bluuebunny/crossref_metadata_2025 huggingface dataset:
Nimotuzumab, an Antitumor Antibody that Targets the Epidermal Growth Factor Receptor, Blocks Ligand Binding while Permitting the Active Receptor Conformation
Authors: Talavera Ariel , Friemann Rosmarie , Gómez-Puerta Silvia , Martinez-Fleites Carlos , Garrido Greta , Rabasa Ailem , López-Requena Alejandro , Pupo Amaury , Johansen Rune F. , Sánchez Oliberto , Krengel Ute , Moreno Ernesto
Published: July 2009

Overexpression of the epidermal growth factor (EGF) receptor (EGFR) in cancer cells correlates with tumor malignancy and poor prognosis for cancer patients. For this reason, the EGFR has become one of the main targets of anticancer therapies. Structural data obtained in the last few years have revealed the molecular mechanism for ligand-induced EGFR dimerization and subsequent signal transduction, and also how this signal is blocked by either monoclonal antibodies or small molecules. Nimotuzumab (also known as h-R3) is a humanized antibody that targets the EGFR and has been successful in the clinics. In this work, we report the crystal structure of the Fab fragment of Nimotuzumab, revealing some unique structural features in the heavy variable domain. Furthermore, competition assays show that Nimotuzumab binds to domain III of the extracellular region of the EGFR, within an area that overlaps with both the surface patch recognized by Cetuximab (another anti-EGFR antibody) and the binding site for EGF. A computer model of the Nimotuzumab-EGFR complex, constructed by docking and molecular dynamics simulations and supported by mutagenesis studies, unveils a novel mechanism of action, with Nimotuzumab blocking EGF binding while still allowing the receptor to adopt its active conformation, hence warranting a basal level of signaling. [Cancer Res 2009;69(14):5851–9]
Genomic Characterization of Esophageal Squamous Cell Carcinoma from a High-Risk Population in China
Authors: Hu Nan , Wang Chaoyu , Ng David , Clifford Robert , Yang Howard H. , Tang Ze-Zhong , Wang Quan-Hong , Han Xiao-You , Giffen Carol , Goldstein Alisa M. , Taylor Philip R. , Lee Maxwell P.
Published: July 2009

Genomic instability plays an important role in most human cancers. To characterize genomic instability in esophageal squamous cell carcinoma (ESCC), we examined loss of heterozygosity (LOH), copy number (CN) loss, CN gain, and gene expression using the Affymetrix GeneChip Human Mapping 500K (n = 30 cases) and Human U133A (n = 17 cases) arrays in ESCC cases from a high-risk region of China. We found that genomic instability measures varied widely among cases and separated them into two groups: a high-frequency instability group (two-thirds of all cases with one or more instability category of ≥10%) and a low-frequency instability group (one-third of cases with instability of <10%). Genomic instability also varied widely across chromosomal arms, with the highest frequency of LOH on 9p (33% of informative single nucleotide polymorphisms), CN loss on 3p (33%), and CN gain on 3q (48%). Twenty-two LOH regions were identified: four on 9p, seven on 9q, four on 13q, two on 17p, and five on 17q. Three CN loss regions—3p12.3, 4p15.1, and 9p21.3—were detected. Twelve CN gain regions were found, including six on 3q, one on 7q, four on 8q, and one on 11q. One of the most gene-rich of these CN gain regions was 11q13.1-13.4, where 26 genes also had RNA expression data available. CN gain was significantly correlated with increased RNA expression in over 80% of these genes. Our findings show the potential utility of combining CN analysis and gene expression data to identify genes involved in esophageal carcinogenesis. [Cancer Res 2009;69(14):5908–17]
MiR-122/Cyclin G1 Interaction Modulates p53 Activity and Affects Doxorubicin Sensitivity of Human Hepatocarcinoma Cells
Authors: Fornari Francesca , Gramantieri Laura , Giovannini Catia , Veronese Angelo , Ferracin Manuela , Sabbioni Silvia , Calin George Adrian , Grazi Gian Luca , Croce Carlo Maria , Tavolari Simona , Chieco Pasquale , Negrini Massimo , Bolondi Luigi
Published: July 2009

The identification of target genes is a key step for assessing the role of aberrantly expressed microRNAs (miRNA) in human cancer and for the further development of miRNA-based gene therapy. MiR-122 is a liver-specific miRNA accounting for 70% of the total miRNA population. Its down-regulation is a common feature of both human and mouse hepatocellular carcinoma (HCC). We have previously shown that miR-122 can regulate the expression of cyclin G1, whose high levels have been reported in several human cancers. We evaluated the role of miR-122 and cyclin G1 expression in hepatocarcinogenesis and in response to treatment with doxorubicin and their relevance on survival and time to recurrence (TTR) of HCC patients. We proved that, by modulating cyclin G1, miR-122 influences p53 protein stability and transcriptional activity and reduces invasion capability of HCC-derived cell lines. In addition, in a therapeutic perspective, we assayed the effects of a restored miR-122 expression in triggering doxorubicin-induced apoptosis and we proved that miR-122, as well as cyclin G1 silencing, increases sensitivity to doxorubicin challenge. In patients resected for HCC, lower miR-122 levels were associated with a shorter TTR, whereas higher cyclin G1 expression was related to a lower survival, suggesting that miR-122 might represent an effective molecular target for HCC. Our findings establish a basis toward the development of combined chemo- and miRNA-based therapy for HCC treatment. [Cancer Res 2009;69(14):5761–7]
Long-lived Min Mice Develop Advanced Intestinal Cancers through a Genetically Conservative Pathway
Authors: Halberg Richard B. , Waggoner Jesse , Rasmussen Kristen , White Alanna , Clipson Linda , Prunuske Amy J. , Bacher Jeffery W. , Sullivan Ruth , Washington Mary Kay , Pitot Henry C. , Petrini John H.J. , Albertson Donna G. , Dove William F.
Published: July 2009

