bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2092: Control of histone 3 lysine 27 methylation by microRNA 520b in medulloblastoma
Authors: Alimova Irina , Venkataraman Sujatha , Patel Purvi , Harris Peter , Vibhakar Rajeev
Published: February 2011

MicroRNAs are small non-coding RNAs with gene expression regulation at posttranscriptional and transcriptional level. MicroRNAs are widely known to play a key role in carcinogenesis. We investigated the role of microRNAs in medulloblastoma, the most common malignant brain tumor of childhood. We found that microRNA 520b is over expressed in medulloblastoma compared to normal cerebellum. We next examined the biological significance of microRNA 520b. Transfection of medulloblastoma cells (DAOY and ONS76) with microRNA 520b significantly increased cell proliferation and enhanced anchorage-independent growth in soft agar compared to parental cells. Conversely, over expression of anti-microRNA 520b in medulloblastoma cells decreases cell proliferation and colony formation in soft agar. Bio-informatics analysis revealed several chromatin-remodeling proteins as potential targets of microRNA 520b. One of these, SUV39H1 is a histone methyl transferase that methylates histone 3 lysine 27 and suppresses activation of associated promoters. It is known that histone methylation is a major determinant for the formation of active and inactive regions of the genome and is crucial for the proper programming of the genome during development. We found that inhibition of microRNA 520b results in increased expression of SUV39H1 and subsequent methylation of Histone 3 Lys27 in medulloblastoma cells. SUV39H1 is known to be a tumor suppressor and is mutated in a subset of medulloblastoma. These data indicate that microRNA 520b can function as an oncogene in medulloblastoma by regulating histone modifications.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2092.
Abstract 2097: microRNA profile and identification of miR-29 as a prognostic marker and pathogenetic factor by targeting CDK6 in mantle cell lymphoma
Authors: Zhao Jian-jun , Lin Jianhong , Lwin Tint , Yang Hua , Guo Jianping , Kong William , Dessureault Sophie , Moscinski Lynn , Rezania Dorna , Dalton William S. , Sotomayor Eduardo , Tao Jianguo , Cheng Jin Q.
Published: February 2011

ABSTRACT: Mantle cell lymphoma (MCL) is one of the most aggressive B-cell lymphomas. While a number of protein-coding genes are altered, expression signature and importance of microRNA (miRNA) have not been well documented in this malignancy. Here, we performed miRNA expression profile in 30 patients with MCL using a platform containing 515 human miRNAs. Eighteen miRNAs were downregulated and 21 were upregulated in MCL when compared with normal B lymphocytes. The most frequently altered miRNAs are decrease of miR-29a/b/c, miR-142-3p/5p and miR-150 and increase of miR-124a and miR-155. Notably, expression levels of miR-29 family are associated with prognosis. The patients with significant downregulated miR-29 had short survival as compared to those who express relative high level of miR-29. Prognostic value of miR-29 is comparable with MIPI (MCL International Prognostic Index). Furthermore, we demonstrate miR-29 inhibition of CDK6 protein and mRNA levels by direct binding to 3′UTR. Inverse correlation between miR-29 and CDK6 was observed in MCL. Since cyclin D1 overexpression is a primary event and exerts its function through activation of CDK4/6, our results in primary MCL cells indicates that downregulation of miR-29 could cooperate with cyclin D1 in MCL pathogenesis. Thus, our findings provide not only miRNA expression signature but also a novel prognostic marker and pathogenetic factor for this malignancy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2097.
Abstract 2098: The receptor tyrosine kinase AXL is a target for the human miR-34a microRNA
Authors: Mackiewicz Mark , Huppi Konrad , Cornelison Robert , Caplen Natasha J.
Published: February 2011

Determining the protein targets of microRNAs (miRNAs) will be critical to understanding the role these non-coding RNAs play in regulating gene function and what impact they have in cancer biology. In this study, we show by a combination of microarray, Northern, and PCR-based analyses that the human miR-34a miRNA is significantly under-expressed in basal-like breast cancer cell lines exhibiting triple receptor negative characteristics, as compared to a non-tumorigenic mammary epithelial cell line control. Introduction of a synthetic mimic of hsa-miR-34a into MDA-MB-231 cells resulted in a substantial alteration in both the mRNA and protein levels of the receptor tyrosine kinase AXL, 48 hours post-transfection. Furthermore, the transfection of the hsa-miR-34a mimic decreased the migratory potential of these cells in a manner consistent with the function of AXL as a promoter of cell proliferation, invasion, and metastasis in a number of cancer cell types including breast cancer. This observed phenotypic effect supports the hypothesis that miR-34a functions as a tumor suppressor, with the direct translational repression of AXL representing just one of many possible targets that this miRNA regulates.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2098.
Abstract 210: Glionitrin A causes apoptosis and DNA damage-induced S phase arrest in human prostate cancer cells
Authors: Kim Young-Joo , Kwon Hak Cheol , Yoo Ji-Hye , Yang Hyun Ok
Published: February 2011

