bluuebunny/crossref_metadata_2025 huggingface dataset:
Plakophilin 3 Oncogene as Prognostic Marker and Therapeutic Target for Lung Cancer
Authors: Furukawa Chiyuki , Daigo Yataro , Ishikawa Nobuhisa , Kato Tatsuya , Ito Tomoo , Tsuchiya Eiju , Sone Saburo , Nakamura Yusuke
Published: September 2005

We investigated gene expression profiles of non–small cell lung carcinomas (NSCLC) to screen candidate molecules that might be useful as diagnostic markers or for development of novel molecular-targeting therapies. Here we report evidence that a member of the armadillo protein family, plakophilin 3 (PKP3), is a potential molecular target for treatment of lung cancers and might also serve as a prognostic indicator. We documented elevated expression of PKP3 in the great majority of NSCLC samples examined. Treatment of NSCLC cells with small interfering RNAs of PKP3 suppressed growth of the cancer cells; on the other hand, induction of exogenous expression of PKP3 conferred growth-promoting activity on COS-7 cells and enhanced their mobility in vitro. To investigate its function, we searched for PKP3-interacting proteins and identified dynamin 1-like, which was also activated in NSCLC. In addition, a high level of PKP3 expression was associated with poor survival as well as disease stage and node status for patients with lung adenocarcinoma, suggesting an important role of the protein in development and progression of this disease. As our data imply that up-regulation of PKP3 is a frequent and important feature of lung carcinogenesis, we suggest that targeting the PKP3 molecule might hold promise for development of a new therapeutic and diagnostic strategy for clinical management of lung cancers.
Soluble Tumor Necrosis Factor-Like Weak Inducer of Apoptosis Overexpression in HEK293 Cells Promotes Tumor Growth and Angiogenesis in Athymic Nude Mice
Authors: Ho David H. , Vu Hong , Brown Sharron A. N. , Donohue Patrick J. , Hanscom Heather N. , Winkles Jeffrey A.
Published: September 2005

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a member of the tumor necrosis factor superfamily of structurally related cytokines. TWEAK acts on responsive cells via binding to a cell surface receptor named Fn14. Recent studies have demonstrated that TWEAK can stimulate numerous cellular responses including cell proliferation, migration, and proinflammatory molecule production. It has also been reported that TWEAK can stimulate blood vessel formation in the rat cornea angiogenesis assay, but it is presently unknown whether this cytokine could play a role in the pathological angiogenesis associated with human diseases such as cancer, rheumatoid arthritis, and diabetic retinopathy. In the present study we investigated whether TWEAK was expressed in human tumors and whether it could promote tumor growth and angiogenesis in vivo. TWEAK mRNA expression was detected in many tumor types by cDNA array hybridization analysis, and TWEAK protein expression was confirmed in human colon cancer tissue by immunohistochemistry. As an initial approach to address whether TWEAK might act as a tumor angiogenesis factor, we established several human embryonic kidney cell lines that constitutively secrete a soluble TWEAK protein and examined their growth properties in vitro and in vivo. We found that although TWEAK-overexpressing cells do not have a growth advantage in vitro, they form larger and more highly vascularized tumors in athymic mice when compared with control, vector-transfected cells. This result suggests that the TWEAK-Fn14 signaling system may be a potential regulator of human tumorigenesis.
Hydrophilic Camptothecin Analogs That Form Extremely Stable Cleavable Complexes with DNA and Topoisomerase I
Authors: Wadkins Randy M. , Bearss David , Manikumar Govindarajan , Wani Mansukhlal C. , Wall Monroe E. , Von Hoff Daniel D.
Published: September 2005

Camptothecin (CPT) analogs that form more stable ternary complexes with DNA and topoisomerase I (termed cleavable complexes) show greater activity in their ability to inhibit tumor cell line growth in preclinical studies. Based on our earlier work, we hypothesized that analogs bearing hydrogen bonding moieties at the 7- through 10-position of CPT would result in more stable cleavable complexes. Consequently, we synthesized analogs with 7-mono-, 7-di-, and 7-trihydroxymethylaminomethyl groups. These analogs showed increasing cleavable complex stability as the number of hydroxyl groups was increased. The 7-trihydroxymethylaminomethyl analog of 10,11-methylenedioxycamptothecin (THMAM-MD) showed remarkable ternary complex stability with a half-life of 116 minutes. This is an order of magnitude more stable than any previously examined analog. Our in vitro analysis demonstrated that these analogs were all potent topoisomerase I poisons and could inhibit tumor cell growth in culture. We studied the effects of THMAM-MD in vivo in severe combined immunodeficient mice bearing HT-29 colon cancer and MiaPaCa-2 pancreatic cancer tumors. The THMAM-MD analog showed excellent, persisting activity in inhibiting tumor growth with both lines. Taken together, our results suggest that CPTs with hydrophilic, hydrogen-bonding groups at the 7-position hold the promise of excellent clinical activity.
Role of Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand in Interferon-Induced Apoptosis in Human Bladder Cancer Cells
Authors: Papageorgiou Angela , Lashinger Laura , Millikan Randall , Grossman H. Barton , Benedict William , Dinney Colin P. N. , McConkey David J.
Published: September 2005

