bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 48: Activation of c-Met receptor signaling by carcinoma-associated fibroblasts in the tumor microenvironment
Authors: Zillhardt Marion , Lengyel Ernst
Published: August 2012

Objective: Carcinoma-associated fibroblasts (CAFs) play a key role in tumorigenesis by promoting tumor growth, invasion, and metastasis. At sites of intra-abdominal ovarian cancer metastases, more than half of the tumor microenvironment is composed of CAFs, however, the mechanism for how CAFs promote metastatic disease in ovarian cancer is not known. Because of the prominent role that the receptor tyrosine kinase c-Met plays in metastasis, we hypothesized that CAFs within the metastatic tumor microenvironment may activate c-Met signaling in ovarian cancer cells leading to tumor progression at a metastatic site. Methods: Human cancer cell lines and primary human carcinoma-associated fibroblasts isolated from patients’ ovarian cancer metastases were used in multiple biochemical assays to analyze the role of CAFs in c-Met receptor signaling. Results: Using a receptor tyrosine kinase array to analyze phosphorylation of 42 different receptor tyrosine kinases (RTKs), we found that c-Met was the only RTK that was phosphorylated in cancer cells upon co-culture with CAFs. Further analysis of the co-culture revealed that direct contact between cancer cells and CAFs was required for activation of c-Met. Interestingly, the unique ligand for c-Met, hepatocyte growth factor (HGF), could not be detected in conditioned media from CAFs by ELISA and addition of HGF neutralizing antibodies to the co-culture did not prevent activation of the receptor. Furthermore, CAFs transiently transfected with HGF siRNA were still able to activate c-Met in the human ovarian cancer cell line SKOV3ip1 suggesting that c-Met activation by CAFs is HGF-independent. This effect was not limited to ovarian cancer cell lines as co-culture of CAFs with the cervical cancer cell line, HeLa, which expresses wild-type c-Met, and the breast cancer cell line, T47D, showed the same results as the two ovarian cancer cell lines, SKOV3ip1 and OVCAR-5. Remarkably, membrane fragments isolated from CAFs were sufficient to stimulate phosphorylation of c-Met in cancer cells in an HGF-independent manner. Several candidate factors were identified in the CAF membrane proteome which can activate c-Met signaling. Conclusions: Collectively, these results suggest a potentially novel mechanism whereby CAFs activate c-Met in cancer cells through direct contact which provides an additional rationale for the therapeutic targeting of c-Met in cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 48. doi:1538-7445.AM2012-48
Abstract 4802: Metabolic distinctions in high grade epithelial ovarian cancer
Authors: Zand Behrouz , Bottsford-Miller Justin , Hu Wei , Stone Rebecca , Nick Alpa M. , Zhang Xinna , Geest Koen De , Lutgendorf Susan K. , Sood Anil K.
Published: August 2012

Objective: To identify differences in overall metabolite profile of human high grade serous epithelial ovarian cancers (EOC) compared to normal ovarian tissues. Methods: Forty-five patient tissues were obtained (n= 30 EOC, n= 15 normal). Samples were analyzed by Gas Chromatography (GC)/mass spectrometry (MS) and Liquid Chromatography (LC)/MS platforms. The difference between the groups was reported in fold change (FC). Welch's two sample t-tests were used to identify metabolites that differed significantly between cancer and normal ovarian tissues and p<0.05 was defined as significant. Results: Using an existing library containing >2000 metabolites, there were 315 metabolites that were identified between EOC and normal ovaries. Of these 315 metabolites, 204 were significantly altered with 180 up regulated metabolites, and 24 down regulated. EOC had altered metabolism in the glycolytic, amino acid, lipid, nucleotide and eicosanoid pathways. There was an increase dependence on glycolysis consistent with the “Warburg effect,” as noted by elevated glucose-6-phosphate (FC= 2.8, p<0.003), fructose-6-phosphate (FC=2.9, p<0.001) and lactate (FC= 1.7, p=0.013). Ovarian cancer cells showed significantly elevated levels of long-chain fatty acids (e.g, docosatrienoate (22:3n3) [FC= 9.06, p < 0.001), including those derived from the diet (linoleate [FC=2.25, p<0.001] and linolenate [FC=2.37, p<0.001]). Furthermore, significantly high levels of components needed for phospholipid biosynthetic pathway (e.g. cytidine 5′-diphosphocholine [16.86, p < 0.001], choline [FC = 1.5, p<0.001]) were seen which along with the altered lipid metabolism supports membrane biosynthesis in cancer cells. The eicosanoid metabolism pathway had significantly higher levels of pro-inflammatory compounds such as prostaglandins E2 (FC= 17.11, p<0.001), I2 (FC=16.35, p<0.001) and 6-keto prostaglandin F1-α (FC= 16.77, p<0.001). Interestingly, compounds best characterized only in the central nervous system, were also significantly elevated in ovarian cancer tissues. Conclusion: High grade EOC shows distinct metabolic alterations compared to normal ovarian tissues, forming the basis for targeting key metabolic pathways for biomarker and/or therapeutic approaches in ovarian cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4802. doi:1538-7445.AM2012-4802
Abstract 4806: Association of changes in 4-aminobutyrate aminotransferase (ABAT) and beta-alanine metabolism with breast cancer and the more aggressive estrogen receptor negative subtype
Authors: Budczies Jan , Lehmann Annika , Griffin Jules L. , Oresic Matej , Fiehn Oliver , Denkert Carsten
Published: August 2012

