bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4627: Generation and characterization of non-competitive furin-inhibiting nanobodies
Authors: Declercq Jeroen , Zhu Jingjing , Lindberg Iris , Ghassabeh Gholamreza H. , Meulemans Sandra , Vermorken Alphons J.M. , Van de Ven Wim J.M. , Muyldermans Serge , Creemers John W.M.
Published: August 2012

Furin belongs to a family of seven closely related subtilisin-like serine endoproteases, known as proprotein convertases (PCs), PC1/3, PC2, PC4, PC5/6, PACE4 and PC7. The physiological role of furin is to cleave and hence activate a large variety of proproteins. Therefore, it is not surprising that it plays a major role in many pathologies such as cancer and infectious diseases. Furin inhibition might be a good strategy for therapeutic intervention, and several furin inhibitors have been generated, although thus far none are entirely furin-specific. To reduce potential side effects caused by cross-reactivity with other proteases, dromedary heavy chain antibodies against catalytically active furin were developed as specific furin inhibitors. Nanobodies derived from these antibodies bind only to mouse and human furin but not to other PCs. In addition, upon overexpression in cell lines, these nanobodies can inhibit the cleavage of three different furin substrates, TGFβ, BAFF and GPC3. The purified nanobodies can also inhibit the cleavage of diphtheria toxin into its enzymatically active A fragment, but do not inhibit cleavage of a small synthetic peptide-based substrate, suggesting a mode of action based on steric hindrance. The inhibitory constant of the purified nanobodies was shown to be in the micromolar range and the nanobodies are non-competitive inhibitors as demonstrated by Dixon plot. Furthermore, anti-furin nanobodies can protect HEK293T cells from diphtheria toxin-induced cytotoxicity as efficiently as the well characterized PC inhibitor nona-D-arginine. In conclusion, these antibody-based single-chain nanobodies represent the first generation of highly specific, non-competitive furin inhibitors.

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 4627. doi:1538-7445.AM2012-4627
Abstract 4637: Cell penetrating antibodies for intracellular targets: Expanding the antibody-based treatments for cancer
Authors: Agnes Richard S. , Hitomi Masahiro , Basilion James P.
Published: August 2012

Antibody based therapeutics have gained significant interest in recent years. Herceptin, which targets cell surface protein, have been successful for the treatment of cancer. While antibodies are promising platforms for therapy, antibodies have been limited to targeting cell surface proteins because they cannot penetrate cells. Recently, we have developed cell penetrating antibodies engineered from the conjugation of a cell penetrating peptide (CPP) and an antibody. The CPP-mIgG and CPP-anti-RPA1 IgGs were internalized in cells as observed by fluorescence microscopy. Biological activities of these conjugates were determined and the expected anti-proliferative effects of the CPP-anti-RPA1 were subtly distinguishable from the control CPP-mIgG. While RPA1 is a valid anti-cancer target, its function depends on complicated pathways and cell cycle where dosing may have to be optimized. This demonstrates the difficulties in establishing CPP-IgG as an efficient therapeutic. To demonstrate that CPP-IgG bioconjugates are capable of inducing biological effects, we sought a different intracellular target. Antimitotic drugs are standard cancer chemotherapeutic agents. Recently, other proteins involved in the mitotic machinery have gained much attention. For example, kinesin motor protein Eg5 plays an important role in mitosis and inhibiting Eg5 prevents the formation of bipolar spindles resulting to an unmistakable change of morphology during mitosis_a star-like structure called “monoastral”. Small molecules, such as monastrol and (S)-trityl cysteine (STLC), lead to monastral formation. Thus, we chose Eg5 protein as an intracellular target to demonstrate that CPP-IgG bioconjugates are able to penetrate cells and have neutralizing activity. We expect that CPP-anti-Eg5 conjugates will penetrate cells and produce the canonical inhibition of Eg5 protein by observing for the monoastral morphology during mitosis. We tested whether CPP-anti-Eg5 IgG will present the same biological activity as STLC. We bioconjugated the anti-Eg5 with a CPP. Cell cycle synchronized cells were treated with the positive control STLC and CPP-anti-Eg5 showed an atypical star-like structure similar to monoastral. In whole, these studies show that CPP-IgG against intracellular targets are able to enter the cells and show inhibitory activities. We will describe our progress and discuss approaches to expanding the utility of antibodies as therapeutics for intracellular cancer targets.

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 4637. doi:1538-7445.AM2012-4637
Abstract 4642: Combined glycolytic inhibition and anti-vascular treatment in retinoblastoma: Novel strategy for tumor control avoids chemotherapy
Authors: Murray Timothy G. , Houston Steve , Decatur Christina , Pina Yolanda , Lampidis Ted
Published: August 2012