C57BL/6J mice carrying the Min allele of Adenomatous polyposis coli (Apc) develop numerous adenomas along the entire length of the intestine and consequently die at an early age. This short lifespan would prevent the accumulation of somatic genetic mutations or epigenetic alterations necessary for tumor progression. To overcome this limitation, we generated F1 ApcMin/+ hybrids by crossing C57BR/cdcJ and SWR/J females to C57BL/6J ApcMin/+ males. These hybrids developed few intestinal tumors and often lived longer than 1 year. Many of the tumors (24-87%) were invasive adenocarcinomas, in which neoplastic tissue penetrated through the muscle wall into the mesentery. In a few cases (3%), lesions metastasized by extension to regional lymph nodes. The development of these familial cancers does not require chromosomal gains or losses, a high level of microsatellite instability, or the presence of Helicobacter. To test whether genetic instability might accelerate tumor progression, we generated ApcMin/+ mice homozygous for the hypomorphic allele of the Nijmegen breakage syndrome gene (Nbs1ΔB) and also treated ApcMin/+ mice with a strong somatic mutagen. These imposed genetic instabilities did not reduce the time required for cancers to form nor increase the percentage of cancers nor drive progression to the point of distant metastasis. In summary, we have found that the ApcMin/+ mouse model for familial intestinal cancer can develop frequent invasive cancers in the absence of overt genomic instability. Possible factors that promote invasion include age-dependent epigenetic changes, conservative somatic recombination, or direct effects of alleles in the F1 hybrid genetic background. [Cancer Res 2009;69(14):5768–75]
Activating Transcription Factor 2 and c-Jun–Mediated Induction of FoxP3 for Experimental Therapy of Mammary Tumor in the Mouse
Authors: Liu Yan , Wang Yin , Li Weiquan , Zheng Pan , Liu Yang
Published: July 2009

FOXP3 is inactivated in breast cancer cells by a number of mechanisms, including somatic mutations, deletion, and epigenetic silencing. Because the mutation and deletion are usually heterozygous in the cancer samples, it is of interest to determine whether the gene can be induced for the purpose of cancer therapy. Here, we report that anisomycin, a potent activator of activating transcription factor (ATF) 2, and c-Jun-NH2-kinase, induces expression of FoxP3 in both normal and malignant mammary epithelial cells. The induction is mediated by ATF2 and c-Jun. Targeted mutation of ATF2 abrogates both constitutive and inducible expression of FoxP3 in normal epithelial cells. Both ATF2 and c-Jun interact with a novel enhancer in the intron 1 of the FoxP3 locus. Moreover, shRNA silencing of ATF2 and FoxP3 reveals an important role of ATF2-FoxP3 pathway in the anisomycin-induced apoptosis of breast cancer cells. A low dose of anisomycin was also remarkably effective in treating established mammary tumor in the mice. Our data showed that FoxP3 can be reactivated for cancer therapy. [Cancer Res 2009;69(14):5954–60]
MicroRNA-661, a c/EBPα Target, Inhibits Metastatic Tumor Antigen 1 and Regulates Its Functions
Authors: Reddy Sirigiri Divijendra Natha , Pakala Suresh B. , Ohshiro Kazufumi , Rayala Suresh K. , Kumar Rakesh
Published: July 2009

MicroRNAs (miR) have been identified as posttranscriptional modifiers of target gene regulation and control the expression of gene products important in cancer progression. Here, we show that miR-661 inhibits the expression of metastatic tumor antigen 1 (MTA1), a widely up-regulated gene product in human cancer, by targeting the 3′ untranslated region (UTR) of MTA1 mRNA. We found that endogenous miR-661 expression was positively regulated by the c/EBPα transcription factor, which is down-regulated during cancer progression. c/EBPα directly interacted with the miR-661 chromatin and bound to miR-661 putative promoter that contains a c/EBPα-consensus motif. In addition, we found that the level of MTA1 protein was progressively up-regulated, whereas that of miR-661 and its activator, c/EBPα, were down-regulated in a breast cancer progression model consisting of MCF-10A cell lines whose phenotypes ranged from noninvasive to highly invasive. c/EBPα expression in breast cancer cells resulted in increased miR-661 expression and reduced MTA1 3′UTR-luciferase activity and MTA1 protein level. We also provide evidence that the introduction of miR-661 inhibited the motility, invasiveness, anchorage-independent growth, and tumorigenicity of invasive breast cancer cells. We believe our findings show for the first time that c/EBPα regulates the level of miR-661 and in turn modifies the functions of the miR661-MTA1 pathway in human cancer cells. Based on these findings, we suggest that miR-661 be further investigated for therapeutic use in down-regulating the expression of MTA1 in cancer cells. [Cancer Res 2009;69(14):5639–42]
Platelets: Guardians of Tumor Vasculature
Authors: Ho-Tin-Noé Benoit , Goerge Tobias , Wagner Denisa D.
Published: July 2009

Solid tumors generate a prothrombotic environment capable of platelet activation. Recent findings indicate that the activated platelets are crucial regulators of tumor vascular homeostasis in that they prevent tumor hemorrhage. Surprisingly, this effect is independent of platelets' capacity to form thrombi and instead relies on the secretion of their granule content. Thus, targeting platelet secretory activities may represent a new approach to specifically destabilize tumor vasculature. [Cancer Res 2009;69(14):5623–6]
Color Fluorescence Ratio for Detection of Bronchial Dysplasia and Carcinoma In situ
Authors: Lee Pyng , van den Berg Remco M. , Lam Stephen , Gazdar Adi F. , Grunberg Katrien , McWilliams Annette , LeRiche Jean , Postmus Pieter E. , Sutedja Tom G.
Published: July 2009

Background: Autofluorescence bronchoscopy is more sensitive than conventional bronchoscopy for detecting early airway mucosal lesions. Decreased specificity can lead to excessive biopsy and increased procedural time. Onco-LIFE, a device that combines fluorescence and reflectance imaging, allows numeric representation by expressing red-to-green ratio (R/G ratio) within the region of interest. The aim of the study was to determine if color fluorescence ratio (R/G ratio) added to autofluorescence bronchoscopy could provide an objective means to guide biopsy.

