bluuebunny/crossref_metadata_2025 huggingface dataset:
Insulin-Like Growth Factor-I, Regulating Aromatase Expression through Steroidogenic Factor 1, Supports Estrogen-Dependent Tumor Leydig Cell Proliferation
Authors: Sirianni Rosa , Chimento Adele , Malivindi Rocco , Mazzitelli Ignazio , Andò Sebastiano , Pezzi Vincenzo
Published: September 2007

The aim of this study was to investigate the role of estrogens in Leydig cell tumor proliferation. We used R2C rat Leydig tumor cells and testicular samples from Fischer rats with a developed Leydig tumor. Both experimental models express high levels of aromatase and estrogen receptor α (ERα). Treatment with exogenous 17β-estradiol (E2) induced proliferation of R2C cells and up-regulation of cell cycle regulators cyclin D1 and cyclin E, the expression of which was blocked by addition of antiestrogens. These observations led us to hypothesize an E2/ERα–dependent mechanism for Leydig cell tumor proliferation. In determining the molecular mechanism responsible for aromatase overexpression, we found that total and phosphorylated levels of transcription factors cyclic AMP–responsive element binding protein and steroidogenic factor 1 (SF-1) were higher in tumor samples. Moreover, we found that tumor Leydig cells produce high levels of insulin-like growth factor I (IGF-I), which increased aromatase mRNA, protein, and activity as a consequence of increased total and phosphorylated SF-1 levels. Specific inhibitors of IGF-I receptor, protein kinase C, and phosphatidylinositol 3-kinase determined a reduction in SF-1 expression and in IGF-I–dependent SF-1 recruitment to the aromatase PII promoter. The same inhibitors also inhibited aromatase expression and activity and, consequently, R2C cell proliferation. We can conclude that one of the molecular mechanisms determining Leydig cell tumorigenesis is an excessive estrogen production that stimulates a short autocrine loop determining cell proliferation. In addition, cell-produced IGF-I amplifies estrogen signaling through an SF-1–dependent up-regulation of aromatase expression. The identification of this molecular mechanism will be helpful in defining new therapeutic approaches for Leydig cell tumors. [Cancer Res 2007;67(17):8368–77]
In vivo Role of NAD(P)H:Quinone Oxidoreductase 1 in Metabolic Activation of Mitomycin C and Bone Marrow Cytotoxicity
Authors: Adikesavan Anbu Karani , Barrios Roberto , Jaiswal Anil K.
Published: September 2007

NAD(P)H:quinone oxidoreductase 1−/− (NQO1−/−), NQO1+/− along with NRH:quinone oxidoreductase 2−/− (NQO2−/−), and wild-type (WT) mice were exposed to five once weekly doses of mitomycin C. The mice were euthanized 15 weeks after the first dose. Blood cell counts and histologic analyses were done. WT and NQO2−/− mice showed hypocellularity and a significant increase in adipocytes in bone marrow. They also showed anemia because of the loss of RBC and hemoglobin. The neutrophils and platelets were reduced, whereas other blood cell types and tissues were normal. Interestingly, NQO1−/− mice showed a complete resistance to mitomycin C–induced bone marrow cytotoxicity and reduction in RBC, hemoglobin, and neutrophils. NQO1+/− mice also showed limited resistance to mitomycin C–induced bone marrow cytotoxicity. These data show a major in vivo role of NQO1 in metabolic activation of mitomycin C with implications in mitomycin C chemotherapy. [Cancer Res 2007;67(17):7966–71]
Enrichment of a Population of Mammary Gland Cells that Form Mammospheres and Have In vivo Repopulating Activity
Authors: Liao Mai-Jing , Zhang Cheng Cheng , Zhou Beiyan , Zimonjic Drazen B. , Mani Sendurai A. , Kaba Megan , Gifford Ann , Reinhardt Ferenc , Popescu Nicholas C. , Guo Wenjun , Eaton Elinor Ng , Lodish Harvey F. , Weinberg Robert A.
Published: September 2007

