bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 123: Cooperative regulation between proliferative signals by activated Ras and inhibitors of apoptosis (IAPs) in gliomagenesis
Authors: Mukherjee Joydeep , Wolf Amparo , Hawkins Cynthia , Guha Abhijit
Published: February 2011

Transformation requires not only aberrant proliferation through signaling pathways such as activated Ras as demonstrated by our lab in GBMs, but also aberrant inhibition of regulators of apoptosis. Towards the latter, little is known about the expression and function of a family of proteins known as Inhibitors of Apoptosis Proteins (IAPs), which includes cIAP1, cIAP2, XIAP and Survivin, in GBMs. Human tumors which are prevalent in activating Ras mutations, such as colon and pancreatic cancers, produce high amounts of Survivin. We hypothesize that elevated activity of Ras in GBMs, as previously described by us, leads to aberrant expression of IAPs in GBMs and through its anti-apoptotic functions plays a role in glioma transformation.

Our previously described GFAP:12V-HaRas (RasB8) transgenic mouse glioma model was utilized. Elevated expression of activated Ras in the mouse gliomas was accompanied with increased expression of XIAP and Survivin. This was also prevalent in expression in human GBM cells, with elevated Ras activity. Knockdown of Ha-Ras in human and mouse GBM cells, by transfecting with couple of siRNA targeted against Ha-Ras gene, significantly decreased XIAP and Survivin levels. Knockdown of Ha-Ras in the mouse and human GBM cells increased their sensitivity to apoptosis inducing chemotherapy.

Current experiments include a genetic approach to down regulate Ras activity, by expression of a dominant-negative form of the Ha-Ras (Ha-Ras N17), in GBM cells and evaluating regulation of XIAP and Survivin expression. These experiments will be complemented with farnesyl transferase inhibitors to also inhibit Ras activity in the glioma cells. A Tet-inducible strategy to over express activated Ha-Ras in both transformed and non-transformed mouse and human astrocytes is being undertaken. These in vitro studies will be complemented by in vivo studies involving breeding our RasB8 glioma model to double transgenics with GFAP-Cre regulated decreased expression of Survivinflox/flox in astrocytes, with a postulated decrease in gliomagenic potential. The results to date and those underway are highly suggestive of the thesis that elevated Ras activity leads to glial transformation by not only mitogenic signals, but also by cooperative expression of anti-apoptotic proteins, such as IAPS.

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 123.
Abstract 1233: AP-1 transcription factor is involved in breast cancer cell proliferation mediated by progestins
Authors: Flaqué María C. Díaz , Beguelin Wendy , Rosemblit Cinthia , Proietti Cecilia , Rivas Martin A. , Tkach Mercedes , Charreau Eduardo H. , Schillaci Roxana , Elizalde Patricia
Published: February 2011

Accumulating evidence has shown the involvement of the progesterone receptor (PR) in breast cancer development. We and others have also shown that progestin are able to induce cyclin D1 expression, a key regulatory molecule that does not contain a progesterone response element (PRE) in the promoter region. In the present study, we propose a novel mechanism in which progestin controls breast cancer growth through the integration of rapid PR signaling and a transcriptional mechanism (tethering), that involves progesterone-bound PR interaction with AP-1 transcription factor (composed of Jun and Fos family members), at specific AP-1 binding sites (TRE) in the cyclin D1 promoter. MPA treatment of breast cancer cells induced an increase in the levels of cyclin D1 protein. We have also shown that MPA treatment of breast cancer cells induced an increase in the levels of c-jun and c-fos phosphorylation. To study the effect of MPA on AP-1-mediated transcriptional activity, cells were transiently transfected with a luciferase reporter plasmid containing three copies of TRE. We found that MPA enhanced AP-1 transcriptional activity and this effect was abolished by the antiprogestin RU486. We assessed the specific association of AP-1 and PR to the TRE region of the cyclin D1 gene in the context of living cells, by performing Chromatin Immunoprecipitation Assays. We found that MPA treatment induced c-jun, c-fos and PR recruitment to the cyclin D1 promoter. These data identify, for the first time, the interaction between AP-1 and PR regulating cyclin D1 transcription by tethering to DNA-bound at TRE site. Furthermore, we found that the inhibition of c-fos and c-jun activation by the use of dominant negative forms of these proteins (A-Fos and TAM-67 respectively) completely blocked progestin-induced cyclin D1 expression and in vitro breast cancer cell growth. Finally, we addressed the effect of targeting AP-1 in in vivo MPA-dependent growth of C4HD progestin-dependent murine mammary tumor. Transfection of C4HD cells with TAM-67 or A-Fos DN expression vectors significantly inhibited these cells’ ability to form tumors in syngeneic mice. Histological examination determined a striking decrease in histological grade in both groups in comparison to control group.

