bluuebunny/crossref_metadata_2025 huggingface dataset:
Targeted Delivery of IFNγ to Tumor Vessels Uncouples Antitumor from Counterregulatory Mechanisms
Authors: Curnis Flavio , Gasparri Anna , Sacchi Angelina , Cattaneo Angela , Magni Fulvio , Corti Angelo
Published: September 2005

Because of its immunomodulatory and anticancer activities, IFNγ has been used as an anticancer drug in several clinical studies, unfortunately with modest results. Attempts to increase the response by increasing the dose or by repeated continuous injection often resulted in lower efficacy, likely due to counterregulatory effects. We show here that targeted delivery of low doses of IFNγ to CD13, a marker of angiogenic vessels, can overcome major counterregulatory mechanisms and delay tumor growth in two murine models that respond poorly to IFNγ. Tumor vascular targeting was achieved by coupling IFNγ to GCNGRC, a CD13 ligand, by genetic engineering technology. The dose-response curve was bell-shaped. Maximal effects were induced with a dose of 0.005 μg/kg, about 500-fold lower than the dose used in patients. Nontargeted IFNγ induced little or no effects over a range of 0.003 to 250 μg/kg. Studies on the mechanism of action showed that low doses of targeted IFNγ could activate tumor necrosis factor (TNF)-dependent antitumor mechanisms, whereas high doses of either targeted or nontargeted IFNγ induced soluble TNF-receptor shedding in circulation, a known counterregulatory mechanism of TNF activity. These findings suggest that antitumor activity and counterregulatory mechanisms could be uncoupled by tumor vascular targeting with extremely low doses of IFNγ.
EMP3, a Myelin-Related Gene Located in the Critical 19q13.3 Region, Is Epigenetically Silenced and Exhibits Features of a Candidate Tumor Suppressor in Glioma and Neuroblastoma
Authors: Alaminos Miguel , Dávalos Verónica , Ropero Santiago , Setién Fernando , Paz Maria F. , Herranz Michel , Fraga Mario F. , Mora Jaume , Cheung Nai-Kong V. , Gerald William L. , Esteller Manel
Published: September 2005

The presence of common genomic deletions in the 19q13 chromosomal region in neuroblastomas and gliomas strongly suggests the presence of a putative tumor suppressor gene for these neoplasms in this region that, despite much effort, has not yet been identified. In an attempt to address this issue, we compared the expression profile of 89 neuroblastoma tumors with that of benign ganglioneuromas by microarray analysis. Probe sets (637 of 62,839) were significantly down-regulated in neuroblastoma tumors, including, most importantly, a gene located at 19q13.3: the epithelial membrane protein 3 (EMP3), a myelin-related gene involved in cell proliferation and cell-cell interactions. We found that EMP3 undergoes hypermethylation-mediated transcriptional silencing in neuroblastoma and glioma cancer cell lines, whereas the use of the demethylating agent 5-aza-2-deoxycytidine restores EMP3 gene expression. Furthermore, the reintroduction of EMP3 into neuroblastoma cell lines displaying methylation-dependent silencing of EMP3 induces tumor suppressor–like features, such as reduced colony formation density and tumor growth in nude mouse xenograft models. Screening a large collection of human primary neuroblastomas (n = 116) and gliomas (n = 41), we observed that EMP3 CpG island hypermethylation was present in 24% and 39% of these tumor types, respectively. Furthermore, the detection of EMP3 hypermethylation in neuroblastoma could be clinically relevant because it was associated with poor survival after the first 2 years of onset of the disease (Kaplan-Meier; P = 0.03) and death of disease (Kendall τ, P = 0.03; r = 0.19). Thus, EMP3 is a good candidate for being the long-sought tumor suppressor gene located at 19q13 in gliomas and neuroblastomas.
A Survivin Gene Signature Predicts Aggressive Tumor Behavior
Authors: Salz Whitney , Eisenberg Dan , Plescia Janet , Garlick David S. , Weiss Robert M. , Wu Xue-Ru , Sun Tung-Tien , Altieri Dario C.
Published: September 2005

Gene signatures that predict aggressive tumor behavior at the earliest stages of disease, ideally before overt tissue abnormalities, are urgently needed. To search for such genes, we generated a transgenic model of survivin, an essential regulator of cell division and apoptosis overexpressed in cancer. Transgenic expression of survivin in the urinary bladder did not cause histologic abnormalities of the urothelium. However, microarray analysis revealed that survivin-expressing bladders exhibited profound changes in gene expression profile affecting extracellular matrix and inflammatory genes. Following exposure to a bladder carcinogen, N-butyl-N-(4-hydroxybutyl) nitrosamine (OH-BBN), survivin transgenic animals exhibited accelerated tumor progression, preferential incidence of tumors as compared with premalignant lesions, and dramatically abbreviated survival. Conversely, transgenic expression of a survivin Thr34→Ala dominant-negative mutant did not cause changes in gene expression or accelerated tumor progression after OH-BBN treatment. Therefore, survivin expression induces global transcriptional changes in the tissue microenvironment that may promote tumorigenesis. Detection of survivin or its associated gene signature may provide an early biomarker of aggressive tumor behavior before the appearance of tissue abnormalities.
β1A Integrin Expression Is Required for Type 1 Insulin-Like Growth Factor Receptor Mitogenic and Transforming Activities and Localization to Focal Contacts
Authors: Goel Hira Lal , Breen Michael , Zhang Jianzhong , Das Ishita , Aznavoorian-Cheshire Sadie , Greenberg Norman M. , Elgavish Ada , Languino Lucia R.
Published: September 2005

