bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3054: P1446A-05: a new oral cyclin-dependent kinase inhibitor with potent preclinical antitumor activity
Authors: Joshi Kalpana S. , Padgaonkar Amol , Rathos Maggie , Wagh Vilas , Manohar Sonal , Bhatia Dimple , Damre Anagha , Sivakumar Meenakshi , Sharma Somesh
Published: August 2012

Cell cycle deregulation is one of the first steps that transform normal cells into tumor cells. Cyclin-dependent kinases (Cdks) related alterations such as overexpression and amplification of cell-cycle activators or inactivation of cell cycle inhibitors are among the most common genetic changes in human tumors. The pursuit for drugs that inhibit Cdks has been an intense area of research for more than 15 years. A number of Cdk inhibitors are being evaluated in clinical trials for the treatment of cancer. P1446A-05 has been identified as a Cdk4-D1, Cdk1-B and Cdk9-T inhibitor. P1446A-05 exhibited greater selectivity to Cdk4-D1 and Cdk1-B that are involved in the G1 and G2/M phase of the cell cycle respectively. The IC50 values were 0.09 and 0.025 μM for Cdk4-D1 and Cdk1-B respectively. P1446A-05 also inhibited Cdk9-T, which plays a role in transcription, with an IC50 of 0.022 μM. P1446A-05 was inactive against many non-Cdks with an IC50 of more than 50 μM. In cytotoxicity assay using 30 human cancer cell lines P1446A-05 showed IC50 in the range of 0.5 to 3.9 μM, whereas the cytotoxicity for unstimulated normal human PBMNCs was > 10 μM. P1446A-05 arrested cell cycle progression at G1 and G2 phases and induced apoptosis in human cancer cell lines such as non-small-cell lung cancer (H-460), colorectal carcinoma (HCT-116) and prostate cancer (PC-3) in a dose and time dependent manner. Furthermore, P1446A-05 affected the proteins involved in cell cycle and apoptosis. It down-regulated cyclin D1, inhibited Cdk4 specific phosphorylation (ser780 of retinoblastoma), induced the tumor suppressor protein p53, and reduced the levels of the anti-apoptotic protein Bcl-2. Pharmacokinetic studies revealed that P1446A-05 exhibited an absolute oral bioavailability of 82.48% in mice and 79.07% in rats. It showed dose-dependent increase in 1446A-05 levels in plasma of mice and rats. In in vivo efficacy studies, orally administered P1446A-05 significantly (p<0.001) inhibited the tumor growth in two xenograft models that of colon cancer (Colo-205) and non-small cell lung cancer (H-460) in SCID mice. These data demonstrated that P1446A-05 is an orally active inhibitor of Cdk with a unique target profile that may have therapeutic potential in human malignancies.

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 3054. doi:1538-7445.AM2012-3054
Abstract 3060: DYRK2-mediated phosphorylation of c-Jun and c-Myc is requisite for proper control of the G1/S transition
Authors: Taira Naoe , Mimoto Rei , Miki Yoshio , Yoshida Kiyotsugu
Published: August 2012

Transcription factors c-Jun and c-Myc are indispensable regulators for the G1/S transition and their expressions were tightly regulated at transcriptional and post-translational levels. Dysregulation of these expressions leads to tumor development and progression. Degradation of c-Jun/c-Myc, a critical event for the G1/S transition, is triggered by sequential phosphorylations by unknown priming kinase(s) and GSK3-beta. Here we show that DYRK2 (dual-specificity tyrosine-phosphorylation-regulated kinase 2) functions as a priming kinase for c-Jun/c-Myc and modifies its expression at the by post-translational level. In the absence of priming phosphorylation, c-Jun and c-Myc escape from GSK-3-mediated sequential phosphorylation and Fbw-7-mediated ubiquitination. Therefore, depletion of DYRK2 results in aberrant expression of c-Jun and c-Myc. Furthermore, in DYRK2 knockdown cells, up-regulated c-Jun and c-Myc facilitate cell proliferation and shorten the length of the G1 phase and prompts the G1/S transition. Furthermore, DYRK2 inactivation contributes to cell proliferation and tumor progression in vitro and in vivo. Pathological analysis demonstrated that DYRK2 expression is decreased in multiple cancerous tissues. Taken together, DYRK2 controls cell cycle progression at G1/S phase via phosphorylation of c-Jun and c-Myc

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 3060. doi:1538-7445.AM2012-3060
Abstract 3118: The proteasome inhibitor Bortezomib rescues clinically identified unstable missense variants of the DNA mismatch repair protein Msh2
Authors: Arlow Tim , Gammie Alison
Published: August 2012

