bluuebunny/crossref_metadata_2025 huggingface dataset:
Specific Targeting of Tumor Angiogenesis by RGD-Conjugated Ultrasmall Superparamagnetic Iron Oxide Particles Using a Clinical 1.5-T Magnetic Resonance Scanner
Authors: Zhang Chunfu , Jugold Manfred , Woenne Eva C. , Lammers Twan , Morgenstern Bernd , Mueller Margareta M. , Zentgraf Hanswalter , Bock Michael , Eisenhut Michael , Semmler Wolfhard , Kiessling Fabian
Published: February 2007

Angiogenesis is essential for the development of malignant tumors and provides important targets for tumor diagnosis and therapy. To noninvasively assess the angiogenic profile of tumors, novel αvβ3 integrin–targeted ultrasmall superparamagnetic iron oxide particles (USPIOs) were designed and their specific uptake by endothelial cells was evaluated in vitro and in vivo. USPIOs were coated with 3-aminopropyltrimethoxysilane (APTMS) and conjugated with Arg-Gly-Asp (RGD) peptides. Accumulation in human umbilical vein endothelial cells (HUVECs) was evaluated using Prussian blue staining, transmission electron microscopy, magnetic resonance (MR) imaging, and inductively coupled plasma mass spectrometry. Uptake of RGD-USPIO by HUVECs was significantly increased when compared with unlabeled USPIO and could be competitively inhibited by addition of unbound RGD. The ability of the RGD-USPIO to noninvasively distinguish tumors with high (HaCaT-ras-A-5RT3) and lower (A431) area fractions of αvβ3 integrin–positive vessels was evaluated using a 1.5-T MR scanner. Indeed, after RGD-USPIO injection, there was a more pronounced decrease in T2 relaxation times in HaCaT-ras-A-5RT3 tumors than in A431 tumors. Furthermore, T2*-weighted images clearly identified the heterogeneous arrangement of vessels with αvβ3 integrins in HaCaT-ras-A-5RT3 tumors by an irregular signal intensity decrease. In contrast, in A431 tumors with predominantly small and uniformly distributed vessels, the signal intensity decreased more homogeneously. In summary, RGD-coupled, APTMS-coated USPIOs efficiently label αvβ3 integrins expressed on endothelial cells. Furthermore, these molecular MR imaging probes are capable of distinguishing tumors differing in the degree of αvβ3 integrin expression and in their angiogenesis profile even when using a clinical 1.5-T MR scanner. [Cancer Res 2007;67(4):1555–62]
Sp1 Is Required for Transforming Growth Factor-β–Induced Mesenchymal Transition and Migration in Pancreatic Cancer Cells
Authors: Jungert Kerstin , Buck Anita , Wichert Götz von , Adler Guido , König Alexander , Buchholz Malte , Gress Thomas M. , Ellenrieder Volker
Published: February 2007

Transition from a sessile epithelial phenotype to a migrating mesenchymal phenotype is a crucial step in transforming growth factor-β (TGF-β)–induced pancreatic cancer cell migration and invasion. These profound morphologic and functional alterations are associated with characteristic changes in TGF-β–regulated gene expression, defined by rapid repression of epithelial markers and a strong and sustained transcriptional induction of mesenchymal markers such as the intermediate filament vimentin. In this study, we have analyzed the role of the transcription factor Sp1 in TGF-β–induced and Smad-mediated gene regulation during epithelial to mesenchymal transition (EMT) and migration of pancreatic cancer cells. Here, we show that Sp1 is required for TGF-β–induced EMT, and that this function is especially mediated through transcriptional induction of vimentin. Our results emphasize the functional relevance of vimentin in TGF-β–induced EMT because prevention of its induction strongly reduces cell migration. Altogether, this study helps to better understand the role of Sp1 in TGF-β–induced progression of pancreatic cancer. It suggests that Sp1, via transcriptional induction of vimentin, cooperates with activated Smad complexes in mesenchymal transition and migration of pancreatic cancer cells upon TGF-β stimulation. [Cancer Res 2007;67(4):1563–70]
Toll-like Receptor-7 Tolerizes Malignant B Cells and Enhances Killing by Cytotoxic Agents
Authors: Shi Yonghong , White Dionne , He Liwei , Miller Richard L. , Spaner David E.
Published: February 2007

Chronic activation through Toll-like receptors (TLR) occurs in a number of pathologic settings, but has not been studied to the same extent as primary activation. TLR7, expressed by B cells and some dendritic cells, recognizes molecular patterns associated with viruses that can be mimicked by synthetic imidazoquinolines. In response to primary stimulation with the imidazoquinoline, S28690, human mononuclear cells produced tumor necrosis factor-α, but were unable to do so upon restimulation with S28690. This state of “tolerization” lasted at least 5 days. Using chronic lymphocytic leukemia B cells as a model to facilitate biochemical analysis, the tolerized state was found to be associated with altered receptor components, including down-regulated expression of TLR7 mRNA and decreased levels of interleukin-1 receptor-associated kinase 1. Tolerization was characterized by a transcriptionally regulated block in stress-activated protein kinase and nuclear factor κB activation, with relatively preserved activation of extracellular signal-regulated kinase (ERK). Tolerized chronic lymphocytic leukemia cells were found to be more sensitive to cytotoxic chemotherapeutic agents, in part through altered stress-activated protein kinase signaling pathways. This property of the TLR7-tolerized state may potentially be exploited in the treatment of B cell cancers. [Cancer Res 2007;67(4):1823–31
Imaging-Guided Gene Therapy of Experimental Gliomas
Authors: Jacobs Andreas H. , Rueger Maria Adele , Winkeler Alexandra , Li Hongfeng , Vollmar Stefan , Waerzeggers Yannic , Rueckriem Benedikt , Kummer Christiane , Dittmar Claus , Klein Markus , Heneka Michael T. , Herrlinger Ulrich , Fraefel Cornel , Graf Rudolf , Wienhard Klaus , Heiss Wolf-Dieter
Published: February 2007

