bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract LB-296: Synergistic anticancer action of inhibitor of DNA methylation 5-aza-2-deocycytidine and EZH2 histone methylase inhibitor 3-deazaneplanocin A
Authors: Momparler Richard L. , Marquez Victor E. , Momparler Louise F.
Published: February 2011

The purpose of this study was to investigate the antineoplastic action of epigenetic therapy using 5-aza-2′-deoxycytidine (5-AZA-CdR) in combination with 3-deazaneplanocin A (DZNep). Aberrant epigenetic modifications play an important role in oncogenesis. DNA hypermethylation, which can silence tumor suppressor genes (TSGs), can be reversed by treatment with 5-AZA-CdR, a potent inhibitor of DNA methylation. Overexpression of EZH2 methyltransferase (HMT) can also contribute to neoplastic transformation by silencing differentiation genes and TSGs. Recent studies have shown that DZNep, an inhibitor of HMT, can reactivate certain TSGs and has activity as an antineoplastic agent. Inhibitors of DNA methylation and histone deacetylation are reported to produce synergistic reactivation of silent TSGs and synergistic anticancer activity. Our objective was to determine if this type of “cross-talk” also exists between inhibitors of DNA methylation and inhibitors of HMT. Our experimental approach was to treat leukemic and tumor cells with 5-AZA-CdR and/or DZNep and determine cell survival by in vitro colony assays. Treatment of human HL-60 myeloid leukemic cells for 24 h with 50 nM 5-AZA-CdR or 500 nM DZNep produced a loss of clonogenity (LC) of 50.0 ± 5.8% and 75.6 ± 4.15, respectively. 5-AZA-CdR in combination with DZNep at the same concentrations of each agent produced a LC of 99 ± 1% (n =3). The interaction between these two different types of epigenetic agents was clearly synergistic. We also performed colony assays with 5-AZA-CdR plus DZNep on human A549 lung carcinoma cells. These epigenetic agents in combination also produced a synergistic LC on these tumor cells. The epigenetic action of 5-AZA-CdR plus DZNep on malignant cells of different phenotype suggests that this type of treatment may show effectiveness in most types of cancer. Our current objective is to verify the synergistic interaction between inhibitors of DNA methylation and HMT in mouse models with cancer. We are also investigating action of these agents alone and in combination on expression of cancer-related genes in order to understand the molecular basis of the synergistic anticancer activity.

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 LB-296.
Abstract LB-3: All-trans retinoic acid (ATRA) downregulates ALK in neuroblastoma cell lines and induces apoptotic cell death in neuroblastoma cell lines with activated ALK
Authors: Futami Hitoyasu , Sakai Ryuichi
Published: February 2011

Neuroblastoma is the most common extracranial solid tumors in children and high-risk patients with such tumors have a poor prognosis despite recent advances with multimodal therapies. Retinoic acids have been clinically used for some cancers including neuroblastoma although the mechanism of their antitumor effect remains to be fully elucidated. It was recently reported that gene amplification and gain-of-function mutation of ALK were found in tumors from some neuroblastoma patients; and RNAi or small molecule inhibitors of ALK induced apoptotic cell death in neuroblastoma cell lines with these genetic alterations of ALK. In this study, we found that all-trans retinoic acid (ATRA) downregulated ALK expression in neuroblastoma cell lines; and the suppression of ALK by ATRA accompanied apoptotic cell death in neuroblastoma cell lines with activated ALK.

We used four neuroblastoma cell lines: NB-39-nu cells with gene amplification of ALK, SH-SY5Y cells with gain-of-function mutation of ALK as well as TNB-1 and GOTO cells without such genetic alterations. Western blotting analysis demonstrated that the levels of ALK protein were remarkably reduced in all cell lines treated with ATRA at the concentration of 1 μM or 10 μM for 72 h. Analysis by quantitative real-time RT-PCR for mRNA expression levels in ATRA-treated NB-39-nu cells revealed that the ALK mRNA quantitative levels of the cells reduced at 24 h after such treatment. Apoptosis assays demonstrated obvious apoptosis in NB-39-nu and SH-SY5Y cells whereas it was not evident in TNB-1 and GOTO cells. Cleaved caspase-3 was also detected in NB-39-nu and SH-SY5Y cells 72 h after treatment with ATRA, but not in TNB-1 and GOTO cells. In addition, we observed cell growth inhibition that was accompanied with apoptosis in NB-39-nu and SH-SY5Y cells treated with ATRA. These results suggest that ALK downregulation by ATRA might lead to apoptotic cell death in neuroblastoma cells with activated ALK. This finding further suggests that ALK downregulation by retinoic acids could contribute to their therapeutic effect in a subset of neuroblastoma patients.

