bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 208: The MAGE family of cancer-testis antigens are potent oncogenes
Authors: Potts P. Ryan , Doyle Jennifer M. , Hao Yi-Heng , Kleinschmidt Elizabeth
Published: August 2012

Introduction: Cancer-testis antigens (CTAs), including the MAGE protein family, are genes whose expression is typically restricted to the germline, but are aberrantly expressed and presented as antigens in human tumors. Therefore, CTAs constitute excellent targets for cancer immunotherapy. Surprising recent evidence suggests that the aberrant expression of MAGE CTAs in tumors is not simply an inert consequence of widespread genomic deregulation, but rather an important functional event promoting tumorigenesis. Furthermore, the expression of MAGE CTAs correlates with poor prognosis in a variety of cancer types. However, the role of MAGE CTAs as oncogenes driving tumorigenesis has been unexplored and their biochemical activity of MAGEs has been enigmatic. Aims and Methods: In this study, we investigated the function of the large MAGE protein family. First we determined the oncogenic potential of more than 20 MAGE CTAs in 3T3 transformation assays, soft agar anchorage-independent growth assays, and matrigel transwell invasion assays. We then selected several of these oncogenic MAGE CTAs for further in-depth study to identify their biochemical and cellular functions. Using a variety of in vitro and cellular assays, we identified common binding partners of more than ten MAGE proteins, solved the crystal structure of one MAGE protein, and investigated the biochemical activity of several MAGE CTAs. Results and Conclusions: We found that the majority of MAGE CTAs (more than 15) have oncogenic activity in cell transformation, anchorage-independent growth, and/or invasion. Additionally, we showed that a common feature of MAGE proteins is their ability to bind to and enhance the activity of E3 RING ubiquitin ligases, such TRIM28/KAP1, through a conserved tandem winged-helix domain. Importantly, we discovered that several MAGE-TRIM28 ubiquitin ligase complexes directly ubiquitylate and degrade the critical p53 tumor suppressor in vitro and in cancer cells. Thus, our findings clearly establish that MAGE CTAs function as oncogenes through their ability to regulate the activity of E3 RING ubiquitin ligases, resulting in the deregulation of vulnerable signaling pathways (such as the p53 tumor suppressor). These results underscore the importance of MAGE proteins as therapeutic targets for cancer and highlight a novel germline signaling program co-opted by cancer cells to facilitate tumorigenesis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 208. doi:1538-7445.AM2012-208
Abstract 2083: The regulation of anoikis by carcinoma-associated fibroblasts in breast cancer cells
Authors: Jakimenko Ana , Weigel Kelsey , Hyatt Rebecca , Kaliney William , Champion Matthew , Schafer Zachary T.
Published: August 2012

The initial step of the metastatic cascade in most carcinomas occurs when tumor cells break through the basement membrane and invade locally into the surrounding microenvironment. This microenvironment is now appreciated to be an active participant in driving the oncogenic behavior of cancer cells. In particular, evidence is mounting that carcinoma-associated fibroblasts (CAFs) in the microenvironment are a critical player in the promotion of tumor progression and metastasis. However, the precise mechanisms utilized by CAFs to enhance malignancy are only beginning to be understood. Given the importance of CAFs in promoting tumor progression and the fact that cancer cells lack normal extracellular matrix (ECM) attachment after breaking through the basement membrane, we hypothesized that CAFs may be actively involved in the inhibition of anoikis (ECM-detachment-induced cell death). To investigate this question, we utilized NIH-3T3 mouse fibroblasts lacking the caveolin-1 gene (KO 3T3s), a cell line that has previously been demonstrated to share many similarities to human CAFs. Interestingly, the addition of media conditioned by KO 3T3s led to robust anoikis inhibition in detached MCF-10A cells, suggesting that factors secreted by CAFs can block anoikis. We validated these findings by demonstrating that factors secreted from patient-derived human CAFs can inhibit anoikis to a similar degree. Moreover, the ability of CAF-conditioned media to block anoikis was not limited to MCF-10A cells as we obtained similar results in two additional non-tumorigenic cell lines: BPH-1 and HMEC. In addition, when investigating the mechanism by which CAFs block anoikis, we discovered that the release of mitochondrial cytochrome c into the cytosol was robustly inhibited by CAF-conditioned media in MCF-10A cells. Surprisingly, this inhibition was independent of signaling through PI(3)K or MAPK and did not involve alterations in Bim protein levels. To examine if the observed differences in anoikis induction caused by secreted factors from CAFs manifest in a more physiologically relevant context, we employed a 3-dimensional (3D) cell culture model of mammary acinus development. MCF-10A acini exposed to secreted factors from CAFs were more prone to have filled lumen suggesting that factors secreted by CAFs can promote the survival of ECM-detached cells in the luminal space. In aggregate, these data identify a novel mechanism by which CAFs contribute to tumorigenesis (the inhibition of anoikis) and suggest that targeting factors secreted from CAFs could be a novel therapeutic approach to eliminate ECM-detached cells through the selective induction of anoikis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2083. doi:1538-7445.AM2012-2083
Abstract 2085: MMP-2 knockdown leads to anoikis and inhibits migration and invasion by attenuating αvβ3/EGFR/Pak4 survival signaling in human glioma xenograft cells in vitro and in vivo
Authors: Kesanakurti Divya , Chetty Chandramu , Maddirela Dilip , Dinh Dzung H. , Rao Jasti S.
Published: August 2012