The cells' ability to proliferate in response to growth factor stimulation is significantly altered during cancer progression. To investigate the mechanisms underlying these alterations in prostate cancer, the role and expression of β1A integrin and type 1 insulin-like growth factor receptor (IGF-IR), known to contribute to cell proliferation and transformation, were analyzed. Using small interfering RNA oligonucleotides to down-regulate β1A, we show that β1A expression is required for IGF-IR–mediated prostate cancer cell proliferation and anchorage-independent growth. In vivo, using age-matched transgenic adenocarcinoma of mouse prostate (TRAMP) mice at different stages of prostate cancer [prostatic intraepithelial neoplasia, PIN; well-differentiated adenocarcinoma, WD; and poorly differentiated adenocarcinoma, PD], the expression of β1A and of IGF-IR was studied. β1A and IGF-IR expression levels were concurrently up-regulated in high PIN and WD, whereas their expression did not correlate in late-stage PD. In contrast to the up-regulated expression of β1A, the levels of β1C, a β1 cytoplasmic variant that inhibits cell proliferation, were down-regulated in all stages of prostate cancer. A similar expression pattern was observed for a β1C downstream effector, Grb2-associated binder-1 (Gab1) which is known to inhibit IGF-IR phosphorylation. To analyze in vitro the mechanistic implications of β1A, β1C, and Gab1 deregulation in prostate cancer, we investigated whether expression of either β1 variant in β1-null cells affected IGF-IR localization. We found that IGF-IR and β1A were colocalized in highly specialized integrin signaling compartments, designated focal contacts. However, in the presence of β1C, IGF-IR remained diffuse on the cell surface and did not localize to focal contacts. The findings that β1 integrins and IGF-IR are concurrently deregulated and that expression of β1 integrins is necessary to achieve appropriate IGF-IR intracellular distribution point to the important role that the cross-talk between these receptors may have during prostate cancer progression and will be helpful in formulating new therapeutic strategies.
Inability of Transforming Growth Factor-β to Cause SnoN Degradation Leads to Resistance to Transforming Growth Factor-β–Induced Growth Arrest in Esophageal Cancer Cells
Authors: Edmiston Jeffery S. , Yeudall W. Andrew , Chung Theodore D. , Lebman Deborah A.
Published: September 2005

It is well established that loss of a growth inhibitory response to transforming growth factor-β (TGF-β) is a common feature of epithelial cancers including esophageal cancer. However, the molecular basis for the abrogation of this key homeostatic mechanism is poorly understood. In esophageal cancer cell lines that are resistant to TGF-β–induced growth inhibition, TGF-β also fails to decrease transcription of c-myc despite the presence of functional signaling components. Consequently, to gain a better understanding of the mechanisms leading to resistance to TGF-β–induced growth arrest, the basis for the inability to decrease c-myc transcription was investigated. Regardless of sensitivity to TGF-β–induced growth arrest, TGF-β enhanced the ability of Smad3-protein complexes to bind c-myc regulatory elements. However, in a growth inhibition–resistant esophageal cancer cell line, the Smad3-protein complexes contained the SnoN oncoprotein. Furthermore, in esophageal cancer cell lines that are resistant to TGF-β–induced growth arrest, TGF-β does not cause degradation of SnoN. Analyses of the effect of modulating SnoN expression in both growth inhibition–sensitive and growth inhibition–resistant cell lines showed that degradation of SnoN is a prerequisite for both TGF-β–induced repression of c-myc transcription and growth arrest. The data indicate that SnoN-Smad3 complexes do not cause repression of c-myc transcription but rather prevent functionality of active repressor complexes. Thus, these studies reveal a novel mechanism for resistance to TGF-β–induced growth inhibition in esophageal cancer, namely the failure to degrade SnoN. In addition, they show that SnoN can block TGF-β repression of gene transcription.
Plexin D1 Expression Is Induced on Tumor Vasculature and Tumor Cells: A Novel Target for Diagnosis and Therapy?
Authors: Roodink Ilse , Raats Jos , van der Zwaag Bert , Verrijp Kiek , Kusters Benno , van Bokhoven Hans , Linkels Marianne , de Waal Robert M.W. , Leenders William P.J.
Published: September 2005

We previously reported that during mouse embryogenesis, plexin D1 (plxnD1) is expressed on neuronal and endothelial cells. Endothelial cells gradually loose plxnD1 expression during development. Here we describe, using in situ hybridization, that endothelial plxnD1 expression is regained during tumor angiogenesis in a mouse model of brain metastasis. Importantly, we found PLXND1 expression also in a number of human brain tumors, both of primary and metastatic origin. Apart from the tumor vasculature, abundant expression was also found on tumor cells. Via panning of a phage display library, we isolated two phages that carry single-domain antibodies with specific affinity towards a PLXND1-specific peptide. Immunohistochemistry with these single-domain antibodies on the same tumors that were used for in situ hybridization confirmed PLXND1 expression on the protein level. Furthermore, both these phages and the derived antibodies specifically homed to vessels in brain lesions of angiogenic melanoma in mice after i.v. injection. These results show that PLXND1 is a clinically relevant marker of tumor vasculature that can be targeted via i.v. injections.
NBS1 Knockdown by Small Interfering RNA Increases Ionizing Radiation Mutagenesis and Telomere Association in Human Cells
Authors: Zhang Ying , Lim Chang U.K. , Williams Eli S. , Zhou Junqing , Zhang Qinming , Fox Michael H. , Bailey Susan M. , Liber Howard L.
Published: September 2005

