bluuebunny/crossref_metadata_2025 huggingface dataset:
Data from DNA Methylation of Tumor Suppressor Genes in Clinical Remission Predicts the Relapse Risk in Acute Myeloid Leukemia
Authors: Agrawal Shuchi , Unterberg Matthias , Koschmieder Steffen , zur Stadt Udo , Brunnberg Uta , Verbeek Walter , Büchner Thomas , Berdel Wolfgang E. , Serve Hubert , Müller-Tidow Carsten
Published: March 2023

Abstract
Epigenetic changes play an important role in leukemia pathogenesis. DNA methylation is among the most common alterations in leukemia. The potential role of DNA methylation as a biomarker in leukemia is unknown. In addition, the lack of molecular markers precludes minimal residual disease (MRD) estimation for most patients with hematologic malignancies. We analyzed the potential of aberrant DNA promoter methylation as a biomarker for MRD in acute leukemias. Quantitative real-time PCR methods with bisulfite-modified DNA were established to quantify MRD based on estrogen receptor α (ERα) and/or p15INK4B methylation. Methylation analyses were done in >370 DNA specimens from 180 acute leukemia patients and controls. Methylation of ERα and/or p15INK4B occurred frequently and specifically in acute leukemia but not in healthy controls or in nonmalignant hematologic diseases. Aberrant DNA methylation was detectable in >20% of leukemia patients during clinical remission. In pediatric acute lymphoblastic leukemia, methylation levels during clinical remission correlated closely with T-cell receptor/immunoglobulin MRD levels (r = +0.7, P < 0.01) and were associated with subsequent relapse. In acute myelogenous leukemia patients in clinical remission, increased methylation levels were associated with a high relapse risk and significantly reduced relapse-free survival (P = 0.003). Many patients with acute leukemia in clinical remission harbor increased levels of aberrant DNA methylation. Analysis of methylation MRD might be used as a novel biomarker for leukemia patients' relapse risk. [Cancer Res 2007;67(3):1370–7]
Data from EGFR Tyrosine Kinase Inhibitors Decrease VEGF Expression by Both Hypoxia-Inducible Factor (HIF)-1–Independent and HIF-1–Dependent Mechanisms
Authors: Pore Nabendu , Jiang Zibin , Gupta Anjali , Cerniglia George , Kao Gary D. , Maity Amit
Published: March 2023

Abstract
Epidermal growth factor receptor (EGFR) inhibitors can decrease vascular endothelial growth factor (VEGF) expression and tumor angiogenesis. In the current study, we investigate the molecular pathways by which this occurs using two drugs that have been used in the clinic, gefitinib (Iressa) and erlotinib (Tarceva). The decrease in VEGF expression by gefitinib in SQ20B squamous cell carcinoma cells was opposed by adenoviral expression of Akt in these cells. The hypoxia-inducible factor-1 (HIF-1) binding site located at approximately −1 kbp in the VEGF promoter was not required for down-regulation of promoter activity by gefitinib under normoxia. Furthermore, the drug decreased activity of a reporter containing the −88/+54 region. In a gel shift assay, gefitinib led to decreased retardation of a labeled DNA oligonucleotide probe corresponding to the −88/−66 region of the VEGF promoter, which contains Sp1 binding sites. These effects of gefitinib on VEGF promoter activity and DNA binding were both reversed by Akt expression. Phosphorylation of Sp1 was decreased in the presence of gefitinib. Gefitinib also decreases VEGF expression by decreasing HIF-1α expression. This occurs due to decreased protein translation without any change in the level of HIF-1α mRNA. Together, these results suggest that gefitinib decreases VEGF expression both by decreasing Sp1 binding to the proximal core VEGF promoter and by down-regulating HIF-1α expression. Similar results were obtained with erlotinib in SQ20B and gefitinib in HSC3 squamous carcinoma cells. These results indicate that there are at least two separate mechanisms by which EGFR inhibitors decrease VEGF expression. (Cancer Res 2006; 66(6): 3197-204)
Data from The E2F-Regulated Gene Chk1 Is Highly Expressed in Triple-Negative Estrogen Receptor−/Progesterone Receptor−/HER-2− Breast Carcinomas
Authors: Verlinden Lieve , Vanden Bempt Isabelle , Eelen Guy , Drijkoningen Maria , Verlinden Ilse , Marchal Kathleen , De Wolf-Peeters Christiane , Christiaens Marie-Rose , Michiels Luc , Bouillon Roger , Verstuyf Annemieke
Published: March 2023

Abstract
We previously showed that checkpoint kinase 1 (Chk1) and Claspin, two DNA-damage checkpoint proteins, were down-regulated by 1,25-dihydroxyvitamin D3, a known inhibitor of cell proliferation. In the present study, we aimed to investigate the transcriptional regulation of Chk1 and Claspin and to study their expression levels in human breast cancer tissue. Transient transfection experiments in MCF-7 breast cancer cells showed that promoter activities of Chk1 and Claspin were regulated by the E2F family of transcription factors. Subsequently, transcript levels of Chk1, Claspin, and E2F1 were determined by quantitative reverse transcriptase-PCR analysis in 103 primary invasive breast carcinomas and were compared with several clinicopathologic variables in breast cancer. A strong correlation was found between Chk1 and Claspin transcript levels. Transcript levels of Chk1, Claspin, and E2F1 were highest in histologic grade 3 tumors and in tumors in which the expression of estrogen receptor (ER) and progesterone receptor (PR) was lost. Moreover, Chk1 expression was significantly elevated in grade 3 breast carcinomas showing a triple-negative ER−/PR−/HER-2− phenotype compared with other grade 3 tumors. Further research is warranted to validate the use of Chk1 inhibitors in triple-negative breast carcinomas for which treatment strategies are limited at present. [Cancer Res 2007;67(14):6574–81]
Data from Heparanase Induces Vascular Endothelial Growth Factor Expression: Correlation with p38 Phosphorylation Levels and Src Activation
Authors: Zetser Anna , Bashenko Yulia , Edovitsky Evgeny , Levy-Adam Flonia , Vlodavsky Israel , Ilan Neta
Published: March 2023

