bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3841: RBEL1A, a novel GTPase is an important regulator of cell growth and cell survival
Authors: Lui Ki , Montalbano JoAnne , Shi Jingxue , Corcoran Chad , He Qin , Hong Sun , An Jie , Sheikh Saeed , Huang Ying
Published: June 2012

We have recently reported the characterization of RBEL1 proteins, a novel subfamily of the Ras/Rab-like GTPases. Our studies demonstrate that RBEL1 proteins have Ras-like GTPase activity and differentially regulate the ERK signaling pathway. Our studies also demonstrate that RBEL1A is overexpressed in the majority of breast cancers and a large segment of colon cancers. Knockdown of RBEL1A negatively affects cell growth and induces apoptosis, suggesting that RBEL1A is critical for regulation of cell growth and survival. To investigate the molecular mechanisms by which RBEL1A mediates its function, we have studied a number of RBEL1A-interacting proteins. Our results indicate that RBEL1A interacts with p53 and negatively regulates p53 function via a proteasome-dependent pathway. We have also examined the molecular interactions between RBEL1A and p53 and our results demonstrate that RBEL1A is an important negative regulator of p53 that affects p53-mediated functions.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3841. doi:10.1158/1538-7445.AM2011-3841
Abstract 3846: SGEF is over-expressed in advanced glial tumors and mediates glioma cell invasion and survival
Authors: Fortin Shannon P. , Rollo Johnathon , Paquette Kimberly , Chan Amanda , Ross Juli , Symons Marc , Tran Nhan L.
Published: June 2012

Glioblastoma multiforme (GBM) is the most malignant of all primary adult brain tumors, histopathologically characterized by the infiltrative capacity of glioma cells to diffuse into the surrounding normal brain. There are currently no anti-invasion therapies available, and thus the identification and functional understanding of genes that mediate malignant tumor cell dispersion could lead to the discovery of molecular targets which have the potential to respond to therapeutics. We have previously shown that several guanine nucleotide exchange factors for Rho family small GTPases are overexpressed in GBM tumors and play important roles in glioma invasion. Here we report a role for SGEF, a guanine nucleotide exchange factor (GEF) for RhoG, in mediating glioma invasion. SGEF mRNA expression increases in correlation with glioma grade and, within GBM tumors, levels of SGEF expression inversely correlate with patient survival. siRNA-mediated depletion of SGEF decreases in vitro glioma cell migration and ex vivo glioma cell invasion. In addition, genome-wide determination of NF-κB controlled genes in temozolomide-resistant primary GBM xenografts (GBM14-TMZ-R) revealed an increased occupancy of NF-κB on the SGEF gene promoter region via ChIP-on-chip analysis as compared to the parent primary line. In fact, increased phosphorylation of IkBa was detected in GBM-14-TMZ-R. Moreover, inhibition of NF-κB in GBM14-TMZ-R significantly decreases SGEF gene expression, and activation of the NF-κB pathway by TWEAK in GBM cells induces SGEF mRNA expression. Furthermore, siRNA-mediated depletion of SGEF expression enhances chemotherapy-induced cell death in glioma cells. Understanding the role of SGEF in promoting cell motility and chemotherapeutic resistance may direct the development of novel targeted therapeutics for GBM.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3846. doi:10.1158/1538-7445.AM2011-3846
Abstract 3860: Differential gene expression and HPV in penile carcinoma
Authors: de Freitas Calmon Marilia , Babeto Erica , Mota Mânlio Tasso Oliveira , Candido Natália Maria , Girol Ana Paula , Oliani Sônia Maria , Bonilha Jane Lopes , Vassallo José , Rahal Paula
Published: June 2012

Penile cancer (PC) is an invasive epithelium tumor, representing more than 10% of the malignancies in men in some developing countries, mainly in Brazil. Studies on the aetiopathogenesis of this cancer have focused on its association with the HPV. Patients with PC without treatment usually die within 2 years after diagnosis, because of uncontrollable loco regional disease or from distant metastasis. The aim of this study is evaluating the possible role of HPV in the development of PC and the use of the RaSH technique to analyze gene expression in normal tissues and penile tumors. HPV detection was done by PCR with generic primers GP5+/GP6+ and HPV typing was done by direct sequencing. RaSH methodology identified differentially expressed genes, generating subtractive cDNA libraries. The presence of HPV was analyzed in 58 samples of patients with PC. It has been observed high prevalence (85.12%) of HPV. Of the 16 samples sequenced, HPV-16 was found in 14 (87.5%) samples, HPV-11 in 1 (6.3%) and HPV-35 in 1 (6.3%). The RaSH subtractive libraries identified the presence of 57 clones differentially expressed between samples, 30 in tumor samples and 27 in the normal tissue. The genes PBEF1, ANXA1, RPL6 and KIAA1033 were overexpressed in the tumors. On the other hand, the gene p16 was overexpressed in the normal tissue. Finally, the expression of the selected genes was confirmed by qPCR. Only ANXA1 was validated with overexpression in 80% of all samples. It was also analyzed ANXA1 protein expression by immunohistochemistry in 25 samples of penile cancer. It was observed overexpression of ANXA1 protein 100% of PC samples compared to normal tissue. The results obtained are capable of revealing differences in patterns of gene expression between normal and tumor tissues. Moreover, it was observed high prevalence of HPV, suggesting an important role of this virus in penile carcinogenesis. Besides, overexpression of ANXA1 was observed in most of the penile cancer samples and such information will contribute to develop a possible marker for penis tumor diagnostic and prognostic, improving the development of directed therapies against this new putative marker.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3860. doi:10.1158/1538-7445.AM2011-3860
Abstract 3864: Evaluation of expression of antigens cancer/testis (CTAs) in head and neck squamous cell carcinoma
Authors: Zamunér Fernando T. , Karia Bruno Takao Real , Carvalho André Lopes , de Oliveira André Luiz Vettore
Published: June 2012