Hypomorphic mutations which lead to decreased function of the NBS1 gene are responsible for Nijmegen breakage syndrome, a rare autosomal recessive hereditary disorder that imparts an increased predisposition to development of malignancy. The NBS1 protein is a component of the MRE11/RAD50/NBS1 complex that plays a critical role in cellular responses to DNA damage and the maintenance of chromosomal integrity. Using small interfering RNA transfection, we have knocked down NBS1 protein levels and analyzed relevant phenotypes in two closely related human lymphoblastoid cell lines with different p53 status, namely wild-type TK6 and mutated WTK1. Both TK6 and WTK1 cells showed an increased level of ionizing radiation–induced mutation at the TK and HPRT loci, impaired phosphorylation of H2AX (γ-H2AX), and impaired activation of the cell cycle checkpoint regulating kinase, Chk2. In TK6 cells, ionizing radiation–induced accumulation of p53/p21 and apoptosis were reduced. There was a differential response to ionizing radiation–induced cell killing between TK6 and WTK1 cells after NBS1 knockdown; TK6 cells were more resistant to killing, whereas WTK1 cells were more sensitive. NBS1 deficiency also resulted in a significant increase in telomere association that was independent of radiation exposure and p53 status. Our results provide the first experimental evidence that NBS1 deficiency in human cells leads to hypermutability and telomere associations, phenotypes that may contribute to the cancer predisposition seen among patients with this disease.
Identification of Novel Small Molecule Inhibitors of Hypoxia-Inducible Factor-1 That Differentially Block Hypoxia-Inducible Factor-1 Activity and Hypoxia-Inducible Factor-1α Induction in Response to Hypoxic Stress and Growth Factors
Authors: Chau Noan-Minh , Rogers Paul , Aherne Wynne , Carroll Veronica , Collins Ian , McDonald Edward , Workman Paul , Ashcroft Margaret
Published: September 2005

Hypoxia-inducible factor-1 (HIF-1) is a transcriptional complex that is activated in response to hypoxia and growth factors. HIF-1 plays a central role in tumor progression, invasion, and metastasis. Overexpression of the HIF-1α subunit has been observed in many human cancers and is associated with a poor prognostic outcome with conventional treatments. Targeting HIF-1 using novel small molecule inhibitors is, therefore, an attractive strategy for therapeutic development. We have generated U2OS human osteosarcoma cells stably expressing a luciferase reporter construct under the control of a hypoxia response element (U2OS-HRE-luc). The U2OS-HRE-luc cells were robustly and reproducibly sensitive to hypoxic stress in a HIF-1–dependent manner. We developed an automated U2OS-HRE-luc cell-based assay that was used in a high-throughput screen to identify compounds that inhibited HIF-1 activity induced by treatment with the hypoxia mimetic, deferoxamine mesylate. We performed a pilot screen of the National Cancer Institute Diversity Set of 2,000 compounds. We identified eight hit compounds, six of these were also identified by Rapisarda et al. in an independent hypoxia screen. However, there were two novel hit compounds, NSC-134754 and NSC-643735, that did not significantly inhibit constitutive luciferase activity in U2OS cells (U2OS-luc). We showed that both NSC-134754 and NSC-643735 significantly inhibited HIF-1 activity and HIF-1α protein induced by deferoxamine mesylate. Interestingly, NSC-134754 but not NCS-643735 inhibited HIF-1 activity and HIF-1α protein induced by hypoxia and significantly inhibited Glut-1 expression. Finally, we showed that both NCS-134754 and NCS-643735 inhibited HIF-1α protein induced by insulin-like growth factor-1. Our cell-based assay approach has successfully identified novel compounds that differentially target hypoxia and/or growth factor–mediated induction of HIF-1α.
ABCG2 Harboring the Gly482 Mutation Confers High-Level Resistance to Various Hydrophilic Antifolates
Authors: Shafran Assaf , Ifergan Ilan , Bram Eran , Jansen Gerrit , Kathmann Ietje , Peters Godefridus J. , Robey Robert W. , Bates Susan E. , Assaraf Yehuda G.
Published: September 2005

ABCG2 is an ATP-binding cassette transporter that confers resistance to various chemotherapeutic agents. Recent studies have established that an Arg (wild-type) to Gly mutation at amino acid 482 in ABCG2 alters substrate specificity. Here, we explored the role of this G482 mutation in antifolate resistance using a clinically relevant 4-hour drug exposure. Stable transfectants overexpressing the mutant G482 transporter displayed 120-, 1,000-, and >6,250-fold resistance to the antifolates methotrexate, GW1843, and Tomudex, respectively, relative to parental human embryonic kidney cells. Moreover, although overexpressing equal transporter levels at the plasma membrane, G482-ABCG2 cells were 6-, 23-, and >521-fold more resistant to methotrexate, GW1843, and Tomudex, respectively, than R482-ABCG2 cells. In contrast, upon a continuous (72-hour) drug exposure, both the G482- and R482-ABCG2 cells lost almost all their antifolate resistance; this result was consistent with the inability of ABCG2 to extrude long-chain antifolate polyglutamates. Ko143, a specific and potent ABCG2 inhibitor reversed methotrexate resistance in both G482- and R482-ABCG2 cells. Consistently, whereas the pool of free methotrexate in parental human embryonic kidney cells was prominent after 4 hours of transport with 1 μmol/L [3H]methotrexate, in R482- and G482-ABCG2 cells, it was minimal. Furthermore, G482-ABCG2 cells contained marked decreases in the di- and triglutamate species of [3H]methotrexate at 4 hours of incubation with methotrexate and in the tetra- and pentaglutamates at 24 hours. These changes were not associated with any significant decrease in folylypoly-γ-glutamate synthetase activity. These results provide the first evidence that the G482-ABCG2 mutation confers high-level resistance to various hydrophilic antifolates.
Homozygous Deletions and Chromosome Amplifications in Human Lung Carcinomas Revealed by Single Nucleotide Polymorphism Array Analysis
Authors: Zhao Xiaojun , Weir Barbara A. , LaFramboise Thomas , Lin Ming , Beroukhim Rameen , Garraway Levi , Beheshti Javad , Lee Jeffrey C. , Naoki Katsuhiko , Richards William G. , Sugarbaker David , Chen Fei , Rubin Mark A. , Jänne Pasi A. , Girard Luc , Minna John , Christiani David , Li Cheng , Sellers William R. , Meyerson Matthew
Published: September 2005

