bluuebunny/crossref_metadata_2025 huggingface dataset:
Dendritic Cells Recognize Tumor-Specific Glycosylation of Carcinoembryonic Antigen on Colorectal Cancer Cells through Dendritic Cell–Specific Intercellular Adhesion Molecule-3–Grabbing Nonintegrin
Authors: van Gisbergen Klaas P.J.M. , Aarnoudse Corlien A. , Meijer Gerrit A. , Geijtenbeek Teunis B.H. , van Kooyk Yvette
Published: September 2005

Dendritic cells play a pivotal role in the induction of antitumor immune responses. Immature dendritic cells are located intratumorally within colorectal cancer and intimately interact with tumor cells, whereas mature dendritic cells are present peripheral to the tumor. The majority of colorectal cancers overexpress carcinoembryonic antigen (CEA), and malignant transformation changes the glycosylation of CEA on colon epithelial cells, resulting in higher levels of Lewisx and de novo expression of Lewisy on tumor-associated CEA. Dendritic cells express the C-type lectin dendritic cell–specific intercellular adhesion molecule-3–grabbing nonintegrin (DC-SIGN) that has high affinity for nonsialylated Lewis antigens, so we hypothesized that DC-SIGN is involved in recognition of colorectal cancer cells by dendritic cells. We show that immature dendritic cells within colorectal cancer express DC-SIGN and that immature dendritic cells but not mature dendritic cells interact with tumor cells. DC-SIGN mediates these interactions through binding of Lewisx and Lewisy carbohydrates on CEA of colorectal cancer cells. In contrast, DC-SIGN does not bind CEA expressed on normal colon epithelium that contains low levels of Lewis antigens. This indicates that dendritic cells may recognize colorectal cancer cells through binding of DC-SIGN to tumor-specific glycosylation on CEA. Similar to pathogens that target DC-SIGN to escape immunosurveillance, tumor cells may interact with DC-SIGN to suppress dendritic cell functions.
Mechanism of Cell Cycle Regulation by FIP200 in Human Breast Cancer Cells
Authors: Melkoumian Zara K. , Peng Xu , Gan Boyi , Wu Xiaoyang , Guan Jun-Lin
Published: September 2005

FIP200 is a novel protein inhibitor for focal adhesion kinase (FAK), which binds to FAK directly and inhibits its kinase activity and associated cellular functions, such as cell adhesion, spreading, and motility in fibroblasts. Here we show that FIP200 inhibits G1-S phase progression, proliferation, and clonogenic survival in human breast cancer cells. Consistent with the G1 arrest induced by FIP200, we found that FIP200 increased p21 and decreased cyclin D1 protein levels in breast cancer cells. In addition, FIP200 significantly induced p21 promoter activity in MCF-7 cells and this response was abolished upon deletion of p53 binding sites within p21 promoter. Furthermore, we found that FIP200 could interact with exogenous and endogenous p53 protein and significantly increase its half-life compared with the control cells. We also found that the NH2-terminal 154 residues of FIP200 were sufficient to mediate p53 interaction and G1 arrest in cells. The increase in p53 half-life correlated with the increased phosphorylation at Ser15 and decreased proteasomal degradation via ubiquitin and Hdm2-independent mechanism. Stabilization of p53 by FIP200 could be partially reversed by NQO1 inhibitor, dicoumarol. In contrast to p53, FIP200 decreased cyclin D1 protein half-life by promoting proteasome-dependent degradation of cyclin D1. In summary, our results suggest that FIP200 increases p21 protein levels via stabilization of its upstream regulator p53 and decreases cyclin D1 protein by promoting its degradation. Both effects are critical for FIP200-induced G1 arrest and may contribute to the putative antitumor activities of FIP200 in breast cancer.
Inactivation of Myc in Murine Two-Hit B lymphomas Causes Dormancy with Elevated Levels of Interleukin 10 Receptor and CD20: Implications for Adjuvant Therapies
Authors: Yu Duonan , Dews Michael , Park Andrea , Tobias John W. , Thomas-Tikhonenko Andrei
Published: September 2005

Overexpression of c-Myc and inactivation of p53 are hallmarks of human Burkitt's lymphomas. We had previously showed that transduction of murine p53-null bone marrow cells with a Myc-encoding retrovirus is sufficient for B lymphomagenesis. To address the role of Myc in tumor sustenance, we generated lymphomas induced by the Myc-estrogen receptor fusion protein (MycER). Engrafted hosts were continuously treated with the ER ligand 4-hydroxytamoxifen (4-OHT) to allow tumor formation. Subsequent inactivation of MycER via 4-OHT deprivation resulted in tumor stasis but only partial regression. At the cellular level, dormant neoplastic lymphocytes withdrew from mitosis and underwent further B-cell differentiation. Concomitantly, they up-regulated genes involved in lymphocyte proliferation and survival, most notably interleukin 10 receptor α (IL10Rα) and CD20, the target for antibody therapy with Rituxan. We found that overexpression of IL10Rα affords significant proliferative advantages and in 4-OHT–deprived animals correlates with eventual tumor relapse. Both dormant and relapsing tumors maintain IL10Rα expression suggesting that they might be sensitive to emerging drugs targeting the IL-10 pathway. Up-regulation of CD20 following Myc inactivation was also observed in immortalized human lymphocytes. Importantly, in this system, MycOFFCD20HIGH cells were more prone to Rituxan-induced apoptosis than MycONCD20MED. Thus, targeting Myc, while moderately effective on its own, shapes the phenotype of dormant neoplastic cells and sensitizes them to adjuvant molecular therapies.
Ckap2 Regulates Aneuploidy, Cell Cycling, and Cell Death in a p53-Dependent Manner
Authors: Tsuchihara Katsuya , Lapin Valentina , Bakal Christopher , Okada Hitoshi , Brown Lauren , Hirota-Tsuchihara Masami , Zaugg Kathrin , Ho Alexandra , Itie-YouTen Annick , Harris-Brandts Marees , Rottapel Robert , Richardson Christopher D. , Benchimol Samuel , Mak Tak Wah
Published: September 2005