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 1233.
Abstract 1237: WT1, Wnt signaling and E-cadherin in prostate cancer
Authors: Brett Adina , Eisermann Kurtis , Fraizer Gail
Published: February 2011

Prostate carcinoma is the most common malignancy and second leading cause of death among American men. The molecular mechanisms that control the progression of this type of cancer are still poorly understood. Among the genes proposed to play a role in prostate cancer is the zinc finger transcription factor, WT1. Previous studies in our lab have shown WT1 protein in a majority of high grade prostate tumor sections with little or no WT1 staining in non-neoplastic or benign prostatic hyperplasia (BPH) tissues. However, a mechanistic role for WT1 in prostate cancer has not been established. Recently, WT1 has been associated with the Wnt signaling pathway and Beta catenin, the central molecule in the pathway. The cytoplasmic levels of Beta catenin can be regulated through the canonical Wnt signaling or through the intracellular adhesion complex where it binds to E-cadherin. Importantly, E-cadherin has previously been identified as a WT1 target gene in NIH 3T3 cells. The objective of this study was to determine whether WT1 might regulate genes that, in turn, regulate the levels of Beta catenin in prostate cancer cells. First potential WT1 binding sites were identified in the promoters of two candidate genes, GSK3 Beta and E-cadherin, using a bioinformatics approach. Then the effect of transfection of LNCaP prostate cancer cells with GFP-tagged WT1 expression constructs was tested by quantitative real time PCR (QRT-PCR). In vivo evidence of direct binding of these gene promoters by WT1 protein was obtained by Chromatin immunoprecipitation (ChIP) of GFP/WT1 transfected LNCaP cells. Using evolutionary conservation analysis we identified two WT1 binding sites in the E-cadherin promoter conserved among primate species and one that was conserved among primates, rodents and dog. QRT- PCR results showed a decrease in E-cadherin transcripts in GFP/WT1 transfected LNCaP cells, compared with untransfected cells. ChIP was used to demonstrate that this effect on E-cadherin expression was direct and mediated by DNA binding to the E-cadherin promoter in vivo. Overall these results suggest that WT1 modulates E-cadherin and the Wnt signaling pathway in LNCaP cells. These findings suggest a novel mechanism for proliferation and migration of prostate cancer cells and ultimately may contribute towards an improved cancer therapy.

This work was supported by NIHR15CA11360 (GF) and GSS KSU grant(AB)

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 1237.
Abstract 1238: Diagnostic and therapeutic potential of nuclear receptor expression in lung cancer
Authors: Jeong Yangsik , Lee Woochang , Xie Yang , Bookout Angie L. , Kim Bong-hyun , Girard Luc , Minna John D. , Mangelsdorf David J.
Published: February 2011

Lung cancer is the leading cause of cancer related death with the highest mortality among cancers. Although several studies have been published on prognostic biomarkers for lung cancer patients, little attention has been given to identify diagnostic biomarkers and new therapeutic targets. Nuclear receptors are druggable targets due to their ligand-controlled activity, and indeed have become well-established therapeutic tools for treating patients with strong estrogen receptor alpha (ERα)-positive breast cancer or androgen-dependent prostate cancer. Our aims in this study were (1) to investigate if the NR superfamily as a whole or individually showed diagnostic potential, (2) to evaluate NRs as therapeutic targets using both in vitro cell lines and in vivo xenografts.

Utilizing quantitative real-time PCR (QPCR), we profiled the mRNA expression of the NR superfamily in a panel of 56 lung cell lines, including 5 normal human bronchial epithelial cells, 10 immortalized cell lines, 14 small cell lung cancers (SCLCs), and 27 non-SCLCs. All individual cell lines were characterized by DNA fingerprinting and gene expression using the affymetrix U133 A plus B microarray. Unsupervised cluster analysis of the QPCR profile and the microarray signature of the NR superfamily revealed that the NR superfamily was able to segregate normal/immortalized cells from tumor cell lines, and SCLCs from non-SCLCs. Further analysis identified subsets of NRs responsible for this discriminatory ability. In addition, using publicly available microarray datasets, we found that the NR signature has a predictive power with an accuracy of 79% for lung cancer incidence in smokers (n=129), in which a prediction model was constructed in a training set (n=77) using penalized logistic regression (PLR), and validated in a testing cohort (n=52). Finally, preclinical evaluation of a panel of NRs ¬- androgen receptor, ERα, vitamin D receptor, and peroxisome proliferators-activated receptor gamma - for their therapeutic potential showed receptor-specific and ligand-dependent cell growth responses in lung cancer cell lines. Evaluation of one receptor/ligand pair using a mouse xenograft model confirmed receptor-specific tumor growth response in vivo.

Collectively, our study provides preclinical evidence that the expression of the NR superfamily yields theragnostic (therapy plus diagnosis) information for predicting lung cancer type and incidence, while simultaneously offering therapeutic targets for potential lung cancer treatment.

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 1238.
Abstract 1242: Expression of steroid receptor coactivators and HER-2/neu in normal and malignant breast tissue in controls and tamoxifen treated tumors
Authors: Moi Line H. , Flaageng Marianne H. , Gandini Sara , Guerrieri-Gonzaga Aliana , Bonanni Bernardo , Lazzeroni Matteo , Gjerde Jennifer , Lien Ernst , Decensi Andrea , Mellgren Gunnar
Published: February 2011

The complex agonist/antagonist effects of tamoxifen may partly be explained by changes in nuclear receptor coactivator expression and activity. In addition, overexpression of the epidermal growth factor receptor HER-2/neu is associated with poor prognosis and tamoxifen resistance in conjunction with coactivator SRC-3/AIB1.