Purpose: The purpose of this study was to evaluate the combination treatment of glycolytic inhibitors and anti-angiogenic agents on tumor burden and hypoxia in advanced retinoblastoma using the LHbetaTag murine transgenic retinoblastoma model. Methods: Under IACUC protocol, thirty advanced LHbetaTag mice (16 weeks of age) were divided into 5 groups (n=6 per group) and treated with periocular injections of (a) saline, (b) 2-deoxy-glucose (2-DG), (c) anecortave acetate (AA) or (d) 2-DG plus AA (one day post AA treatment) or (e) 2-DG plus AA (one week post AA treatment). Eyes were enucleated at 21 weeks and tumor sections were analyzed for tumor burden and intra-tumoral hypoxia. Results: Combined treatment with 2-DG and AA (both 2-DG one day and one week post AA treatment) showed significant reduction in tumor burden compared to saline control (61% and 56% respectively, p <.001). Eyes treated with 2-DG one day post AA injection showed a significant tumor burden reduction as compared to 2-DG alone or 2-DG plus AA injected after one week (p<01). Further, intra-tumoral hypoxia was eliminated in 2-DG treated eyes at one day post AA injection and was significantly reduced in 2-DG eyes treated one week after AA injection (p<.0001). Conclusion: Combination focal therapy with glycolytic inhibitors and angiogenic inhibitors significantly enhanced tumor control and reduced tumor hypoxia. Treatment timing greatly impacted the effect of combined treatment. Combination therapy with 2-DG and AA was as effective as current chemotherapy regimens in the treatment of LHbetaTag murine transgenic retinoblastoma and may potentially serve as an alternative treatment strategy for advanced pediatric retinoblastoma.

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 4642. doi:1538-7445.AM2012-4642
Abstract 4643: Shedding light on the molecular basis of tumor dormancy using antiangiogenic nanomedicines
Authors: Tiram Galia , Segal Ehud , Bachar Gal , Shreberk Roni , Ofek Paula , Satchi-Fainaro Ronit
Published: August 2012

Based on clinical necessity for improved anticancer drugs and the importance of understanding the mechanism underlying the angiogenic switch phenomenon, we designed a targeted drug delivery system for the treatment of bone-related angiogenesis-dependent malignances. Using multidisciplinary approaches involving novel chemistry and molecular imaging, we synthesized polymer-drug nano-conjugates of HPMA copolymer-alendronate bound to TNP-470 or paclitaxel designed to target the angiogenic switch of bone neoplasms such as osteosarcoma and bone metastases. Antitumor efficacy of the conjugates was evaluated in osteosarcoma- and breast cancer bone metastases-bearing mice revealing improved anti-angiogenic and antitumor activity with reduced toxicity compared with the free drugs. To obtain data regarding the pharmacokinetic profiles of the drugs, we developed a novel prodrug system that enables real-time monitoring of drug release by different fluorescence imaging modalities. To further analyze host-tumor interactions in response to treatment, we developed a model of tumor dormancy based on mCherry-infected human osteosarcoma cells that allows deep tissue signal penetration with an optical readout. This pair of tumor cells that originate from the same parental cell line, enable intravital non-invasive detection of early stages of cancer and evaluation of treatment efficacy with the nanoconjugate. The pair has similar growth rate in vitro. However, when inoculated into mice, Saos-2-E cells established a vascularized and palpable tumor within one month, while Saos-2-D cells remained avascular and non-palpable for a year until they spontaneously escape. Phenotypic and molecular (microRNA-based) differences between these two cell lines were evaluated by in vitro and in vivo methods, with emphasis on changes related to the angiogenesis process. Although slight differences between the Saos-2-D and Saos-2-E cells were shown in vitro, when inoculated in vivo Saos-2-E cells showed increased angiogenic potential compared with the Saos-2-D cells. Our nanoconjugate was able to regress fast growing-angiogenic human tumors to a dormant avascular phenotype.

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 4643. doi:1538-7445.AM2012-4643
Abstract 4655: Aplidin induces a non-canonical ER stress response in HeLa cells response in HeLa cells
Authors: Losada Alejandro , Bejarano Alberto , Palafox Marta , Moreno-Mateos David , Moneo Victoria , Jimenez-Ruiz Antonio , Gago Federico , Cuevas Carmen , Garcia-Fernandez Luis F. , Leal Juan FM , Galmarini Carlos M.
Published: August 2012

Aplidin (APL), a marine cyclic depsipeptide originally found in the Mediterranean tunicate Aplidium albicans, is currently under phase II/III clinical investigation for cancer therapy. The mechanism of action of the compound includes the oxidative-stress mediated activation of Rac1 and JNK1, which rapidly trigger the mitochondrial apoptotic pathway. Comparing the protein expression profiles of HeLa wt cells and an APL-resistant subclone previously generated in our lab (HeLa-APL), we identified a subset of differentially expressed proteins the function of which was related to the unfolded protein response (UPR). HeLa-APL resistant cells showed reduced levels of BiP (GRP78) and relatively higher levels of Ero1α and phospho-eIF2α, indicating a higher basal endoplasmic reticulum (ER) stress. To investigate whether APL was inducing a bona fide ER stress response in HeLa cells and whether this process was essential in the mechanism of action of this compound, we compared the molecular and cellular effects elicited by APL with those induced by two well-known ER-stress inducing agents, thapsigargin and tunicamycin. Basically, while these agents elicited a complete canonical UPR response, APL only triggered part of the stress signaling cascade, including the phosphorylation of eIF2α and JNK1 and a rapid inhibition of protein synthesis. By contrast, CHOP, a transcription factor involved in launching apoptosis by the UPR, was not induced by APL. Rather, it seemed to be slightly reduced in treated cells. Similarly, while tunicamycin induced the alternative splicing of XBP1 by IRE1 and the activation of the ER-related caspase-4, APL failed to induce the same response in HeLa cells. At present, the precise connection between the partial activation of the ER stress signaling pathway and the rapid induction of apoptosis by APL remains poorly understood, although it could represent a new, aberrant UPR response