We used DNA microarray screening to identify Ckap2 (cytoskeleton associated protein 2) as a novel p53 target gene in a mouse erythroleukemia cell line. DNA damage induces human and mouse CKAP2 expression in a p53-dependent manner and p53 activates the Ckap2 promoter. Overexpressed Ckap2 colocalizes with and stabilizes microtubules. In p53-null cells, overexpression of Ckap2 induces tetraploidy with aberrant centrosome numbers, suggesting disturbed mitosis and cytokinesis. In p53-competent cells, Ckap2 does not induce tetraploidy but activates p53-mediated cell cycle arrest and apoptosis. Our data suggest the existence of a functional positive feedback loop in which Ckap2 activates the G1 tetraploidy checkpoint and prevents aneuploidy.
DNA-Dependent Protein Kinase Is a Molecular Target for the Development of Noncytotoxic Radiation–Sensitizing Drugs
Authors: Shinohara Eric T. , Geng Ling , Tan Jiahui , Chen Heidi , Shir Yu , Edwards Eric , Halbrook James , Kesicki Edward A. , Kashishian Adam , Hallahan Dennis E.
Published: September 2005

DNA-dependent protein kinase (DNA-PK)–defective severe combined immunodeficient (SCID) mice have a greater sensitivity to ionizing radiation compared with wild-type mice due to deficient repair of DNA double-strand break. SCID cells were therefore studied to determine whether radiosensitization by the specific inhibitor of DNA-PK, IC87361, is eliminated in the absence of functional DNA-PK. IC87361 enhanced radiation sensitivity in wild-type C57BL6 endothelial cells but not in SCID cells. The tumor vascular window model was used to assess IC87361-induced radiosensitization of SCID and wild-type tumor microvasculature. Vascular density was 5% in irradiated SCID host compared with 50% in C57BL6 mice (P < 0.05). IC87361 induced radiosensitization of tumor microvasculature in wild-type mice that resembled the radiosensitive phenotype of tumor vessels in SCID mice. Radiosensitization by IC87361 was eliminated in SCID tumor vasculature, which lack functional DNA-PK. Irradiated LLC and B16F0 tumors implanted into SCID mice showed greater tumor growth delay compared with tumors implanted into either wild-type C57BL6 or nude mice. Furthermore, LLC tumors treated with radiation and IC87361 showed tumor growth delay that was significantly greater than tumors treated with radiation alone (P < 0.01 for 3 Gy alone versus 3 Gy + IC87361). DNA-PK inhibitors induced no cytotoxicity and no toxicity in mouse normal tissues. Mouse models deficient in enzyme activity are useful to assess the specificity of novel kinase inhibitors. DNA-PK is an important target for the development of novel radiation-sensitizing drugs that have little intrinsic cytotoxicity.
Immune Selection and Emergence of Aggressive Tumor Variants as Negative Consequences of Fas-Mediated Cytotoxicity and Altered IFN-γ-Regulated Gene Expression
Authors: Liu Kebin , Caldwell Sheila A. , Abrams Scott I.
Published: September 2005

Antitumor responses can be induced in patients via active or adoptive immunotherapy, yet complete tumor eradication occurs infrequently. This paradox in tumor immunology led us to address two questions: (a) Does an antitumor response, which is intended to destroy the aberrant target population, also at the same time select for aggressive tumor variants (ATV) in vivo? (b) If this process does occur, what is the contribution of the perforin- or Fas-mediated effector mechanism in the immune selection of such ATV? Here, in an experimental mouse lung metastasis model, we showed that ATV generated either naturally in vivo or in vitro by anti-Fas selection resembled each other biologically and genetically as judged by enhanced tumor growth and genome-scale gene expression profiling, respectively. Furthermore, ATV that survived CTL adoptive immunotherapy displayed an even more profound loss of Fas expression and function as well as enhanced malignant proficiency in vivo. ATV, however, retained sensitivity to perforin-mediated lysis in vitro. Lastly, such ATV displayed a diminished responsiveness in their expression of IFN-γ-regulated genes, including those mechanistically linked to Fas-mediated death (i.e., Fas and caspase-1). Overall, we showed that (a) immune selection did occur in vivo and played an important role in the emergence of ATV, (b) ATV bearing a Fas-resistant phenotype was a chief consequence of immune selection, and (c) an overall diminished responsiveness of IFN-γ-regulated gene expression was characteristic of ATV. Thus, in this model, Fas-mediated cytotoxicity, in concert with IFN-γ-regulated gene expression, mechanistically constituted significant determinants of immune selection of ATV in vivo.
Optimizing Radiolabeled Engineered Anti-p185HER2 Antibody Fragments forIn vivoImaging
Authors: Olafsen Tove , Kenanova Vania E. , Sundaresan Gobalakrishnan , Anderson Anne-Line , Crow Desiree , Yazaki Paul J. , Li Lin , Press Michael F. , Gambhir Sanjiv S. , Williams Lawrence E. , Wong Jeffrey Y.C. , Raubitschek Andrew A. , Shively John E. , Wu Anna M.
Published: September 2005