In recent study, we have isolated a new diketopiperazine disulfide, Glionitrin A (GN A), from co-culture broth of a Aspergillus fungal strain KMC901 and a Sphingomonas bacterial strain KMK001, and demonstrated anti-microbial and anti-tumor effects; however, its mechanisms of action are not understood yet. Here, we investigated anti-tumor activity of GN A and its underlying molecular mechanism in human prostate cancer DU145 cells in vitro and in vivo. GN A showed significant cytotoxicity (IC50, 1.5 μM) against DU145 cells through a caspase-8-dependent apoptotic pathway. GN A treatment (0.75 ∼ 3 μM) activated caspase-8, 9 and 3, and also increased formation of truncated BID and expression of BAX protein in a dose-dependent manner. Furthermore, GN A elevated phospho-H2A.X (Ser139), which is known to be phosphorylated by ATM/ATR in response to DNA damage, and activated S phase checkpoint associated with ATM-Chk1/2-Cdc25A pathway. In vivo study performed in nude mice bearing xenografts of DU145 (1×107 cells/mouse, s.c.), GN A dramatically reduced the tumor volume by an average of 38.2% (5 mg/kg, p.o.) and 71.3% (10 mg/kg, p.o.) at the 27-day time point, respectively. These observations may provide aid in better understanding of the mechanism underlying the anti-tumor activity of GN A, which has the potential to be developed into an anti-prostate cancer agent.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 210.
Abstract 2106: Regulation of Rb1 and caspase 3/7 by miR-17-5p, miR-132/-212, and miR-337-3p: Oncogenic role of miRNAs in pancreatic cancer
Authors: Park Jong Kook , Jiang Jinmai , Gusev Yuriy , Lerner Megan R. , Brackett Daniel J. , Schmittgen Thomas D.
Published: February 2011

Pancreatic ductal adenocarcinoma (PDAC) continues to be one of the most lethal forms of cancer. Reasons for the high mortality associated with this disease include a lack of effective treatments and poor early detection methods. Enhanced understanding of the pathways that are deregulated in PDAC could lead to improved therapies and diagnostics. We and others have reported a microRNA expression signature that is associated with PDAC. The purpose of this study was to identify target genes and pathways that are regulated by miRNAs with altered expression in PDAC. We found several miRNAs including miR-17-5p, −132/-212, and −337-3p that have increased expression in clinical specimens of PDAC compared to normal and adjacent benign pancreas. miR-132 and −212 are located within 472 bp of each other on chromosome 17, have the identical seed sequence and should therefore regulate the identical targets. Through a luciferase reporter assay and western blotting, we found that miR-132/-212 directly regulate the expression of the tumor suppressor Rb1 in the pancreatic tumor cell lines HS766T and PANC-1. Over expression of these miRNAs (miR-132/-212) either through stable expression or pre-miR oligos increased the proliferation rate of HS766T and PANC-1 cells. Expression of 18 genes that are transcriptionally regulated by the E2F including Cyclin A, Cyclin B, PCNA, MCM2, MKI67 and others was increased following miR-132/-212 over expression. Additional genes that are predicted to be targeted by these miRNAs include procaspase 3 (miR-337-3P) and procaspase 7 (miR-132/-212 and miR-17-5p). These caspases are essential in executing programmed cell death in tumors. Western blotting and luciferase reporter assays determined that procaspase 3 and procaspase 7 are regulated by miR-337-3P and miR-132/-212 & miR-17-5p, respectively. Our data strongly suggest that over expression of miR-132/-212 contributes to the enhanced proliferation that is observed in pancreatic cancer. Furthermore, this study is the first to discover that main effector caspases, caspase 3 and 7, are post-transcriptionally regulated by miRNAs. Our findings suggest that resistance to chemotherapeutics in pancreatic tumor tissues may be linked to reduced expression of these procaspases.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2106.
Abstract 2110: Comparing detection of EGFR mutations in lung cancer patients
Authors: Ikeda Takaya , Nakatomi Katsumi , Yamaguchi Hiroyuki , Nakamura Yoichi , Ogawara Taiki , Senju Hiroaki , Tomonaga Nanae , Doi Seiji , Iida Tetsuya , Igawa Tsukasa , Nagayasu Takeshi , Tsukamoto Kazuhiro , Kohno Shigeru
Published: February 2011

INTRODUCTION:

Epidermal growth factor receptor (EGFR) mutations are a strong determinant factor of response to EGFR tyrosine kinase inhibitors in non-small cell lung cancer (NSCLC). Now some methods have been used to detect EGFR mutations and they have approximately the same sensitivity and specificity. In a case, we experienced unmatched results from two methods of detecting EGFR mutations. In this study, we compared data from three methods of detecting mutations.

METHODS:

We obtained 30 samples of operation, transbronchial lung biopsy, bronchoscopic brushing, pleural effusion and lymph nodes from NSCLC patients. EGFR mutation status was determined by two or three methods (mutant-enriched PCR method, PNA-LNA PCR Clamp method and PCR invader method).