Methods: Subjects at risk for lung cancer were recruited at two centers: VU University Medical Centre (Amsterdam) and BC Cancer Agency (Canada). R/G ratio for each site appearing normal or abnormal was measured before biopsy. R/G ratios were correlated with pathology, and a receiver operating characteristic curve of R/G ratio for high-grade and moderate dysplasia was done. Following analysis of the training data set obtained from two centers, a prospective validation study was done.

Results: Three thousand three hundred sixty-two adequate biopsies from 738 subjects with their corresponding R/G ratios were analyzed. R/G ratio 0.54 conferred 85% sensitivity and 80% specificity for the detection of high-grade and moderate dysplasia, area under the curve was 0.90, and 95% confidence interval was 0.88 to 0.92. In another 70 different sites that were assessed, κ measurements of agreement of R/G ratios with visual scores and pathology were 0.66 (P < 0.0001) and 0.61 (P < 0.0001), respectively. R/G ratio combined with visual score improved specificity to 88% (95% confidence interval, 0.73-0.96) for high-grade and moderate dysplasia.

Conclusion: Color fluorescence ratio can objectively guide the bronchoscopist in selecting sites for biopsy with good pathologic correlation.
Combination of Viral Oncolysis and Tumor-Specific Immunity to Control Established Tumors
Authors: Chuang Chi-Mu , Monie Archana , Wu Annie , Pai Sara I. , Hung Chien-Fu
Published: July 2009

Purpose: Advanced-stage cancers are extremely difficult to treat and rarely result in a cure. The application of oncolytic viruses is a potential strategy for controlling advanced-stage cancer because intratumoral (i.t.) injection of an oncolytic virus, such as vaccinia virus, results in tumor cell lysis and subsequent release of tumor antigens into the microenvironment. Furthermore, the viruses can serve as a vehicle for delivering genes of interest to cancer cells.

Experimental Design: In the current study, we hypothesize that in tumor-bearing mice primed with DNA encoding an immunogenic foreign antigen, ovalbumin (OVA) followed by a boost with i.t. administration of vaccinia virus encoding the same foreign antigen, OVA, can generate enhanced antitumor effects through the combination of viral oncolysis and tumor-specific immunity.

Results: We observed that tumor-bearing mice primed with OVA DNA and boosted with vaccinia encoding OVA (Vac-OVA) generated significant therapeutic antitumor effects as well as induced significant levels of OVA-specific CD8+ T cells in two different tumor models. Furthermore, treatment with Vac-OVA not only kills the tumor and stromal cells directly but also renders the tumor cells and surrounding stromal cells susceptible to OVA-specific CD8+ T-cell killing, resulting in enhanced antitumor therapeutic effects.

Conclusions: Thus, the current study may provide a novel therapeutic strategy for the control of advanced-stage cancers.
Activation of Phosphatidylinositol-3′-kinase/AKT Signaling Is Essential in Hepatoblastoma Survival
Authors: Hartmann Wolfgang , Küchler Jan , Koch Arend , Friedrichs Nicolaus , Waha Anke , Endl Elmar , Czerwitzki Jacqueline , Metzger Dagmar , Steiner Susanne , Wurst Peter , Leuschner Ivo , von Schweinitz Dietrich , Buettner Reinhard , Pietsch Torsten
Published: July 2009

Purpose: Hepatoblastoma represents the most frequent malignant liver tumor in childhood. The phosphatidylinositol-3′-kinase (PI3K)/AKT pathway is crucial in downstream signaling of multiple receptor tyrosine kinases of pathogenic importance in hepatoblastoma. Increased PI3K/AKT signaling pathway activity and activating mutations of PIK3CA, encoding a PI3K catalytic subunit, have been reported in different childhood tumors. The current study was done to analyze the role of PI3K/AKT signaling in hepatoblastoma.

Experimental Design: Immunohistochemical stainings of (Ser473)-phosphorylated (p)-AKT protein, its targets p-(Ser9)-GSK-3β and p-(Ser2448)-mTOR, as well as the cell cycle regulators Cyclin D1, p27KIP1, and p21CIP1 were done and the PIK3CA gene was screened for mutations. In vitro, two hepatoblastoma cell lines treated with the PI3K inhibitor LY294002 were analyzed for AKT and GSK-3β phosphorylation, cell proliferation, and apoptosis. Additionally, simultaneous treatments of hepatoblastoma with LY294002 and cytotoxic drugs were carried out.

Results: Most tumors strongly expressed p-AKT, p-GSK-3β, and p-mTOR; subgroups showed significant Cyclin D1, p27KIP1, and p21CIP1 expression. One hepatoblastoma carried an E545A mutation in the PIK3CA gene. In vitro, PI3K inhibition diminished hepatoblastoma cell growth being accompanied by reduced AKT and GSK-3β phosphorylation. Flow cytometry and 4', 6-diamidino-2-phenylindole stainings showed that PI3K pathway inhibition leads to a substantial increase in apoptosis and a decrease in cellular proliferation linked to reduced Cyclin D1 and increased p27KIP1 levels. Simultaneous treatment of hepatoblastoma cell lines with LY294002 and cytotoxic drugs resulted in positive interactions.

Conclusions: Our findings imply that PI3K signaling plays an essential role in growth control of hepatoblastoma and might be successfully targeted in multimodal therapeutic strategies.
MSH6 Mutations Arise in Glioblastomas during Temozolomide Therapy and Mediate Temozolomide Resistance
Authors: Yip Stephen , Miao Jiangyong , Cahill Daniel P. , Iafrate A. John , Aldape Ken , Nutt Catherine L. , Louis David N.
Published: July 2009

Purpose: Over the past few years, the alkylating agent temozolomide has become the standard-of-care therapy for patients with glioblastoma, the most common brain tumor. Recently, large-scale cancer genome sequencing efforts have identified a hypermutation phenotype and inactivating MSH6 mismatch repair gene mutations in recurrent, post-temozolomide glioblastomas, particularly those growing more rapidly during temozolomide treatment. This study aimed to clarify the timing and role of MSH6 mutations in mediating glioblastoma temozolomide resistance.