We have recently described the in vivo properties of an iodinated anti-p185HER2 engineered antibody fragment [minibody (scFv-CH3)2; 80 kDa], made from the internalizing 10H8 monoclonal antibody. Although the 10H8 minibody showed excellent binding to the target in vitro, only modest tumor uptake [5.6 ± 1.7% injected dose per gram (ID/g) of tissue] was achieved in nude mice bearing MCF7/HER2 breast cancer tumors. Here, in an attempt to improve targeting, the 10H8 minibody was conjugated to 1,4,7,10-tetraazacyclododecane-N, N′, N′′, N′′′-tetraacetic acid (DOTA), radiometal labeled, and evaluated in vivo. The tumor uptake of 111In-DOTA 10H8 minibody was 5.7 ± 0.1% ID/g, similar to the radioiodinated 10H8 minibody. However, in addition to the expected liver clearance, the kidneys had unexpectedly high activity (34.0 ± 4.0% ID/g). A minibody derived from a second anti-p185HER2 antibody (trastuzumab; hu4D5v8) was also made. Tumor uptakes, evaluated by quantitative microPET using 64Cu-DOTA hu4D5v8 minibody, were 4.2 ± 0.5% ID/g. Furthermore, in non-tumor-bearing mice, 111In-DOTA hu4D5v8 minibody exhibited similar elevated uptake in the kidneys (28.4 ± 6.5% ID/g). Immunohistochemical staining of kidneys from non-tumor-bearing mice showed strong specific staining of the proximal tubules, and Western blot analysis of kidney lysate confirmed the presence of cross-reactive antigen. To further improve tumor uptake and normal tissue distribution, a larger hu4D5v8 fragment [(scFv-CH2-CH3)2; 105 kDa] was made, engineered to exhibit rapid clearance kinetics. This fragment, when evaluated by microPET, exhibited improved tumor targeting (12.2 ± 2.4% ID/g) and reduced kidney uptake (13.1 ± 1.5% ID/g). Thus, by manipulating the size and format of anti-p185HER2 antibody fragments, the kidney activity was reduced and high or low expression of p185HER2 in xenografts could be distinguished by microPET imaging.
Suppression of Tumor Development and Metastasis Formation in Mice Lacking the S100A4(mts1) Gene
Authors: Grum-Schwensen Birgitte , Klingelhofer Jörg , Berg Christian Hededam , El-Naaman Christina , Grigorian Mariam , Lukanidin Eugene , Ambartsumian Noona
Published: September 2005

The S100A4(mts1) protein stimulates metastatic spread of tumor cells. An elevated expression of S100A4 is associated with poor prognosis in many human cancers. Dynamics of tumor development were studied in S100A4-deficient mice using grafts of CSML100, highly metastatic mouse mammary carcinoma cells. A significant delay in tumor uptake and decreased tumor incidences were observed in S100A4(−/−) mice compared with the wild-type controls. Moreover, tumors developed in S100A4(−/−) mice never metastasize. Immunohistochemical analyses of these tumors revealed reduced vascularity and abnormal distribution of host-derived stroma cells. Coinjection of CSML100 cells with immortalized S100A4(+/+) fibroblasts partially restored the dynamics of tumor development and the ability to form metastasis. These fibroblasts were characterized by an enhanced motility and invasiveness in comparison with S100A4(−/−) fibroblasts, as well as by the ability to release S100A4 into the tumor environment. Taken together, our results point to a determinative role of host-derived stroma cells expressing S100A4 in tumor progression and metastasis.
Distinct B-Cell and T-Cell Lymphoproliferative Disease Prevalence among Dog Breeds Indicates Heritable Risk
Authors: Modiano Jaime F. , Breen Matthew , Burnett Robert C. , Parker Heidi G. , Inusah Seidu , Thomas Rachael , Avery Paul R. , Lindblad-Toh Kerstin , Ostrander Elaine A. , Cutter Gary C. , Avery Anne C.
Published: September 2005

Immunophenotypes in lymphoproliferative diseases (LPD) are prognostically significant, yet causative factors for these conditions, and specifically those associated with heritable risk, remain elusive. The full spectrum of LPD seen in humans occurs in dogs, but the incidence and lifetime risk of naturally occurring LPD differs among dog breeds. Taking advantage of the limited genetic heterogeneity that exists within dog breeds, we tested the hypothesis that the prevalence of LPD immunophenotypes would differ among different breeds. The sample population included 1,263 dogs representing 87 breeds. Immunophenotype was determined by the presence of clonal rearrangements of immunoglobulin heavy chain or T-cell receptor γ chain. The probability of observing the number of B-cell or T-cell tumors in a particular breed or breed group was compared with three reference populations. Significance was computed using χ2 test, and logistic regression was used to confirm binomial predictions. The data show that, among 87 breeds tested, 15 showed significant differences from the prevalence of LPD immunophenotypes seen across the dog population as a whole. More significantly, elevated risk for T-cell LPD seems to have arisen ancestrally and is retained in related breed groups, whereas increased risk for B-cell disease may stem from different risk factors, or combinations of risk factors, arising during the process of breed derivation and selection. The data show that domestic dogs provide a unique and valuable resource to define factors that mediate risk as well as genes involved in the initiation of B-cell and T-cell LPD.
The Antitumor Enediyne C-1027 Alters Cell Cycle Progression and Induces Chromosomal Aberrations and Telomere Dysfunction
Authors: McHugh Mary M. , Gawron Loretta S. , Matsui Sei-Ichi , Beerman Terry A.
Published: September 2005