RESULTS:method (n=31)mutation positive2 method (n=20)m.e./Clamp (n=10)4/4 m.e./invader (n=2)1/0 Clamp/invader (n=8)4/33 method (n=10)m.e./Clamp/invader (n=10)3/2/3m.e.: mutant-enriched PCR, Clamp: PNA-LNA PCR Clamp, invader: PCR invader

EGFR mutations were detected in 12 samples. Mutation statuses of two or three methods were matched in 9 samples, but 3 samples were unmatched.

CONCLUSIONS:

EGFR mutation statuses were unmatched in same samples. EGFR gene status is essential information for NSCLC patients. We need to take false negative into consideration in the clinical setting and need father deliberation about detecting methods of EGFR mutations.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2110.
Abstract 2113: A wild-type-blocking reference sequence enhances COLD-PCR and enables fast amplification and high enrichment of all types of low-prevalence unknown mutations
Authors: Milbury Coren A. , Li Jin , Makrigiorgos G. Mike
Published: February 2011

Background: Molecular profiling of somatic mutations in cancer often requires the identification of low-prevalence DNA mutations in an excess of wild-type (WT) DNA; however, the method selectivity and sensitivity are often limiting factors. COLD-PCR (CO-amplification at Lower Denaturation Temperature) resolves several limitations in low-level mutation detection by using critical denaturation temperatures to enrich mutant-containing amplicons during PCR. However, certain mutation types are not enriched by some COLD-PCR formats. We report a novel enhancement in COLD-PCR that enables an Improved and Complete Enrichment (ice-COLD-PCR) for all mutation types.

Methods: A Reference Sequence (RS) that preferably forms double-stranded structures with WT sequences, but not with mutant sequences, was added to COLD-PCR. A RS was designed to bind to one WT-sequence strand, avoid primer binding and prevent polymerase extension. To validate the use of a RS in COLD-PCR, we evaluated segments of TP53 exon 8. Serial dilutions of mutant cell-line DNA, or human lung tumor DNA, in WT DNA were analyzed. Mutations that increase, decrease, or retain the amplicon melting temperature were tested. Following conventional-PCR, COLD-PCR (full or fast COLD-PCR formats), and ice-COLD-PCR methods amplicons were sequenced and the degree of mutation enrichment was compared. Several clinical lung adenocarcinoma samples with known low-level mutations were also tested with ice-COLD-PCR.

Results: ice-COLD-PCR yielded ∼13-fold enrichment for Tm-increasing and Tm-equivalent mutations, and ∼15-fold enrichment for Tm-reducing mutations. In contrast, Full-COLD-PCR demonstrated ∼5-fold enrichment for all mutations. Further, fast-COLD-PCR, which can only enrich Tm-reducing mutations, exhibited ∼17-fold enrichment for these types of mutations, while the Tm-increasing and Tm-equivalent mutations remained undetectable. Regardless of mutant type and position, after ice-COLD-PCR amplification, all mutation types are strongly enriched and can be reliably sequenced down to a level of 1-3%. Ice-COLD-PCR duration is ∼1hour, compared to several hours for full-COLD-PCR.

Conclusions: The inclusion of appropriately designed RS within COLD-PCR selectively inhibits WT amplification throughout PCR, while preferentially enriching mutants and reducing time-intensive hybridization times. Ice-COLD-PCR combines high sensitivity, speed, and low-cost, and facilitates direct sequencing for all types of unknown low-prevalence mutations in clinical cancer samples.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2113.
Abstract 2118: Rare allele enrichment by ARMS combined with probe base genotyping techniques
Authors: Zhou Luming , Smith Denice , Wittwer Carl
Published: February 2011

Mutation detection plays a key role in several medical areas for diagnosis, treatment and prognosis. Additionally, the ability to detect low level mutations among mostly wild-type DNA is critical to areas such as cancer detection, prenatal testing and infectious diseases. For example, low-level (<10%) cancer mutations can not be sequenced by standard PCR or genotyping techniques. Many moderate- to high-selectivity PCR methods have been developed over the past two decades to enrich minority alleles for known mutations. One of the most widely used approaches is the amplification refractory mutation system (ARMS) which relies on the use of 3’ end terminal nucleotide mismatch to enhance the minor allele. The mutation detection sensitivity ranges from 1:10 to 1:1000. Probe based genotyping techniques such as fluorescent labeled probes, unlabeled probes and Snapback primers depend on short oligonucleotides that hybridize to the template for genotyping. We have combine allele enrichment by ARMS and probe based genotyping techniques to enhance allele discrimination. We designed a 3’ end blocked oligonucleotides to form a perfect match with the wild- type allele (mismatched to other alleles). Therefore, the probe blocks the primer from annealing to the wild-type template, while simultaneously permitting the primer to anneal to, and amplify, the mutant template. Wild-type amplification is completely blocked, and the mutation is enriched. The sensitivity of unlabeled probe-ARMS mutation enrichment detection is 1:30,000. Combining ARMS enrichment of minority alleles and snapback primers has the additional benefit that only two PCR primers are required, with the addition of a short tail of nucleotides on one primer that results in an intra-molecular hybridization probe. The sensitivity of snapback-ARMS mutation enrichment detection is 1:60,000. Fourty-four patients with paired thyroid biopsies and needle samples and 3 additional patients with only needle samples were tested for the BRAF mutation p.V600E (c.1799 T>A) by dual hybridization probes. A blinded test using unlabeled probe-ARMS and Snapback-ARMS enrichment techniques were compared to the dual hybridization probe method. All samples were concordant (47 positive and 42 negative) except 4 samples were positive using unlabeled probe-ARMS and Snapback-ARMS but were negative using hybridization probes. Minority allele enrichment using ARMS combined with probe base genotyping methods increases the mutation detection sensitivity 100 times or more. ARMS combined with an unlabeled probe or a snapback primer is an inexpensive and rapid enrichment technique. No separation, purification or addition steps that increase risk of contamination are necessary.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2118.
Abstract 2121: High-resolution melting for detection of KRAS and EGFR mutations in tumors of the alimentary system
Authors: Li Mei , Liu Lina , Zhou Luming , Liu Zhimin , Feng Lu , Zhang Qiuping , Wang Zhaohui , Lu Shen
Published: February 2011