Experimental Design: MSH6 sequence and microsatellite instability (MSI) status were determined in matched prechemotherapy and postchemotherapy glioblastomas identified by The Cancer Genome Atlas (TCGA) as having posttreatment MSH6 mutations. Temozolomide-resistant lines were derived in vitro through selective growth under temozolomide, and the MSH6 gene was sequenced in resistant clones. The role of MSH6 inactivation in mediating resistance was explored using lentiviral short hairpin RNA knockdown and MSH6 reconstitution.

Results: MSH6 mutations were confirmed in posttreatment TCGA glioblastomas but absent in matched pretreatment tumors. The posttreatment hypermutation phenotype displayed a signature bias toward CpC transitions and was not associated with MSI. In vitro modeling through exposure of an MSH6 wild-type glioblastoma line to temozolomide resulted in resistant clones; one clone showed an MSH6 mutation, Thr1219Ile, that had been independently noted in two treated TCGA glioblastomas. Knockdown of MSH6 in the glioblastoma line U251 increased resistance to temozolomide cytotoxicity and reconstitution restored cytotoxicity in MSH6-null glioma cells.

Conclusions: MSH6 mutations are selected in glioblastomas during temozolomide therapy both in vitro and in vivo and are causally associated with temozolomide resistance.
Regulation of Replicative and Stress-Induced Senescence by RSK4, which is Down-regulated in Human Tumors
Authors: López-Vicente Laura , Armengol Gemma , Pons Berta , Coch Laura , Argelaguet Elisabet , Lleonart Matilde , Hernández-Losa Javier , de Torres Inés , Ramon y Cajal Santiago
Published: July 2009

Purpose: The control of senescence and its biochemical pathways is a crucial factor for understanding cell transformation. In a large RNA interference screen, the RSK4 gene was found to be related to p53-dependent arrest. The purpose of the present study was to investigate the potential role of RSK4 as a tumor suppressor gene.

Experimental Design: RSK4 expression was determined by quantitative real-time PCR and immunoblot in 30 colon and 20 renal carcinomas, and in 7 colon adenomas. Two HCT116 colon carcinoma cell lines (p53 wt and p53 null), IMR90 human fibroblasts, and E1A-expressing IMR90 cells were infected with RSK4 cDNA and/or shRNA. RSK4 expression levels were analyzed in HCT116 p53 wt or p53 null and IMR90 after senescence induction by quantitative real-time PCR and Western blot.

Results: The RSK4 gene was down-regulated in 27 of 30 colon carcinomas (P < 0.001), 16 of 20 renal cell carcinomas (P < 0.01), and 6 of 7 colon adenomas (P < 0.01). In vitro overexpression of RSK4 induced cell arrest and senescence features in normal fibroblasts and malignant colon carcinoma cell lines. Interestingly, in these cell lines RSK4 mRNA levels were increased both in replicative and stress-induced senescence. Moreover, IMR90 partially immortalized by RSK4 shRNA and HCT116 with this short hairpin RNA were more resistant to cisplatin treatment. Finally, cells expressing E1A or Rb short interfering RNA were resistant to RSK4-mediated senescence.

Conclusion: These results support the concept that RSK4 may be an important tumor suppressor gene by modulating senescence induction and contributing to cell proliferation control in colon carcinogenesis and renal cell carcinomas.
PF-00477736 Mediates Checkpoint Kinase 1 Signaling Pathway and Potentiates Docetaxel-Induced Efficacy in Xenografts
Authors: Zhang Cathy , Yan Zhengming , Painter Cory L. , Zhang Qin , Chen Enhong , Arango Maria E. , Kuszpit Kyle , Zasadny Kenneth , Hallin Max , Hallin Jill , Wong Anthony , Buckman Dana , Sun Guizhen , Qiu Ming , Anderes Kenna , Christensen James G.
Published: July 2009

Purpose: Checkpoint kinase 1 (Chk1) plays a critical role in the activation of mitotic spindle checkpoint and DNA damage checkpoint. We examined the preclinical use of the Chk1 inhibitor PF-00477736 as a docetaxel-sensitizing agent. Specifically, we investigated the correlation between PF-00477736–mediated modulation of biomarkers and the sensitization of docetaxel efficacy.

Experimental Design: In vitro and in vivo studies using COLO205 and other cell lines were done to assess PF-00477736–induced enhancement of docetaxel efficacy and effects on associated biomarkers.

Results: PF-00477736 significantly enhanced the docetaxel-induced efficacy in tumor cells and xenografts. Docetaxel induced dose- and time-dependent increase in the levels of phosphorylated Chk1 (Ser345), phosphorylated histone H3 (Ser10), and γH2AX foci and promoted the cytoplasmic localization of phosphorylated Cdc25C (Ser216). PF-00477736 cotreatment suppressed docetaxel-induced changes in phosphorylated histone H3 and cytoplasmic phosphorylated Cdc25C (Ser216) levels and concurrently sensitized the docetaxel-induced apoptosis. Docetaxel alone or in combination with PF-00477736 induced significant antiproliferative activity in xenografts, shown via [18F]FLT-PET imaging. However, changes in [18F]FLT uptake did not reflect the potentiation of docetaxel efficacy. In contrast, bioluminescence imaging showed that PF-00477736 sensitized docetaxel-induced suppression of tumor survival.