Abstract
Heparanase is an endo-β-d-glucuronidase involved in cleavage of heparan sulfate moieties and hence participates in extracellular matrix (ECM) degradation and remodeling. Traditionally, heparanase activity was correlated with the metastatic potential of a variety of tumor-derived cell types. Cloning of the heparanase gene indicated that heparanase expression is up-regulated in a variety of primary human tumors. In some cases, heparanase up-regulation correlated with increased tumor vascularity, an angiogenic feature that could be recapitulated in a number of in vitro and in vivo models. The mechanism by which heparanase enhances angiogenic responses is not entirely clear but is thought to be mediated primarily by release of ECM-resident angiogenic growth factors such as basic fibroblast growth factor and vascular endothelial growth factor (VEGF). Here, we examined the possibility that heparanase directly participates in VEGF gene regulation. We provide evidence that heparanase overexpression in human embryonic kidney 293, MDA-MB-435 human breast carcinoma, and rat C6 glioma cells resulted in a 3- to 6-fold increase in VEGF protein and mRNA levels, which correlated with elevation of p38 phosphorylation. Moreover, heparanase down-regulation in B16 mouse melanoma cells by a specific siRNA vector was accompanied by a decrease in VEGF and p38 phosphorylation levels, suggesting that VEGF gene expression is regulated by endogenous heparanase. Interestingly, a specific p38 inhibitor did not attenuate VEGF up-regulation by heparanase whereas Src inhibitors completely abrogated this effect. These results indicate, for the first time, that heparanase is actively involved in the regulation of VEGF gene expression, mediated by activation of Src family members. (Cancer Res 2006; 66(3): 1455-63)
Data from Spatiotemporal Differences in CXCL12 Expression and Cyclic AMP Underlie the Unique Pattern of Optic Glioma Growth in Neurofibromatosis Type 1
Authors: Warrington Nicole M. , Woerner B. Mark , Daginakatte Girish C. , Dasgupta Biplab , Perry Arie , Gutmann David H. , Rubin Joshua B.
Published: March 2023

Abstract
Astrocytoma (glioma) formation in neurofibromatosis type 1 (NF1) occurs preferentially along the optic pathway during the first decade of life. The molecular basis for this unique pattern of gliomagenesis is unknown. Previous studies in mouse Nf1 optic glioma models suggest that this patterning results from cooperative effects of Nf1 loss in glial cells and the action of factors derived from the surrounding Nf1+/− brain. Because CXCL12 is a stroma-derived growth factor for malignant brain tumors, we tested the hypothesis that CXCL12 functions in concert with Nf1 loss to facilitate NF1-associated glioma growth. Whereas CXCL12 promoted cell death in wild-type astrocytes, it increased Nf1−/− astrocyte survival. This increase in Nf1−/− astrocyte survival in response to CXCL12 was due to sustained suppression of intracellular cyclic AMP (cAMP) levels. Moreover, the ability of CXCL12 to suppress cAMP and increase Nf1−/− astrocyte survival was a consequence of mitogen-activated protein/extracellular signal-regulated kinase kinase–dependent inhibition of CXCL12 receptor (CXCR4) desensitization. In support of an instructive role for CXCL12 in facilitating optic glioma growth, we also show that CXCL12 expression along the optic pathway is higher in infant children and young mice and is associated with low levels of cAMP. CXCL12 expression declines in multiple brain regions with increasing age, correlating with the age-dependent decline in glioma growth in children with NF1. Collectively, these studies provide a mechanism for the unique pattern of NF1-associated glioma growth. [Cancer Res 2007;67(18):8588–95]
Data from Intraprostatic Androgens and Androgen-Regulated Gene Expression Persist after Testosterone Suppression: Therapeutic Implications for Castration-Resistant Prostate Cancer
Authors: Mostaghel Elahe A. , Page Stephanie T. , Lin Daniel W. , Fazli Ladan , Coleman Ilsa M. , True Lawrence D. , Knudsen Beatrice , Hess David L. , Nelson Colleen C. , Matsumoto Alvin M. , Bremner William J. , Gleave Martin E. , Nelson Peter S.
Published: March 2023

Abstract
Androgen deprivation therapy (ADT) remains the primary treatment for advanced prostate cancer. The efficacy of ADT has not been rigorously evaluated by demonstrating suppression of prostatic androgen activity at the target tissue and molecular level. We determined the efficacy and consistency of medical castration in suppressing prostatic androgen levels and androgen-regulated gene expression. Androgen levels and androgen-regulated gene expression (by microarray profiling, quantitative reverse transcription-PCR, and immunohistochemistry) were measured in prostate samples from a clinical trial of short-term castration (1 month) using the gonadotropin-releasing hormone antagonist, Acyline, versus placebo in healthy men. To assess the effects of long-term ADT, gene expression measurements were evaluated at baseline and after 3, 6, and 9 months of neoadjuvant ADT in prostatectomy samples from men with localized prostate cancer. Medical castration reduced tissue androgens by 75% and reduced the expression of several androgen-regulated genes (NDRG1, FKBP5, and TMPRSS2). However, many androgen-responsive genes, including the androgen receptor (AR) and prostate-specific antigen (PSA), were not suppressed after short-term castration or after 9 months of neoadjuvant ADT. Significant heterogeneity in PSA and AR protein expression was observed in prostate cancer samples at each time point of ADT. Medical castration based on serum testosterone levels cannot be equated with androgen ablation in the prostate microenvironment. Standard androgen deprivation does not consistently suppress androgen-dependent gene expression. Suboptimal suppression of tumoral androgen activity may lead to adaptive cellular changes allowing prostate cancer cell survival in a low androgen environment. Optimal clinical efficacy will require testing of novel approaches targeting complete suppression of systemic and intracrine contributions to the prostatic androgen microenvironment. [Cancer Res 2007;67(10):5033–41]
Data from Bortezomib Inhibits Cell-Cell Adhesion and Cell Migration and Enhances Epidermal Growth Factor Receptor Inhibitor–Induced Cell Death in Squamous Cell Cancer
Authors: Lorch Jochen H. , Thomas Tarita O. , Schmoll Hans-Joachim
Published: March 2023