MMP-2 plays a key role in the degradation of the extracellular matrix and thereby enhances the proliferative, invasive and metastatic potential of cancer cells. MMP-2 knockdown using siRNA (pM) in human glioma xenograft cell lines, 4910 and 5310, sensitized cells to anoikis associated with cleavage of PARP, and caspases 8 and 3. pM-inhibited cell adhesion, invasion and migration correlated with attenuation of EGFR, c-Src and Pak4 phosphorylation. High expression of Pak4 in human glioma cell lines and tumor tissues implicated its essential role in increasing glioma malignancy. Co-immunoprecipitation studies implied that MMP-2 directly interacts by complex formation with EGFR, p130CAS and Pak4 along with αvβ3 integrin and pM treatment disrupted this direct binding. Vitronectin-mediated αvβ3 activation and EGF and rhMMP-2 supplementation counteracted pM-inhibited, Pak4-mediated migration and invasion. Conversely, αvβ3 blocking antibody and ErbB-2/EGFR inhibitor (GW2974) further reduced pM-inhibited, Pak4-mediated invasiveness and migration, thereby suggesting a role of αvβ3 and EGFR in the regulation of Pak4 survival signaling. Individual treatments of pM and Pak4.si led to considerable anoikis in both 4910 and 5310 cells and simultaneous co-depletion MMP-2 and Pak4 resulted in robust increase in anoikis levels. In vivo orthotopic experiments in nude mice revealed decreased tumor size with pM treatment. Immunofluorescence analysis of pSV-tumor sections showed high expression and co-localization of MMP-2/αvβ3, which decreased upon treatment with pM along with significant decrease in phospho-EGFR, phospho-Pak4, Bcl2 and Bcl-xL levels. Our results imply a possible crosstalk between MMP-2 and Pak4 and suggest the potential of targeting both MMP-2 and Pak4 as a future therapeutic approach in glioma therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2085. doi:1538-7445.AM2012-2085
Abstract 2086: Evaluation of a promoter methylation panel in plasma cell-free DNA to screen for colorectal cancer in Hispanics
Authors: del Mar Gonzalez-Pons Maria , Rodríguez Heriberto M. , Delgado Patricia , Velazquez Francisco , Lacourt Mercedes Y. Y. , Cardona Alberto , Bernabe Raúl D. , Cruz-Correa Marcia R.
Published: August 2012

Introduction: Detection of quantitative and qualitative changes in cell-free, tumor-derived nucleic acids in cancer patients are being evaluated as potential screening, monitoring and diagnostic tools. Genetic and epigenetic changes in cell-free plasma DNA (cfpDNA) have been studied as non-invasive colorectal cancer (CRC) diagnostic markers, but a gene panel with the diagnostic accuracy necessary for clinical use has not been defined. The CpG island methylator phenotype (CIMP), characterized by hypermethylation in promoter-associated regions, is one of the molecular mechanisms resulting in CRC. We evaluated the detection of promoter hypermethylation in a gene panel previously used to define CIMP in CRC tumors, as plasma biomarkers. Methods: Methylation specific PCR was used to analyze eight CIMP-specific gene promoters (CACNA1G, IGF2, NEUROG1, RUNX3, SOCS1, hMLH1, p16, CRABP1) in cfpDNA. Forty age-and-gender matched plasma samples (20 CRC and 20 controls) were evaluated. Samples were classified as methylated, unmethylated or not detected if not amplified by either primer set. Results: Methylation patterns of eight genes in 40 subjects (20 CRC and 20 controls; mean age 67 ± 12.2 years; 20 females) were examined. Five genes revealed a 100% unmethylation pattern. p16 was found to be more likely methylated in controls (11%) whereas NEUROG1 and CRABP1 were more likely to be methylated in CRC cases (6% and 33%, respectively). 53.3% of CRC and 48% controls had β1 methylated genes (OR = 1.24, 95% CI 0.34-4.46). The use of β1 methylated genes detected CRC with 40% sensitivity and 65% specificity. Discussion: We report the first evaluation of the methylation pattern of an eight-gene panel as CRC biomarkers in Hispanics. Our preliminary findings showed that genes were mostly unmethylated in cases and controls. NEUROG1 and CRAP1 were found to be most likely methylated in CRC patients, but the diagnostic accuracy of this panel was poor as a CRC screening biomarker.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2086. doi:1538-7445.AM2012-2086
Abstract 2088: Detection of PIK3CA somatic mutations in cell-free DNA of breast cancer patients by high-resolution melting curve analysis
Authors: Schiza Christina , Farkona Sofia , Chimonidou Maria , Vorkas Panos , Malamos Nikos , Georgoulias Vasilis , Lianidou Evi S.
Published: August 2012