Background: Among the ten most common cancers are the head and neck tumors with more than 500,000 new cases diagnosed annually worldwide, the majority (90%) represented by squamous cell carcinomas (HNSCC). Despite of the many strategies used in the treatment for HNSCC, the 5 years overall survival rate is close to 50%, and the development of loco-regional recurrence is the main reason for treatment failure. The head and neck cancer surgery is often mutilating and require extensive reconstruction. The development of new therapies and their integration into the current forms of treatment (including surgery, radiotherapy and chemotherapy) is of great interest to improve the therapeutic efficacy. Immunotherapy may provide an alternative for current treatment scheme, since the natural immune response can be stimulated by active immunotherapeutic interventions which may help control tumor progression and recurrence. A requirement for the development of immunotherapeutic approaches is the identification of immunogenic gene products expressed predominantly in tumor cells. Cancer/testis antigens (CTAs) fulfills this prerequisite as their expression is restricted to tumors with no expression in normal tissues, except germ line cells. Methods: In this study we performed a search on Oncomine, SAGE, CTdatabase and PubMed databases to select among 153 CTAs genes those that might be expressed in HNSCC. The expression of selected genes was investigated in 93 HNSCC samples and 10 normal mucosa samples from healthy individuals using reverse-transcription polymerase chain reaction (RT-PCR). The in silico analysis allowed the selection of 30 CTAs genes to be evaluated in tumors and control samples. The RT-PCR assays identified 6 CTAs with high specificity (100%) and frequently expressed in HNSCC samples (39 – 85%). Noteworthy, 99% of the tumor samples expressed at least one of the 6 CTAS, 91% expressed at least two of them, and 71% expressed at least three of these antigens. The six CTAs were expressed simultaneously in 13% of cases. Conclusion: We identified 6 CTAs genes with high expression and specificity in head and neck cancer. This result could be important for the development of immunotherapy strategies for treating HNSCC patients.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3864. doi:10.1158/1538-7445.AM2011-3864
Abstract 3876: Study the role of HEYL in breast tumorigenesis
Authors: Han Liangfeng , Sukumar Saraswati
Published: June 2012

HEYL is a basic Helix-Loop-Helix (bHLH) transcription factor in the HEY family. It is the direct target of Notch pathway and mainly functions in cardiovascular development. The functions of HEYL in cancer have not been studied. In this study, we tested whether HEYL has a direct effect on promoting breast tumorigenesis. Overexpression of HEYL in NIH3T3 cells resulted in transformation of the cells in vivo. Breast cancer cell lines with exogenous HEYL expression showed accelerated cell growth in vitro and in vivo. On the contrary, the reduction of HEYL expression in MDA-MB-231 cells retarded tumor growth in vivo. Consistent with its oncogenic activity, HEYL is found to be overexpressed in 40% of breast cancers. Overall, these data indicated a role of HEYL in enhancing breast cancer development.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3876. doi:10.1158/1538-7445.AM2011-3876
Abstract 388: Inhibition of IL-17 in tumor tissue suppressed tumor growth via anti-angiogenesis and CTL activation
Authors: Hayata Keiji , Iwahashi Makoto , Ojima Toshiyasu , Katsuda Masahiro , Iida Takeshi , Nakamori Mikihito , Ueda Kentaro , Nakamura Masaki , Miyazawa Motoki , Yamaue Hiroki
Published: June 2012