To further develop gene therapy for patients with glioblastomas, an experimental gene therapy protocol was established comprising a series of imaging parameters for (i) noninvasive assessment of viable target tissue followed by (ii) targeted application of herpes simplex virus type 1 (HSV-1) amplicon vectors and (iii) quantification of treatment effects by imaging. We show that viable target tissue amenable for application of gene therapy vectors can be identified by multitracer positron emission tomography (PET) using 2-18F-fluoro-2-deoxy-d-glucose, methyl-11C-l-methionine, or 3′-deoxy-3′-18F-fluoro-l-thymidine ([18F]FLT). Targeted application of HSV-1 amplicon vectors containing two therapeutic genes with synergistic antitumor activity (Escherichia coli cytosine deaminase, cd, and mutated HSV-1 thymidine kinase, tk39, fused to green fluorescent protein gene, gfp) leads to an overall response rate of 68%, with 18% complete responses and 50% partial responses. Most importantly, we show that the “tissue dose” of HSV-1 amplicon vector–mediated gene expression can be noninvasively assessed by 9-[4-18F-fluoro-3-(hydroxymethyl)butyl]guanine ([18F]FHBG) PET. Therapeutic effects could be monitored by PET with significant differences in [18F]FLT accumulation in all positive control tumors and 72% in vivo transduced tumors (P = 0.01) as early as 4 days after prodrug therapy. For all stably and in vivo transduced tumors, cdIREStk39gfp gene expression as measured by [18F]FHBG-PET correlated with therapeutic efficiency as measured by [18F]FLT-PET. These data indicate that imaging-guided vector application with determination of tissue dose of vector-mediated gene expression and correlation to induced therapeutic effect using multimodal imaging is feasible. This strategy will help in the development of safe and efficient gene therapy protocols for clinical application. [Cancer Res 2007;67(4):1706–15]
Tumor-Suppressive Effect of Retinoid Receptor–Induced Gene-1 (RRIG1) in Esophageal Cancer
Authors: Huang Jie , Liang Zheng D. , Wu Tsung-Teh , Hoque Ashraful , Chen Hongli , Jiang Yan , Zhang Hongfu , Xu Xiao-chun
Published: February 2007

We previously showed that induction of retinoid receptor–induced gene-1 (RRIG1) expression inhibited RhoA activation and tumor cell colony formation, invasion, and proliferation, and these effects are associated with the suppression of extracellular signal-regulated protein kinases 1 and 2 phosphorylation and cyclooxygenase-2 expression. To further elucidate its role in tumor cell growth, gene expression, and tumorigenesis, we determined RRIG1 expression in breast and esophageal tissue specimens and then stably transfected RRIG1 into a TE-8 esophageal squamous cell carcinoma (SCC) cell line. We found that RRIG1 was expressed in normal mammary glands (10 of 10) but not all ductal carcinoma in situ [11 of 19 (57.9%), P = 0.018] and invasive cancer [14 of 30 (46.7%), P = 0.0023] tissues. Similarly, RRIG1 was expressed in normal esophageal epithelium (22 of 22) but not all dysplastic [6 of 43 (14%), P = 0.0001] and SCC [50 of 122 (41%), P = 0.0001] tissues. Furthermore, RRIG1 expression correlated positively with tumor differentiation but inversely with lymph node metastasis of esophageal SCC. Finally, the stable transfection of RRIG1 inhibited esophageal SCC cell growth and the expression of extracellular signal-regulated protein kinases 1 and 2 and cell cycle–related genes (e.g., cyclin D1, phosphorylated Rb, and E2F). RRIG1-transfected sublines also inhibited tumor development in nude mice. The results of this study indicate that RRIG1 plays a role in suppressing tumorigenesis. [Cancer Res 2007;67(4):1589–93]
Activation of TLX3 and NKX2-5 in t(5;14)(q35;q32) T-Cell Acute Lymphoblastic Leukemia by Remote 3′-BCL11B Enhancers and Coregulation by PU.1 and HMGA1
Authors: Nagel Stefan , Scherr Michaela , Kel Alexander , Hornischer Klaus , Crawford Gregory E. , Kaufmann Maren , Meyer Corinna , Drexler Hans G. , MacLeod Roderick A.F.
Published: February 2007

In T-cell acute lymphoblastic leukemia, alternative t(5;14)(q35;q32.2) forms effect dysregulation of either TLX3 or NKX2-5 homeobox genes at 5q35 by juxtaposition with 14q32.2 breakpoints dispersed across the BCL11B downstream genomic desert. Leukemic gene dysregulation by t(5;14) was investigated by DNA inhibitory treatments with 26-mer double-stranded DNA oligonucleotides directed against candidate enhancers at, or near, orphan T-cell DNase I hypersensitive sites located between 3′-BCL11B and VRK1. NKX2-5 down-regulation in t(5;14) PEER cells was almost entirely restricted to DNA inhibitory treatment targeting enhancers within the distal breakpoint cluster region and was dose and sequence dependent, whereas enhancers near 3′-BCL11B regulated that gene only. Chromatin immunoprecipitation assays showed that the four most effectual NKX2-5 ectopic enhancers were hyperacetylated. These enhancers clustered ∼1 Mbp downstream of BCL11B, within a region displaying multiple regulatory stigmata, including a TCRA enhancer motif, deep sequence conservation, and tight nuclear matrix attachment relaxed by trichostatin A treatment. Intriguingly, although TLX3/NKX2-5 promoter/exon 1 regions were hypoacetylated, their expression was trichostatin A sensitive, implying extrinsic regulation by factor(s) under acetylation control. Knockdown of PU.1, known to be trichostatin A responsive and which potentially binds TLX3/NKX2-5 promoters, effected down-regulation of both homeobox genes. Moreover, genomic analysis showed preferential enrichment near ectopic enhancers of binding sites for the PU.1 cofactor HMGA1, the knockdown of which also inhibited NKX2-5. We suggest that HMGA1 and PU.1 coregulate ectopic homeobox gene expression in t(5;14) T-cell acute lymphoblastic leukemia by interactions mediated at the nuclear matrix. Our data document homeobox gene dysregulation by a novel regulatory region at 3′-BCL11B responsive to histone deacetylase inhibition and highlight a novel class of potential therapeutic target amid noncoding DNA. [Cancer Res 2007;67(4):1461–71]
NGEP, a Prostate-Specific Plasma Membrane Protein that Promotes the Association of LNCaP Cells
Authors: Das Sudipto , Hahn Yoonsoo , Nagata Satoshi , Willingham Mark C. , Bera Tapan K. , Lee Byungkook , Pastan Ira
Published: February 2007