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 LB-3.
Abstract LB-30: Smac mimetic sensitizes antitumor activity of bacillus Calmette-Guerin stimulated neutrophils against bladder cancer cells
Authors: Jinesh Goodwin G. , Chunduru Srinivas K. , Kamat Ashish M.
Published: February 2011

Bacillus Calmette-Guerin (BCG) is the current gold standard therapy for bladder cancer. It is believed to work via recruitment of neutrophils to the tumor microenvironment and releasing cytokines, specifically TRAIL. However, BCG mediated therapy is hampered by relapse and resistance problems indicating the need for alternative or combination therapeutic options. Smac mimetics provide an alternative platform to rescue cells from TRAIL resistance by circumventing the IAP mediated inhibition of caspases. Herein we show that the KU-7 and RT4v6 bladder cancer cell lines are resistant to cell death with BCG stimulated neutrophils alone in vitro but a Smac mimetic kills bladder cancer cells in combination with BCG stimulated neutrophils. The sensitization of bladder cancer cells to Smac mimetic and BCG stimulated neutrophils is associated with the dowregulation of cIAP2 and cIAP1. TNF-α, TRAIL or Smac mimetic compound could not induce significant cell death as single agents in these cell lines, where as Smac mimetic compound in combination with TNF-α or TRAIL induced potent and massive cell death supporting our hypothesis. Our results suggest that Smac mimetics present a viable mechanism based approach to combination therapy with BCG for bladder cancer.

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 LB-30.
Abstract LB-319: A novel cancer-testis antigen, BORIS-based vaccine delivered by dendritic cells is effective against metastatic disease
Authors: Mkrtichyan Mikayel , Ghochikyan Anahit , Loukinov Dmitry , Movsesyan Nina , Davtyan Hayk , Laust Amanda K. , Lobanenkov Victor , Nelson Edward L. , Agadjanyan Michael
Published: February 2011

Although broad clinical benefits of anti-tumor immunotherapy have not been demonstrated, the current data suggest such strategies could be beneficial if capable of inducing strong anti-cancer cellular immune responses. We report testing for the first time, the therapeutic efficacy of a dendritic cell (DC)-based immunotherapy strategy targeting a novel cancer testis (CT) antigen, Brother of Regulator of Imprinted Sites (BORIS), which is an epigenetically acting tumor promoting transcription factor expressed in various human and mouse cancer cells.

In addition to therapeutic potency, we evaluated the cellular immune responses by assessing BORIS-specific CD4 T cell proliferation, T cell cytokine production both by intracellular cytokine staining and ELISpot analysis, and CTL responses. We evaluated the impact of this therapy on the development of metastatic disease, by analyzing the number of spontaneous clonogenic metastases after a 10-day culture of lung homogenates in presence of 6-thioguanine. The presence of CD4, CD8, Treg and myeloid derived suppressor cells (MDSCs) in the tumor and in the spleen was analyzed by flow cytometry.

DC loaded with zink-finger deleted (mutated) BORIS vaccine (DC/mBORIS) was evaluated in the non-immunogenic, very aggressive and highly metastatic mouse model of 4T1 mammary carcinoma. Vaccination of mice after 4T1 tumor implantation inhibited tumor growth (18.75% mice remained tumor-free) and dramatically lowered number of spontaneous clonogenic metastases (50% mice remained metastases-free). Higher numbers of immune-effector CD4 and CD8 T cells infiltrated the tumors of vaccinated mice compared to control animals. Therapeutic vaccination with DC/mBORIS significantly decreased the numbers of MDSC infiltrating the tumor, but not splenic MDSC, likely associated with the effect of the immunotherapy on tumor burden rather than a direct effect on MDSCs. Currently, we are testing the suppressive activity of splenocytes from tumor-bearing mice as well as MDSC isolated from these splenocytes. These data suggest that mBORIS is an attractive target for immunotherapy having substantial efficacy in this highly aggressive tumor model when incorporated into a DC-based immunotherapy, and which may be enhanced even further in combination with agents to attenuate tumor-associated immune suppression.

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 LB-319.
Abstract LB-327: Targeted microbeads for attraction and induction of specific innate immune response in the tumor microenvironment
Authors: Pitcovski Jacob , Shahar Ehud , Aizenshtein Elina , Gorodetsky Raphael
Published: February 2011

Anti-tumor activity of molecules and cells of the innate immune system has been reported. Here we propose a method for targeting preferred innate immune cells and magnifying their tumoricidal effect at the tumor microenvironment, by modular multiple-component complexes. As a model, micro-complexes carried monoclonal anti-HER2 antibodies, lipopolysaccharide and mannose. The complexes showed high binding capacity to HER2-positive cancer cells in vitro, high induction of interleukin-1 RNA transcription by the activated monocytes and ability to mediate monocytes’ attachment to HER2-positive cells. We thus present a new vision of targeting a desired innate immune response to the tumor microenvironment.