[Purpose]It remains controversial whether IL-17 promotes or inhibits cancer progression in tumor bearing mice. We hypothesized that IL-17 locally produced in tumor microenvironment had an important role on tumor growth. Therefore, we investigated inhibition of IL-17 by siRNA in only tumor sites using wild type mice have an effect on tumor progression.[Methods]Adenoviral vector encoding the siRNA against mouse IL-17 gene (Ad-IL-17 siRNA) was generated by the COS-TPC method. MC38 cells or B16 cells were inoculated subcutaneously into C57BL/6 mice, and at day 5, 8, 11 or at day 7,10,13 Ad-IL-17 siRNA with 1 × 109 MOI were injected intratumorally into mice. [Results]Intratumoral injection of Ad-IL-17 siRNA significantly suppressed tumor growth in both tumor models compared with control mice. We confirmed that intratumoral administration of Ad-IL17 siRNA completely suppressed endogenous IL-17 protein expression in tumor sites. To investigate the mechanisms of tumor growth suppression by inhibition of IL-17, in term of the angiogenesis we performed immunohistochemical staining of CD31 and MMP-9. Intratumoral injection of Ad-IL-17 siRNA significantly suppressed CD31 expression and MMP-9 expression in tumor tissue. Next, to investigate the effect of the inhibition of IL-17 in tumor tissue on systemic and local anti-tumor immunity, we assessed CTL activities of CD8+ T cells from splenocytes or tumor-infiltrating lymphocytes (TILs). The cytotoxic activities in CD8+ T cells from TILs in mice treated with intratumoral injection of Ad-IL-17 siRNA were significantly higher than those in control mice, but not splenocytes. To identify why the mechanism of the enhanceing effect on CTL activity by the inhibition of IL-17, especially in tumor microenvironment, we examined the Th1/Th2 phenotype from splenocytes or TILs. The levels of IFNγ+ CD4+ T cells from TILs were significantly higher in Ad-IL-17 siRNA-injected mice compared with those in control mice, but not splenocytes. On the other hand IL-4+ CD4+ T cells from TILs and splenocytes were similar levels in both mice.[Conclusion]The suppression of IL-17 expression in tumor tissues improved the immunological tumor microenvironment by shifting to Th1 dominant environment, resulting in CTL activation in tumor sites. It was suggested that the blockage of IL-17 in tumor tissue contributed to suppressing tumor growth via the inhibition of angiogenesis as well as CTL activation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 388. doi:10.1158/1538-7445.AM2011-388
Abstract 3880: Synthesis of complex oligonucleotide libraries enables flexible and precise high-resolution fluorescence imaging of cellular nucleic acids
Authors: Ach Robert A. , Yamada N. Alice , Tsang Peter , Scheffer-Wong Alicia , Bruhn Laurakay
Published: June 2012

Fluorescence in situ Hybridization (FISH) is a powerful technique for studying chromosome structure, organization, and stability. To date, the use of FISH has been largely dependent upon access to cloned template DNA for the generation of probes. Clones for specific regions may be unavailable, or the genomic region of interest may contain repetitive and other non-informative sequences which can be problematic for FISH analysis. We have leveraged our ability to chemically synthesize DNA in massively parallel reactions to produce libraries of oligonucleotides up to 200 bases in length that can be used to generate FISH probes. The sequences of the oligonucleotides in these libraries are selected in silico using empirically determined criteria so as to avoid repetitive elements or regions homologous to other non-targeted loci, thus targeting only the most informative elements in the region of the genome to be analyzed. We demonstrate that using our FISH technique, human genomic regions as small as 1.8 kb and as large as whole chromosomes can be visualized in both metaphase and interphase cells using the same simple assay protocol. In addition, specific and robust signals can readily be obtained for chromosomal regions rich in repeats and/or GC content. These libraries can also be used to determine the localization of cellular RNAs. Co-detection of both nuclear proteins by immunocytochemistry and fluorescent imaging of chromosome domains has also been demonstrated.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3880. doi:10.1158/1538-7445.AM2011-3880
Abstract 3883: Multiplexed formats for detection of SNPs for cell line authentication and cancer related mutations
Authors: Fang Rixun , Furtado Manohar R.
Published: June 2012

Rapid and accurate identification of SNPs and mutations are useful in ensuring that the correctly identified cell lines are being used in cancer research studies and for accurate identification of SNPs and indels in cancer cells. In this study we report on a method that uses nucleic acid isolation methods, multiplexed PCR to amplify up to 48 regions within the human genome followed by an oligonucleotide ligation assay that can detect SNPs, mutations and indels. The protocol takes about 4 hrs to complete and uses panels optimized for human cell identification and detection of mutations in EGFR and KRAS genes.

We typed the NCI-60 panel and several cell lines from the ATCC using two panels. One is a panel of SNPs that has been tested with samples from over 46 human populations to identify a set of SNPs that would discriminate globally. This SNP set has 48 markers with high heterozygosity (>0.4) and low Fst values (<0.06) across multiple human populations. The panel has a high power of discrimination. (Hum Genet; 127 (3) 315-324; 2010) comparable to that obtained with STR kits. A panel of 31 samples from closely related individuals within families was typed to test the power of discrimination. In another study, we applied this system to a cancer mutation study, in which an assay for detecting 29 EGFR and 5 KRAS gene mutations was developed. These mutations are frequently found in human cancers. DNA preparations from 48 ATCC and 60 NCI tumor cell lines were genotyped by this assay. These SNPs and mutations were further confirmed by the Sanger sequencing chemistry, showing 100% concordance between two technologies. The information generated from this study will be definitely useful when these cell lines are used as models for cancer study and cancer drug screening.