Genome-wide copy number changes were analyzed in 70 primary human lung carcinoma specimens and 31 cell lines derived from human lung carcinomas, with high-density arrays representing ∼115,000 single nucleotide polymorphism loci. In addition to previously characterized loci, two regions of homozygous deletion were found, one near the PTPRD locus on chromosome segment 9p23 in four samples representing both small cell lung carcinoma (SCLC) and non–small cell lung carcinoma (NSCLC) and the second on chromosome segment 3q25 in one sample each of NSCLC and SCLC. High-level amplifications were identified within chromosome segment 8q12-13 in two SCLC specimens, 12p11 in two NSCLC specimens and 22q11 in four NSCLC specimens. Systematic copy number analysis of tyrosine kinase genes identified high-level amplification of EGFR in three NSCLC specimens, FGFR1 in two specimens and ERBB2 and MET in one specimen each. EGFR amplification was shown to be independent of kinase domain mutational status.
Shared Epigenetic Mechanisms in Human and Mouse Gliomas Inactivate Expression of the Growth SuppressorSLC5A8
Authors: Hong Chibo , Maunakea Alika , Jun Peter , Bollen Andrew W. , Hodgson J. Graeme , Goldenberg David D. , Weiss William A. , Costello Joseph F.
Published: September 2005

Tumors arise in part from the deleterious effects of genetic and epigenetic mechanisms on gene expression. In several mouse models of human tumors, the tumorigenic phenotype is reversible, suggesting that epigenetic mechanisms also contribute significantly to tumorigenesis in mice. It is not known whether these are the same epigenetic mechanisms in human and mouse tumors or whether they affect homologous genes. Using an integrated approach for genome-wide methylation and copy number analyses, we identified SLC5A8 on chromosome 12q23.1 that was affected frequently by aberrant methylation in human astrocytomas and oligodendrogliomas. SLC5A8 encodes a sodium monocarboxylate cotransporter that was highly expressed in normal brain but was significant down-regulated in primary gliomas. Bisulfite sequencing analysis showed that the CpG island was unmethylated in normal brain but frequently localized methylated in brain tumors, consistent with the tumor-specific loss of gene expression. In glioma cell lines, SLC5A8 expression was also suppressed but could be reactivated with a methylation inhibitor. Expression of exogenous SLC5A8 in LN229 and LN443 glioma cells inhibited colony formation, suggesting that it may function as a growth suppressor in normal brain cells. Remarkably, 9 of 10 murine oligodendroglial tumors (from p53+/− or ink4a/arf+/− animals transgenic for S100β-v-erbB) showed a similar tumor-specific down-regulation of mSLC5A8, the highly conserved mouse homologue. Taken together, these data suggest that SLC5A8 functions as a growth suppressor gene in vitro and that it is silenced frequently by epigenetic mechanisms in primary gliomas. The shared epigenetic inactivation of mSLC5A8 in mouse gliomas indicates an additional degree of commonality in the origin and/or pathway to tumorigenesis between primary human tumors and these mouse models of gliomas.
Association of DNA Methylation of Phosphoserine Aminotransferase with Response to Endocrine Therapy in Patients with Recurrent Breast Cancer
Authors: Martens John W.M. , Nimmrich Inko , Koenig Thomas , Look Maxime P. , Harbeck Nadia , Model Fabian , Kluth Antje , Bolt-de Vries Joan , Sieuwerts Anieta M. , Portengen Henk , Meijer-Van Gelder Marion E. , Piepenbrock Christian , Olek Alexander , Höfler Heinz , Kiechle Marion , Klijn Jan G.M. , Schmitt Manfred , Maier Sabine , Foekens John A.
Published: September 2005

To understand the biological basis of resistance to endocrine therapy is of utmost importance in patients with steroid hormone receptor–positive breast cancer. Not only will this allow us prediction of therapy success, it may also lead to novel therapies for patients resistant to current endocrine therapy. DNA methylation in the promoter regions of genes is a prominent epigenetic gene silencing mechanism that contributes to breast cancer biology. In the current study, we investigated whether promoter DNA methylation could be associated with resistance to endocrine therapy in patients with recurrent breast cancer. Using a microarray-based technology, the promoter DNA methylation status of 117 candidate genes was studied in a cohort of 200 steroid hormone receptor–positive tumors of patients who received the antiestrogen tamoxifen as first-line treatment for recurrent breast cancer. Of the genes analyzed, the promoter DNA methylation status of 10 genes was significantly associated with clinical outcome of tamoxifen therapy. The association of the promoter hypermethylation of the strongest marker, phosphoserine aminotransferase (PSAT1) with favorable clinical outcome was confirmed by an independent quantitative DNA methylation detection method. Furthermore, the extent of DNA methylation of PSAT1 was inversely associated with its expression at the mRNA level. Finally, also at the mRNA level, PSAT1 was a predictor of tamoxifen therapy response. Concluding, our work indicates that promoter hypermethylation and mRNA expression of PSAT1 are indicators of response to tamoxifen-based endocrine therapy in steroid hormone receptor–positive patients with recurrent breast cancer.
K-ras Codon-Specific Mutations Produce Distinctive Metabolic Phenotypes in Human Fibroblasts
Authors: Vizan Pedro , Boros Laszlo G. , Figueras Agnes , Capella Gabriel , Mangues Ramon , Bassilian Sara , Lim Shu , Lee Wai-Nang P. , Cascante Marta
Published: September 2005

