bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3127: Expression and sub-cellular localization of claudin-4 in MCF-7 breast and HEC-1A endometrial cancer cell lines
Authors: Cuevas Maria E. , Sheller Rebecca A. , Holland Andrea C. , Piana Brytanie N. , Todd Maria C.
Published: June 2012

The claudin (CLDN) family consists of 24 tetraspan membrane proteins that are essential for the formation of tight junctions (TJs). Members of the CLDN family display tissue-specific expression and are involved in multiple normal cellular processes. In addition, alterations in CLDN gene expression have been shown to be associated with a variety of cancers. For example, CLDN-3 and CLDN-4 have been found to be upregulated in prostate, ovarian and breast cancer. We have previously shown gross overexpression of CLDN-3 in the breast cancer cell lines MCF-7 and MDA-MB-415. Furthermore, using sequential protein extraction techniques we have consistently observed expression of CLDN-3 in all four subcellular fractions of MCF-7 cells with the bulk of expression in the membrane fraction. CLDN-3 and CLDN-4 genes are located within 50 kb on chromosome 7 and their coordinated regulation has been reported in normal, neoplastic tissues and various cancer cell lines. The present study reports the expression and sub-cellular localization of CLDN-4 in a panel of cancer cell lines using immunoblot analysis. Like CLDN-3, overexpression of claudin-4 was observed in the breast cancer cell lines MCF-7 and MDA-MB-415. In addition, we observed expression of CLDN-3 and CLDN-4 in both endometrial (HEC-1A and RL95-2) and prostate (DU-145) cancer cell lines. Sub-cellular localization analysis of MCF-7 cells demonstrated the expression of CLDN-4 in the membrane, nuclear, cytosolic and to a lesser extent in the cytoskeletal fractions. Currently we are investigating the sub-cellular localization of CLDN-3 and 4 in the endometrial and prostate cancer cell lines described above. Recent studies have shown CLNDs to be involved in signaling to and from TJs, conveying important chemical messages for cell proliferation and differentiation. The presence of CLDN-3 and 4 in the cytosol and nucleus observed in this study may indicate their involvement in cell signaling. Alternatively, their unusual localization may be a characteristic of breast cancer cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3127. doi:10.1158/1538-7445.AM2011-3127
Abstract 3178: Identification and validation of markers for colon adenoma and adenocarcinoma
Authors: Sewda Kamini , Enkemann Steven , Gillies Robert J. , Morse David L.
Published: June 2012

Colorectal cancer (CRC) is the second leading cause of cancer related deaths in the United States and Europe. The early detection and diagnosis of dysplastic and early neoplastic colonic lesions will improve survival and make possible curative treatments. Currently, this is best done with colonoscopy, which is expensive and requires a cathartic bowel prep that has led to low compliance. The goal of this study is to develop targeted molecular imaging agents for the early detection of colon cancer via targeted and potentially prepless colonography using CT or MRI contrast. Such agents can also be used to improve detection of lesions for removal during traditional colonoscopy. By gene expression profiling of DNA microarray data from patient samples of colon adenoma (n =39), adenocarcinoma (n = 432), inflammatory bowel disease (IBD) (n = 16) and unaffected gastrointestinal (GI) tract tissues (normal colon, n = 178 and other gastrointestinal tissues n = 32), we have identified 6 potential cell-surface targets that are highly expressed in colon adenomas and adenocarcinomas relative to unaffected GI tissues: TLR4, LY6G6D, GRM8, GPR56, SLCO1B3 and CLDN1. None of these markers alone cover 100% of the colon adenoma and adenocarcinoma samples surveyed, although combinations did. Nearly all (99.7%) of the adenoma and adenocarcinoma samples express at least one of the three markers TLR4, LY6G6D and CLDN1. One tumor did not express any of these markers. CSF3R and SLC6A14 are expressed exclusively in IBD, which is a risk factor for colon cancer. DNA microarray expression data for colon tumor cell lines (DIFI, SW-620, SW-480, HT-29, COLO-205, KM12, KM12S, HCT116, and HCT115) were also analyzed, and SW-620 and SW-480 cell lines were identified as expressing TLR4, LY6G6D, GPR56 and CLDN1. COLO-205 cells highly express GRM8 and KM12 cells express SLCO1B3. GPR56 and CLDN1 were broadly expressed amongst all cell lines. Validation of these markers for protein expression in patient tissue samples by immunohistochemistry is underway. Molecular imaging agents against validated targets are being developed for the potentially prepless and non-invasive detection of colon adenomas and adenocarcinomas using molecular colonography.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3178. doi:10.1158/1538-7445.AM2011-3178
Abstract 3285: Ang-2-VEGF CrossMab, a novel bispecific human IgG1 antibody blocking VEGF-A and Ang-2 function mediates potent anti-tumoral and anti-angiogenic efficacy
Authors: Thomas Markus , Scheuer Werner , Regula Jörg Thomas , Bähner Monika , Schanzer Jürgen Michael , Croasdale Rebecca , Gassner Christian , Georges Guy , Kettenberger Hubert , Dürr Harald , Imhof-Jung Sabine , Schwaiger Manfred , Stubenrauch Kay-Gunnar , Weidner Michael K. , Schäfer Wolfgang , Klein Christian
Published: June 2012

VEGF-A blockade has been validated clinically as a treatment for human cancers. Angiopoietin-2 (Ang-2) expression has been shown to function as a key regulator of blood vessel remodeling and tumor angiogenesis. In tumors Ang-2 is up-regulated and a bad prognostic factor. Recent data demonstrated that Ang-2 inhibitors, both as a single agent or in combination with chemo- or anti-VEGF therapy mediate anti-tumoral effects.

