bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3128: Mxd3 is required for proliferation of human neuroblastoma cells.
Authors: Barisone Gustavo , Satake Noriko , Lewis Carly , Duong Connie , Chan Cathy , Lam Kit , Nolta Jan , Diaz Elva
Published: August 2014

Neuroblastoma (NB) is the most common extracranial solid tumor in children. Up to 45% of the cases are high risk at diagnosis, representing very aggressive, usually metastasized tumors that are refractory or recurrent after all available therapies. Prognosis for these patients is very poor with only 30% survival. MXD3 is a transcription factor previously shown in our lab to be a novel member of the Hedgehog (Hh) pathway (Yun, Rust, Ishimaru, Diaz, Mol Cell Biol, 2007) in granule neuron precursors. In these cells, overexpression of MXD3 results in higher MYCN expression, suggesting a functional interaction between the two transcription factors. MXD3 phenocopies MYCN expression in mouse models of medulloblastoma; we have also shown that it is expressed in human medulloblastoma and that its knockdown reduces proliferation of human medulloblastoma cell lines (Barisone et al., PLoS One, 2012). More than 20% of NBs have MYCN amplification, which is considered a marker of poor prognosis. In the current study, we investigated a possible role for MXD3 in NB cell proliferation. Using qRT-PCR we observed high levels of MXD3 mRNA expression in 5 NB cell lines: SK-N-DZ, IMR-32, SH-SY5Y, SK-N-BE and SK-N-SH. Immunoblot analysis with anti-MXD3 monoclonal antibodies confirmed that the protein was present in these cells. Furthermore, immunohistochemistry analysis showed strong immunoreactivity in a cohort of 16 primary NB tissue specimens. We investigated the role of MXD3 in NB cell proliferation by silencing MXD3 in the SK-N-DZ cell line. We used lentiviral delivery to knockdown MXD3 using an RNA interference approach. Upon transduction by the viral vector, MXD3 knockdown was confirmed at both the RNA and protein level. Within 48 hours, MXD3 protein levels were reduced >90% in cells infected with the shMXD3 virus but not with the control virus. MXD3 knockdown resulted in decreased proliferation in NB cells, supporting our hypothesis that it may be involved in the maintenance of NB. We analyzed cell cycle progression after knockdown using flow cytometry. We observed no significant differences in the G0/G1, S or G2/M populations between experimental and control samples. Our results suggest that MXD3 is important for NB cell proliferation, possibly by acting as an anti-apoptotic factor. Therefore, MXD3 is a potential candidate for targeted therapies against neuroblastoma.

Citation Format: Gustavo Barisone, Noriko Satake, Carly Lewis, Connie Duong, Cathy Chan, Kit Lam, Jan Nolta, Elva Diaz. Mxd3 is required for proliferation of human neuroblastoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3128. doi:10.1158/1538-7445.AM2013-3128
Abstract 3129: COX2 overexpression increases malignant potential of human glioma cells through Id1.
Authors: Xu Kaiming , Shu Hui-Kuo G.
Published: August 2014

Increased COX2 expression directly correlates with glioma grade and is also associated with shorter survival in malignant glioma patients. Id1 is similarly overexpressed in higher grade gliomas. Id1 is a member of the helix-loop-helix (HLH) family of transcriptional repressors that act as dominant-negative inhibitors of basic-HLH factors. Recent studies indicate that Id1 may be involved in the genesis/progression of gliomas. Here, we find that COX2 overexpression in human glioma cells correlates with increased Id1 protein levels. We further find that forced expression of COX2 and Id1 enhances soft agar colony formation. In cell invasion assay, both COX2 and Id1 expression in LN229 human glioma cells (LN/COX2 and LN/Id1) increase migration through matrigel. These cells also showed more tumorigenic potential with development of significantly larger subcutaneous tumors compared with controls. Of note, tumors expressing COX2 or Id1 had higher microvessel density than control tumors. Next, the effect of COX2 and Id1 overexpression was assessed in an orthotopic intracranial model. Here, nude mice injected with LN/COX2 or LN/Id1 cells had significantly decreased survival times compared with corresponding controls (Table 1). To assess the role of Id1 induction on the malignant potential of COX2-expressing glioma cells, LN/COX2 cells with shRNA against Id1 were intracranially injected and found to have a statistically significant increase in survival compared to those injected with control shRNA LN/COX2 cells (Table 1). Finally, to test a COX2-targeting therapy, we used the COX2 inhibitor celecoxib in our xenograft model and found inhibition of growth of flank tumors derived from glioma cells overexpressing COX2. In conclusion, COX2 overexpression in human glioma cells results in elevated Id1 protein level, which, in turn, enhances the transformation and invasiveness of human glioma cells, promotes microvessel formation and enhances xenograft tumor growth subcutaneously and intracranially.