Immunomodulators such as Bacillus Calmette-Guerin and interferon are clinically active in transitional cell carcinoma of the bladder, but their mechanisms of action remain unclear. Here we investigated the effects of IFNα on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expression and apoptosis in a panel of 20 human bladder cancer cell lines. Six (30%) displayed significant DNA fragmentation in response to increasing concentrations of IFNα (10–100,000 units/mL). In these lines IFNα induced early activation of caspase-8, and DNA fragmentation was blocked by a caspase-8-selective inhibitor (IETDfmk), consistent with the involvement of death receptor(s) in cell death. IFNα stimulated marked increases in TRAIL mRNA and protein in the majority of IFN-sensitive and IFN-resistant cell lines. A blocking anti-TRAIL antibody significantly inhibited IFN-induced DNA fragmentation in four of six IFN-sensitive cell lines, confirming that TRAIL played a direct role in cell death. Bortezomib (PS-341, Velcade), a potent TRAIL-sensitizing agent, increased sensitivity to IFNα in two of the IFN-resistant cell lines that produced large amounts of TRAIL in response to IFN treatment. Our data show that IFN-induced apoptosis in bladder cancer cells frequently involves autocrine TRAIL production. Combination therapy strategies aimed at overcoming TRAIL resistance may be very effective in restoring IFN sensitivity in a subset of human bladder tumors.
Inhibitor of Apoptosis Proteins: Translating Basic Knowledge into Clinical Practice
Authors: Schimmer Aaron D.
Published: September 2005

The inhibitor of apoptosis proteins (IAPs) are a family of antiapoptotic proteins that bind and inhibit caspases 3, 7, and/or 9, but not caspase 8. Growing evidence also indicates that IAPs also modulate cell division, cell cycle progression, and signal transduction pathways. As our basic understanding of IAPs has increased, the knowledge is being translated into clinically useful applications in the diagnosis and treatment of malignancy. For example, IAPs such as survivin are being investigated as diagnostic markers for the presence of occult malignancy. In addition, IAP overexpression is a poor prognostic marker in a variety of solid tumors and hematologic malignancies. Finally, IAPs are attractive therapeutic targets, and efforts are under way to develop antisense and chemical IAP inhibitors that may be useful for the treatment of a variety of malignancies. For all of these potential clinical applications, however, the challenge remains to incorporate these findings into actual clinical practice.
Role of the Human ST6GalNAc-I and ST6GalNAc-II in the Synthesis of the Cancer-Associated Sialyl-Tn Antigen
Authors: Marcos Nuno T. , Pinho Sandra , Grandela Catarina , Cruz Andrea , Samyn-Petit Bénédicte , Harduin-Lepers Anne , Almeida Raquel , Silva Filipe , Morais Vanessa , Costa Julia , Kihlberg Jan , Clausen Henrik , Reis Celso A.
Published: September 2005

The Sialyl-Tn antigen (Neu5Acα2–6GalNAc-O-Ser/Thr) is highly expressed in several human carcinomas and is associated with carcinoma aggressiveness and poor prognosis. We characterized two human sialyltransferases, CMP-Neu5Ac:GalNAc-R α2,6-sialyltransferase (ST6GalNAc)-I and ST6GalNAc-II, that are candidate enzymes for Sialyl-Tn synthases. We expressed soluble recombinant hST6GalNAc-I and hST6GalNAc-II and characterized the substrate specificity of both enzymes toward a panel of glycopeptides, glycoproteins, and other synthetic glycoconjugates. The recombinant ST6GalNAc-I and ST6GalNAc-II showed similar substrate specificity toward glycoproteins and GalNAcα-O-Ser/Thr glycopeptides, such as glycopeptides derived from the MUC2 mucin and the HIVgp120. We also observed that the amino acid sequence of the acceptor glycopeptide contributes to the in vitro substrate specificity of both enzymes. We additionally established a gastric cell line, MKN45, stably transfected with the full length of either ST6GalNAc-I or ST6GalNAc-II and evaluated the carbohydrate antigens expression profile induced by each enzyme. MKN45 transfected with ST6GalNAc-I showed high expression of Sialyl-Tn, whereas MKN45 transfected with ST6GalNAc-II showed the biosynthesis of the Sialyl-6T structure [Galβ1–3 (Neu5Acα2–6)GalNAc-O-Ser/Thr].

In conclusion, although both enzymes show similar in vitro activities when Tn antigen alone is available, whenever both Tn and T antigens are present, ST6GalNAc-I acts preferentially on Tn antigen, whereas the ST6GalNAc-II acts preferentially on T antigen. Our results show that ST6GalNAc-I is the major Sialyl-Tn synthase and strongly support the hypothesis that the expression of the Sialyl-Tn antigen in cancer cells is due to ST6GalNAc-I activity.
Activating Mutations of the Noonan Syndrome-Associated
SHP2/PTPN11
Gene in Human Solid Tumors and Adult Acute Myelogenous Leukemia
Authors: Bentires-Alj Mohamed , Paez J. Guillermo , David Frank S. , Keilhack Heike , Halmos Balazs , Naoki Katsuhiko , Maris John M. , Richardson Andrea , Bardelli Alberto , Sugarbaker David J. , Richards William G. , Du Jinyan , Girard Luc , Minna John D. , Loh Mignon L. , Fisher David E. , Velculescu Victor E. , Vogelstein Bert , Meyerson Matthew , Sellers William R. , Neel Benjamin G.
Published: September 2005

The SH2 domain-containing protein-tyrosine phosphatase PTPN11 (Shp2) is required for normal development and is an essential component of signaling pathways initiated by growth factors, cytokines, and extracellular matrix. In many of these pathways, Shp2 acts upstream of Ras. About 50% of patients with Noonan syndrome have germ-line PTPN11 gain of function mutations. Associations between Noonan syndrome and an increased risk of some malignancies, notably leukemia and neuroblastoma, have been reported, and recent data indicate that somatic PTPN11 mutations occur in children with sporadic juvenile myelomonocytic leukemia, myelodysplasic syndrome, B-cell acute lymphoblastic leukemia, and acute myelogenous leukemia (AML). Juvenile myelomonocytic leukemia patients without PTPN11 mutations have either homozygotic NF-1 deletion or activating RAS mutations. Given the role of Shp2 in Ras activation and the frequent mutation of RAS in human tumors, these data raise the possibility that PTPN11 mutations play a broader role in cancer. We asked whether PTPN11 mutations occur in other malignancies in which activating RAS mutations occur at low but significant frequency. Sequencing of PTPN11 from 13 different human neoplasms including breast, lung, gastric, and neuroblastoma tumors and adult AML and acute lymphoblastic leukemia revealed 11 missense mutations. Five are known mutations predicted to result in an activated form of Shp2, whereas six are new mutations. Biochemical analysis confirmed that several of the new mutations result in increased Shp2 activity. Our data demonstrate that mutations in PTPN11 occur at low frequency in several human cancers, especially neuroblastoma and AML, and suggest that Shp2 may be a novel target for antineoplastic therapy.
A Novel Peptide Specifically Binding to Nasopharyngeal Carcinoma For Targeted Drug Delivery
Authors: Lee Tong-Young , Wu Han-Chung , Tseng Yun-Long , Lin Chin-Tarng
Published: September 2005

