically heterogeneous neuroblastoma. Further NGS analysis provided important candidates of indicators for diagnostic and therapeutic targets for NBLs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2502. doi:1538-7445.AM2012-2502
Abstract 2504: Defining the contribution of the extracellular domain of ICAM-2 to the non metastatic phenotype of neuroblastoma cells using chimeric fusion proteins
Authors: Feduska Joseph M. , Yoon Karina J.
Published: August 2012

Neuroblastoma (NB) is the most common extracranial pediatric cancer, accounting for 15% of all childhood cancer deaths. Recently, our lab reported the novel finding of ICAM-2 expression in NB cell lines and primary NB tumor cells. Interestingly, ICAM-2-expressing NB cells exhibited decreased motility in vitro and decreased metastatic potential in vivo. In these ICAM-2-expressing non-metastatic NB cells, the cytoplasmic domain of ICAM-2 interacted with β-actinin, which in turn interacted with actin, leading to subsequent redistribution of actin fibers to a non-motile state. The goal of the current study is determining the potential role of the ICAM-2 extracellular domain in NB metastasis development. Therefore, we constructed a series of chimeric fusion proteins, one of which consists of the extra cellular domain of CD89 with the transmembrane and cytoplasmic domains of ICAM-2 (CD89αED). CD89 is the receptor for IgA, and bears striking similarity to ICAM-2 in terms of size (287AA vs. 275AA, respectively) and structure. Both are immunoglobulin superfamily members with two Ig-like domains, six extracellular N-glycosylation sites, and short cytoplasmic domains. Furthermore, there is no literature implicating CD89 in cancer progression. This construct was stably transfected into a non-ICAM-2 expressing SK-N-AS NB cell line, and protein expression was confirmed by flow cytometry and western blot. Our preliminary data showed distinct phenotypic differences between NB cells stably transfected with the CD89αED construct compared to wild type ICAM-2. In scratch assays, CD89αED cells showed faster migration ability than ICAM-2 wt (75% vs. 50% wound closure) at 72 hour time points. Furthermore, in anchorage-independent colony growth in soft agar, CD89αED cells formed a significantly (P < 0.001) higher number of colonies compared to ICAM-2 wt. Ongoing work focuses on determining the effect of CD89αED on F-actin modification and regulation of potential downstream signaling pathways. This work was supported by the UAB Comprehensive Cancer Center Young Supporters Board - New Faculty Development Award 2010

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2504. doi:1538-7445.AM2012-2504