This study examined the extent of chromosome instability induced in cultured human colon carcinoma HCT116 cells by the antitumor radiomimetic enediyne antibiotic C-1027. Spectral karyotype analysis showed frequent intrachromosomal fusions and fragmentations 26 hours after addition of as little as 0.035 nmol/L C-1027. When the concentration was increased to 0.14 nmol/L C-1027, 92% of cells showed chromosomal aberrations compared with only 2.9% after treatment with an equivalent growth inhibitory dose of ionizing radiation (20 Gy). Thus, chromosome misrejoining was associated to a much greater extent with C-1027–induced than with ionizing radiation–induced cell growth inhibition. Despite these aberrations, a large fraction of C-1027–treated cells progressed into G1. Comet analysis showed that these extensive chromosomal anomalies were not due to increased induction or reduced repair of C-1027–induced compared with ionizing radiation–induced strand breaks. Fluorescence in situ hybridization analysis showed that misrejoining of telomere repeats (i.e., chromosomes joined end to end at their telomeres or fused together after complete loss of telomere sequences) was observed within 26 hours of C-1027 addition. The extreme cytotoxicity of C-1027 may reflect both induction and erroneous repair of DNA double-strand break in the whole genome and/or in subgenomic targets such as telomere sequences.
Immunoselective Pressure and Human Leukocyte Antigen Class I Antigen Machinery Defects in Microsatellite Unstable Colorectal Cancers
Authors: Kloor Matthias , Becker Christina , Benner Axel , Woerner Stefan M. , Gebert Johannes , Ferrone Soldano , von Knebel Doeberitz Magnus
Published: September 2005

In colorectal cancer, the immune response is particularly pronounced against tumors displaying the high microsatellite instability (MSI-H) phenotype. MSI-H tumors accumulate mutations affecting microsatellites located within protein encoding regions (coding microsatellites, cMS), which lead to translational shifts of the respective reading frames. Consequently, novel tumor-specific frameshift-derived neopeptides (FSP) are generated and presented by MSI-H tumor cells, thus eliciting effective cytotoxic immune responses. To analyze whether the immunoselective pressure was reflected by the phenotype of MSI-H colorectal cancer cells, we compared here the expression of antigen processing machinery (APM) components and human leukocyte antigen (HLA) class I antigen subunits in 20 MSI-H and 20 microsatellite-stable (MSS) colorectal cancer using a panel of newly developed APM component-specific monoclonal antibodies. In addition, we did a systematic analysis of mutations at cMS located within APM genes and β2-microglobulin (β2m). Total HLA class I antigen loss was observed in 12 (60.0%) of the 20 MSI-H lesions compared with only 6 (30.0%) of the 20 MSS colorectal cancer lesions. Moreover, total loss of membraneous HLA-A staining was significantly more frequent in MSI-H colorectal cancer (P = 0.0024). Mutations at cMS of β2m and genes encoding APM components (TAP1 and TAP2) were detected in at least 7 (35.0%) of 20 MSI-H colorectal cancers but in none of the MSS colorectal cancers (P = 0.0002). These data show that defects of HLA class I antigen processing and presentation seem to be significantly more frequent in MSI-H than in MSS colorectal cancer, suggesting that in MSI-H colorectal cancer the immunoselective pressure leads to the outgrowth of cells with defects of antigen presentation.
Real-time In vivo Dual-color Imaging of Intracapillary Cancer Cell and Nucleus Deformation and Migration
Authors: Yamauchi Kensuke , Yang Meng , Jiang Ping , Yamamoto Norio , Xu Mingxu , Amoh Yasuyuki , Tsuji Kazuhiko , Bouvet Michael , Tsuchiya Hiroyuki , Tomita Katsuro , Moossa A.R. , Hoffman Robert M.
Published: September 2005

The mechanism of cancer cell deformation and migration in narrow vessels is incompletely understood. In order to visualize the cytoplasmic and nuclear dynamics of cells migrating in capillaries, red fluorescent protein was expressed in the cytoplasm, and green fluorescent protein, linked to histone H2B, was expressed in the nucleus of cancer cells. Immediately after the cells were injected in the heart of nude mice, a skin flap on the abdomen was made. With a color CCD camera, we could observe highly elongated cancer cells and nuclei in capillaries in the skin flap in living mice. The migration velocities of the cancer cells in the capillaries were measured by capturing images of the dual-color fluorescent cells over time. The cells and nuclei in the capillaries elongated to fit the width of these vessels. The average length of the major axis of the cancer cells in the capillaries increased to approximately four times their normal length. The nuclei increased their length 1.6 times in the capillaries. Cancer cells in capillaries over 8 μm in diameter could migrate up to 48.3 μm/hour. The data suggests that the minimum diameter of capillaries where cancer cells are able to migrate is approximately 8 μm. The use of the dual-color cancer cells differentially labeled in the cytoplasm and nucleus and associated fluorescent imaging provide a powerful tool to understand the mechanism of cancer cell migration and deformation in small vessels.
A Role for ADAM12 in Breast Tumor Progression and Stromal Cell Apoptosis
Authors: Kveiborg Marie , Fröhlich Camilla , Albrechtsen Reidar , Tischler Verena , Dietrich Nikolaj , Holck Peter , Kronqvist Pauliina , Rank Fritz , Mercurio Arthur M. , Wewer Ulla M.
Published: September 2005

As in developmental and regenerative processes, cell survival is of fundamental importance in cancer. Thus, a tremendous effort has been devoted to dissecting the molecular mechanisms involved in understanding the resistance of tumor cells to programmed cell death. Recently, the importance of stromal fibroblasts in tumor initiation and progression has been elucidated. Here, we show that stromal cell apoptosis occurs in human breast carcinoma but is only rarely seen in nonmalignant breast lesions. Furthermore, we show that ADAM12, a disintegrin and metalloprotease up-regulated in human breast cancer, accelerates tumor progression in a mouse breast cancer model. ADAM12 does not influence tumor cell proliferation but rather confers both decreased tumor cell apoptosis and increased stromal cell apoptosis. This dual role of ADAM12 in governing cell survival is underscored by the finding that ADAM12 increases the apoptotic sensitivity of nonneoplastic cells in vitro while rendering tumor cells more resistant to apoptosis. Together, these results show that the ability of ADAM12 to influence apoptosis may contribute to tumor progression.
Disruption of the Inhibitor of Apoptosis Protein Survivin Sensitizes Bcr-abl–Positive Cells to STI571-Induced Apoptosis
Authors: Wang Zhanxiang , Sampath Janardhan , Fukuda Seiji , Pelus Louis M.
Published: September 2005

