bluuebunny/crossref_metadata_2025 huggingface dataset:
AKT Regulation of Estrogen Receptor β Transcriptional Activity in Breast Cancer
Authors: Duong Bich N. , Elliott Steven , Frigo Daniel E. , Melnik Lilia I. , Vanhoy Lyndsay , Tomchuck Suzanne , Lebeau Helena P. , David Odile , Beckman Barbara S. , Alam Jawed , Bratton Melyssa R. , McLachlan John A. , Burow Matthew E.
Published: September 2006

Growth factor activation of the phosphatidylinositol 3-kinase (PI3K)-AKT pathway has been shown to activate the estrogen receptor (ER) α and to mediate tamoxifen resistance in breast cancer. Here, we investigated the regulation of the transcriptional activity of the newer ERβ by PI3K-AKT signaling. Tissue arrays of breast cancer specimens showed a positive association between the expressions of AKT and ERβ in the clinical setting. Reporter gene assays using pharmacologic and molecular inhibitors of AKT and constitutively active AKT revealed for the first time the ability of AKT to (a) potentiate ERβ activity and (b) target predominantly the activation function-2 (AF2) domain of the receptor, with a requirement for residue K269. Given the importance of coactivators in ER transcriptional activity, we further investigated the possible involvement of steroid receptor coactivator 1 (SRC1) and glucocorticoid receptor-interacting protein 1 (GRIP1) in AKT regulation of ERβ. Mammalian two-hybrid assays revealed that AKT enhanced both SRC1 and GRIP1 recruitment to the ERβ-AF2 domain, and reporter gene analyses revealed that AKT and GRIP1 cooperatively potentiated ERβ-mediated transcription to a level much greater than either factor alone. Investigations into AKT regulation of GRIP with mammalian one-hybrid assays showed that AKT potentiated the activation domains of GRIP1 itself, and in vitro kinase assays revealed that AKT directly phosphorylated GRIP1. The cross-talk between the PI3K-AKT and ERβ pathways, as revealed by the ability of AKT to regulate several components of ERβ-mediated transcription, may represent an important aspect that may influence breast cancer response to endocrine therapy. (Cancer Res 2006; 66(17): 8373-81)
Analysis of Genetic Alterations and Clonal Proliferation in Children Treated for Acute Lymphocytic Leukemia
Authors: Kendall Heather E. , Vacek Pamela M. , Rivers Jami L. , Rice Sederick C. , Messier Terri L. , Finette Barry A.
Published: September 2006

The development of risk-directed treatment protocols over the last 25 years has resulted in an increase in the survival rates of children treated for cancer. As a consequence, there is a growing population of pediatric cancer survivors in which the long-term genotoxic effects of chemotherapy is unknown. We previously reported that children treated for acute lymphocytic leukemia have significantly elevated somatic mutant frequencies at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene in their peripheral T cells. To understand the molecular etiology of the increase in mutant frequencies following chemotherapy, we investigated the HPRT mutation spectra and the extent of clonal proliferation in 562 HPRT T cell mutant isolates of 87 blood samples from 47 subjects at diagnosis, during chemotherapy, and postchemotherapy. We observed a significant increase in the proportion of CpG transitions following treatment (13.6-23.3%) compared with healthy controls (4.0%) and a significant decrease in V(D)J-mediated deletions following treatment (0-6.8%) compared with healthy controls (17.0%). There was also a significant change in the class type percentage of V(D)J-mediated HPRT deletions following treatment. In addition, there was a >5-fold increase in T cell receptor gene usage–defined mean clonal proliferation from diagnosis compared with the completion of chemotherapeutic intervention. These data indicate that unique genetic alterations and extensive clonal proliferation are occurring in children following treatment for acute lymphocytic leukemia that may influence long-term risks for multifactorial diseases, including secondary cancers. (Cancer Res 2006; 66(17): 8455-61)
Breast Cancer Growth Prevention by Statins
Authors: Campbell Michael J. , Esserman Laura J. , Zhou Yamei , Shoemaker Mark , Lobo Margaret , Borman Elizabeth , Baehner Frederick , Kumar Anjali S. , Adduci Kelly , Marx Corina , Petricoin Emanuel F. , Liotta Lance A. , Winters Mary , Benz Stephen , Benz Christopher C.
Published: September 2006

Statins are cholesterol-lowering drugs with pleiotropic activities including inhibition of isoprenylation reactions and reduction of signals driving cell proliferation and survival responses. The objectives of this study were to examine the effects of statins on breast cancer cells, both in vitro and in vivo, and to begin to determine their mechanism of action. We evaluated the effects of statins on breast cancer cell growth, phosphoprotein signaling intermediates, survival/apoptosis regulators, cell cycle regulators, and activated transcription factors. We also examined the in vivo effect of statin administration in a mouse ErbB2+ breast cancer model. Only lipophilic statins had direct anticancer activity in vitro. Breast cancer cells with activated Ras or ErbB2 pathways seemed to be more sensitive than those overexpressing estrogen receptor, and this correlated with endogenous levels of activated nuclear factor κB (NF-κB). Key intermediates regulating cell survival by NF-κB activation, as well as cell proliferation by the mitogen activated protein kinase cascade, were among the earliest phosphoproteins influenced by statin treatment. These early effects were followed by declines in activator protein-1 and NF-κB activation and concordant changes in other mediators of proliferation and apoptosis. In vivo results showed that oral dosing of statins significantly inhibited the growth of a mouse mammary carcinoma. Lipophilic statins can exert direct anticancer activity in vitro by reducing proliferation and survival signals in susceptible breast cancer phenotypes. Tumor growth inhibition in vivo using a clinically relevant statin dose also seems to be associated with reduced tumor cell proliferation and survival. These findings provide supporting rationale for future statin trials in breast cancer patients. (Cancer Res 2006; 66(17): 8707-13)
Ephrin-B3 Ligand Promotes Glioma Invasion through Activation of Rac1
Authors: Nakada Mitsutoshi , Drake Kelsey L. , Nakada Satoko , Niska Jared A. , Berens Michael E.
Published: September 2006