Metabolic changes are the final answer of the cell to environmental or genetic changes. While gene expression in breast cancer has extensively been studied, studies on the breast cancer metabolome and its correlation to transcriptional changes are rare. Therefore, a combined metabolomics and transcriptomics study was carried out by the METAcancer consortium. In this project, 275 breast cancer tissues collected as fresh-frozen samples in the METAcancer tumor bank were analyzed using gas chromatography combined with time of flight mass spectrometry (GC-TOF-MS). Out of these, 156 tumor samples were transcriptionally profiled using whole genome DASL. Before, we identified 10 increased and 9 decreased metabolites in the more aggressive estrogen receptor negative (ER-) compared to the estrogen receptor positive (ER+) breast cancer (poster #5573, AACR Annual Meeting 2010). Among these changes, the increase of beta-alanine (fold change = 2.4, p = 1.7E-20) was the strongest and most significant alteration. Comparing breast cancer and normal tissues, beta-alanine turned out to be strongly increased in the cancer tissues (fold change = 2.6, p = 1.1E-34). Using the Kyoto Encyclopedia of Genes and Genomes (KEGG) we identified 135 enzymes that catalyze reactions of the 19 regulated metabolites. These enzymes were subjected to correlation analysis with their substrates and products. As most significant result, a negative relationship between 4-aminobutyrate aminotransferase (ABAT) expression and beta-alanine concentration was detected (Pearson correlation = –0.62, p = 1.1E-17). ABAT was significantly down-regulated in the more aggressive ER- subtype (fold change = –1.9, p = 1.6E-08). The differential expression of ABAT was confirmed in three publicly available breast cancer microarray data sets. The effect of ABAT inhibition was investigated in estrogen receptor positive MCF-7 and triple-negative MDA-MB-231 breast cancer cells. In both cell lines, treatment with vigabatrin, a small molecule that binds irreversibly to ABAT and inhibits the enzymatic activity, increased proliferation. In summary, we detected an accumulation of beta-alanine in breast cancer and an increase of beta-alanine and a down-regulation of ABAT in the more aggressive ER- subtype. The regulation of beta-alanine metabolism that underlies these changes is currently investigated in cell culture models.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4806. doi:1538-7445.AM2012-4806
Abstract 4809: Elucidating acquired drug resistance in multiple myeloma using expression proteomics and phosphorylation profiling
Authors: Fang Bin , Xiang Yun , Mirza Sayeef , Remily-Wood Elizabeth , Mathews Linda , Dalton William , Shain Kenneth , Koomen John M.
Published: August 2012

Introduction: Multiple myeloma (MM) is an incurable malignancy of the plasma cell. While patients may initially respond to treatment, the development of acquired drug resistance (ADR) is a consistent feature of the disease. In order to understand the processes involved in ADR, proteomics is used to examine multiple phenotypic outcomes associated with drug resistance in cell line models. Both protein expression and phosphorylation data will be mined for biological mechanisms and candidate biomarkers for detection of ADR in MM patients. Experimental Procedures: Using cell line models of melphalan and doxorubicin resistance, protein expression is compared using spectral counts and extracted ion chromatograms from GeLC-MS/MS data. In addition, phosphorylation profiles are created using SCX peptide fractionation with IMAC phosphopeptide enrichment prior to LC-MS/MS cataloging. Additional hypothesis-driven experiments are conducted using liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) for targeted quantification. In addition to steady state comparisons between naïve and drug resistant cells, initial responses to therapy are also evaluated. Assays developed for interrogation of cell line models can be translated to patient specimens. Data Summary: GeLC-MS/MS expression measurements typically produce semi-quantitative data for > 2,000 proteins (2+ peptides per protein). Phosphorylation analysis has produced a catalog of > 1,000 sites across the entire group of cell lines. Data are manually evaluated for proteins relevant to MM and mapped to pathways using Metacore GeneGO. Because both melphalan and doxorubicin produce DNA damage, common processes include DNA damage repair, cell cycle regulation, and apoptosis. Each drug also produces unique phenotypes. For example, doxorubicin resistance is mediated in large part by multidrug resistance proteins; melphalan resistance is marked by increases in redox proteins. LC-MRM of heat shock proteins and Bcl-2 family apoptosis regulators supplements existing data. LC-MRM is also used to examine the response in the expression of proteins involved in ADR at the onset of treatment in naïve MM cells. Conclusions: Proteomics can further elucidate the biological processes involved with ADR in MM to generate candidate biomarkers. LC-MRM is used in cell line models to develop a platform for assessing panels of proteins related to ADR, which can ultimately be translated to assist in clinical decisions for tailoring therapy to the individual patient.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4809. doi:1538-7445.AM2012-4809
Abstract 4810: Multiplexed immunoprecipitation-liquid chromatography-multiple reaction monitoring mass spectrometry quantification of protein expression and phosphorylation for breast cancer biomarkers
Authors: Chen Yi , Britton David J. , Wood Elizabeth R. , Pike Ian , Koomen John M.
Published: August 2012

