bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2338: Functional investigation of the CRD-BP hnRNP-K-Homology domains for C-myc messenger RNA interaction
Authors: Rensburg Gerrit van , Barnes Mark , Lee Chow
Published: May 2015

The coding region determinant binding protein (CRD-BP) is an oncofetal protein, being ubiquitously expressed during embryogenesis, yet highly repressed in adult tissues, except in cancerous tissues [Bell et al, 2013; Mueller-Pillasch et al, 1999]. CRD-BP was inherently identified by its ability to physically associate with c-myc mRNA, and has been shown to protect the transcript from degradation, hence prolonging its mRNA half-life [Prokipcak et al, 1994, Noubissi et al, 2006]. In addition to c-myc mRNA, several other mRNAs bound by CRD-BP have gene products implicated in the development and progression of cancer [Bell et al, 2013]. As an RNA binding protein (RBP), CRD-BP is the Mus musculus variant and homologous to the Homo sapiens IMP-1/Insulin-Like Growth Factor 2 with 99.31% similarity, both categorized into a larger family named VICKZ (Vera/Vg1 RBP, Insulin-Like Growth Factor 1,2,3/IMP-1,2,3, CRD-BP, KOC, and ZBP-1). The VICKZ family of RBPs contains two RNA Recognition Motifs (RRM) and four heterogeneous nuclear ribonucleoprotein [hnRNP]-K-Homology (KH) domains. Deletion analysis had shown that the KH domains, and not the RRM domains, of IMP1 are important for binding RNA substrates. To understand which of the KH domains are critical for interaction with RNA, site-directed mutagenesis was performed to introduce an aspartic acid (D) at the first glycine (G) of the G-X-X-G region in the KH domains of CRD-BP. This D-X-X-G variation was individually introduced to the four KH domains of CRD-BP. These four CRD-BP KH domain variants were expressed, purified and assessed for ability to bind c-myc mRNA using electromobility shift assays (EMSA). In addition, plasmids carrying these variants of FLAG-CRD-BP were transfected into HeLa cells. Immunoprecipitation (IP) using anti-FLAG was performed, followed by RT-qPCR to measure the relative amounts of c-myc mRNA associated with each of the variants as compared to the controls. The in vitro EMSA and IP cell data were all consistent with the notion that all KH domain(s) are critical for CRD-BP-c-myc RNA interaction. Using EMSA, additional binding assessments were carried out in a systematic fashion to determine the smallest region of c-myc mRNA bound by CRD-BP. These results will be presented and discussed, as they provide insight into the role played by the KH domains of CRD-BP in binding RNA, as well as what RNA characteristics are important for binding to CRD-BP.

Citation Format: Gerrit van Rensburg, Mark Barnes, Chow Lee. Functional investigation of the CRD-BP hnRNP-K-Homology domains for C-myc messenger RNA interaction. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2338. doi:10.1158/1538-7445.AM2014-2338
Abstract 2341: CAPG improves breast cancer metastasis through competing with PRMT5 to activate STC-1 transcription
Authors: Huang Sheng , Chi Yayun , Xue Jingyan , Shao Zhimin , Wu Zhaohui , Wu Jiong
Published: May 2015

Recent studies have shown that over-expression of macrophage-capping protein (CapG)improves cancer cell metastasis. Previously, we demonstrated that CAPG was highly expressed in MDA-MB-231 cell line with high metastatic potency compared with its maternal cell line. Sequencing of CapG-cDNA complex showed some overlap with the sequence of a group of DNA-binding proteins, the basic helix-loop-helix family, including the c-myc oncogene. We supposed that CAPG inhibited cancer cell metastasis through modulating gene transcription as a transcription factor.

To examine whether CAPG played a functional role in breast cancer metastasis, three kinds of breast cancer cell lines were applied, including overexpression and knockdown of CAPG. Total RNA from MCF-7 CAPG overexpression cells versus control was obtained. Microarray analysis of global genomic changes was conducted in transcriptional level of these cells. Stanniocalcin 1 (stc-1), an oncogene, was observed significantly up-regulated by over expression of CAPG. Mass spectra and co-immunopreciptation were used to find the potential transcription factors of CAPG.

In breast cancer cell lines, we found that CAPG expression is much higher in relatively aggressive cell line than the mild-nature one on both RNA and protein level. CAPG is also higher in cancer tissues than its adjacent noncancerous tissues (ANCT). Furthermore, it was confirmed that CAPG could directly bind to the promoter of stc-1 to activate stc-1 transcription by luciferase report assay and chip experiments. In CAPG overexpression cell line, knock-down of stc-1 could restrain the CAPG-induced migration and invasion. Interestingly, protein arginine methyltransferase 5 (PRMT5) was found as transcription repressor to compete with CAPG in modulating stc-1 transcription.

In this study, CAPG was approved to play a direct role in participating transcription for the first time. We also supposed that by combining with PRMT5, CAPG could compete with PRMT5, and prevent PRMT5 from repressing stc-1 transcriptional process.