Among K-ras mutations, codon 12 mutations have been identified as those conferring a more aggressive phenotype. This aggressiveness is primarily associated with slow proliferation but greatly increased resistance to apoptosis. Using transfected NIH3T3 fibroblasts with a mutated K-ras minigene either at codon 12 (K12) or at codon 13 (K13), and taking advantage of [1,2-13C2]glucose tracer labeling, we show that codon 12 mutant K-ras (K12)-transformed cells exhibit greatly increased glycolysis with only a slight increase in activity along pathways that produce nucleic acid and lipid synthesis precursors in the oxidative branch of the pentose phosphate pathway and via pyruvate dehydrogenase flux. K13 mutants display a modest increase in anaerobic glycolysis associated with a large increase in oxidative pentose phosphate pathway activity and pyruvate dehydrogenase flux. The distinctive differences in metabolic profiles of K12 and K13 codon mutated cells indicate that a strong correlation exists between the flow of glucose carbons towards either increased anaerobic glycolysis, and resistance to apoptosis (K12), or increased macromolecule synthesis, rapid proliferation, and increased sensitivity to apoptosis.
Characterization of the Mismatch Repair Defect in the Human Lymphoblastoid MT1 Cells
Authors: Szadkowski Marta , Iaccarino Ingram , Heinimann Karl , Marra Giancarlo , Jiricny Josef
Published: September 2005

Mutations in mismatch repair (MMR) genes predispose to hereditary nonpolyposis colon cancer. Those leading to truncated proteins bring about a MMR defect, but phenotypes of missense mutations are harder to predict especially if they do not affect conserved residues. Several systems capable of predicting the phenotypes of MMR missense mutations were described. We deployed one of these to study the MMR defect in MT1 cells, which carry mutations in both alleles of the hMSH6 gene. In one, an A→T transversion brings about an Asp(1213)Val amino acid change in the highly conserved ATP binding site, whereas the other carries a G→A transition, which brings about a Val(1260)Ile change at a nonconserved site. The hMSH2/hMSH6 (hMutSα) heterodimers carrying these mutations were expressed in the baculovirus system and tested in in vitro MMR assays. As anticipated, the Asp(1213)Val mutation inactivated MMR by disabling the variant hMutSα from translocating along the DNA. In contrast, the recombinant Val(1260)Ile variant displayed wild-type activity. Interestingly, partial proteolytic analysis showed that this heterodimer was absent from MT1 extracts, although both hMSH6 alleles in MT1 cells could be shown to be transcribed with an efficiency similar to each other and to that seen in control cells. The MMR defect in MT1 cells is thus the compound result of one mutation that inactivates the ATPase function of hMutSα and a second mutation that apparently destabilizes the Val(1260)Ile hMSH6 protein in human cells in vivo.
SV40-Dependent AKT Activity Drives Mesothelial Cell Transformation after Asbestos Exposure
Authors: Cacciotti Paola , Barbone Dario , Porta Camillo , Altomare Deborah A. , Testa Joseph R. , Mutti Luciano , Gaudino Giovanni
Published: September 2005

Human malignant mesothelioma is an aggressive cancer generally associated with exposure to asbestos, although SV40 virus has been involved as a possible cofactor by a number of studies. Asbestos fibers induce cytotoxicity in human mesothelial cells (HMC), although cell survival activated by key signaling pathways may promote transformation. We and others previously reported that SV40 large T antigen induces autocrine loops in HMC and malignant mesothelioma cells, leading to activation of growth factor receptors. Now we show that SV40 induces cell survival via Akt activation in malignant mesothelioma and HMC cells exposed to asbestos. Consequently, prolonged exposure to asbestos fibers progressively induces transformation of SV40-positive HMC. As a model of SV40/asbestos cocarcinogenesis, we propose that malignant mesothelioma originates from a subpopulation of transformed stem cells and that Akt signaling is a novel therapeutic target to overcome malignant mesothelioma resistance to conventional therapies.
A Ras Homologue Member I Directly Inhibits Signal Transducers and Activators of Transcription 3 Translocation and Activity in Human Breast and Ovarian Cancer Cells
Authors: Nishimoto Arata , Yu Yinhua , Lu Zhen , Mao Xiang , Ren Zhiyong , Watowich Stephanie S. , Mills Gordon B. , Liao Warren S-L. , Chen Xiaomin , Bast Robert C. , Luo Robert Z.
Published: September 2005

A Ras homologue member I (ARHI) is a novel imprinted tumor suppressor gene whose expression is frequently lost in breast and ovarian cancers. This small GTP-binding protein is a member of the Ras superfamily with significant homology to both Ras and Rap. Unlike the Ras oncogene, however, ARHI inhibits tumor cell growth. To elucidate the mechanisms by which ARHI inhibits cancer growth, we screened a human breast epithelial cell cDNA library using a yeast two-hybrid system for ARHI-interacting proteins. ARHI was found to interact with signal transducers and activators of transcription (STAT) 3, a latent transcription factor that transduces signals from the cell surface to the nucleus and activates gene transcription. STAT3 is frequently phosphorylated and activated in breast and ovarian cancers, where cytokines and growth factors up-regulate STAT3 and stimulate proliferation. The ARHI-STAT3 interaction was confirmed by coimmunoprecipitation in mammalian cells and shown to be specific for STAT3 but not STAT1 or STAT5a. When ARHI and STAT3 were coexpressed in SKOv3 cells, ARHI formed a complex with STAT3 in the cytoplasm and prevented interleukin-6–induced STAT3 accumulation in the nucleus. ARHI markedly reduced STAT3 binding to DNA and STAT3-dependent promoter activity while only moderately affecting STAT3 phosphorylation. Deletion of the NH2 terminus of ARHI significantly compromised its inhibitory activity, suggesting that this unique NH2-terminal extension contributes to ARHI's inhibition of STAT3-mediated transcriptional activity. Thus, the physical association between STAT3 and ARHI as well as the functional inhibition of STAT3 transcriptional activity by ARHI suggests a novel mechanism through which a putative tumor suppressor gene can inhibit STAT3 activity in breast and ovarian cancers.
Brain Tumor Oncolysis with Replication-Conditional Herpes Simplex Virus Type 1 Expressing the Prodrug-Activating Genes, CYP2B1 and Secreted Human Intestinal Carboxylesterase, in Combination with Cyclophosphamide and Irinotecan
Authors: Tyminski Edyta , LeRoy Stanley , Terada Kinya , Finkelstein Dianne M. , Hyatt Janice L. , Danks Mary K. , Potter Philip M. , Saeki Yoshinaga , Chiocca E. Antonio
Published: September 2005

