bluuebunny/crossref_metadata_2025 huggingface dataset:
Data from Signal Transducers and Activators of Transcription Mediate Fibroblast Growth Factor–Induced Vascular Endothelial Morphogenesis
Authors: Yang Xinhai , Qiao Dianhua , Meyer Kristy , Friedl Andreas
Published: March 2023

Abstract
The fibroblast growth factors (FGF) play diverse roles in development, wound healing, and angiogenesis. The intracellular signal transduction pathways, which mediate these pleiotropic activities, remain incompletely understood. We show here that the proangiogenic factors FGF2 and FGF8b can activate signal transducers and activators of transcription (STAT) in mouse microvascular endothelial cells (EC). Both FGF2 and FGF8b activate STAT5 and to a lesser extent STAT1, but not STAT3. The FGF2-dependent activation of endothelial STAT5 was confirmed in vivo with the Matrigel plug angiogenesis assay. In tissue samples of human gliomas, a tumor type wherein FGF-induced angiogenesis is important, STAT5 is detected in tumor vessel EC nuclei, consistent with STAT5 activation. By forced expression of constitutively active or dominant-negative mutant STAT5A in mouse brain ECs, we further show that STAT5 activation is both necessary and sufficient for FGF-induced cell migration, invasion, and tube formation, which are key events in vascular endothelial morphogenesis and angiogenesis. In contrast, STAT5 is not required for brain EC mitogenesis. The cytoplasmic tyrosine kinases Src and Janus kinase 2 (Jak2) both seem to be involved in the activation of STAT5, as their inhibition reduces FGF2- and FGF8b-induced STAT5 phosphorylation and EC tube formation. Constitutively active STAT5A partially restores tube formation in the presence of Src or Jak2 inhibitors. These observations show that FGFs use distinct signaling pathways to induce angiogenic phenotypes. Together, our findings implicate the FGF-Jak2/Src-STAT5 cascade as a critical angiogenic FGF signaling pathway. [Cancer Res 2009;69(4):1668–77]
Data from Peroxisome Proliferator-Activated Receptor-δ Induces Cell Proliferation by a Cyclin E1–Dependent Mechanism and Is Up-regulated in Thyroid Tumors
Authors: Zeng Lingchun , Geng Yan , Tretiakova Maria , Yu Xuemei , Sicinski Peter , Kroll Todd G.
Published: March 2023

Abstract
Peroxisome proliferator-activated receptors (PPARs) are lipid-sensing nuclear receptors that have been implicated in multiple physiologic processes including cancer. Here, we determine that PPARδ induces cell proliferation through a novel cyclin E1–dependent mechanism and is up-regulated in many human thyroid tumors. The expression of PPARδ was induced coordinately with proliferation in primary human thyroid cells by the activation of serum, thyroid-stimulating hormone/cyclic AMP, or epidermal growth factor/mitogen-activated protein kinase mitogenic signaling pathways. Engineered overexpression of PPARδ increased thyroid cell number, the incorporation of bromodeoxyuridine, and the phosphorylation of retinoblastoma protein by 40% to 45% in just 2 days, one usual cell population doubling. The synthetic PPARδ agonist GW501516 augmented these PPARδ proliferation effects in a dose-dependent manner. Overexpression of PPARδ increased cyclin E1 protein by 9-fold, whereas knockdown of PPARδ by small inhibitory RNA reduced both cyclin E1 protein and cell proliferation by 2-fold. Induction of proliferation by PPARδ was abrogated by knockdown of cyclin E1 by small inhibitory RNA in primary thyroid cells and by knockout of cyclin E1 in mouse embryo fibroblasts, confirming a cyclin E1 dependence for this PPARδ pathway. In addition, the mean expression of native PPARδ was increased by 2-fold to 5-fold (P < 0.0001) and correlated with that of the in situ proliferation marker Ki67 (R = 0.8571; P = 0.02381) in six different classes of benign and malignant human thyroid tumors. Our experiments identify a PPARδ mechanism that induces cell proliferation through cyclin E1 and is regulated by growth factor and lipid signals. The data argue for systematic investigation of PPARδ antagonists as antineoplastic agents and implicate altered PPARδ–cyclin E1 signaling in thyroid and other carcinomas. [Cancer Res 2008;68(16):6578–86]
Data from Baicalein Overcomes Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand Resistance via Two Different Cell-Specific Pathways in Cancer Cells but not in Normal Cells
Authors: Taniguchi Hiroya , Yoshida Tatsushi , Horinaka Mano , Yasuda Takashi , Goda Ahmed E. , Konishi Masako , Wakada Miki , Kataoka Keisho , Yoshikawa Toshikazu , Sakai Toshiyuki
Published: March 2023

Abstract
Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) is one of the most promising candidates for new cancer therapeutics. A current problem is that some cancers still remain resistant to TRAIL. We show for the first time that a naturally occurring flavonoid, baicalein, overcomes TRAIL resistance in cancer cells. The combination of baicalein and TRAIL effectively induced apoptosis in TRAIL-resistant colon cancer SW480 cells. Baicalein up-regulated the expression of death receptor 5 (DR5) among TRAIL receptors at the mRNA and protein levels. Suppression of this up-regulation with small interfering RNA (siRNA) efficiently reduced the apoptosis induced by TRAIL and baicalein, suggesting that the sensitization was mediated through DR5 induction. Moreover, baicalein also overcame TRAIL resistance with DR5 up-regulation in prostate cancer PC3 cells. Of note, the combination of TRAIL and baicalein hardly induced apoptosis in normal human cells, such as blood cells and hepatocytes. Baicalein increased DR5 promoter activity, and this enhanced activity was diminished by mutation of a CCAAT/enhancer-binding protein homologous protein (CHOP)–binding site in SW480 cells. In SW480 cells, CHOP siRNA blocked both functions of baicalein. CHOP expression was induced by baicalein in SW480 cells; however, in PC3 cells, baicalein scarcely induced CHOP and mutation of the CHOP-binding site did not abrogate the DR5 promoter activation by baicalein. Interestingly, baicalein induced reactive oxygen species (ROS) and a ROS scavenger prevented DR5 expression and TRAIL sensitization in PC3 but not SW480 cells. These results indicate that, using two different pathways, baicalein exposes cancer surveillance of TRAIL and overcomes TRAIL resistance in cancer cells. [Cancer Res 2008;68(21):8918–27]
Data from Targeting XIAP Bypasses Bcl-2–Mediated Resistance to TRAIL and Cooperates with TRAIL to Suppress Pancreatic Cancer Growth In vitro and In vivo
Authors: Vogler Meike , Walczak Henning , Stadel Dominic , Haas Tobias L. , Genze Felicitas , Jovanovic Marjana , Gschwend Jürgen E. , Simmet Thomas , Debatin Klaus-Michael , Fulda Simone
Published: March 2023