We have recently described a novel generic method for the production of bivalent bispecific human IgG1 antibodies (CrossMabs) based on the crossover of the CH1 and CL domains within the Fab region of one half of a bispecific antibody combined with the knobs-into-holes technology to enforce heterodimerization of the Fc portion. Subsequently, we have applied the CrossMab technology for the generation of a bispecific antibody recognizing VEGF-A with one arm based on bevacizumab and recognizing Ang-2 with the other arm based on LC06, a highly Ang-2 selective human IgG1 antibody. The Ang-2-VEGF CrossMAb could be produced with good yields and purity by eukaryotic.

Surface Plasmon resonance studies showed that the two different arms of the Ang-2-VEGF CrossMab retained their antigen binding affinity for VEGF-A and Ang-2 and interfered with VEGF-induced HUVEC proliferation and Ang-2 induced Tie2-phosphorylation in a similar manner than the parental antibodies. Crossmab showed very potent tumor growth inhibition in orthotopic (KPL-4) and in subcutaneous xenograft tumors (Colo205). In the orthotopic KPL-4 and the s.c. Colo205 xenograft we observed a strong inhibition of angiogenesis by ex vivo analysis. In the VEGF-induced cornea pocket assay Crossmab resulted in a complete shutdown of angiogenesis.

We have generated a bispecific human IgG1 antibody blocking VEGF-A and Ang-2 function simultaneously. Our data indicate that the Ang-2-VEGF CrossMab mediates potent anti-tumoral, anti-metastatic and anti-angiogenic efficacy and represents a promising therapeutic agent for the therapy of cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3285. doi:10.1158/1538-7445.AM2011-3285
Abstract 3350: Inhibition of breast cancer and cancer stem cells via a novel micheliolide analogue CY-9
Authors: Newman Bryan W. , Zhang Quan , Lu Yaxin , Zhai Jiadai , Ding Yahui , Ma Weiwei , Fan Hongxia , Long Jing , Zhang Haoliang , Wang Miao , Chen Yue , Sun Duxin
Published: June 2012

Natural products that target cancer stem cells (CSCs) have the potential to treat cancers and reduce the occurrence of relapse. The sesquiterpene lactone parthenolide has received much attention for its ability to preferentially target CSCs over their normal equivalents. However, poor pharmacokinetics have severely limited the clinical use of parthenolide, and prompted synthesis of a water soluble analogue, LC-1. Although currently in Phase I trials for disseminated cancers, LC-1 suffers from a short half-life, which could potentially limit its efficacy. In order to identify new sesquiterpene lactones which function similarly to parthenolide and LC-1 but with improved pharmacokinetics, we tested a new micheliolide analogue CY-9 for activity against both breast cancer and CSCs. Using the MTS assay, SUM159 breast cancer cells displayed sensitivity to CY-9 with an IC50 of 5 μM, which is similar to reported inhibitory concentrations of parthenolide. SUM159 cells assayed for Aldefluor expression after a three day treatment with CY-9 (1, 2.5, 5 μM) showed a 27%, 64%, and 83% reduction in their CSC populations, respectively. This CSC depletion was largely reversed in SUM159 cells that were pre-treated with the antioxidant N-Acetyl-Cysteine (NAC) before exposure to CY-9. Inhibition of SUM159 CSC self-renewal after CY-9 treatment was assayed using tumorsphere formation of single SUM159 Aldefluor-positive cells. A seven day treatment of 5 μM CY-9 reduced SUM159 Aldefluor-positive cell tumorsphere formation by 74%. Under drug-free conditions, tumorsphere formation was reduced by 26% in the second passage, but returned to control levels by the third passage. Western blotting also showed that 5 μM CY-9 reduced the levels of nuclear NF-κB. These results demonstrate the effectiveness of our new micheliolide analogue CY-9 at targeting populations of both bulk breast tumor cells and CSCs. The slight reduction in tumorsphere formation under drug-free conditions suggests that inhibition of self-renewal plays a minor role in CY-9's activity against CSCs. Loss of CY-9 activity with cells pre-treated with NAC suggests that the inhibitory effect of CY-9 is partially mediated by its interaction with biologically relevant cysteines and/or ROS generation. Additionally, as NF-κB is vital for defense against cellular stressors such as ROS, depletion of nuclear NF-κB might also explain the observed inhibition with CY-9. Together, these results warrant further study of CY-9 as a potential therapy for breast cancer and CSCs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3350. doi:10.1158/1538-7445.AM2011-3350
Abstract 3374: Effect of Ginsenoside Rg3 on 17β-estradiol-induced epithelial mesenchymal transition through HuR protein in MCF-7 human breast cancer cells
Authors: Lee Chang Ki , Chung Won-Yoon , Kim Jin , Park Kwang-Kyun
Published: June 2012