Msh2 is required for DNA mismatch repair in eukaryotes. Deleterious mutations in human MSH2 account for approximately half of the alleles associated with Hereditary Nonpolyposis Colorectal Cancer (HNPCC) or Lynch Syndrome. Previously, we characterized clinically-identified MSH2 missense mutations, using yeast as a model system and found that the most common cause of defective DNA mismatch repair was low levels of the variant Msh2 proteins. Here, we measured the half-lives of the Msh2 variants and found that increased protein turnover was responsible for the low cellular levels. Additionally, restoring levels with increased gene dosage fully restored mismatch repair function in 10 of the 21 variants in a DNA polymerase proofreading defective strain background in which mismatch repair is essential. Using immunoprecipitation, we detected high molecular weight ubiquitinated species of unstable Msh2 variants when over-expressed. Additionally, genetic and chemical inhibition of the proteasome stabilized low-level Msh2 missense variants. We identified San1, an ubiquitin ligase known to target misfolded nuclear proteins, as responsible for targeting the MSH2 missense variants. Deletion of San1 restored protein levels for all but one variant, but did not elevate wild-type Msh2 levels. Additionally, mismatch repair function was significantly improved when the Msh2 variants that had displayed high-copy restoration of function were expressed in a san1α strain. Taken together, these results indicate that the ubiquitin-mediated proteasome degradation pathway is the major mechanism for increased turnover of the clinically identified Msh2 variants. Human MutSα, comprised of Msh2 and Msh6, is also regulated by the ubiquitin-mediated pathway, underscoring the conservation of regulation as well as function for mismatch repair in eukaryotes. Additionally, it is well established that mismatch repair defective tumors are resistant to standard chemotherapeutic regimens. Of therapeutic significance, the clinically approved drug Bortezomib partially restored levels and mismatch repair function in two different msh2 missense variants. In this regard, Bortezomib may prove to be a valuable adjuvant chemotherapeutic for individuals with particular alleles of MSH2. Our results provide the foundation for an innovative therapeutic regime for certain mismatch repair defective cancers that are refractory to conventional chemotherapies. Currently we are testing the rescue phenotype in human 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 3118. doi:1538-7445.AM2012-3118
Abstract 3127: Cigarette smoke condensate (CSC) induces differential expression and promoter methylation profiles of critical genes involved in lung cancer in NL-20 lung cells in vitro: Short-term and chronic exposure
Authors: Lyn-Cook Lascelles E. , Word Beverly , Mwamba Bibi , Lyn-Cook Beverly D. , Hammons George
Published: August 2012

Lung cancer is the leading cause of cancer deaths in the United States. Smoking accounts for a large number of those deaths. Recently, the Food and Drug Administration has been given authority to regulate any new cigarette products coming to market and further demonstrate the harm caused by constituents in cigarettes or those chemicals created from smoking. Establishing early diagnostic markers are critical to effective prevention programs. This study examined the effects of cigarette smoke condensate on expression and promoter methylation profile of critical genes involved in various stages of lung cancer development, such as E-cadherin (ECAD), O6-mehyl-guanine-DNA methyltransferase (MGMT), and RASSF1A. NL-20 cells were treated with 0.1, 1, 10 or 100 µg/ml of CSC for 24, 48 and 72 hrs for short-term exposures. ECAD showed a significant decrease in expression after 72 hrs with both 10 and 100 µg/ml CSC. This decrease in expression correlated to hypermethylation of the ECAD promoter at both concentrations using MSP analysis. MGMT expression increased only with the concentration of 100 µg/ml CSC at all time points. This correlated to an unmethylated profile of the promoter at this concentration. RASSF1A promoter profile was methylated in the control samples. However, when treated with 10 or 100 µg/ml CSC, an unmethylated promoter profile was noted. This correlated to increased expression of RASSF1A at both the 10 and 100 µg/ml concentrations. No changes were noted at 0.1 or 1 µg/ml of CSC in NL-20 lung cells. For chronic studies, the cells were exposed for 30 days with 10 or 100 µg/ml CSC, with media changes every three days adding new CSC. After 30 days, cells showed morphological changes associated with transformation. Foci were noted. These cells were further analyzed for further changes in critical genes involved in lung cancer. This study provides critical data showing epigenetic regulation of critical genes involved in DNA repair, invasion and tumor suppressor with treatment of lung cells with CSC.

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 3127. doi:1538-7445.AM2012-3127
Abstract 313: K-ras- and EGFR-mediated lung tumor formation require epithelial NF-κB signaling to modulate the inflammatory microenvironment
Authors: Ausborn Jamie A. , Sherrill Taylor , Zaynagetdinov Rinat , Polosukhin Vasiliy , McMahon Frank , Yull Fiona , Blackwell Timothy S.
Published: August 2012