Abstract
The lack of cell-cell adhesion and increased migration are key characteristics of cancer cells. The loss of expression of cell adhesion components and overexpression of components critical for cell migration, such as focal adhesion kinase (FAK), correlate with poor prognosis. Because alteration of protein turnover affects the expression levels and, in turn, may influence protein function, we investigated the effects of the proteasome inhibitor bortezomib on cell adhesion and migration in oral squamous cell cancer cell lines SCC68 and SCC15. Following treatment with bortezomib, protein levels of adherens junction components such as E-cadherin were unchanged. The desmosomal linker protein desmoplakin level was increased, whereas the protein level of the desmosomal cadherin, desmoglein 2, was diminished. Reduced desmoglein 2 levels correlated with the diminished strength of mechanical cell-cell adhesion. The protein level of the epidermal growth factor receptor (EGFR) increased after proteasome inhibition and EGFR inhibition with the EGFR-specific tyrosine kinase inhibitor PKI166 was able to restore cell-cell adhesion. Furthermore, we found that the combination of PKI166 with bortezomib enhanced the rate of cell death. Although the FAK protein level was unchanged following bortezomib treatment, recruitment of FAK phosphorylated at tyrosine residue 397 to the periphery of the cell was induced. Migration was reduced following treatment with bortezomib, which could potentially be explained by a prominent but disorganized actin fiber network revealed through immunofluorescence. Collectively, our results suggest that proteasome inhibition using bortezomib affects cell adhesion and cell migration profoundly and provides a rationale for its clinical use in conjunction with an EGFR inhibitor. [Cancer Res 2007;67(2):727–34]
Data from High Expression of Ligands for Chemokine Receptor CXCR2 in Alveolar Epithelial Neoplasia Induced by Oncogenic Kras
Authors: Wislez Marie , Fujimoto Nobukazu , Izzo Julie G. , Hanna Amy E. , Cody Dianna D. , Langley Robert R. , Tang Hongli , Burdick Marie D. , Sato Mitsuo , Minna John D. , Mao Li , Wistuba Ignacio , Strieter Robert M. , Kurie Jonathan M.
Published: March 2023

Abstract
CXCL8, a ligand for the chemokine receptor CXCR2, was recently reported to be a transcriptional target of Ras signaling, but its role in Ras-induced tumorigenesis has not been fully defined. Here, we investigated the role of KC and MIP-2, the murine homologues of CXCL8, in KrasLA1 mice, which develop lung adenocarcinoma owing to somatic activation of the KRAS oncogene. We first investigated biological evidence of CXCR2 ligands in KrasLA1 mice. Malignant progression of normal alveolar epithelial cells to adenocarcinoma in KrasLA1 mice was associated with enhanced intralesional vascularity and neutrophilic inflammation, which are hallmarks of chemoattraction by CXCR2 ligands. In in vitro migration assays, supernatants of bronchoalveolar lavage samples from KrasLA1 mice chemoattracted murine endothelial cells, alveolar inflammatory cells, and the LKR-13 lung adenocarcinoma cell line derived from KrasLA1 mice, an effect that was abrogated by pretreatment of the cells with a CXCR2-neutralizing antibody. CXCR2 and its ligands were highly expressed in LKR-13 cells and premalignant alveolar lesions in KrasLA1 mice. Treatment of KrasLA1 mice with a CXCR2-neutralizing antibody inhibited the progression of premalignant alveolar lesions and induced apoptosis of vascular endothelial cells within alveolar lesions. Whereas the proliferation of LKR-13 cells in vitro was resistant to treatment with the antibody, LKR-13 cells established as syngeneic tumors were sensitive, supporting a role for the tumor microenvironment in the activity of CXCR2. Thus, high expression of CXCR2 ligands may contribute to the expansion of early alveolar neoplastic lesions induced by oncogenic KRAS. (Cancer Res 2006; 66(8): 4198-207)
Data from Novel Chemical Enhancers of Heat Shock Increase Thermal Radiosensitization through a Mitotic Catastrophe Pathway
Authors: Sekhar Konjeti R. , Sonar Vijayakumar N. , Muthusamy Venkatraj , Sasi Soumya , Laszlo Andrei , Sawani Jamil , Horikoshi Nobuo , Higashikubo Ryuji , Bristow Robert G. , Borrelli Michael J. , Crooks Peter A. , Lepock James R. , Roti Roti Joseph L. , Freeman Michael L.
Published: March 2023