Table 1. Survival of nude mice injected intracranially with the indicated glioma cells. Expt. 1 Expt. 2 Expt. 3 Cell type N Median Survival (days) N Median Survival (days) N Median Survival (days) LN/Ctrl 10 71 8 78 LN/COX2 10 40 9 53 LN/Id1 9 22 11 21 LN/COX2 shCtrl 10 75 LN/COX2 shId1 10 56 p-value by logrank <0.001 <0.001 0.011

Citation Format: Kaiming Xu, Hui-Kuo G. Shu. COX2 overexpression increases malignant potential of human glioma cells through Id1. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3129. doi:10.1158/1538-7445.AM2013-3129
Abstract 313: ATDC drives bladder cancer formation by promoting methylation of the PTEN promoter and inhibition of p53 function.
Authors: Palmbos Phillip , Wang Lidong , Yang Huibin , Detzler Taylor , Ney Gina , Hart Justin , Daignault-Newton Stephanie , Kunju L. Priya , Kumar-Sinha Chandan , Liebert Monica , Ljungman Mats , Simeone Diane
Published: August 2014

Bladder cancer is the 5th most common malignancy and a significant cause of morbidity and mortality, but the molecular events leading to its development are incompletely understood. We have identified an oncogene, Ataxia-Telangiectasia Group D Complementing (ATDC) gene, which drives formation and progression of pancreatic and bladder tumors. To better characterize the oncogenic function of ATDC, we generated transgenic (tg) mice which overexpress ATDC and the predominant phenotype of these mice was the development of non-invasive and muscle-invasive urothelial carcinomas. Tg bladder tumors were histologically identical to human tumors and displayed a similar gene expression signature to human invasive bladder tumors. ATDC expression was assessed in a tissue microarray and was highly up-regulated in 70% of human invasive bladder tumors (173/252). High expression of ATDC correlated with more advanced stage disease and worse survival after chemotherapy (p = 0.002). ATDC knockdown in human bladder cancer cell lines correlated with increased sensitivity to chemotherapy in vitro and decreased proliferation and invasion both in vitro and in vivo in a human bladder cancer xenograft model. To understand the mechanism of ATDC-mediated tumor formation, we modulated ATDC expression in normal and bladder cancer cells lines. ATDC overexpression down-regulated PTEN levels via DNA promoter methylation by DNMT3A in both tg mouse tumors and human cell lines. IHC analysis of ATDC, PTEN and DNMT3A expression in human bladder tumors specimens demonstrated similar findings. ATDC was also found to bind to p53 and inhibit p53 mediated functions. These findings establish ATDC as a novel determinant of bladder cancer initiation, progression and resistance to treatment which mediates tumorigenesis by down-regulation of PTEN expression and inhibition of p53.

Citation Format: Phillip Palmbos, Lidong Wang, Huibin Yang, Taylor Detzler, Gina Ney, Justin Hart, Stephanie Daignault-Newton, L. Priya Kunju, Chandan Kumar-Sinha, Monica Liebert, Mats Ljungman, Diane Simeone. ATDC drives bladder cancer formation by promoting methylation of the PTEN promoter and inhibition of p53 function. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 313. doi:10.1158/1538-7445.AM2013-313
Abstract 3137: Functional analysis of the chr13q22.1 pancreatic cancer risk locus suggests allele-specific effects on DIS3 expression.
Authors: Hoskins Jason , Ibrahim Abdisamad , Jia Jinping , Collins Irene , Parikh Hemang , Petersen Gloria M. , Amundadottir Laufey
Published: August 2014

Pancreatic cancer is the 10th most common cancer and 4th most common cause of cancer mortality in the United States. A genome wide association study has revealed pancreatic cancer susceptibility loci on chromosomes 13q22.1, 1q32.1 and 5p15.33. The chr13q22.1 region contains the most significant of all risk SNPs, rs9543325, (P= 3.27 x 10−11) which is located in a gene desert. The nearest genes are KLF5, KLF12, PIBF1, DIS3 and BORA, which range in distance from 265kb to 586kb, respectively, from the most significant variants. Given the high linkage disequilibrium across this risk locus, there are many candidate functional variants to consider. Imputation in the region did not improve the signal but gave a set of highly correlated SNPs which likely includes the functional variant(s). In an effort to identify the functional variant(s) we performed eQTL analyses to test the association between the genotypes of these candidate SNPs and expression of nearby genes. Among 100 normal pancreatic tissue samples, DIS3 showed the strongest association with SNPs in our risk locus (P-values as low as 0.0004). Mutations in DIS3 have been identified in acute myeloid leukemia and multiple myeloma, and its expression has been correlated with metastatic potential in colorectal cancer, suggesting this gene could be important in pancreatic cancer biology. Chromosome Conformation Capture (3C) was performed to test for physical interactions between the risk locus and nearby genes. This assay confirmed the three dimensional proximity of the risk locus and DIS3 promoter. Sub-regions of the risk locus were then cloned upstream of a minimal promoter controlling luciferase expression to assay for potential allele specific enhancer/silencer activity. This assay revealed a sub-region downstream of rs9543325 that causes allele-specific silencing. These results suggest that a sub-region of the chr13q22.1 risk locus has allele-specific effects on DIS3 expression, which may have implications in the susceptibility to pancreatic cancer.

Citation Format: Jason Hoskins, Abdisamad Ibrahim, Jinping Jia, Irene Collins, Hemang Parikh, Gloria M. Petersen, Laufey Amundadottir. Functional analysis of the chr13q22.1 pancreatic cancer risk locus suggests allele-specific effects on DIS3 expression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3137. doi:10.1158/1538-7445.AM2013-3137
Abstract 3138: Development of a tetracycline-inducible lentiviral system for RNAi screen in PDX tumors.
Authors: Chen Lei , Geng Yi , Fruhling Dave , Zhong Wenyan , Diesl Veronica , Pan Jing , Loreth Christine , Wang Elizabeth , Bulynko Yaroslava , Arndt Kim , Follettie Maximillian
Published: August 2014