The transcription factor nuclear factor-κB (NF-κB) is a key mediator of the inflammatory response, and its activation correlates with poor prognosis in non-small cell lung cancer patients. Oncogenic K-ras and epidermal growth factor receptor (EGFR) mutations occur frequently in lung adenocarcinoma and are associated with an inflammatory response in lung tumors. Recent papers have shown that inhibition of epithelial NF-κB signaling in a mouse model of K-ras-mediated lung tumorigenesis decreased tumor formation. However, the mechanisms linking NF-κB signaling to tumor formation remain unclear. We hypothesize that epithelial NF-κB signaling is a common requirement for lung tumorigenesis, causing activation and/or recruitment of inflammatory cells that promote lung tumor formation. For our studies, we used mice that inducibly express oncogenic K-rasG12D or EGFRT790M+L858R as well as a dominant negative inhibitor of κB signaling (DN-IκB) specifically in the lung epithelium. Mice expressing DN-IκB together with K-rasG12D or EGFRT790M+L858R developed significantly fewer tumors than mice expressing K-rasG12D or EGFRT790M+L858R alone after two months of doxycycline administration. However, two months of K-rasG12D or EGFRT790M+L858R expression did not significantly alter total inflammatory cell numbers in bronchoalveolar lavage fluid (BAL). EGFRT790M+L858R expression led to an increase in BAL neutrophils that was lost when NF-κB signaling was inhibited, while no changes in BAL macrophage, lymphocyte, or neutrophil subpopulations were observed in K-rasG12D-expressing mice with or without NF-κB signaling. At later time points, both K-rasG12D- and EGFRT790M+L858R-expressing mice demonstrated a profound inflammatory response, which was associated with an increased tumor burden. Using a model of long term alveolar macrophage depletion by intratracheal administration of liposomal clodronate, we found that depletion of alveolar macrophages decreased tumor burden in K-rasG12D-expressing mice. Together, these results suggest that, rather than causing a profound inflammatory influx, epithelial NF-κB signaling may be important for activating resident inflammatory cell populations that aid in K-ras- and EGFR-mediated tumor formation. From these studies, we hope to broaden understanding of the role of inflammatory signaling pathways in regulation of lung carcinogenesis.

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 313. doi:1538-7445.AM2012-313
Abstract 3130: Targeted DNA methylation reprogrammed differentiation and tumorigenesis of mesenchymal stem cells
Authors: Leu Yu-Wei , Hsiao Shu-Huei
Published: August 2012

Mesenchymal stem cells (MSCs) are multipotent stem cells that can be isolated from various sources and induced into different cell types. Although MSCs possess immune privilege and are more easily obtained, their propensity to tumorigenesis and immune responses they might bring to patients have not been fully explored. Epigenomic revolution like changes in DNA methylation and chromatin modification have been hypothesized to be critical in the determination of lineage-specific differentiation and tumorigenesis of MSCs but has not been forwardly proved. We identified the Polycomb group-governed loci in MSCs and tracked their differential methylation changes during the MSC-to-liver or MSC-to-adipocyte differentiation. These loci were also differentially methylated in breast cancer (n=93) and liver cancer (n=40). In an independent collection of liver cancer (n=100), the methylation states of these Polycomb-governed loci like Trip10, Casp8AP2, ENSA and ZNF484 were clustered together in contrast to the non-Polycomb targets like HIC1 and RassF1A. We then developed and applied a targeted DNA methylation method to methylate the Polycomb group protein-governed gene, Trip10, in MSCs which accelerated the cell fate determination of MSCs. Targeted methylation of Casp8AP2, another Polycomb group protein-governed gene, increased cellular survival and its resistance to drugs. The targeted methylation of ENSA and ZNF484 reveal the epigenomic evolution. Targeted methylation of HIC1 and RassF1A, both are tumor suppressors but not the Polycomb target genes, transformed MSCs into tumor stem cell-like cells. With this new method, we proved that DNA methylation within critical loci is sufficient to steer the cell fate of MSCs. (Supported in part by NSC 100-2320-B-194-001)

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 3130. doi:1538-7445.AM2012-3130
Abstract 3139: Frequent downregulation of NGB/GTPBP in lung cancer
Authors: Kim Donghwa , Park Sungman , Lee Sang Haak , Park Jong Y. , Cheng Jin Q.
Published: August 2012

GTP-binding protein NGB (GTPBP4) is located at chromosome 10p14-p15, a region common loss in various types of human malignancy. Here, we demonstrated that NGB expression was frequently down-regulated in human lung cancer cell lines and primary tumors. Furthermore, we found hypermethylation in NGB promoter region. Treatment with trichostatin A (TSA) and 5-Aza-2′-deoxycytidine (5-Aza) restored NGB expression. In addition, the reintroduction of NGB into the cells, in which it is downregulated, represses cell growth, colony formation, angiogenesis and tumor growth in nude mouse. These findings indicate that NGB is a putative tumor suppressor gene in lung cancer and could play a pivotal role in lung carcinogenesis.

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 3139. doi:1538-7445.AM2012-3139
Abstract 3142: Hypermethylation of the tissue inhibitor of metalloproteinases-2 gene in multiple myeloma
Authors: Wilop Stefan , Jost Edgar , Schubert Claudia , Herman James G. , Brümmendorf Tim H. , Galm Oliver
Published: August 2012