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 LB-327.
Abstract LB-329: Defining rgulatory T-cells as CD4+CD25hiCD127- or as CD4+CD25hiFoxP3+ in immune monitoring of cancer vaccine clinical trials
Authors: Clifton Guy T. , Patil Ritesh , Clive Kevin , Tyler Josh , Zacharia Athina , Jama Yusuf , Mursal Mohamed , Ciano Kathy , Holmes Jarrod P. , Mittendorf Elizabeth A. , Peoples George E. , Ponniah Sathibalan
Published: February 2011

Background: Regulatory T cells (Treg) are widely accepted to play a key role in the immune tolerance and escape of cancer cells. Originally Tregs were identified as being CD4+ T cells expressing high levels of CD25 (IL2 receptor). The identification of the transcription factor FoxP3 resulted in defining Tregs as CD4CD25hiFoxP3+ T cells by staining permeablized cells. Recently Tregs have been identified as CD4CD25hi T cells with low levels of CD127 (IL7 receptor) expressing a CD4+CD25hiCD127- cell surface phenotype. In an ongoing clinical trial with a MHC class II HER2/neu pep tide vaccine we have evaluated these various definitions of Treg phenotypes for immunological monitoring.

Methods: Tregs were quantified in disease-free, high risk BCa patients enrolled in adjuvant peptide-based breast cancer vaccine trials. Peripheral blood mononuclear cells (PBMCs) were both permeabilized and stained with CD4, CD25, and FoxP3 monoclonal antibodies and surface stained with CD4, CD25, and CD127. Samples were then analyzed by flow cytometry. The means are compared with t-test and correlation is determined with Pearson's correlation coefficient.

Results: 196 PBMC samples from 72 patients were available for Treg quantification. There was a greater mean percentage of cells identified as Treg in permeabilized than nonpermeabilized cells by staining with CD4+CD25hi (5.9±0.1% vs 4.8 ±0.1%, p<0.01) and excellent correlation between the tests (r=0.78, p<0.01). There was a greater mean percentage of cells identified as Tregs by CD4+CD25hi than CD4+CD25hiFoxP3+ (5.9±0.1% vs 3.6±0.1%, p<0.01) with good correlation (r=0.68, p<0.01). There was a greater mean percentage of Tregs by CD4+CD25+ than CD4+CD25+CD127- (4.8±0.1 vs 4.0±0.1, p<0.01) with excellent correlation (r=0.89, p<0.01). There was a greater mean percentage of Tregs by CD4+CD25hiCD127- than Tregs by CD4+ CD25hiFoxP3+ although levels were similar (4.0±0.1 vs 3.6±0.1, p<0.01) and the two tests had good correlation (r=0.58, p<0.01).

Conclusion: There was good correlation between various methods of defining Tregs with and without cell membrane permeabilization. Defining Treg cells as either CD4+CD25hiFoxP3+ or CD4+CD25+CD127- appear to be more specific than defining Tregs as CD4+CD25hi alone. Furthermore, defining Treg populations by surface staining as CD4+CD25hiCD127- yields similar levels with good correlation as CD4+CD25hiFoxP3+ and may be the preferred method for immune monitoring.

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 LB-329.
Abstract LB-338: Increased apoptosis and p53 expression in sclerotome of COX-2 transgenic embryo
Authors: Shim Minsub , Foley Julie , Anna Colleen , Mishina Yuji , Eling Thomas E.
Published: February 2011

Cyclooxygenases (COXs) have important functions in various physiological and pathological processes. COX-2 expression is highly induced by a variety of stimuli and is observed during certain periods of embryonic development. We examined the direct effect of COX-2 expression on embryonic development using a novel COX-2 transgenic mouse model which ubiquitously expresses human COX-2 from the early stages of embryonic development. COX-2 transgenic fetuses exhibit severe skeletal malformations and die shortly after birth. Skeletal malformations are localized along the entire vertebral column and rib cage and are linked to defective formation of cartilage anlagen. The cartilage anlagen of axial skeleton fail to properly develop in transgenic embryos because of impaired pre-cartilaginous sclerotomal condensation, which results from the reduction of cell number in the sclerotome. Despite the ubiquitous expression of COX-2, the number of apoptotic cells is highly increased in the sclerotome of transgenic embryos, but not in other tissues, suggesting that it is a tissue-specific response. Therefore, the loss of sclerotomal cells due to an increased apoptosis is likely responsible for axial skeletal malformations in transgenic fetuses. In addition, the sclerotomal accumulation of p53 protein is observed in transgenic embryos. Our result suggests that COX-2 signaling may act upstream of p53 and positively regulate the level of p53.

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 LB-338.
Abstract LB-342: Matrix metalloproteinase-9 is a novel pathogenic factor in B-cell chronic lymphocytic leukemia
Authors: Redondo-Muñoz Javier , Ugarte-Berzal Estefanía , Terol María-José , Van den Steen Philippe E. , del Cerro Mercedes Hernández , Roderfeld Martin , Roeb Elke , Opdenakker Ghislain , García-Marco José A. , García-Pardo Angeles
Published: February 2011