In this study, 64 patients with ER positive breast cancer were randomly treated with 20, 5 or 1 mg tamoxifen/day for 4 week preoperatively. 28 ER positive patients were included as not-treated controls. Specimens at the time of surgery were collected from tumor tissue and normal breast tissue below 1 cm from the tumor. Real-time PCR analyses were performed to measure mRNA expression of the members of the steroid receptor coactivator (SRC) family; SRC-1, SRC-2/TIF-2, SRC-3/AIB1 and the receptor HER-2/neu. The mRNA levels were analyzed with the focus on effects of tamoxifen dose, tissue type, as well as potential correlation to the plasma levels of the biomarkers IGF-I, SHBG and immunohistochemical expression of Ki-67.

The mRNA-levels of SRC-1, SRC-2/TIF-2, SRC-3/AIB1 and HER-2/neu were all significantly higher in tumors compared to normal tissue (P < 0.001). Tamoxifen significantly increased mRNA expression level of the coactivators in a dose-independent manner in both tissue-types, with the clearest effect for SRC-3/AIB1 (P < 0.001). There was no difference in HER-2/neu mRNA levels in tamoxifen treated samples compared to controls. We observed no significant correlation between mRNA levels of the coactivators and HER-2/neu with IGF-1, SHBG or Ki-67.

In conclusion, nuclear receptor coactivators were significantly higher both in breast tumors and normal tissue after tamoxifen treatment; however there was no difference in mRNA levels between the tamoxifen doses of 1, 5 or 20 mg/day. Whether such an increase in expression of coactivators could be a sign of effective endocrine treatment or an attempt of the cells to shift back to a more estrogen sensitive state, remains to be elucidated.

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 1242.
Abstract 1244: RXR agonist induced changes in gene expression in liver and mammary cancers: Confirmation by RT-PCR on independent samples collected years apart
Authors: Cvetkovic Dusica , Clapper Margie , Wang Yang , You Ming , Muccio Donald , Grubbs Clinton , Lubet Ronald
Published: February 2011

Several years ago, our laboratries examined the effects of short-term treatment with the RXR agonist Targretin, a highly effective preventive agent in rodent mammary models, on gene expression. Oligonucleotide microarrays were employed both in normal tissues (liver, lung and mammary gland) and in ER+ mammary cancers (Wang, et al., Molecular Cancer Therapeutics, 2006). It was thought that the gene expression changes observed might serve as potential pharmacodynamic markers, as well as giving mechanistic insights into the efficacy of Targretin. Numerous changes in Targretin treated livers and literally hundreds of changes in Targretin treated mammary cancers were observed by microarray analysis. Pathway alterations in Targretin treated mammary cancers included those for MAPK and TGF-beta signaling. In the present RT-PCR studies, a separate laboratory employed different samples collected approximately four years after the originals. Roughly 20 genes whose expression was modulated in the original liver arrays and a similar number of genes from the original mammary cancer arrays were examined. Of the genes examined in the rat liver, which we expected to be relatively homogeneous, roughly 75% were confirmed by RT-PCR (e.g., cytosolic acyl-CoA thioesterase, CYP 2B2, IGFBP2). More surprisingly, approximately two-thirds of the gene changes observed in the rat mammary cancers (e.g., MMP12, apolipoprotein D, CYP4B1) were confirmed. These results imply that even in different samples collected years apart one can confirm gene expression changes observed in arrays in a relatively homogeneous tissue such as liver. Furthermore, one can confirm gene expression changes observed in mammary cancers, a result potentially more surprising since one might expect substantial heterogeneity in those samples. We are also currently examining expression changes of these same genes following treatment with a series of RXR agonists that are structurally dissimilar to Targretin. Targretin is a cyclic compound, while the analogs have a linear 9-cis-backbone (Muccio et al, Carcinogenesis, 2006). In conclusion, the present data support the use of arrays to identify pharmacodynamic markers that may be utilized with agents of the same class.

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 1244.
Abstract 1246: Mechanism of endostatin action in prostate cancer in dependent on androgen receptor signaling
Authors: Isayeva Tatyana , Chanda Diptiman , Tsuladze George , Moore Lakisha , Ponnazhagan Selvarangan
Published: February 2011