The treatment of malignant glioma is currently ineffective. Oncolytic viruses are being explored as a means to selectively lyse tumor cells in the brain. We have engineered a mutant herpes simplex virus type 1 with deletions in the viral UL39 and γ134.5 genes and an insertion of the two prodrug activating genes, CYP2B1 and secreted human intestinal carboxylesterase. Each of these can convert the inactive prodrugs, cyclophosphamide and irinotecan (CPT-11), into their active metabolites, respectively. This new oncolytic virus (MGH2) displays increased antitumor efficacy against human glioma cells both in vitro and in vivo when combined with cyclophosphamide and CPT-11. Importantly, cyclophosphamide, CPT-11, or the combination of cyclophosphamide and CPT-11 does not significantly affect oncolytic virus replication. Therefore, MGH2 provides effective multimodal therapy for gliomas in preclinical models when combined with these chemotherapy agents.
Bisphosphonates and Cancer-Induced Bone Disease: Beyond Their Antiresorptive Activity
Authors: Clézardin Philippe , Ebetino Frank H. , Fournier Pierrick G.J.
Published: September 2005

Bisphosphonates are primarily known for their ability to inhibit osteoclast-mediated bone resorption. They are an indispensable part of therapy for patients with cancers that cause osteolysis. However, there is now a growing body of evidence from preclinical research showing that bisphosphonates also exhibit antitumor activity, both in vitro and in vivo. They can affect molecular mechanisms of tumor cell adhesion, invasion, and proliferation; reinforce the effects of cytotoxic agents in a synergistic manner; and exhibit antiangiogenic and immunomodulatory effects. These preclinical findings reveal exciting ways of optimizing bisphosphonate therapy in oncology to fully exploit their antitumor potential.
Malignant Transformation but not Normal Cell Growth Depends on Signal Transducer and Activator of Transcription 3
Authors: Schlessinger Karni , Levy David E.
Published: September 2005

Signal transducer and activator of transcription 3 (STAT3) has been indirectly implicated in numerous fundamental cellular processes, including proliferation, survival, and differentiation. We provide genetic evidence from studies of STAT3-null cells that STAT3 is dispensable for normal growth of mouse fibroblasts in culture. STAT3 contributed to the full induction of some (typified by c-fos) but not all (typified by c-myc) immediate early gene expression, but STAT3-independent processes were sufficient to support full cell growth and survival. However, STAT3 was required to manifest a transformed state following expression of v-src, and STAT3-null cells were impaired for anchorage-independent growth as colonies in soft agar and as tumors in mice. The data suggest that STAT3 mediates the maintenance of focal adhesion kinase activity in the absence of cell adhesion by suppressing the action of an inhibitory phosphatase.
Systemic Gene-Directed Enzyme Prodrug Therapy of Hepatocellular Carcinoma Using a Targeted Adenovirus Armed with Carboxypeptidase G2
Authors: Schepelmann Silke , Hallenbeck Paul , Ogilvie Lesley M. , Hedley Douglas , Friedlos Frank , Martin Janet , Scanlon Ian , Hay Carl , Hawkins Lynda K. , Marais Richard , Springer Caroline J.
Published: September 2005

Hepatocellular carcinoma is the fifth most common cancer worldwide, and there is no effective therapy for unresectable disease. We have developed a targeted systemic therapy for hepatocellular carcinoma. The gene for a foreign enzyme is selectively expressed in the tumor cells and a nontoxic prodrug is then given, which is activated to a potent cytotoxic drug by the tumor-localized enzyme. This approach is termed gene-directed enzyme prodrug therapy (GDEPT). Adenoviruses have been used to target cancer cells, have an intrinsic tropism for liver, and are efficient gene vectors. Oncolytic adenoviruses produce clinical benefits, particularly in combination with conventional anticancer agents and are well tolerated. We rationalized that such adenoviruses, if their expression were restricted to telomerase-positive cancer cells, would make excellent gene vectors for GDEPT therapy of hepatocellular carcinoma. Here we use an oncolytic adenovirus to deliver the prodrug-activating enzyme carboxypeptidase G2 (CPG2) to tumors in a single systemic administration. The adenovirus replicated and produced high levels of CPG2 in two different hepatocellular carcinoma xenografts (Hep3B and HepG2) but not other tissues. GDEPT enhanced the adenovirus-alone therapy to elicit tumor regressions in the hepatocellular carcinoma models. This is the first time that CPG2 has been targeted and expressed intracellularly to effect significant therapy, showing that the combined approach holds enormous potential as a tumor-selective therapy for the systemic treatment of hepatocellular carcinoma.
Enhancement of 5-Fluorouracil Sensitivity by an rTS Signaling Mimic in H630 Colon Cancer Cells
Authors: Dolnick Ree , Wu Qi , Angelino Norman J. , Stephanie Lawrence V. , Chow Kuan-Chih , Sufrin Janice R. , Dolnick Bruce J.
Published: September 2005