Eph receptor tyrosine kinases are involved in nervous system development. Eph ligands, termed ephrins, are transmembrane proteins that bind to Eph receptors, the mutual activation of which causes repulsive effects in reciprocally contacting cells. Previously, we showed that overexpression of EphB2 in glioma cells increases cell invasion. Here, expression profiles of ephrin-B family members were determined in four glioma cell lines and in invading glioblastoma cells collected by laser capture microdissection. Ephrin-B3 mRNA was up-regulated in migrating cells of four of four glioma cell lines (1.3- to 1.7-fold) and in invading tumor cells of eight of eight biopsy specimens (1.2- to 10.0-fold). Forced expression of ephrin-B3 in low expressor cell lines (U87, T98G) stimulated cell migration and invasion in vitro and ex vivo, concomitant with tyrosine phosphorylation of ephrin-B3. In high expressor cell lines (U251, SNB19), ephrin-B3 colocalized with Rac1 to lamellipodia of motile wild-type cells. Cells transfected with ephrin-B3 small interfering RNA (siRNA) showed significant morphologic change and decreased invasion in vitro and ex vivo. Depletion of endogenous ephrin-B3 expression abrogated the increase of migration and invasion induced by EphB2/Fc, indicating increased invasion is dependent on ephrin-B3 activation. Furthermore, using a Rac1-GTP pull-down assay, we showed that ephrin-B3 is associated with Rac1 activation. Reduction of Rac1 by siRNA negated the increased invasion by addition of EphB2/Fc. In human glioma specimens, ephrin-B3 expression and phosphorylation correlated with increasing tumor grade. Immunohistochemistry revealed robust staining for phosphorylated ephrin-B and ephrin-B3 in invading glioblastoma cells. These data show that ephrin-B3 expression and signaling through Rac1 are critically important to glioma invasion. (Cancer Res 2006; 66(17): 8492-500)
The Role of the Vascular Endothelial Growth Factor–Delta-like 4 Ligand/Notch4-Ephrin B2 Cascade in Tumor Vessel Remodeling and Endothelial Cell Functions
Authors: Hainaud Patricia , Contrerès Jean-Olivier , Villemain Aude , Liu Lang-Xia , Plouët Jean , Tobelem Gérard , Dupuy Evelyne
Published: September 2006

Vascular endothelial growth factor (VEGF) and Delta-like 4 ligand (DLL4) are the only genes whose haploinsufficiency results in vascular abnormalities. Although many common pathways are up-regulated in both vascular development and tumor angiogenesis and in vascular remodeling, the role of the Delta/Notch pathway has not been clearly defined in tumor angiogenesis. In this study, we assessed the expression of DLL4, Notch4, and ephrin B2 in transgenic mice developing hepatocarcinoma characterized by a strong remodeling of the tumor sinusoids. We also investigated the role of VEGF in the expression and biological functions of these molecules on human venous endothelial cells. In transgenic livers, we showed that DLL4, active Notch4, and ephrin B2 were gradually up-regulated within the hepatocarcinoma progression and expressed on tumor sinusoidal endothelial cells. In venous endothelial cells, we showed that VEGF up-regulates DLL4 and presenilin, and increased the activation of Notch4, leading to an up-regulation of ephrin B2 with a down-regulation of Eph B4. We also showed that the activation of Notch4 is required for VEGF-induced up-regulation of ephrin B2 and the differentiation of human venous endothelial cells in vitro. Accordingly, the disruption of Notch4 signaling by pharmacologic inhibition of presenilin or addition of soluble DLL4 inhibited the effect of VEGF on human venous endothelial cell migration and differentiation. Our study strongly suggests that a coordinated activation of DDL4/Notch4 and ephrin B2 pathways downstream of VEGF plays a key role in the abnormal remodeling of tumor vessels. (Cancer Res 2006; 66(17): 8501-10)
Calreticulin, a Molecular Chaperone in the Endoplasmic Reticulum, Modulates Radiosensitivity of Human Glioblastoma U251MG Cells
Authors: Okunaga Tomohiro , Urata Yoshishige , Goto Shinji , Matsuo Takayuki , Mizota Shingo , Tsutsumi Keisuke , Nagata Izumi , Kondo Takahito , Ihara Yoshito
Published: September 2006

Radiotherapy is the primary and most important adjuvant therapy for malignant gliomas. Although the mechanism of radiation resistance in gliomas has been studied for decades, it is still not clear how the resistance is related with functions of molecular chaperones in the endoplasmic reticulum. Calreticulin (CRT) is a Ca2+-binding molecular chaperone in the endoplasmic reticulum. Recently, it was reported that changes in intracellular Ca2+ homeostasis play a role in the modulation of apoptosis. In the present study, we found that the level of CRT was higher in neuroglioma H4 cells than in glioblastoma cells (U251MG and T98G), and was well correlated with the sensitivity to γ-irradiation. To examine the role of CRT in the radiosensitivity of malignant gliomas, the CRT gene was introduced into U251MG cells, which express low levels of CRT, and the effect of overexpression of CRT on the radiosensitivity was examined. The cells transfected with the CRT gene exhibited enhanced radiation-induced apoptosis compared with untransfected control cells. In CRT-overexpressing cells, cell survival signaling via Akt was markedly suppressed. Furthermore, the gene expression of protein phosphatase 2Acα (PP2Acα), which is responsible for the dephosphorylation and inactivation of Akt, was up-regulated in CRT-overexpressing cells, and the regulation was dependent on Ca2+. Thus, overexpression of CRT modulates radiation-induced apoptosis by suppressing Akt signaling through the up-regulation of PP2Acα expression via altered Ca2+ homeostasis. These results show the novel mechanism by which CRT is involved in the regulation of radiosensitivity and radiation-induced apoptosis in malignant glioma cells. (Cancer Res 2006; 66(17): 8662-71)
Ionizing Radiation Enhances Matrix Metalloproteinase-2 Secretion and Invasion of Glioma Cells through Src/Epidermal Growth Factor Receptor–Mediated p38/Akt and Phosphatidylinositol 3-Kinase/Akt Signaling Pathways
Authors: Park Chang-Min , Park Myung-Jin , Kwak Hee-Jin , Lee Hyung-Chahn , Kim Mi-Suk , Lee Seung-Hoon , Park In-Chul , Rhee Chang Hun , Hong Seok-Il
Published: September 2006