Patient-derived xenograft (PDX) tumors are believed to be more clinically relevant pre-clinical models for cancer target identification and validation. We used a tetracycline-inducible lentiviral vector that delivers shRNA expression which is tightly linked to a GFP reporter in PDX cells. Using shRNAs against PLK1 (polo-like kinase 1) as positive controls, we demonstrated that three of the four PLK1 hairpins resulted in growth inhibition of HT29 and A549 cells upon dox induction. Correspondingly, these 3 hairpins showed significant knockdown of PLK1 mRNA. To facilitate RNAi in PDX tumors, we have established an in vitro system to culture PDX tumor cells. Gene expression and IHC study demonstrated that tumors regenerated from colon PDX cells grown in vitro retained features of their parental tumors. PDX cells cultured in vitro also remained tumorigenic. When GFP/shRNA-transduced colon PDX cells were re-implanted in mice to form tumors, approximately 30% of the new tumor cells was GFP-positive, enabling us to capture shRNA-expressing cells by flow cytometry. Initial shRNA drop-out screens were carried out in HT29 and colon PDX cells transduced with a pool of ∼600 shRNAs targeting a set of potential cancer associated genes. Included amongst the hairpins demonstrating statistically significant depletion in HT29 were the exact same three PLK1 shRNAs previously functionally validated. These results demonstrated that we have established a robust in vivo shRNA drop-out screening platform in PDX tumors for identification of novel cancer drug targets.

Citation Format: Lei Chen, Yi Geng, Dave Fruhling, Wenyan Zhong, Veronica Diesl, Jing Pan, Christine Loreth, Elizabeth Wang, Yaroslava Bulynko, Kim Arndt, Maximillian Follettie. Development of a tetracycline-inducible lentiviral system for RNAi screen in PDX tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3138. doi:10.1158/1538-7445.AM2013-3138
Abstract 3139: Identification and functional studies of p53 mutants detected in breast cancers after chemotherapy treatment.
Authors: Huun Johanna , Berge Elisabet O. , Lillehaug Johan R. , Lonning Per Eystein , Knappskog Stian
Published: August 2014

Background: Approximately 4300 different TP53 mutations have been reported in human cancers. TP53 mutations, in particular those affecting the L2/L3 domains, are associated with resistance to anthracycline or mitomycin treatment in breast cancer patients. While many mutations have been characterised functionally, novel TP53 mutations are continuously reported. Here, we characterise 10 p53 protein variants encoded by mutated TP53 (5 within and 5 outside L2/L3) detected in locally advanced or metastatic breast cancers. Each tumour was previously characterised for response to therapy, allowing comparison between in vivo and in vitro findings.

Methods: Mutated p53 variants were analysed for their ability to oligomerise with the wild-type protein and their subcellular localisation by immunoprecipitation and immunofluorescence, respectively. Their ability to induce transcription of target genes was determined by qPCR. Cellular growth rate, apoptosis and senescence were monitored by WST-1, TUNEL and beta-galactosidase assays, respectively.

Results: Immunoprecipitation assays revealed each mutant protein to retain binding capacity for wild-type p53, thus potentially acting in a dominant negative manner. Even though each p53 variant located predominantly in the nucleus, the percentage of cells with only nuclear p53 localisation varied between 60-90%. None of the p53 variants were able to induce target genes to levels similar to wild-type p53, nor where they able to reduce cellular growth rate, induce apoptosis or senescence similar to wild-type p53 after anthracycline treatment in vitro.

Conclusions/General Significance: Our data add further information characterising the effects of somatic TP53 mutations on p53 protein function and anthracycline resistance in breast cancer.

Citation Format: Johanna Huun, Elisabet O. Berge, Johan R. Lillehaug, Per Eystein Lonning, Stian Knappskog. Identification and functional studies of p53 mutants detected in breast cancers after chemotherapy treatment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3139. doi:10.1158/1538-7445.AM2013-3139
Abstract 3143: A functional interaction between alpha-fetoprotein and miRNA-29 modulates the HCC epigenome.
Authors: Parpart Sonya , Roessler Stephanie , Dong Fei , Rao Vinay , Loffredo Christopher , Wang Xin Wei
Published: August 2014

Globally, hepatocellular carcinoma (HCC) accounts for 70-85% of primary liver cancers and ranks second in the leading cause of male cancer death. Serum alpha-fetoprotein (AFP), normally expressed during fetal development, is reactivated in 60% of HCC tumors and associated with poor patient outcome. We hypothesize that AFP+ and AFP- tumors may differ biologically and have different prognosis in HCC patients. Using microarray-based global microRNA and mRNA profiling, we found that members of the miR-29 family are the most significantly (p<1E-5) down-regulated miRs in AFP+ tumors (n=242). Physiologically, during mouse embryonic development, miR-29 family expression is low but gradually increases after birth, contrary to AFP expression which dramatically decreases after birth. In addition, a member of the DNA methyltransferase (DNMT) family, DNMT3A, is one of the most significantly (p=8E-6) up-regulated genes in AFP+ HCC. Interestingly, there is a significant inverse correlation (p<1E-4) between miR-29 and DNMT gene expression, suggesting that they may be functionally antagonistic, which is consistent with the finding showing that DNMT3A and DNMT3B are direct targets of miR-29. Experimentally, we found that increased AFP expression or the addition of AFP+ media to AFP- cells inhibits miR-29a expression in HCC cell lines. We also found that AFP transcriptionally regulates the promoter of miR-29a. Moreover, global DNA methylation array profiling reveals increased methylation in HCC patients with high levels of serum AFP. These results support our hypothesis that AFP inhibits miR-29, which modulates epigenetic changes that contribute to poor outcome. Taken together, this study elucidates the mechanisms that link AFP and miR-29 expression to epigenetic alterations and may aid in the development of novel therapeutic agents to improve survival of HCC patients.