Epithelial-mesenchymal transition (EMT) is a cellular mechanism long recognized as a central feature of normal development, such as neural crest formation and heart morphogenesis. More recent studies have reported that physiopathological transitions occur during the progression of epithelial tumors, endowing cancer cells with increased motility and invasiveness. EMT implicated that epithelial cells become elongated, lose their polarity and cellular junctions, and show mesenchymal fibroblast-like properties. The role of estrogen and estrogen receptor (ER) in breast cancer cell motility and invasion is controversial. Although ER-positive breast tumors are considered less aggressive and more differentiated, they still undergo metastasis. Human antigen R (HuR) represents one of the best characterised RNA-binding proteins. HuR is involved in mRNA shuttling between nuclear and cytoplasmic compartments and has been shown to increase stability of many mRNA. In recent reports, HuR might serve as a central step in the control of the expression of factors that are involved in tumor growth and angiogenesis, and also plays a critical role in the control of ER mRNA stability. In this study, we investigated whether ginsenoside Rg3 could inhibit the 17β-estradiol (E2)-induced EMT via HuR in MCF-7 human breast cancer cell. Upon E2 stimulation, the levels of epithelial markers, E-cadherin and γ-catenin were strikingly suppressed, whereas those of mesenchymal markers, vimentin, α-SMA, β-catenin and HuR were significantly up-regulated. E2 stimulated the transfer of HuR protein from nucleus into cytoplasm. HuR down-regulated (HuR-DR) MCF-7 cells were established using the HuR shRNA plasmid. Down regulation of HuR did not change the levels of E-cadherin and ER-α, but decreased the levels of β-catenin and vimentin in E2-stimulated MCF-7 cells. Cell proliferation, migration and invasion was not affected by E2 in HuR-DR MCF-7 cells. Rg3 decreased the viability, migration and invasion of MCF-7 cells in the absence or presence of E2. Rg3 treatment inhibited EMT, evidenced by the increased expression of E-cadherin and ER-α, and the decreased expression of β-catenin and vimentin as compared with E2 stimulation. Rg3 inhibited E2-induced expression and cytoplasmic translocalization of HuR protein. In a mouse xenograft model, Rg3 attenuated tumor growth of MCF-7 cells and blocked expressions of EMT marker proteins and HuR, whereas HuR-DR MCF-7 cells did not result in tumor growth. Therefore, Rg3 inhibited E2-stimulated migration, invasion and growth by blocking E2-induced EMT via HuR as well as tumor growth in MCF-7 cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3374. doi:10.1158/1538-7445.AM2011-3374
Abstract 3387: Deletion of NR4A orphan nuclear receptors in adult mice leads to development of acute myeloid leukemia due to disrupted HSC homeostasis
Authors: Ramirez-Herrick Ashley M. , Zhang Shuo , Conneely Orla M.
Published: June 2012

The NR4A subfamily of orphan nuclear receptors (NR4A1, NR4A2, and NR4A3) function as nuclear transcription factors that transduce diverse extracellular and stress signals into altered gene transcription to coordinate apoptosis, proliferation, cell cycle arrest and DNA repair. We previously discovered that two of these receptors, NR4A1 and NR4A3, are potent tumor suppressors of acute myeloid leukemia (AML); they are silenced in human AML and abrogation of both genes in mice results in extremely rapid early postnatal development of AML.

To address the cellular and molecular mechanisms by which NR4As regulate myeloid homeostasis we have generated tamoxifen regulated conditional knockout mice to interrogate the preleukemic effects of NR4A1/3 deletion in hematopoietic cells. We show that deletion of NR4As in adult mice leads to development of transplantable AML disease with a mean survival latency of 15 weeks. Retroviral rescue of NR4A1 or NR4A3 expression after AML development is sufficient to prevent long-term reconstitution of leukemia initiating cells after transplantation into sub-lethally irradiated NOD-SCIDγ mice.

AML development in the conditional knockout mice is associated with loss of hematopoietic stem cell (HSC) homeostasis due to unscheduled HSC proliferation and abnormal skewing of the lineage development program of HSC subpopulations. Skewing of HSC developmental potential leads to a decrease in lymphoid-biased HSCs and relative overproduction of myeloid-biased HSCs, resulting in excessive generation of myeloid progenitor progeny. Our data indicate that NR4As are intrinsic transcriptional regulators of HSC homeostasis that are essential for coordination of lineage development programming of HSCs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3387. doi:10.1158/1538-7445.AM2011-3387
Abstract 3410: Regulation of pluripotency and lineage differentiation in human male germ cell tumors
Authors: Jagadish Nirmala , Kustagi Manjunath , Mendiratta Geetu , Kushwaha Ritu , Bansal Mukesh , Hyunjae Kim R. , Sumazin Pavel , Alvarez Mariano , Lefebvre Celine , Viale Agnes , Korkola James E. , Houldsworth Jane , Bosl George J. , Califano Andrea , Chaganti Raju SK
Published: June 2012