Citation Format: Sheng Huang, Yayun Chi, Jingyan Xue, Zhimin Shao, Zhaohui Wu, Jiong Wu. CAPG improves breast cancer metastasis through competing with PRMT5 to activate STC-1 transcription. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2341. doi:10.1158/1538-7445.AM2014-2341
Abstract 2343: Role of calreticulin in the regulation of MALAT-1 expression in mouse adenocarcinoma cells
Authors: Massaeli Hamid , Viswanathan Divya , Pillai Dhanya , Mesaeli Nasrin
Published: May 2015

Calreticulin (CRT) is a ubiquitously expressed protein, with both calcium binding and chaperone activity. CRT is involved in quality control process during the folding and maturation of proteins in ER. Recently, we showed that overexpression of CRT resulted in the development of metastatic lung adenocarcinoma. In order to examine genes involved in the development of lung cancer we carried out microarray analysis on RNA isolated from mouse lung tumors versus control. In this screen we observed a significant increase in long noncoding RNA (LncRNA) MALAT-1 in the lungs of CRT overexpressing mouse models. MALAT-1 is a LncRNA that has been documented to be upregulated in the human lung adenocarcinoma and associated with metastasis. Our study was to investigate the mechanism of regulation of MALAT-1 expression and role of CRT in this process. We hypothesized that CRT as a regulator of intracellular calcium regulated MALAT-1 expression thus inducing the development of metastatic lung adenocarcinoma in our mice model. Our data showed a significant correlation between intracellular calcium levels and MATAT-1 RNA expression and stability. Treatment of adenocarcinoma cells with BAPTA-AM resulted in a significant reduction in MALAT-1 level. Treating the cells with thapsigargin induced a 2 fold increase in MALAT-1 expression. We also demonstrated that knock down of MALA-1 expression using specific siRNA reduces the proliferation of adenocarcinoma cell. Our data demonstrate for the first time involvement of calcium binding protein CRT and intracellular level of calcium on expression and stability of MALAT-1 involved in development of lung adenocarcinoma.

Funded by NPRP4-043-3-016

Citation Format: Hamid Massaeli, Divya Viswanathan, Dhanya Pillai, Nasrin Mesaeli. Role of calreticulin in the regulation of MALAT-1 expression in mouse adenocarcinoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2343. doi:10.1158/1538-7445.AM2014-2343
Abstract 2346: The expression of ASPP2 and its new isoform delta-ASPP2 in HCC
Authors: Shi Ying , Liu Kai , Ding Huiguo , Lv Fudong , Lopez Charles , Li Ning , Chen Dexi
Published: May 2015

Purpose: Although full-length ASPP2 is necessary to stimulate p53-dependent apoptosis efficiently, the precise mechanisms and cell contexts that enable this function remain to be clarified. In recent study, we found a new ASPP2 isoform (ΔN-ASPP2) may play an important role in regulating the ASPP2-p53 axis. The present study was undertaken to investigate the expression of isoform of ASPP2 in liver tumor and it would be a domain negative regulation of ASPP2 and would be prognostic significance in HCC.

Experimental Design: 120 HCC tissues and same amount of nonneoplastic liver tissues were analyzed for ASPP2 alone and ASPP2 plus ΔN-ASPP2 mRNA expression by real-time RT-PCR. The ΔN-ASPP2 mRNA was analyzed by RT-PCR and real-time RT-PCR with ΔN-ASPP2 specific forward primers. ASPP2 plus ΔN-ASPP2 P53 downstream effectors of the apoptotic and cell cycle arrest pathway (BAX and P21) were studied in 100 HCC specimens.

Results: The expression of ASPP2 and ΔN-ASPP2 mRNA levels were highly variable in HCC tissues. There was no association between the ASPP2/ ΔN-ASPP2 mRNA level and the p53 status. Expression of full length ASPP2 in pairs were detected in 62 HCC and its adjacent tissue.ASPP2-full mRNA level is significant lower in HCC than that in the tumor surrounding tissue (P=0.0159). Expressions of ΔN-ASPP2 mRNA, ASPP2-total (ASPP2, ΔN-ASPP2 and 53BP2), BAX and P21 in pairs were compared between 72 of HCC and their adjacent tissue. The results showed that ΔN-ASPP2 mRNA (P=0.0435) is significantly higher and ASPP2-total (P=0.0212), BAX (P=0.0057) and P21 (P=0.002) are significantly lower in HCC than that in adjacent tissue. CONCLUSIONS: The high level ΔASPP2 which is in contrast to ASPP2 function and low level ASPP2-full which is a p53 activator in HCC would play an important role in tumor development and tumor senescence.

Citation Format: Ying Shi, Kai Liu, Huiguo Ding, Fudong Lv, Charles Lopez, Ning Li, Dexi Chen. The expression of ASPP2 and its new isoform delta-ASPP2 in HCC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2346. doi:10.1158/1538-7445.AM2014-2346
Abstract 2353: A comparative meta-analysis of prognostic gene signatures for late-stage ovarian cancer
Authors: Waldron Levi , Riester Markus , Birrer Michael , Parmigiani Giovanni
Published: May 2015