We recently reported that stable expression of endostatin and angiostatin arrested the progression of well differentiated prostate carcinoma to poorly-differentiated state and distant metastasis in TRAMP mice when given prior to the onset of metastasis switch. Analysis of the expression of angiogenesis-related genes in the prostate tissue of endostatin treated mice indicated a significant downregulation of genes involved in cell motility, proliferation, metastasis and angiogenesis when therapy was initiated at early stage-disease. In vitro experiments demonstrated that recombinant endostatin treatment significantly downregulated the expression of growth factors, receptor tyrosine kinases and proteases in androgen sensitive cells. A similar pattern was confirmed in prostate tissues obtained from in vivo studies. Targeted downregulation of androgen receptor (AR) prior to endostatin treatment indicated that the effect of endostatin through AR downregulation is by a non-genotypic mechanism on MAPK/ERK pathway, and independently of Src kinase. Further analysis indicated the kinetics of endostatin internalization for androgen sensitive prostate cancer cells to be much higher than in androgen independent cells. Co-immunoprecipitation studies in human prostate cancer cells, and yeast two-hybrid studies with endostatin and AR demonstrated a direct binding of endostatin protein with AR. Based on the results so far, we predict that direct interaction of endostatin with AR possibly eliminates ligand binding during early stages of prostate cancer arresting and delaying the progression of the disease. However, once metastatic switch occurs, when the cells grow in androgen independent manner, a limitation in endostatin internalization or binding of non-androgen ligands at other domains in AR impairs the tumoristatic ability of endostatin. Ongoing studies are aimed in identifying interaction domains of endostatin with AR by NMR as well as testing the combination of endostatin therapy with therapies targeting AR signaling.

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 1246.
Abstract 1249: The role of ATM-dependent Chk2 activation on post-transcriptional gene regulation by RNA-binding protein, HuR: implications for lymphoma development in AT patients
Authors: Mazan-Mamczarz Krystyna , Hagner Patrick , Dai Bojie , Liu Zhenqiu , Gartenhaus Ronald B.
Published: February 2011

Maintenance of genomic stability depends on the DNA damage response a biological tumorigenesis barrier in early stages of cancer development. In response to DNA double strand breaks, ATM phosphorylates multiple proteins involved in both cell cycle checkpoints and DNA repair including the checkpoint kinase Chk2. The human disease ataxia-telangiectasia (AT) in which the ATM gene is deleted or inactivated is characterized by progressive neurodegeneration, immunodeficiency and genomic instability. AT patients show a hypersensitivity to radiotherapy and an extraordinary high risk of developing pediatric lymphomas. Recently it was reported that Chk2 interacts and phosphorylates the RNA-binding protein HuR, leading to alteration of it RNA-binding ability. HuR is a key post-transcriptional regulator of genes involved in stress response, cell cycle, immunoresponse, tumorigenesis, and senescence.

To gain a better understanding of the complexity of the ATM/Chk2/HuR regulatory pathway we investigated the influence of ATM/Chk2 signaling on HuR binding activity in the DNA damage response by global microarray examination of mRNAs associated with HuR in response to ionizing radiation (IR) in both ATM wild type and ATM null lymphocytes. Importantly, pharmacological inhibition either ATM or Chk2 proteins, or knockdown of Chk2 expression using RNA interference, reduced the HuR-mRNA association with a number of validated target genes. Mutation of Chk2 phosphorylated residues in HuR suggests Ser100 site to be important for increased association and Thr118 dissociation of HuR-mRNA complexes after IR.

Our data demonstrated that low dose (1Gy) of IR activates the ATM signaling cascade, but not ATR pathway, resulting in the sequential phosphorylation of Chk2 and HuR, subsequently modulating the association of HuR with target mRNAs and thereby regulating these gene's expression. Functional network analysis revealed signaling pathways, which are frequently deregulated in lymphoid malignancies to be predominantly represented by transcripts differentially associated with HuR after exposure to IR in ATM wild type compared to ATM null lymphocytes. A better understanding of the mechanism and affected species of mRNA could help to develop more effective targeted treatments in ATM null 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 1249.
Abstract 1253: Combination of chloroquine to inhibitors of the HER family receptors leads to synergistic growth inhibition of breast cancer cell lines
Authors: Shah Apurva N. , Ling Yi-He , Perez-Soler Roman
Published: February 2011

Autophagy is emerging as a new mechanism of cellular escape from different forms of cellular stress including the cytotoxic effects of chemotherapeutic agents. There is preclinical evidence that this mechanism may explain tumor resistance to chemotherapy as well as to biologic agents. In this study, we investigated the effects of the combination of inhibitors of autophagy and inhibitors of the HER family of receptors in breast cancer cell lines. Chloroquine (CQ) is a 4-aminoquinolone with antimalarial, immunosuppressive, and anti-autophagy activity. It raises the intra-lysosomal pH thereby impairing protein degradation and causing accumulation of autophagosomes. Erlotinib (E) is an inhibitor of the EGFR tyrosine kinase and trastuzumab (T) is a monoclonal antibody against the HER2 receptor.

We used the SK-BR-3 cell line (high ErbB1 and ErbB2 expression), and the MCF-7 cell line (low ErbB1 and ErbB2 expression) and studied the effect of T alone, E alone, and in combination with CQ on cell growth inhibition using the MTT assay. Our results indicate that combinations of CQ and T or E for 7 days cause synergistic cell growth inhibition in SK-BR-3 cells (combination index 0.17-0.31) but have no effect on cell growth inhibition in MCF-7 cells.