NGEP is a prostate-specific gene identified by analysis of expressed sequence tag databases. RNA analysis revealed two spliced forms of NGEP mRNA: a short form encoding a soluble protein (NGEP-S) and a long form encoding a polytopic membrane protein (NGEP-L). Transient expression of myc epitope–tagged NGEP-L showed that it was localized to the plasma membrane. We have now produced a specific antibody to the COOH terminus of NGEP-L and showed that it detects an ∼100-kDa protein in extracts of normal prostate and prostate cancers that contain high levels of NGEP mRNA. The antibody detects a protein that is highly expressed on the apical and the lateral surfaces of normal prostate and prostate cancer cells by immunohistochemistry. The antibody does not detect a protein in the prostate cancer cell line LNCaP, which has very low NGEP mRNA levels. To study NGEP function, two stable LNCaP cell lines were prepared by transfection with NGEP-L and shown to contain similar amounts of NGEP-L protein as human prostate. Confocal immunofluorescence showed that NGEP-L is present on the plasma membrane of the transfected LNCaP cells and is highly concentrated at cell:cell contact regions. Furthermore, as the cell density increased, the cells formed large aggregates. A specific RNA interference that lowered NGEP-L levels prevented formation of cell aggregates. Our results suggest that NGEP-L has a role in promoting cell contact–dependent interactions of LNCaP prostate cancer cells and also that NGEP is a promising immunotherapy target for prostate cancer. [Cancer Res 2007;67(4):1594–601]
The Triterpenoid 2-Cyano-3,12-dioxooleana-1,9-dien-28-oic Acid and Its Derivatives Elicit Human Lymphoid Cell Apoptosis through a Novel Pathway Involving the Unregulated Mitochondrial Permeability Transition Pore
Authors: Brookes Paul S. , Morse Kimberly , Ray Denise , Tompkins Andrew , Young Sara M. , Hilchey Shannon , Salim Suhail , Konopleva Marina , Andreeff Michael , Phipps Richard , Bernstein Steven H.
Published: February 2007

2-Cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) and its C28 imidazole and dinitrile derivatives are novel oleanane triterpenoids exhibiting promise as both therapeutic and preventative agents for cancer. Herein we show that these triterpenoids induce normal and malignant B-lymphoid cell apoptosis, with the C28 derivatives being more potent than CDDO, through a novel mitochondrial mechanism. We show using both normal and malignant human B cells, as well as isolated rat mitochondria, that CDDO directly interacts with a limited number of as yet undefined mitochondrial proteins. Such an interaction results in the loss of mitochondrial thiol status and the secondary modification of numerous mitochondrial protein thiols. Our data further suggest that such modifications result in the formation of high molecular weight protein aggregates that form “unregulated,” constitutively open, cyclosporin A–insensitive permeability transition (PT) pores. The formation of such PT pores results in the subsequent generation of mitochondrial superoxide and cell death. In total, our studies (a) suggest a novel mechanism of action for triterpenoid-induced cell death; (b) are among the first to directly support the existence of an unregulated PT pore formed by mitochondrial protein aggregates, as first proposed by Lemasters and colleagues; and (c) validate such an unregulated PT pore as a viable target for the development of new cancer therapeutics. [Cancer Res 2007;67(4):1793–802]
Suppression of the Hypoxia-Inducible Factor-1 Response in Cervical Carcinoma Xenografts by Proteasome Inhibitors
Authors: Birle Diana C. , Hedley David W.
Published: February 2007

Experimental data suggest therapeutic advantage from selective disruption of the hypoxia response. We recently found that the proteasome inhibitor bortezomib decreases tumor carbonic anhydrase IX (CAIX) expression in colon cancer patients and herein report a companion laboratory study to test if this effect was the result of hypoxia-inducible factor (HIF) inhibition. Human cervical (SiHa and Me180) and colon (RKO) carcinoma cell lines were treated with bortezomib or the structurally unrelated proteasome inhibitor MG132 in normoxic and hypoxic conditions in vitro. Two different in vivo experiments investigated bortezomib effects after single dose (2 mg/kg, 24 h) or longer exposure in severe combined immunodeficient mice bearing SiHa xenografts. Treatment with either drug produced accumulation of HIF-1α in vitro but strongly inhibited the production of CAIX and vascular endothelial growth factor (VEGF) under hypoxia. This correlated with more than 10-fold reduction in HIF-1 transcriptional activity under hypoxic conditions. A similar effect of bortezomib was seen in vivo, using the nitroimidazole probe EF5 to define regions of tumor hypoxia and a triple immunofluorescence technique to measure the spatial distributions of HIF-1α and CAIX. Plasma VEGF levels decreased by ∼90% during treatment with bortezomib, indicating that this agent can potently inhibit the hypoxia response in tumors. [Cancer Res 2007;67(4):1735–43]
Inhibition of CCAAT/Enhancer Binding Protein Family DNA Binding in Mouse Epidermis Prevents and Regresses Papillomas
Authors: Oh Won Jun , Rishi Vikas , Orosz Andras , Gerdes Michael J. , Vinson Charles
Published: February 2007