Numerous gene signatures of patient prognosis for late-stage, high-grade serous ovarian cancer (HSOC) have been proposed, but diverse data and methods make these difficult to compare or prioritize for clinical application. However, the corresponding volume of publicly available expression data creates an opportunity to validate previous findings and to develop more robust signatures. To establish the current state of HSOC prognostic gene signatures, we undertook a systematic review and meta-analysis of the publicly available microarray data and published prognostic gene signatures. This effort generated a curated database of 1,319 clinically annotated HSOC microarray assays from 10 studies (Ganzfried et al., DATABASE 2013) and implementations of 14 published prognostic signatures for evaluation in independently available data. For each published prognostic signature we evaluated 1) its reproducibility, 2) its prognostic accuracy for overall survival in independent datasets, and 3) the prognostic value of the genes used in each signature relative to random gene sets. Twelve published models performed better than 97.5% of randomized risk scores, and six out-performed 97.5% of random signatures of the same size trained on the same data. The four top-ranked models achieved overall validation C-Indices of 0.56 to 0.60, and shared anti-correlation with expression of immune response pathways. Most models demonstrated lower accuracy in new datasets than in validation sets presented in their publication. This analysis provides definitive support for several prognostic models, but confirms that these require improvement to be of clinical value. This work addresses outstanding controversies in the ovarian cancer literature, including whether the prognostic signature proposed by The Cancer Genome Atlas Consortium (Nature 2011) improved on existing signatures, the impact of batch effects on a frequently re-used, high-profile dataset (Dressman et al., J Clin Oncol 2007), and the prognostic value of a recently proposed DNA damage repair signature (Kang et al., J Natl Cancer Inst 2012). Finally, this work provides a reproducible framework for meta-analytic evaluation of gene signatures.

Citation Format: Levi Waldron, Markus Riester, Michael Birrer, Giovanni Parmigiani. A comparative meta-analysis of prognostic gene signatures for late-stage ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2353. doi:10.1158/1538-7445.AM2014-2353
Abstract 2357: Gene expression profiling robustly predicts the outcome of patients diagnosed with early stage lung adenocarcinoma
Authors: Gaston-Mathé Yann , FERTE CHARLES , gauthier benoit , bateson mathilde , planchard david , besse benjamin , Armand Jean-Pierre , soria jean-charles
Published: May 2015

Background: Although the management of metastatic lung adenocarcinoma has been profoundly modified by the identification of actionnable molecular traits, decision making for early-stage lung adenocarcinoma still relies on the tumor stage (TNM) only. Most of these patients recur within 5 years after tumor resection and are given platinum based adjuvant chemotherapy. The recent availability of multiple gene expression data and the concomitant developments of novel algorithms - such as the Hypercube algorithm - could dramatically improve such predictive challenges.

Methods: Datasets were selected from public repositories (Director's challenge (DC) n=274, Rousseaux n=86, Hou n=35, JBR.21 n=31) upon the following criteria: lung adenocarcinoma, stage IA-3A, no adjuvant therapy, R0 tumor resection. To generate robust predictors of 3-year overall survival, an iterative feature selection framework (Hypercube algorithm) was applied successively on DC and Hou. Only highly predictive rules in both datasets (AUC >.7) were then included in a subsequent logistic regression model and finally assessed for external validation. The predictive performance of the models was evaluated by ROC-AUC and by Kaplan Meier curves.

Results: Our best model combining gene expression (10 genes) and clinical data (TNM) was robust across three lung adenocarcinoma datasets (AUC= .85, .80 and .59). Particularly, it was able to better predict the 3 year of survival compared to the TNM-based clinical model only (100% vs. 77%). Kaplan Meier estimates of overall survival in the validation datasets were dramatically discriminated according to the high- and low-risk groups predicted by our model (all P values =.01). Interestingly, this predictive classifier was specific to lung adenocarcinoma and has no predictive value in lung squamous datasets.

Conclusion: We developed a robust and highly performing gene expression predictive signature of 3-year overall survival, specific to the lung adenocarcinoma setting. These promising results have the potential to change the decision making at bedside for early stage lung adenocarcinoma patients.

Citation Format: Yann Gaston-Mathé, CHARLES FERTE, benoit gauthier, mathilde bateson, david planchard, benjamin besse, Jean-Pierre Armand, jean-charles soria. Gene expression profiling robustly predicts the outcome of patients diagnosed with early stage lung adenocarcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2357. doi:10.1158/1538-7445.AM2014-2357
Abstract 2358: Dissecting the cellular response to cisplatin from RNA transcription to translation
Authors: Mackintosh Carlos , Konovalov Sergiy , Garcia-Bassets Ivan
Published: May 2015

Platinum-based antineoplastic drugs, such as cisplatin, are DNA damaging agents typically utilized in cancer treatment. In ovarian cancer, however, these drugs have a relatively low therapeutic index (which is a measure of clinical benefit over toxicity of these drugs), thus even a small change (reduction) in the cellular sensitivity to these chemotherapeutic agents leads to clinical treatment failure, also known as ‘drug resistance’. To increase the clinical efficacy of platinum-based antineoplastic drugs, we need to develop therapies that are less likely to result in drug resistance, or we need to modify the current therapies to prevent or reduce drug resistance.