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation in peripheral blood of CD5+ B lymphocytes, which ‘ progressively infiltrate lymphoid tissues. Several molecules, including matrix metalloproteinase-9 (MMP-9), are involved in these processes and could represent therapeutic targets. We previously showed that MMP-9 plays an essential role in B-CLL cell migration through basement membranes or endothelial cells (Redondo-Muñoz et al., Blood 108: 3143, 2006). We also recently demonstrated that alpha4beta1 integrin and a 190 kDa CD44v isoform are receptors for MMP-9 in B-CLL but not in normal B cells and that the MMP-9 hemopexin domain is essential for these interactions. Binding of MMP-9 to B-CLL cells inhibited cell migration and this required MMP-9 proteolytic activity (Redondo-Muñoz et al., Blood 112: 169, 2008). To further define the role of surface-bound MMP-9 in B-CLL we have employed several techniques including western blot, zymography, immunofluorescence, immunoprecipitation, siRNA transfection, cell adhesion/soluble binding assays, apoptosis, and cell migration assays. B-CLL cells cultured on pro- or mature MMP-9, proMMP-9 hemopexin domain or proMMP-9 catalytically-inactive mutant, showed decreased spontaneous apoptosis. This was due to induction of a survival pathway involving Lyn activation, STAT3 phosphorylation and Mcl-1 upregulation and did not require catalytic activity. This pathway was induced in all B-CLL cases and was active in B-CLL lymphoid tissues. Our results highlight the role of MMP-9 in B-CLL pathogenesis and indicate that MMP-9 hemopexin domain could constitute a therapeutic target in this malignancy. Supported by grants PI060400 and SAF2009-07035 from the Ministerio de Ciencia e Innovación and by the Fundación de Investigación Médica Mutua Madrileña.

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 LB-342.
Abstract LB-345: Functional genomics reveals acute dependencies on polyadenylation for tumor cell mitotic progression
Authors: Cappell Kathryn , Sciaky Noah , Larson Brittany , Whitehurst Angelique
Published: February 2011

We are using genome-wide synthetic lethal paclitaxel chemosensitizer screens to understand the protein networks that support mitosis in lung tumor cells. Using this approach, we have identified several components of the polyadenylation machinery that are required for a high-fidelity mitosis in the presence of a synthetic lethal dose of paclitaxel. Here, we show depletion of multiple polyadenylation proteins in combination with low-dose paclitaxel causes a G2/M cell cycle block. We show depletion of one of these polyadenylation proteins, symplekin, has profound effects on mitotic spindle assembly resulting in over-duplicated centrosomes and impaired microtubule growth. Furthermore, we use a panel of lung lines to show symplekin is uniquely required for tumor cell mitosis as compared to normal cell mitosis. Our mechanistic studies suggest symplekin may mediate these effects on tumor cell mitosis by interacting with the centrosomally-associated protein transforming acidic coiled coil 3 protein (TACC3). Interestingly, TACC3 is typically expressed during development but becomes aberrantly upregulated in tumor cells. We propose TACC3 may become upregulated during tumorigenesis and engage symplekin to support mitosis in tumor cells. These results suggest polyadenylation components may represent an attractive therapeutic target in combination with already available anti-mitotic agents.

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 LB-345.
Abstract LB-353: From worms to humans: understanding the role of microRNAs in cancer progression
Authors: Esquela-Kerscher Aurora
Published: February 2011

MicroRNAs (miRNAs) regulate important developmental events and are often misexpressed in human cancers, but little is known regarding how these small RNAs contribute to tumor formation. Our laboratory uses a combination of nematode and mammalian model systems to functionally characterize miRNAs and determine the role these factors play in controlling cancer progression pathways. We have successfully used the nematode to analyze how members of the lin-4 and let-7 miRNA families functionally overlap to direct common developmental events associated with cell cycle progression and differentiation. Specifically, animals that carry a triple deletion for the lin-4 homologue, mir-237, and the let-7 homologues, mir-48 and mir-84, exhibit morphological defects and fertility problems related to abnormal mitotic and meiotic abnormalities in the germline that were not found in the single or double deletion combinations. We have also begun to determine how the let-7 miRNA family functions as tumor suppressor genes in mammals. Our experiments showing that exogenous let-7 miRNA administration in the lung using a conditionally activated KRAS lung cancer mouse model result in reduced lung tumor load in these animals highlight the therapeutic potential of miRNAs. We are also studying the role of let-7 together with other miRNA families in directing cancer progression pathways in human tissues of urothelial origin. For instance, we find that let-7 is dysregulated in human prostate cancer cell lines differing in their metastatic status and indicates a novel role for this miRNA in prostate cancer progression. Due to our access to a vast repository of human prostate specimens from the Virginia Prostate Center at Eastern Virginia Medical School, we have begun to analyze the miRNA expression profiles in FFPE specimens taken from the primary prostate tumor, normal adjacent tissue and the lymph node metastasis of individual prostate cancer patients as well as in tumors from patients presenting insignificant and organ confined cancer. We are also determining the “miRNA signatures” in blood, urine, and expressed prostatic secretions (EPS) that were obtained non-invasively from both normal and cancer patients. Our work investigating the role of miRNAs during cancer progression promises to reveal novel therapeutic approaches and diagnostic and prognostic biomarkers for human cancer.