Introduction: Somatic mutations in the gene encoding for the phosphatidyl-inositol 3-kinase (PI3K) catalytic subunit, PIK3CA, have been discovered in many different human cancers. We have recently developed a high-resolution melting curve analysis method for the detection of the three hotspot mutations of PIK3CA gene (Vorkas et al, J of Mol Diagn 2010). The aim of our study was to evaluate this method for the presence of PIK3CA mutations in cell-free DNA (cfDNA) circulating in plasma of breast cancer patients and healthy individuals. Patients and methods: cfDNA was isolated from plasma (200μL) of 30 patients with operable breast cancer, 45 patients with metastatic breast cancer and 12 healthy donors. After DNA extraction, real-time PCR was performed in triplicate for all samples in the LightCycler (Roche), in the presence of LC-Green Plus saturating dye (Idaho Technology Inc.). High-resolution melting curves were obtained with HR-1 High Resolution Melter (Idaho Technology Inc.). Results: We identified 15 cfDNA samples mutated out of 75 tested (20%). 3/75 (4%) cfDNA samples were mutated within the exon 9 amplified region, and 12/75 (16%) cfDNA samples were mutated within exon 20 (31.6%). In operable breast cancer 1/30 (3.3%) cfDNA sample was mutated in exon 9 and 3/30 (10%) cfDNA samples were mutated in exon 20, while in metastatic breast cancer 2/45 (4.4%) cfDNA samples wer mutated in exon 9 and 9/45 (20%) cfDNA samples were mutated in exon 20. PIK3CA mutations were not detected in any of the 12 cfDNA samples from healthy donors, used as negative controls. Conclusions: Using High-Resolution Melting curve Analysis (HRMA) PIK3CA gene mutations were detected in cfDNA circulating in plasma of patients with operable and metastatic breast cancer, but not in plasma of healthy individuals. The prognostic significance of this finding will be evaluated when a longer follow up period will be completed for these patients and a larger number of clinical samples will be analyzed.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2088. doi:1538-7445.AM2012-2088
Abstract 2090: Mutational analysis in circulating tumor cells (CTC):ScreenCell MB Filtration Unit and ICE COLD-PCR
Authors: Richardson Katherine A. , Wu Grant , Kuang Yanan , Wang Lilin , Legendre Benjamin , Lin Rui , Shi Yanggu , Eastlake Phil , Cayre Yvon , Janne Pasi , Distel Robert
Published: August 2012

The aim of this study was to validate the use of the ScreenCell® MB Filtration Unit for detection of mutations in Circulating Tumor Cells. This device allows physicians to easily isolate any CTCs using a simple blood draw which can be performed at a local doctor's office. This is especially important when trying to analyze CTCs in elderly patient populations and in regions where the large medical centers are far away. Once isolated, the filter containing the CTCs can be sent to a laboratory for DNA isolation and the mutation analysis. Mutational analysis using ICE COLD-PCR can then be performed without the use of expensive or specialized equipment. The mutation was characterized using standard Sanger DNA sequencing with a detection limit of 0.5 - 0.1% mutant sequence in the wild-type background. Results will be presented from a mock experiment where PC9 cells (EGFR exon 19 deletion: p.E746_A750del) or H1975 cells (EGFR exon 21: p.L858R) were spiked into blood and then these mock CTCs were collected on the ScreenCell Molecular Filter Device. Following DNA isolation, the mutations were easily detected at the lowest number of cells spiked in to the blood samples (10 cells). Additional data from CTCs collected from NSCLC patients detected the L858R point mutation, an exon 19 E746_A750del mutation, and an exon 19 E746_S752delinsV. Mutations in these same samples could not be detected in DNA sequencing following standard PCR amplification. These data show that the ScreenCell MB Filtration Unit coupled to mutational analysis using ICE COLD-PCR can be an easy, cost-effective method for collection and analysis of CTCs in patient blood samples. Moreover, the device is simple enough to be used in any doctor's office.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2090. doi:1538-7445.AM2012-2090
Abstract 2092: Integrated genetic analysis of clear cell renal cell carcionoma
Authors: Sato Yusuke , Mekawa Shigekatsu , Nagata Yasunobu , Yoshida Kenichi , Sato Aiko , Okuno Yusuke , Shiraishi Yuichi , Yoshizato Tetsuichi , Suzuki Hiromichi , Sanada Masashi , Kume Haruki , Homma Yukio , Ogawa Seishi
Published: August 2012