Nasopharyngeal carcinoma (NPC) is a common cancer among Chinese living in southern China, Taiwan, and Singapore. The 5-year survival rate in the early stage of NPC has been reported as high as 90 to 95% with the use of radiotherapy, but in the advanced cases, even with the use of both chemotherapy and radiotherapy, the survival rate is still <50%. To improve the survival rate, we identify a 12-mer peptide (l-peptide) specifically binding to NPC cells with a phage displayed random peptide library. The l-phage and synthetic l-peptide bound to the tumor cell surfaces of most NPC cell lines and biopsy specimens, but not normal nasal mucosal cells, and the l-peptide–linked liposomes containing fluorescent substance (l-peptide-Lipo-HPTS) were capable of binding to and translocating across plasma membranes. l-Peptide–linked liposomes that carried doxorubicin (l-peptide-Lipo-Dox) caused marked cytotoxicity in NPC cells. In SCID mice bearing NPC xenografts, the l-phages specifically bound to the tumor mass, an effect that was inhibited by competition with synthetic l-peptide. In addition, the l-peptide-Lipo-Dox suppressed tumor growth better than Lipo-Dox. These results indicate that the novel l-peptide specifically binds NPC cells and is a good candidate for targeted drug delivery to NPC solid tumors.
Microarray-based Detection of Multidrug Resistance in Human Tumor Cells by Expression Profiling of ATP-binding Cassette Transporter Genes
Authors: Gillet Jean-Pierre , Efferth Thomas , Steinbach Daniel , Hamels Jacques , de Longueville Françoise , Bertholet Vincent , Remacle José
Published: September 2005

Different mechanisms of drug resistance, including ATP-binding cassette (ABC) transporters, are responsible for treatment failure of tumors. We developed a low-density DNA microarray which contains 38 genes of the ABC transporter gene family. This tool has been validated with three different multidrug-resistant sublines (CEM/ADR5000, HL60/AR, and MCF7/CH1000) known to overexpress either the ABCB1 (MDR1), ABCC1 (MRP1), or ABCG2 (MXR and BCRP) genes. When compared with their drug-sensitive parental lines, we observed not only the overexpression of these genes in the multidrug-resistant cell lines but also of other ABC transporter genes pointing to their possible role in multidrug resistance. These results were corroborated by quantitative real-time reverse transcription-PCR. As the microarray allows the determination of the expression profile of many ABC transporters in a single hybridization experiment, it may be useful as a diagnostic tool to detect drug resistance in clinical samples.
Tissue Inhibitors of Metalloproteinase 2 Inhibits Endothelial Cell Migration through Increased Expression of RECK
Authors: Oh Junseo , Seo Dong-Wan , Diaz Tere , Wei Beiyang , Ward Yvona , Ray Jill M. , Morioka Yoko , Shi Shuliang , Kitayama Hitoshi , Takahashi Chiaki , Noda Makoto , Stetler-Stevenson William G.
Published: September 2005

The antiangiogenic function of the tissue inhibitors of metalloproteinases (TIMPs) has been attributed to their matrix metalloproteinase inhibitory activity. Here we demonstrate that TIMP-1 but not Ala+TIMP-1 inhibits both basal and vascular endothelial growth factor (VEGF)-stimulated migration of human microvascular endothelial cells (hMVECs), suggesting that this effect is dependent on direct inhibition of matrix metalloproteinase (MMP) activity. In contrast, TIMP-2 and mutant Ala+TIMP-2, which is devoid of MMP inhibitory activity, block hMVEC migration in response to VEGF-A stimulation. TIMP-2 and Ala+TIMP-2 also suppress basal hMVEC migration via a time-dependent mechanism mediated by enhanced expression of RECK, a membrane-anchored MMP inhibitor, which, in turn, inhibits cell migration. TIMP-2 treatment of hMVECs increases the association of Crk with C3G, resulting in enhanced Rap1 activation. hMVECs stably expressing Rap1 have increased RECK expression and display reduced cell migration compared with those expressing inactive Rap1(38N). RECK-null murine embryo fibroblasts fail to demonstrate TIMP-2–mediated decrease in cell migration despite activation of Rap1. TIMP-2–induced RECK decreases cell-associated MMP activity. Anti-RECK antibody increases MMP activity and reverses the TIMP-2–mediated reduction in cell migration. The effects of TIMP-2 on RECK expression and cell migration were confirmed in A2058 melanoma cells. These results suggest that TIMP-2 can inhibit cell migration via several distinct mechanisms. First, TIMP-2 can inhibit cell migration after VEGF stimulation by direct inhibition of MMP activity induced in response to VEGF stimulation. Secondly, TIMP-2 can disrupt VEGF signaling required for initiation of hMVEC migration. Third, TIMP-2 can enhance expression of RECK via Rap1 signaling resulting in an indirect, time-dependent inhibition of endothelial cell migration.
p53-Binding Protein 1 Is Fused to the Platelet-Derived Growth Factor Receptor β in a Patient with a t(5;15)(q33;q22) and an Imatinib-Responsive Eosinophilic Myeloproliferative Disorder
Authors: Grand Francis H. , Burgstaller Sonja , Kühr Thomas , Baxter E. Joanna , Webersinke Gerald , Thaler Josef , Chase Andrew J. , Cross Nicholas C. P.
Published: September 2005