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 4655. doi:1538-7445.AM2012-4655
Abstract 4660: Antiproliferative effects of antagonists of Inhibitor of Apoptosis Proteins (IAPs) in human cancer cells
Authors: Suh Young-Ah , Lee Gi-Young , Lee Sun-Hae , Bae InHwan , Kim Maeng Sup , Jin Dong-Hoon , Kim Tae Won , Jang Se Jin
Published: August 2012

The inhibitor of apoptosis (IAP) proteins are a family of proteins containing one or more conserved, cysteine and histidine-rich baculoviral IAP repeat (BIR) N-terminal domains and a C-terminal RING domain The BIR domains of the IAPs form the zinc-figure-like structures that bind to active caspases to block caspase activity. The RING domain acts as an ubiquitin ligase to facilitate proteasomal degradation of caspases. Upregulation of IAPs is found in many tumor cell lines as well as in most primary tumor tissues. Increased expression of IAPs has been associated with apoptosis resistance and low sensitivity to chemotherapy drugs in several tumor types. Recent studies have shown that inhibition of levels or functions of survivin and/XIAP with anti-sense, siRNA, and dominant negative mutant survivin genes induces the apoptotic cell death selectively in human tumor cells but not in normal cells. Like such, the importance of IAPs in regulating the apoptotic response and as molecular targets for production of therapeutic effects selectively in tumor cells have attracted a great attention to identify peptide antagonists or small molecule inhibitors for those proteins. In the effort to develop efficient antagonists of IAPs into chemotherapy drug, we analyzed the apoptotic effect of HM antagonists, novel small molecules inhibiting IAP activities, on non-small-cell lung cancer cells and colon cells. We also analyzed changes in cell signaling pathways after HM antagonists treatment to determine the mechanisms of the apoptotic response in cancer cells. Finally, the feasibility of using HM antagonists to sensitize human cancer cells to chemotherapy drugs was examined by combining HM antagonists with two commonly used agents, cisplatin and MK2206. In the present study, we have demonstrated that this reagent efficiently decreased IAPs expression in several NSCLC cells, especially on EGFR activated cells, as well as colon cells. It showed cleaved caspase 3 and PARP, and decreasing phosphor-Akt and phosphor-Erk, indicating that the reagents exerted antiapoptotic activity through inhibition of Akt and MAPK signaling pathways. Ongoing xenograft experiments using NSCLC cells and colon cells would manifest the potential chemotherapeutic agents.

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 4660. doi:1538-7445.AM2012-4660
Abstract 467: Mitochondrial dynamics regulates migration and invasion of breast cancer cells
Authors: Tu Yaping , Zhao Jing , Zhang Juan , Yu Meifang , Wolff Dennis W. , Abel Peter W.
Published: August 2012

Mitochondrial structure is dynamically regulated by fusion and fission events that are essential for maintaining physiological functions of cells. Although dysfunction of mitochondria has been implicated in tumorigenesis, little is known about the roles of mitochondrial dynamics in metastasis, the major cause of cancer death. In the present study, we found that mitochondria were more fragmented in metastatic breast cancer cells that express higher levels of mitochondrial fission protein dynamin-related protein 1 (Drp1) and less mitochondrial fusion protein 1 (Mfn1). Silence of Drp1 or overexpression of Mfn1 resulted in mitochondria elongation and significantly suppressed metastatic abilities of breast cancer cells. In contrast, silence of Mfn proteins led to mitochondrial fragmentation and enhanced metastatic abilities of breast cancer cells. Interestingly, these manipulations of mitochondrial dynamics had no significant effects on overall mitochondrial bioenergetics but altered the subcellular distribution of mitochondria in breast cancer cells. Silence of Drp1 or overexpression of Mfn1 inhibited lamellipodia formation, a key step for cancer metastasis, and suppressed chemoattractant-induced recruitment of mitochondria to lamellipodial regions. Conversely, silence of Mfn proteins caused accumulation of more mitochondria in lamollipodia regions with higher mitochondrial membrane potential, an indication of higher ATP synthesis. Treatment with the ATP uncoupling agent m-chlorophenylhydrazone (CCCP) or mitochondrial ATP synthesis inhibitor oligomycin A caused disappearance of lamellipodia and decreased breast cancer cell migration and invasion. Together, our findings show a new mechanism for regulation of cancer cell migration and invasion by mitochondrial dynamics. Thus targeting mitochondrial dynamics may provide a novel strategy for suppressing breast cancer metastasis. This work was supported by the National Institutes of Health (CA125661), State of Nebraska Research Fund (Grant LB595), the National Natural Science Foundation of China (31100973) and the National Basic Research Program (2010CB833701, 2012CB934003).