Renal cell carcinoma (RCC) is the most common form of adult kidney cancer and accounts for 2-3% of all adult malignancies, in which clear cell carcinoma accounts for more than 80% of the cases. As for the pathogenesis of clear cell RCC, inactivation of the VHL gene has been reported in 80% of clear cell RCC and more recently, frequent mutations of epigenetic regulators, including PBRM1, SETD2, KDM5C and UTX, have been demonstrated through high-throughput mutation studies, including PBRM1 has been demonstrated in ∼40% of the cases. Nevertheless, probably, our knowledge of the full spectrum of gene mutations in RCC is still incomplete. In this study, to obtain a better understanding of molecular pathogenesis of clear cell RCC, we performed an integrated genetic study of clear cell RCC, where a total of 93 paired specimens were analyzed by massively parallel sequencing of SureSelect (Agilent)-enriched whole exomes, SNP array-based copy number analysis (Affymetrix), as well as gene expression profiling (Agilent). In whole exome sequencing, 42 somatic mutations per sample were identified on average, which involved not only previously reported genes, but also a number of novel gene targets. Among these, mutations of genes involved in chromatin regulation or histone modification were preferentially found in advanced cases. To understand whole picture of gene mutations of epigenetic mechanism in clear cell RCC, mutation analysis of 85 genes involved in chromatin regulation or histone modification were performed in 180 cases using multiplexed barcode sequencing. A total of 201 somatic mutations were validated and 74% cases had at least one somatic mutation. PBRM1 mutations were found in 43% cases and SETD2 were mutated in 10% of cases. When comparing clinical picture with mutation status, SETD2 mutation was associated with the risk of metastasis, while PBRM1 mutations had no impact on prognosis. Our results showed that in clear cell RCC, multiple component of complexes involved in epigenetic regulation undergo gene mutations, confirming that deregulated epigenetic apparatus play important roles in pathogenesis of clear cell RCC. In this meeting, we will present the result of our integrated genetic analysis of RCC and discuss the genetic basis of RCC in terms of copy number alterations, gene mutations, as well as gene expression profiles.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2092. doi:1538-7445.AM2012-2092
Abstract 2093: Next-gen deep sequencing improves FGFR3 mutation detection in the urine of bladder cancer patients
Authors: Millholland John M. , Li Shuqiang , Fernandez Cecilia A. , Shuber Anthony P.
Published: August 2012

FGFR3 mutations have been identified in ∼60-70% of low-stage, non-invasive tumors. Our group and others have developed assays to detect FGFR3 mutations in the urine of bladder cancer patients. However, urine-based assays have been limited by the technical ability to detect rare events in a dilute medium where there is a high background of normal DNA. In these assays, FGFR3 mutations are generally found in ∼30% of the urine samples, which is < 50% concordance with the expected detection in tissue. We have now developed an ultra-deep amplicon sequencing technique that increases FGFR3 mutation detection in urine to ∼67%, near the expected detection if every mutation found in tissue could be detected in urine. Amplicons were designed against FGFR3 exons 7, 10, and 15 using PCR primers containing the adapter sequences for unidirectional sequencing. Taqman probes were used to determine if sufficient DNA was present in each sample. Primary amplification was performed from DNA isolated from 4 ml of urine. The resulting PCR products were used as template for emulsion PCR and these were then sequenced using the Roche 454 GS Junior. Samples were analyzed for total DNA reads per sample and number of mutant sequencing reads to determine percent mutation. Urine samples from 29 patients with stage Ta bladder cancer were analyzed by both our previously described qPCR method and the new ultra-deep sequencing approach. Of the 29 samples, 2 did not have sufficient DNA for analysis by sequencing. Using ultra-deep amplicon sequencing, 18 out of 27 (66.7%) were positive for FGFR3 mutations, while only 3 out of 27 (11.1%) were positive for mutations by qPCR. The urine samples from the 15 newly identified mutations using deep sequencing contained FGFR3 mutations as low as 0.05%. Using matched tissue and urine samples, we demonstrate that the sensitivity in urine approximates the FGFR3 mutations observed in tissue. We have developed a highly sensitive non-invasive urine based assay that can detect FGFR3 mutant DNA when present at < 1% of the sample and that achieves > 90% concordance with tumor tissues. To our knowledge, this is the first practical application of next generation sequencing technology for diagnostic use.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2093. doi:1538-7445.AM2012-2093
Abstract 2095: A virtual pyrogram generator to resolve ambiguity of pyrosequencing results
Authors: Chen Guoli , Olson Matthew T. , O'Neill Alan , Norris-Kirby Alexis , Beierl Katie , Harada Shuko , Debeljak Marija , Rivera-Roman Keila , Finley Samantha , Stafford Amanda , Gocke Christopher D. , Lin Ming-Tseh , Eshleman James R.
Published: August 2012

