bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1131: ING4 attenuates breast cancer cell growth in Matrigel by altering Wnt/beta-catenin signaling pathway
Authors: Raymond-Whish Stefanie L. , Kim Suwon
Published: February 2011

ING4 is a member of the INhibitor of Growth (ING) tumor suppressor family, which plays a role in various cellular processes and suppresses hallmarks of cancer. ING4 contains a plant homeodomain (PHD) finger motif that has been implicated in transcriptional regulation via chromatin remodeling. However, the molecular mechanism(s) of ING4 tumor suppressor function is not known. In breast cancer, the ING4 locus is deleted in 10-20% of primary tumors, suggesting its role as a tumor suppressor. To better understand tumor suppressive function of ING4 in breast cancer, we tested whether ING4 can influence cell growth in three-dimensional culture environment using Matrigel. We found that ING4 retarded proliferation of T47D human breast cancer cells in Matrigel. In order to identify downstream target genes of ING4 that may be responsible for this activity, we employed gene expression profiling assays. The results showed that ING4 down-regulated several genes in the Wnt/β-catenin pathway, including Wnt4, Wnt6, dishevelled 1, dishevelled 3, and frizzled 4. Deregulation of the Wnt/β-catenin signaling pathway has been shown in human breast cancer as well as to induce mammary tumors in mice. We conclude that ING4 attenuates T47D cell growth in Matrigel by down-regulating the Wnt/β-catenin signaling pathway. Our data suggests that ING4 may function as a tumor suppressor in breast cancer by suppressing Wnt/β-catenin signals.

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 1131.
Abstract 1145: Analysis of circulating tumor cells from castrate resistant prostate cancer patients captured via a microfluidic device using the prostate specific membrane antigen antibody
Authors: Loftus Matthew , Gleghorn Jason , Pratt Erica , Bander Neil , Nanus David , Kirby Brian , Giannakakou Paraskevi
Published: February 2011

Prostate cancer (PC) is the most common type of cancer in males and the second leading cause of male cancer deaths in the United States. Circulating tumor cells (CTCs) are commonly found in the blood of patients suffering from mestatic disease and the capture of these cells is important in the study of disease progression. Existing devices use the EpCAM antibody which can capture circulating epithelial cancer cells, but also leads to a large number of leukocyte contamination. We have developed a microfluidic chip functionalized with an antibody to the prostate-specific membrane antigen (PSMA), which captures prostate CTCs with high purity and efficiency. This chip is designed with a staggered obstacle array that maximizes the CTC-wall interactions while minimizing the interactions with the other cells in the blood. PSMA-positive or -negative PC cells were spiked into blood from healthy donors, and we calculated capture rates to be ∼80%. Next, blood samples from patients (n = 20) with castrate resistant prostate cancer (CRPC) were run through the device and CTCs were captured and enumerated. CTCs were found in 90% of samples tested with the number captured averaging 27 ± 4 cells per mL of blood with a capture purity of 62 ± 2%. We plan to use this device to elucidate the molecular basis of CRPC clinical response to taxane treatment, which is the only class of chemotherapy drugs shown to improve survival in this type of PC. Previous data from our laboratory has shown that taxane treatment of PC cells inhibits the ligand-induced androgen receptor (AR) nuclear accumulation and transcriptional activation of androgen response element (ARE) containing genes including the prostate-specific antigen (PSA). These results implicate microtubules (MTs) in AR trafficking and suggest that the clinical activity of taxanes in CRPC may be mediated in part by the drugs’ ability to stabilize MTs and inhibit AR transcriptional activity. We plan to capture CTCs using the PSMA-specific microfluidic device from CRPC patients receiving taxane treatment and analyze them for AR subcellular distribution and correlate clinical response to taxane treatment. Our microfluidic device is a novel, specific, and improved technology to capture prostate CTCs that will allow us to better define the molecular basis of taxane treatment in CRPC 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 1145.
Abstract 1159: Single molecule sequencing to detect and characterize somatic mutations in cancer genomes
Authors: Mardis Elaine , Maupin Rachel , Travers Kevin , Chin Chen-Shan , Eid John , Chau Benson , Londry Jason , Baybayan Primo , Magrini Vince , McLellan Mike , Wylie Todd
Published: February 2011

One result of large-scale cancer genome characterization is the identification of sets of genes that are commonly mutated in specific tumor types or subtypes and have clinical relevance, e.g. are prognostic or diagnostic. In this paradigm, we have investigated the use of a novel, single molecular real time (SMRT) sequencing technology from Pacific Biosciences that enables targeted regions to be sequenced in real time as single molecules in a mixed population. Several experiments were performed to evaluate the performance of this technology in the context of testing for cancer-specific mutations in previously characterized samples. In the first experiment we assessed whether SMRT sequencing could detect the known mutations of PCR products derived from genomic DNA of tumor cells compared to normal cells. In the second experiment we investigated the impact of different neoplastic cellularity percentages on the ability to detect known mutations. The final experiment involved producing deep read count SMRT sequencing data from PCR products containing known variants to ascertain their different levels of prevalence in a discrete tumor cell population, and then comparing these results to deep read counts for the same variants obtained with the Illumina instrument. Our results indicate that the Pacific Biosciences instrument offers exquisite sensitivity and speed in detecting somatic single base mutations in tumor-derived genomic DNAs.