The rTSβ protein has been hypothesized to synthesize signaling molecules that can down-regulate thymidylate synthase. These molecules share biological and chemical properties with acyl-homoserine lactones (AHL), suggesting some AHLs might act as rTS signaling mimics and down-regulate thymidylate synthase. We have determined that the AHL, 3-oxododecanoyl homoserine lactone (3-oxo-C12-(l)-HSL) can down-regulate thymidylate synthase protein at 10 μmol/L and reduce H630 (human colorectal cancer) growth by 50% at 23 μmol/L (IC50) in cell culture. At its IC50 concentration, 3-oxo-C12-(l)-HSL reduces the apparent IC50 of 5-fluorouracil (5-FU) from 1 μmol/L to 80 nmol/L (12-fold) in a colony formation assay. 3-Oxo-C12-(l)-HSL enhances the activity of 5-fluorodeoxyuridine, tomudex, and taxol but not the activity of 5-fluorouridine, methotrexate or Adriamycin. The unexpected interaction with taxol probably results from effects of the AHL on tubulin expression. Differences in taxol sensitivity, tubulin, and cellular morphology between H630 and the thymidylate synthase and rTSβ-overproducing, 5-FU-resistant H630-1 cell line as determined by colony formation assays, Western analysis of one-dimensional and two-dimensional gels, and photomicroscopy confirm that cytoskeletal changes are induced by the AHL or by rTS signaling. Isozyme differences in thymidylate synthase and rTSβ also exist in the two cell lines. Phosphorylation of rTSβ amino acid S121 is shown to occur and is decreased at least 10-fold in the drug-resistant cells. The data presented provide support for further investigations of rTS signaling mimics as enhancers to thymidylate synthase–directed chemotherapy, evidence that the phosphorylation state of rTSβ may be a marker for 5-FU resistance and a previously unrealized relationship between rTS signaling and the cytoskeleton.
Activation of the Canonical Wnt Pathway during Genital Keratinocyte Transformation: A Model for Cervical Cancer Progression
Authors: Üren Aykut , Fallen Shannon , Yuan Hang , Usubütün Alp , Küçükali Türkan , Schlegel Richard , Toretsky Jeffrey A.
Published: September 2005

Cervical carcinoma, the second leading cause of cancer deaths in women worldwide, is associated with human papillomavirus (HPV). HPV-infected individuals are at high risk for developing cervical carcinoma; however, the molecular mechanisms that lead to the progression of cervical cancer have not been established. We hypothesized that in a multistep carcinogenesis model, HPV provides the initial hit and activation of canonical Wnt pathway may serve as the second hit. To test this hypothesis, we evaluated the canonical Wnt pathway as a promoting factor of HPV-induced human keratinocyte transformation. In this in vitro experimental cervical carcinoma model, primary human keratinocytes immortalized by HPV were transformed by SV40 small-t (smt) antigen. We show that smt-transformed cells have high cytoplasmic β-catenin levels, a hallmark of activated canonical Wnt pathway, and that activation of this pathway by smt is mediated through its interaction with protein phosphatase-2A. Furthermore, inhibition of downstream signaling from β-catenin inhibited the smt-induced transformed phenotype. Wnt pathway activation transformed HPV-immortalized primary human keratinocytes even in the absence of smt. However, activation of the Wnt pathway in the absence of HPV was not sufficient to induce transformation. We also detected increased cytoplasmic and nuclear staining of β-catenin in invasive cervical carcinoma samples from 48 patients. We detected weak cytoplasmic and no nuclear staining of β-catenin in 18 cases of cervical dysplasia. Our results suggest that the transformation of HPV expressing human keratinocytes requires activation of the Wnt pathway and that this activation may serve as a screening tool in HPV-positive populations to detect malignant progression.
Cyclin D1 Antagonizes BRCA1 Repression of Estrogen Receptor α Activity
Authors: Wang Chenguang , Fan Saijun , Li Zhiping , Fu Maofu , Rao Mahadev , Ma Yongxian , Lisanti Michael P. , Albanese Chris , Katzenellenbogen Benita S. , Kushner Peter J. , Weber Barbara , Rosen Eliot M. , Pestell Richard G.
Published: September 2005

The cyclin D1 gene is frequently overexpressed in human breast cancer and is capable of inducing mammary tumorigenesis when overexpressed in transgenic mice. The BRCA1 breast tumor susceptibility gene product inhibits breast cancer cellular growth and the activity of several transcription factors. Herein, cyclin D1 antagonized BRCA1-mediated repression of estrogen receptor α (ERα)–dependent gene expression. Cyclin D1 repression of BRCA1 function was mediated independently of its cyclin-dependent kinase, retinoblastoma protein, or p160 (SRC-1) functions in human breast and prostate cancer cells. In vitro, cyclin D1 competed with BRCA1 for ERα binding. Cyclin D1 and BRCA1 were both capable of binding ERα in a common region of the ERα hinge domain. A novel domain of cyclin D1, predicted to form a helix-loop-helix structure, was required for binding to ERα and for rescue of BRCA1-mediated ERα transcriptional repression. In chromatin immunoprecipitation assays, 17β-estradiol (E2) enhanced ERα and cyclin D1 recruitment to an estrogen response element (ERE). Cyclin D1 expression enhanced ERα recruitment to an ERE. E2 reduced BRCA1 recruitment and BRCA1 expression inhibited E2-induced ERα recruitment at 12 hours. Cyclin D1 expression antagonized BRCA1 inhibition of ERα recruitment to an ERE, providing a mechanism by which cyclin D1 antagonizes BRCA1 function at an ERE. As cyclin D1 abundance is regulated by oncogenic and mitogenic signals, the antagonism of the BRCA1-mediated ERα repression by cyclin D1 may contribute to the selective induction of BRCA1-regulated target genes.
BAD Ser128 Is Not Phosphorylated by c-Jun NH2-Terminal Kinase for Promoting Apoptosis
Authors: Zhang Jiyan , Liu Jing , Yu Chenfei , Lin Anning
Published: September 2005