Abstract
Resistance to apoptosis is a hallmark of pancreatic cancer, a leading cause of cancer deaths. Therefore, novel strategies are required to target apoptosis resistance. Here, we report that the combination of X-linked inhibitor of apoptosis (XIAP) inhibition and tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) is an effective approach to trigger apoptosis despite Bcl-2 overexpression and to suppress pancreatic cancer growth in vitro and in vivo. Knockdown of XIAP by RNA interference cooperates with TRAIL to induce caspase activation, loss of mitochondrial membrane potential, cytochrome c release, and apoptosis in pancreatic carcinoma cells. Loss of mitochondrial membrane potential and cytochrome c release are extensively inhibited by a broad range or caspase-3 selective caspase inhibitor and by RNAi-mediated silencing of caspase-3, indicating that XIAP inhibition enhances TRAIL-induced mitochondrial damage in a caspase-3–dependent manner. XIAP inhibition combined with TRAIL even breaks Bcl-2–imposed resistance by converting type II cells that depend on the mitochondrial contribution to the death receptor pathway to type I cells in which TRAIL-induced activation of caspase-3 and caspase-9 and apoptosis proceeds irrespective of high Bcl-2 levels. Most importantly, XIAP inhibition potentiates TRAIL-induced antitumor activity in two preclinical models of pancreatic cancer in vivo. In the chicken chorioallantoic membrane model, XIAP inhibition significantly enhances TRAIL-mediated apoptosis and suppression of tumor growth. In a tumor regression model in xenograft-bearing mice, XIAP inhibition acts in concert with TRAIL to cause even regression of established pancreatic carcinoma. Thus, this combination of XIAP inhibition plus TRAIL is a promising strategy to overcome apoptosis resistance of pancreatic cancer that warrants further investigation. [Cancer Res 2008;68(19):7956–65]
Data from Conditional Deletion of Insulin-like Growth Factor-I Receptor in Prostate Epithelium
Authors: Sutherland Brent W. , Knoblaugh Sue E. , Kaplan-Lefko Paula J. , Wang Fen , Holzenberger Martin , Greenberg Norman M.
Published: March 2023

Abstract
Insulin-like growth factor-I (IGF-I) is a polypeptide hormone that can influence growth, differentiation, and survival of cells expressing the cognate type 1 receptor (IGF-IR). To better understand cell autonomous IGF-IR signaling in the epithelial compartment of the prostate gland, we generated a conditional (Cre/loxP) prostate-specific IGF-IR knockout mouse model. In contrast to epidemiologic studies that established a correlation between elevated serum IGF-I and the risk of developing prostate cancer, we show that abrogation of IGF-IR expression in the dorsal and lateral prostate could activate extracellular signal-regulated kinase 1/2 signaling and cause cell autonomous proliferation and hyperplasia. Moreover, persistent loss of IGF-IR expression in dorsal and ventral lobes induced p53-regulated apoptosis and cellular senescence rescue programs, predicting that titration of IGF-IR signaling might facilitate growth of tumors with compromised p53 activity. Therefore, we crossed the mice carrying the prostate-specific IGF-IR knockout alleles into the transgenic adenocarcinoma of the mouse prostate model that is driven, in part, by T antigen–mediated functional inactivation of p53. Consistent with our prediction, prostate epithelial–specific deletion of IGF-IR accelerated the emergence of aggressive prostate cancer when p53 activity was compromised. Collectively, these data support a critical role for IGF-IR signaling in prostate tumorigenesis and identify an important IGF-IR–dependent growth control mechanism. [Cancer Res 2008;68(9):3495–504]
Data from Early Therapy Evaluation of Combined Anti–Death Receptor 5 Antibody and Gemcitabine in Orthotopic Pancreatic Tumor Xenografts by Diffusion-Weighted Magnetic Resonance Imaging
Authors: Kim Hyunki , Morgan Desiree E. , Buchsbaum Donald J. , Zeng Huadong , Grizzle William E. , Warram Jason M. , Stockard Cecil R. , McNally Lacey R. , Long Joshua W. , Sellers Jeffrey C. , Forero Andres , Zinn Kurt R.
Published: March 2023