Introduction: Estrogen receptor alpha (ER alpha), progesterone receptor B (PR B), and the receptor tyrosine kinase, Her2, are tumor markers classified by the American Society of Clinical Oncology (ASCO) as level of evidence I in breast cancer. The quantitative evaluation of protein expression and phosphorylation status of these targets in tumor tissues will enable subtype classification and support clinical decision-making and selection of targeted therapy in breast cancer. Experimental Procedures: Breast cancer cell lines were used to develop peptide-based quantitative assays for protein expression and phosphorylation using immunoprecipitation (IP) and liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM). Stable isotope-labeled standard peptides were synthesized and developed as internal standards. Multiplexed IP for ER alpha, PR B, and HER2 will be developed to enable the enrichment of the proteins to enable quantification of the expression and phosphorylation of these biomarkers in the same sample. The sensitivity of each assay will be tested using serial dilutions of cell lysate to evaluate requirements for analysis of clinical specimens. Data Summary: LC-MRM assays for both unmodified and phosphorylated peptides have been developed and characterized for each protein. Recovery using multiplexed IP for these proteins is similar to recovery of individual IP. Data have been generated for cell line models of stimulation and treatment. For example, LC-MRM quantification of HER2 in cell line models stimulated with EGF or Heregulin shows that protein expression is unchanged, but phosphorylation increases; on the other hand, LC-MRM shows clearly that phosphorylation of Her2 is inhibited with Lapatinib. Conclusions: The ability to examine the protein expression as well as the phosphorylation status in these biomarkers with quantitative mass spectrometry will produce improved understanding of their role in the development and progression of breast cancer and has the potential to improve the selection of targeted therapeutics.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4810. doi:1538-7445.AM2012-4810
Abstract 4811: Transient telomere dysfunction induces chromosomal instability and promotes carcinogenesis in telomerase-proficient mice
Authors: Hartmann Daniel , Begus-Nahrmann Yvonne , Eshraghi Parisa , Rasche Volker , Schirmacher Peter , Lee Han-Woong , Lechel André , Rudolph K. Lenhard
Published: August 2012

Introduction: A current concept of carcinogenesis indicates that telomere dysfunction can increase tumor initiation by induction of chromosomal instability, but initiated tumor cells need to reactivate telomerase for genome stabilization and tumor progression. So far, this concept has not been proven in vivo since appropriate mouse models were lacking. Methods: Here, we analyzed hepatocarcinogenesis (i) in a novel mouse model of inducible telomere dysfunction on a telomerase-proficient background, (ii) in telomerase knockout mice with chronic telomere dysfunction (G3 mTerc-/-), and (iii) in wild-type mice with functional telomeres and telomerase. Results: Transient or chronic telomere dysfunction enhanced the rates of chromosomal aberrations during hepatocarcinogenesis, but only telomerase-proficient mice exhibited significantly increased rates of macroscopic tumor formation and cancer cell proliferation in response to telomere dysfunction. In contrast, telomere dysfunction resulted in pronounced accumulation of DNA damage, cell cycle arrest and apoptosis in telomerase-deficient liver tumors. Conclusion: Together, these data provide the first in vivo evidence that telomere dysfunction can promote chromosomal instability and carcinogenesis in telomerase-proficient mice in the absence of additional, genetic checkpoint defects at germline level.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4811. doi:1538-7445.AM2012-4811
Abstract 4812: p53 acts during total-body irradiation to promote lymphomagenesis
Authors: Lee Chang-Lung , Blum Jordan M. , Moding Everett J. , Sullivan Julie M. , Jeffords Laura B. , Rodrigues Rafaela C. , Ma Yan , Kim Yongbaek , Kirsch David G.
Published: August 2012