Abstract
Radiation therapy combined with adjuvant hyperthermia has the potential to provide outstanding local-regional control for refractory disease. However, achieving therapeutic thermal dose can be problematic. In the current investigation, we used a chemistry-driven approach with the goal of designing and synthesizing novel small molecules that could function as thermal radiosensitizers. (Z)-(±)-2-(1-Benzenesulfonylindol-3-ylmethylene)-1-azabicyclo[2.2.2]octan-3-ol was identified as a compound that could lower the threshold for Hsf1 activation and thermal sensitivity. Enhanced thermal sensitivity was associated with significant thermal radiosensitization. We established the structural requirements for activity: the presence of an N-benzenesulfonylindole or N-benzylindole moiety linked at the indolic 3-position to a 2-(1-azabicyclo[2.2.2]octan-3-ol) or 2-(1-azabicyclo[2.2.2]octan-3-one) moiety. These small molecules functioned by exploiting the underlying biophysical events responsible for thermal sensitization. Thermal radiosensitization was characterized biochemically and found to include loss of mitochondrial membrane potential, followed by mitotic catastrophe. These studies identified a novel series of small molecules that represent a promising tool for the treatment of recurrent tumors by ionizing radiation. [Cancer Res 2007;67(2):695–701]
Data from The Oncogenic Epidermal Growth Factor Receptor Variant Xiphophorus Melanoma Receptor Kinase Induces Motility in Melanocytes by Modulation of Focal Adhesions
Authors: Meierjohann Svenja , Wende Elisabeth , Kraiss Anita , Wellbrock Claudia , Schartl Manfred
Published: March 2023

Abstract
One of the most prominent features of malignant melanoma is the fast generation of metastasizing cells, resulting in the poor prognosis of patients with this tumor type. For this process, cells must gain the ability to migrate. The oncogenic receptor Xmrk (Xiphophorus melanoma receptor kinase) from the Xiphophorus melanoma system is a mutationally activated version of the epidermal growth factor receptor that induces the malignant transformation of pigment cells. Here, we show that the activation of Xmrk leads to a clear increase of pigment cell motility in a fyn-dependent manner. Stimulation of Xmrk induces its interaction with the focal adhesion kinase (FAK) and the interaction of active, receptor-bound fyn with FAK. This results in changes in FAK activity and induces the modulation of stress fibers and focal adhesions. Overexpression of dominant-negative FAK shows that the activity of innate FAK and a receptor-induced focal adhesion turnover are a prerequisite for pigment cell migration. Our findings show that in our system, Xmrk is sufficient for the induction of pigment cell motility and underlines a role of the src family protein tyrosine kinase fyn in melanoma development and progression. (Cancer Res 2006; 66(6): 3145-52)
Data from Adoptive Transfer of Type 1 CTL Mediates Effective Anti–Central Nervous System Tumor Response: Critical Roles of IFN-Inducible Protein-10
Authors: Nishimura Fumihiko , Dusak Jill E. , Eguchi Junichi , Zhu Xinmei , Gambotto Andrea , Storkus Walter J. , Okada Hideho
Published: March 2023

Abstract
The development of effective immunotherapeutic strategies for central nervous system (CNS) tumors requires a firm understanding of factors regulating the trafficking of tumor antigen–specific CTLs into CNS tumor lesions. Using C57BL/6 mice bearing intracranial (i.c.) ovalbumin-transfected melanoma (M05), we evaluated the efficacy and tumor homing of i.v. transferred type 1 or 2 CTLs (Tc1 or Tc2, respectively) prepared from ovalbumin-specific T-cell receptor–transgenic OT-1 mice. We also tested our hypothesis that intratumoral (i.t.) delivery of dendritic cells that had been transduced with IFN-α cDNA (DC-IFN-α) would enhance the tumor-homing and antitumor effectiveness of adoptively transferred Tc1 via induction of an IFN-γ-inducible protein 10 (IP-10). In vitro, DC-IFN-α induced IP-10 production by M05 and enhanced the cytolytic activity of Tc1. In vivo, i.v. transferred Tc1 trafficked efficiently into i.c. M05 and mediated antitumor responses more effectively than Tc2, and their effect was IP-10 dependent. I.t. injections of DC-IFN-α remarkably enhanced the tumor homing, therapeutic efficacy, and in situ IFN-γ production of i.v. delivered Tc1, resulting in the long-term survival and persistence of systemic ovalbumin-specific immunity. These data suggest that Tc1-based adoptive transfer therapy may represent an effective modality for CNS tumors, particularly when combined with strategies that promote a type 1 polarized tumor microenvironment. (Cancer Res 2006; 66(8): 4478-87)
Data from Human Tumor-Derived Exosomes Selectively Impair Lymphocyte Responses to Interleukin-2
Authors: Clayton Aled , Mitchell J. Paul , Court Jacquelyn , Mason Malcolm D. , Tabi Zsuzsanna
Published: March 2023

Abstract
Exosomes are nanometer-sized vesicles, secreted by normal and neoplastic cells. The outcome following interaction between the cellular immune system and cancer-derived exosomes is not well understood. Interleukin-2 (IL-2) is a key factor supporting expansion and differentiation of CTL and natural killer (NK) cells but can also support regulatory T cells and their suppressive functions. Our study examined whether tumor-derived exosomes could modify lymphocyte IL-2 responses. Proliferation of healthy donor peripheral blood lymphocytes in response to IL-2 was inhibited by tumor exosomes. In unfractionated lymphocytes, this effect was seen in all cell subsets. Separating CD4+ T cells, CD8+ T cells, and NK cells revealed that CD8+ T-cell proliferation was not inhibited in the absence of CD4+ T cells and that NK cell proliferation was only slightly impaired. Other exosome effects included selective impairment of IL-2–mediated CD25 up-regulation, affecting all but the CD3+CD8− T-cell subset. IL-2–induced Foxp3 expression by CD4+CD25+ cells was not inhibited by tumor exosomes, and the suppressive function of CD4+CD25+ T cells was enhanced by exosomes. In contrast, exosomes directly inhibited NK cell killing function in a T-cell–independent manner. Analysis of tumor exosomes revealed membrane-associated transforming growth factor β1 (TGFβ1), which contributed to the antiproliferative effects, shown by using neutralizing TGFβ1-specific antibody. The data show an exosome-mediated mechanism of skewing IL-2 responsiveness in favor of regulatory T cells and away from cytotoxic cells. This coordinated “double hit” to cellular immunity strongly implicates the role of exosomes in tumor immune evasion. [Cancer Res 2007;67(15):7458–66]
Data from Identification of Differentially Regulated Splice Variants and Novel Exons in Glial Brain Tumors Using Exon Expression Arrays
Authors: French Pim J. , Peeters Justine , Horsman Sebastiaan , Duijm Elza , Siccama Ivar , van den Bent Martin J. , Luider Theo M. , Kros Johan M. , van der Spek Peter , Sillevis Smitt Peter A.
Published: March 2023