We describe the fusion of TP53BP1 to PDGFRB in a patient with a chronic myeloid leukemia-like disorder associated with eosinophilia and a t(5;15)(q33;q22). TP53BP1 encodes 53BP1, a p53-binding protein that plays a role in cellular responses to DNA damage. The 53BP1-PDGFRβ fusion protein is predicted to retain the kinetochore-binding domain of 53BP1 fused to the transmembrane and intracellular tyrosine kinase domain of PDGFRβ. The presence of the fusion was confirmed by two-color fluorescence in situ hybridization, reverse transcription-PCR, and by characterizing the genomic breakpoints. The reciprocal fusion, which would contain the p53-binding 53BP1 BRCA1 COOH-terminal domains, was not detectable by fluorescence in situ hybridization or nested PCR. Imatinib, a known inhibitor of PDGFRβ, blocked the growth of patient colony-forming unit, granulocyte-macrophage in vitro and produced a clinically significant response before relapse and subsequent death with imatinib-resistant disease. We conclude that TP53BP1-PDGFRB is a novel imatinib target in atypical chronic myeloid leukemia.
Insulin Receptor, Insulin Receptor Substrate-1, Raf-1, and Mek-1 during Hormonal Hepatocarcinogenesis by Intrahepatic Pancreatic Islet Transplantation in Diabetic Rats
Authors: Evert Matthias , Sun Jianping , Pichler Sabine , Slavova Nadia , Schneider-Stock Regine , Dombrowski Frank
Published: September 2005

Low-number transplantation of pancreatic islets into the livers of diabetic rats leads to transformation of the downstream liver acini into clear-cell foci of altered hepatocytes (FAHs). These FAHs correspond to the glycogen-storing (clear-cell) phenotype of hepatocellular preneoplasias and develop into hepatocellular adenomas (HCAs) and hepatocellular carcinomas (HCCs) within 6 to 24 months. In addition, they show metabolic alterations that resemble well-known insulin effects, most likely constituting the result of the local hyperinsulinemia. Thus, we investigated FAHs, HCAs, and HCCs for altered expression of insulin receptor, insulin receptor substrate-1 (IRS-1), Raf-1 and Mek-1. Light and electron microscopic immunohistochemistry revealed a translocation of insulin receptor from the plasma membrane (normal tissue) into the cytoplasm in clear-cell FAHs and an increase in insulin receptor expression in HCAs and HCCs. FAHs also showed an increase in IRS-1 gene expression, investigated by in situ hybridization and quantitative reverse transcription-PCR. IRS-1, Raf-1, and Mek-1 proteins were strongly overexpressed in FAHs and tumors, as compared with the unaltered liver tissue. These overexpressions were closely linked to the clear-cell phenotype of preneoplastic and neoplastic hepatocytes, because basophilic FAHs (later stages) and basophilic tumors showed no overexpressions. In this endocrine model of hepatocarcinogenesis, severe alterations of insulin signaling were induced by the pathological local action of islet hormones in the livers and may substantially contribute to the carcinogenic process.
International Meeting on Cancer Vaccines
Authors: Belardelli Filippo , Ferrantini Maria , Parmiani Giorgio , Schlom Jeffrey , Garaci Enrico
Published: September 2005

The major aims of the International Meeting on Cancer Vaccines were to review the state-of-the-art research on cancer vaccines, to compare different experimental approaches of therapeutic vaccination and to discuss critical issues and perspectives. The results from recent clinical trials in patients treated with different types of cancer vaccines were presented. Reasons for the limited response and possible modalities for enhancing efficacy of therapeutic vaccines were subjects of major discussion. A consensus was achieved on the need of combining cancer vaccines with other anticancer treatments. Of note, evidence stemming from studies in animal models pointed out new rationales for a selective combination of cancer vaccines with chemotherapy. In addition, some main presentations focused on new adjuvants (CpG oligonucleotides) and on the role of cytokines (i.e., type I IFN, interleukin 12, and interleukin 15) in promoting an antitumor immune response to vaccines. A considerable attention was given to regulatory T cells and to strategies for suppressing their function, thus enhancing vaccine efficacy. An entire session was devoted to the use of dendritic cells for the development of cancer vaccines. The results of clinical studies and the advantages of using new modalities for preparing dendritic cell-based vaccines were discussed.
Mutations of BRAF and KRAS Precede the Development of Ovarian Serous Borderline Tumors
Authors: Ho Chung-Liang , Kurman Robert J. , Dehari Reiko , Wang Tian-Li , Shih Ie-Ming
Published: September 2005

Molecular genetic changes that are associated with the initiating stage of tumor development are important in tumorigenesis. Ovarian serous borderline tumors (SBTs), putative precursors of low-grade serous carcinomas, are among the few human neoplasms with a high frequency of activating mutations in BRAF and KRAS genes. However, it remains unclear as to how these mutations contribute to tumor progression. To address this issue, we compared the mutational status of BRAF and KRAS in both SBTs and the adjacent epithelium from cystadenomas, the presumed precursor of SBTs. We found that three of eight SBTs contained mutant BRAF, and four SBTs contained mutant KRAS. All specimens with mutant BRAF harbored wild-type KRAS and vice versa. Thus, seven (88%) of eight SBTs contained either BRAF or KRAS mutations. The same mutations detected in SBTs were also identified in the cystadenoma epithelium adjacent to the SBTs in six (86%) of seven informative cases. As compared to SBTs, the cystadenoma epithelium, like ovarian surface epithelium, lacks cytological atypia. Our findings provide cogent evidence that mutations of BRAF and KRAS occur in the epithelium of cystadenomas adjacent to SBTs and strongly suggest that they are very early events in tumorigenesis, preceding the development of SBT.
Coagulation Facilitates Tumor Cell Spreading in the Pulmonary Vasculature during Early Metastatic Colony Formation
Authors: Im Jae Hong , Fu Weili , Wang Hui , Bhatia Sujata K. , Hammer Daniel A. , Kowalska M. Anna , Muschel Ruth J.
Published: September 2005