Multiple myeloma (MM) is a B-cell neoplasm that is characterized by the accumulation of malignant plasma cells in the bone marrow (BM) as well as abundant BM angiogenesis. Aberrant methylation of CpG islands near gene promoter regions is the most widely studied epigenetic abnormality in human malignancies and is associated with loss of gene function. There is increasing evidence for a role of the tissue inhibitor of metalloproteinases 2 (TIMP-2) gene as a tumor suppressor. Overexpression of TIMP-2 may result in decreased invasive potential, suppression of tumor growth and vascularization as well as inhibition of angiogenesis. We could previously show that TIMP-2 may become hypermethylated in association with transcriptional silencing in indolent and aggressive non-Hodgkin's lymphomas. Recently, downregulation of TIMP-2 was also reported in MM, and this is thought to contribute to increased BM angiogenesis. In this study, we determined the methylation status of the promoter-associated CpG island of TIMP-2 in primary MM samples and investigated correlations between TIMP-2 methylation and clinical parameters. Methylation of the promoter-associated CpG island of TIMP-2 was analyzed by methylation-specific polymerase chain reaction (MSP) in samples from 84 MM patients (median age 65 years [range 40-94]; 48 males, 36 females; 79 BM samples, 5 peripheral blood samples). Overall survival curves were plotted according to the method of Kaplan and Meier and compared using the log-rank test. Correlations between variables were analyzed using the Fisher's exact two-sided test and independent t-test, respectively. MSP analysis revealed aberrant methylation of the TIMP-2 promoter region in 4/84 MM patient samples. We found an association of TIMP-2 hypermethylation with plasma cell leukemia (p<0.0001) and decreased platelet counts (111 G/l vs. 258 G/l; p=0.04). There was a trend towards an inferior overall survival in MM patients with a hypermethylated TIMP-2 gene, but this difference was not statistically significant (p=0.09). We conclude that promoter hypermethylation of TIMP-2 is a novel epigenetic event in MM that may contribute to disease progression and could serve as a prognostic biomarker. Further studies are warranted to elucidate the functional consequences of epigenetic dysregulation of TIMP-2 in the pathogenesis of MM. Additionally, the increasing evidence for the role of DNA methylation changes in MM may serve as a basis for the use of epigenetically targeted therapeutic approaches in malignant plasma cell disorders.

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 3142. doi:1538-7445.AM2012-3142
Abstract 3143: Epigenetic regulation of PTEN by miR-185 and miR-200b in liver cancer
Authors: Qadir Ximena V. , Han Chang , Wu Tong
Published: August 2012

PTEN is one of the most commonly lost tumor suppressors in human cancers. Deletion of PTEN in mice has been shown to induce steatohepatitis and the development of primary liver cancers (hepatocellular carcinoma and cholangiocarcinoma). This study provides novel evidence for epigenetic regulation of PTEN in both hepatocellular carcinoma and cholagniocarcinoma cells. Treatment of the hepatocellular carcinoma cell line (Huh-7) and cholangiocarcinoma cells line (TFK-1) with the DNA methyltransferase (DNMT) inhibitor, Decitabine (1 mM), and the histone deacetylase inhibitor, Trichostatin A (TSA, 10mM), increased the expression of PTEN protein. Methylation specific qPCR assay shows that Decitabine/TSA treatment significantly reduced the methylation of the CpG islands located at the PTEN gene promoter. Furthermore, we have shown that miR-185 and miR-200b target DNMT-1 and -3A/3B, respectively, thus upregulating the expression of PTEN in hepatic cancer cells. Forced overexpression of miR-185 or treatment with mimic miR-185 abolished the expression of DNMT1 protein and enhanced the level of PTEN protein in both Huh-7 and TFK-1 cells. Accordingly, miR-200b overexpression or mimic treatment significantly decreased DNMT3A and DNMT3B protein levels and enhanced the expression of PTEN. These results demonstrate a novel epigenetic regulation of PTEN by miR-185 and -200b which is critical in hepatic carcinogenesis

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 3143. doi:1538-7445.AM2012-3143
Abstract 3144: A role for DOK2 methylation in platinum resistance and tumor suppression in ovarian cancer
Authors: Lum Elena , Vigliotti Michele , Cutter Noelle , Khan Sohail , Dimitrov Nevenka , Levine Douglas , Lucito Robert
Published: August 2012

Ovarian cancer is the 5th leading cause of cancer in women, affecting close to 22,000 women in the year 2011, of which nearly 15,500 will die. It is difficult to detect until it reaches advanced stages and becomes malignant. Currently, the standard treatment for ovarian cancer is platinum-based therapeutics, such as Carboplatin or Cisplatin, combined with Taxol. Unfortunately, approximately 25% of patients are inherently platinum-resistant and all patients who suffer from recurrence will have developed acquired platinum resistance. The genetic/epigenetic causes of this resistance are poorly understood. Epigenetic events are reversible and the identification of genes altered by this mechanism may lead to studies on how to reprogram the process leading up to resistance. To examine the ovarian epigenome, we utilized an array based method, Methylation Oligonucleotide Microarray Analysis (MOMA), to analyze a set of 50 primary ovarian tumors and 12 ovarian normal samples. We identified epigenetic differences that segregated with platinum response and then associated this with expression data to identify gene candidates transcriptionally repressed and methylated in patients resistant to platinum. Next, a pooled shRNA screen was performed on candidate genes to identify those that were functionally relevant to platinum resistance. One of the validated candidate genes identified through the pooled shRNA screen was DOK2, an adapter protein downstream of tyrosine kinase, which has been shown by others to be a lung cancer tumor suppressor. We show that suppression of DOK2 by short hairpin RNAs in ovarian cell lines conferred resistance to platinum treatment. To elucidate the mechanism for resistance, we measured the influx of platinum into the cells using C-14 tagged carboplatin. As a result, uptake of carboplatin was found to be decreased with DOK2 suppression. Consistent with DOK2 having tumor suppressor activity, knockdowns in ovarian cell lines increases growth and migration. Furthermore, loss of DOK2 induces invasive and tumorigenic phenotypes in ovarian cell lines. DOK2 is already a proven tumor suppressor in lung cancer, and our experiments indicate DOK2 has tumor suppressor features in ovarian cancer as well. We show that DOK2 is a tumor suppressor in ovarian cancer and that the loss of DOK2 also contributes to platinum resistance. Understanding DOK2 function will help us understand ovarian cancer development, progression as well as therapy resistance.