Conclusions: Docetaxel triggers mitotic spindle checkpoint activation at low concentrations and activates both the DNA damage checkpoint and the spindle checkpoint at high concentrations. In combination with docetaxel, PF-00477736 abrogates the mitotic checkpoint, as well as the DNA damage checkpoint, and results in sensitization to docetaxel. Chk1 inhibitor PF-00477736 offers a therapeutic potential for the enhancement of taxane therapy.
Receptor-Targeted Nanoparticles for In vivo Imaging of Breast Cancer
Authors: Yang Lily , Peng Xiang-Hong , Wang Y. Andrew , Wang Xiaoxia , Cao Zehong , Ni Chunchun , Karna Prasanthi , Zhang Xinjian , Wood William C. , Gao Xiaohu , Nie Shuming , Mao Hui
Published: July 2009

Purpose: Cell-surface receptor-targeted magnetic iron oxide nanoparticles provide molecular magnetic resonance imaging contrast agents for improving specificity of the detection of human cancer.

Experimental Design: The present study reports the development of a novel targeted iron oxide nanoparticle using a recombinant peptide containing the amino-terminal fragment of urokinase-type plasminogen activator (uPA) conjugated to magnetic iron oxide nanoparticles amino-terminal fragment conjugated-iron oxide (ATF-IO). This nanoparticle targets uPA receptor, which is overexpressed in breast cancer tissues.

Results: ATF-IO nanoparticles are able to specifically bind to and be internalized by uPA receptor–expressing tumor cells. Systemic delivery of ATF-IO nanoparticles into mice bearing s.c. and i.p. mammary tumors leads to the accumulation of the particles in tumors, generating a strong magnetic resonance imaging contrast detectable by a clinical magnetic resonance imaging scanner at a field strength of 3 tesla. Target specificity of ATF-IO nanoparticles showed by in vivo magnetic resonance imaging is further confirmed by near-IR fluorescence imaging of the mammary tumors using near-IR dye-labeled amino-terminal fragment peptides conjugated to iron oxide nanoparticles. Furthermore, mice administered ATF-IO nanoparticles exhibit lower uptake of the particles in the liver and spleen compared with those receiving nontargeted iron oxide nanoparticles.

Conclusions: Our results suggest that uPA receptor–targeted ATF-IO nanoparticles have potential as molecularly targeted, dual modality imaging agents for in vivo imaging of breast cancer.
Identification of a New Panel of Serum Autoantibodies Associated with the Presence of In situ Carcinoma of the Breast in Younger Women
Authors: Desmetz Caroline , Bascoul-Mollevi Caroline , Rochaix Philippe , Lamy Pierre-Jean , Kramar Andrew , Rouanet Philippe , Maudelonde Thierry , Mangé Alain , Solassol Jerome
Published: July 2009

Purpose: We examined the feasibility of using a panel of autoantibodies to multiple tumor-associated proteins as a method for early detection of breast cancer and, more particularly, carcinoma in situ (CIS).

Experimental Design: PPIA, PRDX2, and FKBP52 were identified as early-stage breast cancer autoantigens by proteomic approaches. The seroreactivity of a panel of antibodies consisting of these three antigens and two previously described autoantigens, HSP60 and MUC1, was tested on 235 samples (60 from primary breast cancer patients, 82 from CIS patients, and 93 from healthy controls) with the use of specific ELISAs. FKBP52, PPIA, and PRDX2 mRNA and protein expression levels were evaluated by reverse transcription-PCR and immunohistochemistry in early-stage breast tumors.

Results: Three of five autoantibodies, FKBP52, PPIA, and PRDX2, showed significantly increased reactivity in primary breast cancer and CIS compared with healthy controls. When combined, the five markers significantly discriminated primary breast cancer [receiver operating characteristic area under the curve, 0.73; 95% confidence interval (95% CI), 0.60-0.79] and CIS (receiver operating characteristic area under the curve, 0.80; 95% CI, 0.71-0.85) from healthy individuals. Importantly, the receiver operating characteristic–area under the curve value of the autoantibody panel was able to distinguish CIS, including high grades, from healthy controls in women under the age of 50 years (receiver operating characteristic area under the curve, 0.85; 95% CI, 0.61-0.92). Finally, only FKBP52 mRNA and protein levels were found to be increased in CIS and primary breast cancer compared with healthy breast tissue.

Conclusions: This autoantibody assay against a panel of five antigens allows for an accurate discrimination between early-stage breast cancer, especially CIS, and healthy individuals. These results could be of interest in detecting early breast cancer as an aid to mammography, especially in women under the age of 50 years with aggressive cancers.
Targeted Delivery of Tumor Antigens to Activated Dendritic Cells via CD11c Molecules Induces Potent Antitumor Immunity in Mice
Authors: Wei Huafeng , Wang Suhui , Zhang Dapeng , Hou Sheng , Qian Weizhu , Li Bohua , Guo Huaizu , Kou Geng , He Jinqiu , Wang Hao , Guo Yajun
Published: July 2009

Purpose: CD11c is an antigen receptor predominantly expressed on dendritic cells (DC), to which antigen targeting has been shown to induce robust antigen-specific immune responses. To facilitate targeted delivery of tumor antigens to DCs, we generated fusion proteins consisting of the extracellular domain of human HER or its rat homologue neu, fused to the single-chain fragment variable specific for CD11c (scFvCD11c-HER2/neu).

Experimental Design: Induction of cellular and humoral immune responses and antitumoral activity of the fusion proteins admixed with DC-activating CpG oligonucleotides (scFvCD11c-HER2/neuCpG) were tested in transplantable HER2/neu-expressing murine tumor models and in transgenic BALB-neuT mice developing spontaneous neu-driven mammary carcinomas.

Results: Vaccination of BALB/c mice with scFvCD11c-HER2CpG protected mice from subsequent challenge with HER2-positive, but not HER2-negative, murine breast tumor cells, accompanied by induction of strong HER2-specific T-cell and antibody responses. In a therapeutic setting, injection of scFvCD11c-HER2CpG caused rejection of established HER2-positive tumors. Importantly, antitumoral activity of such a fusion protein vaccine could be reproduced in immunotolerant BALB-neuT mice, where scFvCD11c-neuCpG vaccination significantly protected against a subsequent challenge with neu-expressing murine breast tumor cells and markedly delayed the onset of spontaneous mammary carcinomas.