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 1159.
Abstract 1163: Sensitive and specific detection of clinically relevant proteins in human testicular germ cell tumors using proximity ligation and quantitative PCR
Authors: Looijenga Leendert H. , Gillis Ad J. , Stoop Hans , Bierman Katharina , Swartzman Elana , Ferlinz Astrid , Oosterhuis Wolter J. , Shannon Mark
Published: February 2011

Human testicular germ cell tumors (TGCTs) are the most frequent solid cancer in Caucasian males between the ages of 15 and 45 years. In spite of the high success rate of current treatment strategies (surgery, irradiation and chemotherapy), TGCTs are the second leading cause of death in this age group. The initiating event, leading to carcinoma in situ (CIS), occurs during embryonal development, offering a window of opportunity for early detection and intervention, thereby preventing the need for intensive (systemic) treatment. CIS is the malignant counterpart of a primordial germ cell, and can be effectively diagnosed using immunohistochemisty for OCT3/4, NANOG, c-KIT and its ligand (stem cell factor, SCF). In addition, SOX17 and SOX2 are informative for the diagnosis of the invasive components seminoma and embryonal carcinoma. Currently, in a clinical setting, these proteins are detected using predominantly immunohistochemistry, an approach that is labor-intensive and non-quantitative. Here we present data on the detection of these proteins using a novel qPCR-based method, Taqman Protein Expression Assay, which is based on the Proximity Ligation Assay (PLATM) technology. The method was first validated on negative controls and a series of TGCT-derived cell lines with efficient siRNA-induced down-regulation of the targets, an informative system for investigating stem cell regulatory circuits. Subsequently, the assay was further developed to enable analysis of both snap frozen and formalin-fixed paraffin-embedded (FFPE) tissues. The assays were found to be fast (results in less than 1 day), highly reproducible (SD less than 0.2 CTs), sensitive (able to detect target protein from as few as 10-100 cells, depending on the level of expression of the protein under investigation), and quantitative. Taqman Protein Expression Assays therefore allow the systematic analysis of multiple proteins within a single small sample in a high throughput set up, and offer unique opportunities for sensitive, specific and quantitative protein detection in both experimental and clinical studies.

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 1163.
Abstract 1172: RARB is a biomarker of poor prognosis in prostate carcinomas
Authors: Alves Flavia C M C , Drigo Sandra A. , Rainho Cláudia A. , Camargo João L V , Fonseca Francisco P. , Soares Fernando A. , Trindade-Filho José C S , Domingues Maria A C , Rogatto Silvia R.
Published: February 2011

Purpose: Despite the advances in prostate carcinomas (PCa) management, about 30% of the patients who undergo radical prostatectomy presented biochemical recurrence (BCR). BCR is considered an unfavorable prognostic factor followed by clinical disease progression. The current prognostic markers in PCa are unable detect correctly who is at risk for BCR. Hypermethylation at RARB and RASSF1A genes have been associated with worse prognosis in PCa. The current study aimed to evaluate RARB, and RASSF1A hypermethylation frequencies in prostate carcinoma (PCa) and to correlate these alterations with down-regulation at transcriptional and protein levels and with clinical outcome.

Methods: Methylation patterns of RARB and RASSF1A were determined by methylation-specific PCR in 68 PCa samples and 27 non-neoplastic prostate tissues. Gene expression was evaluated by quantitative RT-PCR in 73 PCa, 15 adjacent non-neoplastic prostate tissues (AdjP) and two normal prostate (N). Protein expression was evaluated in an independent PCa cohort (n=141) with long-term follow-up period. The findings were associated with clinicopathological parameters and BCR-free survival (BRFS).

Results: RARB and RASSF1A genes were hypermethylated in PCa (P <0.0001 and P = 0.0017, respectively). RASSF1A mRNA and protein levels were similar in PCa and AdjP samples. Down-expression of RARβ in transcript (P=0.001) and protein (P=0.0007) levels were detected in PCa. Lower mRNA levels were associated with RARB hypermethylation. Nuclear and cytoplasmic RARβ protein expression was detected in tumor tissues. Multivariate analysis demonstrated that cytoplasmic RARβ immunostaining was independently associated with decreased BRFS (P=0.005, HR=2.314).

Conclusions: RARB hypermethylation seems to be one of the mechanisms involved in RARβ down-expression in PCa. Cytoplasmic RARβ protein is an independent predictor of poor prognosis that may be considered a new biomarker in prostate cancer providing, therefore, relevant information for patient management.