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 3144. doi:1538-7445.AM2012-3144
Abstract 3154: Sequencing of the small RNA transcriptome in cytologically normal bronchial airway epithelium of smokers with and without lung cancer
Authors: Campbell Joshua D. , Luo Lingqi , Xiao Ji , Liu Gang , Aleksyev Yuriy , Elashoff David , Dubinett Steven M. , Lenburg Marc E. , Spira Avrum
Published: August 2012

Introduction: Based on the concept that cigarette smoke creates a molecular field of injury in epithelial cells throughout the respiratory tract, we have previously shown that mRNA expression differences in cytologically normal airway epithelial cells in patients with and without lung cancer can serve as a clinically-relevant lung cancer biomarker. MicroRNAs are short, non-coding RNAs that can each regulate the expression of hundreds of target genes and can be reliably detected in clinical samples with variable quality. The goal of this study is to generate a microRNA biomarker in the airway epithelium of smokers for the diagnosis of lung cancer. Methods: Cytologically-normal bronchial airway epithelial cells were collected via brushings of the mainstem bronchus of smokers undergoing fiberoptic bronchoscopy for suspicion of lung cancer (n=128). Subjects were followed after their bronchoscopy until a final diagnosis of lung cancer (n=75) or an alternate benign diagnosis (n=53) was made. The small RNA transcriptome (<40 nucleotides) was sequenced using the Illumina HiSeq 2000 with multiplexing (8 samples/lane). Reads were aligned to hg19 using Bowtie allowing up to 3 mismatches and 10 locations to the genome. Linear models were used to associate expression profiles with disease-related phenotypes while controlling for differences in the total number of reads and other clinical covariates. Results: An average of 10.6 million reads was sequenced per sample with 77% of these reads having at least one alignment to the human genome. In addition, 75% of the aligned reads mapped to known microRNA precursors. 508 novel microRNA precursors were predicted using the miRDeep algorithm. 362 mature microRNAs were considered consistently expressed (Median Absolute Deviation > 20). 98 of these microRNAs were differentially expressed between current and former smokers and 56 microRNAs differentially expressed in the airway epithelium of smokers with and without lung cancer (p<0.05; 18 expected by chance at this threshold). Conclusions: We have sequenced the small RNA transcriptome in the cytologically-normal proximal airway epithelium of smokers and have identified microRNA expression profiles associated with smoking status and/or a final diagnosis of lung cancer. In the future, a microRNA biomarker will be developed on these samples using procedures outlined in the MicroArray Quality Control (MAQC)-II project and tested on an independent cohort in order to confirm their ability to serve as an early diagnostic tool for lung 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 3154. doi:1538-7445.AM2012-3154
Abstract 3160: Expression of apoptosis-regulating miRNAs and target mRNAs in oral squamous cell carcinoma
Authors: Coutinho-Camillo Cláudia M. , Lourenço Silvia V. , Nishimoto Ines N. , Kowalski Luiz P. , Soares Fernando A.
Published: August 2012

Apoptosis is a genetically programmed form of cell death, which primarily functions to eliminate senescent or altered cells that are useless or harmful for the multicellular organism. In contrast, aberrations of the apoptotic mechanisms that cause excessive or deficient programmed cell death have been linked to a wide array of pathologic conditions. MicroRNAs (miRNAs) are approximately 22 nucleotide non-coding RNA molecules that regulate gene expression post-transcriptionally and recent studies have indicated the dysregulation of miRNAs involved in apoptosis. In this study, using Real Time RT-PCR we have analyzed the expression of apoptosis-regulating miRNAs (miR-15a, miR-16, miR-29a, miR-34a, miR-17-5p, miR20a, miR21) in a series of 20 oral squamous cell carcinoma samples and a pool of 5 normal oral mucosa tissue samples. Using TargetScan algorithms, target genes of these miRNAs were identified and the expression of mRNA transcripts of these target genes was also evaluated (Bcl-2, Caspase-2, Caspase-7, Bid, SMAC/DIABLO) in a series of 50 oral squamous cell carcinoma samples and a pool of 10 normal oral mucosa tissue samples. Considering up- or down-regulation as a 2-fold difference in expression relative to the normal tissue, we observed down-regulation of miR-15a, miR29a and miR34a in 50%, 75% and 70% of the samples, respectively. Normal-like expression was observed for miR-16, miR-17-5p, miR-20a and miR-21 in 80%, 75%, 90% and 60% of the samples, respectively. Down-regulation of the BCL-2 transcripts was observed in 60% of the samples and normal-like expression was observed for BID, CASPASE-2, CASPASE-7 and SMAC/DIABLO transcripts in 66%, 82%, 68% and 60% of the samples, respectively. Expression of BID, CASPASE-2 and CASPASE-7 might be regulated by miR-17-5p and miR-20a and we found no alteration in expression of both mRNAs and miRNAs. Expression of BCL-2 might be regulated by miR-15a, miR-16, miR-21 and miR-34. We found down-regulation of BCL-2 transcripts and miRNAs-15a and -34, suggesting that other mechanisms might be involved in the regulation of BCL-2. Expression of SMAC/DIABLO might be regulated by miR-29 and we found normal-like expression of SMAC/DIABLO transcripts and down-regulation of miRNA-29 also suggesting that other mechanisms might be involved in the regulation of SMAC/DIABLO. Our study provides evidence of alterations in the expression of apoptosis-regulating miRNAs and alterations in the expression of genes involved in the apoptotic pathway demonstrating that regulation of apoptosis is an important feature in OSCC behavior. FAPESP research grants 98/14335-2 and 07/50608-4.