We report a freely available software program, Pyromaker, which generates simulated pyrosequencing traces based on user inputs and aids in analysis of complex pyrosequencing results. To achieve this goal, simulated pyrograms are generated based on the hypothesized mutations and matched with the actual pyrogram. We validated the software testing common KRAS gene mutations and several mutations in the BRAF, GNAS and, p53 genes. We demonstrate that all eighteen possible single base mutations within codons 12 and 13 of KRAS gene generate unique simulated pyrosequencing traces and highlight the distinctions between them. We further verify that all reported codon 12 and 13 complex mutations produce unique pyrograms. However, some complex pyrograms are indistinguishable from single base mutations, using a standard dispensation order. Pyromaker was used in two strategies, one in which hypothesis-based simulated pyrograms were pattern-matched with the actual pyrograms. In a second mode, with only the pyrogram, Pyromaker was used to identify the underlying mutation by iteratively reconstructing the mutant pyrogram. Either strategy was able to successfully identify the complex mutations, which were confirmed by cloning and subsequent sequencing. With two examples of KRAS codon 12 mutations, (GGTαTTT, G12F and GGTαGAG, G12E), we report optimal combinations of the five analysis approaches that permit unambiguous mutation identification. The most efficient one is pyrosequencing with Pyromaker.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2095. doi:1538-7445.AM2012-2095
Abstract 2097: In situ mutation detection in cancer tissue sections for research and diagnostics in clinical oncology
Authors: Grundberg Ida , Kiflemariam Sara , Imgenberg-Kreuz Juliana , Edlund Karolina , Micke Patrick , Sundström Magnus , Botling Johan , Nilsson Mats
Published: August 2012

Activating oncogenic mutations are targets for a new generation of cancer drugs. Current mutation assays for clinical diagnostics are based on DNA-extracts from tissue sections that often contain morphologically heterogeneous neoplastic regions with variable mixtures of normal stromal and inflammatory cells. We have developed an RNA-based in situ mutation assay that can target oncogenic mutations in a multiplex fashion on tissue slides by the use of multiple mutation-specific padlock probes and rolling-circle amplification. For EGFR therapy prediction, we demonstrate reliable in situ detection of KRAS mutations in codon 12 and 13 in colon and lung cancer - on cytologic tumor imprints as well as on fresh frozen and formalin-fixed, paraffin-embedded tissue sections. High throughput screening of KRAS mutation status was successfully performed on a tissue microarray. Moreover, we show how the pattern of expressed mutated and wild-type alleles can be studied in situ from tumors with complex combinations of mutated EGFR, KRAS and TP53. This in situ method holds great promise as a tool to investigate the role of somatic mutations during tumor progression and for prediction of response to targeted therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2097. doi:1538-7445.AM2012-2097
Abstract 21: Gastrokine 1 functions as a tumor suppressor by regulating inflammatory mediators
Authors: Yoon Junghwan , Choi Yoo Jin , Han Lin , Lee Suk Woo , Park Won Sang
Published: August 2012

Gastrokine 1 (GKN1) plays an important role in the gastric mucosal defense mechanisms and also acts as a functional gastric tumor suppressor. The specific aim of this study was to determine the inflammatory mediator regulation underlying GKN1 activity in the tumor development/progression of gastric cancer. We examined the effect of GKN1 on expression of inflammatory mediators including NF-κB, COX-2, and cytokines in GKN1-transfected and recombinant GKN1-treated AGS gastric cancer cells by western blot and real time RT-PCR. Lymphocyte migration and cell viability were also analyzed in AGS cells after being treated with GKN1 and inflammatory mediators by transwell chemotaxis and MTT assay, respectively. In GKN1-transfected AGS cells, we observed inactivation and reduced expression of NF-κB and COX-2. Ectopic GKN1 expression induced the production of inflammatory mediators including IL-8 and -17A, but decreased IL-6 and IL-10 expressions. GKN1 also functions as a chemoattractant in migration of Jurkat T cells and peripheral B lymphocytes in transwell assay. In addition, GKN1 significantly inhibited cell viability, whereas treatment of IL-10, IL-17 and TNF-α in AGS cells did not show significant difference in cell viability. These data suggest that the GKN1 gene may play an important role in the regulation of inflammatory mediators in the tumor development/progression of gastric cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 21. doi:1538-7445.AM2012-21
Abstract 2100: Cancer biomarker research using castPCR technology
Authors: Bao Yun , Ching Bonnie , Mouanoutoua Mokang , Yu Wang , Keys David , Desai Sejal , Stevens Junko
Published: August 2012

Cancer Biomarkers have applications in the diagnosis, staging, prognosis and monitoring of disease progression, as well as in the predication and monitoring of drug response. Profiling and validation research tools are needed that exhibit the combined features of high sensitivity and high specificity for cancers. However, the sensitivity of molecular methods such as DNA sequencing and conventional genotyping in tumor samples is limited, typically ranging from 5-20%. We have recently developed TaqMan® Mutation Detection Assays using our competitive allele specific TaqMan® PCR (castPCR) technology for cancer biomarker research. castPCR assays were tested with >300 tumor research samples (either fresh/frozen or formalin-fixed, paraffin-embedded samples) and cell lines to assess mutation status. The results showed that castPCR technology can robustly detect mutations as low as 0.1% and has >99% concordance to other technologies including PCR-based technology and sequencing. In this study, a large panel of castPCR assays for AKT1, APC, BRAF, CTNNB1, HRAS, KRAS, NRAS, PIK3CA, PTEN and TP53 genes were used for investigating somatic mutations in breast tumor research samples. Initially, 4 model FFPE cell lines were used to validate the assays. Mutant DNAs were titrated in the wild type DNAs from 50% to 0.1%. Mutations were identified down to 0.1% titration with high reproducibility. No false positives were found in non-tumor samples. The results obtained by castPCR assays for 20 breast tumor samples were concordant to those reported by other methods. Our data showed that castPCR technology provides an excellent tool for identifying cancer biomarkers or confirming potential cancer markers such as those obtained by next-generation sequencing and other technologies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2100. doi:1538-7445.AM2012-2100
Abstract 2104: Molecular alterations in JAK1 and JAK2 genes in head and neck squamous cell carcinoma
Authors: Carvalho Thaís G. , Carvalho André Lopes , Vettore André Luiz
Published: August 2012