To gain mechanistic insights into the process of drug resistance that may help us to develop new or modified therapies that are less prone to clinical drug resistance, we have focused our studies on the comprehensive analysis of two still poorly studied cisplatin-associated processes at genome-wide scale: the processes of RNA nascent transcription and RNA translation. In particular, we investigate by global run-on combined with next-generation sequencing (GRO-seq) how cisplatin-sensitive A2780 ovarian cancer cells and its cisplatin-resistance derivative, A2780cis cells, respond to acute cisplatin treatment at the level of the nascent transcriptome. We also investigate, in this case by ribosome profiling, how the same cells respond to acute cisplatin treatment at the level of the ribosome-associated transcriptome (also known as translatome). These two massive analyses, complemented with conventional profiling of steady-state RNA (profiled by RNA-seq) provide the first multidimensional analysis of the actions induced by any chemotherapeutic agent at the level of the three basic states of the transcriptome (nascent RNA, steady-state RNA, and translated RNA). Interestingly, this analysis reveals an unexpected large fraction of uncoupled events between these three transcriptomic states, which challenges the conventional one-dimensional approach based exclusively on RNA-seq for the study of drug resistance.

Note: This abstract was not presented at the meeting.

Citation Format: Carlos Mackintosh, Sergiy Konovalov, Ivan Garcia-Bassets. Dissecting the cellular response to cisplatin from RNA transcription to translation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2358. doi:10.1158/1538-7445.AM2014-2358
Abstract 2365: Analysis of angiogenesis pathway-related gene expression impacted by MDM2 using PCR array
Authors: Venkatesan Thiagarajan , Stinson Corine , Dhandayuthapani Sivanesan , Rathinavelu Appu
Published: May 2015

The Murine Double Minute 2 (MDM2) gene encodes an oncogenic MDM2 protein, which is a negative regulator of the p53 tumor suppressor. MDM2 amplification or over expression is found in many tumors that eventually lead to the inactivation of the cell cycle control and loss of pro-apoptotic functions that are typically exerted by wild type p53. Many of the oncogenic effects of MDM2 are resulting from the inactivation of p53 through direct binding or degradation of p53 following E3 ligase mediated ubiquitination. However, our recent findings have indicated that MDM2 over expression can also promote tumor angiogenesis through p53 independent pathways and thereby enhance the metastatic potential of cancer cells. In this respect increasing the levels of pro-angiogenic factors such as VEGF has been confirmed as one of the mechanisms regulated by MDM2. However, the actual cluster of genes or pathways regulated by MDM2 leading to such increase in the metastatic potentials of cancer cells have not been clearly identified. Therefore, the main purpose of our study was to understand the impact of MDM2 over expression on the levels of angiogenesis related genes that can also increase growth rate and metastatic potential. Hence, for our study we used the Human Angiogenesis PCR Array to compare the gene expression profile of LNCaP (prostate cancer cells) and LNCaP-MST (MDM2 transfected prostate cancer) cells. The MST cells expressed at least 10 fold increase in the levels of MDM2 after transfection. As a result of the elevated levels of MDM2 in LNCaP-MST cells the expression of genes such as THBS1 (125 fold), TNF-α (14 fold), MMP-9 (1.4 fold) were found to be significantly up-regulated compared to the MDM2 non-transfected cells. Cluster analysis further confirmed that these gene expression profiles are positively correlated with the pro-angiogenic potential of the LNCaP-MST cells compared to the non-transfected cells. In addition, the levels of EREG, TIMP1 and CXCL3 were down regulated significantly that favors the suppression of anti-angiogenic mechanisms. Our results offer a conclusive evidence that MDM2 can impact the cancer growth and metastatic potential of cancer cells by shifting the balance towards pro-angiogenic mechanisms (The financial support from the Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida is gratefully acknowledged).

Citation Format: Thiagarajan Venkatesan, Corine Stinson, Sivanesan Dhandayuthapani, Appu Rathinavelu. Analysis of angiogenesis pathway-related gene expression impacted by MDM2 using PCR array. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2365. doi:10.1158/1538-7445.AM2014-2365
Abstract 2367: High expression of GAPDH is relevant to lung adenocarcinoma with low expression of the tumor suppressor gene Deleted in Liver Cancer-1 (DLC1)
Authors: Wang Dunrui , Zhang Guangxian
Published: May 2015

Although glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is often considered to be a stable housekeeping marker gene, we recently determined that high levels of GAPDH mRNA expression in solid tumors was highly correlated with the expression of a group of genes, most of which are classified as cell cycle-dependent (Wang et al. PLoS One, 2013). In the current project, we have further evaluated the role of GAPDH in lung adenocarcinoma. Using data from an integrated microarray gene expression dataset and The Cancer Genome Atlas (TCGA) Project, we have now found a correlation between high mRNA expression of GAPDH and low expression of the tumor suppressor gene Deleted in Liver Cancer-1 (DLC1). DLC1 encodes a protein with strong RhoGAP (GTPase activating protein) activity and is inactivated in various human malignancies. In lung adenocarcinoma, low DLC1 expression occurred frequently together with up-regulation of GAPDH. The correlation coefficient of GAPDH and DLC1 expression in the integrated gene expression dataset was -0.6. In addition, high GAPDH and low DLC1 expression in lung cancer was associated with unfavorable prognosis. The combination of DLC1 expression level and GAPDH level improved the predictive prognostic power compared with that of the individual gene level alone. Pathway analysis indicated that low DLC1 in lung cancer was accompanied by increased expression of genes implicated in glycolysis and reduced expression of fructose-1,6-bisphosphatase (FBP1) in gluconeogenesis. Our findings suggest that GAPDH may contribute to the regulation of cell cycle genes and of tumor suppressor genes in cancer.