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 467. doi:1538-7445.AM2012-467
Abstract 4805: Distinctive phospho-proteome signatures in non-small cell lung carcinoma tumors
Authors: Wei Yuhong , Taylor Paul , Tong Jiefei , Pham Nhu-An , Li Ming , Panchal Devang , To Christine , Chui Michael H. , Tsao Ming-Sound , Kislinger Thomas , Moran Michael F.
Published: August 2012

Non-small cell lung carcinoma (NSCLC) is a major, lethal cancer worldwide. Various molecular and cellular abnormalities are found in NSCLC, and an established theme is the alteration of protein phosphotyrosine (pY), as exemplified by elevated epidermal growth factor receptor (EGFR) activity recognized in a subset of NSCLC. In order to gain a broad perspective of pY-mediated signaling networks in NSCLC, mass spectrometry was used to analyze pY-containing peptides affinity purified from protease-digested xenograft tumors. Seventeen tumors representing the major NSCLC subtypes adenocarcinoma (ADC) and squamous cell carcinoma (SCC) were characterized. The major classes of pY proteins detected were cytoskeletal, protein kinases, and adhesion proteins. Pathways and cellular functions enriched in the data set included focal adhesion, adherens junction, and ErbB signaling. Five receptor tyrosine kinases (RTKs) were detected, with EGFR and EPHA2 most prevalent. Src-family kinases (YES, LYN, FRK and LCK) and focal adhesion kinase (FAK) comprised the majority of non-receptor tyrosine kinases measured. Analysis of individual pY sites on 14 tyrosine kinases indicates their activation by kinase domain activation loop phosphorylation, but also revealed additional pY sites suggestive of regulation by trafficking/endocytosis and inter- and intra-molecular protein-protein interactions involving, for example, SH2 and SH3 domains, and extra-kinase domain regions. Analysis of tumors based on protein-pY suggested subtypes distinguished by their activated tyrosine kinase networks. Genetic analysis of 3 tumors that displayed EGFR-associated phospho-proteome signatures confirmed that they carried either EGFR activating mutations (in two ADCs) or increased gene copy number (in one SCC). Western blot analysis of protein pY confirmed different patterns of cellular protein pY among the tumor phospho-types in agreement with the phospho-proteomic analysis. Quantification of kinase and substrate pY sites, according to their integrated extracted ion currents, revealed a set of phosphorylations that constituted a molecular signature that differentiated between the ADC and SCC subtypes. In summary, quantitative phospho-proteomic profiling of NSCLC tumors is feasible and may have utility in the categorization of individual tumors according to their networks of activated tyrosine kinases and substrates.

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 4805. doi:1538-7445.AM2012-4805
Abstract 4807: Identification of pre-invasive pancreatic cancer serum biomarkers using genetically engineered mouse models and mass spectrometry techniques
Authors: Ludwig Michael R. , Kojima Kyoko , Grizzle William , Mobley James A. , Klug Christopher A.
Published: August 2012

Background: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death in the United States. Surgery is the only curative treatment approach if the tumor is diagnosed early at an organ confined stage. Therefore, serum biomarkers specific for early stage pancreatic neoplasia would be of tremendous value in the treatment of this disease. Genetically engineered mouse models (GEM) of pancreatic neoplasia have been generated that closely recapitulate the molecular and biological properties of human PDAC. Here we take advantage of these GEMs and a high-throughput proteomics approaches to “dig deep” into the serum proteome of these mice to identify representative biomarkers of early stage pancreatic neoplasia. Methods: Serum was collected from 9 Pdx1-Cre (control) and 9 Pdx1-Cre;LSL-RasG12D (pre-invasive PDAC model) mice at 4 months of age. Serum was then pooled into 3 sets of 3 samples for each arm. The top 7 most abundant proteins were depleted from each pooled sample prior to labeling with a tandem mass tag (TMT) sixplex isobaric labeling system and long-column MudPIT analysis. Data were searched in SEQUEST, and quantified in ProteoIQ. Candidate proteins were accepted only with 97-99% confidence, and at least 2 unique TMT tagged peptides. Only biomarkers with >2 fold change and passing 2 non-parametric statistical tests were further evaluated. The same samples were also analyzed with a 1D MALDI-TOF MSMS approach to survey the low-mass serum proteome. In addition, a multi-dimensional MALDI-TOF approach was carried out on whole protein serum samples using spectral counting to quantify proteins instead of TMT labeling. Results/Discussion: Long-column MudPIT analysis resulted in identification of 3001 proteins with high confidence. Seventy of these proteins passed fold change and statistical filters, with 25 of these having known biological connections to pancreatic disease or function. A number of these proteins are currently being validated by immune assays. Multi-dimensional MALDI-TOF analysis resulted in identification of 8 differentially abundant proteins. Two of these proteins have been validated by Western blot. Taken together, these mass spectrometry techniques allow for a relatively in-depth survey of the serum proteome with accurate quantitation of potentially novel early stage pancreatic neoplasia biomarkers. Ultimately, the biomarkers identified in this study will be validated in human studies with the expectation that high risk patients will benefit from the application of these biomarkers for early diagnosis and improved treatment of disease.