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 LB-353.
Abstract LB-356: Characterization of microRNA expression of clear cell renal cell carcinoma by whole genome small RNA deep sequencing using human tissue cohort
Authors: Wu Huiqing , Weng Lihong , Wu Xiwei , Gao Hanlin , Mu Bing , Li Xuejun , Jin Jennifer , Wang Jin-hui , Guo Chao , Chen Zhuo , Weiss Lawrence
Published: February 2011

Introduction: MicroRNA (miRNA) is a group of small non-coding regulating RNA and has shown altered expression in cancer. With great advantages in test specificity and capability to detect novel (unknown) miRNA and other small RNA targets, the next-generation deep sequencing technology is replacing microarray platforms, at least in the discovery phase. Clear cell renal cell carcinoma (ccRCC) is the most common type of kidney malignancies and one of the leading causes of death. Characterizing miRNA expression of ccRCC will enhance the understanding of its tumorigenesis and progression and will, therefore, lead to the development of cancer-specific molecular therapy. In this study, we analyzed miRNA expression using a human ccRCC cohort by whole genome small RNA deep sequencing. We characterized the miRNA expression in association with CCRCC and further validated the altered miRNA expression in a larger clinical cohort by RT-PCR. A group of tumor associated novel miRNAs have been discovered.

Design: (1) Deep sequencing of whole genome small RNA (17-52 nucleotides in size) in a 6-sample frozen CCRCC cohort (3 benign and 3 ccRCC samples) using Illumina system (Solexa) was performed. (2) The miRNA expression of benign kidney and ccRCC samples was quantitatively compared. (3) 8 miRNAs, which were randomly selected from the list of top 20 aberrantly expressed miRNAs detected by deep sequencing, were tested using ABI RT-PCR technology in a 38-sample ccRCC cohort (9 benign kidney and 29 different stage ccRCC samples) for validation. (4) Analysis of unknown small RNAs was performed to find tumor associated novel miRNAs.

Results: (1) Using a deep sequencing technology, we detected miRNAs and other small RNAs in frozen human samples of ccRCC cohort and successfully profiled miRNA expression in association with tumor. (2) We validated the altered expression of 8 selected miRNA targets in the 38-sample clinical ccRCC cohort using an RT-PCR method. (3) We discovered a group of novel miRNAs associated with ccRCC.

Conclusions: We performed whole genome small RNA deep sequencing of human ccRCC tissue with further RT-PCR validation in a clinical sample cohort. We have characterized miRNA expression and discovered a group of novel miRNAs in association with ccRCC.

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 LB-356.
Abstract LB-357: Efficient identification of tumor of unknown primary origin by its microRNA profile
Authors: Søkilde Rolf , Møller Anne K. , Vincent Martin , Nielsen Boye S. , Hansen Alastair , Daugaard Gedske , Møller Søren , Litman Thomas
Published: February 2011

Background: Carcinomas of unknown primary origin (CUP) constitute 3-5% of all newly diagnosed metastatic cancers, the primary source of which is hard to classify with current histological methods, often because CUPs are poorly differentiated. Since effective cancer treatment depends on early and proper identification of the tumor, CUP patients have poor prognosis and short survival rates. Increasing evidence suggests that microRNAs play an important role in the initiation and progression of cancer, and because the expression of microRNAs is highly tissue specific, the microRNA profile of an unknown tumor can be used to identify its tissue of origin. Here, we present a microRNA based classifier, which based on a sample's microRNA profile, can predict its tissue origin with high accuracy.

Methods: Total RNA was extracted from 1115 formalin fixed paraffin embedded (FFPE) samples, and subjected to expression analysis on an LNA (Locked Nucleic Acid) enhanced microarray platform, which allows for very sensitive and specific detection of 2090 microRNAs. Based on the microRNA expression profiles, we tested and cross-validated several classification algorithms, including KNN (K nearest neighbors), LDA (linear discriminant analysis), SVM (support vector machines) and LASSO (least absolute shrinkage and selection operator) to assess, which method is best suited for the current multiclass classification task.

Results: The currently best classifier for tumors of unknown primary origin is based on the LASSO algorithm, which consistently is able to identify 15 different tumor histologies with 90% or higher accuracy based on a small set of microRNAs. We are currently assessing the classifier's performance on true unknown primary tumors, where the correct diagnosis has been obtained at autopsy.

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 LB-357.
Abstract LB-359: Plasma microRNAs as novel biomarkers for lung cancer
Authors: Zheng Dali , Wang Michael X.
Published: February 2011

Objective: Lung cancer is the leading cause of cancer-related deaths in both men and women. Since there is no validated population-based screening procedure, most patients with lung cancer are diagnosed at advanced stages with an overall five-year survival rate of only 15%. Therefore, diagnosis of lung cancer at an early stage is important for improving the outcome of patients. MicroRNAs (miRNAs), a family of 19- to 25-nucleotide and noncoding small RNAs that primarily function as gene regulators at post-transcriptional level, are frequently dysregulated in cancer. Recent studies demonstrate that the miRNAs in blood are present in notably stabile form and are readily detectible by various sensitive methods. This new finding has raised a concept that circulating miRNAs could be novel non-invasive biomarkers for cancer detection. The objective of this study is to investigate the potential of circulating microRNAs for lung cancer detection.