Abstract
Aberrant splice variants are involved in the initiation and/or progression of glial brain tumors. We therefore set out to identify splice variants that are differentially expressed between histologic subgroups of gliomas. Splice variants were identified using a novel platform that profiles the expression of virtually all known and predicted exons present in the human genome. Exon-level expression profiling was done on 26 glioblastomas, 22 oligodendrogliomas, and 6 control brain samples. Our results show that Human Exon arrays can identify subgroups of gliomas based on their histologic appearance and genetic aberrations. We next used our expression data to identify differentially expressed splice variants. In two independent approaches, we identified 49 and up to 459 exons that are differentially spliced between glioblastomas and oligodendrogliomas, a subset of which (47% and 33%) were confirmed by reverse transcription-PCR (RT-PCR). In addition, exon level expression profiling also identified >700 novel exons. Expression of ∼67% of these candidate novel exons was confirmed by RT-PCR. Our results indicate that exon level expression profiling can be used to molecularly classify brain tumor subgroups, can identify differentially regulated splice variants, and can identify novel exons. The splice variants identified by exon level expression profiling may help to detect the genetic changes that cause or maintain gliomas and may serve as novel treatment targets. [Cancer Res 2007;67(12):5635–8]
Data from Mesenchymal-to-Epithelial Transition Facilitates Bladder Cancer Metastasis: Role of Fibroblast Growth Factor Receptor-2
Authors: Chaffer Christine L. , Brennan Janelle P. , Slavin John L. , Blick Tony , Thompson Erik W. , Williams Elizabeth D.
Published: March 2023

Abstract
Epithelial-to-mesenchymal transition (EMT) increases cell migration and invasion, and facilitates metastasis in multiple carcinoma types, but belies epithelial similarities between primary and secondary tumors. This study addresses the importance of mesenchymal-to-epithelial transition (MET) in the formation of clinically significant metastasis. The previously described bladder carcinoma TSU-Pr1 (T24) progression series of cell lines selected in vivo for increasing metastatic ability following systemic seeding was used in this study. It was found that the more metastatic sublines had acquired epithelial characteristics. Epithelial and mesenchymal phenotypes were confirmed in the TSU-Pr1 series by cytoskeletal and morphologic analysis, and by performance in a panel of in vitro assays. Metastatic ability was examined following inoculation at various sites. Epithelial characteristics associated with dramatically increased bone and soft tissue colonization after intracardiac or intratibial injection. In contrast, the more epithelial sublines showed decreased lung metastases following orthotopic inoculation, supporting the concept that EMT is important for the escape of tumor cells from the primary tumor. We confirmed the overexpression of the IIIc subtype of multiple fibroblast growth factor receptors (FGFR) through the TSU-Pr1 series, and targeted abrogation of FGFR2IIIc reversed the MET and associated functionality in this system and increased survival following in vivo inoculation in severe combined immunodeficient mice. This model is the first to specifically model steps of the latter part of the metastatic cascade in isogenic cell lines, and confirms the suspected role of MET in secondary tumor growth. (Cancer Res 2006; 66(23): 11271-8)
Data from Selenium Regulates Cyclooxygenase-2 and Extracellular Signal-Regulated Kinase Signaling Pathways by Activating AMP-Activated Protein Kinase in Colon Cancer Cells
Authors: Hwang Jin-Taek , Kim Young Min , Surh Young-Joon , Baik Haing Woon , Lee Seong-Kyu , Ha Joohun , Park Ock Jin
Published: March 2023

Abstract
Epidemiologic and experimental evidences indicate that selenium, an essential trace element, can reduce the risk of a variety of cancers. Protection against certain types of cancers, particularly colorectal cancers, is closely associated with pathways involving cyclooxygenase-2 (COX-2). We found that AMP-activated protein kinase (AMPK), which functions as a cellular energy sensor, mediates critical anticancer effects of selenium via a COX-2/prostaglandin E2 signaling pathway. Selenium activated AMPK in tumor xenografts as well as in colon cancer cell lines, and this activation seemed to be essential to the decrease in COX-2 expressions. Transduction with dominant-negative AMPK into colon cancer cells or application of cox-2−/−-negative cells supported the evidence that AMPK is an upstream signal of COX-2 and inhibits cell proliferation. In HT-29 colon cancer cells, carcinogenic agent 12-O-tetradecanoylphorbol-13-acetate (TPA) activated extracellular signal-regulated kinase (ERK) that led to COX-2 expression and selenium blocked the TPA-induced ERK and COX-2 activation via AMPK. We also showed the role of a reactive oxygen species as an AMPK activation signal in selenium-treated cells. We propose that AMPK is a novel and critical regulatory component in selenium-induced cancer cell death, further implying AMPK as a prime target of tumorigenesis. (Cancer Res 2006; 66(20): 10057-63)
Data from Comparative Genome Analysis Identifies the Vitamin D Receptor Gene as a Direct Target of p53-Mediated Transcriptional Activation
Authors: Maruyama Reo , Aoki Fumio , Toyota Minoru , Sasaki Yasushi , Akashi Hirofumi , Mita Hiroaki , Suzuki Hiromu , Akino Kimishige , Ohe-Toyota Mutsumi , Maruyama Yumiko , Tatsumi Haruyuki , Imai Kohzoh , Shinomura Yasuhisa , Tokino Takashi
Published: March 2023