The phosphorylation and regulation of the proapoptotic Bcl-2 family protein BAD by c-Jun NH2-terminal kinase (JNK) is controversial. JNK can suppress interleukin-3 withdrawal-induced apoptosis via phosphorylation of BAD at Thr201. However, it has also been reported that JNK promotes apoptosis through phosphorylation of BAD at Ser128. Here, we report that JNK is not a BAD Ser128 kinase. JNK phosphorylates murine BAD (mBAD), but not human BAD (hBAD), in which Ser91 is equivalent to Ser128 in mBAD. In contrast, Cdc2, which phosphorylates Ser128, phosphorylates both mBAD and hBAD. Replacement of Ser128 by alanine has no effects on BAD phosphorylation by JNK in vitro and in vivo. Two-dimensional phosphopeptide mapping in combination with phosphoamino acid analysis reveals that JNK does not phosphorylate BAD at Ser128. Elimination of Ser128 phosphorylation has no effects on the proapoptotic activity of BAD in apoptosis induced by UV via JNK or growth factor withdrawal. Thus, our results show that Ser128 is not phosphorylated by JNK for promoting cell death.
Side Population Is Enriched in Tumorigenic, Stem-Like Cancer Cells, whereas ABCG2+ and ABCG2− Cancer Cells Are Similarly Tumorigenic
Authors: Patrawala Lubna , Calhoun Tammy , Schneider-Broussard Robin , Zhou Jianjun , Claypool Kent , Tang Dean G.
Published: September 2005

Recently, several human cancers including leukemia and breast and brain tumors were found to contain stem-like cancer cells called cancer stem cells (CSC). Most of these CSCs were identified using markers that identify putative normal stem cells. In some cases, stem-like cancer cells were identified using the flow cytometry-based side population technique. In this study, we first show that ∼30% of cultured human cancer cells and xenograft tumors examined (∼30 in total) possess a detectable side population. Purified side population cells from two cell lines (U373 glioma and MCF7 breast cancer) and a xenograft prostate tumor (LAPC-9) are more tumorigenic than the corresponding non–side population cells. These side population cells also possess some intrinsic stem cell properties as they generate non–side population cells in vivo, can be further transplanted, and preferentially express some “stemness” genes, including Notch-1 and β-catenin. Because the side population phenotype is mainly mediated by ABCG2, an ATP-binding cassette half-transporter associated with multidrug resistance, we subsequently studied ABCG2+ and ABCG2− cancer cells with respect to their tumorigenicity in vivo. Although side population cells show increased ABCG2 mRNA expression relative to the non–side population cells and all cancer cells and xenograft tumors examined express ABCG2 in a small fraction (0.5-3%) of the cells, highly purified ABCG2+ cancer cells, surprisingly, have very similar tumorigenicity to the ABCG2− cancer cells. Mechanistic studies indicate that ABCG2 expression is associated with proliferation and ABCG2+ cancer cells can generate ABCG2− cells. However, ABCG2− cancer cells can also generate ABCG2+ cells. Furthermore, the ABCG2− cancer cells form more and larger clones in the long-term clonal analyses and the ABCG2− population preferentially expresses several “stemness” genes. Taken together, our results suggest that (a) the side population is enriched with tumorigenic stem-like cancer cells, (b) ABCG2 expression identifies mainly fast-cycling tumor progenitors, and (c) the ABCG2− population contains primitive stem-like cancer cells.
Concurrent Induction of Antitumor Immunity and Autoimmune Thyroiditis in CD4+CD25+ Regulatory T Cell–Depleted Mice
Authors: Wei Wei-Zen , Jacob Jennifer B. , Zielinski John F. , Flynn Jeffrey C. , Shim K. David , Alsharabi Ghazwan , Giraldo Alvaro A. , Kong Yi-chi M.
Published: September 2005

When CD4+CD25+ regulatory T cells are depleted or inactivated for the purpose of enhancing antitumor immunity, the risk of autoimmune disease may be significantly elevated because these regulatory T cells control both antitumor immunity and autoimmunity. To evaluate the relative benefit and risk of modulating CD4+CD25+ regulatory T cells, we established a new test system to measure simultaneously the immune reactivity to a tumor-associated antigen, neu, and an unrelated self-antigen, thyroglobulin. BALB/c mice were inoculated with TUBO cells expressing an activated rat neu and treated with anti-CD25 monoclonal antibody to deplete CD25+ cells. The tumors grew, then regressed, and neu-specific antibodies and IFN-γ–secreting T cells were induced. The same mice were also exposed to mouse thyroglobulin by chronic i.v. injections. These mice produced thyroglobulin-specific antibody and IFN-γ–secreting T cells with inflammatory infiltration in the thyroids of some mice. The immune responses to neu or thyroglobulin were greater in mice undergoing TUBO tumor rejection and thyroglobulin injection than in those experiencing either alone. To the best of our knowledge, this is the first experimental system to assess the concurrent induction and possible synergy of immune reactivity to defined tumor and self-antigens following reduction of regulatory T cells. These results illustrate the importance of monitoring immune reactivity to self-antigens during cancer immunotherapy that involves immunomodulating agents, and the pressing need for novel strategies to induce antitumor immunity while minimizing autoimmunity.