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 4807. doi:1538-7445.AM2012-4807
Abstract 4815: Cytomegalovirus enhances glioblastoma via PDGF-B/STAT3 pathway activation
Authors: Price Richard L. , Song Jieun , Bingmer Katherine , Yi JiYeun , Rivera Andreana , Ogelsbee Michael , Cook Charles , Kwon Chang-Hyuk , Chiocca E. Antonio
Published: August 2012

Recently several groups have demonstrated cytomegalovirus (CMV) protein and DNA in a large majority of glioblastomas (GBMs), though the exact role of CMV in tumors remains controversial. Although CMV is capable of activating several oncogenic pathways, it has never been shown to be a transforming virus. Because of this, we hypothesize that CMV infection can modify the rate of gliomagenesis in the context of genetic mutations known to predispose to tumor formation. To test this, we used the Smith strain mouse CMV (MCMV) to infect Mut3 (GFAP-cre; Nf1loxP/+; Trp53-/+) mice that develop normally but eventually develop spontaneous gliomas at an adult age After intraperitoneal viral inoculation, mice exhibited multisystemic infection throughout the body, including the brain. Mut3 mice infected perinatally (103 plaque forming units) developed gliomas significantly sooner than mock-infected Mut3 mice. MCMV-infected mice developed GBMs (WHO grade IV) at a higher rate than less severe tumors, whereas, mock-infected mice developed less severe gliomas (WHO grade III). Since MCMV preferentially infected the neural stem cell (NSC) niche, an area shown to generate gliomas, we interrogated changes in signaling in this population via microarray of cultured NSCs in MCMV- versus Mock-infected mice. Ingenuity Pathway Analysis identified a PDGF-B network as the highest scoring differentially regulated network between MCMV-and Mock-infected neurospheres. In vitro infection of mouse tumorspheres and human GBM tumorspheres with CMV validated the observed upregulation of PDGF-B, a molecule shown to cause gliomas de novo. Additionally, in vitro infection of human glioma stem cells with HCMV (Towne strain) or exogenous PDGF-B activated STAT3, a key regulator of gliomas. In mice infected with MCMV, PDGF-B is upregulated in CA2/3 neurons and STAT3 is activated in the subventricular zone and dentate gyrus suggesting a paracrine effect of MCMV-induced PDGF-B. Also, infected human brain tumor stem cells demonstrated an increase in proliferation via flow cytometry. Taken together, our data suggest that CMV in gliomas may accelerate GBM progression by activating PDGF-B/STAT3 signaling in the NSC population.

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 4815. doi:1538-7445.AM2012-4815
Abstract 4821: Biomarker evaluation In phase I clinical trials of selective PI3K and PI3K/mTOR inhibitors
Authors: Spoerke Jill , O'Brien Carol , Wu Jenny , Desai Rupal , Patel Rajesh , Raja Rajiv , Koeppen Hartmut , Amler Lukas , Hampton Garret , Yan Yibing , Lackner Mark R.
Published: August 2012

Background: The PI3K/Akt pathway is frequently activated in cancer by multiple mechanisms including PI3K activating mutations, PTEN loss, RTK activation, and other means. GDC-0941 and GDC-0980 are selective PI3K and dual PI3K/mTOR inhibitors, respectively, which are currently in phase II clinical development. Assays for candidate predictive and pharmacodynamic biomarkers were conducted on patient samples collected from phase I studies of GDC-0941 and GDC-0980. The purpose was to confirm pathway inhibition at tolerable doses, as well as look for association between anti-tumor activity and candidate predictive biomarkers. Methods: Archival tumor tissue samples were analyzed using a 6 gene mutation assay (PIK3CA, EGFR, KRAS, BRAF, NRAS, AKT1), an immunohistochemistry assay for PTEN, and a fluorescence in situ hybridization (FISH) assay for PIK3CA. Select samples were analyzed for an expanded mutation panel and genome-wide copy number alterations using the Oncoscan platform (Affymetrix). For pharmacodynamic biomarker assays, analyses were initially limited to pre and post treatment phospho-AKT levels in platelet rich plasma. Once substantial downmodulation of pAKT in surrogate tissue was observed, pre- and post treatment biopsies were collected from a subset of patients and analyzed for phospho-S6 levels by immunohistochemistry. Results: Predictive biomarker assays were conducted on over 150 samples from the phase I study. Overall we found a prevalence of 7% PIK3CA mutations and 15% loss of PTEN in these samples. PIK3CA amplification was observed in several samples from ovarian cancer patients. Overall there were five confirmed partial responses in the studies, and two of these occurred in patients with activating mutations in PIK3CA. Robust PD knockdown of pAKT in surrogate tissue was observed in the early stage of dose-escalation. Moreover, substantial knockdown of pS6 was seen in tumor tissues in patients treated at or below the recommended phase II dose. Conclusions: Pharmacodynamic assays confirmed effective pathway knockdown at safe and tolerated clinical doses of GDC-0941 and GDC-0980. Anti-tumor activity was observed in patients with PIK3CA mutations, as well as some patients whose tumors did not harbor pathway alterations. These data support patient stratification in phase II clinical studies to determine whether a predictive biomarker will be useful in identifying responsive patients.