Flow cytometry analysis revealed that in SK-BR-3 cells exposure to T or E induces G1-phase accumulation and S-phase blockade. However, combinations of T or E with CQ did not further inhibit cell cycle progression. Analysis of apoptosis using the TUNEL reaction assay or by FACS analysis indicated that the combined treatment increases apoptotic cell death associated with increased caspase-3 activity as compared with the treatment with T or E alone, suggesting that activation of apoptotic signal pathways may be involved in the synergistic effect.

Finally, we determined the effect of the combination of CQ and T or E on receptor activation as well as downstream signaling and found that combined treatment enhanced the inhibition of ErbB2 as well as Akt phosphorylation, but had no effect on Erk activation as compared with T or E alone in SK-BR-3 cells.

Conclusion: The combination of CQ with T or E in breast cancer cell lines with high ErbB1 and ErbB2 expression results in synergistic growth inhibition, enhanced apoptosis, and increased inhibition of ErbB signaling via the PI-3K/AKT pathway. This enhanced cytotoxic activity and its potential clinical utility should be explored further. This work was supported in part by NIH grant CA84119 and CA96515.

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 1253.
Abstract 1262: Bif-1 is required for keeping mitochondrial and lysosomal positive feedback loops in check to prevent apoptosis induced by death receptors in type II cells
Authors: Park Sungman , Komine Kimiyo , Takahashi Yoshinori , Wang Hong-Gang
Published: February 2011

Bif-1, also known as endophilin B1 and SH3GLB1, is involved in the regulation of apoptosis, mitochondrial morphogenesis, and autophagy. We have previously shown that increasing the levels of Bif-1 accelerates Bax conformational change, caspase-3 activation, and apoptotic cell death in response to a variety of intrinsic apoptotic stimuli, whereas the loss of Bif-1 delays all these processes. Here, we report the role of Bif-1 as a negative regulator in the extrinsic pathway of apoptosis by controlling mitochondrial and lysosomal amplification loops in type II cells. There are two different types of cells distinguished by their distinct extrinsic apoptotic signaling pathways. In type I cells, the activation of caspase-8 by death receptor ligation is sufficient to activate downstream effector caspases (e.g. caspaspe-3) for cell killing, whereas in type II cells, caspase cascade amplification through mitochondria and/or lysosome is required. We found that depletion of Bif-1 dramatically enhances not only caspase-3 but also caspase-8 activation and cell death in type II cell lines including MEF, HeLa, and Jurkat, but not in type I H9 cells, following treatment by the extrinsic apoptotic stimuli TNFα/cycloheximide, TRAIL or Fas ligand. Overexpression of Bcl-XL or XIAP in Bif-1 knockout MEFs significantly diminishes the enhanced caspase activation, indicating that a mitochondrial feedback loop is elevated by the loss of Bif-1. Moreover, while treatment with cathepsin inhibitors does not suppress TNFα/cycloheximide-induced caspase-3 activation in Bif-1 knockout MEFs, it completely blocks the activation of caspase-3 in Bif-1 knockout MEFs overexpressing Bcl-XL or XIAP, implying that lysosomal pathway is also involved. Furthermore, inhibition of caspase-8 is able to completely block the activation of caspase-3 induced by TNFα/cycloheximide, indicating that caspase-8 initiates the mitochondrial and lysosomal amplification loops observed in Bif-1 deficient type II cells. Taken together, these studies suggest that Bif-1 plays an important role in the regulation of caspase-8-initiated mitochondrial and lysosomal positive feedback loops for killing type II cells by death receptors.

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 1262.
Abstract 1265: RbBP6 Isoform 3 (DWNN) is a p53 stabilizer in arsenic trioxide-induced apoptosis in human cancer cell lines
Authors: Dlamini Zodwa , Mbita Zukile , Skepu Amanda , Pugh David , Rees Jasper , Meyer Mervin
Published: February 2011

Domain With No Name (DWNN) has a ubiquitin-like fold and shares 22% similarity with ubiquitin. This suggests its significant importance in cell homeostasis and protein degradation in a human body. The presence of a ubiquitin-like fold suggests a role similar to ubiquitin and RbBP6 may be involved in ubiquitin ligase-like activities. Furthermore RbBP6 interacts with both p53 and pRb which are key components of cell cycle regulation. These two tumour suppressors are key to the understanding the function of the RbBP6. It has been found that upon arsenic trioxide-induced treatment RbBP6 isoform 3 (DWNN) is up-regulated. In this study the role of the DWNN on p53-dependent apoptosis was investigated.

Over-expressions of RbBP6 isoforms was achieved by transfection of recombinant isoforms using Metafectene Easy. Apoptosis was induced using arsenic trioxide and staurosporine. Apoptosis was then measured using APOPercentage and flow cytometry. The expression of RbBP6 gene products and p53 was determined using both real-time PCR and western blotting.