Citation Format: Dunrui Wang, Guangxian Zhang. High expression of GAPDH is relevant to lung adenocarcinoma with low expression of the tumor suppressor gene Deleted in Liver Cancer-1 (DLC1). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2367. doi:10.1158/1538-7445.AM2014-2367
Abstract 2373: Deep computational analysis of human and mouse specific next-generation sequencing data generated from PDX specimen
Authors: Menon Roopika , Schneider Petra , Peifer Martin , Leenders Frauke , Heuckmann Johannes M.
Published: May 2015

Patient derived xenograft (PDX) samples are powerful tools to analyze tumor biology and drug sensitivity of primary tumors in animal models. However, sequencing studies on those samples face the challenge of mouse stromal contamination, which varies in each of the explanted tumor specimen. Thus, without applying mouse and human specific filters on sequencing data, species specific genomic alterations and differences in expression are impossible to detect. To allow mouse and human specific transcriptome/genome analyses, we adapted our NGS data analysis pipelines to filter for mouse and human specific reads. By applying the analysis algorithms on hybrid-capture and transcriptome sequencing data from 60 lung cancer PDX samples, we were able to generate a comprehensive overview on tumor specific alterations, including point mutations, deletions and insertions, copy number alterations, gene fusions and isoform specific expression levels, as well as species specific genomic alterations and transcriptome wide expression levels. From 5781 nonsynonymous substitutions which were detected by hybrid-capture sequencing of 333 cancer relevant genes (CAGE-Rx Scanner), 863 human specific nonsynonymous substitutions were detected after filtering for mouse specific variations. The mean sequencing coverage of the CAGE analysis across all targeted bases in all 60 samples was 662x. An integrated analysis of this data set and corresponding transcriptome sequencing data revealed TP53 and KRAS as the most frequently mutated genes which were expressed. As expected, due to the relative high tumor content in all samples (ranging from 30 to 90% with a median of 90% as determined by pathological review), 80% of all mutations called from transcriptome sequencing in an unfiltered fashion were human specific and called after species specific filtering. In addition, the detection of mouse specific transcriptome sequencing reads identified several growth factors expressed by tumor surrounding tissue, which would have been discarded as unmapped reads in the normal analysis setting. Thus, our analysis alorithms not only enable to remove mouse specific false positive single nucleotide variants (SNVs), but also a detailed analysis of mouse specific expression patterns which might strongly influence tumor growth via the tumor microenvironment.

Citation Format: Roopika Menon, Petra Schneider, Martin Peifer, Frauke Leenders, Johannes M. Heuckmann. Deep computational analysis of human and mouse specific next-generation sequencing data generated from PDX specimen. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2373. doi:10.1158/1538-7445.AM2014-2373
Abstract 2376: MutationValidator: A computational method for variant cross-validation in next-generation sequencing data
Authors: Rosenberg Mara , Getz Gad , Kiezun Adam , Sivachenko Andrey
Published: May 2015

Background:

Massively parallel next-generation sequencing (NGS) technologies already enable comprehensive genome-wide detection of germline and somatic variants in thousands of individuals, and the pace of data production keeps accelerating. However, with increased sequencing techniques and variant calling algorithms, proper validation and determination of ground truth is essential for benchmarking, comparisons, and confirmation of novel discoveries. Datasets generated on different sequencing platforms, derived from distinct biological samples and/or using different library construction protocols can provide at least partially independent sources for crosschecking variants. While methods for validation exist to date, they fail to account for the inherent variability in the datasets, and thereby propose biased estimates for the validation rates.

Results:

Here we present a method for variant cross-validation and present software implementation (written in Matlab) that generates validation metrics and diagnostics to characterize a mutation as true somatic, germline, or artifactual. Our method adjusts for the power to observe variant-supporting evidence in the validation dataset, which allows for accurate assessment of validation rates across wide ranges of sequencing coverages and variant allelic fractions. When this method is applied to a test cohort of 172 patients from The Cancer Genome Atlas Lung Adenocarcenoma dataset, we observe a SNP and Indel validation rate of 95% and 96% respectively with a 99% power for detection. A previously implemented naïve method of >8% supporting alleles in the tumor and <3% alternate allele in the normal for a validated event would have yielded rates of 72% and 95% for SNPs and Indels, values we claim to be underestimations of the true rates as they misclassify underpowered sites as a false positives.

Conclusions:

Our proposed method, MutationValidator, enables confident classification of mutations using orthogonal next-generation sequencing technologies for validation as it takes into consideration the variability in and across the datasets.