The identification of mammary gland stem cells (MGSC) or progenitors is important for the study of normal breast development and tumorigenesis. Based on their immunophenotype, we have isolated a population of mouse mammary gland cells that are capable of forming “mammospheres” in vitro. Importantly, mammospheres are enriched for cells that regenerate an entire mammary gland on implantation into a mammary fat pad. We also undertook cytogenetic analyses of mammosphere-forming cells after prolonged culture, which provided preliminary insight into the genomic stability of these cells. Our identification of new cell surface markers for enriching mammosphere-initiating cells, including endoglin and prion protein, will facilitate the elucidation of the cell biology of MGSC. [Cancer Res 2007;67(17):8131–8]
Lipid Raft–Specific Knockdown of Src Family Kinase Activity Inhibits Cell Adhesion and Cell Cycle Progression of Breast Cancer Cells
Authors: Hitosugi Taro , Sato Moritoshi , Sasaki Kazuki , Umezawa Yoshio
Published: September 2007

Src family kinase (SFK) is known to control various cell functions, but the significance of the location of its activation was largely unknown. We herein revealed that SFK activation occurs in lipid rafts. Based on this finding, we have developed a lipid raft–targeted SFK inhibitory fusion protein (LRT-SIFP) that inhibits the SFK activity in lipid rafts. LRT-SIFP has a peptide inhibitor of SFK and a lipid raft–targeting sequence in which two cysteine residues are palmitoylated for clustering in lipid rafts. LRT-SIFP was found to inhibit cell adhesion and cell cycle progression of human breast cancer cell lines MCF-7 and MDA-MB231. On the other hand, the cell functions of MCF-7 cells were found to be not affected with a previously developed peptide inhibitor of SFK that lacks the lipid raft–targeting sequence. In addition, when we replaced the targeting sequence of LRT-SIFP with the consensus sequence for geranylgeranylation to make LRT-SIFP unable to cluster in lipid rafts, this mutated LRT-SIFP did not show any effect on the above cell functions of MCF-7 cells. Furthermore, in contrast to the breast cancer cell lines, LRT-SIFP did not show any inhibitory effect on cell adhesion and cell cycle progression of human normal cell line HEK293. The present lipid raft–specific knockdown of SFK activity would potentially be useful for selective cancer therapy to prevent tumorigenesis and metastasis of breast cancer cells. [Cancer Res 2007;67(17):8139–48]
Ribosomal S6 Kinase 2 Is a Key Regulator in Tumor Promoter–Induced Cell Transformation
Authors: Cho Yong-Yeon , Yao Ke , Kim Hong-Gyum , Kang Bong Seok , Zheng Duo , Bode Ann M. , Dong Zigang
Published: September 2007

The ribosomal S6 kinase 2 (RSK2), a member of the p90RSK (RSK) family of proteins, is a widely expressed serine/threonine kinase that is activated by extracellular signal-regulated kinase 1/2 and phosphoinositide-dependent kinase 1 in response to many growth factors and peptide hormones. Its activation signaling enhances cell survival. However, the roles of RSK2 in cell transformation have not yet been elucidated. Here, we found that RSK2 is a critical serine/threonine kinase for the regulation of cell transformation. When cells were stimulated with tumor promoters, such as epidermal growth factor (EGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA), phosphorylation of RSK was increased within 5 min. Cell proliferation was suppressed in RSK2−/− mouse embryonic fibroblasts (MEFs) compared with RSK2+/+ MEFs. Moreover, RSK2−/− MEFs accumulated at the G1 phase of the cell cycle under normal cell culture conditions as well as after stimulation with EGF or TPA. In the anchorage-independent cell transformation assay (soft agar), stable expression of RSK2 in JB6 cells significantly enhanced colony formation in either the presence or absence of tumor promoters. Furthermore, knockdown of RSK2 with small interfering RNA-RSK2 suppressed constitutively active Ras (RasG12V)-induced foci formation in NIH3T3 cells. In addition, kaempferol, an inhibitor of RSK2, suppressed EGF-induced colony formation of JB6 Cl41 cells in soft agar, which was associated with inhibition of histone H3 phosphorylation (Ser10). These results showed that RSK2 is a key regulator for cell transformation induced by tumor promoters such as EGF and TPA. [Cancer Res 2007;67(17):8104–12]
Taxol Increases the Amount and T Cell–Activating Ability of Self-Immune Stimulatory Multimolecular Complexes Found in Ovarian Cancer Cells
Authors: Tsuda Naotake , Chang David Z. , Mine Takashi , Efferson Clay , García-Sastre Adolfo , Wang Xinhui , Ferrone Soldano , Ioannides Constantin G.
Published: September 2007