Methods: First, we searched the miRNA microarray data of lung cancer from published literature, and selected 15 of miRNAs (miR-17, 21, 24, 106a, 125b, 128, 155, 182, 183, 197, 199b, 203, 205, 210 and 221) that were most frequently up-regulated in lung cancer tissues. Total RNA including miRNAs were isolated with mirVana PARIS kit (Ambion, TX), then polyadenylated and reverse-transcribed with a poly(T) adapter into cDNAs for real-time PCR using the miRNA-specific forward primer and the sequence complementary to the poly(T) adapter as the reverse primer. The levels of miRNAs were determined in 28 plasma samples from lung cancer patients and 16 of controls.

Results: We found that the levels of miR-155, miR-182 and miR-197 in plasma of lung cancer patients were significantly elevated compared with controls. MiR-155 yielded an AUC (the areas under the ROC curve) of 0.8739 (95% CI:0.7489 to 0.9989, P<0.001), miR-182 yielded an AUC of 0.8426 (95% CI:0.7239 to 0.9614, P<0.001) and miR-197 yielded an AUC of 0.8037 (95% CI:0.6705 to 0.9369, P<0.001) in discriminating lung cancer from controls.

Conclusions: MiR-155, miR-182 and miR-197 are significantly elevated in patient plasma with lung cancer and can be a potential non-invasive molecular biomarker for lung cancer screening and clinical follow-up.

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 LB-359.
Abstract LB-360: Fluorescence-based co-registration with protein markers reveals distinct cellular compartments for altered microRNA expression in solid tumors
Authors: Sempere Lorenzo F. , Preis Meir , Yezefski Todd , Ouyang Haoxu , Suriawinata Arief A. , Silahtaroglu Asli , Conejo-Garcia Jose R. , Kauppinen Sakari , Wells Wendy , Korc Murray
Published: February 2011

MicroRNAs (miRNAs) are a class of short non-coding regulatory RNA genes, which have been implicated in the control of developmental, physiological and pathological processes. High-throughput profiling experiments have linked altered expression of miRNAs to different types of cancer. To clarify the clinical significance of these expression changes, we developed a sensitive fluorescence-based in situ hybridization (ISH) method to visualize miRNA accumulation within individual cells in formalin-fixed paraffin-embedded tissues. This ISH method was implemented to be compatible with routine automated clinical immunohistochemical (IHC) assays to enable co-detection with protein markers on the same tissue section for co-localization and functional studies. Here, we utilized this robust and rapid combined ISH/IHC assay to study a subset of commonly highlighted miRNAs including tumor suppressive miRNAs (let-7 and miR-34) and oncogenic miRNAs (miR-21 and miR-155) in a panel of breast, colon, lung, pancreas and prostate carcinomas, which collectively are the leading causes of cancer-related mortality in the United States. Despite the idiosyncrasies of each particular cancer type, general trends emerged that pinpointed to distinct cellular sources of altered miRNA expression. While altered expression of miR-21 and miR-34 was manifested within cancer cells, that of miR-126 and miR-155 was predominantly confined to endothelial cells and immune cells, respectively. These findings suggest a heterogeneous and complex participation of miRNAs in carcinogenesis occurring through cancer cell-autonomous effects as well as through the modulation of stromal, vascular and immune responses. We discuss potential applications of miRNA-based ISH/IHC assays as novel diagnostic and prognostic tools, as well as clinical implications of our findings for miRNA-based therapeutic strategies.

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 LB-360.
Abstract LB-364: Dovitinib (TKI258), a multikinase inhibitor of FGFR, PDGFR, and VEGFR tyrosine kinases, induces growth suppression in mouse models of human hepatocellular carcinoma (HCC)
Authors: Huynh Hung T. , Chow Pierce , Soo Chee Khee , Shi Micheal
Published: February 2011

Background: Vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF) and their receptors are over-expressed and involved in the neovascularization, invasiveness, and metastatic potential of HCC. Therefore, suppression of the VEGFR/FGFR/PDGFR by TKI258 may represent a novel approach for treatment of HCC. The aims of this study were 1) to determine the in vivo antineoplastic activity of dovitinib (TKI258), a multikinase inhibitor of FGF, VEGF, and PDGF signaling pathways, and 2) to compare the efficacy of TKI258 with sorafenib in mouse models of human HCC.

Experimental Design: We treated six patient-derived HCC xenografts with i) 50 or 75 mg/kg TKI258 and ii) 50 mg/kg sorafenib. Western blotting was employed to determine pharmacodynamic changes in biomarkers relevant to FGF/VEGF/PDGF signaling pathways. Apoptosis, microvessel density, and cell proliferation were analyzed by immunohistochemistry.