Glioblastoma is a severe type of primary brain tumor, and its highly invasive character is considered to be a major therapeutic obstacle. Several recent studies have reported that ionizing radiation (IR) enhances the invasion of tumor cells, but the mechanisms for this effect are not well understood. In this study, we investigated the possible signaling mechanisms involved in IR-induced invasion of glioma cells. IR increased the matrix metalloproteinase (MMP)-2 promoter activity, mRNA transcription, and protein secretion along with the invasiveness of glioma cells lacking functional PTEN (U87, U251, U373, and C6) but not those harboring wild-type (WT)-PTEN (LN18 and LN428). IR activated phosphatidylinositol 3-kinase (PI3K), Akt, and mammalian target of rapamycin, and blockade of these kinases by specific inhibitors (LY294002, Akt inhibitor IV, and rapamycin, respectively) and transfection of dominant-negative (DN) mutants (DN-p85 and DN-Akt) or WT-PTEN suppressed the IR-induced MMP-2 secretion in U251 and U373 cells. In addition, inhibitors of epidermal growth factor receptor (EGFR; AG490 and AG1478), Src (PP2), and p38 (SB203580), EGFR neutralizing antibody, and transfection of DN-Src and DN-p38 significantly blocked IR-induced Akt phosphorylation and MMP-2 secretion. IR-induced activation of EGFR was suppressed by PP2, whereas LY294002 and SB203580 did not affect the activations of p38 and PI3K, respectively. Finally, these kinase inhibitors significantly reduced the IR-induced invasiveness of these cells on Matrigel. Taken together, our findings suggest that IR induces Src-dependent EGFR activation, which triggers the p38/Akt and PI3K/Akt signaling pathways, leading to increased MMP-2 expression and heightened invasiveness of PTEN mutant glioma cells. (Cancer Res 2006; 66(17): 8511-9)
Inducible Resistance of Tumor Cells to Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand Receptor 2–Mediated Apoptosis by Generation of a Blockade at the Death Domain Function
Authors: Li Yang , Wang Hong , Wang Zheng , Makhija Sharmila , Buchsbaum Donald , LoBuglio Albert , Kimberly Robert , Zhou Tong
Published: September 2006

Induction of tumor cell resistance to therapeutics has been a major obstacle in cancer therapy. Targeting of the death receptors by a natural ligand, tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), or agonistic monoclonal antibodies against TRAIL receptor 1 (TRAIL-R1) or TRAIL receptor 2 (TRAIL-R2) has been thought to be a promising cancer therapy. To determine whether tumor cells are able to generate a resistance to apoptosis induced by an anti-TRAIL-R2 antibody, TRA-8, we examined the apoptotic response of human breast and ovarian cancer cell lines after treatment with TRA-8. Our results show that tumor cell resistance to TRA-8 can be induced by repeated treatment of tumor cells with low, non-apoptosis-inducing doses of TRA-8. Interestingly, the induced resistance to apoptosis was not due to a global apoptotic defect in tumor cells but rather a selective defect in the TRAIL-R2 signaling pathway. Whereas TRA-8-treated tumor cells developed a selective resistance to TRAIL-R2-mediated apoptosis, the apoptotic responses induced by TRAIL, an anti-TRAIL-R1 antibody (2E12), and other apoptotic stimuli were not impaired. The expression levels of cell surface TRAIL-R2 were not altered and mutations of TRAIL-R2 were not found in the resistant cells. The induced TRA-8 resistance was due to a selective blockade at the level of the death domain and could be reversed by a wide array of chemotherapeutic agents. Proteomic analysis of death-inducing signaling complex formation during TRA-8 treatment shows that the translocation of TRAIL-R2-associated apoptotic proteins was significantly altered. Our results suggest that the prevention of tumor cell resistance to therapeutic agents that target the death receptors must be taken into consideration. (Cancer Res 2006; 66(17): 8520-8)
Prognostic Significance of Nuclear Factor-κB p105/p50 in Human Melanoma and Its Role in Cell Migration
Authors: Gao Kai , Dai Derek L. , Martinka Magdalena , Li Gang
Published: September 2006

Transcriptional factor nuclear factor-κB (NF-κB) family has been shown to play an important role in tumor pathogenesis and serve as a potential target in cancer therapy. However, it is necessary to clarify the specific functions of NF-κB members, which would provide the basis for the selective blockade and reduction of therapeutic side effects resulting from unspecific inhibition of NF-κB members. In this study, we explored the role of NF-κB p105/p50 in melanoma pathogenesis in vitro and in vivo. We found that the expression of NF-κB p105/p50 significantly increased in dysplastic nevi, primary melanoma, and metastatic melanoma compared with normal nevi (P = 0.0004, χ2 test). Furthermore, NF-κB p105/p50 nuclear staining increased with melanoma progression and strong NF-κB p105/p50 nuclear staining was inversely correlated with disease-specific 5-year survival of patients with tumor thickness >2.0 mm (P = 0.014, log-rank test). Multivariate Cox regression analysis revealed that nuclear expression of NF-κB p105/p50 is an independent prognostic factor in this subgroup. Moreover, we found that up-regulation of NF-κB p50 enhanced melanoma cell migration, whereas small interfering RNA knockdown inhibited cell migration. In addition, overexpression of NF-κB p50 induced RhoA activity and Rock-mediated formation of stress fiber in melanoma cells. Taken together, our data indicate that NF-κB p105/p50 may be an important marker for human melanoma progression and prognosis as well as a potentially selective therapeutic target. (Cancer Res 2006; 66(17): 8382-8)
Chk1- and Claspin-Dependent but ATR/ATM– and Rad17-Independent DNA Replication Checkpoint Response in HeLa Cells
Authors: Rodríguez-Bravo Verónica , Guaita-Esteruelas Sandra , Florensa Roger , Bachs Oriol , Agell Neus
Published: September 2006