It has been proposed that chemotherapy enhances tumor antigen (TA)–specific immunity. The molecular form of TA from ovarian tumor that activates cellular immunity is unknown. We report here identification of a novel molecular form of immunogenic TA for CD8+ cells named self-immune stimulatory multimolecular complexes (ISMMC). ISMMC consist of a molecular complex of polyosome/ribosome-bound ubiquitinated nascent HER-2 polypeptides. This complex is chaperoned by heat shock protein Gp96, which mediates ISMMC uptake by antigen-presenting cells through the scavenger receptor CD91. RNAs in ISMMC stimulate immature dendritic cells to secrete interleukin 12 and induce IFN-γ in peripheral blood mononuclear cells. ISMMC dissociate, retrotranslocate from the lysosome to cytoplasm, and are processed to peptides by the proteasome. At subpharmacologic doses, Taxol increased the amount of ISMMC by three to four times and modified their composition by inducing the attachment of cochaperones of HSP70, such as the mitotic-phase phosphoprotein 11J. On a total protein basis, Taxol induced ISMMC, expanded more CD8+ cells, activated more CD56+ NKG2D+ cells to produce IFN-γ, and were more potent inducers of high T-cell receptor density Perforin+ cells than native ISMMC and peptide E75. Elucidation of the composition of ISMMC and identification of adducts formed by Taxol should be important for developing molecular cancer vaccines. [Cancer Res 2007;67(17):8378–87]
Silibinin Sensitizes Human Glioma Cells to TRAIL-Mediated Apoptosis via DR5 Up-regulation and Down-regulation of c-FLIP and Survivin
Authors: Son Yong-gyu , Kim Eun Hee , Kim Jin Yeop , Kim Seung U. , Kwon Taeg Kyu , Yoon A-Rum , Yun Chae-Ok , Choi Kyeong Sook
Published: September 2007

Silibinin, a flavonoid isolated from Silybum marianum, has been reported to have cancer chemopreventive and therapeutic effects. Here, we show that treatment with subtoxic doses of silibinin in combination with tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) induces rapid apoptosis in TRAIL-resistant glioma cells, but not in human astrocytes, suggesting that this combined treatment may offer an attractive strategy for safely treating gliomas. Although the proteolytic processing of procaspase-3 by TRAIL was partially blocked in glioma cells, cotreatment with silibinin efficiently recovered TRAIL-induced caspase activation in these cells. Silibinin treatment up-regulated DR5, a death receptor of TRAIL, in a transcription factor CHOP-dependent manner. Furthermore, treatment with silibinin down-regulated the protein levels of the antiapoptotic proteins FLIPL, FLIPS, and survivin through proteasome-mediated degradation. Taken together, our results show that the activity of silibinin to modulate multiple components in the death receptor–mediated apoptotic pathway is responsible for its ability to recover TRAIL sensitivity in TRAIL-resistant glioma cells. [Cancer Res 2007;67(17):8274–84]
Tissue-Specific Consequences of Cyclin D1 Overexpression in Prostate Cancer Progression
Authors: He Yue , Franco Omar E. , Jiang Ming , Williams Karin , Love Harold D. , Coleman Ilsa M. , Nelson Peter S. , Hayward Simon W.
Published: September 2007

The cyclin D1 oncogene encodes the regulatory subunit of a holoenzyme that phosphorylates and inactivates the Rb protein and promotes progression through G1 to S phase of the cell cycle. Several prostate cancer cell lines and a subset of primary prostate cancer samples have increased cyclin D1 protein expression. However, the relationship between cyclin D1 expression and prostate tumor progression has yet to be clearly characterized. This study examined the effects of manipulating cyclin D1 expression in either human prostatic epithelial or stromal cells using a tissue recombination model. The data showed that overexpression of cyclin D1 in the initiated BPH-1 cell line increased cell proliferation rate but did not elicit tumorigenicity in vivo. However, overexpression of cyclin D1 in normal prostate fibroblasts (NPF) that were subsequently recombined with BPH-1 did induce malignant transformation of the epithelial cells. The present study also showed that recombination of BPH-1 + cyclin D1–overexpressing fibroblasts (NPFcyclin D1) resulted in permanent malignant transformation of epithelial cells (BPH-1NPF-cyclin D1 cells) similar to that seen with carcinoma-associated fibroblasts (CAF). Microarray analysis showed that the expression profiles between CAFs and NPFcyclin D1 cells were highly concordant including cyclin D1 up-regulation. These data indicated that the tumor-promoting activity of cyclin D1 may be tissue specific. [Cancer Res 2007;67(17):8188–97]
Epidermal Growth Factor Receptor (EGFR) Ubiquitination as a Mechanism of Acquired Resistance Escaping Treatment by the Anti-EGFR Monoclonal Antibody Cetuximab
Authors: Lu Yang , Li Xinqun , Liang Ke , Luwor Rodney , Siddik Zahid H. , Mills Gordon B. , Mendelsohn John , Fan Zhen
Published: September 2007