Results: Body weight was maintained up to 75 mg/kg TKI258 and 50 mg/kg sorafenib. TKI258 treatment resulted in suppression of tumor growth in a dose-dependent manner. Potent antitumor activity was observed in all six HCC xenograft lines studied. The T/C ratio's for 50 mg/kg TKI258 and 50 mg/kg sorafenib for xenograft lines 06-0606, 26-0808A, 26-1004, 28-1108, 5-1318, and 25-0705A, were 0.04 and 0.22; 0.11 and 0.18; 0.13 and 0.26; 0.23 and 0.37; 0.14 and 0.30, and 0.28 and 0.19, respectively, suggesting that in general the antitumor effect of TKI258 was greater than that of sorafenib. TKI258-induced growth inhibition was associated with inhibition of angiogenesis and cell proliferation and induction of tumor cell death. Inhibition of p-FGFR, p-PDGFR, p-VEGFR-2, p-ERK1/2, and upregulation of Bim and p-histone H2A-X Ser139 were observed.

Conclusion: TKI258 demonstrated significant antitumor effect in a mouse model of human HCC xenograft. This study provides a strong rationale for clinical investigation of TKI258 in patients with HCC.

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 LB-364.
Abstract LB-366: A role for hNDPK-B in breast cancer metastasis in vivo.
Authors: Yokdang Nucharee
Published: February 2011

Cancer cells promote angiogenesis early in tumorigenesis under mutagenic/hypoxic conditions that drive oncogene expression of pro-angiogenic proteins. Clinical data suggest that by the time women are diagnosed with breast cancer, micrometastases are extant but suppressed by the primary tumor. It is imperative that we discover how to suppress metastases at or before breast biopsy, lumpectomy or mastectomy. We have shown in vitro that human breast cancers secrete nucleoside diphosphate kinase (sNDPK), which generates ATP from ADP locally to act at ATP-P2Y receptors locally to regulate blood flow, cell migration, and vascular permeability consistent with intravasation and extravasation of cancer cells and angiogenic stimulation at the metastatic niche. The ability of ATP-P2 receptor stimulation to transactivate the endothelial VEGF receptor in the absence of VEGF suggests the possibility that cancer cell-secreted NDPK, endothelial P2Y receptors, and VEGF receptors interact as partners in tumor angiogenesis in vitro. Our in vitro data strongly suggest a role for NDPK blockers and P2-receptor antagonists in adjuvant therapy for breast cancer (US Patent No. 10/331,375). We have now extended our studies of purinergic regulation of angiogenesis in vivo in a breast cancer metastasis model in mice carrying luciferase-tagged human breast carcinoma cells (MDA-MB-231Luc+) to permit tracking of primary tumor development and tumor metastases. We hypothesized that orthotopic-human tumors growing in SCID mice modulate extracellular sNDPK-B levels to attract angiogenic growth, development and metastasis. Examination of serum using Western blot and an ELISA assay of our own design revealed that tumors secreted sNDPK-B in large amounts two weeks after subcutaneous injection of cancer cells in the mammary fat pad. There is a correlation between secreted NDPK-B and tumor size. We confirmed the activity of sNDPK-B as a phosphoprotein trans-phosphorylase. PCR studies suggest that in more aggressive tumors, expression of sNDPK-B mRNA is increased (r2=0.97), while there is no correlation between intracellular NDPK-B and extracellular sNDPK-B expression. Differential mRNA expression may be seen between primary and metastatic cells and could underscore the role of sNDPK-B in mediating metastatic tumor development. Studies underway with mice carrying tumors and implanted with osmotic drug-delivery pumps will determine the effect of NDPK-B blockers, purinergic receptor antagonists and combined therapy on tumor burden and metastasis development and regression.

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 LB-366.
Abstract LB-367: Wnt-dependent transcription factor LEF-1 controls endothelial cell invasion through changes of MMP-2 expression
Authors: Planutis Kestutis , Planutiene Marina , Holcombe Randall F.
Published: February 2011

Background: Wnt signaling is involved in many events including embryogenesis, stem cell regulation, morphogenesis, cell fate determination, and oncogenesis. The role of Wnt pathway in angiogenesis is proposed, however the mechanism is still unclear. Most cancers express elevated levels of different Wnts when compared to normal cells. LEF-1 is a participant in and a target gene of the Wnt/ -catenin pathway. LEF-1 increases the in vitro invasiveness of cancer cells.

Hypothesis: We suggest that LEF-1 controls the invasion also in endothelial cells. We hypothesize further that cancer affects local tumor angiogenesis via Wnt pathway-dependent up-regulation of LEF-1 in endothelial cells (EC).

Methods: To test this hypothesis we construct a model system: endothelial cell line EaHy926 treated with Wnt3a (mostly known as a canonical Wnt signal) conditioned medium or grown in co-culture with L-cells producing Wnt3a.

Results: Wnt3a increases nuclear ß-catenin in human endothelial cell line, and up-regulates LEF/TCF-dependent promoter activity. Wnt3a augments its target gene's LEF-1 promoter activity and LEF-1 mRNA concentration, demonstrating involvement of some transcription factors with E-tails. Both Wnt3a treatment and LEF-1 overexpression elevate MMP-2 mRNA level. Elimination of Wnt-dependent induction of MMP-2 gene expression by LEF-1 siRNA confirms the LEF-1 role in the MMP-2 control mechanism. Loosening up the extracellular matrix MMPs allows the cells to move around. These enzymes are integral part of invasion process. Indeed, LEF-1 up-regulates endothelial cells invasiveness in Matrigel matrix. LEF-1 increases also EC proliferation, in agreement with published data for parental primary endothelial culture HUVEC.