Citation Format: Mara Rosenberg, Gad Getz, Adam Kiezun, Andrey Sivachenko. MutationValidator: A computational method for variant cross-validation in next-generation sequencing data. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2376. doi:10.1158/1538-7445.AM2014-2376
Abstract 2383: Translesion DNA synthesis by DNA polymerase kappa somatic variants found in prostate cancer
Authors: Yadav Santosh , Mukhopadhyay Sudurika , Makridakis Nick
Published: May 2015

Prostate cancer is one of the most prevalent malignancies affecting men worldwide, and is the most frequent cancer among men in the USA, with an estimated 238,590 new cases and 29720 deaths in 2013 (NCI). DNA damage found in prostate cancer promotes tumor progression. Repair of DNA lesions that occur endogenously or in response to diverse genotoxic stresses is indispensable for genome integrity. DNA lesions activate checkpoint pathways that regulate specific DNA-repair mechanisms. During replication, DNA polymerases frequently encounter unrepaired DNA lesions and posses 3′→5′ exonuclease proofreading activities. Such polymerases tend to stall at sites of damage, which can lead to replication fork collapse and which, unless rescued, can eventually cause large-scale genomic rearrangements and cell death. As a preventative measure, most organisms have alternative polymerases that are capable of ‘damage tolerance’ and can synthesize past DNA lesions by a mechanism called translesion synthesis (TLS). Polymerase kappa (Polκ) a Y-family polymerase participates in the process of TLS. Current study was designed to investigate the TLS of somatic missense mutations of Polκ such as p.F155S, p.L442F, p.G154E, p.E29K, p.E449K and p.E430G found after sequence analysis of prostate tumor DNA from 30 prostate cancer patients. We studied these variants to identify the nucleotide incorporated opposite to the apurinic (AP) site template, in vitro using site-specific tetrahydrofuran (AP site analog). We report that missense somatic mutations of Polκ at p.F155S, p.L442F, p.G154E, p.E29K, p.E449K mutants significantly decreases catalytic efficiency (k(cat)/Km). The biological consequence of an unrepaired DNA lesion is determined by whether it is bypassed or not, bypassed error prone. Our results provide evidence that mutations in the polymerase kappa gene can causes interference in Translesion DNA synthesis, this interference in pol

κ function may play a role in prostate tumor progression.

We acknowledge to Prof. Dr. Guengerich, for providing Polκ construct.

This work was supported by DOD grant PC094628, and NIH grant 8 P20 GM103518.

Citation Format: Santosh Yadav, Sudurika Mukhopadhyay, Nick Makridakis. Translesion DNA synthesis by DNA polymerase kappa somatic variants found in prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2383. doi:10.1158/1538-7445.AM2014-2383
Abstract 2388: Deciphering the role of APE1 protein variants in disease etiology
Authors: Illuzzi Jennifer , Harris Nicole A. , Manvilla Brittney A. , Kim Daemyung , Li Mengxia , Drohat Alexander C. , Wilson David M.
Published: May 2015

Base excision repair (BER) is necessary for removal of DNA damage that has occurred from spontaneous decomposition, alkylation and oxidation. Defects in BER have been associated with cancer predisposition, neurodegeneration and premature aging. BER is initiated by a damage-specific DNA glycosylase that excises the damaged base, creating an apurinic/apyrimidinic (AP) site. The AP site is recognized by the multifunctional AP endonuclease 1 (APE1), which cleaves the phosphodiester backbone, producing a DNA strand break. DNA polymerase beta incorporates the correct nucleotide, and BER is completed via ligation by a protein complex of Ligase III and XRCC1. Like many BER proteins, APE1 is essential for survival, as deletion of both alleles in mice leads to embryonic lethality. We are pursuing the hypothesis that more mild reductions in APE1 activity will contribute to disease risk and development. To address this issue, we have engaged two strategies: (1) identification and characterization of APE1 missense mutations and (2) design and characterization of genetically-altered mutant human cell lines. In our first study, we have characterized eight APE1 variants found within the population or identified in endometrial cancer. Our studies reveal that while most of these proteins are normal for protein stability, biochemical activities, and intracellular localization, the endometrial cancer-associated variant Arg237Cys has decreased 3′-functions and AP-DNA binding. In our second study, we have created a heterozygous knockout HCT116 cell line, in which we have deleted one allele of APE1. The heterozygous knockout cells show a loss in AP endonuclease activity and are hypersensitive to DNA damaging drugs. Current cellular strategies are determining the essential nature of APE1 activity in HCT116 and other cell lines

Citation Format: Jennifer Illuzzi, Nicole A. Harris, Brittney A. Manvilla, Daemyung Kim, Mengxia Li, Alexander C. Drohat, David M. Wilson. Deciphering the role of APE1 protein variants in disease etiology. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2388. doi:10.1158/1538-7445.AM2014-2388
Abstract 2389: PTEN localizes to the nucleus and interacts with Rad52 in response to DNA damage
Authors: Choi Byeong Hyeok , Dai Wei
Published: May 2015

PTEN is a tumor suppressor with a dual phosphatase activity. Mutations of PTEN gene has been associated with many sporadic cancers and heritable neoplastic syndromes. PTEN also exhibits nuclear localization. However, the exact function of nuclear PTEN remains unclear. Here we show that PTEN exhibits a time-dependent nuclear translocation after DNA damage, co-localizing with DNA damage foci on chromatin, and that the nuclear translocation of PTEN is closely associated with its phosphorylation on S366/T370. Deletional analysis reveals that C2 domain of PTEN is responsible for its nuclear translocation after exposure to genotoxin. Both casein kinase 2 and GSK3β are involved in the phosphorylation of S366/T370, as well as PTEN's association with chromatin during DNA damage response. Significantly, PTEN specifically interacts with Rad52 and positively regulates its sumoylation. Taken together, these observations strongly suggest that PTEN may mediate DNA damage repair through interacting with and regulating Rad52 in the nucleus.