Abstract
Early therapeutic efficacy of anti–death receptor 5 antibody (TRA-8) combined with gemcitabine was measured using diffusion-weighted magnetic resonance imaging (DWI) in an orthotopic pancreatic tumor model. Groups 1 to 4 of severe combined immunodeficient mice (n = 5–7 per group) bearing orthotopically implanted, luciferase-positive human pancreatic tumors (MIA PaCa-2) were subsequently (4–5 weeks thereafter) injected with saline (control), gemcitabine (120 mg/kg), TRA-8 (200 μg), or TRA-8 combined with gemcitabine, respectively, on day 0. DWI, anatomic magnetic resonance imaging, and bioluminescence imaging were done on days 0, 1, 2, and 3 after treatment. Three tumors from each group were collected randomly on day 3 after imaging, and terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling staining was done to quantify apoptotic cellularity. At just 1 day after starting therapy, the changes of apparent diffusion coefficient (ADC) in tumor regions for group 3 (TRA-8) and group 4 (TRA-8/Gem) were 21 ± 9% (mean ± SE) and 27 ± 3%, respectively, significantly higher (P < 0.05) than those of group 1 (−1 ± 5%) and group 2 (−2 ± 4%). There was no statistical difference in tumor volumes for the groups at this time. The mean ADC values of groups 2 to 4 gradually increased over 3 days, which were concurrent with tumor volume regressions and bioluminescence signal decreases. Apoptotic cell densities of tumors in groups 1 to 4 were 0.7 ± 0.4%, 0.6 ± 0.2%, 3.1 ± 0.9%, and 4.7 ± 1.0%, respectively, linearly proportional to the ADC changes on day 1. Further, the ADC changes were highly correlated with the previously reported mean survival times of animals treated with the same agents and doses. This study supports the clinical use of DWI for pancreatic tumor patients for early assessment of drug efficacy. [Cancer Res 2008;68(20):8369–76]
Data from Dual Function of Pancreatic Endoplasmic Reticulum Kinase in Tumor Cell Growth Arrest and Survival
Authors: Ranganathan Aparna C. , Ojha Shishir , Kourtidis Antonis , Conklin Douglas S. , Aguirre-Ghiso Julio A.
Published: March 2023

Abstract
Pancreatic endoplasmic reticulum kinase (PERK)-eIF2α signaling, a component of the endoplasmic reticulum (ER) stress response, has been proposed as a therapeutic target due to its importance to cell survival in hypoxic tumors. In this study, we show that in addition to promoting survival, PERK can also suppress tumor growth of advanced carcinomas. Our results show that in squamous carcinoma T-HEp3 cells, which display low PERK-eIF2α signaling, inducible activation of an Fv2E-PERK fusion protein results in a strong G0-G1 arrest in vitro. Most importantly, Fv2E-PERK activation, in addition to promoting survival in vitro, inhibits T-HEp3 and SW620 colon carcinoma growth in vivo. Increased PERK activation is linked to enhanced p-eIF2α levels, translational repression, and a decrease in Ki67, pH 3, and cycD1/D3 levels, but not to changes in angiogenesis or apoptosis. Experimental reduction of PERK activity, or overexpression of GADD34 in a spontaneously arising in vivo quiescent variant of HEp3 cells that displays strong basal PERK-eIF2α activation, reverts their quiescent phenotype. We conclude that the growth-inhibitory function of PERK is preserved in tumors and upon proper reactivation can severely inhibit tumor growth through induction of quiescence. This is an important consideration in the development of PERK-based therapies, as its inhibition may facilitate the proliferation of slow-cycling or dormant tumor cells. [Cancer Res 2008;68(9):3260–8]
Data from Multimodal Control of Cdc25A by Nitrosative Stress
Authors: Tomko Robert J. , Lazo John S.
Published: March 2023

Abstract
Cdc25A propels cell cycle progression, is overexpressed in numerous human cancers, and possesses oncogenic and antiapoptotic activities. Reactive oxygen species, such as hydrogen peroxide, regulate Cdc25A, but the physiologic and pathologic effects of nitric oxide (•NO) and •NO-derived reactive species are not well defined. Herein, we report novel independent mechanisms governing Cdc25A in response to nitrosative insult. We observed direct and rapid inhibition of Cdc25A phosphatase activity after in vitro treatment with the low molecular mass cell-permeable S-nitrosothiol S-nitrosocysteine ethyl ester (SNCEE). In addition, treatment of cancer cells with SNCEE induced nitrosative stress and decreased Cdc25A protein levels in a time-dependent and concentration-dependent manner. Similarly, iNOS-derived •NO was sufficient to suppress Cdc25A expression, consistent with its role in mediating nitrosative stress. Whereas a decrease in Cdc25A half-life was not observed in response to SNCEE, we found the translational regulator eukaryotic initiation factor 2α (eIF2α) was hyperphosphorylated and total protein translation was decreased with kinetics consistent with Cdc25A loss. Inhibition of eIF2α decreased Cdc25A levels, supporting the hypothesis that SNCEE suppressed Cdc25A translation through inhibition of eIF2α. Nitrosative stress decreased the Cdc25A-bound fraction of apoptosis signal-regulating kinase-1 (ASK-1) and sensitized cells to apoptosis induced by the ASK-1–activating chemotherapeutic cis-diaminedichloroplatinum (II), suggesting that nitrosative stress–induced suppression of Cdc25A primed cells for ASK-1–dependent apoptosis. Together these data reveal novel •NO-dependent enzymatic and translational mechanisms controlling Cdc25A, and implicate Cdc25A as a mediator of •NO-dependent apoptotic signaling. [Cancer Res 2008;68(18):7457–65]
Data from Tnk1/Kos1 Knockout Mice Develop Spontaneous Tumors
Authors: Hoare Sarasija , Hoare Kishalay , Reinhard Mary K. , Lee Young J. , Oh S. Paul , May W. Stratford
Published: March 2023