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 3160. doi:1538-7445.AM2012-3160
Abstract 3167: miR-138 and its role in melanoma progression
Authors: da Cruz Adriana T. , Hunger Aline , Morais Alice Santana , Meliso Fabiana Marcelino , Staruss Brayn , Jasiulionis Miriam Galvonas
Published: August 2012

miRNAs are non-coding RNAs that bind to mRNA targets preventing their translation. It is predicted that over 30% of mRNAs are regulated by miRNAs. Therefore these molecules are considered essential to many biological responses, such as cell proliferation, apoptosis and stress responsiveness. Loss or gain of miRNAs function contributes to development of many diseases, included cancer. Melanoma rises from malignant transformation of melanocytes. Despite being rare, it is responsible for the greatest number of deaths promoted by all skin cancers. One reason for this scenario is that melanoma metastatic is usually incurable. To determine the role of miRNAs in melanoma progression, the expression of several miRNAs were evaluated in a murine model that mimics different stages of the genesis of this tumor. In that model, several cell lines were obtained after submitting melan-a melanocytes to sequential cycles of anchorage blockade. Among miRNAs in which expression were altered, miR-138 has decreased expression in intermediate phases of melanoma genesis and was selected for further investigation. It was verified that mRNA of p53, Ezh2, MyoD and Sirt-1 are putative targets of miR-138. Interestingly, these mRNAs also have inverse expression of miR-138 in the distinct lineages of this model. Luciferase assay was done to confirm if miR-138 directly regulate such mRNAs. Super expression of miR-138 in the cell lineages that have lower levels of this miRNA was also induced. Functional experiments assessing proliferation rate, migration and invasiveness capacity in these cells expressing ectopic miR-138 were performed. The results indicate that miR-138 deregulation is an important event of melanoma progression. The targets of this miRNA are key components in pathways frequently disrupted in cancer, like cell cycle and apoptosis control, epithelial to mesenchymal transition regulation, and differentiation process. Thus, miR-138 represents a strong candidate to therapeutic target. The exploration of its role can contribute to improve the comprehension about melanoma progression and elucidate new ways of interventions against cancer. Supported by FAPESP.

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 3167. doi:1538-7445.AM2012-3167
Abstract 3169: MicroRNA-144 induces proliferation and invasion in nasopharyngeal carcinoma through repression of PTEN
Authors: Zhang Li Yi , Fu Li , Guan Xin Yuan
Published: August 2012

Nasopharyngeal carcinoma (NPC) is a characteristic type of head and neck cancer which is frequent in Southern China. Different from other head and neck cancers, tumor suppressor genes (TSGs) mutation and deletion in NPC is uncommon. Conversely, downregulation of TSGs expression by miRNA is increasingly recognized as an important mechanism of nasopharyngeal tumorigenesis. In this study, we report that miR-144 is commonly upregulated in NPC specimens and NPC cell lines, its repression decreased cell proliferation, clonogenicity, migration, invasion and tumor growth, establishing an important oncogenic role for this miRNA. Further, we verified tumor suppressor gene PTEN as a direct target through luciferase reporter assay in NPC cell lines. Similar to the restoring miR-144 expression in miR-144 attenuated cells, PTEN downregulation in miR-144 attenuated cells increase cell growth and invasion. Mechanistic investigations revealed that miR-144 suppressed the expression of PTEN to downregulate p53 while suppressing the expression of pAKT and cyclin D1 induce G1-phase cell-cycle arrest. Taken together, our results indicate that miR-144 functions as an onco-miRNA in NPC, and its onco-effects are mediated chiefly by repressing PTEN expression.