Citation Format: Byeong Hyeok Choi, Wei Dai. PTEN localizes to the nucleus and interacts with Rad52 in response to DNA damage. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2389. doi:10.1158/1538-7445.AM2014-2389
Abstract 2391: USP7 deubiquitinates XPC in response to ultraviolet light irradiation
Authors: He Jinshan , Zhu Qianzheng , Sharma Nidhi , Wani Gulzar , Han Chunhua , Qian Jiang , Pentz Kyle , Wang Qi-en , Wani Altaf A
Published: May 2015

Ultraviolet light (UV)-induced Xeroderma pigmentosum complementation group C (XPC) protein ubiquitination is mediated by an E3 ubiquitin ligase complex containing UV damaged-DNA binding protein. Here, we report that ubiquitin specific protease 7 (USP7) deubiquitinates XPC during NER. We have demonstrated that transiently compromising cellular USP7, by siRNA or specific inhibitor HBX41108, leads to accumulation of ubiquitinated forms of XPC. However, complete USP7 disruption causes an ubiquitin-mediated XPC degradation upon cellular irradiation. We show that USP7 interacts with XPC in vitro and in vivo. Overexpression of wild-type USP7, but not its catalytically inactive or interaction-defective mutants, reduces ubiquitinated forms of XPC. Importantly, USP7 efficiently deubiquitinates XPC-ubiquitin conjugates in deubiquitination assays in vitro. We further showed that valosin-containing protein (VCP)/p97 is required for UV-induced XPC degradation in USP7-deficient cells. VCP/p97 is readily recruited to DNA damage sites and co-localizes with XPC. Inhibition of VCP/p97 causes an accumulation of ubiquitinated XPC on DNA damaged chromatin. Moreover, USP7 disruption severely impairs the repair of cyclobutane pyrimidine dimers (CPD) and, to a lesser extent, affects the repair of 6-4 photoproducts (6-4PP). Taken together, our findings have uncovered an important role of USP7 in regulating NER via deubiquitinating XPC and by preventing its VCP/p97-regulated proteolysis (This work was supported by grants from NIH).

Citation Format: Jinshan He, Qianzheng Zhu, Nidhi Sharma, Gulzar Wani, Chunhua Han, Jiang Qian, Kyle Pentz, Qi-en Wang, Altaf A Wani. USP7 deubiquitinates XPC in response to ultraviolet light irradiation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2391. doi:10.1158/1538-7445.AM2014-2391
Abstract 2393: The BARD1 BRCT domains are essential for maintenance of telomere integrity
Authors: Irminger-Finger Irmgard , Pilyugin Maxim , Ratajska Magdalena
Published: May 2015

BARD1 has tumor suppressor functions by binding to BRCA1 and to p53 via RING finger and ankyrin repeats, respectively. The BARD1-BRCA1 dimer has E3 ligase activity, and BARD1-p53 interaction induces apoptosis. The C-terminus of BARD1 is binding to many proteins that are targets of the BRCA1-BARD1 ubiquitin ligase, and expression of N-terminally truncated RING-less BARD1 isoforms was correlated with poor prognosis in various cancers, consistent with their oncogenic functions by antagonistic binding to target proteins.

Expression and interaction assays showed that the BARD1 BRCT domains were critically involved in telomere maintenance and interacted with the telomere binding protein TRF2. Overexpression of BARD1 or BRCA1 lead to TRF2 degradation, suggesting that TRF2 presents a novel target of the BARD1-BRCA1 E3 ubiquitination ligase. Repression of BARD1 or BRCA1 by siRNA leads to genetic instability, as shown before, and telomere alterations.

We observed distinct telomeric alterations in blood cells of patients with a BARD1 germline mutation that causes a translation stop and translation of a truncated protein lacking the BRCT domains. Cells of carriers of a BRCA1 mutation that disrupts the BARD1-BRCA1 interaction show similar telomere alterations. These in vitro and in vivo observations suggest that BARD1 and BRCA1 play distinct roles in maintaining telomere integrity in a dosage dependent manner.

BARD1δ, an isoform lacking RING and ankyrin motifs, but retaining the BRCT domains, is abundantly expressed in all cancers investigated so far. Overexpression of BARD1δ in vitro blocked cell cycle progression and induced chromosomal and telomere abnormalities, suggesting that BARD1δ also affected telomere integrity.

We conclude that the correct dose of BARD1 and/ or its BRCT domains is critically important for maintaining telomere integrity. Expression of BARD1δ in cancer, or reduced expression of BARD1 in mutation carriers causes chromosomal instability and promotes carcinogenesis and tumor progression.