Radiation therapy can cause acute toxicity and long-term side effects including radiation-induced cancer. Because some of the short-term side effects of radiation are due to p53-mediated apoptosis, blocking p53 during radiation can protect some normal tissues from acute radiation injury and might improve the therapeutic ratio of radiation therapy. However, temporarily blocking p53 could also increase late effects of radiation because mice in which p53 is permanently deleted are sensitized to radiation-induced carcinogenesis. Experiments using a mouse model where p53 can be temporarily turned on during irradiation suggest that radiation-induced apoptosis does not contribute to p53-mediated tumor suppression. Here, we perform reciprocal experiments and temporarily turn p53 off during total-body irradiation (TBI) using transgenic mice with reversible RNA interference (shp53). Our results show that temporary p53 knockdown during TBI suppressed death from bone marrow failure by protecting hematopoietic stem and progenitor cells (HSPCs) against radiation. Remarkably, we found that temporary knockdown of p53 during TBI not only ameliorated acute hematological toxicity, but also prevented lymphoma development. In contrast, p53 knockdown during and permanently after TBI sensitized mice to radiation-induced carcinogenesis, which recapitulates the tumor spectrum of p53+/− mice exposed to irradiation. Mechanistic studies show that, by blocking apoptosis in hematopoietic cells, p53 knockdown during TBI suppressed accelerated repopulation of HSPCs, a critical step that facilitates expansion of pre-malignant clones with mutations after irradiation. Taken together, our results demonstrate that, although p53 is indispensible to suppress tumor formation after radiation, p53 functions during TBI to promote lymphoma formation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4812. doi:1538-7445.AM2012-4812
Abstract 4814: Hepatocarcinogenesis induced by transgenic expression of hepatitis B virus in mice
Authors: Na Bing , Huang Zhiming , Sun Bing , Wang Qian , Ou J.-H. James , Liu Limin
Published: August 2012

Hepatocellular carcinoma (HCC) is the third leading cause of cancer death worldwide and chronic hepatitis B virus (HBV) infection is the most important risk factor of HCC. However, it remains unclear whether HBV plays any direct role in carcinogenesis. We have established two independent transgenic mouse lines expressing a mutant HBV (“preS2 mutant”) that is found at a significant higher frequency in people with HCC than those without. These mice do not show any evidence of chronic liver injury, but by 2 years of age a majority of the male mice developed hepatocellular neoplasms, including HCC. We also found a significant increase in hepatocarcinogenesis independent of inflammation in a transgenic mouse line expressing wildtype HBV. The significant increase of HCC incidence in all three HBV transgenic lines argues that HBV contributes directly to the development of HCC in the absence of chronic inflammation. Endoplasmic reticulum stress-induced unfolded protein response activation and β-catenin truncation/mutation were found in the HCC samples, which may contribute to the hepatocacinogenesis. To screen other candidate genes critical in the HCC development, we performed mouse whole genome microarray analysis and identified a group of genes whose expression levels increased in the livers of the HBV transgenic mice and often further increased in HCC compared to that of the non-transgenic littermates. Using quantitative RT-PCR, we confirmed the expression pattern in 8 out of 10 selected genes that are known to involve in the control of cell survival/proliferation, mobility and intercellular signaling. Among the eight genes, Fabp5 encodes a retinoic acid-binding protein that is known to be overexpressed in various types of human tumors, including HCC, and is implicated in tumor cell survival and proliferation. We also found that Fabp5 is highly expressed in HCC cell lines and in the HCC of our HBV transgenic mice by Western Blot. Immunohistochemistry/immunofluorescence staining confirmed that hepatocytes in HCC overexpress Fabp5. More importantly, Fabp5 expression is significantly elevated in the liver of 4-month old HBV transgenic mice long before HCC development. Therefore our findings suggest that HBV expression may disrupt regulation of proteins important for cell survival and proliferation and contribute directly to hepatocarcinogenesis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4814. doi:1538-7445.AM2012-4814
Abstract 482: Comparison of the actin-binding proteins actinin-1 and actinin-4: Their role in cancer
Authors: Foley Kate S. , Young Paul
Published: August 2012