Citation Format: Sonya Parpart, Stephanie Roessler, Fei Dong, Vinay Rao, Christopher Loffredo, Xin Wei Wang. A functional interaction between alpha-fetoprotein and miRNA-29 modulates the HCC epigenome. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3143. doi:10.1158/1538-7445.AM2013-3143
Abstract 3150: Characterizing the effects of somatic mutations in splice factors on the transcriptome.
Authors: Brooks Angela N. , Wan Youzhong , Choi Peter , Jing Rui , DeLuca David S. , Sougnez Carrie , Chmielecki Juliann , Imielinski Marcin , Getz Gad , Wu Catherine J. , Meyerson Matthew
Published: August 2014

Recent studies have identified recurrent somatic mutations in splice factors, including SF3B1 and U2AF1, in myelodysplastic syndromes, chronic lymphocytic leukemias, breast cancer, and lung adenocarcinoma. In one study, 91 tumor and matched normal whole-exomes were sequenced from patients with CLL and the second most frequently mutated gene was SF3B1. Analysis of 187 whole-exomes in lung adenocarcinoma and matched normals revealed recurrent somatic mutations in the splice factor U2AF1 in 3% of cases. In addition to U2AF1 mutations, recurrent truncating mutations were observed in a putative splice factor, RBM10, in 4% of lung adenocarcinomas. The recurrence of somatic mutations in splice factors in multiple tumor types suggests that alterations in RNA splicing may play an important role in tumorigenesis.

Although these mutations have been identified in cancer genomes, the effect on the cancer transcriptome has yet to be fully explored; therefore, we do not understand how mutations in splice factors can contribute to tumorigenesis. We hypothesize that somatic mutations in splice factors cause alterations in mRNA splicing of important cancer genes.

We are using a computational tool called JuncBASE to identify alternative splicing in tumor transcriptomes from RNA-Seq data. We are then associating somatic mutations in splice factors, found from whole-exome sequencing, with splicing signatures identified through RNA-Seq.

Preliminary results on the identification of splice events associated with SF3B1 mutations in CLL reveal novel isoforms of multiple genes specifically expressed in SF3B1 mutant samples. Additionally, we examined the effects of SF3B1, U2AF1, and RBM10 mutations on tumor transcriptomes using RNA-Seq data from The Cancer Genome Atlas.

Citation Format: Angela N. Brooks, Youzhong Wan, Peter Choi, Rui Jing, David S. DeLuca, Carrie Sougnez, Juliann Chmielecki, Marcin Imielinski, Gad Getz, Catherine J. Wu, Matthew Meyerson. Characterizing the effects of somatic mutations in splice factors on the transcriptome. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3150. doi:10.1158/1538-7445.AM2013-3150
Abstract 3156: New candidate oncogenes discovered in microsatellite unstable colorectal cancer.
Authors: Gylfe Alexandra E. , Kondelin Johanna , Turunen Mikko , Ristolainen Heikki , Katainen Riku , Pitkänen Esa , Kaasinen Eevi , Rantanen Ville , Tanskanen Tomas , Lehtonen Heli J. , Palin Kimmo , Taipale Minna , Taipale Jussi , Renkonen-Sinisalo Laura , Järvinen Heikki , Böhm Jan , Mecklin Jukka-Pekka , Ristimäki Ari , Tuupanen Sari , Karhu Auli , Kilpivaara Outi , Vahteristo Pia , Aaltonen Lauri A.
Published: August 2014

Around 15% of colorectal cancers (CRCs) show microsatellite instability (MSI). A characteristic high passenger mutation load has discouraged systematic mutation screens in MSI CRCs. To systematically search for novel MSI CRC oncogenes, the exomes of 25 MSI CRC-normal pairs were sequenced as the discovery set. Observed hot spots were confirmed by Sanger sequencing and further validated in a set of 86 MSI CRCs. Mutational hot spots were confirmed in 15 genes and three genes showed additional hot spot mutations in the validation set. These three sites were highly conserved across species. The hot spot sites of these three genes were screened in 75 microsatellite stable CRCs and 12 MSI CRC cell lines with negative results. Next, we analyzed the subcellular localization of wild-type and mutant proteins. The findings of this study support the idea that cancer genomes are heterogeneous and characterized by few, frequently mutated “mountains” (e.g. BRAF and KRAS) and numerous, less frequently mutated “hills”. The identified mutational hot spots may prove important in developing personalized tumor profiling and therapy.

Citation Format: Alexandra E. Gylfe, Johanna Kondelin, Mikko Turunen, Heikki Ristolainen, Riku Katainen, Esa Pitkänen, Eevi Kaasinen, Ville Rantanen, Tomas Tanskanen, Heli J. Lehtonen, Kimmo Palin, Minna Taipale, Jussi Taipale, Laura Renkonen-Sinisalo, Heikki Järvinen, Jan Böhm, Jukka-Pekka Mecklin, Ari Ristimäki, Sari Tuupanen, Auli Karhu, Outi Kilpivaara, Pia Vahteristo, Lauri A. Aaltonen. New candidate oncogenes discovered in microsatellite unstable colorectal cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3156. doi:10.1158/1538-7445.AM2013-3156
Abstract 3157: Loss of heterozygosity as a molecular ‘second hit’ In familial pancreatic cancer.
Authors: Kanji Zaheer S. , Serra Stefano , Holter Spring , Borgida Ayelet , Grant Robert , Gallinger Steven
Published: August 2014

BACKGROUND/HYPOTHESIS: Familial Pancreatic Cancer (FPC) has an autosomal dominant, variable penetrant mode of inheritance with >80% of its genetic cause yet to be discovered. We hypothesize that a high density DNA microarray analysis of Formalin-Fixed Paraffin Embedded (FFPE) FPC tumors will yield novel regions of genomic loss harboring disease causing FPC gene(s).