The EGFR/Ras/Raf/MAPK pathway is one of the most important signal transduction pathways in cells. Dysregulation of this pathway is a common event in many cancers, including cancers of the alimentary system. EGFR and KRAS are two common genes in which mutations caused abnormal activation of the pathway. EGFR mutations are closely associated with a favorable response to treatment with tyrosine kinase inhibitors. KRAS mutations are associated with unresponsiveness to EGFR inhibitors. EGFR and KRAS mutation detection is important for carcinogenesis research and clinical therapy. Tumor tissue samples included 13 esophagus cancer, 52 gastric cancer and 67 colon cancer from a Chinese population. These were obtained from Dalian Medical University collection. High resolution melting (on a LightScanner™) was used to scan the mutations of KRAS exon 2, EGFR exon 19 and exon 21. All mutations detected by HRM were sequenced to identify the genotype. We found KRAS mutations in 5 (9.6%) patients with gastric cancer (2 with G12D; 1 with G12V; 2 with G13D) and 19 (28.4%) patients with colon cancers (11 with G12D, 3 with G12V, 4 with G13D; 1 with G13V). No KRAS mutations were found in esophagus cancer patients. The frequency of KRAS mutations in colon cancer was significantly higher than those in esophagus and gastric cancer (P<0.01). In 19 colon cancer patients with KRAS mutations, there were 6 (20.7%, 6/29) cases in the right colon (ascending colon and proximal 2/3 transverse colon) and 13 (34.2%, 13/38) cases in the left colon (distal 1/3 transverse colon, sigmoid colon and rectum). There is no significant difference in KRAS mutation frequencies between the right and the left colon. KRAS mutations were not associated with genders, ages, the invasive depth, lymph node metastasis and histopathological types in gastric and colon cancers. There were no EGFR exon 19 mutations found in any cases. EGFR exon 21 mutations were found in 2 gastric cancer patients (1 with L858R; 1 with G863D). In cancers of the alimentary system, KRAS mutations were more common than EGFR mutations, and KRAS mutations could be associated with the original tissues in the embryonic development. Namely, KRAS mutations were more common in cancers of organs from the midgut and hintgut than of those from the foregut, while EGFR mutations were all found in organs from the foregut. High resolution melting was a valuable method both in research and clinic.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2121.
Abstract 2122: KRAS mutation detection using high resolution melting analysis and its prognostic value in archival colorectal cancer tissues
Authors: Deschoolmeester Vanessa , Boeckx Carolien , Wuyts Wim , Marck Eric Van , Vermeulen Peter , Pauwels Patrick , Peeters Marc , Lardon Filip , Vermorken Jan B. , Baay Marc
Published: February 2011

Introduction: The development of targeted therapies has created a pressing clinical need for the rapid and robust molecular characterization of cancers. High resolution melting analysis (HRMA) provides a valid approach to efficiently detect DNA mutations. In the current study, HRMA was validated and implemented for screening of 164 colorectal cancer (CRC) patients to detect hot spot mutations (codon 12/13) in the KRAS oncogene. Direct sequencing was used to confirm and characterize HRMA results. In addition, the prognostic potential of KRAS in CRC was evaluated.

Materials and Methods: Initially, the HRMA sensitivity was established using a cell line dilution model, enabling us to detect 3% mutant DNA in a background of wildtype DNA. Next, HRMA was validated on 7 cell lines with or without a known KRAS mutation. All mutations were readily detectable using HRMA. Subsequently, inter- and intravariation was analyzed.

Results: HRMA revealed abnormal melting patterns in 57/164 (34.8%) CRC samples with G12D substitution the most prevalent type. However, only 50/164 (30.5%) samples could be confirmed by sequencing. The presence of KRAS mutation was associated with proximal location, advanced stage and microsatellite stability (MSS). Kaplan-Meier survival analysis revealed a significantly shorter overall and disease free survival for CRC patients harboring a KRAS mutation, especially for the G12C substitution. In Cox regression, KRAS mutation was still found to be a negative predictor of survival, however, significance was lost.