When DNA synthesis is inhibited, DNA replication checkpoint is activated to prevent mitosis entry without fully replicated DNA. In Xenopus, caffeine-sensitive kinases [ataxia telangiectasia mutated (ATM) and ATM-related protein (ATR)] are essential in this checkpoint response, but in mammal cells an ATR/ATM–independent checkpoint response to DNA synthesis inhibition exists. Using HeLa cells, which have a caffeine-insensitive checkpoint response, we have analyzed here which molecules known to be involved in the DNA replication checkpoint participate in the caffeine-insensitive response. When DNA synthesis was inhibited in the presence of UCN01 or after knocking down Chk1 expression [Chk1 small interfering RNA (siRNA)], HeLa cells entered into aberrant mitosis. Consequently, Chk1 is essential for both the ATR/ATM–dependent and ATR/ATM–independent checkpoint response in HeLa cells. Neither wortmannin, Ly294002, nor SB202190 abrogated the caffeine-insensitive checkpoint response, indicating that DNA-PK and p38α,β are not involved in the ATR/ATM–independent Chk1 activation upon DNA synthesis inhibition. Using siRNA to knock down Rad17 and claspin, two molecules involved in sensing stalled replication forks, we also showed that claspin but not Rad17 is essential for the ATR/ATM–independent checkpoint response. Inhibition of DNA synthesis in HeLa cells led to a decrease in cyclin B1 protein accumulation that was abrogated when UCN01 was added or when claspin was knocked down. We conclude that upon DNA synthesis inhibition, Chk1 can be activated in a claspin-dependent manner independently of ATR and ATM, leading to cyclin B1 down-regulation and providing the cells of an additional mechanism to inhibit mitosis entry. (Cancer Res 2006; 66(17): 8672-9)
Class II Histone Deacetylases Are Associated with VHL-Independent Regulation of Hypoxia-Inducible Factor 1α
Authors: Qian David Z. , Kachhap Sushant K. , Collis Spencer J. , Verheul Henk M.W. , Carducci Michael A. , Atadja Peter , Pili Roberto
Published: September 2006

Hypoxia-inducible factor 1α (HIF-1α) plays a critical role in transcriptional gene activation involved in tumor angiogenesis. A novel class of agents, the histone deacetylase (HDAC) inhibitors, has been shown to inhibit tumor angiogenesis and HIF-1α protein expression. However, the molecular mechanism responsible for this inhibition remains to be elucidated. In the current study, we investigated the molecular link between HIF-1α inhibition and HDAC inhibition. Treatment of the VHL-deficient human renal cell carcinoma cell line UMRC2 with the hydroxamic HDAC inhibitor LAQ824 resulted in a dose-dependent inhibition of HIF-1α protein via a VHL-independent mechanism and reduction of HIF-1α transcriptional activity. HIF-1α inhibition by LAQ824 was associated with HIF-1α acetylation and polyubiquitination. HIF-1α immunoprecipitates contained HDAC activity. Then, we tested different classes of HDAC inhibitors with diverse inhibitory activity of class I versus class II HDACs and assessed their capability of targeting HIF-1α. Hydroxamic acid derivatives with known activity against both class I and class II HDACs were effective in inhibiting HIF-1α at low nanomolar concentrations. In contrast, valproic acid and trapoxin were able to inhibit HIF-1α only at concentrations that are effective against class II HDACs. Coimmunoprecipitation studies showed that class II HDAC4 and HDAC6 were associated with HIF-1α protein. Inhibition by small interfering RNA of HDAC4 and HDAC6 reduced HIF-1α protein expression and transcriptional activity. Taken together, these results suggest that class II HDACs are associated with HIF-1α stability and provide a rationale for targeting HIF-1α with HDAC inhibitors against class II isozymes. (Cancer Res 2006; 66(17): 8814-21)
Hyperplasia and Carcinomas in Pten-Deficient Mice and Reduced PTEN Protein in Human Bladder Cancer Patients
Authors: Tsuruta Hiroshi , Kishimoto Hiroyuki , Sasaki Takehiko , Horie Yasuo , Natsui Miyuki , Shibata Yoshiko , Hamada Koichi , Yajima Nobuyuki , Kawahara Koichi , Sasaki Masato , Tsuchiya Norihiko , Enomoto Katsuhiko , Mak Tak Wah , Nakano Toru , Habuchi Tomonori , Suzuki Akira
Published: September 2006