Pluripotent embryonal carcinoma (EC) cells derived from germ cell tumors (GCTs) are an excellent model system to study the interface between malignancy, pluripotency, and lineage differentiation. We previously characterized the gene expression profile (GEP) of a panel of 135 germ cell tumor (GCT) biopsies comprising all described differentiation lineages and 6 normal testes as controls, using the Affymetrix U133A+B arrays (Korkola et al. Cancer Res., 66: 820-7, 2006 and J. Clin. Oncol., 27: 5240-7, 2009). We applied the ARACNE reverse engineering algorithm to this GEP to obtain the first de novo and context-specific transcriptional interactome (GCTNet) comprising 1,310 transcription factors (TFs). GCTnet recapitulated 830 regulatory targets together of POU5F1, NANOG, and SOX2, the core pluripotency TFs. ChIP-seq and ChIP-PCR analysis of NT2/D1 embryonal carcinoma and H9 ES cells provided significant validation of ARACNe-inferred targets, besides providing the first detailed mapping of DNA binding sites of these 3 genes in human cells. Functional validation of ARACNe-inferred targets by stable knockdown of the POU5F1, NANOG and SOX2 in NT2/D1 cells also showed a significant enrichment of ARACNe targets. Analysis of GEP in a time-course of SOX2 knockdown by principal component analysis (PCA) identified 3 orthogonal gene expression programs: (1) genes with monotonic change (PC1), (2) genes showing a strong and focal gene expression at day 5 (PC2), and (3) genes which show oscillatory pattern of expression (PC3). Ingenuity and GO annotation analysis of PCA provided new insights into the pathways regulated by this TF. Our results provide a novel, comprehensive, and accurate regulatory networks responsible for pluripotency and lineage differentiation in GCTs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3410. doi:10.1158/1538-7445.AM2011-3410
Abstract 346: Integrative genomic analysis of sporadic clear cell renal cell carcinoma
Authors: Dondeti Vijay R. , Wubbenhorst Bradley , Lal Priti , Gordan John D. , D'Andrea Kurt , Attiyeh Edward F. , Simon M Celeste , Nathanson Katherine L.
Published: June 2012

Sporadic clear cell Renal Cell Carcinoma (ccRCC) is the most common type of adult kidney cancer. Using high-density copy-number profiling and gene expression profiles, we performed an integrated genomic analysis of 54 sporadic tumor samples. We took a two pronged approach, focusing on 1) all samples and 2) comparing ccRCCs that express both HIF1α and HIF2α (H1H2) and those that express only HIF2α (H2), sub-types of ccRCC that we previously described. For all samples at a frequency over 10%, we identified regions of amplification on 1q, 5q, 7q, 8q, 12p, and 20q, and regions of deletion on 1p, 3p, 6q, 8p, 9p, and 14q, as previously described. We used our integrative analysis to identify target genes that had consistent copy-number and gene expression changes (e.g. amplified and upregulated or deleted and downregulated) within regions on chromosomes 5 and 7. Combining our data with an independent dataset (Beroukhim et al., 2009) and including a literature based analysis, we were able to narrow down to three target genes, which we subsequently validated using a renal cancer cell line. We then compared genomic changes in H1H2 and H2 ccRCCs. Prior data suggested that H2 ccRCCs have increased expression of genes involved in double strand break repair, thus potentially greater genomic stability. Using high-density copy-number data, we have confirmed that H2 tumors have significantly fewer genomic aberrations compared to H1H2 tumors (for amplifications p=0.032 and for deletions p=0.003). In addition, using GISTIC, we found that deletion of chromosome 6 is significantly more common in H1H2 tumors (p<0.001), whereas deletion of chromosome 9 is significantly more common in H2 tumors (p<0.001). Of note, deletion of chromosome 9 previously has been associated with poor prognosis. Thus, correlations between the copy-number data and gene expression profiles indicate that the H1H2 and H2 classes of ccRCC tumors may each have a distinct set of drivers of tumorigenesis; target gene validation for these groups is underway. These experiments provide novel insights into the biology of clear cell Renal Cell Carcinoma (ccRCC).

Beroukhim et al. Patterns of Gene Expression and Copy-Number Alternations in von-Hippel Lindau Disease-Associated and Sporadic Clear Cell Carcinoma of the Kidney. Can Res 2009; 69: (11):4674-81.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 346. doi:10.1158/1538-7445.AM2011-346
Abstract 3462: PARP inhibition sensitizes high grade childhood brain tumors to radiation
Authors: van Vuurden Dannis G. , Meijer Olga L.M. , Hulleman Esther , Würdinger Thomas , Noske David P. , Kaspers Gertjan L. , Cloos Jacqueline
Published: June 2012

Background: Poly ADP-ribose polymerases (PARPs) are proteins involved in single strand break repair. Recently, PARP inhibitors have shown considerable promise in the treatment of several cancers, both in monotherapy as in combination with cytotoxic agents. Synthetic lethal action of PARP inhibitors has been observed in tumors with mutations in genes in double strand break repair pathways. Besides this, PARP inhibition enhances sensitivity of tumor cells to DNA damaging agents, such as chemo- and radiotherapy.