The CCAAT/enhancer binding proteins (C/EBP) are a family of B-ZIP DNA binding proteins that act as transcription factors to regulate growth and differentiation of many cell types, including keratinocytes. To examine the consequences of inhibiting the C/EBP family of transcription factors in skin, we generated transgenic mice that use the tetracycline system to conditionally express A-C/EBP, a dominant negative that inhibits the DNA binding of C/EBP family members. We expressed A-C/EBP in the basal layer of the skin epidermis during a two-step skin carcinogenesis protocol. A-C/EBP expression caused hyperplasia of the basal epidermis and increased apoptosis in the suprabasal epidermis. The mice developed fewer papillomas and had systemic hair loss. A-C/EBP expression caused C/EBPβ protein to disappear whereas C/EBPα, p53, Bax, and caspase-3 protein levels were dramatically up-regulated in the suprabasal layer. Primary keratinocytes recapitulate the A-C/EBP induction of cell growth and increase in p53 protein. A-C/EBP expression after papilloma development caused the papillomas to regress with an associated increase in apoptosis and up-regulation of p53 protein. Furthermore, A-C/EBP–expressing mice heterozygous for p53 were more susceptible to papilloma formation, suggesting that the suppression of papilloma formation has a p53-dependent mechanism. These results implicate DNA binding of C/EBP family members as a potential molecular therapeutic target. [Cancer Res 2007;67(4):1867–76]
Prostate-Derived ETS Factor Is a Mediator of Metastatic Potential through the Inhibition of Migration and Invasion in Breast Cancer
Authors: Turner David P. , Moussa Omar , Sauane Moira , Fisher Paul B. , Watson Dennis K.
Published: February 2007

Cell migration and invasion are critical events during the progression to metastasis. Without motile function, cancer cells are unable to leave the primary tumor site, invade through the basement membrane, and form secondary tumors. Expression of the epithelial-specific ETS factor prostate-derived ETS factor (PDEF) is reduced in human invasive breast tissue and lost in invasive breast cancer cell lines. Gain-of-function studies that examine different aspects of cell migration show that constitutive or inducible PDEF reexpression inhibits migration and invasion in multiple breast cancer cell lines, and loss-of-function studies show a stimulation of migration in noninvasive breast cancer cells. Furthermore, the introduction of PDEF into invasive breast cancer cells led to a remodeling of the actin cytoskeleton and altered focal adhesion localization and adherence levels. Cells expressing PDEF no longer form the defined morphologic polarity required for efficient, directional migration. Collectively, these data indicate that PDEF down-regulation in invasive breast cancer may promote actin-mediated cell migration through the extracellular matrix. [Cancer Res 2007;67(4):1618–25]
Ectopic Expression of Vascular Cell Adhesion Molecule-1 as a New Mechanism for Tumor Immune Evasion
Authors: Lin Ken-Yu , Lu Dan , Hung Chien-Fu , Peng Shiwen , Huang Lanqing , Jie Chunfa , Murillo Francisco , Rowley Jesse , Tsai Ya-Chea , He Liangmei , Kim Dae-Jin , Jaffee Elizabeth , Pardoll Drew , Wu T.-C.
Published: February 2007

Immune escape is an important reason why the immune system cannot control tumor growth, but how escape variants emerge during immunotherapy remains poorly understood. Here, we identify a new mechanism of tumor immune escape using an in vivo selection strategy. We generated a highly immune-resistant cancer cell line (P3) by subjecting a susceptible cancer cell line (P0/TC-1) to multiple rounds of in vivo immune selection. Microarray analysis of P0 and P3 revealed that vascular cell adhesion molecule-1 (VCAM-1) is up-regulated in the P3-resistant variant. Retroviral transfer of VCAM-1 into P0 significantly increased its resistance against a vaccine-induced immune response. Analysis of tumors showed a dramatic decrease in the number of tumor-infiltrating cluster of differentiation 8+ (CD8+) T cells in the tumors expressing VCAM-1. In vitro transwell migration assays showed that VCAM-1 can promote the migration of CD8+ T cells through its interaction with the α4β1 integrin. Site-directed mutagenesis of VCAM-1 at amino acid residues required for interaction with α4β1 integrin completely abolished the immune resistance conferred by VCAM-1 in vivo. Surface staining showed that most renal cell carcinomas (RCC) express VCAM-1, whereas an RCC that responded to vaccination was VCAM-1 negative. These data provide evidence that tumor expression of VCAM-1 represents a new mechanism of immune evasion and has important implications for the development of immunotherapy for human RCC. [Cancer Res 2007;67(4):1832–41]
Determination of Cancer Risk Associated with Germ Line BRCA1 Missense Variants by Functional Analysis
Authors: Carvalho Marcelo A. , Marsillac Sylvia M. , Karchin Rachel , Manoukian Siranoush , Grist Scott , Swaby Ramona F. , Urmenyi Turan P. , Rondinelli Edson , Silva Rosane , Gayol Luis , Baumbach Lisa , Sutphen Rebecca , Pickard-Brzosowicz Jennifer L. , Nathanson Katherine L. , Sali Andrej , Goldgar David , Couch Fergus J. , Radice Paolo , Monteiro Alvaro N.A.
Published: February 2007