Abstract
Tnk1/Kos1 is a non–receptor protein tyrosine kinase implicated in negatively regulating cell growth in a mechanism requiring its intrinsic catalytic activity. Tnk1/Kos1 null mice were created by homologous recombination by deleting the catalytic domain. Both Tnk1+/− and Tnk1−/− mice develop spontaneous tumors, including lymphomas and carcinomas, at high rates [27% (14 of 52) and 43% (12 of 28), respectively]. Tnk1/Kos1 expression is silenced in tumors that develop in Tnk1+/− mice but not in adjacent uninvolved tissue, and silencing occurs in association with Tnk1 promoter hypermethylation. Tissues and murine embryonic fibroblasts derived from Tnk1/Kos1-null mice exhibit proportionally higher levels of basal and epidermal growth factor–stimulated Ras activation that results from increased Ras-guanine exchange factor (GEF) activity. Mechanistically, Tnk1/Kos1 can directly tyrosine phosphorylate growth factor receptor binding protein 2 (Grb2), which promotes disruption of the Grb2-Sos1 complex that mediates growth factor–induced Ras activation, providing dynamic regulation of Ras GEF activity with suppression of Ras. Thus, Tnk1/Kos1 is a tumor suppressor that functions to down-regulate Ras activity. [Cancer Res 2008;68(21):8723–32]
Data from CCCTC-Binding Factor Confines the Distal Action of Estrogen Receptor
Authors: Chan Chang S. , Song Jun S.
Published: March 2023

Abstract
Distal enhancers have recently emerged as a common mode of gene regulation for several transcription factors, including estrogen and androgen receptors, the two key regulators of breast and prostate cancer major subtypes. Despite the rapid success in genome-wide annotation of estrogen receptor-α (ERα) binding sites in cell lines, the precise mechanism governing the gene-to-enhancer association is still unknown and no quantitative model that can predict the estrogen responsiveness of genes has been hitherto proposed. This article presents an integrative genomics approach to construct a predictive model that can explain more than 70% of estrogen-induced expression profiles. The proposed method combines a recent map of the insulator protein CCCTC-binding factor (CTCF) with previous ER location studies and expression profiling in the presence of the translation inhibitor cycloheximide, providing evidence that CTCF partitions the human genome into distinct ER-regulatory blocks. It is shown that estrogen-responsive genes with a decreased transcription level (down-regulated genes) have a markedly different relative distribution of ER binding sites compared with those with an increased transcription level (up-regulated genes). Finally, Bayesian belief networks are constructed to quantify the effects of ER-binding distance from genes as well as the insulating effects of CTCF on the estrogen responsiveness of genes. This work thus represents a stride toward understanding and predicting the distal activities of steroid hormone nuclear receptors. [Cancer Res 2008;68(21):9041–9]
Data from Common Transcriptional Signature of Tumor-Infiltrating Mononuclear Inflammatory Cells and Peripheral Blood Mononuclear Cells in Hepatocellular Carcinoma Patients
Authors: Sakai Yoshio , Honda Masao , Fujinaga Haruo , Tatsumi Isamu , Mizukoshi Eishiro , Nakamoto Yasunari , Kaneko Shuichi
Published: March 2023

Abstract
Hepatocellular carcinoma (HCC) is frequently associated with infiltrating mononuclear inflammatory cells. We performed laser capture microdissection of HCC-infiltrating and noncancerous liver-infiltrating mononuclear inflammatory cells in patients with chronic hepatitis C (CH-C) and examined gene expression profiles. HCC-infiltrating mononuclear inflammatory cells had an expression profile distinct from noncancerous liver-infiltrating mononuclear inflammatory cells; they differed with regard to genes involved in biological processes, such as antigen presentation, ubiquitin-proteasomal proteolysis, and responses to hypoxia and oxidative stress. Immunohistochemical analysis and gene expression databases suggested that the up-regulated genes involved macrophages and Th1 and Th2 CD4 cells. We next examined the gene expression profile of peripheral blood mononuclear cells (PBMC) obtained from CH-C patients with or without HCC. The expression profiles of PBMCs from patients with HCC differed significantly from those of patients without HCC (P < 0.0005). Many of the up-regulated genes in HCC-infiltrating mononuclear inflammatory cells were also differentially expressed by PBMCs of HCC patients. Analysis of the commonly up-regulated or down-regulated genes in HCC-infiltrating mononuclear inflammatory cells and PBMCs of HCC patients showed networks of nucleophosmin, SMAD3, and proliferating cell nuclear antigen that are involved with redox status, the cell cycle, and the proteasome system, along with immunologic genes, suggesting regulation of anticancer immunity. Thus, exploring the gene expression profile of PBMCs may be a surrogate approach for the assessment of local HCC-infiltrating mononuclear inflammatory cells. [Cancer Res 2008;68(24):10267–79]
Data from Nitric Oxide Induces Early Viral Transcription Coincident with Increased DNA Damage and Mutation Rates in Human Papillomavirus–Infected Cells
Authors: Wei Lanlan , Gravitt Patti E. , Song Hebin , Maldonado Anastacia M. , Ozbun Michelle A.
Published: March 2023

Abstract
High-risk human papillomavirus (HPV) infections are necessary but insufficient causes of cervical cancers. Other risk factors for cervical cancer (e.g., pregnancy, smoking, infections causing inflammation) can lead to high and sustained nitric oxide (NO) concentrations in the cervix, and high NO levels are related to carcinogenesis through DNA damage and mutation. However, the effects of NO exposure in HPV-infected cells have not been investigated. In this study, we used the NO donor DETA-NO to model NO exposure to cervical epithelium. In cell culture media, 24-hour exposure to 0.25 to 0.5 mmol/L DETA-NO yielded a pathologically relevant NO concentration. Exposure of cells maintaining episomal high-risk HPV genomes to NO increased HPV early transcript levels 2- to 4-fold but did not increase viral DNA replication. Accompanying increased E6 and E7 mRNA levels were significant decreases in p53 and pRb protein levels, lower apoptotic indices, increased DNA double-strand breaks, and higher mutation frequencies when compared with HPV-negative cells. We propose that NO is a molecular cofactor with HPV infection in cervical carcinogenesis, and that modifying local NO cervical concentrations may constitute a strategy whereby HPV-related cancer can be reduced.[Cancer Res 2009;69(11):4878–84]
Data from The Promyelocytic Leukemia Zinc Finger–MicroRNA-221/-222 Pathway Controls Melanoma Progression through Multiple Oncogenic Mechanisms
Authors: Felicetti Federica , Errico M. Cristina , Bottero Lisabianca , Segnalini Patrizia , Stoppacciaro Antonella , Biffoni Mauro , Felli Nadia , Mattia Gianfranco , Petrini Marina , Colombo Mario P. , Peschle Cesare , Carè Alessandra
Published: March 2023