Actinins are cytoskeleton proteins that crosslink actin filaments. Evolution of the actinin family resulted in the formation of calcium-insensitive muscle isoforms (actinin-2 and- 3) and calcium-sensitive non-muscle isoforms (actinin-1 and -4) with regard to their actin-binding function. Although the non-muscle actinin isoforms show 87% amino acid identity, they are currently viewed as distinct entities. Each isoform is reported to exert distinct effects on cell migration, adhesion and proliferation depending on the cell type studied. Actinin-4 is the predominant isoform reported to be associated with the cancer phenotype. Actinin-4 protein levels are elevated in a number of cancers including breast, ovarian, colorectal, bladder, pancreatic, ovarian and glioblastomas. Actinin-4 enhances the motility and metastatic potential of various carcinoma cell lines. This suggests that actinin-4 may have some unique characteristic that facilitates its role in cancer. We aimed to compare the actin-binding affinities, actin-bundling capacities and calcium sensitivities of the non-muscle actinins. We found that the non-muscle actinins have similar actin-binding affinities and calcium sensitivities with regard to their actin-binding and actin-bundling functions. Actinin structure consists of two anti-parallel monomers that form a homodimer. Heterodimer formation is reported to occur between the muscle actinin isoforms however this has not been systematically examined for the non-muscle isoforms. Through yeast two-hybrid analysis and in vitro binding assays we have shown that non-muscle actinin homodimers and heterodimers form with equal affinity. Using native gel electrophoresis we have shown that non-muscle actinin heterodimer formation occurs in a number of cancer cell lines studied. Comparison of non-muscle actinin protein levels indicates that the heterodimer represents the most abundant form of actinin present in the majority of cell lines studied. siRNA- mediated knockdown of actinin-4 resulted in a decrease in heterodimer levels and an increase in actinin-1 homodimer levels. Taken together this data suggests that actinin-1 and actinin-4 cannot be viewed as distinct entities from each other but rather as proteins that can exist in both homodimeric and heterodimeric forms. The ability to behave in this manner may have functional implications. This may be of importance considering that these proteins are central to such processes as cell migration and adhesion.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 482. doi:1538-7445.AM2012-482
Abstract 4822: Identifiying the anti-Tr antigen in paraneoplastic cerebellar degeneration
Authors: Smitt Peter A.E. Sillevis , de Graaff Esther , Maat Peter , Hulsenboom Esther , van den Berg Robert , van den Bent Martin , Demmers Jeroen , Hoogenraad Casper
Published: August 2012

Introduction: Paraneoplastic cerebellar degeneration (PCD) is characterized by subacute cerebellar ataxia which coincides with the presence of specific tumour types and antineuronal antibodies. One particular form of PCD is associated with Hodgkin Lymphoma (HL) and the presence of antibodies against cerebellar Purkinje cells in several of these patients’ sera. These later called anti-Tr antibodies recognize a specific punctuate immunoreactivity in the large dendritic tree as well as the soma of the Purkinje cells, but not in the axons. Although this characteristic immunoreactive pattern is considered to be a good hallmark for the presence of anti-Tr antibodies, further diagnosis is elaborate. In order to aid this diagnosis, and to understand the pathogenic nature of the PCD we aimed to identify the antigen recognized by anti-Tr antibodies. Objective: We aimed to identify the long sought antigen recognized by anti-Tr antibodies. Methods: To identify the antigen we performed immunoprecipitation using four anti-Tr positive sera on total rat brain extract followed by mass spectrometry. By Western blotting and cell-based assays we subsequently determined the region of the epitope recognized by the anti-Tr antibodies. Deletion and mutant constructs were generated to further map the antigenic region. Results: We identified Delta/Notch-like epidermal growth factor (EGF)-related Receptor (DNER) as the Tr antigen. In a cell-based screening assay 191 control samples were negative, whereas all of the 12 anti-Tr positive sera stained HA-tagged-DNER expressing cells. Using deletion constructs we pinpointed the main epitope to the extracellular domain. Glycosylation inhibitor tunicamycin and N-glycosylation mutations in DNER abolished the anti-Tr staining, indicating that DNER must be glycosylated to be recognized by anti-Tr antibodies. Conclusion: Anti-Tr positive sera recognize DNER. Using the cell based assay, presence of anti-Tr antibodies in patients with PCD and HL can now be screened both quickly and reliably.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4822. doi:1538-7445.AM2012-4822
Abstract 4826: A therapeutic sphingosine 1-phosphate antibody improves intratumoral oxygenation and sensitizes to chemotherapy in prostate cancer animal model
Authors: Ader Isabelle , Bouquerel Pierre , Golzio Muriel , Malavaud Bernard , Zalvidea Santiago , Richard Sylvain , Sabbadini Roger A. , Cuvillier Olivier
Published: August 2012