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 1172.
Abstract 121: Characterization of an extracellular, cell permeable pool of the inhibitor of apoptosis (IAP) protein survivin
Authors: Khan Salma , Slater Jessica M. , Neidigh Jonathan W. , Wall Nathan R.
Published: February 2011

Inhibitor of apoptosis (IAP) and Heat shock proteins (HSPs) provide assistance in protecting cells from the stresses of hypoxia, imbalanced pH, and altered metabolic and redox states commonly found in the microenvironmental mixture of tumor and nontumor cells. HSPs are upregulated, cell-surface displayed and released extracellularly in some types of tumors, a finding that until now was not shared by members of the IAP family. The IAP, Survivin, has been implicated in apoptosis inhibition and the regulation of mitosis in cancer cells. Survivin exists in a number of subcellular locations such as the mitochondria, cytoplasm, nucleus, and most recently, the extracellular space. Our previous work showing that extracellular survivin was able to enhance cellular proliferation, survival and tumor cell invasion provided evidence that survivin might be secreted via an unidentified exocytotic pathway. In the present study, we describe for the first time the exosome-release of survivin to the extracellular space both basally and after proton irradiation-induced stress. To examine whether exosomes contributed to survivin release from cancer cells, exosomes were purified from HeLa cervical carcinoma cells and exosome quantity and survivin content were determined. We demonstrate that although proton irradiation does not influence the exosomal secretory rate, the survivin content of exosomes isolated from HeLa cells treated with a sublethal dose of proton irradiation (3Gy) is significantly higher than control. These data identify a novel secretory pathway by which survivin can be actively released from cells in both the basal and stress-induced state. The quantitation of exosomal survivin from blood or sera may be used to identify the presence of cancer. It may also be feasible to positively impact cancer therapy by targeting exosome-associated protein release, and thus prohibit stress-activated, anti-apoptotic proteins like survivin from impacting the tumor microenvironment and ultimately a patient's response to 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 121.
Abstract 1213: Cytotoxic T lymphocyte antigen-2 alpha (CTLA-2α) induces apoptosis in lymphoma cells and is regulated by the cAMP/PKA signaling pathway
Authors: Zhang Lingzhi , Hwang Xia , Insel Paul A.
Published: February 2011

The second messenger cAMP promotes apoptosis of lymphoma and leukemia cells but mechanisms that mediate this apoptosis are poorly understood. We thus used wild-type (WT) murine S49 T lymphoma cells and S49 variants that lack PKA and cAMP-promoted responses (kin-) or have normal PKA activity but lack cAMP-promoted-apoptosis (“Deathless”, D-) to examine such mechanisms. Results from DNA microarrays revealed that the largest increase in gene expression induced by 8-CPT-cAMP treatment of WT cells is that of cytotoxic T lymphocyte antigen (CTLA-2α), an inhibitor of cathepsin L-like cysteine proteinases. By contrast, CTLA-2α expression was not increased in kin- S49 cells and only slightly increased in D- S49 cells treated with 8-CPT-cAMP. Real-time PCR and immunoblotting confirmed the prominent cAMP-promoted increase in CTLA-2α expression in WT cells. The β-adrenergic receptor agonist isoproterenol and the adenylyl cyclase activator forskolin also induced CTLA-2α gene expression in WT cells, thus indicating that increased CTLA-2α expression can occur via activation of endogenous activators of the cAMP/PKA signaling pathway. Treatment with an adenoviral construct, Adv-CTLA-2α, induced apoptosis in both WT and D- S49 cells but enhanced cAMP-promoted apoptosis only in WT cells. WT, but not D-, S49 cells treated with 8-CPT-cAMP had increased cathepsin activity. Taken together, these results indicate that the cAMP/PKA pathway up-regulates CTLA-2α expression and based on the contrasting findings obtained with WT vs. D- S49 cells, CTLA-2α may be a key contributor to cAMP/PKA-promoted apoptosis. CTLA-2α and cysteine proteases thus appear to have previously unappreciated roles in apoptosis of lymphoid cells by agents that increase cellular cAMP levels.

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 1213.
Abstract 1218: SMAC modulates gemcitabine-induced apoptosis in head and neck cancer cells
Authors: Sun Quanhong , Zhang Lin , Yu Jian
Published: February 2011