METHODS: 158 FFPE FPC tumor specimens with matched normal tissue were reviewed by a pancreatic pathologist and tumors with <70% cellularity underwent laser capture microdissection. The samples with >70% cellularity were microdissected directly from marked unstained slides. A total of 74 samples were DNA extracted, whole genome amplified and processed on the Affymetrix 660K Oncoscan Microarray. Copy number analysis was performed using Nexus Copy Number Variation Version 6.1 software employing the SNP-FASST2 segmentation and Allele Specific Copy Number Analysis of Tumors (ASCAT) algorithms.

PRELIMINARY RESULTS: A pair-wise analysis of 55 FPC samples with matched normal tissue was performed. Recurrent regions of loss of heterozygosity (LOH) were known loci of importance in pancreatic tumorigenesis, including CDKN2A, p53 and SMAD4. Copy neutral/gain LOH was observed throughout the genome and may account for >35% of chromosomal loss. Recurrent novel genetic loci displaying LOH was observed on chromosomes: 3p(25%), 5q(18%), 6p(22%), 12q(18%) and Xq (42%). Examination of 2 FPC siblings elucidates shared loss regions spanning the 3p22.2-3p22.3 and 17p13.3 loci. Candidate tumor suppressor genes (TSGs) of interest include DCLK3, SERPINF1, SERPINF2 and SMYD4.

CONCLUSION: We have described for the first time the presence of copy neutral/gain LOH in FPC highlighting previously unknown regions displaying genetic loss. Further confirmation with quantitative PCR, integrating with germline exome sequencing data, and validation of putative TSGs shared by members of high risk FPC kindreds will help uncover novel genes associated with this disease.

Citation Format: Zaheer S. Kanji, Stefano Serra, Spring Holter, Ayelet Borgida, Robert Grant, Steven Gallinger. Loss of heterozygosity as a molecular ‘second hit’ In familial pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3157. doi:10.1158/1538-7445.AM2013-3157
Abstract 3158: Transcription factor SOX11 decreases viability in ovarian carcinoma cells.
Authors: St. Clair Caryn M. , Wethington Stephanie L. , Bisogna Maria , Dao Fanny , Jelinic Petar , Levine Douglas A.
Published: August 2014

Objective: The Cancer Genome Atlas (TCGA) evaluated nearly 500 advanced serous ovarian carcinomas and identified four gene expression subclasses. The transcription factor SOX11 was markedly overexpressed in the proliferative subtype and associated with phosphorylation of other cell cycle related proteins including CHEK2, WWTR1, and EIF4EBP1. We tested the hypothesis that over-expression of SOX11 will decrease proliferation of ovarian cancer cells through transcriptional repression.

Methods: SOX11 was overexpressed via lipofectamine transfection in the TP53 wild-type ovarian cancer cell line SKOV3 and the TP53 mutated ovarian cancer cell line OVCAR3. Cell proliferation was assessed 48 hours after transfection using the VisionBlue fluorescence-based viability assay. Cell cycle analysis was performed utilizing a propidium iodide (PI) assay followed by fluorescence-activated cell sorting (FACS). Finally migration of SOX11-transfected cells was analyzed in comparison to mock-transfected controls via monolayer scratch assay.

Results: Successful overexpression of the SOX11 protein was confirmed using GFP fluoroscopy and western blot 24 and 48 hours following transfection. Cell viability was reduced by 14-63% at 48 hours in SOX11-transfected SKOV3 cells compared to mock controls (P = 0.01) in a dose dependent fashion. Cell cycle was unaffected by SOX11 overexpression. Finally, the monolayer scratch assay demonstrated that cells overexpressing SOX11 migrated more slowly than mock controls (median velocity 3.92 vs. 6.19 um/hr, P = 0.001). Data from OVCAR3 cells are currently being analyzed and will be presented.

Conclusions: Overexpression of SOX11 resulted in decreased cell viability without affecting cell cycle. Further studies will assess the mechanism of this decreased viability which may involve diversion toward an apoptotic pathway when SOX11 is overexpressed or regulation by transcription repressors such as WWTR1. SOX11 suppression has been shown in lymphoma to be associated with more aggressive tumors through mechanisms associated with lysophosphatidic acid growth regulation, offering mechanistic associations with ovarian cancer. Ongoing work will determine the relationship between SOX11 and TP53 mutation frequently found in high-grade serous carcinoma. These studies will help to explain the differential outcomes seen across high-grade serous ovarian carcinoma.