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 4821. doi:1538-7445.AM2012-4821
Abstract 4827: Humanized hepsin neutralizing antibody RO5486055 inhibits tumor growth and leads to accumulation of hepsin substrate laminin-332
Authors: Daouti Sherif , Filipovic Zoran , Stern Alvin , Tardell Christine , Zhang Xiaolei , Lucas-McGady Debra , Higgins Brian , Huang Kuo-Sen , Gubler Ueli , Li Ying , Berkofsky-Fessler Windy , Hang Julie , Samokhin Gennady , Seidl Annette , Niu Huifeng , Cai James , Hilton Holly , Thomas Markus , Pamulapati Chandra , Mundigl Olaf , Hansen Silke , Heimbrook David C. , Packman Kathryn E.
Published: August 2012

Hepsin is overexpressed in prostate and other cancers where it is implicated in promoting tumor growth, invasion, and metastasis. To further our understanding of the role of hepsin in prostate and breast cancer, a fully humanized monoclonal antibody that recognizes human and cynomolgus hepsin has been developed. RO5486055 selectively binds to hepsin but not related type II transmembrane serine proteases, and neutralizes hepsin serine protease activity with an IC50 in the single digit nM range. In LNCaP prostate cancer cells, both shRNA knockdown of the hepsin gene and RO5486055 treatment cause a similar accumulation of the β3 chain of laminin-332, a known hepsin substrate. With ip administration in mice, RO5486055 demonstrates dose-dependent exposure and a long serum half-life of 168-406 hours. RO5486055 attenuates tumor growth in the LNCaP prostate cancer and T-47D breast cancer xenograft models, but not in the CRW-22Rv1 prostate or MCF-7 breast cancer xenografts. In these models, it has been shown that the level of the β3 chain of laminin-332 detected by Western blot analysis predicts sensitivity to RO5486055-mediated growth inhibition. Moreover, accumulation of the β3 chain during RO5486055 treatment correlates with antitumor activity. In 8/10 hepsin-expressing patient-derived prostate tumors, an inverse correlation between hepsin and β3 chain expression is observed. The β3 chain of laminin-332 may therefore be useful as both a predictive and response biomarker for anti-hepsin therapy. RO5486055-mediated tumor growth inhibition is enhanced by combination with the EGFR-targeted antibody cetuximab in LNCaP prostate cancer xenografts, and by combination with hormone withdrawal in T-47D breast cancer xenografts. These preclinical results suggest that hepsin-directed therapy could be effective in prostate and breast 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 4827. doi:1538-7445.AM2012-4827
Abstract 4844: A novel mechanism of immune escape in cancer and its regulation by nitric oxide
Authors: Barsoum Ivraym B. , Hamilton Thomas K. , Miles Ellen , Siemens Robert , Graham Charles H.
Published: August 2012

One key to malignant progression is the acquired ability of tumor cells to escape immune-mediated lysis. Whereas tumor hypoxia is known to play a causal role in cancer metastasis and resistance to therapy, the link between hypoxia and immune escape in cancer remains poorly understood. Previously, we showed that hypoxia induces resistance to natural killer (NK) cell-mediated lysis in tumour cells through a mechanism that involves the shedding of the NK cell-activating ligand, MHC Class I Chain Related molecule A (MICA), from the cell surface. Additionally, we reported that activation of NO signalling in the tumour cells was able to block this hypoxia-induced shedding of MICA, as well as resistance to NK-mediated lysis. Here, we show that hypoxia-induced tumor resistance to lysis is mediated by a HIF-1-dependent increased expression of the metalloproteinase ADAM10, which is required for the hypoxia-induced shedding of MICA. Our findings demonstrate that HIF-1α knock down attenuated the hypoxia-induced expression of ADAM10 and MICA in tumour cells. Also, siRNA-mediated knockdown of HIF-1α or ADAM10 prevented the hypoxia-induced resistance of DU145 cells to innate immune effector cell-mediated lysis. Treatment with nitric oxide mimetic agents interfered with the hypoxia-induced accumulation of HIF-1α, and with the hypoxia-induced up-regulation of ADAM10 expression required for decreased surface MICA expression and resistance to lysis. To validate the in vitro results, we adopted a model in which human DU145 cells were injected subcutaneously into Swiss nude mice (T and B cell-deficient). Continuous transdermal delivery of nitroglycerine (1.8 µg/h for up to 62 days) significantly attenuated attenuated the tumor growth (P < 0.05). This tumor growth-inhibitory effect of nitroglycerine was absent in mice depleted of innate immune effector cells. Within the tumours, areas of hypoxia co-localized with the metalloproteinase ADAM10 expression. Our results demonstrate a novel mechanism by which hypoxia contributes to immune escape in tumor cells and provide evidence that activation of nitric oxide signalling interferes with this mechanism. The findings described here are important because they indicate that nitric oxide mimetics could potentially be used as immunosensitizers in the treatment and/or prevention of 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 4844. doi:1538-7445.AM2012-4844
Abstract 4846: Manipulation of autophagy during tumor cell killing regulates the growth of neighboring tumor cells
Authors: Thorburn Jacqueline , Morgan Michael , Thorburn Andrew
Published: August 2012