In conclusion, we have developed a sensitive, highly accurate, high throughput genotyping system. Studies have demonstrated its effectiveness for successful SNP/InDel detection and mutation screening. This system has been successfully used for oncogen mutation studies in cancer cell lines. Overall, the system can become a valuable tool for molecular and clinical diagnostics.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3883. doi:10.1158/1538-7445.AM2011-3883
Abstract 3884: DNA mismatch repair defects, MSI, does not play major role in colon carcinogenesis in young African Americans
Authors: Ashktorab Hassan , Lee Edward L. , Razjouan Hadi , Mohammed Roshila , Hassanzadeh Hassan , Qadwai Saad , Smoot Duane T. , Ali Waleed El-sayed Mohammed , Elnour Hassan , Sanderson Andrew , Horton Sara , Singleton Daniella , Brim Hassan
Published: June 2012

Back ground: African Americans (AAs) are known to display a higher rate and even earlier age of colorectal cancer (CRC) than in the general population. Genetically, 10-12% microsatellite instability (MSI) cases involved in CRC development. However, if it occurs below the age of 50 it could be suspicious of Lynch syndrome.

Aims and methods: We aimed to assess the role of MSI in pathogenesis of sporadic colorectal cancer in young African American. The CRC patients in this study were less than 50 years and referred to Howard University Hospital from 2005 to 2008. The medical records (n=50) as well as the pathology reports were reviewed. The MSI status was assessed in a multiplex PCR targeting 5 markers: BAT25, BAT26, NR21, NR27 and NR24. The PCR products were analysed in an Applied Biosystems Gene Scan 3130 machine. Samples with two or more instable markers were labeled MSI-H, with one instable marker: MSI-L and no stable marker: MSS. The presence or absence of expression of four DNA MMR proteins: MLH1, PMS2, MSH2 and MSH6 were evaluated using immunohistochemistry on a Tissue Microarray (TMA, n=26, duplicate) consisting (stage1: 3; stage 2: 6; stage3: 5; stage4: 4).TMA IHC staining were read by two different pathologists who were not aware of the patient clinical status and were scored thus in an objective manner. Two patients were MSI-L and one MSI-H. The response to 5-FU based therapy was reported.

Results: Twenty six patients [age, median: 44.5; age range: 26-49; male, 17 (65.4%)] were enrolled in the study. Seven out of 13 available data had family history of CRC. Fourteen CRC cases were left sided. MSI analysis revealed that out of 24 patients: one was MSI-H and two were MIS-L and the rest were MSS. The MSI high patient and one of the MSI low had family history of CRC. MLH1, PMS2, MSH2 and MSH6 were not expressed in 7 (26.9%), 1 (3.8%), 0 (0%) and 5 (19.2%) of tumors, respectively. IHC confirmed the MSI-H tumor. There was a recurrence with lung and brain metastases at 72 months in one of the MSI-L patients following adjuvant chemotherapy with 5 Fluorouracil and Leucovorin.

Discussion: Our results show that MSI is not a major pathway in tumorgenesis of sporadic CRCs in young AAs and we may need to study chromosomal instability or CpG Island methylator phenotype as alternative pathways which occur in African Americans and make them present the disease at younger age and advanced stage.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3884. doi:10.1158/1538-7445.AM2011-3884
Abstract 3886: The G-quadruplex induced DNA damage at telomeres is regulated by ATM
Authors: Islam Ashraful , Thoma Shelia D. , Miller Donald M.
Published: June 2012

Quadruplex-forming DNA sequences are present throughout the genome, including in the telomeres. Our group has shown that quadruplex-forming oligonucleotides selectively inhibit proliferation of transformed cells. One of these oligonucleotides, AS1411, is currently in a Phase IIb clinical trial. We have recently shown that the c-myc promoter quadruplex-forming sequence, Pu-27, also inhibits growth of malignant cells. We have hypothesized that one potential mechanism of this effect is competition for telomere-binding proteins. If this is correct, we would expect this effect to be mediated by the DNA repair response. Telomere integrity is maintained by the shelterin protein complex which is required to prevent chromosomal ends from being recognized as DNA double strand breaks. Like many other DNA damage response proteins, ATM has been shown to play a role in the maintenance of telomere integrity. We have shown that ATM deficient cells are more sensitive to growth inhibition by Pu-27 whereas ATM proficient cells were relatively resistant to Pu-27. Co-immunostaining with γ-H2AX and TRF1 revealed that the sensitivity to Pu-27 in ATM deficient cells was associated with phosphorylation of H2AX, an indicator of DNA double strand break and induction of TIFs (Telomere-dysfunction Induced Foci). There was induction of γ-H2AX and downregulation of shelterin proteins TRF2 and TRF1 in Pu-27 treated ATM deficient cells (as determined by western blotting). U937 leukemia cells, which were sensitive to Pu-27, became relatively resistant when they were stably transfected with a vector expressing a dominant negative mutant of the shelterin protein TRF2 (dn-TRF2). This suggests that Pu-27 may act by altering the telomere-shelterin complex. This idea was further substantiated by the fact that ALT (Alternative Lengthening of Telomere) cells were also relatively resistant to Pu27. A549 cells, which were sensitive to Pu-27, also showed abundant presence of TIF compared to untreated cells. Lastly, we showed that both p53 wild type and deficient colorectal cells (HCT116 cell line) were relatively resistant to Pu-27. Taken together, this data suggests that ATM plays a pivotal role in Pu-27 induced DNA damage at telomere ends, which occurs in response to a Pu-27 induced alteration in telomere-shelterin homeostasis. It is likely that this response is induced in a p53 independent manner.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3886. doi:10.1158/1538-7445.AM2011-3886
Abstract 3890: Regulation of mediator of DNA damage checkpoint 1 (MDC1) during mitosis
Authors: Yu Bing , Dalton William B. , Yang Vincent W.
Published: June 2012