PTEN is a tumor suppressor gene mutated in many human cancers. We used the Cre-loxP system to generate an urothelium-specific null mutation of Pten in mice [FabpCrePtenflox/flox (FPtenflox/flox) mice]. Histologic examination revealed that all FPtenflox/flox mice exhibited urothelial hyperplasia in which component cells showed enlarged nuclei and increased cell size. With time, 10% of FPtenflox/flox mice spontaneously developed pedicellate papillary transitional cell carcinomas (TCC). This type of tumor also arose in FPtenflox/flox mice treated with the chemical carcinogen N-butyl-N-(4-hydroxybutyl) nitrosamine. FPtenflox/flox urothelial cells were hyperproliferative and showed increased activation of the survival signaling molecules Akt and extracellular signal-regulated kinase. In humans, 53% of primary bladder cancer patients exhibited decreased or absent expression of PTEN protein in either the cytoplasm or nucleus of tumor cells. In early bladder cancers, PTEN expression was repressed in 42% of superficial papillary TCC but in only 8% of cases of carcinoma in situ (CIS). In advanced bladder cancers, PTEN protein was significantly reduced (particularly in the nucleus) in 94% of cases, and this decrease in PTEN correlated with disease stage and grade. Thus, PTEN deficiency may contribute to bladder cancer both by initiating superficial papillary TCC and by promoting the progression of CIS to advanced invasive and metastatic forms. (Cancer Res 2006; 66(17): 8389-95)
AMG 706, an Oral, Multikinase Inhibitor that Selectively Targets Vascular Endothelial Growth Factor, Platelet-Derived Growth Factor, and Kit Receptors, Potently Inhibits Angiogenesis and Induces Regression in Tumor Xenografts
Authors: Polverino Anthony , Coxon Angela , Starnes Charlie , Diaz Zobedia , DeMelfi Thomas , Wang Ling , Bready James , Estrada Juan , Cattley Russell , Kaufman Stephen , Chen Danlin , Gan Yongmei , Kumar Gondi , Meyer James , Neervannan Sesha , Alva Gonzalo , Talvenheimo Jane , Montestruque Silvia , Tasker Andrew , Patel Vinod , Radinsky Robert , Kendall Richard
Published: September 2006

The growth of solid tumors is dependent on the continued stimulation of endothelial cell proliferation and migration resulting in angiogenesis. The angiogenic process is controlled by a variety of factors of which the vascular endothelial growth factor (VEGF) pathway and its receptors play a pivotal role. Small-molecule inhibitors of VEGF receptors (VEGFR) have been shown to inhibit angiogenesis and tumor growth in preclinical models and in clinical trials. A novel nicotinamide, AMG 706, was identified as a potent, orally bioavailable inhibitor of the VEGFR1/Flt1, VEGFR2/kinase domain receptor/Flk-1, VEGFR3/Flt4, platelet-derived growth factor receptor, and Kit receptors in preclinical models. AMG 706 inhibited human endothelial cell proliferation induced by VEGF, but not by basic fibroblast growth factor in vitro, as well as vascular permeability induced by VEGF in mice. Oral administration of AMG 706 potently inhibited VEGF-induced angiogenesis in the rat corneal model and induced regression of established A431 xenografts. AMG 706 was well tolerated and had no significant effects on body weight or on the general health of the animals. Histologic analysis of tumor xenografts from AMG 706–treated animals revealed an increase in endothelial apoptosis and a reduction in blood vessel area that preceded an increase in tumor cell apoptosis. In summary, AMG 706 is an orally bioavailable, well-tolerated multikinase inhibitor that is presently under clinical investigation for the treatment of human malignancies. (Cancer Res 2006; 66(17): 8715-21)
Multiple Endocrine Neoplasia Type 1 Interacts with Forkhead Transcription Factor CHES1 in DNA Damage Response
Authors: Busygina Valeria , Kottemann Molly C. , Scott Kenneth L. , Plon Sharon E. , Bale Allen E.
Published: September 2006

Multiple endocrine neoplasia type 1 (MEN1) is a cancer susceptibility syndrome affecting several endocrine tissues. Investigations of the biochemical function of the MEN1 protein, menin, have suggested a role as a transcriptional comodulator. The mechanism by which MEN1 inactivation leads to tumor formation is not fully understood. MEN1 was implicated to function in both regulation of cell proliferation and maintenance of genomic integrity. Here, we investigate the mechanism by which MEN1 affects DNA damage response. We found that Drosophila larval tissue and mouse embryonic fibroblasts mutant for the MEN1 homologue were deficient for a DNA damage-activated S-phase checkpoint. The forkhead transcription factor CHES1 (FOXN3) was identified as an interacting protein by a genetic screen, and overexpression of CHES1 restored both cell cycle arrest and viability of MEN1 mutant flies after ionizing radiation exposure. We showed a biochemical interaction between human menin and CHES1 and showed that the COOH terminus of menin, which is frequently mutated in MEN1 patients, is necessary for this interaction. Our data indicate that menin is involved in the activation of S-phase arrest in response to ionizing radiation. CHES1 is a component of a transcriptional repressor complex, that includes mSin3a, histone deacetylase (HDAC) 1, and HDAC2, and it interacts with menin in an S-phase checkpoint pathway related to DNA damage response. (Cancer Res 2006; 66(17): 8397-402)
p27Kip1 Repression of ErbB2-Induced Mammary Tumor Growth in Transgenic Mice Involves Skp2 and Wnt/β-Catenin Signaling
Authors: Hulit James , Lee Richard J. , Li Zhiping , Wang Chenguang , Katiyar Sanjay , Yang Jianguo , Quong Andrew A. , Wu Kongming , Albanese Chris , Russell Robert , Di Vizio Dolores , Koff Andrew , Thummala Saiinder , Zhang Hui , Harrell Jennifer , Sun Hong , Muller William J. , Inghirami Giorgio , Lisanti Michael P. , Pestell Richard G.
Published: September 2006