Many anti-cancer agents induce macroautophagy (hereafter “autophagy”) and it is widely thought that autophagy can regulate tumor chemosensitivity. Most work in this area asks if autophagy increases or decreases tumor cell killing. Using a targeted anti-cancer agent (DT-EGF, an EGF receptor-targeted diphtheria toxin), we demonstrated that autophagy can also control the characteristics of dying glioma cells. DT-EGF induces autophagy in glioma cells, however this autophagy has little impact on whether or not the cells die. Instead its major effect is to determine how the cells die. When autophagy is allowed, the cells die by a non-apoptotic mechanism that has little caspase activation, whereas if autophagy is inhibited, the glioma cells die by inducing caspase-dependent apoptosis. Here, we present data showing that the effects of this switch in death mechanism change the growth of other tumor cells that did not die in response to the drug. By mixing DT-EGF-sensitive and DT-EGF-resistant cells such that 99% of the tumor cells in the population die, we found that manipulation of autophagy in the dying cells affects the growth rates of the surviving 1% of tumor cells. If caspase-dependent apoptosis is allowed by inhibiting autophagy, the surviving tumor cells grow faster to repopulate the tumor. Conversely if autophagy is allowed in the dying cells, the resistant cells grow more slowly. We propose that cancer therapy should take account not just of how many tumor cells are killed but also the way that they are killed and that manipulating autophagy during cancer treatment could be used to improve outcomes by regulating the mechanism of tumor cell death as well as the amount of tumor cell death.

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 4846. doi:1538-7445.AM2012-4846
Abstract 4854: Genomic deletion of PTEN is associated with tumor progression and early PSA recurrence in ERG fusion positive and fusion negative prostate cancer
Authors: Krohn Antje , Stahl Phillip , Burkhardt Lia , Gerhäuser Clarissa , Huang Joseph , Mader Malte , Sirma Hüseyin , Plass Christoph , Sauter Guido , Minner Sarah , Simon Ronald , Schlomm Thorsten
Published: August 2012

The PTEN (Phospatase and Tensin Homolog deleted on chromosome Ten) gene is frequently altered in prostate cancer. To determine the prevalence and clinical significance of different mechanisms of PTEN inactivation, we analyzed 4,699 hormone-naïve and 57 hormone-refractory prostate cancers by fluorescence in situ hybridization for PTEN deletion. In addition, PTEN mutations and promoter methylation were analyzed in subsets of 149 and 34 tumors. PTEN deletions were present in 20.2% (458/2,266) of prostate cancers harboring and heterozygous (8.1%) and homozygous (12.1%) deletions. Deletions of PTEN were linked to advanced tumor stage (p<0.0001), high Gleason grade (p<0.0001), presence of lymph-node metastasis (p=0.0002), hormone refractory disease (p<0.0001), presence of ERG gene fusion (p<0.0001), and nuclear p53 accumulation (p<0.0001). PTEN deletions were also associated with early PSA recurrence in univariate (p<0.0001) and multivariate analysis (p=0.0158) if compared to the established risk factors pT stage, Gleason grade, and preoperative PSA level. The PTEN deletion had the identical prognostic impact in both in ERG fusion-positive and -negative tumors. PTEN mutations were found in 4 of 26 (15%) of cancers with heterozygous PTEN deletion, but in only 1 of 59 (2%) of cancers without PTEN deletion (p=0.0183). Analysis of the PTEN promoter for methylation changes in 34 tumors revealed no aberrations. The results of this study demonstrate that biallelic inactivation of PTEN, either by homozygous deletion or by deletion of one allele and mutation of the second one, occurs in the majority of prostate cancers with PTEN alteration and is characteristic feature for a particularly aggressive subset of metastatic and hormone-refractory prostate cancers.

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 4854. doi:1538-7445.AM2012-4854
Abstract 4924: Dysregulated transcriptional mechanisms that control crypt cell maturation: A mechanism that contributes to stem cell overpopulation during colon cancer development
Authors: Padmanabhan Jayasree , Gonye Greg , Zhang Tao , Boman Bruce M.
Published: August 2012

Our goal is to determine how transcriptional regulatory mechanisms that control crypt cell maturation in normal colonic epithelium, when dysregulated, lead to maturation arrest, tumor stem cell (SC) overpopulation and colorectal cancer (CRC) development. Accordingly, we studied changes in maturation during colon cancer development caused by APC mutations. Quantitative mapping of crypt cell subpopulations was done on colon tissues from FAP patients using markers for stem cells (SC), proliferating cells, cell-cycle arrest, and apoptosis. In normal human colon, mapping defined the sequential transitions in colonic crypts that enterocyte subpopulations undergo during their maturation from stem-to-proliferating-to-differentiated-to-apoptotic cells, illustrating the point that maturation is a process involving both proliferation and differentiation. In normal-appearing (heterozygous-mutant) and adenomatous (homozygous-mutant) FAP crypts, SC and proliferating cell subpopulations increasingly expanded towards the crypt top, while differentiated/apoptotic subpopulations in the upper crypt contracted, indicating that crypt cell maturation becomes progressively delayed. These data indicate that there are coordinated mechanisms that regulate maturation of crypt cells in normal crypts which become dysregulated in premalignant crypts. To investigate possible transcriptional regulation of a network of genes underlying maturation, we used bioinformatic tools to identify (i) genes that are co-expressed in the proliferating cell population and (ii) transcriptional regulatory elements (TREs) statistically enriched in the promoter regions among those genes. Computational analysis revealed (i) a specific cassette of sixty-six genes that are co-expressed with the markers used for mapping the proliferating cell population in the lower colonic crypt and (ii) eleven TREs that are significantly enriched in the promoter regions among genes in this gene set. Quantitative PCR and immunohistochemistry was then used to validate that the transcription factors (TFs), which correspond to the common TREs, are differentially expressed along the normal crypt axis and in normal colonic epithelium versus malignant colon tissues. Our results indicate that dysregulation of a transcriptional regulatory network contributes to delayed crypt cell maturation, SC overpopulation, and initiation and promotion of CRC. Accordingly, restoring maturation by targeting transcriptional pathways that control expression of multiple genes involved in crypt cell maturation may be a therapeutically effective approach to CRC 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 4924. doi:1538-7445.AM2012-4924
Abstract 4928: Deregulation of asymmetry genes in oligodendrogliomas and glioblastomas
Authors: Andor Noemi , Petritsch Claudia
Published: August 2012