Apoptosis induction is an important anti-tumor mechanism of many conventional chemotherapeutic agents. Biochemical data indicate that SMAC promotes caspase activation by antagonizing IAPs. However, SMAC-knockout mice do not exhibit obvious apoptotic deficiencies in response to a wide range of stimuli. Work for us and others demonstrate that SMAC plays an essential role in apoptosis induced by Nonsteroidal Anti-inflammatory Drug (NSAID), TRAIL or BH3 only proteins in colon cancer cells. In this study we documented a critical role of SMAC in mediating apoptosis induced by chemotherapeutic agent gemcitabine in head and neck squamous cell carcinoma (HNSCC) cells. Using RNA interference, we found that that SMAC mediates gemcitabine-induced apoptosis through the intrinsic/mitochondrial pathway. SMAC Knockdown led to impairment in caspase-3 activation, mitochondrial membrane depolarization and release of cytochrome c. Small-molecule SMAC mimetics at nanomolar concentrations significantly sensitized HNSCC cells to gemcitabine-induced apoptosis, and overcame gemcitabine resistance in SMAC-knockdown cells by restoring caspase-3 activation and cytochrome c release. Furthermore, in xenograft models, SMAC knockdown significantly blocked the therapeutic responses of HNSCC tumors to gemcitabine by inhibiting apoptosis, while SMAC overexpression dramatically enhanced that. Together, these results suggest that SMAC mediates apoptosis in cancer cells, and provide a strong rationale for combining SMAC mimetics with other anticancer agents to treat apoptosis-resistant tumors.

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 1218.
Abstract 1225: Regulation of ARE-mediated mRNA decay during colorectal tumorigenesis
Authors: Blanco Fernando F. , Sanduja Sandhya , Young Lisa , Dixon Dan
Published: February 2011

Colon cancer is mediated through various genetic alterations that promote the initiation and progression of tumorigenesis. As a consequence of these defects, overexpression of many oncogenic factors promoting enhanced cell growth and inflammation are observed. In the normal intestinal epithelium, oncogenic factor gene expression is controlled through AU-rich mRNA elements (AREs) that target mRNA for rapid decay. However, ARE-mRNA turnover is compromised during colon tumorigenesis resulting in a 3- to 4-fold enrichment in ARE-containing genes. To better understand how loss of ARE-mRNA decay contributes to colon tumorigenesis, the role of the mRNA decay factor tristetraprolin (TTP) in normal and tumor cells was examined. The RNA-binding protein TTP binds ARE motifs and promotes rapid mRNA decay by sequestering the mRNA to cytoplasmic sites known as processing bodies (P-bodies). Here, we demonstrate that treatment of non-transformed intestinal epithelial cells with TGF-B inhibited ARE-mRNA expression. This effect of TGF-B occurred through a Smad-dependent induction of TTP expression and this led to a 2-fold increase in cytoplasmic P-body assembly. These findings indicate that induction of post-transcriptional regulation is a novel growth-inhibitory feature of TGF-B in normal cells. Colon cancer progression is frequently characterized by the presence of activating mutations in the RAS proto-oncogene, and, in this context, the tumor-promoting effects of TGF-B signaling are observed. Using an intestinal epithelial cell model stably transfected with an inducible oncogenic Ras cDNA, we determined the impact of oncogenic cellular transformation on TTP expression. In cells expressing oncogenic Ras, the expression of TTP was decreased and these cells were rendered refractory to TGF-B-mediated TTP induction. The functional significance of this loss of TTP expression in transformed cells is observed with a 2-fold decrease in P-body formation. Consistent with this, colon cancer cells and tumor tissue show decreased TTP expression compared to normal tissue and a respective loss of P-body formation. Taken together, these results demonstrate that loss of the mRNA decay factor TTP occurs during colon tumorigenesis, thereby compromising ARE-mRNA decay and resulting in stabilization of various cancer-associated mRNAs. Through its ability to modulate expression of a majority of oncogenic factors, these findings define a role for TTP in tumor-suppressor capacity in the context of colon 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 1225.
Abstract 1227: Inhibition of the RSK2-ATF1 signaling axis by eriodictyol suppresses neoplastic cell transformation
Authors: Liu Kangdong , Cho Yong-Yeon , Yao Ke , Pugliese Angelo , Zheng Duo , Zhang Jishai , He Zhiwei , Malakhova Margarita , Ma Wei-Ya , Bode Ann , Dong Ziming , Dong Zigang
Published: February 2011

The ribosomal S6 kinase 2 (RSK2), a member of the p90RSK (RSK) family of proteins, is a widely expressed serine/threonine kinase and its activation enhances cell proliferation. Here we report that activating transcription factor 1(ATF1) is a novel substrate of RSK2 and the RSK2-ATF1 signaling axis plays an important role in neoplastic cell transformation. RSK2 phosphorylates ATF1 at Ser63 and enhances the transactivation and transcriptional activities of ATF1. Computational modeling, high-through put screening and in vitro pull down assays demonstrated that eriodictiol, a flavanone found in fruits, binds with the N-terminal kinase domain and linker region of RSK2 and inhibits RSK2 N-terminal kinase activity. In a cell culture system, eriodictyol treatment suppressed phosphorylation of ATF1, but did not affect phosphorylation of RSK, MEK1/2, ERK1/2, p38 or JNKs, indicating that eriodictyol specifically inhibits RSK2 signaling. Furthermore, eriodictyol inhibited RSK2-mediated ATF1 transactivation activity and cell transformation induced by tumor promoters in JB6 Cl41 mouse skin epidermal cells. In a foci formation assay, knockdown of RSK2 or ATF1 suppressed foci formation compared with RasG12V, RasG12V/RSK2, RasG12V/ATF1 and RasG12V/RSK2/ATF1 expressing cells. In addition, eriodictyol treatment showed the same effect as RSK2 knockdown in foci formation. Taken together, these results indicated that the RSK2-ATF1 signaling axis plays an important role in neoplastic cell transformation and eriodictyol is a new natural compound for selectively inhibiting RSK2 kinase 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 1227.
Abstract 1232: Aberrant JAK/STAT signaling confers epigenetic silencing of STAT3 targets in gastric cancer by integrated microarray and bioinformatic analysis
Authors: Chang Alex L.Y. , Dittner Claudia , Chou Jian-Liang , Cheng Alfred S.L. , Lin Jiayuh , Yeh Kun-Tu , Chan Michael W.Y.
Published: February 2011