Hypoxia promotes neovascularization, metastasis, and resistance to treatments. The activation of the transcription factor HIF-1α has been identified as the master mechanism of adaptation to hypoxia. We recently identified the sphingosine kinase 1/sphingosine 1-phosphate (SphK1/S1P) pathway as a new modulator of HIF-1α activity under hypoxia in multiple cancer cell models including prostate cancer (Ader et al, Cancer Res, 2008). S1P elicits proliferation, survival, or angiogenesis, and is believed to exert most of its actions as a ligand for a family of specific GPCRs to elicit paracrine or autocrine signaling. We have suggested that inhibiting SphK1/S1P signaling, which is upregulated under hypoxia, may normalize the microenvironment and increase sensitivity to chemotherapy, in the broader concept of “normalization of tumor vessels” as tumor oxygenation is known to enhance response to chemotherapy (Ader et al., Cancer Res, 2009). Methods: Quantification of intratumoral hypoxia and angiogenesis, and treatment efficacy (primary tumor, metatasis dissemination) using an orthotopic (o.t) xenograft model of fluorescent hormone refractory prostate cancer cells. Results: We first provide in vitro evidence that inhibition of the S1P exogenous signaling, through pharmacological inhibition of its receptors or by taking advantage of a monoclonal antibody neutralizing S1P, blocks HIF-1α accumulation and its activity in prostate cancer cells under to hypoxia. Second, using an o.t model of prostate cancer, we show that an anti-S1P antibody inhibits intratumoral hypoxia and modifies vessel architecture and improves tumor perfusion within 5 days of treatment. Third, we show for the first time that an anti-S1P strategy sensitizes to docetaxel, the ‘gold standard’ treatment for hormone-refractory prostate cancer. A 5-day anti-S1P antibody pretreatment markedly sensitizes to docetaxel in an o.t. PC-3/GFP model established in nude mice. The combination anti-S1P antibody together with docetaxel was not only accompanied by a smaller primary tumor volume compared to docetaxel treatment, but also significantly reduced the occurrence and number of metastases. Conclusion: These preclinical data establish the proof-of-concept that blocking the exogenous action of S1P induces vascular normalization, improves intratumoral oxygenation and sensitizes to chemotherapy in prostate cancer animal model.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4826. doi:1538-7445.AM2012-4826
Abstract 483: Serine phosphorylation of vasodilator-stimulated phosphoprotein defines the malignant cell phenotype in colon cancer
Authors: Ali Mehboob , Pitari Giovanni M.
Published: August 2012

Metastasis is the principal pathological process underlying mortality for colorectal cancer, the third deadly cancer worldwide. During tumor invasion, cancer cells assume a mesenchymal morphology that confers migratory and proliferative advantages. One of the mechanisms regulating malignant cell shape is the activity of the vasodilator-stimulated phosphoprotein (VASP) on the actin cytoskeleton. Here, human colon carcinoma cells were treated with cell permeant analogs of cAMP or cGMP, to preferentially induce VASP Ser157 (pVASP-Ser157) or Ser239 (pVASP-Ser239) phosphorylation, and examined by confocal microscopy, western blotting and colony formation. Confirming previous results, cancer dynamic membrane regions formed by the actin cytoskeleton were differentially modulated by VASP Ser phosphorylation. However, treatments with cAMP, which promoted a mesenchymal cell phenotype, reduced E-cadherin at tumor cell-cell junctions and enhanced vimentin and vinculin levels. In contrast cGMP induced an opposite cell morphology with increased E-cadherin-rich cell-cell contacts and decreased cellular vimentin and vinculin. Moreover, the cAMP-dependent phenotype increased the cancer cell ability to form tumor colonies, while cGMP compromised that metastatic potential. Importantly, cyclic nucleotide effects were mediated by selective VASP Ser phosphorylation, because VASP phosphomutants at Ser157 and Ser239 blocked cAMP or cGMP actions, respectively. Together, these findings suggest that in colon cancer cells pVASP-Ser157 and pVASP-Ser239 are critical determinants of the malignant phenotype, which define the function of actin cytoskeletal regulators, invasive cell morphology and tumorigenic potential.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 483. doi:1538-7445.AM2012-483
Abstract 4831: Treatment of brain inflammatory diseases by delivering exosome encapsulated anti-inflammatory drugs from the nasal region to the brain
Authors: Zhuang Xiaoying , Zhang Huang-Ge
Published: August 2012

In this study, exosomes used to encapsulate curcumin (Exo-cur) or a Stat3 inhibitor, i.e., JSI124, (Exo-JSI124) were delivered non-invasively to microglila cells via an intranasal route. The results generated from three inflammation mediated disease models, i.e., a LPS induced brain inflammation model, experimental autoimmune encephalitis and a GL26 brain tumor model, showed that mice treated intranasally with Exo-cur or Exo-JSI124 are protected from LPS induced brain inflammation, the progression of MOG peptide induced EAE, and had significantly delayed brain tumor growth in the GL26 tumor model. Intranasal administration of Exo-cur or Exo-JSI124 led to rapid delivery of exosome encapsulated drug to the brain that was selectively taken up by microglial cells, and subsequently induced apoptosis of microglial cells. Our results demonstrate that this strategy may provide a non-invasive and novel therapeutic approach for treating brain inflammatory related diseases.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4831. doi:1538-7445.AM2012-4831
Abstract 484: A role for mDia formins in epithelial ovarian cancer spheroid invasion
Authors: Pettee Krista , Eisenmann Kathryn M.
Published: August 2012