Coagulation has long been known to facilitate metastasis. To pinpoint the steps where coagulation might play a role in the metastasis, we used three-dimensional visualization of direct infusion of fluorescence labeled antibody to observe the interaction of tumor cells with platelets and fibrinogen in isolated lung preparations. Tumor cells arrested in the pulmonary vasculature were associated with a clot composed of both platelets and fibrin(ogen). Initially, the cells attached to the pulmonary vessels were rounded. Over the next 2 to 6 hours, they spread on the vessel surface. The associated clot was lysed coincident with tumor cell spreading. To assess the importance of clot formation, we inhibited coagulation with hirudin, a potent inhibitor of thrombin. The number of tumor cells initially arrested in the lung of hirudin-treated mice was essentially the same as in control mice. However, tumor cell spreading and subsequent retention of the tumor cells in the lung was markedly inhibited in the anticoagulated mice. These associations of the tumor cells with platelets were independent of tumor cell expression of P-selectin ligands. This work identifies tumor cell spreading onto the vascular surface as an important component of the metastatic cascade and implicates coagulation in this process.
Role of Synaptojanin 2 in Glioma Cell Migration and Invasion
Authors: Chuang Ya-yu , Tran Nhan L. , Rusk Nicole , Nakada Mitsutoshi , Berens Michael E. , Symons Marc
Published: September 2005

The small GTPase Rac1 is thought to play an important role in cell migration and invasion. We have previously identified synaptojanin 2, a phosphoinositide phosphatase, as an effector of Rac1. Here, we show that small interfering RNA-mediated depletion of either Rac1 or synaptojanin 2 inhibits invasion of SNB19 and U87MG glioblastoma cells through Matrigel and rat brain slices. Depletion of Rac1 or synaptojanin 2 also inhibits migration of SNB19 and U87MG cells on glioma-derived extracellular matrix. In addition, we found that depletion of Rac1 or synaptojanin 2 inhibits the formation of lamellipodia and invadopodia, specialized membrane structures that are thought to be involved in extracellular matrix degradation. These results suggest that synaptojanin 2 contributes to the role of Rac1 in cell invasion and migration by regulating the formation of invadopodia and lamellipodia. This study also identifies synaptojanin 2 as a novel potential target for therapeutic intervention in malignant tumors.
Conditional Overexpression of Active Transforming Growth Factor β1
In vivo
Accelerates Metastases of Transgenic Mammary Tumors
Authors: Muraoka-Cook Rebecca S. , Kurokawa Hirokazu , Koh Yasuhiro , Forbes James T. , Roebuck L. Renee , Barcellos-Hoff Mary Helen , Moody Susan E. , Chodosh Lewis A. , Arteaga Carlos L.
Published: September 2005

To address the role of transforming growth factor (TGF) β in the progression of established tumors while avoiding the confounding inhibitory effects of TGF-β on early transformation, we generated doxycycline (DOX)-inducible triple transgenic mice in which active TGF-β1 expression could be conditionally regulated in mouse mammary tumor cells transformed by the polyomavirus middle T antigen. DOX-mediated induction of TGF-β1 for as little as 2 weeks increased lung metastases >10-fold without a detectable effect on primary tumor cell proliferation or tumor size. DOX-induced active TGF-β1 protein and nuclear Smad2 were restricted to cancer cells, suggesting a causal association between autocrine TGF-β and increased metastases. Antisense-mediated inhibition of TGF-β1 in polyomavirus middle T antigen-expressing tumor cells also reduced basal cell motility, survival, anchorage-independent growth, tumorigenicity, and metastases. Therefore, induction and/or activation of TGF-β in hosts with established TGF-β-responsive cancers can rapidly accelerate metastatic progression.
Phosphorylation of Estrogen Receptor α Blocks Its Acetylation and Regulates Estrogen Sensitivity
Authors: Cui Yukun , Zhang Mao , Pestell Richard , Curran Edward M. , Welshons Wade V. , Fuqua Suzanne A. W.
Published: September 2005

Estrogen receptor (ER) α is mutated (lysine 303 to arginine, K303R) in approximately one third of premalignant breast hyperplasias, which renders breast cancer cells expressing the mutant receptor hypersensitive for proliferation in response to low doses of estrogen. It is known that ERα is posttranslationally modified by protein acetylation and phosphorylation by a number of secondary messenger signaling cascades. The K303R ERα mutation resides at a major protein acetylation site adjacent to a potential protein kinase A (PKA) phosphorylation site at residue 305 within the hinge domain of the receptor. Mutation of this phosphorylation site to aspartic acid to mimic constitutive phosphorylation blocks acetylation of the K303 ERα site and generates an enhanced transcriptional response similar to that seen with the naturally occurring K303R mutant receptor. Activation of PKA signaling by the cell-permeable cyclic AMP (cAMP) analog 8-bromo-cAMP further enhances estrogen sensitivity of the mutant receptor, whereas a specific PKA inhibitor antagonizes this increase. We propose that the hypersensitive ERα mutant breast cancer phenotype involves an integration of coupled acetylation and phosphorylation events by upstream signaling molecules.
Evidence that TRPM8 Is an Androgen-Dependent Ca2+ Channel Required for the Survival of Prostate Cancer Cells
Authors: Zhang Lei , Barritt Gregory John
Published: September 2005