Aim of this study is to investigate the expression of PARP1 in normal brain tissues and high grade childhood brain tumors, medulloblastoma, ependymoma and high grade glioma (HGG), and to analyse sensitivity and radiosensitizing properties of PARP inhibitor Olaparib in cell lines of these tumors.

Methods: Expression of PARP1 has been determined in medulloblastoma, ependymoma and HGG patient material and normal brain tissues by in silico analysis of PARP1 mRNA expression arrays, Western blot and immunohistochemistry. In addition, we performed clonogenic and viability assays, combining radiation with PARP inhibition in medulloblastoma, ependymoma and HGG cell lines.

Results: Increased PARP1 expression was observed in medulloblastoma, ependymoma and HGG, as compared to non-neoplastic brain tissue. Moreover, medulloblastoma gene expression analysis revealed high PARP1 expression to be associated with poor prognosis. Growth inhibition with PARP inhibitor Olaparib resulted in differential sensitivity, with IC50 values ranging from 1.8 to 30 µM in cell lines, irrespective of PARP1 protein expression. HGG cell line SF-188, which harbors a focal deletion of NF1 gene, showed a 15-fold higher sensitivity to Olaparib compared to other HGG cell lines. This high sensitivity may be due to synthetic lethality and warrants further investigation. The combination of subcytotoxic concentrations of Olaparib and radiation showed increased cytotoxicity and reduced clonogenic capacity in medulloblastoma, ependymoma and HGG cell lines.

Conclusion: PARP1 is overexpressed in high grade childhood brain tumors. In medulloblastoma, high PARP expression is correlated with poor prognosis. PARP inhibition sensitizes ependymoma, medulloblastoma and HGG cells to radiation. These results provide a basis for clinical translation, combining PARP inhibitors with radiotherapy in clinical studies in childhood high grade brain tumors.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3462. doi:10.1158/1538-7445.AM2011-3462
Abstract 3466: Tumor pericytes: A novel target for immune therapy in renal cell carcinoma
Authors: Chi Nina , Taylor Jennifer , Maranchie Jodie , Storkus Walter
Published: June 2012

Tumor vasculature is classified by leaky, chaotic blood vessels consisting of several components including vascular endothelial cells (VEC) and mural cells (smooth muscle cells and pericytes), as well as immune regulatory cells, stem cells and vascular progenitors. Irregular tumor vasculature limits immune effector cell access to the tumor, and may in part be responsible for the modest success observed in many current anti-cancer immunotherapies. Certain drugs targeting tyrosine kinase receptors expressed on tumor stromal cells induce a Type-1 proinflammatory skewing of the tumor microenvironment (TME) with a coordinate reduction in the number of immune regulatory cell populations. A transient normalization of the vasculature may also occur in association with enhanced leukocyte recruitment. We have shown that vaccination against tumor-associated stromal antigens leads to the activation of VCAM-1 expression on tumor vascular endothelial cells and the enhanced recruitment of protective Type-1 T cells into the TME. Currently little is known regarding pericyte biology and their behavior in the TME, with no existing treatments specifically targeting pericytes. We hypothesize that pericytes residing in the TME differentially express antigens that distinguish them from pericytes in healthy tissue, and vaccination specifically targeting tumor pericyte antigens will result in vascular normalization and improved delivery of protective Type-1 T cells into the TME. To address these hypotheses, we examined mRNA expression of pericytes and VEC that have been flow-sorted from human renal cell carcinoma (RCC) tumors and patient-matched normal adjacent kidney (NAK) tissue. We have identified antigens that are differentially expressed by pericytes and/or by VEC in the TME versus NAK (such as RGS5). We are currently investigating the therapeutic efficacy of genetic (lentiviral) vaccines encoding tumor-associated stromal antigens in murine tumor models.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3466. doi:10.1158/1538-7445.AM2011-3466
Abstract 3477: Radiation impaired cell migration and wound healing after postoperative radiotherapy is mediated through inhibition of Shp2 tyrosine phosphatase
Authors: Natarajan Mohan , Mohan Sumathy , Zheng Xiangpeng , Aravindan Natarajan
Published: June 2012