Gastric cancer is the 2nd leading cause of cancer worldwide. One of the major risk factors of gastric cancer is infection of H. pylori. Patients infected with CagA-positive H. pylori would have increased risk of gastric cancer. Previous studies including ours have demonstrated that infection of CagA-positive H. pylori induced activation of JAK/STAT3 signaling pathway. However, the role of aberrant activation of this signaling pathway in gastric cancer is not clear understood. We hypothesized that aberrant activation of JAK/STAT signaling may lead to epigenetic silencing of STAT3 target genes in gastric cancer. In this study, we transfected a constitutively activated mouse STAT3 mutant (STAT3c) into MKN28 gastric cancer cell line in which the JAK/STAT signaling pathway is not active. STAT3c stable transfectant MKN28/STATc-16 showing hyper-phosphorylation of STAT3 demonstrated increased cell proliferation as compared to vector control MKN28/C-9. In order to identify STAT3 target genes that are epigenetically down-regulated in MKN28/STATc-16 cells, expression microarray using Agilent 44K oligonucleotide microarray coupled bioinformatic analysis were performed. Our result showed that there are 2945 genes showing at least 1.5 fold down-regulation in MKN28/STATc-16 cells as compared with vector control. By whole genome bioinformatic analysis, 397 genes with STAT3 binding sites within 5kb of a CpG island were discovered. Integrated analysis revealed that there are 18 downregulated genes with a STAT3 binding site around the promoter CpG island. Further characterization of two STAT3 target, NR4A3 and LOXL4 demonstrated that promoter hypermethylation of these 2 genes were observed in MKN28/STATc-16 cells but not the vector control. Demethylation treatment using 5azaDC also restored expression of these 2 genes in MKN28/STATc-16 cells. These phenomena may be partially related to the up-regulation of DNMT1 in MKN28/STATc-16 cells. In conclusion, our result demonstrated that aberrant JAK/STAT3 signaling may lead to epigenetic silencing of its target gene in gastric cancer. The role of STAT3 in mediating epigenetic silencing of NR4A3 and LOXL4 and its clinical significance in gastric cancer patients is currently under investigation.

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 1232.
Abstract 1256: Protein kinase G type Iα activity in human ovarian cancer cells contributes to enhanced Src activation and DNA synthesis/cell proliferation
Authors: Wong Janica C. , Leung Elaine L. , Johlfs Mary G. , Tsang Benjamin K. , Fiscus Ronald R.
Published: February 2011

We have previously shown that basal activity of the nitric oxide (NO)/cyclic GMP (cGMP)/protein kinase G (PKG) signaling pathway protects human ovarian cancer cells against spontaneous apoptosis and confers resistance to cisplatin-induced apoptosis. The present study determines whether basal PKG activity up-regulates Src and induces cell proliferation in human ovarian cancer cells. PKG-Iα was identified as the predominant isoform of PKG expressed in both cisplatin-sensitive OV2008 and cisplatin-resistant A2780cp human ovarian cancer cells. In both cell lines, both ODQ, an inhibitor of endogenous-NO-induced cGMP biosynthesis, and DT-2, highly-specific inhibitor of PKG-Iα activity, as well as PKG siRNA caused concentration-dependent inhibition of DNA synthesis (assessed by BrdU incorporation) under normal (plus-serum) growing conditions, indicating an important role of basal cGMP/PKG-Iα activity in promoting cell proliferation. DNA synthesis in OV2008 cells was dependent on Src activation, determined using selective Src inhibitor, 4-(4′-phenoxyanilino)-6,7-dimethoxyquinazoline (SKI-1). In vitro co-incubation of human recombinant Src and PKG-Iα resulted in Src-mediated tyrosine-phosphorylation of PKG-Iα and enhanced Src autophosphorylation at tyrosine-416, suggesting that Src directly tyrosine-phosphorylates PKG-Iα and the interaction between PKG-Iα and Src also leads to enhanced Src activation. The result was further confirmed by using immunoprecipitation of PKG, showing a concentration-dependent decrease in PKG tyrosine-phosphorylation by SKI in both ovarian cancer cell lines. In intact OV2008 cells, inhibition of basal PKG-Iα activity with DT-2 dramatically reduced endogenous Src activation and completely blocked the elevation of DNA synthesis stimulated by EGF. The data suggest that Src activation and DNA synthesis/cell proliferation in human ovarian cancer cells are dependent on endogenous basal PKG-Iα activity. The NO/cGMP/PKG-Iα signaling pathway may provide a novel therapeutic target for disrupting the proliferation of ovarian cancer 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 1256.
Abstract 1263: Induction of nonsteroidal anti-inflammatory drug-activated gene-1 (Nag-1) by NSAIDs: role of p75NTR
Authors: Wynne Shehla , Djakiew Daniel
Published: February 2011