The Bcr-abl oncogene induces hematopoietic cell transformation and protects cells from apoptosis; however, the mechanisms whereby Bcr-abl blocks apoptosis are poorly defined. We examined whether the inhibitor of apoptosis protein (IAP) family, in particular survivin, are regulated by Bcr-abl. Overexpression of Bcr-abl in Mo7e or BaF3 hematopoietic cells elevated survivin mRNA and protein concomitant with a 4-fold increase in survivin promoter activity. The region of the survivin promoter responding to Bcr-abl was narrowed down to a 116 bp fragment between nucleotides −1,194 and −1,078. The IAP family member IAP-like protein-2 was also up-regulated by Bcr-abl. Disruption of Bcr-abl in Bcr-abl–transduced BaF3 cells by small interfering RNA resulted in 3- to 4-fold reduction in survivin protein confirming the link between Bcr-abl and survivin. Survivin disruption in Bcr-abl–transduced Mo7e cells, or in K562 cells that endogenously express Bcr-abl, by transfection with dominant-negative or antisense survivin constructs promoted apoptosis induced by the Bcr-abl tyrosine kinase inhibitor STI571, which was accompanied by caspase-dependent cleavage of Bcr-abl, mitochondrial membrane potential disruption, and enhanced mitochondrial cytochrome c release. Although ectopic survivin protected K562 cells from apoptosis induced by STI571, it did not protect cells from apoptosis induced either by tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) or the combination of TRAIL plus Hemin. Our results identify a new signal pathway downstream of Bcr-abl, in addition to the Bcl-2 family involved in the antiapoptotic effects of Bcr-abl, and suggest that anti-survivin therapy may have utility in patients with chronic myelogenous leukemia.
Identification of a Novel c-Myc Protein Interactor, JPO2, with Transforming Activity in Medulloblastoma Cells
Authors: Huang Annie , Ho Cynthia S.W. , Ponzielli Romina , Barsyte-Lovejoy Dalia , Bouffet Eric , Picard Daniel , Hawkins Cynthia E. , Penn Linda Z.
Published: September 2005

c-myc oncogene activation is critical in the pathogenesis of a spectrum of human malignancies. The c-Myc NH2-terminal domain (MycNTD) is essential for cellular transformation, and mediates critical protein interactions that modulate c-Myc oncogenic properties. In medulloblastoma, the most common malignant pediatric brain tumor, deregulated c-myc expression is linked with poorer disease phenotypes and outcomes. The biological basis for these associations is, however, not well understood. To better understand mechanisms underlying Myc-mediated transformation of medulloblastoma, we sought to identify novel MycNTD protein interactors from a medulloblastoma cell line library using a unique two-hybrid system. We identified a novel MycNTD binding protein, JPO2, which shows nuclear colocalization with c-Myc, and interacts with c-Myc both in vitro and in mammalian cells. In Rat1a transformation assays, JPO2 potentiates c-Myc transforming activity, and can complement a transformation-defective Myc mutant. Immunohistochemical studies indicate tumor-specific JPO2 expression in human medulloblastoma, and an association of JPO2 expression with metastatic tumors. Significantly, JPO2 expression induces colony formation in UW228, a medulloblastoma cell line, whereas RNAi-mediated JPO2 knockdown impairs colony formation in UW228, and in Myc-transformed UW228 cells. These data provide evidence for biochemical and functional interaction between c-Myc and JPO2 in medulloblastoma transformation. JPO2 is closely related to JPO1, a Myc transcriptional target with transforming activity. As tumor-specific JPO1 expression in human and murine medulloblastoma has also been reported; these collective observations suggest important functional links between the novel JPO protein family and c-Myc in medulloblastoma transformation.
Plasminogen Kringle 5–Engineered Glioma Cells Block Migration of Tumor-Associated Macrophages and Suppress Tumor Vascularization and Progression
Authors: Perri Sabrina R. , Nalbantoglu Josephine , Annabi Borhane , Koty Zafiro , Lejeune Laurence , François Moïra , Di Falco Marcos R. , Béliveau Richard , Galipeau Jacques
Published: September 2005

Angiostatin, a well-characterized angiostatic agent, is a proteolytic cleavage product of human plasminogen encompassing the first four kringle structures. The fifth kringle domain (K5) of human plasminogen is distinct from angiostatin and has been shown, on its own, to act as a potent endothelial cell inhibitor. We propose that tumor-targeted K5 cDNA expression may act as an effective therapeutic intervention as part of a cancer gene therapy strategy. In this study, we provide evidence that eukaryotically expressed His-tagged human K5 cDNA (hK5His) is exported extracellularly and maintains predicted disulfide bridging conformation in solution. Functionally, hK5His protein produced by retrovirally engineered human U87MG glioma cells suppresses in vitro migration of both human umbilical vein endothelial cells and human macrophages. Subcutaneous implantation of Matrigel-embedded hK5His-producing glioma cells in nonobese diabetic/severe combined immunodeficient mice reveals that hK5His induces a marked reduction in blood vessel formation and significantly suppresses the recruitment of tumor-infiltrating CD45+Mac3+Gr1− macrophages. Therapeutically, we show in a nude mouse orthotopic brain cancer model that tumor-targeted K5 expression is capable of effectively suppressing glioma growth and promotes significant long-term survival (>120 days) of test animals. These data suggest that plasminogen K5 acts as a novel two-pronged anticancer agent, mediating its inhibitory effect via its action on host-derived endothelial cells and tumor-associated macrophages, resulting in a potent, clinically relevant antitumor effect.
Therapeutic EphA2 Gene Targeting In vivo Using Neutral Liposomal Small Interfering RNA Delivery
Authors: Landen Charles N. , Chavez-Reyes Arturo , Bucana Corazon , Schmandt Rosemarie , Deavers Michael T. , Lopez-Berestein Gabriel , Sood Anil K.
Published: September 2005