Germ line inactivating mutations in BRCA1 confer susceptibility for breast and ovarian cancer. However, the relevance of the many missense changes in the gene for which the effect on protein function is unknown remains unclear. Determination of which variants are causally associated with cancer is important for assessment of individual risk. We used a functional assay that measures the transactivation activity of BRCA1 in combination with analysis of protein modeling based on the structure of BRCA1 BRCT domains. In addition, the information generated was interpreted in light of genetic data. We determined the predicted cancer association of 22 BRCA1 variants and verified that the common polymorphism S1613G has no effect on BRCA1 function, even when combined with other rare variants. We estimated the specificity and sensitivity of the assay, and by meta-analysis of 47 variants, we show that variants with <45% of wild-type activity can be classified as deleterious whereas variants with >50% can be classified as neutral. In conclusion, we did functional and structure-based analyses on a large series of BRCA1 missense variants and defined a tentative threshold activity for the classification missense variants. By interpreting the validated functional data in light of additional clinical and structural evidence, we conclude that it is possible to classify all missense variants in the BRCA1 COOH-terminal region. These results bring functional assays for BRCA1 closer to clinical applicability. [Cancer Res 2007;67(4):1494–501]
Lack of Extracellular Signal-Regulated Kinase Mitogen-Activated Protein Kinase Signaling Shows a New Type of Melanoma
Authors: Shields Janiel M. , Thomas Nancy E. , Cregger Melissa , Berger Aaron J. , Leslie Michael , Torrice Chad , Hao Honglin , Penland Shannon , Arbiser Jack , Scott Glynis , Zhou Tong , Bar-Eli Menashe , Bear James E. , Der Channing J. , Kaufmann William K. , Rimm David L. , Sharpless Norman E.
Published: February 2007

The majority of human melanomas harbor activating mutations of either N-RAS or its downstream effector B-RAF, which cause activation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase and the ERK MAPK cascade. The melanoma-relevant effectors of ERK activation, however, are largely unknown. In this work, we show that increased ERK activation correlates strongly with mutational status of N-RAS or B-RAF in 21 melanoma cell lines. Melanoma lines that were wild-type for RAS/RAF showed low levels of ERK activation comparable with primary human melanocytes. Through supervised analysis of RNA expression profiles, we identified 82 genes, including TWIST1, HIF1α, and IL-8, which correlated with ERK activation across the panel of cell lines and which decreased with pharmacologic inhibition of ERK activity, suggesting that they are ERK transcriptional targets in melanoma. Additionally, lines lacking mutations of N-RAS and B-RAF were molecularly distinct and characterized by p53 inactivation, reduced ERK activity, and increased expression of epithelial markers. Analysis of primary human melanomas by tissue microarray confirmed a high correlation among expression of these epithelial markers in a heterogeneous sample of 570 primary human tumors, suggesting that a significant frequency of primary melanomas is of this “epithelial-like” subtype. These results show a molecularly distinct melanoma subtype that does not require ERK activation or epithelial-mesenchymal transformation for progression. [Cancer Res 2007;67(4):1502–12]
Clioquinol, a Therapeutic Agent for Alzheimer's Disease, Has Proteasome-Inhibitory, Androgen Receptor–Suppressing, Apoptosis-Inducing, and Antitumor Activities in Human Prostate Cancer Cells and Xenografts
Authors: Chen Di , Cui Qiuzhi Cindy , Yang Huanjie , Barrea Raul A. , Sarkar Fazlul H. , Sheng Shijie , Yan Bing , Reddy G. Prem Veer , Dou Q. Ping
Published: February 2007

Tumor growth and metastasis depend on angiogenesis that requires the cofactor copper. Consistently, high levels of copper have been found in many types of human cancers, including prostate, breast, colon, and lung. Recent studies suggest that copper could be used as a novel selective target for cancer therapies. Clioquinol is capable of forming stable complexes with copper and currently used in clinics for treatment of Alzheimer's disease. Most recently, it has been reported that clioquinol possesses antitumor effects. However, the underlying molecular mechanism is unclear. We report here that after binding to copper, clioquinol can inhibit the proteasomal chymotrypsin-like activity, repress androgen receptor (AR) protein expression, and induce apoptotic cell death in human prostate cancer LNCaP and C4-2B cells. In addition, clioquinol alone exhibits similar effects in prostate cancer cell lines with elevated copper at concentrations similar to those found in patients. Addition of dihydrotestosterone did not affect clioquinol-mediated proteasome inhibition in both prostate cancer cell lines. However, dihydrotestosterone partially inhibited clioquinol-induced AR suppression and apoptosis only in androgen-dependent LNCaP cells. Animal studies show that clioquinol treatment significantly inhibits the growth of human prostate tumor C4-2B xenografts (by 66%), associated with in vivo proteasome inhibition, AR protein repression, angiogenesis suppression, and apoptosis induction. Our study provides strong evidence that clioquinol is able to target tumor proteasome in vivo in a copper-dependent manner, resulting in formation of an active AR inhibitor and apoptosis inducer that is responsible for its observed antiprostate tumor effect. [Cancer Res 2007;67(4):1636–44]
Xrcc3 and Nbs1 Are Required for the Production of Extrachromosomal Telomeric Circles in Human Alternative Lengthening of Telomere Cells
Authors: Compton Sarah A. , Choi Jun-Hyuk , Cesare Anthony J. , Özgür Sezgin , Griffith Jack D.
Published: February 2007