Epidemiologic studies have shown that patients with long term consumption of certain nonsteroidal anti-inflammatory drugs (NSAIDs) exhibit a reduced incidence of prostate cancer. Treatment of prostrate cancer cell lines with NSAIDs leads to caspase mediated apoptosis. Previously, our laboratory has demonstrated that R-flurbiprofen and Ibuprofen induce the expression of the pro-apoptotic protein p75NTR in PC-3, DU-145 and LNCaP prostate cancer cell lines, with a subsequent decrease in cell survival. The p75NTR is a member of the Tumor Necrosis Factor Receptor superfamily of death receptors. It acts as a tumor suppressor in the normal prostate. The expression of p75NTR is lost as prostate cancer progresses and is minimal in established metastatic prostate cancer cell lines such as PC-3, DU-145, and LNCaP. The observed increase in p75NTR protein due to R-flurbiprofen and Ibuprofen treatment occurs as a result of activation of the p38 mitogen-activated protein kinase (MAPK) pathway. Here we show that the upregulation of p75NTR following treatment with NSAIDs corresponds to a concomitant upregulation of the NSAID activated gene-1 (Nag-1) protein, a pro-apoptotic member of the TGF beta family, in PC-3 cells. Nag-1 has been linked to decreased cell proliferation in many studies, and has been shown to play a pro-apoptotic role in breast, colorectal, gastric and prostate cancer cells. Nag-1 is also known by the names GDF −15, MIC-1, PDF, PLAB and PTGFB. This study also shows that Nag-1 protein induction following NSAID treatment is mediated by the p38 MAPK pathway, and is downstream of the p75NTR induction. This is the first report of a relationship between p75NTR and Nag-1.

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 1263.
Abstract 1270: Elevated levels of poly(ADP-ribose) lead to novel apoptotic signaling and the induction of caspase-independent cell death
Authors: Feng Xiaoxing , Zhou Yiran , Koh David W.
Published: February 2011

The metabolism of poly(ADP-ribose) (PAR) is activated by DNA damage, where PAR polymerase-1 (PARP-1) transfers ADP-ribose onto nuclear proteins to form PAR and PAR glycohydrolase (PARG) catalyzes its degradation. Previous findings show that high levels of PAR lead to its presence in the cytoplasm, where it signals the release of the proapoptotic protein, apoptosis inducing factor (AIF). Our studies suggest that inhibiting PAR glycohydrolase (PARG), and thus promoting increased levels of PAR, will increase the efficacy of anti-cancer agents by promoting this alternative cell death pathway mediated by AIF. The objective of this study is to elucidate the pathway of cytoplasm PAR cell death signaling in cancer cells. We utilized MDA-MB-231 breast cancer cells to reveal the ability of AIF to induce cancer cell death following caspase inhibition. In addition, PARG null embryonic trophoblast stem cells were utilized to determine the cytoplasmic PAR signaling pattern and to purify the PAR-modified nuclear proteins released in the cytoplasm. FACS analysis demonstrated equivalent levels of cell death with or without caspases and Western blot revealed a robust release of AIF from the mitochondria following DNA damage. Following low-dose DNA-damaging agent treatment in PARG null cells, cytoplasmic PAR was detected concurrently with AIF translocation to the nucleus, as shown by Western blot and confocal immunofluorescence. In PARG null cells in the absence of stress, cytoplasmic PAR levels were maximal after 3 days as shown by cell fractionations and Western blot. The cytoplasmic fraction containing PAR was then purified using dihydroxyboronyl Bio-Rex affinity chromatography. The purified PAR-modified proteins were treated with phosphodiesterase to remove all PAR and SDS-PAGE analysis demonstrated several proteins ranging from 20-50 kDa, which together serve as the cytoplasmic signal that causes AIF release from the mitochondria. These proteins potentially reveal novel nuclear-to-mitochondrial cell signaling ligands that are necessary for the induction of the AIF pathway of caspase-independent cell death. Taken together, the results demonstrate the importance of AIF in cancer cell death and novel insight into the cell death signaling by PAR that produces AIF-mediated cell death. Therefore, ability to increase PAR levels by inhibiting PARG is potentially a unique method to induce the death of cancer cells by an alternative cell death pathway.