Abstract
p53 is the most frequently mutated tumor suppressor gene in human neoplasia and encodes a transcriptional coactivator. Identification of p53 target genes is therefore key to understanding the role of p53 in tumorigenesis. To identify novel p53 target genes, we first used a comparative genomics approach to identify p53 binding sequences conserved in the human and mouse genome. We hypothesized that potential p53 binding sequences that are conserved are more likely to be functional. Using stringent filtering procedures, 32 genes were newly identified as putative p53 targets, and their responsiveness to p53 in human cancer cells was confirmed by reverse transcription-PCR and real-time PCR. Among them, we focused on the vitamin D receptor (VDR) gene because vitamin D3 has recently been used for chemoprevention of human tumors. VDR is induced by p53 as well as several other p53 family members, and analysis of chromatin immunoprecipitation showed that p53 protein binds to conserved intronic sequences of the VDR gene in vivo. Introduction of VDR into cells resulted in induction of several genes known to be p53 targets and suppression of colorectal cancer cell growth. In addition, p53 induced VDR target genes in a vitamin D3-dependent manner. Our in silico approach is a powerful method for identification of functional p53 binding sites and p53 target genes that are conserved among humans and other organisms and for further understanding the function of p53 in tumorigenesis. (Cancer Res 2006; 66(9): 4574-83)
Data from Neurospheres Enriched in Cancer Stem–Like Cells Are Highly Effective in Eliciting a Dendritic Cell–Mediated Immune Response against Malignant Gliomas
Authors: Pellegatta Serena , Poliani Pietro Luigi , Corno Daniela , Menghi Francesca , Ghielmetti Francesco , Suarez-Merino Blanca , Caldera Valentina , Nava Sara , Ravanini Maria , Facchetti Fabio , Bruzzone Maria Grazia , Finocchiaro Gaetano
Published: March 2023

Abstract
Cancer stem–like cells (CSC) could be a novel target for cancer therapy, including dendritic cell (DC) immunotherapy. To address this, we developed experiments aimed at DC targeting of neurospheres (NS) from GL261 glioma cells because neurospheres can be enriched in CSC. We obtained murine neurospheres by growing GL261 cells in epidermal growth factor/basic fibroblast growth factor without serum. GL261-NS recapitulated important features of glioblastoma CSC and expressed higher levels of radial glia stem cell markers than GL261 cells growing under standard conditions (GL261 adherent cells, GL261-AC), as assessed by DNA microarray and real-time PCR. GL261-NS brain gliomas were highly infiltrating and more rapidly lethal than GL261-AC, as evidenced by survival analysis (P < 0.0001), magnetic resonance imaging and histology. DC from the bone marrow of syngeneic mice were then used for immunotherapy of GL261-NS and GL261-AC tumors. Strikingly, DC loaded with GL261-NS (DC-NS) cured 80% and 60% of GL261-AC and GL261-NS tumors, respectively (P < 0.0001), whereas DC-AC cured only 50% of GL261-AC tumors (P = 0.0022) and none of the GL261-NS tumors. GL261-NS expressed higher levels of MHC and costimulatory molecules (CD80 and CD86) than GL261-AC; the JAM assay indicated that DC-NS splenocytes had higher lytic activity than DC-AC splenocytes on both GL261-NS and GL261-AC, and immunohistochemistry showed that DC-NS vaccination was associated with robust tumor infiltration by CD8+ and CD4+ T lymphocytes. These findings suggest that DC targeting of CSC provides a higher level of protection against GL261 gliomas, a finding with potential implications for the design of clinical trials based on DC vaccination. (Cancer Res 2006; 66(21): 10247-52)
Data from OSI-930: A Novel Selective Inhibitor of Kit and Kinase Insert Domain Receptor Tyrosine Kinases with Antitumor Activity in Mouse Xenograft Models
Authors: Garton Andrew J. , Crew Andrew P.A. , Franklin Maryland , Cooke Andrew R. , Wynne Graham M. , Castaldo Linda , Kahler Jennifer , Winski Shannon L. , Franks April , Brown Eric N. , Bittner Mark A. , Keily John F. , Briner Paul , Hidden Chris , Srebernak Mary C. , Pirrit Carrie , O'Connor Matthew , Chan Anna , Vulevic Bojana , Henninger Dwight , Hart Karen , Sennello Regina , Li An-Hu , Zhang Tao , Richardson Frank , Emerson David L. , Castelhano Arlindo L. , Arnold Lee D. , Gibson Neil W.
Published: March 2023

Abstract
OSI-930 is a novel inhibitor of the receptor tyrosine kinases Kit and kinase insert domain receptor (KDR), which is currently being evaluated in clinical studies. OSI-930 selectively inhibits Kit and KDR with similar potency in intact cells and also inhibits these targets in vivo following oral dosing. We have investigated the relationships between the potency observed in cell-based assays in vitro, the plasma exposure levels achieved following oral dosing, the time course of target inhibition in vivo, and antitumor activity of OSI-930 in tumor xenograft models. In the mutant Kit–expressing HMC-1 xenograft model, prolonged inhibition of Kit was achieved at oral doses between 10 and 50 mg/kg and this dose range was associated with antitumor activity. Similarly, prolonged inhibition of wild-type Kit in the NCI-H526 xenograft model was observed at oral doses of 100 to 200 mg/kg, which was the dose level associated with significant antitumor activity in this model as well as in the majority of other xenograft models tested. The data suggest that antitumor activity of OSI-930 in mouse xenograft models is observed at dose levels that maintain a significant level of inhibition of the molecular targets of OSI-930 for a prolonged period. Furthermore, pharmacokinetic evaluation of the plasma exposure levels of OSI-930 at these effective dose levels provides an estimate of the target plasma concentrations that may be required to achieve prolonged inhibition of Kit and KDR in humans and which would therefore be expected to yield a therapeutic benefit in future clinical evaluations of OSI-930. (Cancer Res 2006; 66(2): 1015-24)
Data from New Potential Ligand-Receptor Signaling Loops in Ovarian Cancer Identified in Multiple Gene Expression Studies
Authors: Castellano Giancarlo , Reid James F. , Alberti Paola , Carcangiu Maria Luisa , Tomassetti Antonella , Canevari Silvana
Published: March 2023