Citation Format: Irmgard Irminger-Finger, Maxim Pilyugin, Magdalena Ratajska. The BARD1 BRCT domains are essential for maintenance of telomere integrity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2393. doi:10.1158/1538-7445.AM2014-2393
Abstract 2394: The role of DNA repair in breast cancer risk and recurrence
Authors: Matta Jaime L. , Suarez Erick , Vargas Wanda , Ortiz Carmen , Bayona Manuel , Morales Luisa
Published: May 2015

BACKGROUND: Breast cancer (BC) is a complex disease that is caused by multiple factors. Deficits in DNA repair capacity (DRC) are known to cause certain familial BC, and dysregulation of DRC develops with progressive carcinogenesis. However, the effect of DRC on carcinogenesis of sporadic breast tumors has not been well characterized_and the factors associated with DRC variability are still poorly understood. DNA repair is integral to maintain genomic integrity, and carcinogenesis occurs when efficient, effective DNA repair is impaired. Our laboratory has just finished the recruitment of nearly 1,200 women in a seven-year molecular epidemiology study focused on the role of DRC as a risk factor for BC. OBJECTIVE: Our aim is to present key findings on the role of DRC as a risk factor for BC and its potential association with BC recurrence. METHODS: A personal interview was conducted for soliciting information on BC risk factors. Among the 493 BC cases and 683 controls the DRC was measured in lymphocytes using a host cell reactivation assay with a luciferase reporter gene. We stratified the women with BC using the median of the DRC level and selected 70 women with a low DRC level (<2.8%) who had been enrolled in the study for at least 5 years in order to assess the BC recurrence. RESULTS: Our 7 year study of DRC of female cohorts in Puerto Rico has shown that women with BC have a

DRC that, on average, is 51% lower than women without BC (P < 0.001, Mann Whitney U-test). Women with low DRC have 6.8 (95%CI 5.2, 9.0) times the odds for BC when compared with women with high DRC level. So far, the overall BC recurrence was 17% and the 5-year mortality was 10%. Recurrence was primarily in the lung, liver, bone, or occurred as generalized metastatic disease. CONCLUSIONS: Using a large sample size (n=1,183 participants), we show that women with BC had on an average a 51% lower DRC than controls (P<0.001). We also present the first data on the potential between a low DRC and risk of BC recurrence. With a larger sample size, we will test the hypothesis that low DRC can predict risk of BC recurrence. Supported by grants from the NCI Center to Reduce Health Disparities through the NIH-MBRS Program grants S06 GM008239-20, 9SC1CA182846-04 to PSMHS through JM and MBRS-RISE GM082406 through CO.

Citation Format: Jaime L. Matta, Erick Suarez, Wanda Vargas, Carmen Ortiz, Manuel Bayona, Luisa Morales. The role of DNA repair in breast cancer risk and recurrence. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2394. doi:10.1158/1538-7445.AM2014-2394
Abstract 2395: Progression of structural change in breast cancer metastasis
Authors: Hartmaier Ryan J. , Lee Adrian V.
Published: May 2015

Structural variations (SVs) have been shown to be a major source of genetic instability in cancer and metastasis but remains understudied. This is due to: (1) the limited power of paired-end sequencing (PES) in detecting SVs that frequently occur around repetitive regions and (2) the large amount (4ug) of high-molecular weight DNA required for mate-pair sequencing (MPS), which is specifically designed to detect SVs even in repetitive regions. Importantly, DNA obtained from FFPE tissue is heavily sheared and therefore not suitable for MPS - excluding most metastatic tissues in tumor banks.

To overcome the barriers of MPS, we leveraged the University of Pittsburgh Rapid Autopsy Program and the Health Sciences Tissue Bank to obtain matched primary/metastatic frozen tumor samples from 6 patients (19 total tumor samples).

Multiple large-insert (3-5, 5-8, & 8-12 kb) mate-pair sequencing, was performed for each sample to obtain a complete catalogue of SVs. Additionally, whole exome sequencing was performed to identify point mutations and to de-convolute clonal evolution within each patient.

After removing SVs found in matched germline DNA, we identified extensive somatic SVs within the primary tumor that tended to decrease in metastatic samples. This is likely due to extensive genetic heterogeneity in the primary tumor that underwent clonal selection due to metastasis and/or treatment. Well-known hotspots (such as 17q), showed extensive intra- and inter- chromosomal SVs in both primary and metastatic tumors indicating that these are likely early and essential events in tumorgenesis. However, metastatic specific events also clustered with 17q suggesting that this region continues to play a role during metastasis. Somatic single-nucleotide variations (SNVs) revealed obvious high density clustering of mutations (kataegis) that overlapped with SV breakpoints. Further analysis revealed that these SNV clusters were primarily TT-TC, which is a signature of the deaminase APOBEC3B recently been shown to bind around dsDNA breaks and cause high levels of mutations. We have observed examples of kataegis as early, shared events and metastatic specific events indicating that kataegis is an ongoing process through metastasis.

Together these data show that extensive SV occurs during breast cancer tumorigenesis and metastasis and these SVs often co-occur with kataegis. As more samples and distant metastases are sequenced, RNA-seq will be used to help prioritize specific metastatic specific SVs for functional studies demonstrating the role in the development of the metastatic phenotype.

Citation Format: Ryan J. Hartmaier, Adrian V. Lee. Progression of structural change in breast cancer metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2395. doi:10.1158/1538-7445.AM2014-2395