This research was supported by funding from the American Cancer Society and the Pharmaceutical Research Manufacturers of America.

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 1270.
Abstract 128: Role of ΔNp63α and its effect in cisplatin-mediated chemoresistance
Authors: Sen Tanusree , Chatterjee Aditi , Sen Nilkantha , Hoque Mohammad Obaidul , Sidransky David
Published: February 2011

ΔNp63α, an important epithelial developmental gene, has significant homology to p53. Unlike p53, the expression of p63 is regulated by two different promoters resulting in proteins with opposite functions: the full-length transcriptionally active TAp63 and the dominant-negative ΔNp63. Increasing evidence show that ΔNp63α is upregulated in various cancers, and plays a central role in both development and tumorigenesis by promoting cell survival. The aim of our current study is to provide insight into the molecular mechanisms accounting for the role of ΔNp63α in tumorogenesis and its involvement in chemoresistance induced by cisplitin. We have investigated the downstream mechanisms by which ΔNp63α elicits tumorigenesis via AKT pathway. Both overexpression and RNAi experiments with ΔNp63α revealed that it can regulate the expression of AKT. These results demonstrate a link between ΔNp63α and the anti-apoptotic pathway. Of clinical relevance is the fact that ΔNp63α protein level is increased in chemoresistant cell line and that inhibitition of ΔNp63α leads to chemosensitivity. Thus, beyond its importance in development and differentiation, we describe the mechanism by which ΔNp63α exerts cell survival and the induction of tumorogenesis and chemoresistance.

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 128.
Abstract 1286: Endothelin-1-induced angiogenesis is regulated by PHD2 in a HIF-1α-dependent manner
Authors: Spinella Francesca , Rosanò Laura , Caprara Valentina , DiCastro Valeriana , Nicotra Maria Rita , Natali Pier Giorgio , Bagnato Anna
Published: February 2011

The endothelin B receptor (ETBR) promotes tumorigenesis and melanoma progression through activation by endothelin (ET)-1, thus representing a promising therapeutic target. The stability of hypoxia-inducible factor (HIF)-1α is essential for melanomagenesis and progression. In melanoma cells cultured in normoxic conditions, ET-1, ET-2, and ET-3 through ETBR enhance HIF-1α and HIF-2α expression, and activity and both transcriptional factors regulate the expression of target genes, such as vascular endothelial growth factor (VEGF) and ET-1 in response to ETs or hypoxia. In turn, ETs control HIF-1α stability by inhibiting its degradation. In particular, ETs through ETBR markedly decrease prolyl hydroxylase domain (PHD)2 mRNA and protein levels and promoter activity. In addition, we found that activation of phosphatidylinositol 3-kinase (PI3K)-dependent integrin linked kinase (ILK)-AKT-mammalian target of rapamycin (mTOR) pathway is required for ETBR-mediated PHD2 inhibition, HIF-1α, HIF-2α, and VEGF expression. At functional level, PHD2 knockdown does not further increase ETs-induced angiogenesis and melanoma cell invasiveness demonstrating that these processes are regulated in a PHD2-dependent manner. In human primary and metastatic melanomas, that express high level of HIF-1α, ETBR expression is associated with low PHD2 levels. In melanoma xenografts, ETBR blockade by ETBR antagonist results in a concomitant reduction of angiogenesis, HIF-1α, HIF-2α and VEGF expression, and an increase in PHD2 levels. In conclusion, we identified the underlying mechanism by which ET-1 controls HIF-1α stability and thereby regulates angiogenesis and tumor invasion through the regulation of PHD2. These results further indicate that targeting ETBR may represent a potential therapeutic treatment of melanoma by impairing HIF-1α stability. Supported by AIRC.

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 1286.
Abstract 1287: Anastellin, the fibronectin-derived angiogenesis inhibitor, activates a proangiogenic signature in stromal fibroblasts
Authors: You Ran , Zheng Mingzhe , McKeown-Longo Paula J.
Published: February 2011