Expression of the cyclin-dependent kinase (Cdk) inhibitor (p27Kip1) is frequently reduced in human tumors, often correlating with poor prognosis. p27Kip1 functions as a haploinsufficient tumor suppressor; however, the mechanism by which one allele of p27Kip1 regulates oncogenic signaling in vivo is not well understood. We therefore investigated the mechanisms by which p27Kip1 inhibits mammary tumor onset. Using the common background strain of FVB, p27Kip1 heterozygosity (p27+/−) accelerated ErbB2-induced mammary tumorigenesis. We conducted microarray analyses of mammary tumors developing in mice with genetic haploinsufficiency for p27Kip1 expressing a mammary-targeted ErbB2 oncogene. Global gene expression profiling and Western blot analysis of ErbB2/p27+/− tumors showed that the loss of p27Kip1 induced genes promoting lymphangiogenesis, cellular proliferation, and collaborative oncogenic signaling (Wnt/β-catenin/Tcf, Cdc25a, Smad7, and Skp2). Skp2 expression was induced by ErbB2 and repressed by p27Kip1. Degradation of p27Kip1 involves an SCF-type E3 ubiquitin ligase, including Skp2. The Skp2 component of the SCFSKP2 complex that degrades p27Kip1 was increased in ErbB2 tumors correlating with earlier tumor onset. In both murine and human ErbB2-overexpressing breast cancers, p27Kip1 levels correlated inversely with Skp2. p27Kip1 haploinsufficiency activated Wnt/β-catenin/hedgehog signaling. Reintroduction of p27Kip1 inhibited β-catenin induction of Tcf-responsive genes (Siamosis, c-Myc, and Smad7). p27Kip1 is haploinsufficient for ErbB2 mammary tumor suppression in vivo and functions to repress collaborative oncogenic signals including Skp2 and Wnt/β-catenin signaling. (Cancer Res 2006; 66(17): 8529-41)
Chromosomal Instability Correlates with Genome-wide DNA Demethylation in Human Primary Colorectal Cancers
Authors: Rodriguez Jairo , Frigola Jordi , Vendrell Elisenda , Risques Rosa-Ana , Fraga Mario F. , Morales Cristina , Moreno Victor , Esteller Manel , Capellà Gabriel , Ribas Maria , Peinado Miguel A.
Published: September 2006

DNA hypomethylation is a common trait of colorectal cancer. Studies in tumor cell lines and animal models indicate that genome-wide demethylation may cause genetic instability and hence facilitate or accelerate tumor progression. Recent studies have shown that DNA hypomethylation precedes genomic damage in human gastrointestinal cancer, but the nature of this damage has not been clearly established. Here, we show a thorough analysis of DNA methylation and genetic alterations in two series of colorectal carcinomas. The extent of DNA demethylation but not of hypermethylation (both analyzed by amplification of intermethylated sites in near 200 independent sequences arbitrarily selected) correlated with the cumulated genomic damage assessed by two different techniques (arbitrarily primed PCR and comparative genomic hybridization). DNA hypomethylation–related instability was mainly of chromosomal nature and could be explained by a genome-wide effect rather than by the concurrence of the most prevalent genetic and epigenetic alterations. Moreover, the association of p53 mutations with genomic instability was secondary to DNA hypomethylation and the correlation between DNA hypomethylation and genomic instability was observed in tumors with and without mutation in the p53 gene. Our data support a direct link between genome-wide demethylation and chromosomal instability in human colorectal carcinogenesis and are consistent with the studies in model systems demonstrating a role of DNA demethylation in inducing chromosomal instability. (Cancer Res 2006; 66(17): 8462-8)
Human β-Defensin-1, a Potential Chromosome 8p Tumor Suppressor: Control of Transcription and Induction of Apoptosis in Renal Cell Carcinoma
Authors: Sun Carrie Q. , Arnold Rebecca , Fernandez-Golarz Carina , Parrish Amanda B. , Almekinder Tara , He Ju , Ho Shuk-mei , Svoboda Pavel , Pohl Jan , Marshall Fray F. , Petros John A.
Published: September 2006

Human β-defensin-1 (hBD-1) is a candidate tumor suppressor gene located on chromosome 8p23. Previously, we showed that cancer-specific loss of hBD-1 was found in 90% of renal clear cell carcinomas and in 82% of prostate cancers. To investigate the possible mechanisms of decreased gene expression and determine the function of hBD-1 protein in urological cancers, we sequenced hBD-1 gene coding regions in prostatic and renal cancer samples. We then analyzed the frequency distribution of promoter polymorphisms and determined the effect of these base changes on transcriptional activity of the hBD-1 promoter. A polymorphism at −688 bases upstream of the ATG start codon affects hBD-1 promoter activity, leading to a rate of reporter gene transcription that is 40% to 50% lower than the wild-type sequence when tested in either DU145 or TSU-Pr1 cell lines. In addition, a polymorphism at −44 bases was shown to enhance transcription up to 2.3 times more than the wild-type sequence in the same cell lines. In addition, three novel hBD-1 promoter mutations were found in renal and prostate cancer clinical samples. An iso-5-aza-2′-deoxycytidine treatment was effective in transcription up-regulation in DU145, suggesting a possible upstream methylation-dependent effect. Synthetic hBD-1 peptide inhibited bladder cancer cell TSU-Pr1 proliferation. Overexpression of the hBD-1 gene in renal cancer cells SW156 resulted in caspase-3-mediated apoptosis. These data support the hypothesis that hBD-1 is a potential tumor suppressor gene for urological cancers. Promoter point mutations may be responsible for cancer-specific loss of hDB-1 expression. (Cancer Res 2006; 66(17): 8542-9)
A Small-Molecule Inhibitor of Bcl-XL Potentiates the Activity of Cytotoxic DrugsIn vitroandIn vivo
Authors: Shoemaker Alex R. , Oleksijew Anatol , Bauch Joy , Belli Barbara A. , Borre Tony , Bruncko Milan , Deckwirth Thomas , Frost David J. , Jarvis Ken , Joseph Mary K. , Marsh Kennan , McClellan William , Nellans Hugh , Ng ShiChung , Nimmer Paul , O'Connor Jacqueline M. , Oltersdorf Tilman , Qing Weiguo , Shen Wang , Stavropoulos Jason , Tahir Stephen K. , Wang Baole , Warner Robert , Zhang Haichao , Fesik Stephen W. , Rosenberg Saul H. , Elmore Steven W.
Published: September 2006