Abstract
The incidence of cutaneous melanoma is steadily increasing. Although several molecular abnormalities have been associated with melanoma progression, the mechanisms underlying the differential gene expression are still largely unknown and targeted therapies are not yet available. Noncoding small RNAs, termed microRNAs (miR), have been recently reported to play important roles in major cellular processes, including those involved in cancer development and progression. We have identified the promyelocytic leukemia zinc finger (PLZF) transcription factor as a repressor of miR-221 and miR-222 by direct binding to their putative regulatory region. Specifically, PLZF silencing in melanomas unblocks miR-221 and miR-222, which in turn controls the progression of the neoplasia through down-modulation of p27Kip1/CDKN1B and c-KIT receptor, leading to enhanced proliferation and differentiation blockade of the melanoma cells, respectively. In vitro and in vivo functional studies, including the use of antisense “antagomir” oligonucleotides, confirmed the key role of miR-221/-222 in regulating the progression of human melanoma; this suggests that targeted therapies suppressing miR-221/-222 may prove beneficial in advanced melanoma. [Cancer Res 2008;68(8):2745–10]
Data from Topology of NGEP, a Prostate-Specific Cell:Cell Junction Protein Widely Expressed in Many Cancers of Different Grade Level
Authors: Das Sudipto , Hahn Yoonsoo , Walker Dawn A. , Nagata Satoshi , Willingham Mark C. , Peehl Donna M. , Bera Tapan K. , Lee Byungkook , Pastan Ira
Published: March 2023

Abstract
New gene expressed in prostate (NGEP) is a prostate-specific polytopic membrane protein found at high concentrations at cell:cell contact regions. To determine if NGEP is a useful target for antibody-based therapy of prostate cancer, we performed an immunohistochemical analysis of 126 human prostate carcinoma samples using polyclonal anti-NGEP sera and found that 91% of the cancers express NGEP protein. To elucidate the topology of NGEP and guide the development of monoclonal antibodies (mAb) reacting with the extracellular regions of NGEP, a hemagglutinin epitope tag was inserted at several positions within the NGEP sequence. The tagged proteins were expressed in 293T cells and locations of the tags were determined by immunofluorescence in intact or permeabilized cells. The results indicate that NGEP contains eight transmembrane domains with both the NH2 and COOH termini of NGEP located inside the cell. We produced mAb to three regions that are predicted to be intracellular based on the epitope tag data (amino acids 1-352, 441-501, and 868-933), and as predicted, the mAb only detected the protein in permeabilized cells. NGEP is a glycoprotein with predicted glycosylation sites at N809 and N824. When these residues were converted to glutamine, glycosylation was abolished, confirming that the residues are extracellular. Our findings on the expression and the orientation of the NGEP protein serve as an important framework for the development of mAb targeting the extracellular regions of NGEP that could be used for prostate cancer immunotherapy. [Cancer Res 2008;68(15):6306–12]
Data from Wnt1 Expression Induces Short-Range and Long-Range Cell Recruitments That Modify Mammary Tumor Development and Are Not Induced by a Cell-Autonomous β-Catenin Effector
Authors: Kim Young Chul , Clark Rod J. , Ranheim Erik A. , Alexander Caroline M.
Published: March 2023

Abstract
Xenograft model studies have shown that tumor-associated, or genetically modified, activated stromal cells can promote tumor cell growth. Here, we examined mammary tumors arising in response to two different transgene-mediated Wnt signaling effectors: Wnt1 (a ligand with cell-nonautonomous effects) and ΔNβ-catenin (a constitutively active form of the intracellular effector). Although the route of tumor development has been shown to be similar for these two models, histologic analysis shows that Wnt1-induced tumors are associated with tracts of activated stroma, whereas most ΔNβ-catenin–induced tumors are solid adenocarcinomas. Furthermore, quantification of the “reactive stroma index” indicates that abundant activated stroma correlates with accelerated tumor progression. Wnt1-expressing mammary epithelial cells induce Wnt-specific target gene expression in local stromal cells (Wnt1-induced secreted protein 1/CCN4) but also induce long-range effects. Thus, mice with rapid tumor progression have 2-fold more circulating endothelial progenitor cells in peripheral blood than control or ΔNβ-catenin transgenic mice. Using tagged bone marrow (BM) transplants, we show that BM-derived cells are massively recruited to infiltrate the stroma of Wnt1-induced tumors where they differentiate into multiple cell types. Thus, localized ectopic expression of the proto-oncogene Wnt1 in mammary glands induces systemic responses, and we propose that this response modifies the tumorigenic outcome. [Cancer Res 2008;68(24):10145–53]
Data from Macrophage-Derived SPARC Bridges Tumor Cell-Extracellular Matrix Interactions toward Metastasis
Authors: Sangaletti Sabina , Di Carlo Emma , Gariboldi Silvia , Miotti Silvia , Cappetti Barbara , Parenza Mariella , Rumio Cristiano , Brekken Rolf A. , Chiodoni Claudia , Colombo Mario P.
Published: March 2023