Citation Format: Caryn M. St. Clair, Stephanie L. Wethington, Maria Bisogna, Fanny Dao, Petar Jelinic, Douglas A. Levine. Transcription factor SOX11 decreases viability in ovarian carcinoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3158. doi:10.1158/1538-7445.AM2013-3158
Abstract 3160: Open-source RNAi genetic screen platform: discovery of drug targets in cancer cell models.
Authors: Komarov Andrei , Makhanov Mikhail , Komarova Elena , Novototzkaja Lilya , Yeluashvili Michael , Suchkov Dmitry , Bonneau Kyle , Tedesco Donato , Frangou Costa G. , Diehl Paul , Deng Debbie , Hyder Karim , Komarov Pavel , Gudkov Andrei , Chenchik Alex
Published: August 2014

We have developed an open-source RNAi genetic screening technology, funded in part by NIH grants and freely available to the scientific community. This platform can be used for functional discovery of drug targets associated with viability and regulation of signaling pathways in cancer cells. Here we present a novel, free public resource for genome-wide RNAi screens: DECIPHER pooled lentiviral shRNA libraries. Results are shown from two RNAi screens with DECIPHER shRNA libraries: one “drop-out” screen to identify genes essential for viability in a panel of leukemic cells, and a second “rescue” screen to identify genes required for FAS induced apoptosis. Both screens found a combination of known and novel signaling pathway and regulatory genes whose functions were confirmed to be required to produce the biological responses. From the viability screens in the panel of leukemia cell lines, subsequent validation using single shRNA-expressing constructs showed that about 80% of the shRNAs identified in each complex library screen did in fact lead to cell death when transduced in cells. Analysis of the identified essential genes for known biological interactions revealed non-random clusters of interacting proteins that provide a useful strategy for prioritization of potential targets. Analysis of the lethal combinations indicates redundant, complementary, and compensatory responses in cancer cells. In the case of the FAS-induced apoptosis, in vitro screening data also enabled us to select targets that protected mice from FAS-induced hepatic failure. These results demonstrate that complex pooled shRNA libraries provide a highly efficient, flexible, and cost-effective alternative to array-based RNAi screening methods for identifying genes regulating biological responses and possible new therapeutic targets.

Citation Format: Andrei Komarov, Mikhail Makhanov, Elena Komarova, Lilya Novototzkaja, Michael Yeluashvili, Dmitry Suchkov, Kyle Bonneau, Donato Tedesco, Costa G. Frangou, Paul Diehl, Debbie Deng, Karim Hyder, Pavel Komarov, Andrei Gudkov, Alex Chenchik. Open-source RNAi genetic screen platform: discovery of drug targets in cancer cell models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3160. doi:10.1158/1538-7445.AM2013-3160
Abstract 3165: Metastatic tumor evolution delineates clinically important oncogenic drivers in diffuse gastric cancer.
Authors: Nadauld Lincoln D. , Garcia Sarah , Natsoulis Georges , Bell John M. , Hopmans Erik , Xu Hua , Pai Reetesh K. , Regan John F. , Ford James M. , Kuo Calvin J. , Ji Hanlee
Published: August 2014

We conducted a detailed genetic and functional analysis of personalized tumor evolution in a case of diffuse gastric cancer. Leveraging the unique genetics of an individual with a confirmed germline mutation in the causative CDH1 tumor suppressor gene, we identified candidate drivers for a primary tumor in the stomach and a metastatic recurrence in the left ovary, referred to as a Krukenberg tumor. Using genome sequencing, we demonstrate that the primary and metastatic tumors share the majority of cancer driver events including biallelic CDH1 and TP53 mutations consistent with a common genetic origin. The primary gastric cancer displayed amplification of FGFR2, a gene that encodes the fibroblast growth factor (FGF) receptor 2 with multiple small molecule inhibitors in clinical therapy development. Tumor evolution and genetic divergence was clearly evident with the ovarian metastasis lacking the FGFR2 amplification and displaying its own subset of mutations and genomic aberrations. Namely, the metastatic tumor had unique biallelic events affecting TGFBR2, a critical gene in the TGF-β (tumor growth factor) signaling pathway that has not previously implicated in diffuse gastric cancer. To determine the independent oncogenic attributes of these separate pathways, we used a diffuse gastric cancer cell line and organoid model system to replicate and study the context of the known and candidate drivers found in the index patient's primary versus metastatic sites. We demonstrated that in the context of biallelic CDH1 and TP53 functional loss, the addition of either FGFR2 or TGFBR2 drivers independently lead to gastric cancer. This result supports the independent cancer driver roles of the FGF and TGF-β signaling pathways in metastatic tumor evolution in diffuse gastric cancer. Our study has implications regarding personalized cancer medicine approaches in patients with metastatic gastric cancer.

Citation Format: Lincoln D. Nadauld, Sarah Garcia, Georges Natsoulis, John M. Bell, Erik Hopmans, Hua Xu, Reetesh K. Pai, John F. Regan, James M. Ford, Calvin J. Kuo, Hanlee Ji. Metastatic tumor evolution delineates clinically important oncogenic drivers in diffuse gastric cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3165. doi:10.1158/1538-7445.AM2013-3165
Abstract 317: Role of ATM and PTEN in prostatic carcinogenic dog prostate: validation of aCGH results.
Authors: Fonseca Alves Carlos E. , Pellicciari Patricia R. , Rodrigues Marcela M. P. , Rogatto Silvia R. , Amorim Renee L.
Published: August 2014