Conclusions: In conclusion, HRMA was found to be a fast, efficient and reproducible screening method for KRAS mutation detection by which degraded DNA from formalin-fixed, paraffin-embedded tissues can be tested. Additionally, accurate detection of KRAS mutations in CRC shows potential as a prognostic marker and evidence is accumulating that poor outcome could be linked to specific mutations. These results might have an impact on patient selection for adjuvant treatment.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2122.
Abstract 2123: Novel integration sites for Friend murine leukemia virus
Authors: Haeri Mehran , Ben-David Yaacov
Published: February 2011

Background: Friend murine leukemia virus (F-MuLV), a type C retrovirus, induces erythroleukemia when injected into certain strains of mice. The Induction of erythroleukemia is associated with viral integration into Friend leukemia integration site-1 (Fli-1). In some F-MuLV-infected mice we did not observe viral integration into Fli-1 region by Southern blot analysis. We hypothesize that induction of leukemia in these mice is due to insertion of F-MuLV into a novel integration site distinct from Fli-1.

Methods: The genomic DNA of F-MuLV-induced leukemic cells is digested by StuI restriction enzyme. DNA sequences of about 50 base pairs in length, termed adaptors, are then ligated to both ends of the digested fragments. Using primers for the adaptors and F-MuLV sequences, we performed PCR, followed by nested PCR to amplify those fragments that contain F-MuLV sequence. The PCR product is gel purified, cloned into a TA vector and sequenced. The sequences are compared to mouse genome databank to determine the F-MuLV flanking sequences.

Results: We identified seven novel integration sites for F-MuLV. Three of these sites are located inside genes and the remaining four do not interrupt any genes. We performed PCR to check if any of these integration sites were also targets of F-MuLV in twenty other tumors of the same type. We found that one of these integration sites which is close to Myb gene occurred in twenty five percent of the tested tumors in an area 1 kb in length.

Conclusion: We identified seven novel F-MuLV integration sites for F-MuLV. We showed that the integration site on chromosome 10 close to the Myb gene occurs in 25% of F-MuLV- induced tumors and is therefore a common integration site.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2123.
Abstract 2126: Quantitative trait locus analysis of tumor morphology in a mouse model of human colorectal cancer
Authors: Bautz David J. , Uronis Joshua M. , Threadgill David W.
Published: February 2011

Colorectal cancer (CRC) is the second leading cause of cancer death in the United States. Early detection has frequently been cited as contributing to a favorable outcome. However, detection of CRC is impacted by tumor morphology, with the classical polypoid lesions being detected at a much higher rate than flat lesions. Recently, mouse models of CRC that consistently and almost exclusively develop flat lesions have been developed, indicating that specific host genetic modifiers exist that influence tumor morphology. Using the azoxymethane (AOM) model of human CRC we find that I/LNJ mice develop approximately 85% flat tumors while KK/HlJ mice develop approximately 85% polypoid tumors. To determine what genetic factors contribute to these phenotypic differences, we established an F2 genetic cross between these two mouse strains consisting of 119 animals. After a standardized AOM treatment regimen, these mice developed approximately 40% flat tumors. The mice were genotyped at 80 SNP's evenly spaced across the genome. Statistical analysis revealed two suggestive QTL for tumor morphology, one located on chromosome 12 (LOD 3.45) and the other on chromosome 9 (LOD 2.67). Follow up work has begun to fine map and identify candidate genes at these QTL through high dense SNP genotyping in an additional 300 F2 animals and eQTL analysis of normal colon tissue. These results indicate that tumor morphology is controlled by host genetic factors and we anticipate future studies focusing on the identification of these factors for development of therapeutic interventions.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2126.
Abstract 2132: Cigarette smoking increases copy number alterations in non-small cell lung cancer
Authors: Huang Yen-Tsung , Lin Xihong , Chirieac Lucian R. , McGovern Ray , Wain John C. , Heist Rebecca S. , Skaug Vidar , Zienolddiny Shanbeh , Haugen Aage , Su Li , Christiani David C.
Published: February 2011

BACKGROUND: Cigarette smoking is a well-established risk factor of lung cancer. How smoking induces oncogenesis in lung remains not fully understood. We conducted a genome-wide study of copy number and smoking pack-years in a large collection of non-small cell lung cancer (NSCLC) tumors on three different scales: whole genome, chromosome and focal region.

METHODS: Genome-wide analyses of copy number and pack-years of cigarette smoking were performed on NSCLC tumors (n=264), which was randomly divided into a discovery set (n=134) and a validation set (n=130). Copy numbers were inferred from the dense single nucleotide polymorphisms array by dChip. On the scales of whole genome and chromosome, four indices were proposed to test large-scale copy number alterations (CNAs) patterns between heavy and light smokers: total CNAs events, continuous CNAs pattern, discrete CNAs pattern, and selecting effect with respect to gene locations. On the scale of focal region, genome-wide association (GWA) analyses of focal copy number and smoking pack-years were performed on 256,554 loci.