Inducing destruction of specific mRNA using small interfering RNA (siRNA) is a powerful tool in analysis of protein function, but its use as a therapeutic modality has been limited by inefficient or impractical delivery systems. We have used siRNA incorporated into the neutral liposome 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) for efficient in vivo siRNA delivery. In nude mice bearing i.p. ovarian tumors, nonsilencing siRNA tagged with the fluorochrome Alexa 555 was encapsulated into DOPC liposomes and shown to be taken up by the tumor as well as many major organs. Furthermore, DOPC-encapsulated siRNA targeting the oncoprotein EphA2 was highly effective in reducing in vivo EphA2 expression 48 hours after a single dose as measured by both Western blot and immunohistochemistry. Therapy experiments in an orthotopic mouse model of ovarian cancer were initiated 1 week after injection of either HeyA8 or SKOV3ip1 cell lines. Three weeks of treatment with EphA2-targeting siRNA-DOPC (150 μg/kg twice weekly) reduced tumor growth when compared with a nonsilencing siRNA (SKOV3ip1: 0.35 versus 0.70 g; P = 0.020; HeyA8: 0.98 versus 1.51 g; P = 0.16). When EphA2-targeting siRNA-DOPC was combined with paclitaxel, tumor growth was dramatically reduced compared with treatment with paclitaxel and a nonsilencing siRNA (SKOV3ip1: 0.04 versus 0.22 g; P < 0.001; HeyA8: 0.21 versus 0.84 g; P = 0.0027). These studies show the feasibility of siRNA as a clinically applicable therapeutic modality.
Protein Kinase Cε Is a Predictive Biomarker of Aggressive Breast Cancer and a Validated Target for RNA Interference Anticancer Therapy
Authors: Pan Quintin , Bao Li Wei , Kleer Celina G. , Sabel Michael S. , Griffith Kent A. , Teknos Theodoros N. , Merajver Sofia D.
Published: September 2005

Tumor metastasis is the major cause of morbidity and mortality in patients with breast cancer. It is critical to identify metastasis enabling genes and understand how they are responsible for inducing specific aspects of the metastatic phenotype to allow for improved clinical detection and management. Protein kinase Cε (PKCε), a member of a family of serine/threonine protein kinases, is a transforming oncogene that has been reported to be involved in cell invasion and motility. In this study, we investigated the role of PKCε in breast cancer development and progression. High-density tissue microarray analysis showed that PKCε protein was detected in 73.6% (106 of 144) of primary tumors from invasive ductal breast cancer patients. Increasing PKCε staining intensity was associated with high histologic grade (P = 0.0206), positive Her2/neu receptor status (P = 0.0419), and negative estrogen (P = 0.0026) and progesterone receptor status (P = 0.0008). Kaplan-Meier analyses showed that PKCε was significantly associated with poorer disease-free and overall survival (log-rank, P = 0.0478 and P = 0.0414, respectively). RNA interference of PKCε in MDA-MB231 cells, an aggressive breast cancer cell line with elevated PKCε levels, resulted in a cell phenotype that was significantly less proliferative, invasive, and motile than the parental or the control RNA interference transfectants. Moreover, in vivo tumor growth of small interfering RNA-PKCε MDA-MB231 clones was retarded by a striking 87% (P < 0.05) and incidence of lung metastases was inhibited by 83% (P < 0.02). PKCε-deficient clones were found to have lower RhoC GTPase protein levels and activation. Taken together, these results revealed that PKCε plays a critical and causative role in promoting an aggressive metastatic breast cancer phenotype and as a target for anticancer therapy.
High Mobility Group A1 Is a Molecular Target for MYCN in Human Neuroblastoma
Authors: Giannini Giuseppe , Cerignoli Fabio , Mellone Massimiliano , Massimi Isabella , Ambrosi Cinzia , Rinaldi Christian , Dominici Carlo , Frati Luigi , Screpanti Isabella , Gulino Alberto
Published: September 2005

High mobility group A1 (HMGA1) is an architectural transcription factor and a putative protoncogene. Deregulation of its expression has been shown in most human cancers. We have previously shown that the expression of the HMGA family members is deregulated in neuroblastoma cell lines and primary tumors. On retinoic acid (RA) treatment of MYCN-amplified neuroblastoma cell lines, HMGA1 decreases with a kinetics that strictly follows MYCN repression. In addition, MYCN constitutive expression abolishes HMGA1 repression by RA. Here we explored the possibility that HMGA1 expression might be sustained by MYCN in amplified cells. Indeed, MYCN transfection induced HMGA1 expression in several neuroblastoma cell lines. HMGA1 expression increased in a transgene dose–dependent fashion in neuroblastoma-like tumors of MYCN transgenic mice. In addition, it was significantly more expressed in MYCN-amplified compared with MYCN single-copy primary human neuroblastomas. MYCN cotransfection activated a promoter/luciferase reporter containing a 1,600 bp region surrounding the first three transcription start sites of the human HMGA1 and eight imperfect E-boxes. By heterodimerizing with its partner MAX, MYCN could bind to multiple DNA fragments within the 1,600 bp. Either 5′ or 3′ deletion variants of the 1,600 bp promoter/luciferase reporter strongly decreased luciferase activity, suggesting that, more than a single site, the cooperative function of multiple cis-acting elements mediates direct HMGA1 transactivation by MYCN. Finally, HMGA1 repression by RNA interference reduced neuroblastoma cell proliferation, indicating that HMGA1 is a novel MYCN target gene relevant for neuroblastoma tumorigenesis.
Tobacco Smoke Stimulates the Transcription of Amphiregulin in Human Oral Epithelial Cells: Evidence of a Cyclic AMP-Responsive Element Binding Protein–Dependent Mechanism
Authors: Du Baoheng , Altorki Nasser K. , Kopelovich Levy , Subbaramaiah Kotha , Dannenberg Andrew J.
Published: September 2005