The canis lupus familiares is the only species besides human that spontaneously develop prostatic carcinoma (PCa). In addition, the metastatic sites are similar to those frequently reported in men. For these reasons, the dog is the best natural model to study the molecular mechanisms in PCa development providing a natural animal model for treatment by molecular targets. Previously, we investigated copy number alterations by arrayCGH (Canine Genome CGH Microarray 4x44K-G2519F, Agilent Technologies) in canine prostatic lesions: 3 benign prostatic hyperplasias (BPH), 4 proliferative inflammatory atrophies (PIA), and 14 PCa. Five histologically normal prostatic tissues were used as reference. Genomic alterations were evaluated using Genomic Workbench Standard Edition 5.0.14. This previous study revealed significant copy number losses of Atm and Pten exclusively in PCa. In the present study, ATM and PTEN immunoexpression were investigated using a tissue microarray (TMA) containing 149 canine prostatic paraffin-embedded lesions (BPH, PIA and PCa) collected from 67 animals. Immunohistochemical reactions were performed using the polyclonal rabbit antibody anti-PTEN (Santa Cruz Biotech, 1:50) and anti-ATM (Abcam, 1:50). The sections were developed with diaminobenzidine (DAB) and peroxidase. The immunohistochemical staining was assessed in each core by the distribution of positive cells for each antibody per lesion (score 1: <25% cells positive, 2: 26% to 50%, 3: being 51% and 75% and 4:> 75%) and intensity (1: weak, 2: moderate, 3: intense). Chi-square or Fisher exact test was used to determine the association between the categorical variables using GraphPad Prism 5 (GraphPad Software Inc., La Jolla, CA). Distribution of positive cells did not differ among lesions. PCa and PIA showed more samples with weak intensity for ATM when compared to normal prostatic tissue and BPH (PCa: p=0,032 and PIA: p=0,025). Benign prostatic hyperplasia and normal samples presented intense PTEN immunostaining than PCa (p=0,021) and PIA (p=0,0013). These results suggest that ATM and PTEN proteins expression in canine prostatic carcinoma are downregulated possibly by copy number losses. These findings are similar from those described in prostate carcinomas from human corroborating for the use of dogs as a natural model to study prostatic disease in men.

Citation Format: Carlos E. Fonseca Alves, Patricia R. Pellicciari, Marcela M. P. Rodrigues, Silvia R. Rogatto, Renee L. Amorim. Role of ATM and PTEN in prostatic carcinogenic dog prostate: validation of aCGH results. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 317. doi:10.1158/1538-7445.AM2013-317
Abstract 3172: Pooled RNAi screens in xenograft mouse models.
Authors: Tedesco Donato , Bonneau Kyle , Makhanov Mikhail , Deng Debbie , Diehl Paul , Sun Peiqing , Chenchik Alex
Published: August 2014

We present results of clonal analysis studies aimed at characterizing the heterogeneity of in vivo cancer cell growth in xenograft models. Using a high-throughput sequencing method, we followed the fate of thousands of individually bar-coded cancer cells grown in vitro, or in vivo upon sub-cutaneous implantation in immunocompromised mice. By precisely and reproducibly quantifying clonal cancer cell growth in vivo, we found that xenograft growth is marked by a phenomenon of clonal dominance, in which a small subset of cancer cell sub-clones account for the bulk of the resulting tumor mass. This phenomenon has important implications for the field of in vivo shRNA screening. Subsequently, we developed a new proprietary clonal RNAi screening platform specifically designed to circumscribe the clonal dominance effect in xenograft model viability screens. We used this technology to perform xenograft “drop-out” screens in a panel of tumorigenic cell lines, including human breast, ovarian and colon carcinomas. Preliminary data will be presented. These results demonstrate that complex pooled shRNA libraries provide a highly efficient, flexible, and unique tool for in vivo screens aimed the discovery of potential cancer therapy targets.

Citation Format: Donato Tedesco, Kyle Bonneau, Mikhail Makhanov, Debbie Deng, Paul Diehl, Peiqing Sun, Alex Chenchik. Pooled RNAi screens in xenograft mouse models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3172. doi:10.1158/1538-7445.AM2013-3172
Abstract 3177: Web-based access using CellMiner for gene expression, DNA copy number, microRNA transcript levels, variant status, drug activity, and their pattern comparisons for the NCI-60.
Authors: Reinhold William C. , Sunshine Margot , Varma Sudir , Liu Hongfang , Abaan Ogan , Meltzer Paul , Morris Joel , Kohn Kurt , Doroshow James , Pommier Yves
Published: August 2014

High-throughput data is increasingly being integrated into the fields of biology, molecular biology, and pharmacology. However, a difficult problem has been the rapid and fluid access to and integration of the data, which tends to reside in huge unwieldy databases. One set of cell lines with substantial potential for benefit from this type of access and integration is the NCI-60 cancerous cell lines. We present here a set of tools within our CellMiner web-application designed to address this need for the areas of transcript expression, microRNA expression, gene DNA copy number, variant status, and drug activity. CellMiner allows the user to rapidly access data for relative levels of transcript expression for 26,065 genes, 360 microRNAs, and 20,602 compounds including 102 Food and Drug Administration (FDA)-approved drugs. These levels in turn create patterns across the NCI-60 that can be compared to one another using our “pattern match” tool. Together, these tools allow one to query the data for potential relationships between these parameters, in a manner specific to a users area of expertise and interest, in a rapid and flexible manner without the need for expertise in computer science or bioinformatics. Comparisons of transcript/drug will be demonstrated with SLFN11/topotecan; variant/drug with BRAF V600E/vemurafinib; colon tissue-of origin specific genes with TRIM15, RNF43, and VIL1; and DNA copy number change with CDKN2A (p16). The data are publicly available at http://discover.nci.nih.gov/cellminer.