Up-regulation of RbBP6 was observed in As2O3-induced apoptosis in cancer cells. Over-expression of the RbBP6 isoform 1 intensified As2O3-induced apoptosis. MTT assay showed an IC50 of 40µM for As2O3. When this concentration was used to induce apoptosis in human cancer cells and this resulted in induced expression of RbBP6 isoform 1 not the isoform 3. Over-expression of RbBP6 isoform 3 though resulted in stabilization of p53 protein. The apoptosis that was observed was also accompanied by the expression of p53. This study suggests that the DWNN may not be directly involved in the prosecution of apoptosis but may be involved through p53 stabilization. This study suggests that the RbBP6 may be a p53 stabilizer in contrast to mdm2, which is a negative regulator of p53. It will be very important to also determine the relationship between RbBP6 and pRb in both cell cycle and apoptosis

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 1265.
Abstract 1267: The RING domain of TRAF2 inhibits TNFα-induced cell death independent of NF-κB activation
Authors: Zhang Laiqun , Blackwell Ken , Shi Zhaohui , Habelhah Hasem
Published: February 2011

TNFα is a pleiotropic cytokine that activates both the pro-survival IKK/NF-κB and the pro-apoptotic FADD/caspase-8 pathways. TNF receptor associated factor 2 and 5 (TRAF2/5) contain N-terminal RING domains, and TRAF2/5 RING domain-mediated RIP1 ubiquitination is believed to be essential for NF-κB activation and inhibition of cell death in response to TNFα stimulation. In support of this notion, TRAF2/5 double knockout (TRAF2/5 DKO) cells are sensitive to TNFα-induced apoptosis. In addition, the RING domain-deleted TRAF2 (TRAF2-ΔR) has been widely used in the field as a dominant negative to block TNFα-induced JNK and IKK activation in transient overexpression systems. Here, we report that stable expression of TRAF2-ΔR in TRAF2/5 DKO cells at a physiological level completely restores normal TNFα-induced NF-κB activation, but that it neither restores RIP1 polyubiquitination nor inhibits cell death. We also found that the basal and inducible expression of anti-apoptotic proteins in TRAF2-ΔR-expressing cells is normal, yet these cells remain sensitive to TNFα-induced cell death because anti-apoptotic proteins were not recruited to the TNFR1 complex efficiently. This suggests that two events are required for the inhibition of TNFα-induced cell death: i) NF-κB-dependent expression of anti-apoptotic proteins such as cIAP1/2 and cFLIP; and ii) TRAF2 RING domain-dependent retention of cIAP1 in the TNFR1 complex. Ablation of either event leads to caspase-8 activation and cell death in response to TNFα stimulation. Our data also suggest that RIP1 ubiquitination is not essential for TNFα-induced NF-κB activation.

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 1267.
Abstract 1273: Involvement of PKCδ and NOX4 in reactive oxygen species-mediated tumor resistance to TRAIL-induced cell death
Authors: Choi Kyungsun , Ryu Seungwook , Song Seungjeong , Choi Hyosook , Choi Chulhee
Published: February 2011

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF family of cytokines, causes apoptosis by caspase activation in various cell types, particularly in transformed cells. Numerous types of tumors are relatively resistant to TRAIL-induced cytotoxicity; however, the reasons for this are not yet fully understood. We report here a new signal transduction pathway involving protein kinase Cδ (PKCδ), NADPH oxidase 4 (NOX4) and reactive oxygen species (ROS), that inhibits caspase-dependent cell death induced by TRAIL ligation in human malignant astrocytoma cells. In our experiments, TRAIL ligation-induced generation of intracellular ROS through caspase-dependent proteolytic activation of PKCδ and subsequent activation of the NOX4 complex. Suppression of intracellular ROS induction using various pharmacological inhibitors or PKCδ- or NOX4-specific RNA interference enhanced the enzymatic activity of caspase-3 by blocking the oxidative modification of its catalytic cysteine residue, resulting in marked augmentation of TRAIL-mediated cell death. In addition, combined treatment with ROS scavengers enhanced TRAIL-induced cytotoxicity in an experimental tumor model. These results collectively indicate that TRAIL-induced activation of PKCδ and NOX4 can modulate TRAIL-mediated apoptosis by promoting oxidative modification of active caspase-3 in a negative-feedback manner.

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 1273.
Abstract 1274: Heterotrimeric stimulatory GTP-binding proteins inhibit cisplatin-induced apoptosis by inducing XIAP in cervical cancer cells
Authors: Cho Eun Ah , Juhnn Yong-Sung
Published: February 2011

Cisplatin (cis-dichlorodiammineplatinum (II)) induces DNA double-strand breaks (DSBs), which are considered as the main cytotoxic damage that induces apoptosis of human cancer cells. Cisplatin was also known to found to inhibit DNA-synthesis and DNA-repair in HeLa cervical carcinoma cells. Recently, we observed that heterotrimeric G proteins modulate apoptosis signaling in several cancer cell lines. In this study, we attempted to investigate the effect of Gαs on apoptosis triggered by cisplatin and its molecular mechanism in HeLa cells. Expression of constitutively active Gαs (GαsQL) decreased the cleavage of caspase-3 and PARP at 24 hr after treatment of HeLa cells with 30 µM cisplatin, indicating that Gαs inhibited cisplatin-induced apoptosis of HeLa cells. Expression of GαsQL increased the expression of XIAP (inhibitor of apoptosis protein), and partially recovered the cisplatin-induced decrease in XIAP. Knock down of Gαs expression by shRNA reduced the expression of XIAP and augmented cisplatin-induced apoptosis. This study shows that Gαs can protect apoptosis triggered by cisplatin by up-regulation of XIAP in HeLa cervical cancer cells. These results suggest that Gαs signaling may contribute carcinogenesis and progression of cervical 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 1274.
Abstract 1275: Expression of Nur77 induced by an n-butylidenephthalide derivative promotes apoptosis and inhibits cell growth in oral squamous cell carcinoma
Authors: Liu Po-Yen , Su Yi-Chang
Published: February 2011