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 3169. doi:1538-7445.AM2012-3169
Abstract 3170: Comprehensive analysis of the microRNA-128 function in head and neck squamous cell carcinoma
Authors: Hauser Belinda , Pang Xiaowu , Fang Yayin , Gu Xinbin
Published: August 2012

MicroRNAs are 22∼nucleotide-long endogenously expressed, highly conserved noncoding RNAs with important regulatory functions in proliferation, apoptosis, and metastasis. miR-128 a brain-enriched, which is transcribes from two gene copies (miR-128-1 and miR-128-2) on chromosome 2 and chromosome 3 which produce identical mature forms. miR-128 is most commonly in gliomas that have shown to function as a tumor suppressor which inhibits cell proliferation. Proteomic alterations of prostate cancer show miR-128 to down regulate cell invasiveness. Here we evaluated the role of miRNA-128 in HNSCC, to identify its putative targets and elucidate the possible mechanisms underlining the function of miR-128 in HNSCC. We established HNSCC cell lines that stably expressed individual members of the miR-128 using a lentiviral delivery system. The levels of miR-128 and their targeted proteins were analyzed by qRT-PCR and Western blot. miR-128 affinity was elucidated by MTT, colony formation, flow cytometry, and a tumor xenograft model. Two kinds of stably transfected HNSCC cell lines, including JHU-22miR-128 transfected with miR-128 and a vector control cell line JHU-22vector, were generated as a testing system. The miR-128 level in the JHU-22miR-128 cells was eleven fold higher than in JHU-22vector cells. We found that overexpression of miR-128 enable to effectively suppress cell viability (30%), decrease of proliferation (55%), and significantly reduced solid tumor growth in tumor xenografts models compared to the control groups. Transfected miR-128 cells exhibited astrocyte morphology. The inhibition mechanism of miR-128 was determined by evaluating five putative targets (BMI-1, BAG2, BAX, H3f3b, and Paip2) of miR-128. Overall, miR-128 enables to directly inhibit all these targets, but the binding affinity of miR-128 is dominantly towards to BMI-1 and BAG-2 targets. Conclusions: miR-128 exhibits anti-proliferative effects and phenotypic alteration in HNSCC by targeting multiple predicted conserved targets activity function in apoptosis, cell cycle, transcription, and translation. Our results provide a better understanding of miR-128 function in HNSCC and the potential targets to develop novel diagnostic markers and 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 3170. doi:1538-7445.AM2012-3170
Abstract 3224: Pre-clinical targeting of the metabolic phenotype of lymphoma by AZD3965, a selective inhibitor of monocarboxylate transporter 1 (MCT1)
Authors: Critchlow Susan E. , Hopcroft Lorna , Mooney Lorraine , Curtis Nicola , Whalley Nicky , Zhong Haihong , Logie Armelle , Revill Mitchell , Xie Liang , Zhang Jingchuan , Yu De-Hua , Murray Clare , Smith Paul D.
Published: August 2012

Introduction - The metabolic phenotype of tumours (relative to most normal tissues) is shifted from oxidative phosphorylation to aerobic (Warburg Effect) or anaerobic glycolysis. This enables tumours to meet their energetic and biosynthetic demands even under conditions of low nutrient and O2. The end-product of glycolysis is lactate, a metabolic dead end, which if allowed to accumulate in the tumour cell may cause feedback inhibition of glycolysis and intra-cellular acidification resulting in inhibition of cell growth or survival. Experimental data - We have studied the effects of a potent, selective and orally available inhibitor of MCT1 in lymphoma cell lines and in vivo models. AZD3965 is a small molecule inhibitor of MCT1 with a binding affinity of 1.6 nM, is 6 fold selective over MCT2 and does not inhibit MCT4 at 10 μM. Both lactate transport and cell growth are potently inhibited by AZD3965 in lymphoma cell lines that preferentially express MCT1. Lactate transport inhibition in some cell lines also induces a cytotoxic effect. In vitro combination studies show that lactate transport inhibition can enhance the induction of cell death by doxorubicin. Blocking lactate transport in vitro also leads to a rapid inhibition of glucose uptake in the Raji Burkitt's lymphoma cell line. In vivo, AZD3965 is well tolerated and induces a dose and time dependent accumulation of lactate in the tumours, suppresses tumour growth and in the Raji model potentiates the effects of Rituxan, doxorubicin and bendamustine. Conclusions - Here we demonstrate that selective inhibition of lactate transport by the MCT1 inhibitor AZD3965 offers an novel mechanism for targeting the metabolic phenotype in tumours that preferentially express MCT1.

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 3224. doi:1538-7445.AM2012-3224
Abstract 3249: Intersectin 1 is required for neuroblastoma tumorigenesis
Authors: Russo Angela , O'Bryan John P.
Published: August 2012

Neuroblastoma (NB) is a malignant tumor deriving from the neural crest and is responsible for 13% of cancer-related deaths in children. Phosphoinositide 3-kinases (PI3Ks) play a central role in NB tumorigenesis. PI3Ks activate AKT, which regulates survival and stabilizes the MYCN oncogene. Both MYCN and AKT are markers for poor prognosis in NBs. Inhibiting PI3Ks with pan-inhibitors blocks malignant progression in vivo; however, these inhibitors have high toxicity that limits their use in NB treatment. Though there are three classes of PI3Ks, only Class I isoforms have been implicated in NB tumorigenesis. We have previously found that the scaffold protein intersectin 1 (ITSN1) interacts with PI3KC2α and regulates its activation as well as AKT activation in neuronal cell lines. In addition, ITSN1 overexpression is transforming in rodent fibroblasts. Thus, we examined the role of the ITSN1-PI3KC2α pathway in human NB tumorigenesis. ITSN1 and PI3KC2α are highly expressed in primary human neuroblastoma tumors of varying stage and MYCN amplification status. In addition, both ITSN1 and PI3KC2α are expressed in NB tumor cell lines derived from high grade NB tumors. Silencing ITSN1 dramatically inhibited the anchorage-independent growth of NB tumor cells in vitro and tumor formation in xenograft assays in vivo independent of MYCN status. Conversely, heterologous overexpression of ITSN1 increased the formation of soft agar colonies. These results suggest that ITSN1 functions as an oncogene in NB cells. Furthermore, PI3KC2α overexpression restores anchorage-independent growth of ITSN1-depleted NB cells demonstrating that PI3KC2α mediates ITSN1's tumorigenic properties. This study reveals a new pathway, i.e., ITSN1-PI3KC2α, involved in regulating the pathogenesis of NBs and suggests that this pathway may represent a new target for therapeutic intervention in the treatment of NB patients.