Cetuximab is an epidermal growth factor receptor (EGFR)–blocking antibody that has been approved for treatment of patients with metastatic colorectal cancer. In this study, we investigated biochemical changes in signaling pathways of a cetuximab-resistant subline of DiFi colorectal cancer cells (DiFi5) that was developed by exposing the parental sensitive cells to subeffective doses of cetuximab over an extended period of time. Compared with parental DiFi cells that express high levels of EGFR and in which cetuximab induces apoptosis, the cetuximab-resistant DiFi5 cells showed markedly lower protein levels of EGFR, an increased association of EGFR with Cbl, and an increased ubiquitination of EGFR. DiFi5 cells also had a markedly higher level of Src-Y416 phosphorylation both at baseline and on EGF stimulation. Although EGFR levels were low, DiFi5 cells responded to EGF stimulation with robust phosphorylation of EGFR on Y845 and strong phosphorylation of Akt and extracellular signal–regulated kinase, comparable to those of parental cells. Most importantly, inhibition of Src kinase activity with PP2 reversed the resistance of DiFi5 cells to cetuximab-induced apoptosis without affecting the levels of EGFR in the cells. Our results indicate that colorectal cancer cells may develop acquired resistance to cetuximab via altering EGFR levels through promotion of EGFR ubiquitination and degradation and using Src kinase-mediated cell signaling to bypass their dependency on EGFR for cell growth and survival. [Cancer Res 2007;67(17):8240–7]
Activity of Androgen Receptor Antagonist Bicalutamide in Prostate Cancer Cells Is Independent of NCoR and SMRT Corepressors
Authors: Hodgson Myles C. , Astapova Inna , Hollenberg Anthony N. , Balk Steven P.
Published: September 2007

The mechanisms by which androgen receptor (AR) antagonists inhibit AR activity, and how their antagonist activity may be abrogated in prostate cancer that progresses after androgen deprivation therapy, are not clear. Recent studies show that AR antagonists (including the clinically used drug bicalutamide) can enhance AR recruitment of corepressor proteins [nuclear receptor corepressor (NCoR) and silencing mediator of retinoid and thyroid receptors (SMRT)] and that loss of corepressors may enhance agonist activity and be a mechanism of antagonist failure. We first show that the agonist activities of weak androgens and an AR antagonist (cyproterone acetate) are still dependent on the AR NH2/COOH-terminal interaction and are enhanced by steroid receptor coactivator (SRC)-1, whereas the bicalutamide-liganded AR did not undergo a detectable NH2/COOH-terminal interaction and was not coactivated by SRC-1. However, both the isolated AR NH2 terminus and the bicalutamide-liganded AR could interact with the SRC-1 glutamine-rich domain that mediates AR NH2-terminal binding. To determine whether bicalutamide agonist activity was being suppressed by NCoR recruitment, we used small interfering RNA to deplete NCoR in CV1 cells and both NCoR and SMRT in LNCaP prostate cancer cells. Depletion of these corepressors enhanced dihydrotestosterone-stimulated AR activity on a reporter gene and on the endogenous AR-regulated PSA gene in LNCaP cells but did not reveal any detectable bicalutamide agonist activity. Taken together, these results indicate that bicalutamide lacks agonist activity and functions as an AR antagonist due to ineffective recruitment of coactivator proteins and that enhanced coactivator recruitment, rather than loss of corepressors, may be a mechanism contributing to bicalutamide resistance. [Cancer Res 2007;67(17):8388–95]
Cutaneous Human Papillomaviruses Down-regulate AKT1, whereas AKT2 Up-regulation and Activation Associates with Tumors
Authors: O'Shaughnessy Ryan F.L. , Akgũl Baki , Storey Alan , Pfister Herbert , Harwood Catherine A. , Byrne Carolyn
Published: September 2007