Abstract
Other than genetic imprinting and epithelial to mesenchymal transition, cancer cells need interaction with the nearby stroma toward metastasis. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein known to regulate extracellular matrix (ECM) deposition and cell-ECM interaction. Gene expression profiles associate SPARC to malignant progression. Using reciprocal bone marrow chimeras between SPARC knockout and wild-type mice, we show that SPARC produced by inflammatory cells is necessary for spontaneous, but not experimental, i.v. metastasis. Macrophage-derived SPARC induces cancer cell migration and enhances their migration to other ECM proteins at least through αvβ5 integrin. Indeed, RNA interference knockdown of β5 integrin expression reduces cell migration in vitro and metastasis in vivo. Together these results show that macrophage-derived SPARC takes part in metastasis, acting at the step of integrin-mediated migration of invasive cells. [Cancer Res 2008;68(21):9050–9]
Data from Development of a Small-Molecule Serum- and Glucocorticoid-Regulated Kinase-1 Antagonist and Its Evaluation as a Prostate Cancer Therapeutic
Authors: Sherk Andrea B. , Frigo Daniel E. , Schnackenberg Christine G. , Bray Jeffrey D. , Laping Nicholas J. , Trizna Walter , Hammond Marlys , Patterson Jaclyn R. , Thompson Scott K. , Kazmin Dmitri , Norris John D. , McDonnell Donald P.
Published: March 2023

Abstract
Androgens, through their actions on the androgen receptor (AR), are required for the development of the prostate and contribute to the pathologic growth dysregulation observed in prostate cancers. Consequently, androgen ablation has become an essential component of the pharmacotherapy of prostate cancer. In this study, we explored the utility of targeting processes downstream of AR as an alternate approach for therapy. Specifically, we show that the serum and glucocorticoid-regulated kinase 1 (SGK1) gene is an androgen-regulated target gene in cellular models of prostate cancer. Furthermore, functional serum- and glucocorticoid-regulated kinase 1 (SGK1) protein, as determined by the phosphorylation of its target Nedd4-2, was also increased with androgen treatment. Importantly, we determined that RNA interference–mediated knockdown of SGK1 expression attenuates the androgen-mediated growth of the prostate cancer cell line LNCaP. Given these findings, we explored the utility of SGK1 as a therapeutic target in prostate cancer by developing and evaluating a small-molecule inhibitor of this enzyme. From these studies emerged GSK650394, a competitive inhibitor that quantitatively blocks the effect of androgens on LNCaP cell growth. Thus, in addition to androgen ablation, inhibition of pathways downstream of AR is likely to have therapeutic utility in prostate cancer. [Cancer Res 2008;68(18):7475–83]
Data from Targeted Deletion of Prkar1a Reveals a Role for Protein Kinase A in Mesenchymal-to-Epithelial Transition
Authors: Nadella Kiran S. , Jones Georgette N. , Trimboli Anthony , Stratakis Constantine A. , Leone Gustavo , Kirschner Lawrence S.
Published: March 2023

Abstract
Dysregulation of protein kinase A (PKA) activity, caused by loss of function mutations in PRKAR1A, is known to induce tumor formation in the inherited tumor syndrome Carney complex (CNC) and is also associated with sporadic tumors of the thyroid and adrenal. We have previously shown that Prkar1a+/− mice develop schwannomas reminiscent of those seen in CNC and that similar tumors are observed in tissue-specific knockouts (KO) of Prkar1a targeted to the neural crest. Within these tumors, we have previously described the presence of epithelial islands, although the nature of these structures was unclear. In this article, we report that these epithelial structures are derived from KO cells originating in the neural crest. Analysis of the mesenchymal marker vimentin revealed that this protein was markedly down-regulated not only from the epithelial islands, but also from the tumor as a whole, consistent with mesenchymal-to-epithelial transition (MET). In vitro, Prkar1a null primary mouse embryonic fibroblasts, which display constitutive PKA signaling, also showed evidence for MET, with a loss of vimentin and up-regulation of the epithelial marker E-cadherin. Reduction of vimentin protein occurred at the posttranslational level and was rescued by proteasomal inhibition. Finally, this down-regulation of vimentin was recapitulated in the adrenal nodules of CNC patients, confirming an unexpected and previously unrecognized role for PKA in MET. [Cancer Res 2008;68(8):2671–7]
Data from Akt1 and Akt2 Play Distinct Roles in the Initiation and Metastatic Phases of Mammary Tumor Progression
Authors: Dillon Rachelle L. , Marcotte Richard , Hennessy Bryan T. , Woodgett James R. , Mills Gordon B. , Muller William J.
Published: March 2023

Abstract
The phosphatidylinositol 3-kinase (PI3K)/Akt survival pathway is often dysregulated in cancer. Our previous studies have shown that coexpression of activated Akt1 with activated ErbB2 or polyoma virus middle T antigen uncoupled from the PI3K pathway (PyVmT Y315/322F) accelerates mammary tumor development but cannot rescue the metastatic phenotype associated with these models. Here, we report the generation of transgenic mice expressing activated Akt2 in the mammary epithelium. Like the mouse mammary tumor virus-Akt1 strain, mammary-specific expression of Akt2 delayed mammary gland involution. However, in contrast to Akt1, coexpression of Akt2 with activated ErbB2 or PyVmT Y315/322F in the mammary glands of transgenic mice did not affect the latency of tumor development. Strikingly, Akt2 coexpresssion markedly increased the incidence of pulmonary metastases in both tumor models, demonstrating a unique role in tumor progression. Together, these observations argue that these highly conserved kinases have distinct biological and biochemical outputs that play opposing roles in mammary tumor induction and metastasis. [Cancer Res 2009;69(12):5057–64]
Data from Androgen Receptor Inhibits Estrogen Receptor-α Activity and Is Prognostic in Breast Cancer
Authors: Peters Amelia A. , Buchanan Grant , Ricciardelli Carmela , Bianco-Miotto Tina , Centenera Margaret M. , Harris Jonathan M. , Jindal Shalini , Segara Davendra , Jia Li , Moore Nicole L. , Henshall Susan M. , Birrell Stephen N. , Coetzee Gerhard A. , Sutherland Robert L. , Butler Lisa M. , Tilley Wayne D.
Published: March 2023