Citation Format: William C. Reinhold, Margot Sunshine, Sudir Varma, Hongfang Liu, Ogan Abaan, Paul Meltzer, Joel Morris, Kurt Kohn, James Doroshow, Yves Pommier. Web-based access using CellMiner for gene expression, DNA copy number, microRNA transcript levels, variant status, drug activity, and their pattern comparisons for the NCI-60. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3177. doi:10.1158/1538-7445.AM2013-3177
Abstract 3179: The head and neck cancer biomarker wiki: A tool for data mining and biomarker meta-analyses.
Authors: Ceder Rebecca , Kohonen Pekka , Grafström Roland C.
Published: August 2014

Biomarkers that accurately predict outcome or treatment response is lacking for most human tumor types, including for common and well-studied cancers like head and neck squamous cell carcinoma (HNSCC). With the aim to summarize existing genomic analysis and biomarker data into a format analyzable with bioinformatics processing tools, we set out to develop a biomarker wiki for HNSCC, hypothesizing that meta-analyses would reveal novel and potentially clinically useful sets of biomarker genes. We applied the user-friendly MediaWiki interface for collecting data in the public domain, and separated the results under the categories: primary tumor profiling, metastasis profiling, cell line profiling and treatment response. The information was captured applying controlled and standardized vocabularies, including with data storage in the multi-omics Investigation/Study/Assay (ISA-Tab) format. Bioinformatics-driven meta-analyses of results from currently over 50 publications indicate that the wiki serves to accurately identify common gene patterns, gene ontologies, molecular networks and key regulator genes within and among the respective categories. Additionally, independent validations relative other databases such as the Human Gene Expression Map, the Human Protein Atlas and the cBio Cancer Genomics Portal confirm many associations, including with significant correlations to poor outcome for gene sub-sets. Overall, the wiki results so far agree with the hypothesis under testing, suggesting novel biomarker genes/signatures for future clinical assessment. On completion, we foresee a query-enabling update-friendly knowledge platform for head and neck cancer studies in the public domain.

Citation Format: Rebecca Ceder, Pekka Kohonen, Roland C. Grafström. The head and neck cancer biomarker wiki: A tool for data mining and biomarker meta-analyses. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3179. doi:10.1158/1538-7445.AM2013-3179
Abstract 318: Preclinical evaluation of monoclonal antibody H6-11 for prostate cancer imaging.
Authors: Jin Hongjun , Xu Mai , Padakanti Prashanth K. , Tu Zhude
Published: August 2014

Altered post-translational modification (PTM) of glycosylations to various nuclear and cytosolic proteins is a universal feature of cancer cells and these modified glycan plan significant role in immune surveillance, tumor invasion, and increase malignancy. In this study, we reported a novel monoclonal antibody reacting to the tumor glycoproteins with molecular weight around 40-60 kDa. Deglycosylation examination suggested the antigenic isotope of the glycan is O-linked β-N-acetylglucosamine (O-GlcNAc) group. We examined the reactivity of H6-11 to cancer cells including PC-3, MCF-7 cells and implanted PC-3 and EMT-6 tumor tissues using immunofluorescence staining and autoradiography technique. The Zr-89 labeled H6-11 was successfully synthesized and the microPET study was performed in PC-3 xenograft prostate model in nude mice. microPET study showed that significant amount of radioactivity accumulation was observed in the excretory organs during the early period post-injection of radioactivity. At 24 hrs post-injection of radioactivity, significant amount of radioactivity was observed in the xenograft prostate tumor, while the radioactivity intensity was decreased in the liver and kidney. Radioactivity uptake in the tumor was still evident at 120 hrs post-injection of radioactivity. Our work suggested that the H6-11 antibody is capable of detecting prostate tumors both in vitro and in vivo.

Citation Format: Hongjun Jin, Mai Xu, Prashanth K. Padakanti, Zhude Tu. Preclinical evaluation of monoclonal antibody H6-11 for prostate cancer imaging. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 318. doi:10.1158/1538-7445.AM2013-318
Abstract 3191: The Akt-mTOR axis determines cell fate through the regulation of eIF2α phosphorylation pathway.
Authors: Tenkerian Clara , Mounir Zineb , Krishnamoorthy Jothi , Koromilas Antonis E.
Published: August 2014

Metazoans respond to environmental stress by inducing the phosphorylation of the α subunit of translation initiation factor eIF2 at S51 (eIF2αP), a modification that leads to protein synthesis inhibition. We demonstrate that eIF2αP is induced by pharmacological inhibition or genetic ablation of the PI3K-Akt-mTOR pathway (1). Increased eIF2αP is an evolutionary conserved process that involves the endoplasmic reticulum (ER)-resident protein kinase PERK, which is negatively regulated by Akt dependent phosphorylation at T799. The PERK-eIF2αP arm is downregulated by Akt in cells exposed to ER stress or oxidative stress leading to the induction of cell survival or death respectively. In unstressed cells, the PERK-eIF2αP pathway guards survival and facilitates adaptation to the deleterious effects of PI3K, Akt or mTOR inactivation. Inactivation of the PERK-eIF2αP arm sensitizes tumor death from pharmacological inhibition of the PI3K-Akt-mTOR pathway. The PERK-eIF2αP pathway links Akt and mTOR signaling to translational control with implications in tumor treatment with pharmacological inhibitors of PI3K-Akt-mTOR pathway.

Citation Format: Clara Tenkerian, Zineb Mounir, Jothi Krishnamoorthy, Antonis E. Koromilas. The Akt-mTOR axis determines cell fate through the regulation of eIF2α phosphorylation pathway. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3191. doi:10.1158/1538-7445.AM2013-3191

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.