Genetic instability is one of the hallmarks of human cancers. DNA double strand break (DSB), if improperly repaired, results in genetic instability and tumorigenesis. Previous research has established γH2AX as a bona fide marker of DSBs and the hierarchical foci assembly of DNA damage response (DDR) proteins at DSB sites is required for efficient repair. Recent findings from our group demonstrated that during either spontaneous or induced prolonged mitosis, cancer cells acquire DSBs which lead to further chromosomal abnormality [Dalton et al.(2007) Cancer Res. 67:11487]. Deciphering the mechanism of such DSB accumulation may provide new insights into the mechanism of genetic instability. We hypothesize that the increase of DSBs during prolonged mitosis is due to lack of efficient DDR. To this end, we focus on the protein Mediator of DNA damage Checkpoint 1 (MDC1), an important mediator in DSB repair. MDC1 directly binds to γH2AX and serves as a platform to accumulate/retain downstream DDR proteins at DSB sites and concomitantly initiates cell cycle arrest through its multiple interaction domains. We observed that during mitosis, many DDR proteins fail to colocalize with γH2AX in response to DSBs, while MDC1 exhibits a weakened interaction with γH2AX as shown by immunofluorescent microscopy. Inhibition of Cyclin-dependent kinase 1 (CDK1) activity, either by treatment with a specific CDK1 inhibitor RO-3306 or transient knockdown of CDK1 protein by siRNA, strengthened MDC1-γH2AX colocalization in colon cancer cells in mitosis. In vitro phosphorylation assay confirmed MDC1 as a direct substrate of CDK1. Moreover, a deletion mutant of MDC1 exhibits an increased colocalization with γH2AX compared to wild-type MDC1. Considering the published role of MDC1 as a positive regulator in metaphase-anaphase transition, we propose that phosphorylation of MDC1 by CDK1 promotes a partial disassembly of MDC1 from γH2AX foci during mitosis, in order for MDC1 to function as a modulator of mitotic progression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3890. doi:10.1158/1538-7445.AM2011-3890
Abstract 3891: Identification and quantification of DNA repair proteins by liquid chromatography/isotope-dilution tandem mass spectrometry using 15N-labeled whole proteins as internal standards
Authors: Dizdaroglu Miral , Jaruga Pawel , Reddy Prasad
Published: June 2012

Reduced DNA repair capacity is associated with increased risk for a variety of disease processes including carcinogenesis. Thus, DNA repair proteins have the potential to be used as important predictive, prognostic and therapeutic biomarkers in cancer and other diseases. The measurement of the expression level of these enzymes may be an excellent tool for this purpose. Mass spectrometry is becoming the technique of choice for the identification and quantification of proteins. We applied liquid chromatography/ isotope-dilution tandem mass spectrometry (LC-MS/MS) for the identification and quantification of DNA repair proteins human 8-oxoguanine-DNA glycosylase (hOGG1) and E. coli formamidopyrimidine DNA glycosylase (Fpg), which are involved in base-excision repair of DNA damage. We over-expressed, purified and characterized 15N-labeled analogues of these proteins to be used as internal standards. 15N-labeled whole proteins are ideal internal standards to ensure the accuracy of quantification of proteins by mass spectrometry. DNA glycosylase activities of 15N-labeled hOGG1 and 15N-labeled Fpg were determined and found to be essentially identical to those of their respective unlabeled counterparts, ascertaining that the 15N-labeling did not perturb their catalytic sites. hOGG1, Fpg and their 15N-labeled analogues were digested with trypsin and analyzed by LC-MS/MS. A large number of tryptic peptides of both proteins were identified on the basis of their full-scan mass spectra. These peptides matched the theoretical peptide fragments expected from the trypsin digestion and provided statistically significant protein scores that would unequivocally identify these proteins. We also recorded the product ion spectra (MS/MS spectra) of the tryptic peptides and defined the characteristic product ions. Mixtures of the analyte proteins and their 15N-labeled analogues as internal standards were analyzed by selected-reaction monitoring (SRM) on the basis of the previously identified product ions in the MS/MS spectra. The experimental conditions for these analyses were optimized. The methodology was successfully applied to the measurement of human OGG1 in human cells and Fpg in E.coli. The results obtained in this work suggest that the methodology developed would be highly suitable for the positive identification and accurate quantification of DNA repair proteins in vivo as potential biomarkers for cancer and other diseases.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3891. doi:10.1158/1538-7445.AM2011-3891
Abstract 3892: Discovery and analysis of COMMD1 in the DNA damage response network
Authors: Woods Nicholas T. , Nguyen Huey , Carvalho Marcelo , Li Xueli , Dapic Virna , Monteiro Alvaro
Published: June 2012