Fibronectin is an extracellular matrix protein that is important in the regulation of angiogenesis, tumor progression and tumor metastasis. Anastellin, a peptide fragment derived from the first Type III repeat in Fibronectin (III1c), has been shown to prevent tumor angiogenesis in mouse models of human cancer and to inhibit serum-dependent growth of endothelial microvessel cells. To determine whether anastellin might have additional roles in the tumor microenvironment, we evaluated the effect of anastellin on gene expression in human dermal fibroblasts. Microarray analysis (Human Cancer PathwayFinder RT2 Profiler PCR Array) indicated that addition of anastellin to fibroblasts induced the expression of several genes including the proangiogenic inflammatory mediators, IL-8 and TNF-α. PCR analysis confirmed that expression of both TNF-α and IL-8 were markedly increased within 2 hrs suggesting a direct effect of anastellin on fibroblast gene expression. ELISA assay of the conditioned medium of anastellin treated cells indicated that the increased gene expression was accompanied by the secretion of IL-8 and TNF-α protein, which was detected between 2-4 hrs of anastellin treatment. Changes in gene expression were preceded by increased phosphorylation of c-Jun N-terminal kinase (JNK) and p38 MAPK as well as the nuclear translocation of NF-κB. PCR and ELISA analysis showed that the NF-κB inhibitor (BAY 11-7082) completely inhibited the induction of IL-8 and TNF-α. The inhibitor of p38 MAPK activity (SB 203580) and the inhibitor of JNK (SP600125) each partially attenuated the induction of IL-8 and TNFα while the combination of inhibitors completely blocked gene expression and the nuclear translocation of NF-κB in response to anastellin. Taken together, these data indicate that treatment of dermal fibroblasts with anastellin causes activation of the p38 and JNK MAPK pathways which converge downstream to promote the NF-κB dependent expression of IL-8 and TNF-α. These data suggest that fibronectin matrix remodeling induces the expression of proangiogenic cytokines by stromal cells present in the tumor microenvironment. This mechanism may represent a novel pathway of tumor resistance to angiogenesis blockade. This work is supported by NIH grant CA69612.

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 1287.
Abstract 1292: Broad vascular distribution of Neuropilin-1 and its ligands in human tumors
Authors: Mak Judy , Tong Raymond , Koch Alexander , Kowalski Joe , Frantz Gretchen , Peale Franklin , Watts Ryan , Bagri Anil
Published: February 2011

Neuropilin-1 (NRP1) is a multi-domain transmembrane receptor that is important for the development of the nervous and vascular systems and has been implicated in modulating various aspects of tumor biology. We have previously shown that an antibody directed against NRP1 inhibits tumor growth and reduces tumor angiogenesis, as a single agent and more potently in combination with anti-VEGF. Currently, anti-NRP1 is in evaluation in early clinical studies. Therefore, understanding the expression pattern of Nrp1 and its relevant ligands in human tumor samples is of critical importance. To this end, we first evaluated the binding of a number of putative ligands that have been suggested to interact with NRP1 in the literature, and furthermore to understand which of these are biochemically or functionally inhibited by our anti-NRP1. We found that members of the semaphorin and VEGF families, but not HGF bind to NRP1. However, only binding and function of specific ligands from the VEGF family are inhibited by anti-NRP1. Additionally, we evaluated the expression pattern of NRP1 in human tumor samples from a number of different tumor types, including breast, colorectal, lung, ovarian, pancreatic, prostate and renal cancer. NRP1 is broadly expressed in the vasculature of most cases of all these tumor types. Additionally, NRP1 is also variably expressed by tumor cells and other stromal elements in a more variable manner that is tumor type dependent. These results suggest that NRP1 is a potentially important therapeutic target in many different tumor types.

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 1292.
Abstract 1311: ATR inhibition sensitizes p53-deficient cancer cells to chemotherapeutic drugs
Authors: Sangster-Guity Niquiche , Conrad Bogdan H.M. , Bunz Fred
Published: February 2011

PURPOSE: We sought to determine whether genetic inhibition of the ATR kinase would preferentially sensitize p53-deficient cancer cells to therapeutic agents that cause replication stress. EXPERIMENTAL PROCEDURES: Previous studies have shown that p53-deficient cancer cells harboring hypomorphic ATR alleles are highly sensitive to DNA damaging agents and DNA replication inhibitors that are commonly used in the clinic. We hypothesize that p53-dependent cell cycle checkpoints would prevent damaged, ATR-deficient cells from entering S-phase and mitosis, and thereby enhance cell survival. To test this hypothesis, we used a gene targeting approach to knock-in wild type p53 into cells with wild type and hypomorphic ATR alleles. The combined effects of ATR and p53 were determined by clonogenic survival assays, flow cytometry and analysis of apoptotic proteins. SUMMARY: RNAi-mediated knockdown of ATR in p53-deficient cells caused reduced survival after cisplatin treatment, compared with knockdown of ATR in isogenic p53-proficient cells. To unambiguously test whether ATR inhibition preferentially sensitizes p53-deficient cancer cells, we used a knock-in vector to replace a p53-mutant allele with a wild type allele. Homologous recombinant cells expressed wild type p53 protein and the p53 target proteins p21 and p53R2, demonstrating restoration of a functional p53 pathway. Knock-in of wild type p53 restored the G1/S checkpoint induced by ionizing radiation. Most significantly, genetic restoration of the p53 pathway rescued the survival defect of the ATR-deficient cells in response to chemotherapeutic drugs. CONCLUSIONS: Our data provide genetic evidence that p53 and ATR function synergistically to enhance the survival of human cancer cells treated with agents that cause DNA replication stress. We propose that pharmacologic inhibition of ATR would cause enhanced killing of cells with loss of p53 function following treatment with DNA replication inhibitors and DNA crosslinking agents, thereby increasing their therapeutic index.

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 1311.