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 3249. doi:1538-7445.AM2012-3249
Abstract 3394: Identification of PRDX4 and P4HA2 as metastasis-associated proteins in oral cavity squamous cell carcinoma by comparative tissue proteomics of microdissected specimens using iTRAQ technology
Authors: Chang Kai-Ping , Chang Yu-Sun , Yu Jau-Song , Chi Lang-Ming
Published: August 2012

Background Cervical lymph node metastasis represents the major prognosticator for oral cavity squamous cell carcinoma (OSCC). Methods Here, we used an iTRAQ-based quantitative proteomic approach to identify proteins that are differentially expressed between microdissected primary and metastatic OSCC tumors. The selected candidates were examined in tissue sections via immunohistochemistry, and their roles in OSCC cell function investigated using RNA interference. Results Seventy-four differentially expressed proteins in nodal metastases, including PRDX4 and P4HA2, were identified. Immunohistochemical analysis revealed significantly higher levels of PRDX4 and P4HA2 in tumor cells than adjacent non-tumor epithelia (P < 0.0001 and P < 0.0001, respectively), and even higher expression in the 31 metastatic tumors of lymph nodes, compared to the corresponding primary tumors (P = 0.060 and P < 0.0001, respectively). Overexpression of PRDX4 and P4HA2 was significantly associated with positive pN status (P = 0.048 and P = 0.041, respectively). PRDX4 overexpression was a significant prognostic factor for disease-specific survival in both univariate and multivariate analyses (P = 0.034 and P = 0.032, respectively). Additionally, cell migration and invasiveness were attenuated in OEC-M1 cells upon in vitro knockdown of PRDX4 and P4HA2 with specific interfering RNA. Conclusions Novel metastasis-related prognostic markers for OSCC could be identified by our approach.

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 3394. doi:1538-7445.AM2012-3394
Abstract 3398: High-throughput expression profiling of circulating tumor cells from breast cancer patients as potential therapy decision indicator
Authors: Kolostova Katarina , Romzova Marianna , Bobek Vladimir , Kubista Mikael , Kasimir-Bauer Sabine
Published: August 2012

Background: The aim of this study was to assess the molecular profiles of circulating tumor cells (CTCs) from metastatic breast cancer (MBC) patients and to evaluate their potential as prognostic and therapy indicators. Methods: Blood samples (5ml) of 77 MBC patients from Czech and German Gynecological Cancer Centers were analyzed for CTCs using the AdnaTest BreastCancer (AdnaGen AG, Germany) for the detection of EpCAM, MUC-1 and HER-2 transcripts. Obtained cDNA molecules have been gene-specifically pre-amplified for multimarker qPCR analysis measured on Biomark® (Fludigm, USA) microfluidic chip for 48 samples and 48 testing positions (in total: 2304 rxn, 31 tumor specific-genes, 30 MBC samples, respectively). qPCR- results have been analyzed by GENEX v.s. 5.0 software (MultiD, SE). Results: CTCs were found in 42% of the MBC patients. Based on the AdnaTest, two distinct groups of CTC positive-MBC patients have been identified after principal component analysis of the gene expression data. The first CTC-positive group had relatively high EPCAM, MUC1, KRT19, HER2, AURKA, and MCM expression. In contrast, expression of PTEN, SATB1 and CD45 was low. The second CTC-positive group showed lower expressions of AURKA, EPCAM, and KRT19 than the first group. Furthermore, markers typically expressed in CD45 positive cells like SATB1, AKT, PARP, PTEN, mTOR, but also oncogene MYC and tumor suppressor TP53 were highly expressed in this group. The samples of CTC-negative MBC patients were characterized by low expression of the cancer phenotype associated genes EPCAM, KRT19, and MUC1, but showed comparably higher expression of genes associated with a stem cell like phenotype such as TWIST, ALDH1 and CD24. Conclusions: Based on our data, we conclude that CTCs enriched by the AdnaGen-technology are heterogeneous and do not necessarily express EPCAM. Even CTCs with low EPCAM expression but with elevated KRT19 and AURKA levels may represent a subgroup of high risk metastatic BC patients. Follow-up of the patients will show whether specific gene profiles of CTCs will a) identify responders and non-responders to breast cancer related therapies and b) be useful for personalized therapy of MBC in the future.

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 3398. doi:1538-7445.AM2012-3398