Conclusions: In endothelial cells canonical (ß-catenin) Wnt signaling enhances invasiveness of EC via LEF-1 - dependent regulation of MMP-2 expression. There is a number of similarities between primary cells HUVEC, studied by others, and cell line EAhy926 in their response to Wnt3a. This indicates that EAhy926 cell line conserves many components of angiogenesis control by Wnt/β-catenin pathway and could serve as a simplified model to study this regulation, replacing primary endothelial cells.

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 LB-367.
Abstract LB-371: Altered vascular endothelial growth factor mRNA stability may lead to cancer development following immunosuppressive therapy
Authors: Basu Aninda , Datta Dipak , Pal Soumitro
Published: February 2011

The development of cancer is an increasing and major problem in immunosuppressed patients, particularly after solid organ transplantation. In part, the increased cancer risk is associated with the use of immunosuppressive agents, especially calcineurin inhibitors (CNIs). The CNIs are used to prevent inflammatory diseases and allograft rejection. However, little is known about the mechanism(s) underlying their ability to promote the development and recurrence of cancer. We have recently demonstrated that CNI can mediate the transcriptional activation of the angiogenic cytokine vascular endothelial growth factor (VEGF) and promote a rapid progression of human renal cancer. In contrast, the mTOR inhibitor rapamycin (RAPA), which is thought to have anti-cancer effects, has been reported to decrease VEGF expression. Here, we investigated whether the CNI cyclosporine (CsA) and RAPA could alter the mRNA stability of VEGF in 786-0 and Caki-1 renal cancer cells. By using actinomycin-D, we observed that CsA increased, while RAPA decreased the VEGF mRNA stability as observed by real time PCR. It is established that the mRNA-binding protein HuR may play a critical role in VEGF mRNA stability. By using HuR-siRNA, we found that the knockdown of HuR significantly decreased the CNI-induced VEGF mRNA stability. By Western blot analysis, it has been observed that CNI treatment induced the translocation of HuR from the nucleus to the cytoplasm; CNI also induced the association between HuR and protein kinase C (PKC)-δ, and promoted the phosphorylation of HuR. Interestingly, there was no significant change in the cytoplasmic translocation of HuR following RAPA treatment. Finally, we found that the inhibition of PKC-δ using a dominant-negative plasmid significantly decreased the CsA-induced cytoplasmic translocation of HuR and the VEGF mRNA stability. Together, we suggest that targeting the pathways that promote the transcription as well as the mRNA stability (post-transcriptional modification) of VEGF in response to CNI might serve as novel therapeutics for the prevention and treatment of cancer in immunosuppressed patients.

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 LB-371.
Abstract LB-372: Activation of focal adhesion kinase in cancer cells is regulated by crosstalk between PKCα and MDA-9/syntenin
Authors: Hwangbo Cheol , Lee Suhyun , Lee Jeongmin , Kang Jungwon , Lee Jeong-Hyung
Published: February 2011

mda-9/Syntenin, a tandem PDZ-domain containing adaptor protein, functions as a positive regulator of cancer cell progression in several human cancers. Protein kinase Cα (PKCα) is a key intermediate between integrins and focal adhesion kinase (FAK) signaling in response to fibronectin (FN), but the molecular mechanism by which PKCα regulates FN-induced FAK activation remains unclear. Here, we report that mda-9/syntenin links FN-induced activation of FAK and PKCα in human breast cancer and melanoma cells. FN induced mda-9/syntenin expression and PKCα activation in a similar manner prior to FAK activation. PKCα inhibition resulted in the suppression of the endogenous and FN-induced mda-9/syntenin expression, as well as mda-9/syntenin-induced cell migration and invasion toward FN. mda-9/Syntenin expression and PKCα activation was interdependently regulated; e.g., inhibition of mda-9/syntenin suppressed both FN-induced and endogenous phosphorylation of PKCα at Thr638/641, and inhibition of PKCα suppressed both FN-induced and endogenous expression of mda-9/syntenin. Moreover, inhibition of either mda-9/syntenin or PKCα suppressed the FN-induced association of integrin- 1/FAK/c-Src signaling complexes. Consistently, suppression of either mda-9/syntenin or PKCα significantly inhibited FN-induced phosphorylation of FAK at Tyr397 and c-Src at Tyr416, activation of downstream signaling molecules such p38 and ERK mitogen-activated protein kinases (MAPKs), Cdc42 GTPase, and nuclear factor-κB (NF-κB). These findings demonstrate that PKC -dependent mda-9/syntenin up regulation could play a critical role in FN-induced activation of FAK, and mda-9/syntenin may act as a molecular adaptor that links PKCα and FAK during adhesion to FN in human breast cancer and melanoma cells.

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 LB-372.