The influence and mechanism of the postoperative radiotherapy on wound healing process is controversial. Based on the vital role of endothelial cell migration on wound healing, this study investigated the response of endothelial cells to low-dose irradiation and the underlying mechanism. Primary vascular endothelial cells were grown to more than 90% of confluence in complete MCDB-131 medium. A wound was inflicted in the confluent monolayer exposing a cell-free zone on the plate. The cells were then exposed to acute dose of 10 cGy, 2 or 10 Gy at a dose rate of 1.29 Gy/min. The number of cells crossing the wound border and the migration distance in the wounded region were measured. Integrin signaling pathway was examined by the assay of expression of αvβ3 integrins, focal adhesion kinase (FAK) and phosphorylated FAK (tyr397), and actin fibers through immunoblotting. Confocal immunofluorescence was applied to observe the distribution and morphological alteration of FAK tyr397 and actins after radiation exposure. Also RNA samples were extracted from the radiation-treated cells and reverse transcriptase polymerase chain reaction was performed to analyze the transcription level of SHP-2 tyrosine phosphatase, the regulator of FAK. The results revealed a dose-dependent decrease in cell motility in terms of migration distance and the number of migrating cells. The migrating distance in 2 and 10 Gy exposed cells were only 0.5 and 0.1 mm, respectively compared to 3mm seen in mock irradiated control cells. With 10 Gy exposure, the expression of β3 domain of integrins was inhibited compared to the levels in other irradiated groups and the control group while there was no detectable difference in αv domain and α-actin among all groups. Although the level of total FAK was invariable, phosphor-FAK tyr397 was higher in the irradiated groups. RT-PCR analysis of SHP-2 demonstrated a significantly dose-dependent decrease in transcription of SHP-2 mRNA. These results suggested that radiation inhibited endothelial cell migration. Unlike chemical factors inducing chemotaxis through the reaction with integrins receptors and the ensuing signaling cascade, radiation-induced inhibition of cell migration could be attributing to the direct damage to SHP-2 transcription, and the subsequent accumulated phosphor-FAK abrogated the contraction of cytoskeletons.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3477. doi:10.1158/1538-7445.AM2011-3477
Abstract 3482: Hypoxia induces translational regulation of lymphangiogenic growth factors
Authors: Garmy-Susini Barbara H. , Pujol Francoise , Van den Berghe Loic , Prats Anne-Catherine
Published: June 2012

Hypoxia is a major condition for induction of angiogenesis during tumor development, nevertheless its role in lymphangiogenesis remain unclear. In this study, we investigate the role of hypoxia in the translational regulation of lymphangiogenic growth factors. The majority of cellular stresses such as hypoxia leads to an inhibition of mRNA translation in cell by the classical cap-dependent mechanism. However, several mRNAs are translated by an alternative mechanism mediated by internal ribosome entry sites (IRESs) located in their 5’ untranslated regions.

We found IRES elements in mRNAs of lymphangiogenic growth factors such as VEGFA and FGF2, and demonstrated that hypoxia is able to activate IRES-mediated mRNA translation both in vitro and in vivo. Interestingly, our recent data demonstrated a strong induction of VEGFA and FGF2 IRES activities in vivo in pancreas adenocarcinoma CAPAN-1, that correlates with development of hypoxia area and lymphangiogenesis in primary tumors. Such an activation is not observed with the viral EMCV IRES used as a control, indicating a translational activation in response to tumoral hypoxia could be specific of angiogenic/lymphangiogenic factors IRESs.

We are interested in the translational control of more specific of lymphangiogenic growth factors, VEGFC and VEGFD. We've shown, in CAPAN-1 model, that VEGFC and VEGFD translation correlates with hypoxia area development in vivo. To determine the presence of IRESs in VEGFC and -D mRNAs, we cloned the 5’ untranslated regions of murine and human VEGF-C and -D mRNAs in bicistronic lentivectors. First data suggest the presence of an IRES VEGFC and -D mRNAs.

In further investigations, our purpose is to demonstrate that the process of IRES-dependent translation plays a pivotal role in the hypoxic response, and should lead to induction of lymphangiogenic factors, including VEGFC and VEGFD. Thus, cell proteins able to activate IRESs, called IRES trans-acting factors (ITAFs), would be crucial protagonists in the lymphangiogenesis process and potential taget for anti-angiogenic therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3482. doi:10.1158/1538-7445.AM2011-3482
Abstract 3490: Identification and characterization of novel galectin-9 splice variants in endothelial cells
Authors: Heusschen Roy , de Bree Martijn , Griffioen Arjan W. , Thijssen Victor Ljl
Published: June 2012

Evidence is accumulating that endothelial galectins play an important role in tumor angiogenesis. Recently, we reported altered galectin-9 expression during endothelial cell activation. Here, we further assessed the expression and function of galectin-9 in endothelial cells. Using cDNA flanking PCR and subsequent cloning we identified 5 different galectin-9 splice variants in endothelial cells, 2 of which result in truncation of the C-terminal carbohydrate recognition domain and one of which has not been described previously i.e. galectin-9Δ5/6/10. By real-time PCR analysis we confirmed the expression of these 5 splice variants in endothelial cells and found that galectin-9Δ5 is the dominant splice variant in endothelial cells, as has been described for most other cell types. Endothelial cell activation resulted in a decreased expression of total galectin-9 levels which was mainly due to a decrease in galectin-9Δ5 expression. The expression of 2 other splice variants, galectin-9Δ5/6 and galectin-9Δ5/6/10 was retained after cell activation. To study the functional consequences of alternative splicing patterns on endothelial cell function, we cloned the 5 endothelial galectin-9 splice variants into expression constructs. We were able to demonstrate efficient transcription/translation of galectin-9 splice variants by real time PCR and Western blot respectively, following transfection of cell lines from non-endothelial (HEK293T) and endothelial (HMEC) origin. These data confirmed that exclusion of exon 10 causes a frameshift and premature stopcodon, resulting in the generation of protein that lacks the C-terminal CRD. Next, we assessed the function of gal-9 splice variants in endothelial cells by determining the effects on endothelial cell proliferation, migration and sprouting. Finally, we explored whether these splice variants have diverging roles in regulating the interaction between endothelial cells and immune cells. In conclusion, we report that endothelial cells express 5 splice variants of gal-9, one of which has not been described previously, and we show that these splice variants have diverging roles in endothelial cell biology. We hypothesize that interfering with the function of endothelial gal-9 splice variants might be an interesting approach for novel anti-cancer therapies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3490. doi:10.1158/1538-7445.AM2011-3490
Abstract 3497: Benzyl isothiocyanate potentiates the in vivo anti-tumor effects of gemcitabine against pancreatic cancer by targeting NF-kB and STAT3
Authors: Boreddy Srinivas Reddy , Srivastava Sanjay K.
Published: June 2012