The Ca2+-permeable channel TRPM8 is thought to play an important role in the pathophysiology of prostate cancer. We have investigated the intracellular location of TRPM8 and its role as a Ca2+-permeable channel in an androgen-responsive and an androgen-insensitive prostate cancer cell line. We report evidence from immunofluorescence experiments that in the androgen-responsive LNCaP cell line, the TRPM8 protein is expressed in the endoplasmic reticulum and plasma membrane, acts as a Ca2+-permeable channel (assessed using Fura-2 to measure increases in the cytoplasmic Ca2+ concentration) in each of these membranes, and is regulated by androgen. Although TRPM8 was detected in the androgen-insensitive PC-3 cell line, no evidence was obtained for regulation of its expression by androgen. The results of experiments using LNCaP cells, the TRPM8 antagonist capsazepine, and small interference RNA targeted to TRPM8 indicate that TRPM8 is required for cell survival. These results indicate that TRPM8 is an important determinator of Ca2+ homeostasis in prostate epithelial cells and may be a potential target for the action of drugs in the management of prostate cancer.
p73 Isoforms Can Induce T-Cell Factor–Dependent Transcription in Gastrointestinal Cells
Authors: Tomkova Katarina , Belkhiri Abbes , El-Rifai Wael , Zaika Alexander I.
Published: September 2005

A new p53 family member, p73, and its isoform ΔNp73 are increasingly recognized in cancer research as important players in tumorigenesis, as well as in chemotherapeutic drug sensitivity. Despite substantial structural similarities to p53, accumulating evidence suggests that p53 and p73 may play different roles in human tumorigenesis. In this study, we have investigated the role of p73 and ΔNp73 in upper gastrointestinal tumorigenesis. Our results indicate that p73 and ΔNp73 are frequently overexpressed in >60% of primary adenocarcinomas of the stomach and esophagus. We have demonstrated that this overexpression can lead to the suppression of p73 transcriptional and apoptotic activity in gastrointestinal cells. Moreover, it induces β-catenin up-regulation and T-cell factor/lymphocyte enhancement factor–dependent transcription. Wild-type p53, but not mutant p53, can inhibit this effect. Our results demonstrate a novel mechanism for activation of β-catenin in gastrointestinal tumors and support the concept that overexpression of p73 isoforms can play an important role in tumorigenesis.
Nuclear βII-Tubulin Associates with the Activated Notch Receptor to Modulate Notch Signaling
Authors: Yeh Tien-Shun , Hsieh Rong-Hong , Shen Shing-Chuan , Wang Shwu-Huey , Tseng Min-Jen , Shih Chwen-Ming , Lin Jing-Jer
Published: September 2005

The Notch signal pathway plays important roles in proliferation, apoptosis, and differentiation. Abnormalities in Notch signaling are linked to many human diseases. After ligand binding, Notch signaling is activated through the cleavage of Notch receptors to release and translocate the Notch intracellular domain into the nucleus. The Notch1 receptor intracellular domain (N1IC), the activated form of the Notch1 receptor, can modulate downstream target genes via C promoter-binding factor 1–dependent and -independent pathways. To further dissect the Notch1 signaling pathway, we screened the N1IC-associated proteins using a yeast two-hybrid system and identified nuclear βII-tubulin as a candidate for the N1IC-associated proteins. It was suggested that the presence of βII-tubulin in nuclei might be correlated with the cancerous state of cells. However, the function of βII-tubulin locating in the nucleus still is unknown. Herein, we show that the complex of α- and βII-tubulin is associated with N1IC in cancer cells by a coimmunoprecipitation analysis. The ankyrin domain of the Notch1 receptor alone was sufficient to associate with βII-tubulin. Furthermore, α- and βII-tubulin were localized in the nucleus and formed a complex with N1IC. Treatment with Taxol increased the amounts of nuclear α- and βII-tubulin in K562 and HeLa cells and promoted the C promoter-binding factor 1–dependent transactivation activity of N1IC. We also show that nuclear βII-tubulin was bound on the C promoter-binding factor 1 response elements via the association with N1IC. These results suggest that nuclear βII-tubulin can modulate Notch signaling through interaction with N1IC in cancer cells.
Ectopic Expression of Interferon Regulatory Factor-1 Promotes Human Breast Cancer Cell Death and Results in Reduced Expression of Survivin
Authors: Pizzoferrato Eva , Liu Ye , Gambotto Andrea , Armstrong Michaele J. , Stang Michael T. , Gooding William E. , Alber Sean M. , Shand Stuart H. , Watkins Simon C. , Storkus Walter J. , Yim John H.
Published: September 2005

The overexpression of the inhibitor of apoptosis protein, survivin, may provide tumor cells with a distinct survival advantage in situ; hence, therapeutic strategies have been designed to inhibit its expression. In this study, we ectopically expressed the interferon regulatory factor (IRF)-1 protein in the breast carcinoma cell lines MDA-MB-468 and SK-BR-3 using a recombinant adenovirus (Ad-IRF-1). By screening microarray analysis of cDNA from the human breast cancer cell line MDA-MB-468 infected with Ad-IRF-1, we observed a 15-fold down-regulation of the survivin gene when compared with uninfected cells. Consequently, we tested survivin expression in Ad-IRF-1-infected MDA-MB-468 and SK-BR-3 breast cancer cell lines. Immunoblotting analyses supported the contention that ectopic expression of the IRF-1 protein results in down-regulation of survivin protein expression that is independent of p53. In addition, Ad-IRF-1 infection of these human breast cancer cell lines induces the expression of p21. We also report that increased apoptosis is observed in tumor cells infected with Ad-IRF-1 compared with Ad-Ψ5 mock-infected cells and that cell death is further augmented when the IRF-1-infected cells are cultured with Adriamycin. Moreover, in a xenogeneic mouse model of breast carcinoma, in vivo treatment of tumor-bearing mice with intratumoral Ad-IRF-1 injections results in tumor growth inhibition. In resected tumors from mice that had been treated with Ad-IRF-1, tumor cells that express the IRF-1 transgene have a predominant IRF-1-positive, survivin-negative phenotype. Collectively, these observations suggest that therapies designed to enhance IRF-1 expression within tumor cells may represent novel treatment strategies for breast cancer.
HPC1/RNASEL
Mediates Apoptosis of Prostate Cancer Cells Treated with 2′,5′-Oligoadenylates, Topoisomerase I Inhibitors, and Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand
Authors: Malathi Krishnamurthy , Paranjape Jayashree M. , Ganapathi Ram , Silverman Robert H.
Published: September 2005