Among the ten most common cancers are the head and neck tumors, with more than 600,000 new cases diagnosed annually worldwide, the majority (90%) represented by the head and neck squamous cell carcinomas (HNSCC). Somatic mutations occur in the genomes of all dividing cells, both normal and neoplastic. The JAK-STAT signaling pathway controls important cell events such as survival, proliferation and differentiation. The presence of mutations in the phosphorylation dominion of protein kinases as JAK family have been linked to development of different kinds of tumors such as hematological, breast, lung and gastric cancers. Furthermore, inhibitors of protein kinases (Gefitinib and Erlotinib) have demonstrated a remarkable efficacy in treatment of some cancers. To date, we do not succeeded in finding reports linking mutations in members of the JAK-STAT pathway and HNSCC. Therefore it is necessary to plunge into molecular research that could identify failures in the cellular machinery with consequent discovery of potential molecular targets and drug direct delivered to them. The present work aims to evaluate the presence of mutations in the JAK-STAT signaling pathway members (JAK1 and JAK2) and verify the expression level of these genes in HNSCC samples. For the mutation detection, primers were designed to cDNA molecules and allowed the amplification of contiguous 700-bp fragments that cover the entire coding region of each gene. The JAK1 and JAK2 genes were investigated in 20 HNSCC samples and no mutation was detected. Polymorphisms were found in JAK1 at codons 193 (20%), 659 (5%), 683 (25%), 699 (10%), 733 (25%) and 1032 (20%) and in JAK2 codons 163 (60%), 830 (85%) and 1063 (5%). In addition we evaluated the expression level of these genes in 87 HNSCC samples and both were found downreguted in the majority of HNSCC samples (JAK1 in 82% and JAK2 in 81%).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2104. doi:1538-7445.AM2012-2104
Abstract 2109: PIK3CA mutations in hepatocellular carcinoma in Korea
Authors: Kim Heesue
Published: August 2012

PI3K/AKT pathway plays a pivotal role in HCC. Mutant PIK3CA, encoding the p110a catalytic subunit, stimulate the AKT pathway and promotes cell growth in various cancers. PIK3CA mutations have been researched in many cancer types and reported low frequency of PIK3CA mutation in HCC except Korea’ case. To investigated the frequency of PIK3CA mutations in Korean patients with HCC. We sequenced the exon2, 4, 5, 7, 8, 9, 10, 20 and 21 of PIK3CA in 268 HCC samples. In these experiments, the mutation were not detected in exon4, 7, 9, 20 and detected 1 of 268 at unknown SNP (G278A) in exon2 and at unknown SNP (C848G) in exon5, 1 of 265 at unknown SNP (C1306A) and 2 of 265 at unknown SNP (C1373A) in exon8, each of 262 at unknown SNP (T2970A) and SNP (G3026A) in exon21. However, exon10 were detected 1 of 266 at unknown SNP (C1629T), 1 of 268 at G1635T (E545D) and 266 of 266 at A1634C (E545A) in 268 HCC samples. Additional experiments through normal tissue and cloning experiments realized double peak at A1634C of exon10 is not mutation and maybe arose as a result gene duplication on 22q11 partial region because exon10 primer have higher efficiency. Our study suggests no clinical characteristic relationship between HCC and mutated of PIK3CA. In conclusion, our results with using large sample size suggest the mutations in PIK3CA gene of Korean patients’ frequency is low as like other countries have been previously reported.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2109. doi:1538-7445.AM2012-2109
Abstract 2112: CA-microsatellite instability as a factor of branch migration impeding in Holliday junctions
Authors: Karpechenko Natalia Y. , Dolinnaya Nina G. , Popenko Vladimir I. , Yakubovskaya Marianna G. , Gasanova Victoria K.
Published: August 2012