The persistent activation of nuclear factor kB (NF-kB) and signal transducer and activator of transcription 3 (STAT3) contributes to gemcitabine resistance in pancreatic ductal adenocarcinoma. We have previously shown that benzyl isothiocyanate (BITC) down-regulates the activation of both NF-kB and STAT3 in pancreatic cancer in vivo and in vitro. In the present study we investigated whether the inhibition of these molecules by BITC could enhance gemcitabine-induced apoptosis in culture and antitumor activity of gemcitabine in vivo. Our results show that pretreatment of cells with BITC (2.5 µM) for 24 hours followed by gemcitabine (5 nM) for 72 h resulted in enhanced apoptosis in BxPC-3 and PanC-1 pancreatic cancer cells as compared with either agent alone. The IC50 of BITC and gemcitabine is 10 µM and 25 nM, respectively. Furthermore, phosphorylation (activation) of STAT3 at Tyr-705 & Ser-727 and NF-KB (Ser-536), which were increased by gemcitabine treatment, were significantly reduced in the combination treatment in BxPC-3 and PanC-1 cells. Interestingly, gemcitabine resistant BxPC-3 (BxPC-3-GR, IC50: 11µM) cells were equally sensitive (p<0.05) to BITC induced apoptosis as compared to BxPC-3 wild type cells (IC50: 10 µM). In addition, gemcitabine-induced DNA binding activity of NF-kB and STAT3 was significantly reduced in BxPC-3, BxPC-3-GR and PanC-1 cells as measured by EMSA and transcriptional activity. Furthermore, our in vivo results show that BITC (10 µmol; daily, oral) in combination with gemcitabine (40 mg/kg; i.p, every third day) was much more effective in suppressing tumor growth as compared with either agent alone in a BxPC-3 tumor xenograft model. Tumor volume (341 ± 20 vs 155 ± 9 mm3) and tumor weight (0.53 ± 0.01 vs 0.195 ± 0.006 g) were significantly reduced (54 % and 63 %, respectively) in BITC and gemcitabine combination treated mice as compared to control mice. Most importantly, our western blot and immunohistochemistry data shows that gemcitabine-induced activation of NF-KB (Ser-536) and STAT3 (Tyr-705) was completely inhibited in the tumors of mice treated with BITC and gemcitabine combination. These results provide strong evidence that inactivation of NF-KB and STAT3 signaling pathways by BITC could potentiate anti-tumor activity of gemcitabine in pancreatic cancer. [Supported in part by R01 grants CA106953 and CA129038 (to S.K.S) awarded by the National Cancer Institute].

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3497. doi:10.1158/1538-7445.AM2011-3497
Abstract 3502: Activity of combinations of anticancer agents in pancreatic carcinoma cell lines
Authors: Schwarting Christina , Belgardt Julia , Warnecke Andre , Kratz Felix , Graeser Ralph
Published: June 2012

Long term and successful treatment of pancreatic cancer remains a considerable challenge, and there is an urgent need to develop new therapeutic strategies that improve the response rate and overall survival of this disease that responds poorly to monotherapy with anticancer agents such as gemcitabine or 5-fluorouracil. Instead of combining drugs at their respective maximum tolerated doses, a number of recent studies have shown that the “more is better” paradigm may not be the optimal strategy for maximizing the clinical outcome for combination therapy and rather synergistic effects can be achieved if the drugs are dosed at a defined ratio. Such an approach is currently being investigated clinically with liposomal formulations which contain irinotecan and floxuridine at a 1:1 ratio or cytarabine and daunorubicin at a 5:1 ratio. We therefore tested a number of combinations of chemotherapeutic agents using defined ratios ranging from 10:1 to 1:10 for their efficacy against a luciferase transduced pancreatic cell line, MIAPaCa2 ELN, using a 72 h Luciferase proliferation assay. Using this assay, a combination of camptothecin with gemcitabine showed synergy at a ratio of 1:5 over a cell survival range of 10-80% with combination indexes (CIs) between 0.36 and 0.71.