The maintenance of telomere length is essential for the indefinite proliferation of cancer cells. This is most often achieved by the activation of telomerase; however, a substantial number of cancers lack detectable telomerase activity and are classified as using an alternative lengthening of telomeres (ALT) pathway. We showed recently that ALT cells have a high level of extrachromosomal telomeric circles (t circles) that may be a specific marker of the ALT phenotype. The mechanism underlying t circle production and the requirement of t circles in ALT remain unclear. Understanding the specific requirements of ALT is key to developing diagnostic tools and therapies that target this pathway and is critical for the treatment of cancers in which ALT is prevalent, including cancers of neuroepithelial and mesenchymal origin. In this study, we used short hairpin RNAs directed at either Xrcc3 or Nbs1, two proteins involved in the homologous recombination pathway, to determine the role of these proteins in t circle production and the requirement of t circles in maintaining the ALT pathway. We show that Xrcc3 and Nbs1 are indeed required for the production of t circles in human ALT. However, these cells continue to proliferate in the absence of t circles, suggesting that they are not required for the survival of ALT cells. [Cancer Res 2007;67(4):1513–9]
Gene Alterations Identified by Expression Profiling in Tumor-Associated Endothelial Cells from Invasive Ovarian Carcinoma
Authors: Lu Chunhua , Bonome Tomas , Li Yang , Kamat Aparna A. , Han Liz Y. , Schmandt Rosemarie , Coleman Robert L. , Gershenson David M. , Jaffe Robert B. , Birrer Michael J. , Sood Anil K.
Published: February 2007

Therapeutic strategies based on antiangiogenic approaches are beginning to show great promise in clinical studies. However, full realization of these approaches requires identification of key differences in gene expression between endothelial cells from tumors versus their normal counterparts. Here, we examined gene expression differences in purified endothelial cells from 10 invasive epithelial ovarian cancers and 5 normal ovaries using Affymetrix U133 Plus 2.0 microarrays. More than 400 differentially expressed genes were identified in tumor-associated endothelial cells. We selected and validated 23 genes that were overexpressed by 3.6- to 168-fold using real-time reverse transcription-PCR and/or immunohistochemistry. Among these, the polycomb group protein enhancer of Zeste homologue 2 (EZH2), the Notch ligand Jagged1, and PTK2 were elevated 3- to 4.3-fold in tumor-associated endothelial cells. Silencing these genes individually with small interfering RNA blocked endothelial cell migration and tube formation in vitro. The present study shows that tumor and normal endothelium differ at the molecular level, which may have significant implications for the development of antiangiogenic therapies. [Cancer Res 2007;67(4):1757–68]
RETRACTED: mda-9/Syntenin Regulates the Metastatic Phenotype in Human Melanoma Cells by Activating Nuclear Factor-κB
Authors: Boukerche Habib , Su Zao-zhong , Emdad Luni , Sarkar Devanand , Fisher Paul B.
Published: February 2007

mda-9/Syntenin is a scaffolding PDZ domain-containing protein overexpressed in multiple human cancers that functions as a positive regulator of melanoma metastasis. Using a normal immortal human melanocyte cell line and weakly and highly metastatic human melanoma cell lines, we presently show that mda-9/syntenin initiates a signaling cascade that activates nuclear factor-κB (NF-κB) in human melanoma cells. As a consequence of elevated mda-9/syntenin expression, tumor cell growth and motility, fundamental components of tumor cell invasion and metastatic spread of melanoma cells, are enhanced through focal adhesion kinase (FAK)–induced and p38 mitogen-activated protein kinase (MAPK)–induced activation of NF-κB. Inhibiting mda-9/syntenin, using an adenovirus expressing antisense mda-9/syntenin, NF-κB, using an adenovirus expressing a mutant superrepressor of IκBα, or FAK, and using a dominant-negative mutant of FAK (FRNK), blocks melanoma cell migration, anchorage-independent growth, and invasion. Downstream signaling changes mediated by mda-9/syntenin, which include activation of FAK, p38 MAPK, and NF-κB, promote induction of membrane-type matrix metalloproteinase-1 that then activates pro-MMP-2–promoting migration and extracellular matrix invasion of melanoma cells. These results highlight the importance of mda-9/syntenin as a key component of melanoma metastasis providing a rational molecular target for potentially intervening in the metastatic process. [Cancer Res 2007;67(4):1812–22]
The Nuclear Receptor Interacting Factor-3 Transcriptional Coregulator Mediates Rapid Apoptosis in Breast Cancer Cells through Direct and Bystander-Mediated Events
Authors: Das Sharmistha , Nwachukwu Jerome C. , Li Dangsheng , Vulin Anthony I. , Martinez-Caballero Sonia , Kinnally Kathleen W. , Samuels Herbert H.
Published: February 2007

We previously reported that amino acids 20 to 50 of nuclear receptor interacting factor-3 mediates rapid apoptosis in breast cancer cell lines but not in cells derived from other tissues. We refer to this short region as death domain-1 (DD1). Small interfering RNA studies indicated that DD1-mediated apoptosis is caspase-2 dependent. In this study, we examined DD1-mediated apoptosis in more detail and generated stable caspase-2 knockdown breast cancer cells. These cells are resistant to DD1-mediated apoptosis. Time-lapse movies suggested that DD1-mediated apoptosis also leads to a “bystander effect.” We found that within 5 h of DD1 expression, breast cancer cells release a factor(s) into the medium that leads to apoptosis of naive breast cancer cells or DD1-resistant cells (e.g., HeLa). The DD1-expressing caspase-2 knockdown cells also release a factor(s) that kills other cells, indicating that this effect is not dependent on the apoptogenic process. The bystander effect seems dependent on the production of reactive oxygen species (ROS). These and other studies indicate that DD1 expression in breast cancer cells leads to at least two death signals: one involving the rapid production of ROS and/or other soluble factors that directly or indirectly leads to a bystander effect and a second caspase-2–dependent process that leads to apoptosis in cells in which DD1 is expressed. [Cancer Res 2007;67(4):1775–82]
The Human let-7a-3 Locus Contains an Epigenetically Regulated MicroRNA Gene with Oncogenic Function
Authors: Brueckner Bodo , Stresemann Carlo , Kuner Ruprecht , Mund Cora , Musch Tanja , Meister Michael , Sültmann Holger , Lyko Frank
Published: February 2007