RESULTS: On the whole genome scale, heavy cigarette smokers (>60 pack-years) have significantly more copy number gains than non-/light smokers (≤60 pack-years) (p=2.46×10−4). Copy number losses tend to occur away from genes in non-/light smokers (p=5.15×10−5) but not in heavy smokers (p=0.52). Smoking-associated copy number gains are most significant on chromosomes 8 (p=1.19×10−5), 10 (p=4.81×10−4) and 12 (p=3.31×10−3). For the focal region, GWA analyses show six loci with strong association of copy number and cigarette smoking pack-years, including four neighboring loci at 12q23.2 (p=1.79×10−8 to 6.17×10−7) where IGF1 (insulin-like growth factor 1) is located. All above analyses were tested in the discovery set and confirmed in the validation set.

CONCLUSIONS: Heavy smokers have more copy number gains and less selection of gene locations in copy number losses. Copy number increases in 12q23.2 where IGF1 is located are positively associated with cigarette smoking pack-years.

Supported by US National Institutes of Health Grants No. CA092824, CA074386, CA090578; and the Norwegian Cancer Society

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2132.
Abstract 2133: Identification of driver genes for amplification of the narrow amplicon at 2q11 present in oral cancers and pre-cancers
Authors: Lin Mauting , Snijders Antoine M. , Bhattacharya Aditi , Paquette Jesse , Jordan Richard C. K. , Schmidt Brian L. , Albertson Donna G.
Published: February 2011

Genome-wide analysis of copy number aberrations in oral squamous cell carcinoma (SCC) and oral epithelial dysplasia, a pre-malignant lesion, by array CGH has revealed that the genomes of these cancers and pre-cancers frequently harbor narrow (∼3 Mb) regions of amplification. These amplicons are recurrent, but rare, being present in <10% of cases. Nevertheless, candidate driver genes in the amplicon are often up-regulated in tumors without amplification. Thus, these narrow amplicons highlight genes/pathways that are de-regulated in oral cancer. A narrow amplicon at 2q11 has been observed in 3/44 oral dysplasia cases, in 1/89 oral SCC and one primary oral SCC cell line. In one patient with synchronous mild and moderate dysplasia lesions, the 2q11 amplicon was uniquely present in moderate dysplasia, suggesting that the amplified and overexpressed gene(s) at 2q11 contributed to progression. The 2q11 amplicon spans 3.5 Mb and includes 43 genes. Examination of two independent head and neck cancer gene expression datasets retrieved from the NCBI Gene Expression Omnibus database revealed up-regulation of 11 of these genes in one or both datasets. All 11 genes are overexpressed in oral SCC lines SCC4, SCC9 and HSC3. Knockdown of individual genes in a panel of eight oral SCC cell lines was performed with one or more siRNA's targeting each of the genes. These studies revealed that cellular proliferation and morphology were significantly disrupted in cells transfected with siRNA to two of the genes, suggesting there are two potential driver genes for the 2q11 amplicon.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2133.
Abstract 2138: Functional characterization of the 19q12 amplicon in grade 3 breast cancers
Authors: Natrajan Rachael C. , Wetterskog Daniel , Lambros Maryou BK , Felipe Geyer , Mackay Alan , Vatcheva Radost , Palacios Jose , Reis-Filho Jorge
Published: February 2011

Grade III (GIII) invasive ductal carcinomas of no special type (IDC-NST) comprise up to 60% of all invasive breast cancers and have an aggressive clinical behavior. We have previously identified 19q12 amplification in a subgroup of estrogen receptor (ER)-negative HER2 amplified (4%) and basal-like (16%) subtypes of breast cancer. This amplicon comprises 9 genes, including cyclin E1 (CCNE1), which has been proposed as its driver due to a strong association with gene over-expression. The aims of this study were to identify functionally the genes within the 19q12 amplicon whose expression is required for the survival of cancer cells harboring this amplification. We analyzed a series of 48 micro-dissected GIII IDC-NSTs using high-resolution microarray CGH and mRNA expression arrays and identified 19q12 amplification in 7.6% of ER negative GIII tumors. Of the 9 genes mapping to the smallest region of amplification, UQCRFS1, POP4, C19ORF12, CCNE1 and C19ORF2 were significantly over-expressed when amplified (P<0.05). Breast cancer cell lines with (MDAMB157 and HCC1569) and without (Hs578T, MCF7, MDAMB231 and ZR75.1) 19q12 amplification were screened with an RNAi library targeting the 9 genes mapping to the smallest region of amplification on 19q12. Individual short interfering RNA (siRNA) SMARTpools targeting these genes were plated in 96-well plates and cell viability was assessed after 9 days using CellTiter-Glo Luminescent Cell viability assay. Silencing of POP4, PLEKHF1, ZNF536 and ZNF537 selectively inhibited cell viability in 19q12-amplified cells compared to control cells. CCNE1 silencing was neither selectively lethal to cells harboring 19q12 amplification, nor lethal in all cells with this amplification. Our results demonstrate that expression of POP4, PLEKHF1, ZNF536 and ZNF537 is required for the survival of cancer cells displaying their amplification and suggest that the 19q12 amplicon may harbor more than one ‘driver’.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2138.
Abstract 214: Anticancer effect of Compound A, a novel modulator of the glucocorticoid receptor, in acute leukemia cells
Authors: Lesovaya Ekaterina A. , Kirsanov Kirill I. , Budunova Irina V. , Yakubovskaya Marianna G.
Published: February 2011