Activation of epidermal growth factor receptor (EGFR)–mediated signaling has been implicated in the pathogenesis of tobacco smoke–induced cancers. Recently, elevated levels of amphiregulin, a ligand of the EGFR, were found in the oral mucosa of smokers. The main objective of this study was to elucidate the mechanism by which tobacco smoke induces amphiregulin. Treatment of a nontumorigenic human oral epithelial cell line (MSK-Leuk1) with a saline extract of tobacco smoke stimulated amphiregulin (AR) transcription resulting in increased amounts of amphiregulin mRNA and protein. Tobacco smoke stimulated the cyclic AMP (cAMP)→protein kinase A (PKA) pathway leading to increased cAMP-responsive element binding protein–dependent activation of AR transcription. These inductive effects of tobacco smoke were dependent on the aryl hydrocarbon receptor (AhR). In fact, α-naphthoflavone, an AhR antagonist, blocked tobacco smoke–mediated induction of binding of cAMP-responsive element binding protein to the AR promoter and thereby suppressed the induction of amphiregulin. Notably, treatment of MSK-Leuk1 cells with tobacco smoke or exogenous amphiregulin stimulated DNA synthesis. An inhibitor of EGFR tyrosine kinase or a neutralizing antibody to amphiregulin abrogated the increase in DNA synthesis mediated by tobacco smoke. Taken together, these findings suggest that tobacco smoke stimulated a signaling pathway comprised of AhR→cAMP→PKA resulting in enhanced AR transcription and increased DNA synthesis. The ability of tobacco smoke to induce amphiregulin and thereby enhance DNA synthesis is likely to contribute to the procarcinogenic effects of tobacco smoke.
Introduction of Sda Carbohydrate Antigen in Gastrointestinal Cancer Cells Eliminates Selectin Ligands and Inhibits Metastasis
Authors: Kawamura Yuki I. , Kawashima Rei , Fukunaga Ryuko , Hirai Kazunari , Toyama-Sorimachi Noriko , Tokuhara Makoto , Shimizu Toshio , Dohi Taeko
Published: September 2005

The Sda blood group carbohydrate structure is expressed in the normal gastrointestinal mucosa. We reported previously that the expression of Sda carbohydrate structures and β1,4-N-acetylgalactosaminyltransferase (β1,4GalNAcT) activity responsible for Sda synthesis were remarkably decreased in cancer lesions of the gastrointestinal tract. In this study, we found that Sda antigen was expressed mainly in chief cells of normal stomach but not in cancer tissue by immunohistologic staining. In separated gastric mucosal cells, the Sda glycolipids and β1,4GalNAcT activity were concentrated in a fraction that contained chief cells as a major population. We cloned the cDNA encoding the glycosyltransferase that catalyzes the synthesis of Sda (Sda-β1,4GalNAcT). Introduction of this cloned cDNA into KATO III gastric or HT29 colonic cancer cell lines, which originally expressed the E-selectin ligands, sialyl Lewisx and sialyl Lewisa, resulted in a marked increase in cell-surface expression of Sda along with the concomitant total loss of both sialyl Lewisx and sialyl Lewisa. Both KATO III and HT29 cells transfected with the Sda-β1,4GalNAcT gene showed significantly decreased adhesion to activated human umbilical vein endothelial cells when compared with mock-transfected cells. Sda determinants showed no direct binding to Siglec-3, -5, -7, and -9. These Sda-β1,4GalNAcT-transfected cells showed strikingly reduced metastatic potential in vivo when compared with mock-transfected cells. In summary, forced expression of Sda carbohydrate determinant caused remarkable elimination of carbohydrate ligands for selectin and reduced metastasis of human gastrointestinal tract cancer cells.
CaSm-Mediated Cellular Transformation Is Associated with Altered Gene Expression and Messenger RNA Stability
Authors: Fraser Melissa M. , Watson Patricia M. , Fraig Mostafa M. , Kelley Joseph R. , Nelson Peter S. , Boylan Alice M. , Cole David J. , Watson Dennis K.
Published: September 2005

CaSm (cancer-associated Sm-like) was originally identified based on elevated expression in pancreatic cancer and in several cancer-derived cell lines. CaSm encodes a 133 amino acid protein that contains two Sm motifs found in the common small nuclear RNA proteins and the LSm (like-Sm) family of proteins. Compared with normal human prostate tissue and primary prostate epithelial cells, some primary prostate tumors and prostate cancer–derived cell lines have elevated CaSm expression. Expression of antisense CaSm RNA in DU145 cells results in reduced CaSm protein levels and less transformed phenotype, measured by anchorage-independent growth in vitro and tumor formation in severe combined immunodeficient mice in vivo. Additional data shows that adenoviral delivery of antisense CaSm inhibits the growth of prostate cancer cell lines by altering cell cycle progression, and is associated with reduced expression of cyclin B1 and CDK1 proteins. Consistent with failure of antisense-treated cells to enter mitosis, microarray analysis identified altered expression of NEK2 and nucleophosmin/B23. Although the mechanisms by which CaSm contributes to neoplastic transformation and cellular proliferation are unknown, it has been shown that the yeast homologue (spb8/LSm1) of CaSm is required for 5′ to 3′ degradation of specific mRNAs. We provide data consistent with a similar role for CaSm in human cells, supporting the hypothesis that elevated CaSm expression observed in cancer leads to destabilization of multiple gene transcripts, contributing to the mutator phenotype of cancer cells.
Expression of a Natural Tumor Antigen by Thymic Epithelial Cells Impairs the Tumor-Protective CD4+ T-Cell Repertoire
Authors: Bos Rinke , van Duikeren Suzanne , van Hall Thorbald , Kaaijk Patricia , Taubert Richard , Kyewski Bruno , Klein Ludger , Melief Cornelis J.M. , Offringa Rienk
Published: September 2005