Inhibition of the prosurvival members of the Bcl-2 family of proteins represents an attractive strategy for the treatment of cancer. We have previously reported the activity of ABT-737, a potent inhibitor of Bcl-2, Bcl-XL, and Bcl-w, which exhibits monotherapy efficacy in xenograft models of small-cell lung cancer and lymphoma and potentiates the activity of numerous cytotoxic agents. Here we describe the biological activity of A-385358, a small molecule with relative selectivity for binding to Bcl-XL versus Bcl-2 (Ki's of 0.80 and 67 nmol/L for Bcl-XL and Bcl-2, respectively). This compound efficiently enters cells and co-localizes with the mitochondrial membrane. Although A-385358 shows relatively modest single-agent cytotoxic activity against most tumor cell lines, it has an EC50 of <500 nmol/L in cells dependent on Bcl-XL for survival. In addition, A-385358 enhances the in vitro cytotoxic activity of numerous chemotherapeutic agents (paclitaxel, etoposide, cisplatin, and doxorubicin) in several tumor cell lines. In A549 non–small-cell lung cancer cells, A-385358 potentiates the activity of paclitaxel by as much as 25-fold. Importantly, A-385358 also potentiated the activity of paclitaxel in vivo. Significant inhibition of tumor growth was observed when A-385358 was added to maximally tolerated or half maximally tolerated doses of paclitaxel in the A549 xenograft model. In tumors, the combination therapy also resulted in a significant increase in mitotic arrest followed by apoptosis relative to paclitaxel monotherapy. (Cancer Res 2006; 66(17): 8731-9)
Reassessing Epithelial to Mesenchymal Transition as a Prerequisite for Carcinoma Invasion and Metastasis
Authors: Christiansen Jason J. , Rajasekaran Ayyappan K.
Published: September 2006

For most carcinomas, progression toward malignancy is accompanied by loss of epithelial differentiation and a shift towards a mesenchymal phenotype. This process, referred to as epithelial to mesenchymal transition (EMT), exacerbates motility and invasiveness of many cell types and is often considered a prerequisite for tumor infiltration and metastasis. However, there are numerous examples of advanced carcinomas that adopt some mesenchymal features, yet retain characteristics of well-differentiated epithelial cells. We provide a review of these reports and describe mechanisms to explain the morphologic and molecular heterogeneity and plasticity of malignant carcinoma cells, including incomplete EMT, reversion to an epithelial phenotype, and collective migration. We suggest that these mechanisms can manifest in a series of independent and reversible steps and that EMT represents just one mechanism in the global metastatic carcinoma development process. (Cancer Res 2006; 66(17): 8319-26)
A Carbohydrate Recognition–Based Drug Delivery and Controlled Release System using Intraperitoneal Macrophages as a Cellular Vehicle
Authors: Ikehara Yuzuru , Niwa Toru , Biao Le , Ikehara Sanae Kabata , Ohashi Norifumi , Kobayashi Takeshi , Shimizu Yoshitaka , Kojima Naoya , Nakanishi Hayao
Published: September 2006

The lymphoid tissue in the omentum, at the so-called milky spots, is known as an initial place for disseminated cancer cells to develop into solid tumors. In the present study, i.p. macrophages significantly took up oligomannose-coated liposomes (OMLs) that were injected into the peritoneal cavity, and then gradually accumulated in the omentum and the other lymphoid tissues within 24 hours of i.p. injection of OMLs. When 5-fluorouracil (5-FU) was encapsulated in the OMLs, >60% of administered 5-FU accumulated in the omentum. Treatment of macrophages at 39°C for 30 minutes led to the release of 5-FU from the macrophages, suggesting that controlled release from macrophages could be achieved by mild hyperthermia. We encased magnetic nanoparticles, which are known to convert electromagnetic energy to heat in the OMLs to achieve in vivo hyperthermia at the site. Using this system in a mouse i.p. metastasis model, we successfully controlled tumor development by coadministration of OML-encased 5-FU and OML-encased magnetic nanoparticles, followed by treatment with an alternating magnetic field. No apparent reduction was seen in tumor growth with the administration of OML-encased magnetic nanoparticles or OML-encased 5-FU alone. Thus, we have established the use of i.p. macrophages as a novel drug delivery system for the control of cancer metastatic to milky spots. (Cancer Res 2006; 66(17): 8740-8)
Tumor Cell Radiosensitivity Is a Major Determinant of Tumor Response to Radiation
Authors: Gerweck Leo E. , Vijayappa Shashirekha , Kurimasa Akihiro , Ogawa Kazuhiko , Chen David J.
Published: September 2006

Substantial evidence suggests that the radiosensitivity of the tumor cells is the primary determinant of tumor response to radiation. More recent studies suggest that tumor stroma radiosensitivity is the principle determinant of response. To assess the relationship between intrinsic tumor cell radiosensitivity and tumor response, we altered the intrinsic radiosensitivity of a cloned tumor cell line and analyzed the effect of this alteration on tumor response. A cloned tumor cell line derived from DNA double-strand break repair–deficient severe combined immunodeficient mice was transfected with the double-strand break repair gene DNA-PKcs. The intrinsic radiosensitivity of the transfected tumor line was decreased by a factor of ∼1.5. The isogenic lines were used to initiate tumors in NCr-nu/nu mice. When transplanted in the same strain of mice and exposed to the same dose of radiation, the isogenic tumors may be expected to exhibit a similar response to radiation if radiation damage to host stroma is the principle determinant of response. This was not observed. Over the dose range of 20 Gy in four 5-Gy fractions to a single dose of 30 Gy, the 1.5-fold increase in intrinsic tumor cell radioresistance conferred by the introduction of DNA-PKcs caused a 1.5-fold decrease in tumor growth delay. The results show that the intrinsic radiosensitivity of tumor cells is a major determinant of tumor response to radiation. (Cancer Res 2006; 66(17): 8352-5)
Fluorodeoxyuridine Modulates Cellular Expression of the DNA Base Excision Repair Enzyme Uracil-DNA Glycosylase
Authors: Fischer Jennifer A. , Muller-Weeks Susan , Caradonna Salvatore J.
Published: September 2006