Glucocorticoids (GCs) are widely used in chemotherapy of blood cancers, including childhood acute leukemia. The biological effects of GCs are mediated by the glucocorticoid receptor (GR), a well known transcription factor. Therapeutic effects of GCs are mainly mediated via GR transrepression that involves negative interaction between GR and other transcription factors. In contrast, many adverse metabolic effects of glucocorticoids are mediated by GR transactivation that requires GR binding to hormone-responsive elements in gene promoters. Thus, selective GR activators (SEGRA) that modify GR, shift GR functions towards transrepression resulting in improved therapeutic index, hold a great potential for the GR-targeted chemotherapies.

Recently we and others characterized novel non-steroidal GR ligand, 2-(4-acetoxyphenyl)-2-chloro-N-methylethylammonium chloride or Compound A (CpdA), and showed that CpdA prevents GR dimerization and transactivation, but strongly enhances GR transrepression. We also revealed that CpdA possesses anti-cancer properties: it inhibited growth and viability of prostate cancer cells in GR-dependent fashion.

Here we report that CpdA at concentrations 10−6-10−5M exerted cytostatic (evaluated by MTT test) and cytotoxic (evaluated by FACS analysis of DNA fragmentation as sub-G1 peak in propidium iodide-labeled cells) effects in K564, NB-4 and CEM leukemia cell lines. We found that these cell lines express wild type nonmutated GR. Consequently, these cell lines present a good model system for the further comparative analysis of CpdA and GCs effects. Leukemia cell lines were sensitive to apoptotic effect of both GR ligands: Dexamethasone (Dex) and CpdA. Dex induced apoptosis at the concentrations 10−7 to 10−5M; and CpdA affected cell viability at concentrations 5×10−6−10−5M.

As a first step towards the evaluation of toxicological profile of this promising anti-cancer compound, we assessed its mutagenicity using Ames test. CpdA was not mutagenic either in frameshift or in base-substitution indicator strains, TA98 and TA100, respectively, at the wide range of concentrations (10−9-10−4M).

Overall, our data suggest that novel non-mutagenic GR modulator has a high chemotherapeutic potential. The detailed mechanisms of its anti-cancer effects in leukemia cell lines is under investigation.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 214.
Abstract 2141: Analysis of DNA copy number changes in ovarian cancer data reveals differences in progressive vs. complete response samples
Authors: Soheil Shams , Parman Craig , Gregg Jeffrey
Published: February 2011

Data from The Cancer Genome Atlas (TCGA) project, related to Ovarian Cancer was obtained from the TCGA web site. The data comprised a total of 489 samples hybridized on 2-color oligo-arrays with 244K probes per array. Raw log-ratio data representing DNA copy number values relative to a DNA pool of normal samples were used in the analysis. The raw data was processed using Nexus Copy Number version 5 software (BioDiscovery, El Segundo CA) and regions of copy number change were identified using the built-in FASST algorithm with significance threshold of 5.0 E-6. After applying a QC filter to remove hybridizations with excessive noise, 463 samples remained for further analysis. Of these, there were 214 solid tumors and 242 normal samples (either surrounding tissue or blood from same patient). In our analysis we compared copy number differences from samples with Progressive disease (n=29) and those with Complete response to therapy (n=245). Using a Fisher's Exact test and a maximum p-value of 0.05 we identified significant regions of copy number change covering a genome area composed of a total of 1049 genes. Included were regions of significant increase in the number of DNA copy number gains in the for the Complete response samples as compared to Progressive cases. The most significant of these were in the 6p21.1 − 6p12.1 and 5p14.1 − 5p13.3 regions. Other regions include a number of well-know genes related to apoptosis, such as TNF (on 6p21.33) and RHOB (on 2p24.1) that were also significantly gained in Complete response but showed DNA loss in Progressive samples.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2141.
Abstract 2142: Multifaceted genomic analyses reveal novel glioblastoma candidate genes and microbial DNAs
Authors: Duncan Christopher G. , Killela Patrick J. , Yan Hai
Published: February 2011

The glioblastoma (GBM) genome displays remarkable chromosomal aberrations, which harbor critical GBM specific genes contributing to several oncogenetic pathways. However, deciphering further driver genomic changes from passenger gene alterations remains a daunting challenge. In attempt to identify novel GBM genes, we carried out a multifaceted genome-wide analysis to characterize the most significant aberrations of DNA content occurring in GBM patients. First, we performed copy number analysis using Digital Karyotyping (DK) and Illumina BeadChip technologies and identify genomic loci which consistently present as focal copy number alterations. Next, we overlaid copy number altered loci with the somatic mutation data from our genome-wide coding sequencing analysis. Upon expression analyses of these genes, we identify several previously unrecognized genes which are dysregulated in GBM. Most significantly, we identify grade-specific activation of set of genes potentially involved in chromosomal instability of the GBM genome. Finally, we evaluated the contribution of microbial DNA to the GBM genome using an approach, called DK-MICROBE, which we developed to identify genomic DNA of bacteria and viruses in human disease tissues. Our multifaceted genomic evaluation of GBM establishes several candidate oncogenes, tumor suppressors and caretaker genes and provides insight on the molecular mechanisms and pathways to this deadly disease.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2142.
Abstract 2144: An integrated genomic and transcriptomic analysis of human uterine leiomyomas
Authors: Cirilo Priscila D.R , Marchi Fabio A. , Carvalho Alex F. , Pontes Anagloria , Rogatto Silvia R.
Published: February 2011