Background: In spite of numerous advances, the 5-year survival rate for head and neck squamous cell cancer has remained largely stagnant, and few new anti-tumor drugs have been developed for oral squamous cell carcinoma (OSCC). PCH4 ((Z)-N-(2-(dimethyl-amino) ethyl)-2-(3-((3-oxo-isobenzofuran-1(3H)-ylidene) methyl) phenoxy) acetamide), a derivative of n-butylidenephthalide (BP), was investigated for anti-tumor effects on OSCC.

Aim: The aim of this study was to investigated the mechanism of Nur77 translocation induced by PCH4

Methods: OSCC cells were treated by PCH4 in vitro and in vivo. The Nur77 translocation was detection by western blotting, immunohistochemistry and immunocytochemistry.

Results: PCH4 promoted Nur77 translocation from the nucleus to the cytoplasm and induced cell apoptosis in OSCC cells. When Nur77 translocation was prohibited by Nur77 short interfering RNAs, the degree of tumor apoptosis caused by PCH4 was significantly alleviated. Within the MAPK pathway, PCH4 induced only JNK phosphorylation. Furthermore, treatment with a JNK inhibitor significantly reduced PCH4-induced apoptosis (p<0.05) and decreased PCH4-induced Nur77 expression (p<0.05). In a xenograft animal model, administration of PCH4 showed similar anti-tumor effects in OSCC cells that were dose dependent. Immunohistological staining showed similar effects on Nur77 expression and apoptosis in xenografts.

Conclusion: We have demonstrated that OCSS cells are sensitive to PCH4 and that Nur77 protein translocation from the nucleus to the cytoplasm might be associated with the induction of apoptosis by PCH4. These results, both in vitro and in vivo, indicate that PCH4 may serve as a potential anti-tumor drug for OSCC therapy.

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 1275.
Abstract 1276: TRAIL-induced p38 activation responsible for increases of Akt catalytic and Invasive activity
Authors: Sun Bo K. , Kim Joo-Hang , Kim So Y. , Oh Seeun , Choi Hye J. , Sohn Joo-Hyuk , Lee Yong J. , Song Jae J.
Published: February 2011

In this paper, we demonstrate that TRAIL-induced p38 activation is responsible for the increases of Akt catalytic activity and invasive activity. For the confirmation of increases of Akt catalytic and invasive activity induced by TRAIL-induced p38 activation, immune complex kinase assay and invasion assay was used. Previously, we observed that TRAIL induces acquired TRAIL resistance with increased Akt phosphorylation resulting from Src-PI3K-Akt signaling pathways mediating by c-Cbl. c-Cbl, an ubiquitously expressed cytoplasmic adaptor protein was shown to be involved in the rapid degradation of TRAIL receptors and simultaneously in the Akt phosphorylation during TRAIL treatment. However, Akt phosphorylation did not have an exclusive role for acquired TRAIL resistance. Like Akt catalytic activation during metabolic oxidative stress, TRAIL induced dramatically Akt catalytic activation in TRAIL-sensitive cells, whereas TRAIL did not induce Akt catalytic activation in TRAIL-resistant cells, suggesting Akt catalytic activation during TRAIL-induced apoptosis is likely to have a role of compensation for cell homeostasis. Our studies also reveal that p38 activation during TRAIL treatment is responsible for the increases of Akt catalytic and invasive activity.

Bo K. Sun and Joo-Hang Kim are equally contributed to this work.

Correspondence: Jae J Song (jjs109@yuhs.ac)

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 1276.
Abstract 1277: Differential roles of TRAIL death receptors during TRAIL-induced signaling pathways
Authors: Kim So Y. , Oh Seeun , Sun Bo K. , Choi Hye J. , Sohn Joo-Hyuk , Song Jae J. , Kim Joo-Hang
Published: February 2011

In this paper, we demonstrate that TRAIL death receptors (DR4, DR5) have their own distinctive function during TRAIL treatment. For the examination of each receptor's role, FACS analysis, biotin labeling to plasma membrane and siRNA technique were used. Previously, we have shown that TRAIL has induced anti-apoptotic signals as well as apoptotic signals. Here, we investigated whether TRAIL death receptors (DR4 and DR5) have their own unique function. In TRAIL-sensitive cell line (DU-145), TRAIL induced much more rapid degradation of DR4 than that of DR5. On the other hand, in TRAIL-resistance cell line (SK-BR-3) with higher level of ErbB2/pAkt, DR5 was rarely expressed, while DR4 was distinctively expressed. We also have shown that relatively much more amount of c-Cbl in SK-BR-3 has played a role of supplying additional pAkt which can be also persistently provided by ErbB2. These phenomena are confirmed by knock down of c-Cbl expression using adenovirus expressing si-c-Cbl. Taken together, we suggest that DR4 functions to transmit apoptosis signals with a favor for anti-apoptosis signals, whereas DR5 functions to transmit apoptosis signals without a favor for anti-apoptosis signals.