The thymidylate synthase inhibitor 5-fluorouracil (5-FU) continues to play a pivotal role in the treatment of cancer. A downstream event of thymidylate synthase inhibition involves the induction of a self-defeating base excision repair process. With the depletion of TTP pools, there is also an increase in dUMP. Metabolism of dUMP to the triphosphate dUTP results in elevated pools of this atypical precursor for DNA synthesis. Under these conditions, there is a destructive cycle of dUMP incorporation into DNA, removal of uracil by the base excision repair enzyme uracil-DNA glycosylase (UDG), and reincorporation of dUMP during the synthesis phase of DNA repair. The end point is DNA strand breaks and loss of DNA integrity, which contributes to cell death. Evidence presented here indicates that both the nuclear and the mitochondrial isoforms of UDG are modulated by FdUrd (and 5-FU) treatment in certain cell lines but not in others. Modulation occurs at the transcriptional and post-translational levels. Under normal conditions, nUDG protein appears in G1 and is degraded during the S to G2 phase transition. The present study provides evidence that, in certain cell lines, FdUrd mediates an atypical turnover of nUDG. Additional data indicate that, for cell lines that do not down-regulate nUDG, small interfering RNA–mediated knockdown of nUDG significantly increases resistance to the cytotoxic effects of FdUrd. Results from these studies show that nUDG is an additional determinant in FdUrd-mediated cytotoxicity and bolster the notion that the self-defeating base excision repair pathway, instigated by elevated dUTP (FdUTP) pools, contributes to the cytotoxic consequences of 5-FU chemotherapy. (Cancer Res 2006; 66(17): 8829-37)
E6AP Mediates Regulated Proteasomal Degradation of the Nuclear Receptor Coactivator Amplified in Breast Cancer 1 in Immortalized Cells
Authors: Mani Aparna , Oh Annabell S. , Bowden Emma T. , Lahusen Tyler , Lorick Kevin L. , Weissman Allan M. , Schlegel Richard , Wellstein Anton , Riegel Anna T.
Published: September 2006

The steroid receptor coactivator oncogene, amplified in breast cancer 1 (AIB1; also known as ACTR/RAC-3/TRAM-1/SRC-3/p/CIP), is amplified and overexpressed in a variety of epithelial tumors. AIB1 has been reported to have roles in both steroid-dependent and steroid-independent transcription during tumor progression. In this report, we describe that the cellular levels of AIB1 are controlled through regulated proteasomal degradation. We found that serum withdrawal or growth in high cell density caused rapid degradation of AIB1 protein, but not mRNA, in immortalized cell lines. Proteasome inhibitors prevented this process, and high molecular weight ubiquitylated species of AIB1 were detected. Nuclear export was required for proteasomal degradation of AIB1 and involved the ubiquitin ligase, E6AP. AIB1/E6AP complexes were detected in cellular extracts, and reduction of cellular E6AP levels with E6AP short interfering RNA prevented proteasomal degradation of AIB1. Conversely, overexpression of E6AP promoted AIB1 degradation. The COOH terminus of AIB1 interacted with E6AP in vitro and deletion of this region in AIB1 rendered it resistant to degradation in cells. From our results, we propose a model whereby signals promoted by changes in the cellular milieu initiate E6AP-mediated proteasomal degradation of AIB1 and thus contribute to the control of steady-state levels of this protein. (Cancer Res 2006; 66(17): 8680-6)
Agonist and Antagonist of Retinoic Acid Receptors Cause Similar Changes in Gene Expression and Induce Senescence-like Growth Arrest in MCF-7 Breast Carcinoma Cells
Authors: Chen Yuhong , Dokmanovic Milos , Stein Wilfred D. , Ardecky Robert J. , Roninson Igor B.
Published: September 2006

Biological effects of retinoids are mediated via retinoic acid (RA) receptors (RAR) and retinoid X receptors (RXR). The best-characterized mechanism of retinoid action is stimulation of transcription from promoters containing RA response elements (RARE). Retinoids induce senescence-like growth arrest in MCF-7 breast carcinoma cells; this effect is associated with the induction of several growth-inhibitory genes. We have now found that these genes are induced by RAR-specific but not by RXR-specific ligands. Genome-scale microarray analysis of gene expression was used to compare the effects of two pan-RAR ligands, one of which is a strong agonist of RARE-dependent transcription, whereas the other induces such transcription only weakly and antagonizes the inducing effect of RAR agonists. Both RAR ligands, however, produced very similar effects on gene expression in MCF-7 cells, suggesting that RARE-dependent transcription is only a minor component of retinoid-induced changes in gene expression. The effects of RAR ligands on gene expression parallel changes associated with damage-induced senescence, and both ligands induced G1 arrest and the senescent phenotype in MCF-7 cells. The RAR ligands up-regulated many tumor-suppressive genes and down-regulated multiple genes with oncogenic activities. Genes that are strongly induced by RAR ligands encode secreted bioactive proteins, including several tumor-suppressing factors. In agreement with these observations, retinoid-treated MCF-7 cells inhibited the growth of retinoid-insensitive MDA-MB-231 breast carcinoma cells in coculture. These results indicate that RARE-independent transcriptional effects of RAR ligands lead to senescence-like growth arrest and paracrine growth-inhibitory activity in MCF-7 breast carcinoma cells. (Cancer Res 2006; 66(17): 8749-61)