A variety of antigens that display a highly tissue-specific expression pattern have recently found to be also expressed in medullary thymic epithelial cells (mTEC). This unique feature of mTEC plays an important role in preventing hazardous autoimmune responses through thymic tolerization of T-cell subsets directed against autoantigens but could also limit the possibility of exploiting tumor-associated antigens for immune-mediated targeting of cancers. Our present study shows that expression of carcinoembryonic antigen (CEA) in thymic epithelial cells of CEA-transgenic mice results in tolerization of a major fraction of the CD4+ T-cell repertoire against this antigen, thereby markedly limiting the effect of CEA-specific immunization against CEA-overexpressing tumors. The expression of CEA in mTEC of CEA-transgenic mice is mirrored by its expression in human mTEC, arguing that promiscuous gene expression in these thymic stromal cells needs to be considered as a potential hurdle for immunotherapies of cancer that target tissue-specific autoantigens.
Cellular Mechanisms for Low-Dose Ionizing Radiation–Induced Perturbation of the Breast Tissue Microenvironment
Authors: Tsai Kelvin K.C. , Chuang Eric Yao-Yu , Little John B. , Yuan Zhi-Min
Published: September 2005

Radiation exposure is an important form of environmental carcinogen and has been associated with increased risk of breast cancer. Epigenetic events, especially those involving alterations in the breast stromal microenvironment, may play an important role in radiation-induced carcinogenesis but remain not well understood. We here show that human mammary stromal fibroblasts respond to protracted low-dose ionizing radiation exposures by displaying a senescence-like phenotype. Using a three-dimensional coculture system to model the interactions of different mammary cell types with their neighbors and with their environment, we provide a direct experimental proof that ionizing radiation–induced senescence-like fibroblasts significantly perturb the mammary stromal microenvironment, which is highlighted by impaired formation of pseudopodia networks due to marked cytoskeletal alterations in senescence-like fibroblasts and increased extracellular matrix degradation because of the up-regulation of multiple secreted matrix metalloproteinases. Within such a perturbed environment, mammary ductal morphogenesis is completely disrupted and epithelial cells instead grow into enlarged cystic structures, which further develop and become disorganized cell masses on inactivation of cellular death pathways. Breast carcinoma cells growing in such an environment are enabled to fully express their malignant potential as evidenced by the α6β4 integrin/phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin pathway–dependent invasive growth. Our results suggest that ionizing radiation, in addition to causing gene mutations in epithelial cells, can contribute to breast carcinogenesis by perturbing the tissue microenvironment that leads to dysregulated cell-cell and cell-matrix interactions.
Toll-Like Receptors on Tumor Cells Facilitate Evasion of Immune Surveillance
Authors: Huang Bo , Zhao Jie , Li Hongxing , He Kai-Li , Chen Yibang , Mayer Lloyd , Unkeless Jay C. , Xiong Huabao
Published: September 2005

The signal pathways that trigger tumor cell escape from immune surveillance are incompletely understood. Toll-like receptors (TLRs), which activate innate and adaptive immune responses, are thought to be restricted to immune cells. We show here that TLRs, including TLR4, are expressed on tumor cells from a wide variety of tissues, suggesting that TLR activation may be an important event in tumor cell immune evasion. Activation of TLR4 signaling in tumor cells by lipopolysaccharide induces the synthesis of various soluble factors and proteins including interleukin-6, inducible nitric oxide synthase, interleukin-12, B7-H1, and B7-H2, and results in resistance of tumor cells to CTL attack. In addition, lipopolysaccharide-stimulated tumor cell supernatants inhibit both T cell proliferation and natural killer cell activity. Blockade of the TLR4 pathway by either TLR4 short interfering RNA or a cell-permeable TLR4 inhibitory peptide reverses tumor-mediated suppression of T cell proliferation and natural killer cell activity in vitro, and in vivo, delays tumor growth and thus prolongs the survival of tumor-bearing mice. These findings indicate that TLR signaling results in a cascade leading to tumor evasion from immune surveillance. These novel functions of TLRs in tumor biology suggest a new class of therapeutic targets for cancer therapy.
Radiation Damage to the Heart Enhances Early Radiation-Induced Lung Function Loss
Authors: van Luijk Peter , Novakova-Jiresova Alena , Faber Hette , Schippers Jacobus M. , Kampinga Harm H. , Meertens Harm , Coppes Rob P.
Published: September 2005

In many thoracic cancers, the radiation dose that can safely be delivered to the target volume is limited by the tolerance dose of the surrounding lung tissue. It has been hypothesized that irradiation of the heart may be an additional risk factor for the development of early radiation-induced lung morbidity. In the current study, the dependence of lung tolerance dose on heart irradiation is determined. Fifty percent of the rat lungs were irradiated either including or excluding the heart. Proton beams were used to allow very accurate and conformal dose delivery. Lung function toxicity was scored using a breathing rate assay. We confirmed that the tolerance dose for early lung function damage depends not only on the lung region that is irradiated but also that concomitant irradiation of the heart severely reduces the tolerance of the lung. This study for the first time shows that the response of an organ to irradiation does not necessarily depend on the dose distribution in that organ alone.