BACKGROUND. Normal neural stem cells and NG2+ oligodendrocyte progenitors (OPC) undergo asymmetric cell division to generate a balanced number of self-renewing and differentiating progeny. Our recent studies showed that NG2+ OPC are the tumor-forming cells in oligodendrogliomas (ODG), a therapy-sensitive glioma. We provide cellular and molecular evidence that defects in asymmetric division of NG2+ OPC might contribute to tumor-initiation in oligodendrogliomas. In contrast, tumor-initiating cells in the highly resistant glioblastomas (GBM) express stem cell markers and show a high number of asymmetric divisions. We propose that distinct key asymmetry genes might be deregulated in the two types of tumor-initiating cells causing them to generate progeny with distinct therapy-sensitivity. Identifying these asymmetry key regulator networks will elucidate the mechanism by which tumor-initiating cells in gliomas maintain tumors and re-grow them after therapy. METHOD. A knowledge based network, consisting of asymmetry-genes and their transcriptional regulators, was constructed and refined by correlated gene expression. Subsequently, copy number and methylation were mapped onto the network and their impact on expression was assessed. In addition, the expression profiles of asymmetry genes within glioma samples were compared against their counterparts in normal brain samples and putative associations between expression and clinical outcome were tested. RESULTS. Our study has identified cancer- and differentiation associated transcription factors as putative regulators of asymmetry genes. In GBM tumors it was found that asymmetry genes are mostly affected by single copy gains and heterozygous deletions with an apparent influence on gene expression. When considered upon ODG tumors, the pattern of deregulation was distinct from that observed in GBM: for a subset of asymmetry genes a mutually exclusive deregulation was observable in either GBM or ODG, while another gene subset was progressively deregulated in ODG- and GBM. Taken together, our results indicate that asymmetry genes are distinctly regulated in ODG and GBM. We will discuss a model how this could contribute to distinct levels of therapy resistance.

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 4928. doi:1538-7445.AM2012-4928
Abstract 4934: Employing the Berg Interrogative Biology® systems platform to elucidate cellular signaling changes that contribute to the pathophysiology of hepatocellular carcinoma (HCC)
Authors: Du Min , Vishnudas Vivek , Lao Socheata C. , Luan Shen , Jimenez Joaquin J. , Li Jie , Sarangarajan Rangaprasad , Narain Niven R.
Published: August 2012

Hepatocellular Carcinoma (HCC) is one of the leading causes of cancer-related death worldwide, ranked as the third most fatal cancer after lung and stomach carcinomas. The diverse etiology, high morbidity/mortality, lack of diagnostic markers for early diagnosis and the highly variable clinical course of HCC have hindered advances in diagnosis and treatment. After years of studying the HCC, the understanding of molecular mechanism operational in HCC remains incomplete. The genomic, transcriptomic and comparative proteomic profiles have yielded some important insights for HCC research. However, many studies focused on single aspect of the cellular changes associated with HCC, hindering the full understanding of biological systems in their true complexity and dynamics. We describe a novel approach combining the power of cell biology, integrated proteomics platforms and an informatics platform that generates causal protein networks. In order to delineate the role of post-translation modification and enzymes that partake in such mechanisms, we incorporated activity based proteomics employing kinase enrichment probes and phosphoproteome mapping of total proteins in HCC cellular models. Multikinase inhibitor Sorafenib, the first line chemotherapeuitc agent for the advanced HCC patients, was used to probe the global kinase activity and protein phosphorylation changes associated with this treatment. Comparative proteomics, phosphoproteome and kinase activity data was integrated into the AI based REFS™ informatics platform. Causal networks of protein interaction specifically from a functional stand point namely kinase activity and potential targets that kinases can phosphorylate were generated. In addition, using cellular functional read out, proteins/ kinases that modulate phosphorylation of targets and mechanistically drive pathophysiological cellular behavior were determined. The approach outlined here enables global characterization of cellular responses, insights into mechanisms of chemo sensitivity and potential targets/biomarkers for clinical management of HCC.

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 4934. doi:1538-7445.AM2012-4934