In addition, a synergy was found at a 1:10 ratio for methotrexate with doxorubicin with CI values from 0.36 to 0.32 over a 20-90% cell survival range. A third synergistic combination was observed between cytarabine and gemcitabine at a 1:5 ratio with CIs from 0.16 to 0.21 over a 20-90% cell survival range. None of the other combinations tested, including campthotecin with methotrexate, camptotecin with doxorubicin, or cytarabine with daunorubicin showed synergistic effects at ratios from 5:1 to 1:5.

These in vitro results show that the ratio of the respective drug combination is a decisive factor for inducing additive, synergistic or even antagonistic effects.

We are currently testing the most promising combinations in additional pancreatic carcinoma cell lines in order to elucidate whether the synergistic effects of these combinations and specific ratios apply to pancreatic cancer cells in general.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3502. doi:10.1158/1538-7445.AM2011-3502
Abstract 3504: Targeting the Akt pathway and HDAC signaling in prostate cancer
Authors: Paesante Silvia , Miles Marie Kiersten , Nguyen Holly , Vessella Robert , Pili Roberto
Published: June 2012

Loss of the tumor suppressor phosphatase and tensin homolog protein (PTEN), a negative regulator of the survival Akt pathway, and elevated levels of histone deacetylase (HDAC) enzymes which regulate DNA transcription, correlated positively with the prostate cancer progression, recurrence and metastasis. MK-2206 is an allosteric Akt inhibitor with a broad antitumor activity; clinical studies have recently been completed in patients with solid tumors, showing MK-2206 good tolerability and evidences of clinical activity. vorinostat, an HDAC inhibitor, is involved in more than 100 clinical trials in solid tumors and it showed antitumor activity in different prostate cell lines. In this study we hypothesized that combination of MK-2206 and vorinostat may provide a greater antitumor efficacy, targeting different survival pathways activated in prostate cancer.

Different prostate cell lines were tested in both in vitro and in vivo xenograft models. LuCaP23.1. hormone-sensitive tumors were placed subcutaneously into male SCID mice. Tumor growth was assessed twice weekly by using serial caliper measurements. Luciferase-transfected PC3 hormone-independent tumor cells were injected in the right tibiae of male SCID mice and tumor progression was monitored weekly by bioluminescence assay using the Xenogen IVIS Living Imaging System. At the end of the experiment, Faxitron radiographs of the tibiae were taken. Mice were treated with MK-2206 at 120 mg/kg/d by oral gavage and i.p. injection of vorinostat at 100 mg/kg/d, 5 days per week. In vitro treatment of PC3 cells showed that both MK-2206 and vorinostat affected cell proliferation in a dose and time dependent manner, with a greater effect in combination. Western blot experiments validated p-Akt protein level inhibition due to MK-2206 treatment. Preliminary PC3 and LuCaP23.1. xenograft in vivo experiments confirmed the therapeutic activity of MK-2206 and vorinostat as single agents. The combination therapy showed a modest but greater inhibition of tumor growth in the PC3 xenograft bone metastatic model though not statistically significant. The antiproliferative effect of this combination was more evident in the subcutaneously LuCaP 23.1. xenograft model. This study showed for the first time the therapeutic association of MK-2206 and vorinostat in prostate cancer. We expect to further investigate the MK-2206 +/- vorinostat therapy and to provide new insight into the role of HDACs signaling and Akt pathway in prostate cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3504. doi:10.1158/1538-7445.AM2011-3504
Abstract 3533: DNA binding molecules as a new tool to regulate cancer cell fate
Authors: Nagase Hiroki , Kimura Makoto T. , Wang Xiaofei , Watanabe Takayoshi , Fujiwara Kyoko , Mishra Rajeev , Sugiyama Hiroshi , Fukuda Noboru , Terui Tadashi
Published: June 2012

Pyrrole-Imidazole (PI) polyamide molecule was originally designed from structures of natural DNA binding molecule, such as Distamicine and Diocarbamicine and has been discovered as a synthetic molecule which recognizes the minor groove of Watson-Click base pair of double-stranded DNA in a sequence-dependent manner. We have recently developed a semi-automatic synthesis system for PI polyamide, which are able to regulate specific target gene-expression under specific transcription factor binding inhibition for biological functional studies and perhaps patients’ therapy. PI polyamide immediately penetrated the nucleus in vitro and in vivo without any vehicle. After intra venous injection It rapidly reduce the serum concentration, delivered to most of tissue cells, excreted to urine or bile juice and did not metabolize in animals. The PI polyamides, designed for anti-Tgfb1 and anti-MMP9 activity, were well tolerated, reduced target gene expression and showed therapeutic effects in animal models of human diseases. For instance, when performed two studies using I.V. treatment by either of two independent anti-MMP9 polyamides, tumor metastasis was significantly suppressed in both studies using the same mouse model of human liver metastasis of colon cancer. This new auto-synthetic chemicals can be designed for many transcriptional regulation of transcripts and applied to prove many biological hypothesis of transcriptional regulation for cancer research, and may be used for cancer therapy in the future.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3533. doi:10.1158/1538-7445.AM2011-3533