Epithelial tumorigenesis has been linked to AKT up-regulation. Human papillomaviruses (HPV) cause anogenital cancers and anogenital HPV infection up-regulates AKT activity. Mounting evidence points to a role for cutaneous HPVs as etiologic factors in skin tumorigenesis. High-risk cutaneous β HPVs have been linked to carcinogenesis in immunosuppressed patients, and high-risk cutaneous HPV8 genes enhance tumorigenesis in transgenic mice. We find that, in contrast to anogenital HPVs, cutaneous HPV8 early genes down-regulate epidermal AKT activity by down-regulating AKT1 isoform levels. This down-regulation occurs before papilloma formation or tumorigenesis and leads to cutaneous differentiation changes that may weaken the epidermal squame for viral release. We find that, in viral warts (papillomas) and HPV gene-induced epidermal tumors, AKT activity can be activated focally by up-regulation and phosphorylation of the AKT2 isoform. In squamous cell carcinomas (SCC), AKT1 down-regulation is also common, consistent with a viral influence, whereas AKT2 up-regulation is widespread. Activation of up-regulated AKT2 by serine phosphorylation associates with high-grade tumors. Our data suggest that AKT2 up-regulation is characteristic of SCC and that coincident AKT2 activation through serine phosphorylation correlates with malignancy. These findings highlight differences between the effects of anogenital and cutaneous HPV on epithelial AKT activity and furthermore show that AKT isoforms can behave differently during epidermal tumorigenesis. These findings also suggest AKT2 as a possible therapeutic tumor target in SCC. [Cancer Res 2007;67(17):8207–15]
Activation of Double-Stranded RNA–Dependent Protein Kinase, A New Pathway by Which Human Polynucleotide Phosphorylase (hPNPaseold-35) Induces Apoptosis
Authors: Sarkar Devanand , Park Eun Sook , Barber Glen N. , Fisher Paul B.
Published: September 2007

Human polynucleotide phosphorylase (hPNPaseold-35) is a type I IFN-inducible 3′,5′ exoribonuclease that mediates mRNA degradation. In melanoma cells, slow and sustained overexpression of hPNPaseold-35 induces G1 cell cycle arrest ultimately culminating in apoptosis, whereas rapid overexpression of hPNPaseold-35 directly promotes apoptosis without cell cycle changes. These observations imply that inhibition of cell cycle progression and induction of apoptosis by hPNPaseold-35 involve multiple intracellular targets and signaling pathways. We now provide evidence that the apoptosis-inducing activity of hPNPaseold-35 is mediated by activation of double-stranded RNA–dependent protein kinase (PKR). Activation of PKR by hPNPaseold-35 precedes phosphorylation of eukaryotic initiation factor-2α and induction of growth arrest and DNA damage-inducible gene 153 (GADD153) that culminates in the shutdown of protein synthesis and apoptosis. Activation of PKR by hPNPaseold-35 also instigates down-regulation of the antiapoptotic protein Bcl-xL. A dominant-negative inhibitor of PKR, as well as GADD153 antisense or bcl-xL overexpression, effectively inhibits apoptosis induction by hPNPaseold-35. These studies elucidate a novel pathway by which an evolutionary conserved RNA-metabolizing enzyme, hPNPaseold-35, regulates cell growth and viability. [Cancer Res 2007;67(17):7948–53]
Side Population Cells Isolated from Mesenchymal Neoplasms Have Tumor Initiating Potential
Authors: Wu Colleen , Wei Qingxia , Utomo Velani , Nadesan Puviindran , Whetstone Heather , Kandel Rita , Wunder Jay S. , Alman Benjamin A.
Published: September 2007

Although many cancers are maintained by tumor-initiating cells, this has not been shown for mesenchymal tumors, in part due to the lack of unique surface markers that identify mesenchymal progenitors. An alternative technique to isolate stem-like cells is to isolate side population (SP) cells based on efflux of Hoechst 33342 dye. We examined 29 mesenchymal tumors ranging from benign to high-grade sarcomas and identified SP cells in all but six samples. There was a positive correlation between the percentage of SP cells and the grade of the tumor. SP cells preferentially formed tumors when grafted into immunodeficient mice, and only cells from tumors that developed from the SP cells had the ability to initiate tumor formation upon serial transplantation. Although SP cells are able to efflux rhodamine dye in addition to Hoechst 33342, we found that the ability to efflux rhodamine dye did not identify a population of cells enriched for tumor-initiating capacity. Here, we identify a subpopulation of cells within a broad range of benign and malignant mesenchymal tumors with tumor-initiating capacity. In addition, our data suggest that the proportion of SP cells could be used as a prognostic factor and that therapeutically targeting this subpopulation of cells could be used to improve patient outcome. [Cancer Res 2007;67(17):8216–22]
γ-Secretase Inhibitor Prevents Notch3 Activation and Reduces Proliferation in Human Lung Cancers
Authors: Konishi Jun , Kawaguchi Keiko S. , Vo Huan , Haruki Nobuhiro , Gonzalez Adriana , Carbone David P. , Dang Thao P.
Published: September 2007