Abstract
There is emerging evidence that the balance between estrogen receptor-α (ERα) and androgen receptor (AR) signaling is a critical determinant of growth in the normal and malignant breast. In this study, we assessed AR status in a cohort of 215 invasive ductal breast carcinomas. AR and ERα were coexpressed in the majority (80-90%) of breast tumor cells. Kaplan-Meier product limit analysis and multivariate Cox regression showed that AR is an independent prognostic factor in ERα-positive disease, with a low level of AR (less than median of 75% positive cells) conferring a 4.6-fold increased risk of cancer-related death (P = 0.002). Consistent with a role for AR in breast cancer outcome, AR potently inhibited ERα transactivation activity and 17β-estradiol–stimulated growth of breast cancer cells. Transfection of MDA-MB-231 breast cancer cells with either functionally impaired AR variants or the DNA-binding domain of the AR indicated that the latter is both necessary and sufficient for inhibition of ERα signaling. Consistent with molecular modeling, electrophoretic mobility shift assays showed binding of the AR to an estrogen-responsive element (ERE). Evidence for a functional interaction of the AR with an ERE in vivo was provided by chromatin immunoprecipitation data, revealing recruitment of the AR to the progesterone receptor promoter in T-47D breast cancer cells. We conclude that, by binding to a subset of EREs, the AR can prevent activation of target genes that mediate the stimulatory effects of 17β-estradiol on breast cancer cells. [Cancer Res 2009;69(15):6131–40]
Data from Tumor Repressive Functions of Estrogen Receptor β in SW480 Colon Cancer Cells
Authors: Hartman Johan , Edvardsson Karin , Lindberg Karolina , Zhao Chunyan , Williams Cecilia , Ström Anders , Gustafsson Jan-Åke
Published: March 2023

Abstract
Estrogen receptor β (ERβ) is the predominant ER in the colorectal epithelium. Compared with normal colon tissue, ERβ expression is reduced in colorectal cancer. Our hypothesis is that ERβ inhibits proliferation of colon cancer cells. Hence, the aim of this study has been to investigate the molecular function of ERβ in colon cancer cells, focusing on cell cycle regulation. SW480 colon cancer cells have been lentivirus transduced with ERβ expression construct with or without mutated DNA-binding domain or an empty control vector. Expression of ERβ resulted in inhibition of proliferation and G1 phase cell cycle arrest and this effect was dependent on a functional DNA-binding region. c-Myc is overexpressed in an overwhelming majority of colorectal tumors. By Western blot and real-time PCR, we found c-Myc to be down-regulated in the ERβ-expressing cells. Furthermore, the c-Myc target gene p21(Waf1/Cip1) was induced and Cdc25A was reduced by ERβ at the transcriptional level. The second cdk2-inhibitor, p27Kip1, was induced by ERβ, but this regulation occurred at the posttranscriptional level, probably through ERβ-mediated repression of the F-box protein p45Skp2. Expression of the ERβ-variant with mutated DNA binding domain resulted in completely different cell cycle gene regulation. We performed in vivo studies with SW480 cells ± ERβ transplanted into severe combined immunodeficient/beige mice; after three weeks of ERβ-expression, a 70% reduction of tumor volume was seen. Our results show that ERβ inhibits proliferation as well as colon cancer xenograft growth, probably as a consequence of ERβ-mediated inhibition of cell-cycle pathways. Furthermore, this ERβ-mediated cell cycle repression is dependent on functional ERE binding. [Cancer Res 2009;69(15):6100–6]
Data from Analysis of Gene Expression Profiles of Microdissected Cell Populations Indicates that Testicular Carcinoma In situ Is an Arrested Gonocyte
Authors: Sonne Si Brask , Almstrup Kristian , Dalgaard Marlene , Juncker Agnieszka Sierakowska , Edsgard Daniel , Ruban Ludmila , Harrison Neil J. , Schwager Christian , Abdollahi Amir , Huber Peter E. , Brunak Søren , Gjerdrum Lise Mette , Moore Harry D. , Andrews Peter W. , Skakkebaek Niels E. , Meyts Ewa Rajpert-De , Leffers Henrik
Published: March 2023

Abstract
Testicular germ cell cancers in young adult men derive from a precursor lesion called carcinoma in situ (CIS) of the testis. CIS cells were suggested to arise from primordial germ cells or gonocytes. However, direct studies on purified samples of CIS cells are lacking. To overcome this problem, we performed laser microdissection of CIS cells. Highly enriched cell populations were obtained and subjected to gene expression analysis. The expression profile of CIS cells was compared with microdissected gonocytes, oogonia, and cultured embryonic stem cells with and without genomic aberrations. Three samples of each tissue type were used for the analyses. Unique expression patterns for these developmentally very related cell types revealed that CIS cells were very similar to gonocytes because only five genes distinguished these two cell types. We did not find indications that CIS was derived from a meiotic cell, and the similarity to embryonic stem cells was modest compared with gonocytes. Thus, we provide new evidence that the molecular phenotype of CIS cells is similar to that of gonocytes. Our data are in line with the idea that CIS cells may be gonocytes that survived in the postnatal testis. We speculate that disturbed development of somatic cells in the fetal testis may play a role in allowing undifferentiated cells to survive in the postnatal testes. The further development of CIS into invasive germ cell tumors may depend on signals from their postpubertal niche of somatic cells, including hormones and growth factors from Leydig and Sertoli cells. [Cancer Res 2009;69(12):5241–50]
Data from Dual Therapeutic Efficacy of Vinblastine as a Unique Chemotherapeutic Agent Capable of Inducing Dendritic Cell Maturation
Authors: Tanaka Hiroaki , Matsushima Hironori , Nishibu Akiko , Clausen Björn E. , Takashima Akira
Published: March 2023