Abstract
Based on the hypothesis that gene products involved in the same biological process would be coupled at transcriptional level, a previous study analyzed the correlation of the gene expression patterns of ligand-receptor (L-R) pairs to discover potential autocrine/paracrine signaling loops in different cancers (Graeber and Eisenberg. Nat Genet 2001; 29:295). By refining the starting database, a list of 511 L-R pairs was compiled, combined to eight data sets from a single pathology, epithelial ovarian cancer, and examined as a proof-of-principle of the statistical and biological validity of the correlation of the L-R gene expression patterns in cancer. Analysis revealed a Bonferroni-corrected significant correlation of 105 L-R pairs in at least one data set and, by systematic analysis, identified 39 more frequently correlated L-R pairs, 7 of which were already biologically confirmed. In four data sets examined for an L-R correlation associated with patient survival time, 15 L-R pairs were significantly correlated in short surviving patients in two of the data sets. Immunohistochemical analysis of one of the newly identified correlated L-R pairs (i.e., EFNB3-EPHB4) revealed the correlated expression of ephrin-B3 and EphB4 proteins in 45 of 55 epithelial ovarian tumor samples (P < 0.0001). Together, these data not only support the validity of cross-comparison analysis of gene expression data because known and expected correlations were confirmed but also point to the promise of such analysis in identifying new L-R signaling loops in cancer. (Cancer Res 2006; 66(22): 10709-19)
Data from Distinguishing Cancer-Associated Missense Mutations from Common Polymorphisms
Authors: Kaminker Joshua S. , Zhang Yan , Waugh Allison , Haverty Peter M. , Peters Brock , Sebisanovic Dragan , Stinson Jeremy , Forrest William F. , Bazan J. Fernando , Seshagiri Somasekar , Zhang Zemin
Published: March 2023

Abstract
Missense variants are commonly identified in genomic sequence but only a small fraction directly contribute to oncogenesis. The ability to distinguish those missense changes that contribute to cancer progression from those that do not is a difficult problem usually only accomplished through functional in vivo analyses. Using two computational algorithms, Sorting Intolerant from Tolerant (SIFT) and the Pfam-based LogR.E-value method, we have identified features that distinguish cancer-associated missense mutations from other classes of missense change. Our data reveal that cancer mutants behave similarly to Mendelian disease mutations, but are clearly distinct from either complex disease mutations or common single-nucleotide polymorphisms. We show that both activating and inactivating oncogenic mutations are predicted to be deleterious, although activating changes are likely to increase protein activity. Using the Gene Ontology and data from the SIFT and LogR.E-value metrics, a classifier was built that predicts cancer-associated missense mutations with a very low false-positive rate. The classifier does remarkably well in a number of different experiments designed to distinguish polymorphisms from true cancer-associated mutations. We also show that recurrently observed mutations are much more likely to be predicted to be cancer-associated than rare mutations, suggesting that our classifier will be useful in distinguishing causal from passenger mutations. In addition, from an expressed sequence tag–based screen, we identified a previously unknown germ line change (P1104A) in tumor tissues that is predicted to disrupt the function of the TYK2 protein. The data presented here show that this novel bioinformatics approach to classifying cancer-associated variants is robust and can be used for large-scale analyses. [Cancer Res 2007;67(2):465–73]
Data from Phage Display–Derived Human Monoclonal Antibodies Isolated by Binding to the Surface of Live Primary Breast Cancer Cells Recognize GRP78
Authors: Jakobsen Charlotte G. , Rasmussen Nicolaj , Laenkholm Anne-Vibeke , Ditzel Henrik J.
Published: March 2023

Abstract
Clinical trials using monoclonal antibodies (mAb) against cell-surface markers have yielded encouraging therapeutic results in several cancer types. Generally, however, anticancer antibodies are only efficient against a subpopulation of cancers, and there is a strong need for identification of novel targets and human antibodies against them. We have isolated single-chain human mAbs from a large naïve antibody phage display library by panning on a single-cell suspension of freshly isolated live cancer cells from a human breast cancer specimen, and these antibodies were shown to specifically recognize cancer-associated cell-surface proteins. One of the isolated human antibody fragments, Ab39, recognizes a cell-surface antigen expressed on a subpopulation of cancer cell lines of different origins. Immunohistochemical analysis of a large panel of cancerous and normal tissues showed that Ab39 bound strongly to several cancers, including 45% breast carcinomas, 35% lung cancers, and 86% melanomas, but showed no or weak binding to normal tissues. A yeast two-hybrid screen of a large human testis cDNA library identified the glucose-regulated protein of 78 kDa (GRP78) as the antigen recognized by Ab39. The interaction was confirmed by colocalization studies and antibody competition experiments that also mapped the epitope recognized by Ab39 to the COOH terminus of GRP78. The expression of GRP78 on the surface of cancer cells, but not normal cells, makes it an attractive target for cancer therapies including mAb-based immunotherapy. Our results suggest that the human antibody Ab39 may be a useful starting point for further genetic optimization that could render it a useful diagnostic and therapeutic reagent for a variety of cancers. [Cancer Res 2007;67(20):9507–17]
Data from An Improved Bioluminescence Resonance Energy Transfer Strategy for Imaging Intracellular Events in Single Cells and Living Subjects
Authors: De Abhijit , Loening Andreas Markus , Gambhir Sanjiv Sam
Published: March 2023