Spatially distinct domains impart functionality to the proteins and are often under evolutionary pressure to maintain sequence and/or structure. The BRCA1 C-terminal (BRCT) domain has an established role in cancer susceptibility and in the DNA damage response (DDR), as mutations in cases of familial breast cancer often result in a truncated protein lacking the two BRCT domains which are required for interactions with proteins such as p53, ABRAXAS, BACH1, and CtIP. BRCT domains can occur as singletons or as tandem domains (tBRCT) that function as a single cohesive structural unit involved in binding phosphorylated peptides targeted by PI3K-like proteins such as ATM in response to DNA damage. Using the Yeast Two-Hybrid system (Y2H) as well as Tandem Affinity Purification coupled to mass spectrometry (TAP-MS), we have built a domain centric protein-protein interaction network for a subset of tBRCT domains from BRCA1, TP53BP1, LIG4, PAXIP1, ECT2, BARD1, and MDC1. Evaluation of this network has revealed a number of interesting interactions and highlighted the interconnectivity potential found in these domains. Analysis of this network has revealed several interesting proteins that are highly interconnected with tBRCT domains, such as COMMD1 (an important regulator of copper homeostasis, sodium transport, NF-κB, and HIF-1), which has not yet been characterized to participate in the DDR, but was found to interact with the tBRCT domains of BARD1, BRCA1, and LIG4. Furthermore, an earlier Y2H screen conducted by our laboratory found COMMD1 as an interaction partner of CHK2. Therefore, we sought to determine the role of COMMD1 in response DNA damaging agents. Convincingly, the levels of COMMD1 are inversely related to apoptotic induction caused by cisplatin and doxorubicin. Furthermore, CHK2 signaling appears to control the ability of COMMD1 to enhance cisplatin induced apoptosis as wild type but not CHK2-/- HCT116 cells exhibited a repression in cisplatin induced apoptosis when COMMD1 was over expressed. These results indicate the importance of COMMD1 for cellular susceptibility to traditional chemotherapeutics. Currently, we are exploring the mechanisms by which COMMD1 in coordination with tBRCT domain containing proteins and CHK2 sensitizes cells to DNA damaging agents.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3892. doi:10.1158/1538-7445.AM2011-3892
Abstract 3893: Mechanism of radiosensitization by inhibitors of poly(ADP-ribose) polymerase (PARP)
Authors: Hegan Denise C. , Glazer Peter M.
Published: June 2012

Inhibitors of poly(ADP-ribose) polymerase (PARP) are in clinical trials for cancer therapy based on specific lethality to cancer cells deficient in BRCA1 or BRCA2. In addition, pre-clinical work has shown that PARP inhibition induces radiosensitization in cancer cells. To provide a basis for the clinical application of PARP inhibitors as radiosensitizers, we conducted mechanistic studies to elucidate the pathways by which PARP inhibitors influence radiation response.

Cells treated or not with PARP inhibitors and treated or not with siRNAs or other reagents to manipulate gene expression were assayed for radiation response by clonogenic survival assays. DNA double strand break repair was assayed using a chromosomally integrated GFP-based reporter construct. Although it was previously thought that PARP plays a role in DNA repair and radiation response primarily via the base excision repair (BER) pathway and recruitment of BER factors to sites of damage, we found that inhibitors of PARP can disrupt repair of double strand breaks (DSBs) by suppressing expression of BRCA1 and RAD51, key factors in homology-dependent repair (HDR) of DSBs. Treatment of human cancer cells with PARP inhibitors or with siRNAs targeted to PARP-1 yielded reduced levels of BRCA1 and RAD51. Radiosensitization by PARP inhibition was also seen in cells in which BER was already attenuated (via expression of a dominant negative variant of polymerase beta). Direct measurement of HDR using a GFP-based chromosomal reporter assay demonstrated reduced HDR in cells treated with PARP inhibitors. This work demonstrates that PARP inhibition can suppress the HDR pathway of DSB repair by down-regulation of BRCA1 and RAD51 expression. As such, it identifies a novel mechanism by which PARP regulates DNA repair and promotes radiosensitization, providing further rationale for therapeutic strategies combining PARP inhibition and radiation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3893. doi:10.1158/1538-7445.AM2011-3893
Abstract 3905: Identification of new tumor-specific splice variants in NSCL cancers by whole genome exon arrays
Authors: Ghoneim Christelle , Kaoma Tony , Nicot Nathalie , François Bernardin , Nazarov Petr , Muller Arnaud , Birembaut Philippe , Vallar Laurent
Published: June 2012

Although lung cancer is one of the major causes of cancer death in men and women worldwide, the molecular pathogenesis of this disease remains elusive. The contribution of alternative splicing (AS) to cancer pathogenesis and progression is emerging as an area of considerable interest. Cancer-specific splice variants seem to play a key role in disease mechanism and etiology.