Correspondance: Jae J. Song (jjs109@yuhs.ac) and Joo-Hang Kim (kjhang@yuhs.ac)

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 1277.
Abstract 1280: Inhibition of cellular FLICE-inhibitory protein and induction of Death Receptor 5 expression is a major component of δ- tocotrienol-induced apoptosis in pancreatic cancer cells
Authors: Zhang Anying , Francois Rony A. , Hodul Pamela J. , Husain Kazim , Shivers Steven , Coppola Domenico , Sebti Said M. , Malafa Mokenge P.
Published: February 2011

Background: Previous pre-clinical studies have demonstrated that δ-tocotrienol, is the most bioactive natural vitamin E compound against pancreatic cancer. δ-Tocotrienol induces growth arrest and apoptosis in several human pancreatic cancer cell lines; however, the underlying mechanism remains unknown. In this study, we evaluated the role of the extrinsic and intrinsic apoptotic signaling pathways in δ-tocotrienol-induced apoptosis. Also, we investigated the modulation of cellular FLICE-inhibitory protein (c-FLIP) and death receptor 5 (DR5) by δ-tocotrienol, and the involvement of c-FLIP down-regulation and DR5 up-regulation in δ-tocotrienol-induced apoptosis.

Methods: We examined δ-tocotrienol induced apoptosis in human pancreatic cancer cell lines in vitro and in vivo. We used western blot and immunohistochemistry to determine the expression of DR4, DR5, Caspase 8, Caspase 9, Caspase 3, c-FLIP, and PARP. The effect of combination treatment with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and δ-tocotrienol was also determined. We examined the cellular effects of DR5 knockdown using small interfering RNA oligonucleotides, and c-FLIP overexpression using c-FLIP cDNA, on δ-tocotrienol-induced apoptosis using various assays.

Results: Delta-tocotrienol activated caspase-8 and its downstream caspases but not caspase-9. Caspase-8 specific inhibitor abrogated δ-tocotrienol-induced apoptosis whereas caspase-9 specific inhibitor did not, indicating that δ-tocotrienol induces caspase-8 -dependent apoptosis. c-FLIP was rapidly down-regulated by δ-tocotrienol in all of the tested cell lines. Delta-tocotrienol up-regulated DR4 and DR5 significantly and enhanced TRAIL-induced apoptosis. Enforced expression of ectopic c-FLIP, and DR5 knockdown partially abolished δ-tocotrienol-induced apoptosis. δ-Tocotrienol inhibition of pancreatic tumor growth in SCID mice was associated with significant induction of apoptosis, activation of caspase 8, and down-regulation of c-FLIP expression.

Conclusion: We conclude that DR5 up-regulation and c-FLIP down-regulation contributes to δ-tocotrienol-induced apoptosis in pancreatic cancer 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 1280.
Abstract 1283: Chromosome 19 amplification and the consequent NOTCH and VEGF pathways over-activation are associated with malignant glioma poor outcome
Authors: Assem Mahfoud , Sibenaller Zita , Agarwal Supreet , Al-Keilani Maha , Alqudah Mohammad , Marner Joyce , Ryken Timothy C.
Published: February 2011

Malignant gliomas are the most frequent type of primitive brain tumors. Their outcome did not improve despite new therapeutics, resulting mainly from their genetic heterogeneity and complexity. By using genome-wide explorations we have recently identified, for the first time, that chr.19 amplification correlates with a grim therapeutic outcome.

The aim of our current study is to investigate the significance of the chromosome 19 genetic signature. To narrow the search for specific genes or pathways that may be responsible for this adverse outcome, we selected genes located on chromosome 19 and focused on those that were statistically significantly deregulated between tumors and non tumor tissues. We identified 41 genes which mapped to chromosome 19. The most significant genes are NOTCH3 and HIF3α. These genes belong to NOTCH and VEGF pathways and play important roles in angiogenesis. Using quantitative real time PCR, RT-PCR and immunobloting, we further analyzed the level of gene expression of critical elements of these pathways and confirmed that NOTCH3 was amplified in primary glioma cell lines that harbor gains of chr.19. This increase has been shown at both the mRNA and protein levels. Moreover, NOTCH ligand, and other constituents of the pathways (DLL3, SHC3, NOTCH4) were also activated in these particular cell lines. Further, HIF3α was also activated in our conditions whereas HIF1α, the other player of HIF transcriptional pathway, was down regulated. These over-expressions were accompanied by a parallel VEGF activation in these cell lines.

This novel genetic signature may lead to critical insights into diagnosis, classification, prognosis, and prediction in improving therapeutic strategies and outcome based on the genetic makeup of the tumor.

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 1283.