The efficient and accurate repair of DNA double-strand breaks is critical for cell survival. HR provides an “error-free” way for the break repair. There is no loss of genetic information when homolog or sister chromatid are used for repair. They are mainly involved in HR and the frequency of non-sister chromatid interaction is greatly lower. However, when recombination occurs between non-sister chromatids, in the half of these cases it leads to loss of heterozygosity (LOH). This genetic alteration represents an important step in multistage carcinogenesis. Recently, the impact of LOH caused by HR in carcinogenesis of inherited cancer has been demonstrated to exceed impacts of point mutagenesis and non-homologous end joining. HR can be modulated by a variety of factors, and deep knowledge of the molecular background of this process is essential to predict and control corresponding genetic rearrangements. Some microsatellites represent “hot-spots” of recombination. In particular, one of the most abundant microsatellites, CA-repeats, enhances recombination between DNA molecules as observed in bacteria and eukaryotes. Previously we described spontaneous DNA-DNA interaction of homologous duplexes via HJ formation, and based on this investigation we elaborated the model system for HJs. We formed several types of HJs using: (1) random fragments restricted from pUC19; (2) fragments of the same sequence but amplified via PCR; (3) restricted fragments of the pE10 plasmid, harboring (CA/TG)31-repeat; (4) corresponding PCR products of the pE10 plasmid. Using different DNA polymerases we demonstrated that PCR amplification caused repetitive unit deletions/insertions in the (CA/TG)31-tract. It leaded to formation in PCR product of the fragment with (CA/TG)31-repeat of heteroduplexes and homoduplexes, differing mainly by one repetitive unit that is in accordance with previously published data. Corresponding restricted fragments with (CA/TG)31-repeats were significantly more stable. Using electron microscopy we showed that the majority of HJs formed by the PCR fragments with (CA/GT)31-repeats had their cross-points at the repetitive region, while the cross points of HJs formed by restricted fragments with (CA/TG)31-repeats and by entirely random sequences were mainly localized at the ends of the fragments. Demonstrated influence of (CA/TG)n-repeat instability on the branch migration is discussed in respect of the role of CA-microsatellites in homologous recombination.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2112. doi:1538-7445.AM2012-2112
Abstract 2119: DDB2 and XPC influence ATR and ATM recruitment and regulate DNA damage response upon UV irradiation
Authors: Ray Alo , Milum Keisha , Battu Aruna , Wani Altaf
Published: August 2012

DNA damage response (DDR) pathways control the activation of DNA repair mechanisms, the activation and eventual release of cell cycle checkpoints to prevent replication of damaged DNA, and apoptosis as a last resort. These processes suggest the coordination of numerous proteins required to preserve genomic integrity. ATR and ATM kinases are central to checkpoint activation in response to DNA damage and replication stress. Activated ATR and ATM phosphorylate several proteins involved in DNA repair and cell cycle arrest. The nature of the signal, which activates ATR and ATM in response to UV damage, is not clear. Here, we showed that DDB2 and XPC, two early damage recognition factors, promote ATR and ATM recruitment and phosphorylation. ATR and ATM localize to the damage site and physically interact with XPC and DDB2. ATR and ATM recruitment and their phosphorylation are affected in cells defective in DDB2 and XPC function. In contrast, ATR- and ATM-deficiency do not regulate DDB2 and XPC recruitment to the damage site. Consequently, phosphorylation of ATR and ATM substrates Chk1, Chk2, H2AX, and BRCA1 was significantly reduced or abrogated in mutated cells, indicating that defective DDB2 and XPC function impair checkpoint signal transduction cascade in response to UV damage. Furthermore, DDB2 and XPC also regulate BRCA1 and Rad51 recruitment to the damage site, implicating their role in homologous recombination-mediated DNA repair pathway. Collectively, these data showed that ATR and ATM are present in the preincision complex with XPC, and influence DNA repair and checkpoint signaling pathways. In support of this, we revealed that depletion of ATR and ATM influence NER efficiency. These results are consistent with a model in which DDB2 and XPC cross-talk with ATR and ATM during maintenance of genomic integrity.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2119. doi:1538-7445.AM2012-2119
Abstract 2124: Non-homologues end joining mediates fatty acid synthase(FASN)-associated resistance to DNA-damaging chemotherapeutics
Authors: Wu Xi , Serrano Moises , Zou Yue , Zhang Jian-Ting
Published: August 2012

Fatty acid synthase (FASN) is the sole mammalian enzyme capable of de novo lipid synthesis. Except in lipogenic tissues, FASN expression and activity is largely repressed by dietary fat. In contrast, abnormally elevated FASN expression and activity have been found in many types of human cancers. In vivo and in vitro studies showed that inhibition of FASN led to selective cytostatic and cytotoxic effect in cancer tissues and cells, with minimal adverse effect towards normal tissues. Clearly, FASN is no longer just an anabolic protein, but also provides growth and survival advantages to tumor cells. It has also been found that ectopic overexpression of FASN causes resistance to DNA-damaging drugs including doxorubicin, mitoxantrone, and etoposide as well as to ionizing radiation (IR), but not to non-genotoxic drugs such as vinblastine and paclitaxel. This finding suggests that FASN overexpression may regulate the DNA damage/repair pathway to help breast cancer cells survive DNA-damaging treatments. In this study, we tested this hypothesis. We found that FASN expression had profound effect on the rate of γ-H2AX clearance after exposure to IR or doxorubucin. While knocking down FASN delayed γ-H2AX clearance, FASN overexpression increased γ-H2AX clearance. Using neutral comet assay, we also found that double strand DNA break (DSB) induced by doxorubicin was increased by knocking down FASN expression but attenuated by increasing FASN expression. Furthermore, using a host-cell reactivation based assay, we showed that FASN overexpression significantly increased Non-homologous End Joining DNA repair activity. Together, we conclude that FASN likely plays an important role in regulating DSB repair in cancer cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2124. doi:1538-7445.AM2012-2124
Abstract 2138: Tumorigenic and migratory potential of Ral GTPases in non-small cell lung cancers
Authors: Guin Sunny , Ru Yuanbin , Mishra Rangnath , Vasu Vihas T. , Owens Charles , Finigan James H. , Kern Jeffrey A. , Theodorescu Dan
Published: August 2012