Abstract
Our recent unbiased functional screen of 54 chemotherapeutic drugs unveiled striking heterogeneity in their effects on dendritic cells (DC). Most notably, vinblastine (VBL) was found to induce phenotypic and functional maturation of DCs in vitro. Here, we sought to determine whether VBL exhibits “dual” therapeutic efficacy in living animals by directly killing tumor cells and by boosting host immunity via DC maturation. Local injection of VBL in a low dose into the skin of C57BL/6 mice induced in situ maturation of epidermal Langerhans cells. When coinjected with a model antigen, ovalbumin (OVA), VBL enhanced OVA-specific cellular and humoral immune responses. When injected directly into the OVA cDNA–transduced E.G7 tumors, VBL augmented clonal expansion of OVA-reactive CD8 T cells and CTL activities. In B16 melanoma model, intratumor VBL injection induced apoptosis of melanoma cells, phenotypic maturation of tumor-infiltrating DCs, and significant CTL activities. Although complete clearance was never achieved, growth kinetic of B16 melanoma was markedly reduced in C57BL/6 mice by intratumor VBL injection. Importantly, the same treatment was far less efficacious in immunocompromised severe combined immunodeficient mice, indicating the requirement of intact host immunity. Our results introduce a new concept that VBL may be used to design “immunostimulatory” chemotherapy regimens. [Cancer Res 2009;69(17):6987–94]
Data from Anti–Placental Growth Factor Reduces Bone Metastasis by Blocking Tumor Cell Engraftment and Osteoclast Differentiation
Authors: Coenegrachts Lieve , Maes Christa , Torrekens Sophie , Van Looveren Riet , Mazzone Massimiliano , Guise Theresa A. , Bouillon Roger , Stassen Jean-Marie , Carmeliet Peter , Carmeliet Geert
Published: March 2023

Abstract
Treatment of bone metastases is largely symptomatic and is still an unmet medical need. Current therapies mainly target the late phase of tumor-induced osteoclast activation and hereby inhibit further metastatic growth. This treatment method is, however, less effective in preventing initial tumor engraftment, a process that is supposed to depend on the bone microenvironment. We explored whether bone-derived placental growth factor (PlGF), a homologue of vascular endothelial growth factor-A, regulates osteolytic metastasis. Osteogenic cells secrete PlGF, the expression of which is enhanced by bone-metastasizing breast tumor cells. Selective neutralization of host-derived PlGF by anti-mouse PlGF (αPlGF) reduced the incidence, number, and size of bone metastases, and preserved bone mass. αPlGF did not affect metastatic tumor angiogenesis but inhibited osteoclast formation by preventing the upregulation of the osteoclastogenic cytokine receptor activator of NF-κB ligand in osteogenic cells, as well as by blocking the autocrine osteoclastogenic activity of PlGF. αPlGF also reduced the engraftment of tumor cells in the bone and inhibited their interaction with matrix components in the metastatic niche. αPlGF therefore inhibits not only the progression of metastasis but also the settlement of tumor in the bone. These findings identify novel properties of PlGF and suggest that αPlGF might offer opportunities for adjuvant therapy of bone metastasis. Cancer Res; 70(16); 6537–47. ©2010 AACR.
Data from Gene Expression: Protein Interaction Systems Network Modeling Identifies Transformation-Associated Molecules and Pathways in Ovarian Cancer
Authors: Bapat Sharmila A. , Krishnan Anagha , Ghanate Avinash D. , Kusumbe Anjali P. , Kalra Rajkumar S.
Published: March 2023

Abstract
Multiple, dissimilar genetic defects in cancers of the same origin contribute to heterogeneity in tumor phenotypes and therapeutic responses of patients, yet the associated molecular mechanisms remain elusive. Here, we show at the systems level that serous ovarian carcinoma is marked by the activation of interconnected modules associated with a specific gene set that was derived from three independent tumor-specific gene expression data sets. Network prediction algorithms combined with preestablished protein interaction networks and known functionalities affirmed the importance of genes associated with ovarian cancer as predictive biomarkers, besides “discovering” novel ones purely on the basis of interconnectivity, whose precise involvement remains to be investigated. Copy number alterations and aberrant epigenetic regulation were identified and validated as significant influences on gene expression. More importantly, three functional modules centering on c-Myc activation, altered retinoblastoma signaling, and p53/cell cycle/DNA damage repair pathways have been identified for their involvement in transformation-associated events. Further studies will assign significance to and aid the design of a panel of specific markers predictive of individual- and tumor-specific pathways. In the parlance of this emerging field, such networks of gene-hub interactions may define personalized therapeutic decisions. Cancer Res; 70(12); 4809–19. ©2010 AACR.
Data from Estrogen Receptor β1 Expression Is Regulated by miR-92 in Breast Cancer
Authors: Al-Nakhle Hakeemah , Burns Philip A. , Cummings Michele , Hanby Andrew M. , Hughes Thomas A. , Satheesha Sampoorna , Shaaban Abeer M. , Smith Laura , Speirs Valerie
Published: March 2023

Abstract
Estrogen receptor β1 (ERβ1) downregulation occurs in many breast cancers, but the responsible molecular mechanisms remain unclear. Here, we report that levels of ERβ1 expression are negatively regulated by the microRNA miR-92. Expression analysis in a cohort of primary breast tumors confirmed a significant negative correlation between miR-92 and both ERβ1 mRNA and protein. Inhibition of miR-92 in MCF-7 cells increased ERβ1 expression in a dose-dependent manner, whereas miR-92 overexpression led to ERβ1 downregulation. Reporter constructs containing candidate miR-92 binding sites in the 3′-untranslated region (UTR) of ERβ1 suggested by bioinformatics analysis confirmed that miR-92 downregulated ERβ1 via direct targeting of its 3′-UTR. Our results define a potentially important mechanism for downregulation of ERβ1 expression in breast cancer. Cancer Res; 70(11); 4778–84. ©2010 AACR.