Conclusions: CD11c-targeted protein vaccines for in vivo delivery of tumor antigens to DCs induce potent immune responses and antitumoral activities and provide a rationale for further development of this approach for cancer immunotherapy.
Human Leukocyte Antigen-G Protein Expression Is an Unfavorable Prognostic Predictor of Hepatocellular Carcinoma following Curative Resection
Authors: Cai Ming-Yan , Xu Yong-Feng , Qiu Shuang-Jian , Ju Min-Jie , Gao Qiang , Li Yi-Wei , Zhang Bo-Heng , Zhou Jian , Fan Jia
Published: July 2009

Purpose: Human leukocyte antigen-G (HLA-G) is a tumor-associated immunosuppressive molecule involved in tumor escape mechanisms. The aim of this study is to elucidate its prognostic significance in hepatocellular carcinoma (HCC).

Experimental Design: Immunohistochemical staining of HLA-G expression as well as tumor-infiltrating FoxP3+ regulatory (Tregs) and CD8+ cytotoxic T cells was carried out on tissue microarrays containing 173 HCC tissue specimens. Membrane-bound HLA-G1 protein expression in five human HCC cell lines was detected by Western blot.

Results: HLA-G expression was associated with HCC prognosis, especially in early-stage diseases, with high expression independently associated with shortened overall survival (P = 0.041) and increased tumor recurrence (P = 0.023). HLA-G level was positively related to Tregs/CD8+ ratio and their combination served as a better prognosticator, patients having concurrent high levels of both variables at more than three times of risk of death and tumor relapse than those with concurrent low levels (both P < 0.001). In addition, HLA-G1 expression increased in a concordant manner with the increase of metastatic potential in human HCC cell lines.

Conclusions: Overexpression of HLA-G protein in HCC was an independent indicator for poor outcome especially in early-stage disease. The combination of HLA-G expression and Tregs/CD8+ ratio added the prognostic power to both variables, offering a possible strategy of tumor-stroma interaction-oriented cancer immunotherapy.
Inhibition of PI3K and MEK: It Is All about Combinations and Biomarkers
Authors: Rexer Brent N. , Ghosh Ritwik , Arteaga Carlos L.
Published: July 2009

A small molecule inhibitor of MAP/ERK kinase (MEK) was effective against human breast cancer cells with a basal-like gene expression signature. Antitumor activity was limited by both feedback upregulation of phosphatidylinositol-3 kinase (PI3K)/AKT upon inhibition of MEK as well as loss of the phosphatase PTEN. Therefore, MEK inhibitors should preferably be investigated in combination with PI3K inhibitors in basal-like breast cancers.(Clin Cancer Res 2009;15(14) July 2009).
Oncogenic transformation confers a selective susceptibility to the combined suppression of the proteasome and autophagy
Authors: Ding Wen-Xing , Ni Hong-Min , Gao Wentao , Chen Xiaoyun , Kang Jeong Han , Stolz Donna B. , Liu Jinsong , Yin Xiao-Ming
Published: July 2009

The proteasome and the autophagy systems are two evolutionarily conserved mechanisms for degrading intracellular materials. They are functionally coupled and suppression of the proteasome promotes autophagy. Although suppression of the proteasome leads to cell death, suppression of autophagy can be either prodeath or prosurvival. To understand the underlining mechanism of this dichotomy and its potential clinical implications, we treated various transformed and nontransformed human cells with proteasome inhibitors. We found that whether autophagy served a prosurvival role in this scenario was contingent on the cellular oncogenic status. Thus, autophagy suppression enhanced apoptosis induced by proteasome inhibitors in transformed cells, but not in nontransformed cells. Oncogenic transformation enhanced the ability of cells to initiate autophagy in response to stress, reflecting a stronger dependence of transformed cells on autophagy for survival. Indeed, a combined use of bortezomib, the only Food and Drug Administration–approved proteasome inhibitor for clinical use, and chloroquine, which inhibits autophagy by disturbing lysosomal functions, suppressed tumor growth more significantly than either agent alone in a xenograft model. These findings indicate that suppression of both intracellular degradation systems could constitute a novel strategy for enhanced cancer control in a tumor-specific way. [Mol Cancer Ther 2009;8(7):2036–45]
Biological properties of potent inhibitors of class I phosphatidylinositide 3-kinases: from PI-103 through PI-540, PI-620 to the oral agent GDC-0941
Authors: Raynaud Florence I. , Eccles Suzanne A. , Patel Sonal , Alix Sonia , Box Gary , Chuckowree Irina , Folkes Adrian , Gowan Sharon , De Haven Brandon Alexis , Di Stefano Francesca , Hayes Angela , Henley Alan T. , Lensun Letitia , Pergl-Wilson Giles , Robson Anthony , Saghir Nahid , Zhyvoloup Alexander , McDonald Edward , Sheldrake Peter , Shuttleworth Stephen , Valenti Melanie , Wan Nan Chi , Clarke Paul A. , Workman Paul
Published: July 2009