MicroRNAs (miRNAs) are small noncoding RNAs that repress their target mRNAs by complementary base pairing and induction of the RNA interference pathway. It has been shown that miRNA expression can be regulated by DNA methylation and it has been suggested that altered miRNA gene methylation might contribute to human tumorigenesis. In this study, we show that the human let-7a-3 gene on chromosome 22q13.31 is associated with a CpG island. Let-7a-3 belongs to the archetypal let-7 miRNA gene family and was found to be methylated by the DNA methyltransferases DNMT1 and DNMT3B. The gene was heavily methylated in normal human tissues but hypomethylated in some lung adenocarcinomas. Let-7a-3 hypomethylation facilitated epigenetic reactivation of the gene and elevated expression of let-7a-3 in a human lung cancer cell line resulted in enhanced tumor phenotypes and oncogenic changes in transcription profiles. Our results thus identify let-7a-3 as an epigenetically regulated miRNA gene with oncogenic function and suggest that aberrant miRNA gene methylation might contribute to the human cancer epigenome. [Cancer Res 2007;67(4):1419–23]
The NC1 domain of type XIX collagen inhibits in vivo melanoma growth
Authors: Ramont Laurent , Brassart-Pasco Sylvie , Thevenard Jessica , Deshorgue Aurélie , Venteo Lydie , Laronze Jean Yves , Pluot Michel , Monboisse Jean-Claude , Maquart François-Xavier
Published: February 2007

Type XIX collagen is a minor collagen that localizes to basement membrane zones, together with types IV, XV, and XVIII collagens. Because several NC1 COOH-terminal domains of other chains from basement membrane collagens were reported to exhibit antitumor activity, we decided to study the effects of the NC1(XIX) collagen domain on tumor progression using an experimental in vivo model of mouse melanoma. We observed a 70% reduction in tumor volume in NC1(XIX)-treated mice compared with the corresponding controls. Histologic examination of the tumors showed a strong decrease in tumor vascularization in treated mice. In vitro, NC1(XIX) inhibited the migrating capacity of tumor cells and their capacity to invade Matrigel. It also inhibited the capacity of human microvascular endothelial cells to form pseudotubes in Matrigel. This effect was accompanied by a strong inhibition of membrane type-1 matrix metalloproteinase (matrix metalloproteinase-14) and vascular endothelial growth factor expression. Collectively, our data indicate that the NC1 domain of type XIX collagen exerts antitumor activity. This effect is mediated by a strong inhibition of the invasive capacities of tumor cells and antiangiogenic effects. NC1(XIX) should now be considered as a new member of the basement membrane collagen-derived matrikine family with antitumor and antiangiogenic activity. [Mol Cancer Ther 2007;6(2):506–14]
Sensitization of p53-mutated epithelial ovarian cancer to CD95-mediated apoptosis is synergistically induced by cisplatin pretreatment
Authors: Bagnoli Marina , Balladore Emanuela , Luison Elena , Alberti Paola , Raspagliesi Francesco , Marcomini Barbara , Canevari Silvana , Mezzanzanica Delia
Published: February 2007

Epithelial ovarian carcinoma (EOC) remains a highly lethal malignancy. Despite the progress in surgical and therapeutic strategies, resistance to chemotherapy is still a major concern. Cytotoxic therapies mediate killing of cancer cells by activating the intrinsic mitochondrial apoptotic pathway, and p53 status is a key factor in determining the efficacy of apoptotic signaling. The extrinsic (CD95) death receptor–dependent signaling pathway also contributes to the efficacy of cancer therapy. We previously showed that EOC are generally resistant to CD95-dependent apoptosis. In p53 wild-type EOC tumors, CD95-mediated apoptosis is impaired at the receptor level by the long form of cellular FLICE-inhibitory protein, whereas this mechanism does not account for resistance in tumors with mutated p53 (p53mu). In the present study, we examined both intrinsic and death receptor–dependent apoptotic signaling in p53mu OVCAR3 EOC cell line, showing that these cells are less susceptible to cisplatin treatment as compared with p53 wild-type EOC cells and also resist CD95-mediated apoptosis due to inefficient formation of the death-inducing signaling complex and weak mitochondrial signal amplification. However, pretreatment of OVCAR3 cells with clinically relevant cisplatin concentrations significantly improved receptor-dependent apoptotic signaling by up-modulating CD95 receptor expression and increasing death-inducing signaling complex formation efficiency. The synergy of cisplatin pretreatment and CD95 triggering in inducing cell death was also shown in p53mu tumor cells derived from ascitic fluid of advanced-stage EOC patients. These findings support the effectiveness of a combined therapeutic treatment able to sensitize cancer cells to apoptosis even when p53 is functionally inactivated. [Mol Cancer Ther 2007;6(2):762–72]
HIV-Tat protein transduction domain specifically attenuates growth of polyamine deprived tumor cells
Authors: Mani Katrin , Sandgren Staffan , Lilja Johanna , Cheng Fang , Svensson Katrin , Persson Lo , Belting Mattias
Published: February 2007

Polyamines are essential for tumor cell growth, and the polyamine pathway represents an attractive target for cancer treatment. Several polyamine transport proteins have been cloned and characterized in bacteria and yeast cells; however, the mechanism of polyamine entry into mammalian cells remains poorly defined, although a role for proteoglycans has been suggested. Here, we show that the HIV-Tat transduction peptide, which is known to enter cells via a proteoglycan-dependent pathway, efficiently inhibits polyamine uptake. Polyamine uptake–deficient mutant cells with intact proteoglycan biosynthesis (CHO MGBG) displayed unperturbed HIV-Tat uptake activity compared with wild-type cells, supporting the notion that HIV-Tat peptide interferes with polyamine uptake via competition for proteoglycan binding sites rather than a putative downstream transporter. HIV-Tat specifically inhibited growth of human carcinoma cells made dependent on extracellular polyamines by treatment with the polyamine biosynthesis inhibitor α-difluoromethylornithine; accordingly, the Tat peptide prevented intracellular accumulation of exogenous polyamines. Moreover, combined treatment with α-difluoromethylornithine and HIV-Tat efficiently blocked tumor growth in an experimental mouse model. We conclude that HIV-Tat transduction domain and polyamines enter cells through a common pathway, which can be used to target polyamine-dependent tumor growth in the treatment of cancer. [Mol Cancer Ther 2007;6(2):782–8]
The histone deacetylase inhibitor FK228 given prior to adenovirus infection can boost infection in melanoma xenograft model systems
Authors: Goldsmith Merrill E. , Aguila Alian , Steadman Kenneth , Martinez Alfredo , Steinberg Seth M. , Alley Michael C. , Waud William R. , Bates Susan E. , Fojo Tito
Published: February 2007