Ovarian Cancers (OvCa) metastasis is highly unusual, relative to other metastatic cancers, and is typically confined to the peritoneal cavity; metastases are derived from exfoliated epithelial ovarian cancer (EOC) cells that adhere to and invade the mesothelial cell layer and extracellular matrix (ECM) underlying the peritoneal cavity and viscera. Exfoliated ovarian tumor cells are detected in the peritoneal ascites as single cells, small aggregates or highly ordered, compacted multi-cellular spheroids Compacted spheroids are poorly understood yet clinically relevant structures that are highly invasive and chemoresistant. Little is known about the molecular underpinnings of EOC spheroid formation or their cellular invasion into the underlying peritoneal mesothelial lining. Spheroid formation and ensuing invasion requires tight regulation of the actin cytoskeleton; a thorough understanding of the contribution of proteins regulating the spheroid actin cytoskeleton during cell-cell adhesions and adhesion to and transmigration through the peritoneal mesothelium is essential for progress in therapy of metastatic EOC. We showed that a panel of human EOC cells representing serous and clear cell adenocarcinomas express robust levels of mDia1 and mDia2 formins, irrespective of the ability to form compacted spheroids. mDia proteins were localized to the leading edge of migrating (spheroid-forming) serous epithelial OvCa 429 cells and disruption of mDia function inhibited this 2D chemotactic migration; conversely, dominant activated mDia2 in OvCa 429 cells enhanced chemotactic migration. Upon culture in 3D gels, both mDia1 and mDia2 are highly expressed and localized along with F-actin to the leading invasive edges of the compacted spheroids. Confocal analysis of single cell migration away from the spheroids revealed that multiple motility programs are employed by EOC cells- including single cell streaming and amoeboid cell motility. These data indicate a potential role for mDia proteins in driving localized invasion of EOC cells from compacted spheroids, and EOC cells exhibit a morphological plasticity while migrating in 3-dimensions.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 484. doi:1538-7445.AM2012-484
Abstract 4845: Reeducate the tumor microenvironment to improve the anticancer efficacy of adoptive T-cell therapy
Authors: Liang Wei , Walters Ian , Yu Hua , Jove Richard
Published: August 2012

The hostile tumor microenvironment interferes with the function of efficient antitumor immune cells and constitutes a major obstacle for cancer immunotherapies. Here, we provide evidence that the immunosuppressive tumor microenvironment can be reeducated by an antiangiogenic agent to favor the development of adaptive antitumor immunity and to improve the efficacy of adoptive T cell therapy. Our results demonstrate that treatment with Brivanib, a novel small-molecule kinase inhibitor of VEGFRs and FGFRs, significantly enhanced the frequency and effector function of CD8+ cytotoxic T cells in tumor-bearing mice associated with significant reduction of tumor burden. Enhanced intratumoral infiltration of CD8+ T cells induced by Brivanib was associated with increased expression of chemokines (e.g., ccl3, ccl5, ccl11 and cxcl9) that regulates T cell trafficking and cell adhesion molecules (VCAM-1 and ICAM-1) on endothelial cells in the tumor microenvironment. Moreover, Brivanib treatment skewed the polarization of tumor-associated macrophages (TAM) away from the tumor-supporting M2 phenotype and restored the antigen presenting function of macrophages. Finally, Pretreatment of tumor-bearing animals with Brivanib prior to adoptive T cell transfer promotes in vivo proliferation, intratumoral infiltration and effector function of infused CD8+ T cells, resulting in better antitumor activity than either treatment alone. These findings indicate that Brivanib can be used to modulate the immunosuppressive microenvironment and as a potential adjuvant for enhancing the efficacy of cell-based cancer immunotherapy for cancer treatment.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4845. doi:1538-7445.AM2012-4845
Abstract 485: Epidermal growth factor-like repeats of Tenascin C promote melanoma cell invasion, possibly by enabling mesenchymal to ameboid migration transition
Authors: Grahovac Jelena , Wells Alan
Published: August 2012