Notch receptors are key regulators of development by controlling cell-fate determination in many multicellular organisms. Genes that are important for normal differentiation play a role in cancer when their normal functions became dysregulated. Notch signaling has been shown to promote and maintain survival of many types of cancers, and we previously have shown that Notch3 plays an important role in lung cancer. In this study, we showed that a high percentage of lung cancer lines expressed Jagged1, Notch receptors, and their transcriptional target genes (HES1, Hey1), suggesting that the Notch pathway plays an important role in lung cancer biology. Thus, inhibition of Notch receptor activation represents a compelling treatment strategy. Notch activation requires proteolytic cleavage of the receptor by γ-secretase protein complex. In this study, we determined the ability of MRK-003, a γ-secretase inhibitor, to inhibit Notch3 signaling, growth, and apoptosis of lung cancer cell lines in vitro and in vivo using mouse xenograft models. We also found that MRK-003 inhibited Notch3 signaling, reduced tumor cell proliferation, inhibited serum independence, and induced apoptosis. This drug had no effect when Notch3 expression was knocked down using small interfering RNA (siRNA), suggesting that the observed effects were mediated by specific action on this receptor. In conclusion, these results support the hypothesis that inhibition of Notch activation using a γ-secretase inhibitor represents a potential new approach for the targeted therapy of lung cancer. [Cancer Res 2007;67(17):8051–7]
Use of Reverse Genetics to Enhance the Oncolytic Properties of Newcastle Disease Virus
Authors: Vigil Adam , Park Man-Seong , Martinez Osvaldo , Chua Mark A. , Xiao Sa , Cros Jerome F. , Martínez-Sobrido Luis , Woo Savio L.C. , García-Sastre Adolfo
Published: September 2007

Naturally occurring strains of Newcastle disease virus (NDV) have shown oncolytic therapeutic efficacy in preclinical studies and are currently in clinical trials. Here, we have evaluated the possibility to enhance the cancer therapeutic potential of NDV by means of reverse genetics. Mice bearing s.c. implanted CT26 tumors were treated with intratumoral (i.t.) injections of a recombinant NDV modified to contain a highly fusogenic F protein. These treated mice exhibited significant reduction in tumor development compared with mice treated with the unmodified virus. Furthermore, mice in a CT26 metastatic tumor model treated with an i.v. injection of the genetically engineered NDV exhibited prolonged survival compared with wild-type control virus. In addition, we examined whether the oncolytic properties of NDV could be improved by expression of immunostimulatory molecules. In this regard, we engineered several NDVs to express granulocyte macrophage colony-stimulating factor, IFN-γ, interleukin 2 (IL-2), or tumor necrosis factor α, and evaluated their therapeutic potential in an immunocompetent colon carcinoma tumor model. Mice bearing s.c. CT26 tumors treated with i.t. injections of recombinant NDV expressing IL-2 showed dramatic reductions in tumor growth, with a majority of the mice undergoing complete and long-lasting remission. Our data show the use of reverse genetics to develop enhanced recombinant NDV vectors as effective therapeutic agents for cancer treatment. [Cancer Res 2007;67(17):8285–92]
Tumor-Associated Tn-MUC1 Glycoform Is Internalized through the Macrophage Galactose-Type C-Type Lectin and Delivered to the HLA Class I and II Compartments in Dendritic Cells
Authors: Napoletano Chiara , Rughetti Aurelia , Agervig Tarp Mads P. , Coleman Julia , Bennett Eric P. , Picco Gianfranco , Sale Patrizio , Denda-Nagai Kaori , Irimura Tatsuro , Mandel Ulla , Clausen Henrik , Frati Luigi , Taylor-Papadimitriou Joyce , Burchell Joy , Nuti Marianna
Published: September 2007