A major limitation of adenovirus type 5–mediated cancer gene therapy is the inefficient infection of many cancer cells. Previously, we showed that treatment with low doses of the histone deacetylase inhibitor FK228 (FR901228, depsipeptide) increased coxsackie adenovirus receptor (CAR) levels, histone H3 acetylation, and adenovirus infection efficiencies as measured by viral transgene expression in cancer cell lines but not in cultured normal cells. To evaluate FK228 in vivo, the effects of FK228 therapy in athymic mice bearing LOX IMVI or UACC-62 human melanoma xenografts were examined. Groups of mice were treated with FK228 using several dosing schedules and the differences between treated and control animals were determined. In mice with LOX IMVI xenografts (n = 6), maximum CAR induction was observed 24 h following a single FK228 dose of 3.6 mg/kg with a 13.6 ± 4.3-fold (mean ± SD) increase in human CAR mRNA as determined by semiquantitative reverse transcription-PCR analysis. By comparison, mouse CAR levels in liver, kidney, and lung from the same animals showed little to no change. Maximum CAR protein induction of 9.2 ± 4.8-fold was achieved with these treatment conditions and was associated with increased histone H3 acetylation. Adenovirus carrying a green fluorescent protein (GFP) transgene (2 × 109 viral particles) was injected into the xenografts and GFP mRNA levels were determined. A 7.4 ± 5.2-fold increase in GFP mRNA was found 24 h following adenovirus injection into optimally FK228-treated mice (n = 10). A 4-fold increase in GFP protein–positive cells was found following FK228 treatment. These studies suggest that FK228 treatment prior to adenovirus infection could increase the efficiency of adenovirus gene therapy in xenograft model systems. [Mol Cancer Ther 2007;6(2):496–505]
Role of placenta growth factor in malignancy and evidence that an antagonistic PlGF/Flt-1 peptide inhibits the growth and metastasis of human breast cancer xenografts
Authors: Taylor Alice P. , Goldenberg David M.
Published: February 2007

The angiogenic growth factor placenta growth factor (PlGF) is implicated in several pathologic processes, including the growth and spread of cancer. We found by immunohistochemistry that 36% to 60% and 65% of primary breast cancers express PlGF and its receptor Flt-1, respectively. These findings suggest that PlGF may be active in tumor growth and metastasis beyond its role in angiogenesis. It was found that exogenously added PlGF (2 nmol/L), in contrast to vascular endothelial growth factor (2 nmol/L), significantly stimulated in vitro motility and invasion of the human breast tumor lines MCF-7 and MDA-MB-231. A PlGF-2/Flt-1–inhibiting peptide, binding peptide 1 (BP1), that binds Flt-1 at or near the heparin-binding site was identified and synthesized. Both PlGF-stimulated motility and invasion were prevented by treatment with BP1 (P < 0.05), as well as by anti-PlGF antibody. Treatment of mice bearing s.c. MDA-MB-231 with BP1 (200 μg i.p., twice per week) decreased the number of spontaneous metastatic lung nodules by 94% (P < 0.02), whereas therapy of animals with orthotopic mammary fat pad tumors decreased pulmonary metastases by 82% (P < 0.02). These results indicate, for the first time, that PlGF stimulates the metastatic phenotype in these breast cancer cells, whereas therapy with a PlGF-2/Flt-1 heparin-blocking peptide reduces the growth and metastasis of human breast cancer xenografts. [Mol Cancer Ther 2007;6(2):524–31]
Loss of homotypic cell adhesion by epithelial-mesenchymal transition or mutation limits sensitivity to epidermal growth factor receptor inhibition
Authors: Buck Elizabeth , Eyzaguirre Alexandra , Barr Sharon , Thompson Stuart , Sennello Regina , Young David , Iwata Kenneth K. , Gibson Neil W. , Cagnoni Pablo , Haley John D.
Published: February 2007

Overexpression and enhanced activation of the epidermal growth factor receptor (EGFR) is frequently observed in human carcinomas. Inhibitors of EGFR signaling have shown clinical utility; however, understanding response at the molecular level is important to define patient subsets most likely to benefit, as well as to support the rational design of drug combinations. Pancreatic and colorectal tumor cell lines insensitive to EGFR inhibition were those that had lost or mutated the epithelial junction constituents E-cadherin and γ-catenin, had lost homotypic adhesion, and often gained proteins associated with an epithelial to mesenchymal–like transition, such as vimentin, zeb1, or snail. In matched pairs of colorectal tumor cells, the epithelial lines showed an average 7-fold greater sensitivity than mesenchymal-like lines. In human pancreatic and colorectal tumor tissues, gain of mesenchymal characteristics and loss of epithelial characteristics correlated with advancing tumor stage. These data indicate an especially sensitive patient subset as well as a rationale for the combination of EGFR antagonists with agents that affect the epithelial to mesenchymal–like transition process as a mechanism to enhance sensitivity for more advanced mesenchymal-like tumors. [Mol Cancer Ther 2007;6(2):532–41]