Abstract
Bioluminescence resonance energy transfer (BRET) is currently used for monitoring various intracellular events, including protein-protein interactions, in normal and aberrant signal transduction pathways. However, the BRET vectors currently used lack adequate sensitivity for imaging events of interest from both single living cells and small living subjects. Taking advantage of the critical relationship of BRET efficiency and donor quantum efficiency, we report generation of a novel BRET vector by fusing a GFP2 acceptor protein with a novel mutant Renilla luciferase donor selected for higher quantum yield. This new BRET vector shows an overall 5.5-fold improvement in the BRET ratio, thereby greatly enhancing the dynamic range of the BRET signal. This new BRET strategy provides a unique platform to assay protein functions from both single live cells and cells located deep within small living subjects. The imaging utility of the new BRET vector is shown by constructing a sensor using two mammalian target of rapamycin pathway proteins (FKBP12 and FRB) that dimerize only in the presence of rapamycin. This new BRET vector should facilitate high-throughput sensitive BRET assays, including studies in single live cells and small living subjects. Applications will include anticancer therapy screening in cell culture and in small living animals. [Cancer Res 2007;67(15):7175–83]
Data from FRA-1 Proto-Oncogene Induces Lung Epithelial Cell Invasion and Anchorage-Independent Growth In vitro, but Is Insufficient to Promote Tumor Growth In vivo
Authors: Adiseshaiah Pavan , Lindner Daniel J. , Kalvakolanu Dhananjaya V. , Reddy Sekhar P.
Published: March 2023

Abstract
FRA-1 forms activator protein-1 complexes in association with members of the JUN family and drives gene transcription. FRA-1 has been implicated in the development of airway squamous metaplasia and is frequently overexpressed in squamous cell carcinomas of the esophagus and stomach. We and others have shown a high level of persistent induction of FRA-1 by lung carcinogens, such as cigarette smoke and asbestos, in pulmonary epithelial cells. However, the exact roles of FRA-1 in regulating lung epithelial cell growth and invasion are poorly understood. To examine this aspect, we have stably overexpressed FRA-1 in human type-II–like alveolar malignant cell line (A549) and a nonmalignant bronchial epithelial cell line (BEAS-2B). FRA-1 greatly enhanced the rate of proliferation, motility, and invasion of A549 and BEAS-2B cells. In athymic nude mice, FRA-1, but not the control vector, rapidly enhanced tumor formation and metastasis by A549 cells. In contrast, FRA-1 failed to promote tumor formation by BEAS-2B. We suggest that FRA-1 can promote motility, invasion, and anchorage-independent growth of lung epithelial cells in vitro, but is insufficient for tumor formation. [Cancer Res 2007;67(13):6204–11]
Data from Armed Antibodies Targeting the Mucin Repeats of the Ovarian Cancer Antigen, MUC16, Are Highly Efficacious in Animal Tumor Models
Authors: Chen Youjun , Clark Suzanna , Wong Terence , Chen Yongmei , Chen Yvonne , Dennis Mark S. , Luis Elizabeth , Zhong Fiona , Bheddah Sheila , Koeppen Hartmut , Gogineni Alvin , Ross Sarajane , Polakis Paul , Mallet William
Published: March 2023

Abstract
MUC16 is a well-validated cell surface marker for serous adenocarcinomas of the ovary and other gynecologic malignancies that is distinguished by highly repetitive sequences (“mucin repeats”) in the extracellular domain (ECD). We produced and compared two monoclonal antibodies: one (11D10) recognizing a unique, nonrepeating epitope in the ECD and another (3A5) that recognizes the repeats and binds multiple sites on each MUC16 protein. 3A5 conjugated to cytotoxic drugs exhibited superior toxicity against tumor cells in vitro and in tumor xenograft models compared with antibody-drug conjugates of 11D10. Importantly, drug conjugates of 3A5 were well tolerated in primates at levels in excess of therapeutic doses. Additionally, the presence of circulating CA125 in a rat model did not exacerbate the toxicity of 3A5 drug conjugates. We conclude that targeting the repeat MUC16 domains, thereby increasing cell-associated levels of drug-conjugated antibody, provides superior efficacy in vitro and in vivo without compromising safety. [Cancer Res 2007;67(10):4924–32]
Data from Bin1 Ablation Increases Susceptibility to Cancer during Aging, Particularly Lung Cancer
Authors: Chang Mee Young , Boulden Janette , Katz Jessica B. , Wang Liwei , Meyer Thomas J. , Soler Alejandro Peralta , Muller Alexander J. , Prendergast George C.
Published: March 2023

Abstract
Age is the major risk factor for cancer, but few genetic pathways that modify cancer incidence during aging have been described. Bin1 is a prototypic member of the BAR adapter gene family that functions in vesicle dynamics and nuclear processes. Bin1 limits oncogenesis and is often attenuated in human cancers, but its role in cancer suppression has yet to be evaluated fully in vivo. In the mouse, homozygous deletion of Bin1 causes developmental lethality, so to assess this role, we examined cancer incidence in mosaic null mice generated by a modified Cre-lox technology. During study of these animals, one notable phenotype was an extended period of female fecundity during aging, with mosaic null animals retaining reproductive capability until the age of 17.3 ± 1.1 months. Through 1 year of age, cancer incidence was unaffected by Bin1 ablation; however, by 18 to 20 months of age, ∼50% of mosaic mice presented with lung adenocarcinoma and ∼10% with hepatocarcinoma. Aging mosaic mice also displayed a higher incidence of inflammation and/or premalignant lesions, especially in the heart and prostate. In mice where colon tumors were initiated by a ras-activating carcinogen, Bin1 ablation facilitated progression to more aggressive invasive status. In cases of human lung and colon cancers, immunohistochemical analyses evidenced frequent attenuation of Bin1 expression, paralleling observations in other solid tumors. Taken together, our findings highlight an important role for Bin1 as a negative modifier of inflammation and cancer susceptibility during aging. [Cancer Res 2007;67(16):7605–12]