In this study, we analyzed 20 matched pairs of tumor specimens of Adenocarcinomas (AC), Squamous Cell Carcinomas (SCC) and adjacent normal tissue using Affymetrix GeneChip Exon microarrays. With this technology, it is possible to profile simultaneously gene expression and alternative splicing patterns for the entire genome using a single array. At the exon level, multiple probes for each exon enable the discrimination among different isoforms of a gene. Total RNA from tumors or normal tissue samples were amplified using the Ambion whole transcript expression kit and were hybridized onto GeneChip Human Exon 1.0 ST Array®. Data generated were normalized using RMA and were subjected to several layers of filtering (DABG filter, Multiple mRNAs filter). The delta splice index (ΔSI) was calculated, and alternative splicing events were detected using MIDAS and Rank Product method. Probesets were ranked based on their SI p-values.

By comparing tumor samples to the corresponding normal subgroups, we generated a list of 1689 splice variant candidates for SCC and 1010 for AC showing significant differential expression between normal and tumor tissues. Top candidates in each tumor subtype were selected based on their ΔSI for manual inspection and further bioinformatics analysis. Study of alternatively spliced candidates revealed that approximately 30 % of the genes were detected in both types of cancer. Gene function analysis combining text mining and knowledge-based approaches indicated that the largest subset (70 %) of these genes was related to cancer. Within this group, 30% of genes were related to lung cancers. Interestingly the major part (85%) of variant candidates was not previously associated with AS events in cancer. We focused our attention on this latter group and, through validation by RT/PCR, confirmed the existence of new specific alternative splicing events occurring in each cancer type. In addition, we identified biological pathways and gene networks that are specifically altered in AC or SCC. The cancer-specific AS variants and pathways identified in our study are promising biomarkers and targets for diagnostic or therapeutic purposes.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3905. doi:10.1158/1538-7445.AM2011-3905
Abstract 397: LPA promotes ovarian cancer mesothelial invasion by regulating VCAM-1 expression
Authors: Slack-Davis Jill K. , Lynch Kevin R.
Published: June 2012

Ovarian cancers metastasize by attaching to and invading through the mesothelium, a single cell layer of mesothelial cells lining the peritoneal cavity. The presence of invasive peritoneal metastases is associated with a poor prognosis for ovarian cancer (5 yr survival <25%). Lysophosphatidic acid (LPA) is abundant in malignant ascites where it regulates ovarian cancer cell growth, migration and response to chemotherapy. Using a cell culture model system of mesothelial invasion, treatment with a LPA receptor antagonist resulted in a dose-dependent decrease in mesothelial invasion. Eliminating LPA1 expression in mesothelial cells using siRNA blocked mesothelial invasion (70% inhibition relative to control) and resulted in decreased expression of VCAM-1. We have shown previously that VCAM-1 is expressed preferentially on the mesothelium of ovarian cancer patients where it regulates mesothelial invasion and ovarian cancer metastatic progression. Re-expression of VCAM-1 in mesothelial cells lacking LPA1 expression rescued mesothelial invasion. Together, these observations implicate a role for LPA in modulating the tumor microenvironment to regulate ovarian cancer progression. Furthermore, they specifically point to a role for LPA in the regulation of VCAM-1 expression in ovarian cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 397. doi:10.1158/1538-7445.AM2011-397
Abstract 3973: MiR-21 modulates hTERT through a STAT3-dependent manner on glioblastoma cell growth
Authors: Sun Guan , Wang Yingyi , Zhang Junxia , Wang Xiefeng , Sun Lihua , Pu Peiyu , Kang Chunsheng , Liu Ning , Fu Zhen , You Yongping
Published: June 2012

MiRNAs, are small noncoding RNA molecules of approximately 20-22 nucleotides in length, regulate protein expression by cleaving or repressing the translation of targets mRNA. MiR-21, as an oncogene, has been reported to be dyregulated in a variety of cancer. Our recent data have verified that miR-21 is one of the most frequently over-expressed miRNA in human glioblastoma (GBM) cell lines. However, the mechanism of miR-21 involved in gliomagenesis is still unknown. In this study, reduction of miR-21 by antisense oligonucleotide induced cell apoptosis and inhibited cell growth in U87 and LN229 GBM cells, with a decreased expression of human telomerase reverse transcriptase (hTERT). Knockdown of hTERT expression using lentiviral vector mediated delivery of hTERT-siRNA triggered cell apoptosis and cell cycle G1/G0 arrest in U87 and LN229 cells. In order to explore the molecular regulation of hTERT by miR-21, we found that STAT3 could be an important mediator between miR-21 and hTERT. Reduction of miR-21 repressed STAT3 expression and STAT3 phosphorylation. STAT3 inhibitor WP1066 led to a remarkable depletion of hTERT both at mRNA and protein levels. To determine the functional interaction between STAT3 and hTERT, ChIP showed the presence of STAT3-binding sites in the hTERT gene. Luciferase reporter assay revealed that the hTERT gene promoter was direct bound by STAT3. Furthermore, we evaluated the status of STAT3 in hTERT regulation by miR-21. U87 and LN229 cells that treated with miR-21 antisense oligonucleotide and IL-6 which could elevate the expression of STAT3 showed a significant increase of hTERT expression, whereas cells that treated with miR-21 oligonucleotide and STAT3 inhibitor WP1066 displayed a reduction of hTERT expression. Finally, knockdown of miR-21 considerably inhibited tumor growth and diminished the expression of STAT3, pSTAT3 and hTERT in xenograft model. Taken together, we have demonstrated that miR-21 inhibits cell growth through regulating hTERT expression in a STAT3-dependent pathway in GBM cells. These results suggest that modulation of the mechanism responsible for miR-21 in GBM could be used as a therapeutic strategy for GBM treatment and warrants further investigation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3973. doi:10.1158/1538-7445.AM2011-3973
Abstract 3977: Systematic analysis of microRNAs targeting the androgen receptor in prostate cancer cells
Authors: Östling Päivi , Leivonen Suvi-Katri , Aakula Anna , Kohonen Pekka , Mäkelä Rami , Hagman Zandra , Edsjö Anders , Kangaspeska Sara , Edgren Henrik , Nicorici Daniel , Bjartell Anders , Ceder Yvonne , Perälä Merja , Kallioniemi Olli
Published: June 2012