In malignant melanoma, the most dangerous skin cancer, Tenascin-C (TNC) is overexpressed, especially at the invasive fronts of invasive melanomas. We found that the migration and invasiveness of a set of melanoma cell lines correlates with levels of TNC expression suggesting, surprisingly, that the melanoma cells themselves provide much of this matrix component. Adding TNC to matrices enhanced individual cell migration in cells that did not express much TNC, whereas it did not enhance the rapid motility of the cells expressing high levels. As TNC is a multidomain protein, the signaling pathways that promote this motogenic invasiveness are not obvious. Herein, we examined the potential role of the EGF-like repeats of TNC that have been shown to preferentially activate motogenic-signaling cascades. To analyze the effects of EGFL signaling on melanoma migration and invasion, we overexpressed TNC fragment containing the assembly and EGFL domains of TNC (TNCEGFL). Interestingly, this led to impaired cell migration in in vitro wound healing assays and live cell tracking experiments. This was likely due to a shift in the adhesion-contractility balance during cell migration. TNCEGFL expressing cells presented a spindle-shaped morphology and formed cords in culture. Expression of TNCEGFL delayed melanoma cell attachment and spreading, as determined in cell adhesion and inverted centrifugation assays. This anti-adhesive phenotype was concomitant with increased MLC2 phosphorylation. Inhibition of ROCK activity, which drives transcellular contractility, restored adhesion of TNCEGFL expressing cells and improved migration in 2D. In distinction to the findings in 2D, TNCEGFL expressing cells had higher invasion potential in Matrigel invasion assays, with cells expressing high TNC having rounded “ameboid” morphology, underlying the importance of morphological shifts, and possibly ROCK signaling, in 3D movement through barrier matrices during invasion and dissemination. These results imply that TNCEGFL repeats are involved in melanoma invasion both by directly influencing cell migration and indirectly changing the tumor microenvironment composition, possibly allowing transition from mesenchymal to ameboid migrational mode.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 485. doi:1538-7445.AM2012-485
Abstract 4911: Systems level network analysis of NCI60 cell lines by alignment of protein pathway activation modules with multiple -omic data fields and therapeutic response signatures
Authors: Federici Giulia , Maria Ruggero De , Liotta Lance A. , Wulfkuhle Julia D. , Petricoin Emanuel F.
Published: August 2012

BACKGROUND: The NCI-60 cell line set is the most extensively profiled set of human tumor cell lines worldwide and have been characterized through chromosome karyotyping, SNP, mutation analysis, miRNA profiling, transcriptional profiling, metabolomic analysis, protein expression measurements, and functional enzymatic analysis. These types of “-omic” data have been mined for correlations with the tremendous drug response data that populates the dataset and provide a great opportunity for a systems level connectivity analysis between drug response and in-depth molecular characterization. However, a missing component of previous attempts to connect datasets together has been the lack of comprehensive functional protein signaling activation analysis. METHODS: Using Reverse-phase Protein MicroArray (RPMA) analysis, we were able to measure the activation state of nearly 200 key signaling proteins representative of the main pathways involved in cell proliferation, survival, migration and adhesion in all 60 cell lines. We used the nature of the comprehensive pathway activation analysis we performed to focus our analysis on modules of interconnected kinase-substrate protein architecture for the following 7 key signaling pathways known to be involved in cancer tumorigenesis and metastasis: AKT signaling, mTOR signaling, EGFR signaling, IGF1R signaling, integrin signaling, autophagy signaling, and apoptosis signaling. These modules were then used to define a systems level view of functional interactions through the correlation of protein phosphorylation/activation states within each module and the mutational, metabolomic, miRNA, transcriptional and drug sensitivity data available.RESULTS AND DISCUSSION: The integration of individual protein pathway activation measurements into biochemically interconnected modules provided a new means to align the functional protein architecture with multiple -omic data sets and therapeutic response correlations. This approach may provide a deeper understanding of how cellular biochemistry through genetic, genomic, transcriptomic, proteomic and metabolomic signatures predicts and defines therapeutic response. Such -omic portraits could inform rational anticancer agent screenings and drive personalized therapeutic approaches.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4911. doi:1538-7445.AM2012-4911
Abstract 4921: A new method for identifying a data-consistent self-reconfigurable predictive bio-network model of the cell cycle based on time series data and its application in cancer systems biology
Authors: Idowu Michael A. , Bown James , Zhelev Nikolai
Published: August 2012

A new method for (re)constructing interaction networks using a limited number of time series data has been developed. This method may provide better opportunities for immediate identification of data-consistent models of biological systems. It may serve as an alternative or complementary approach to other existing data-driven strategies for modeling or mining time-evolutionary properties of complex biological processes based on the analysis of their time series data. We present some practically useful data-mining techniques for constructing self-reconfigurable predictive models of complex biological systems, which approximate their underlying processes. These parametric models, if well reverse-engineered, may help capture key features of data relevant for the purpose of interest. To demonstrate our network inference method, we focus on analyzing time series data of cdk2, cyclinA1, cyclinD1, cycinD2, cyclinD3, E2F1, p16 and p27 obtained from real experiments. The constructed predictive model of the cell-cycle is found to be data-consistent. This model approximates the underlying biological processes hidden in the data, and may help reveal or identify key processes that may govern G1-S phase progression. Given multiple sets of time series data from a cell line, where some sets represent control conditions and other intervention conditions, the method introduced here can help construct interaction networks for each of these data sets. To investigate areas of the signaling network most affected by the intervention, critical areas that have been identified in those networks could be compared to the effects of real perturbations. This may help inform future experimental design by targeting sensitive areas in the signaling network or avoiding resistant pathways.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4921. doi:1538-7445.AM2012-4921