The phosphatidylinositide 3-kinase pathway is frequently deregulated in human cancers and inhibitors offer considerable therapeutic potential. We previously described the promising tricyclic pyridofuropyrimidine lead and chemical tool compound PI-103. We now report the properties of the pharmaceutically optimized bicyclic thienopyrimidine derivatives PI-540 and PI-620 and the resulting clinical development candidate GDC-0941. All four compounds inhibited phosphatidylinositide 3-kinase p110α with IC50 ≤ 10 nmol/L. Despite some differences in isoform selectivity, these agents exhibited similar in vitro antiproliferative properties to PI-103 in a panel of human cancer cell lines, with submicromolar potency in PTEN-negative U87MG human glioblastoma cells and comparable phosphatidylinositide 3-kinase pathway modulation. PI-540 and PI-620 exhibited improvements in solubility and metabolism with high tissue distribution in mice. Both compounds gave improved antitumor efficacy over PI-103, following i.p. dosing in U87MG glioblastoma tumor xenografts in athymic mice, with treated/control values of 34% (66% inhibition) and 27% (73% inhibition) for PI-540 (50 mg/kg b.i.d.) and PI-620 (25 mg/kg b.i.d.), respectively. GDC-0941 showed comparable in vitro antitumor activity to PI-103, PI-540, and PI-620 and exhibited 78% oral bioavailability in mice, with tumor exposure above 50% antiproliferative concentrations for >8 hours following 150 mg/kg p.o. and sustained phosphatidylinositide 3-kinase pathway inhibition. These properties led to excellent dose-dependent oral antitumor activity, with daily p.o. dosing at 150 mg/kg achieving 98% and 80% growth inhibition of U87MG glioblastoma and IGROV-1 ovarian cancer xenografts, respectively. Together, these data support the development of GDC-0941 as a potent, orally bioavailable inhibitor of phosphatidylinositide 3-kinase. GDC-0941 has recently entered phase I clinical trials. [Mol Cancer Ther 2009;8(7):1725–38] [Mol Cancer Ther 2009;8(7):1725–38]
Inhibition of S-adenosylmethionine decarboxylase by inhibitor SAM486A connects polyamine metabolism with p53-Mdm2-Akt/protein kinase B regulation and apoptosis in neuroblastoma
Authors: Koomoa Dana-Lynn T. , Borsics Tamas , Feith David J. , Coleman Craig C. , Wallick Christopher J. , Gamper Ivonne , Pegg Anthony E. , Bachmann André S.
Published: July 2009

S-adenosylmethionine decarboxylase (AdoMetDC) is an essential enzyme of polyamine (PA) biosynthesis, and both AdoMetDC and PA levels are often up-regulated in cancer cells. The second-generation inhibitor SAM486A inhibits AdoMetDC enzyme activity and has been evaluated in phase II clinical cancer trials. However, little is known about the mechanism of action and potential use of this therapeutic drug in the treatment of the pediatric cancer neuroblastoma (NB). Here, we show that p53 wild-type NB cells are highly sensitive to SAM486A treatment. Most notably, SAM486A treatment resulted in the rapid accumulation of proapoptotic proteins p53 and Mdm2. Concomitant with the increase of proteins at endogenous levels, the in vivo phosphorylation of p53 at residues Ser46/Ser392 and Mdm2 at residue Ser166 was observed. Moreover, the antiapoptotic protein Akt/protein kinase B was down-regulated and also dephosphorylated at residue Ser473 in a dose- and time-dependent manner and NB cells entered apoptotic cell death. The results presented in this study highlight the importance of PA homeostasis and provide a direct link between PA metabolism and apoptotic cell signaling pathways in p53 wild-type NB cells. PA inhibitors such as SAM486A may be effective alternative agents for the treatment of NBs with or without MYCN amplification. [Mol Cancer Ther 2009;8(7):2067–75]
Topoisomerase I levels in the NCI-60 cancer cell line panel determined by validated ELISA and microarray analysis and correlation with indenoisoquinoline sensitivity
Authors: Pfister Thomas D. , Reinhold William C. , Agama Keli , Gupta Shalu , Khin Sonny A. , Kinders Robert J. , Parchment Ralph E. , Tomaszewski Joseph E. , Doroshow James H. , Pommier Yves
Published: July 2009

Topoisomerase I (Top1) is a proven target for cancer therapeutics, and the level of Top1 in tumors has been used as a biomarker for chemotherapeutic efficacy. In this study, we report the development and validation of a two-site enzyme chemiluminescent immunoassay for Top1, which we used to measure Top1 levels in the NCI-60 cancer cell line panel. Top1 levels ranged from 0.9 to 9.8 ng/mL/μg protein extract in these cell lines. Levels varied both within and between cancer types but were generally highest in colon cancer and leukemia cell lines and lowest in central nervous system and renal cancer cell lines. Top1 mRNA levels in the NCI-60 cell lines were also measured by microarray; mRNA values generally showed a good correlation with protein levels (Pearson correlation = 0.8). When these expression levels were compared with the activity of the indenoisoquinoline class of Top1 inhibitors across the NCI-60 cell panel, low levels of Top1 were associated with increased resistance to these drugs. The results of our studies indicate that our Top1 assay can be used to quantify Top1 levels in untreated cells as well as cells treated with Top1 inhibitors and that the assay has the potential to be adapted for use in predicting clinical response to Top1-active antineoplastic agents. [Mol Cancer Ther 2009;8(7):1878–84]
Upregulation of thrombospondin-1 and angiogenesis in an aggressive human pancreatic cancer cell line selected for high metastasis
Authors: McElroy Michele K. , Kaushal Sharmeela , Tran Cao Hop S. , Moossa A.R. , Talamini Mark A. , Hoffman Robert M. , Bouvet Michael
Published: July 2009

Pancreatic cancer remains a leading cause of death despite its relatively low incidence. As in many other solid tumors, angiogenesis is critical to the growth and metastasis of this cancer. Through serial in vivo passages in mice, we have developed a highly aggressive variant of human pancreatic cancer cell line XPA-1 which shows more rapid primary tumor growth, faster time to metastasis, and more rapid lethality than the parental cell line. The high-metastatic variant developed a much denser tumor vasculature early during growth within the pancreas. Interestingly, examination of the in vitro growth of this aggressive variant yielded no significant difference from the parental cell line. Real-time PCR evaluation of genes involved in angiogenesis revealed a 24-fold increase in Thrombospondin-1 expression in cells derived from the high-metastatic variant when compared with the parental cell line. These findings provide direct evidence that elevated capability for angiogenesis, mediated by specific changes in gene expression, can lead to a large increase in cancer aggressiveness and resulting metastasis. These findings have important implications for the treatment of metastatic disease. [Mol Cancer Ther 2009;8(7):1779–45]
EGFRvIII and c-Met pathway inhibitors synergize against PTEN-null/EGFRvIII+ glioblastoma xenografts
Authors: Lal Bachchu , Goodwin C. Rory , Sang Yingying , Foss Catherine A. , Cornet Kathrine , Muzamil Sameena , Pomper Martin G. , Kim Jin , Laterra John
Published: July 2009