The Ral GTPase family, consisting of the Ral A and B isoforms contributes to tumor formation, invasion and metastasis, however their role in non-small cell lung cancer (NSCLC) is yet to be validated. Recently, we investigated the role of Ral GTPases in NSCLC by in vitro expression, colony formation and migration assay, in vivo tumor formation experiments, and analysis of microarray data from NSCLC patients. In vitro, Ral A and Ral B was expressed in all NSCLC cell lines studied (14) to varying levels. Increased expression of Ral A or Ral B was more frequent in Ras mutant cell lines (Ral A elevated in 2/8 cell lines with mutant KRas vs. 0/6 cell lines with WT KRas, Ral B elevated in 5/8 cell lines with mutant KRas vs. 2/6 WT KRas). Soft agar experiments with transient knockdown of Ral A, Ral B and Ral A+B in these cell lines demonstrated significant decrease (p<0.05) in colony formation. Dual knockdown of Ral A and B had a 2 to 3 fold decrease (p<0.05) in NSCLC cell motility in wound healing assays. In vivo, knockdown of Ral GTPases resulted in reduced subcutaneous tumor growth (p<0.05) in mice injected with the knockdown cells. Microarray data from NSCLC patients was analyzed to study the correlation between Ral A and B expression with overall patient survival. High Ral A expression was associated with poor patient survival in these microarray datasets (p<0.05). The above observations demonstrate that Ral GTPases play a role in lung cancer tumorigenesis and migration. In the Ras signaling cascade, Ral GTPases are downstream of Ras along with PI3K and MAPK signaling pathways. KRas mutations in NSCLC carry a poor patient prognosis. Based on our observations, we posit that Ral GTPases can be a valid target for drug development against KRas mutated lung cancers along with PI3K and MEK inhibitors.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2138. doi:1538-7445.AM2012-2138
Abstract 2144: RasGRP1 induces autophagy in primary human epidermal keratinocytes resembling fail-safe mechanisms triggered by oncogenic Ras
Authors: Fonseca Lauren L. , Lorenzo Patricia S.
Published: August 2012

RasGRP1 is a guanyl nucleotide exchange factor for Ras that is abundantly expressed in lymphocytes, regulating their maturation and activation. Its aberrant expression has been associated to transformation in various cell types, although the role in human cancer remains largely unexplored. Our lab has previously demonstrated expression of RasGRP1 in epidermal keratinocytes, followed by studies that showed a critical role in skin carcinogenesis in mouse models of cutaneous squamous cell carcinoma through its ability to increase the dosage of active Ras. To explore whether RasGRP1 could contribute to transformation of human keratinocytes, we utilized primary epidermal keratinocytes (HEK) isolated from neonatal foreskin. In a model of tissue reconstruct, or 3-D culture, HEK manipulated to overexpress RasGRP1 (RasGRP1+) failed at generating a stratified epithelium. In fact, the RasGRP1 + cells underwent changes resembling autophagy and cell death in culture, with vacuolated cytoplasm as well as rounded bodies. Autophagy was confirmed at the molecular level by measuring increases in the LC3-II species by Western blot and the formation of a LC3 punctate pattern by immunofluorescence. Surprisingly, there was no caspase-3 activation in the RasGRP1+ HEK despite the morphological signs of apoptosis, although we noticed an increase in the expression of the BH3-only protein NOXA as early as 48 hours post RasGRP1 overexpression. Since oncogenic signals are known to induce fail-safe mechanisms that restrict cell expansion, we presumed that the changes observed in the HEK cell overexpressing RasGRP1 were due to Ras hyperactivation (oncogenic)-induced cell death. As predicted, levels of active Ras-GTP were 3 times higher in the RasGRP1+ cells compared to the wild type HEK, with a preponderance of the H-Ras isoform being activated. In line with these observations, enforced expression of an oncogenic version of the H-Ras isoform (H-RasQ61L) also resulted in cytoplasmatic vacuolization of HEK and increases in LC3-II levels. In contrast to the RasGRP1+ cells, H-RasQ61L caused activation of caspase-3 while increases in NOXA were delayed compared to those caused by RasGRP1. While RasGRP1 and oncogenic H-RasQ61L differ in some aspect of their downstream molecular pathways, probably as a result of the magnitude of Ras activation, the present findings reveal a novel RasGRP1-Ras axis that can lead to oncogenic signals in human keratinocytes. This warrants further investigation into the potential participation of RasGRP1 in keratinocyte transformation, particularly contributing to cutaneous squamous cell carcinoma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2144. doi:1538-7445.AM2012-2144