Uterine leiomyoma (UL) are tumors that arise from the uterine smooth muscle wall. Despite their nonmalignant nature, UL represent a major concern in women's health through their induction of significant morbidity in more than 25% of reproductive-age. The etiology of UL remains unknown. Cytogenetic studies have been identified recurrent aberrations involving the t(12;14); del(7)(q22q32); trisomy 12; and 6p21 rearrangements. The aim of this study was integrate genomic and gene expression microarray data to identify genes with altered function due to genomic instability. DNA and RNA were extracted from 24 UL from women at proliferative (n=14) and secretory (n=10) phase of the menstrual cycle. Normal control RNA was obtained from adjacent myometrium from all cases. Array CGH and gene expression microarray was done using the Human 44K Agilent oligoarrays according to manufacturer's instructions. Data were extracted and flagged with Feature Extraction software (version 10.1.1.1) and processed using Genomic Workbench Standard (version.5.0.14). Non-supervised analysis of gene expression revealed clustering of all cases independent of the phase of menstrual cycle. It was observed a significant correlation between deletion and down expression at 1q32.2-q44 and 7q11.22-q33. Although deletions at 1q and 7q had been described previously as involved in UL, the genes mapped at these regions were poorly studied. These genes are involved in Wnt signaling pathway, induction of apoptosis, transforming growth factor signaling pathway, fumarate hydratase activity, and fibrinolysis. Furthermore, it was observed correlation between amplification and increased gene expression at 11p15.5, 14q32.33, and 16p13.3. These regions harboring genes with function associated with regulation of cell proliferation and regulation of JNK cascade. Our results provide strong circumstantial evidence that different patterns of genomic imbalances contribute to their specific transcriptomic profiles. In the future, new therapies based on these findings can be applied in a subgroup of patients with specific genomic imbalances.

Supported by the FAPESP (07/51643-8) and CNPq (485032/2007-4), Brazil.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2144.
Abstract 215: CSE1L as a potential target to sensitize ovarian cancer cells to cisplatin
Authors: Lorenzato Annalisa , Olivero Martina , Martino Cosimo , Dani Nadia , Musiani Daniele , Calogero Raffaele , Renzo Maria Flavia Di
Published: February 2011

Human ovarian cancer is frequently (70% of cases) at advanced stage at presentation and requires multimodality treatment, consisting of cytoreductive surgery and chemotherapy based on platinum-drugs and taxanes. Patients are initially responsive to this treatment, but, in most cases, experience disease relapse because tumour cells acquire drug resistance.

We demonstrated that the Hepatocyte Growth Factor (HGF) sensitizes to cisplatin (CDDP) and taxol an ovarian carcinoma cell line known to be resistant to platinum drugs: SK-OV-3, leading to an increased cancer cell death at very low drug doses both in vitro and in an animal model (Bardella et al., 2007; Rasola et al., 2004). This was an unexpected finding as HGF and its tyrosine kinase receptor, encoded by the MET oncogene, drives cell proliferation, survival and invasiveness during development and tumorigenesis. We also demonstrated that this effect is mediated by p38MAPK, which overwhelms the concomitant HGF activation of survival pathways, as the expression of a dominant negative form of p38MAPK abrogates the sensitization effect of HGF (Coltella et al., 2006).

Using oligonucleotide microarrays, we studied the transcriptional responses of cell treated with CDDP in the presence or in the absence of HGF, and found that HGF pre-treatment modifies the transcriptional response to CDDP not only in SK-OV-3, but also in NIH-OVCAR-3, and TOV-21G ovarian cancer cells. The up- or down-modulation of the most differentially expressed genes found in common within these cell lines was reverted when the cells expressed a dominant negative form of p38MAPK, upholding once again the involvement of p38MAPK in this phenomenon.

Among the top-ranked differentially expressed genes, which we identified, we focused functional studies on CSE1L/CAS. This gene is a nuclear transporter involved in both proliferation and apoptosis found to be amplified and over-expressed in ovarian cancer. Knocking down of CSE1L/CAS made ovarian cancer cells sensitized to low doses of cisplatin and triggered apoptosis while had no effect on other cancer and normal cell lines.

In conclusion, we show that HGF and CDDP modulate transcription in ovarian cancer cells and that this transcriptional response is involved in apoptosis regulation. We also provide the proof-of-concept that the identified genes might be targeted to either increase the efficacy of chemotherapeutics or to revert chemotherapy resistance.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 215.