Receptor tyrosine kinase (RTK) systems, such as hepatocyte growth factor (HGF) and its receptor c-Met, and epidermal growth factor receptor (EGFR), are responsible for the malignant progression of multiple solid tumors. Recent research shows that these RTK systems comodulate overlapping and dynamically adaptable oncogenic downstream signaling pathways. This study investigates how EGFRvIII, a constitutively active EGFR deletion mutant, alters tumor growth and signaling responses to RTK inhibition in PTEN-null/HGF+/c-Met+ glioma xenografts. We show that a neutralizing anti-HGF monoclonal antibody (L2G7) potently inhibits tumor growth and the activation of Akt and mitogen-activated protein kinase (MAPK) in PTEN-null/HGF+/c-Met+/EGFRvIII− U87 glioma xenografts (U87wt). Isogenic EGFRvIII+ U87 xenografts (U87-EGFRvIII), which grew five times more rapidly than U87-wt xenografts, were unresponsive to EGFRvIII inhibition by erlotinib and were only minimally responsive to anti-HGF monoclonal antibodies. EGFRvIII expression diminished the magnitude of Akt inhibition and completely prevented MAPK inhibition by L2G7. Despite the lack of response to L2G7 or erlotinib as single agents, their combination synergized to produce substantial antitumor effects (inhibited tumor cell proliferation, enhanced apoptosis, arrested tumor growth, prolonged animal survival), against subcutaneous and orthotopic U87-EGFRvIII xenografts. The dramatic response to combining HGF:c-Met and EGFRvIII pathway inhibitors in U87-EGFRvIII xenografts occurred in the absence of Akt and MAPK inhibition. These findings show that combining c-Met and EGFRvIII pathway inhibitors can generate potent antitumor effects in PTEN-null tumors. They also provide insights into how EGFRvIII and c-Met may alter signaling networks and reveal the potential limitations of certain biochemical biomarkers to predict the efficacy of RTK inhibition in genetically diverse cancers. [Mol Cancer Ther 2009;8(7):1751–60]
Increased antitumor activity of bevacizumab in combination with hypoxia inducible factor-1 inhibition
Authors: Rapisarda Annamaria , Hollingshead Melinda , Uranchimeg Badarch , Bonomi Carrie A. , Borgel Suzanne D. , Carter John P. , Gehrs Bradley , Raffeld Mark , Kinders Robert J. , Parchment Ralph , Anver Miriam R. , Shoemaker Robert H. , Melillo Giovanni
Published: July 2009

Inhibition of hypoxia inducible factor-1 (HIF-1) is an attractive therapeutic strategy to target the tumor microenvironment. However, HIF-1 inhibitors may have limited activity as single agents and combination therapies may be required. We tested the hypothesis that HIF-1 inhibition in a hypoxic-stressed tumor microenvironment, which could be generated by administration of antiangiogenic agents, may result in a more pronounced therapeutic effect. The activity of bevacizumab, either alone or in combination with the HIF-1α inhibitor topotecan, was evaluated in U251-HRE xenografts. Tumor tissue was collected at the end of treatment and changes in tumor oxygenation, angiogenesis, proliferation, apoptosis, HIF-1α levels, HIF-1 target genes, and DNA damage were evaluated. Bevacizumab decreased microvessel-density and increased intratumor-hypoxia, but did not induce apoptosis. Moreover, bevacizumab alone caused a significant increase of HIF-1–dependent gene expression in tumor tissue. Addition of a low dose of daily topotecan to bevacizumab significantly inhibited tumor growth, relative to mice treated with topotecan or bevacizumab alone (P < 0.01). The addition of topotecan to bevacizumab was also associated with profound inhibition of HIF-1 transcriptional activity, significant inhibition of proliferation, and induction of apoptosis. Importantly, DNA damage induced by topotecan alone was not augmented by addition of bevacizumab, suggesting that increased cytotoxic activity did not account for the increased antitumor effects observed. These results strongly suggest that combination of anti–vascular endothelial growth factor antibodies with HIF-1 inhibitors is an attractive therapeutic strategy targeting in the hypoxic tumor microenvironment. [Mol Cancer Ther 2009;8(7):1867–77]
Pertuzumab increases epidermal growth factor receptor down-regulation by counteracting epidermal growth factor receptor-ErbB2 heterodimerization
Authors: Hughes Juliana Bentes , Berger Christian , Rødland Marianne Skeie , Hasmann Max , Stang Espen , Madshus Inger Helene
Published: July 2009

Epidermal growth factor receptor (EGFR) and ErbB2 readily form heterodimers when both are expressed in the same cell and the EGFR is activated by one of its ligands. Our data show that such heterodimers are constitutively formed also in a ligand-independent manner on overexpression of EGFR and ErbB2 in porcine aortic endothelial cells. Interestingly, cross-linking experiments showed that incubation with the antibody pertuzumab, which has been shown to bind the dimerization arm of ErbB2, resulted in dissolution of EGFR-ErbB2 heterodimers. Incubation with pertuzumab also increased the amount of EGF-induced EGFR homodimers, and under these conditions, endocytosis of radiolabeled EGF was increased. This increase was significant, although slightly more EGF was internalized in cells expressing EGFR only compared with pertuzumab-treated cells expressing both EGFR and ErbB2. By confocal microscopy analysis, more EGF was observed in endosomes on incubation with pertuzumab, and under similar conditions, immunoblotting experiments showed increased EGFR degradation on incubation with both EGF and pertuzumab. These results show that pertuzumab enhanced the endocytic down-regulation of EGFR by counteracting EGFR-ErbB2 heterodimerization. Our previous results showing that ErbB2 counteracts EGFR endocytosis can therefore be explained by tethering of EGFR to ErbB2 at the plasma membrane. [Mol Cancer Ther 2009;8(7):1885–92]