Androgen receptor (AR) is expressed in all stages of prostate cancer progression and its levels increase in the castration-resistant tumors. However, the mechanisms regulating AR expression remain poorly understood. Here, our aim was to determine the role of miRNAs in regulating AR protein. To this end, we performed a systematic gain-of function screen for 1129 miRNA molecules in prostate cancer cell lines and quantified changes in AR protein content using protein lysate microarrays. We identified 71 unique miRNAs that influenced the level of AR in LNCaP, LAPC-4, 22Rv1, CWR-1R and MDA-PCa-2B cells. Our RNA-sequencing data showed that the 3’UTRs of many genes, including the AR, are much longer than currently used by target prediction programs. Our analyses predicted that the majority of the miRNA regulation of AR would occur through the extended 6 kb 3’UTR. 3’UTR-binding assays validated 13 miRNAs (miR-135b, miR-185, miR-297, miR-299-3p, miR-34a, miR-34c, miR-371-3p, miR-421, miR-449a, miR-449b, miR-634, miR-654-5p, and miR-9) as direct AR regulators. Fifteen AR down-regulating miRNAs decreased androgen-induced proliferation of prostate cancer cells in vitro. Negative correlation of miR-34a and miR-34c expression with AR levels was also found in clinical prostate cancers. To conclude, our results demonstrate that miRNAs interacting with the long 3’UTR of the AR gene are important regulators of AR protein levels. This could be applied for developing novel therapeutic strategies to inhibit AR function and androgen-dependent cell growth.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3977. doi:10.1158/1538-7445.AM2011-3977
Abstract 3979: Identification of microRNA 885-5p as a novel regulator of tumor metastasis in colorectal cancer
Authors: Lam CSC
Published: June 2012

Introduction: MicroRNAs (miRNAs) are a class of small non-coding RNAs that play a crucial role in cellular proliferation, differentiation and apoptosis by regulating gene expression. They are involved in the regulation of human diseases as well as colorectal cancer (CRC). CRC is the third most common cancer in the world and second leading cause of cancer death in the Hong Kong. Liver is the most common site of distant metastasis with poor prognosis for CRC patients. Recent evidences suggested that miRNAs affect the cell invasiveness of various cancers and regulate key steps in metastatic cascade. Moreover, studies also demonstrated that miRNAs play critical roles in the regulation of drug resistance. The study of miRNAs mechanism in liver metastasis may provide some new strategies for cancer treatment.

Method: Microarrays were used for screening the expression differences of miRNAs between normal colon, CRC and liver metastasis. Lentiviral transduced colon cell lines were sorted with flow cytometer high-speed sorter by green fluorescent protein signal. Functional assay such as wound healing and invasion chamber were used to study the metastatic property (migration and invasion) in vitro. GeneChip expression was used to study the mRNA expression differences between gain-of-function and control. Orthotopic implantation of mi-885-5p-expressing CRC cells into the cecal wall of nude mice was performed to investigate their tumorigenic and metastatic capacity in vivo.

Results: We quantified the expression levels of miRNAs in 14 liver metastasis samples and 12 colorectal cancer tissues, and identified hsa-miR-885-5p whose expression levels were significantly up-regulated in liver metastasis samples compared with the CRC samples. Over-expressed miR-885-5p cell line showed more invasive and migratory when comparing with control. 67% of the mice developed liver metastasis with orthotopic implantation of a CRC cell line overexpressing miR-885-5p, as compared with 33% of metastatic incidence in mice injected with the vector-expressing CRC cell line. GeneChip array showed that VCAM1 may be one of a possible target of miR-885-5p, which was comparable with the online database.

Conclusion: We conclude that over expression of miR-885-5p is observed in liver metastasis has an important role in regulating the metastatic capacity of CRC cells vitro and in vivo.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3979. doi:10.1158/1538-7445.AM2011-3979