The hereditary prostate cancer 1 (HPC1) allele maps to the RNASEL gene encoding a protein (RNase L) implicated in the antiviral activity of interferons. To investigate the possible role of RNase L in apoptosis of prostate cancer cells, we decreased levels of RNase L by severalfold in the DU145 human prostate cancer cell line through the stable expression of a small interfering RNA (siRNA). Control cells expressed siRNA with three mismatched nucleotides to the RNase L sequence. Cells deficient in RNase L, but not the control cells, were highly resistant to apoptosis by the RNase L activator, 2′,5′-oligoadenylate (2-5A). Surprisingly, the RNase L-deficient cells were also highly resistant to apoptosis by combination treatments with a topoisomerase (Topo) I inhibitor (camptothecin, topotecan, or SN-38) and tumor necrosis factor-related apoptosis-inducing ligand [TRAIL (Apo2L)]. In contrast, cells expressing siRNA to the RNase L inhibitor RLI (HP68) showed enhanced apoptosis in response to Topo I inhibitor alone or in combination with TRAIL. An inhibitor of c-Jun NH2-terminal kinases reduced apoptosis induced by treatment with either 2-5A or the combination of camptothecin and TRAIL, thus implicating c-Jun NH2-terminal kinase in the apoptotic signaling pathway. Furthermore, prostate cancer cells were sensitive to apoptosis from the combination of 2-5A with either TRAIL or Topo I inhibitor, whereas normal prostate epithelial cells were partially resistant to apoptosis. These findings indicate that RNase L integrates and amplifies apoptotic signals generated during treatment of prostate cancer cells with 2-5A, Topo I inhibitors, and TRAIL.
Early Changes in Protein Expression Detected by Mass Spectrometry Predict Tumor Response to Molecular Therapeutics
Authors: Reyzer Michelle L. , Caldwell Robert L. , Dugger Teresa C. , Forbes James T. , Ritter Christoph A. , Guix Marta , Arteaga Carlos L. , Caprioli Richard M.
Published: September 2005

Biomarkers that predict therapeutic response are essential for the development of anticancer therapies. We have used matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to directly analyze protein profiles in mouse mammary tumor virus/HER2 transgenic mouse frozen tumor sections after treatment with the erbB receptor inhibitors OSI-774 and Herceptin. Inhibition of tumor cell proliferation and induction of apoptosis and tumor reduction were predicted by a >80% reduction in thymosin β4 and ubiquitin levels that were detectable after 16 hours of a single drug dose before any evidence of in situ cellular activity. These effects were time- and dose-dependent, and their spatial distribution in the tumor correlated with that of the small-molecule inhibitor OSI-774. In addition, they predicted for therapeutic synergy of OSI-774 and Herceptin as well as for drug resistance. These results suggest that drug-induced early proteomic changes as measured by MALDI-MS can be used to predict the therapeutic response to established and novel therapies.
Role of a BCL9-Related β-Catenin-Binding Protein, B9L, in Tumorigenesis Induced by Aberrant Activation of Wnt Signaling
Authors: Adachi Shungo , Jigami Takafumi , Yasui Toshio , Nakano Tetsuhiro , Ohwada Susumu , Omori Yoshihiro , Sugano Sumio , Ohkawara Bisei , Shibuya Hiroshi , Nakamura Tsutomu , Akiyama Tetsu
Published: September 2005

Wnt signaling plays a crucial role in a number of developmental processes and in tumorigenesis. β-Catenin is stabilized by Wnt signaling and associates with the TCF/LEF family of transcription factors, thereby activating transcription of Wnt target genes. Constitutive activation of β-catenin-TCF–mediated transcription resulting from mutations in adenomatous polyposis coli (APC), β-catenin, or Axin is believed to be a critical step in tumorigenesis among divergent types of cancers. Here we show that the transactivation potential of the β-catenin-TCF complex is enhanced by its interaction with a BCL9-like protein, B9L, in addition to BCL9. We found that B9L is required for enhanced β-catenin-TCF–mediated transcription in colorectal tumor cells and for β-catenin–induced transformation of RK3E cells. Furthermore, expression of B9L was aberrantly elevated in about 43% of colorectal tumors, relative to the corresponding noncancerous tissues. These results suggest that B9L plays an important role in tumorigenesis induced by aberrant activation of Wnt signaling.
Shark Cartilage, Cancer and the Growing Threat of Pseudoscience
Authors: Ostrander Gary K. , Cheng Keith C. , Wolf Jeffrey C. , Wolfe Marilyn J.
Published: September 2005

The promotion of crude shark cartilage extracts as a cure for cancer has contributed to at least two significant negative outcomes: a dramatic decline in shark populations and a diversion of patients from effective cancer treatments. An alleged lack of cancer in sharks constitutes a key justification for its use. Herein, both malignant and benign neoplasms of sharks and their relatives are described, including previously unreported cases from the Registry of Tumors in Lower Animals, and two sharks with two cancers each. Additional justifications for using shark cartilage are illogical extensions of the finding of antiangiogenic and anti-invasive substances in cartilage. Scientific evidence to date supports neither the efficacy of crude cartilage extracts nor the ability of effective components to reach and eradicate cancer cells. The fact that people think shark cartilage consumption can cure cancer illustrates the serious potential impacts of pseudoscience. Although components of shark cartilage may work as a cancer retardant, crude extracts are ineffective. Efficiencies of technology (e.g., fish harvesting), the power of mass media to reach the lay public, and the susceptibility of the public to pseudoscience amplifies the negative impacts of shark cartilage use. To facilitate the use of reason as the basis of public and private decision-making, the evidence-based mechanisms of evaluation used daily by the scientific community should be added to the training of media and governmental professionals. Increased use of logical, collaborative discussion will be necessary to ensure a sustainable future for man and the biosphere.
Lymphocyte Transformation by Pim-2 Is Dependent on Nuclear Factor-κB Activation
Authors: Hammerman Peter S. , Fox Casey J. , Cinalli Ryan M. , Xu Anne , Wagner John D. , Lindsten Tullia , Thompson Craig B.
Published: September 2005

Pim-2 is a transcriptionally regulated oncogenic kinase that promotes cell survival in response to a wide variety of proliferative signals. Deregulation of Pim-2 expression has been documented in several human malignancies, including leukemia, lymphoma, and multiple myeloma. Here, we show that the ability of Pim-2 to promote survival of cells is dependent on nuclear factor (NF)-κB activation. Pim-2 activates NF-κB–dependent gene expression by inducing phosphorylation of the oncogenic serine/threonine kinase Cot, leading to both augmentation of IκB kinase activity and a shift in nuclear NF-κB from predominantly p50 homodimers to p50/p65 heterodimers. Blockade of NF-κB function eliminates Pim-2–mediated survival in both cell lines and primary cells, and both Cot phosphorylation and expression are required for the prosurvival effects of Pim-2. Although Pim-2 cooperates with Myc to promote growth factor-independent cell proliferation, this feature is abrogated by NF-κB blockade. The ability of Pim-2 to serve as an oncogene in vivo depends on sustained NF-κB activity. Thus, the transcriptional induction of Pim-2 initiates a novel NF-κB activation pathway that regulates cell survival.
Antiproliferative Effect of Liver X Receptor Agonists on LNCaP Human Prostate Cancer Cells
Authors: Fukuchi Junichi , Kokontis John M. , Hiipakka Richard A. , Chuu Chih-pin , Liao Shutsung
Published: September 2005

Liver X receptors function as central transcriptional regulators for lipid homeostasis, for which agonists have been developed as potential drugs for treatment of cardiovascular diseases and metabolic syndromes. Because dysregulation of lipid metabolism has been implicated in sex hormone-dependent cancers, we investigated the effect of liver X receptor agonists on prostate and breast cancer cell proliferation. Treatment of human prostate cancer LNCaP cell lines with the synthetic liver X receptor agonist T0901317 decreased the percentage of S-phase cells in a dose-dependent manner and increased the expression of cyclin-dependent kinase inhibitor p27Kip-1 (p27). Knockdown of p27 by RNA interference blocks T0901317-induced growth inhibition, suggesting that p27 expression plays a crucial role in this signaling. Liver X receptor agonists also inhibited the proliferation of other prostate and breast cancer cell lines. The level of liver X receptor α expression correlated directly with sensitivity to growth inhibition by liver X receptor agonists. Retroviral expression of liver X receptor α in human breast cancer MDA-MB435S cells, which express low levels of endogenous liver X receptors and are insensitive to T0901317, sensitized these cells to T0901317. Consistent with our observations in LNCaP cells, T0901317 induces dramatic up-regulation of p27 in liver X receptor α–overexpressing MDA-MB435S cells. Furthermore, oral administration of T0901317 inhibited the growth of LNCaP tumors in athymic nude mice. Based on these results, modulation of the liver X receptor signaling pathway is a new target for controlling tumor cell proliferation; therefore, liver X receptor agonists may have utility as antitumorigenic agents.
Insulin Regulates Cleavage of Procaspase-9 via Binding of X Chromosome-Linked Inhibitor of Apoptosis Protein in HT-29 Cells
Authors: Kim Ji-Eun , Tannenbaum Steven R.
Published: September 2005

Insulin significantly reduced tumor necrosis factor (TNF)-α–induced cleavage of procaspase-8, -9, and -3 and poly(ADP-ribose) polymerase when observed for up to 24 hours in a dose-dependent manner. Signaling pathways responsible for the inhibitory effects of insulin were investigated by using protein kinase inhibitors. Both phosphatidylinositol 3′-kinase (PI3K) and mitogen-activated protein kinase kinase pathways mediate the ability of insulin to decrease the TNF-α–induced cleavage of procaspase-8. In contrast, only the PI3K inhibitor reversed the effect of insulin on the TNF-α–induced cleavage of procaspase-9. Moreover, insulin decreased the apoptotic level induced by TNF-α, whereas the PI3K inhibitor enhanced it. The protein level of Apaf-1, an activator of procaspase-9, remained constant with the application of agents affecting the cleavage of procaspase-9. In examining another regulator of cleaved caspase-9, X chromosome-linked inhibitor of apoptosis protein (XIAP), we observed that TNF-α treatment induced fragmentation of XIAP, which was also enhanced by the PI3K inhibitor. In addition, XIAP was coimmunoprecipitated with procaspase-9. The treatment with TNF-α reduced the level of XIAP precipitated with procaspase-9, whereas insulin reversed this effect. Moreover, PI3K and Akt inhibitors, but not mammalian target of rapamycin inhibitor, inhibited the effect of insulin on the coprecipitation of procaspase-9 and XIAP. Our data suggest that insulin decreases the TNF-α–induced cleavage of procaspase-9 and subsequent apoptosis by regulating XIAP via the PI3K/Akt pathway.