The type of interaction between tumor-associated antigens and specialized antigen-presenting cells such as dendritic cells (DCs) is critical for the type of immunity that will be generated. MUC1, a highly O-glycosylated mucin, is overexpressed and aberrantly glycosylated in several tumor histotypes. This results in the expression of tumor-associated glycoforms and in MUC1 carrying the tumor-specific glycan Tn (GalNAcα1-O-Ser/Thr). Glycopeptides corresponding to three tandem repeats of MUC1, enzymatically glycosylated with 9 or 15 mol of GalNAc, were shown to specifically bind and to be internalized by immature monocyte-derived DCs (iDCs). Binding required calcium and the GalNAc residue and was competed out by GalNAc polymer and Tn-MUC1 or Tn-MUC2 glycopeptides. The macrophage galactose-type C-type lectin (MGL) receptor expressed on iDCs was shown to be responsible for the binding. Confocal analysis and ELISA done on subcellular fractions of iDCs showed that the Tn-MUC1 glycopeptides colocalized with HLA class I and II compartments after internalization. Importantly, although Tn-MUC1 recombinant protein was bound and internalized by MGL, the glycoprotein entered the HLA class II compartment, but not the HLA class I pathway. These data indicate that MGL expressed on iDCs is an optimal receptor for the internalization of short GalNAcs carrying immunogens to be delivered into HLA class I and II compartments. Such glycopeptides therefore represent a new way of targeting the HLA class I and II pathways of DCs. These results have possible implications in designing cancer vaccines. [Cancer Res 2007;67(17):8358–67]
Real-time Imaging of Tumor-Cell Shedding and Trafficking in Lymphatic Channels
Authors: Hayashi Katsuhiro , Jiang Ping , Yamauchi Kensuke , Yamamoto Norio , Tsuchiya Hiroyuki , Tomita Katsuro , Moossa A.R. , Bouvet Michael , Hoffman Robert M.
Published: September 2007

In the present report, we show real-time imaging of cancer cell trafficking in lymphatic vessels. Cancer cells labeled with both green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in the cytoplasm or with GFP only or RFP only were injected into the inguinal lymph node of nude mice. The labeled cancer cells trafficked through lymphatic vessels where they were imaged via a skin flap in real time at the cellular level until they entered the axillary lymph node. The bright fluorescence of the cancer cells and the real-time microscopic imaging capability of the Olympus OV100 small-animal imaging system enabled imaging of the trafficking cancer cells in the lymphatics. Using this imaging strategy, two different cancer cell lines, one expressing GFP and the other expressing RFP, were simultaneously injected in the inguinal lymph node. Fluorescence imaging readily distinguished the two color-coded cell lines and their different abilities to survive in the lymphatic system. Using this imaging technology, we also investigated the role of pressure on tumor-cell shedding into lymphatic vessels. Pressure was generated by placing 25- and 250-g weights for 10 s on the bottom surface of a tumor-bearing footpad. Tumor cell fragments, single cells, and emboli shed from the footpad tumor were easily distinguished with the labeled cells and OV100 imaging system. Increasing pressure on the tumor increased the numbers of shed cells, fragments, and emboli. Pressure also deformed the shed emboli, increasing their maximum major axis. Imaging lymphatic trafficking of cancer cells can reveal critical steps of lymph node metastasis. [Cancer Res 2007;67(17):8223–8]
Integrative Analysis of Genomic Aberrations Associated with Prostate Cancer Progression
Authors: Kim Jung H. , Dhanasekaran Saravana M. , Mehra Rohit , Tomlins Scott A. , Gu Wenjuan , Yu Jianjun , Kumar-Sinha Chandan , Cao Xuhong , Dash Atreya , Wang Lei , Ghosh Debashis , Shedden Kerby , Montie James E. , Rubin Mark A. , Pienta Kenneth J. , Shah Rajal B. , Chinnaiyan Arul M.
Published: September 2007

Integrative analysis of genomic aberrations in the context of trancriptomic alterations will lead to a more comprehensive perspective on prostate cancer progression. Genome-wide copy number changes were monitored using array comparative genomic hybridization of laser-capture microdissected prostate cancer samples spanning stages of prostate cancer progression, including precursor lesions, clinically localized disease, and metastatic disease. A total of 62 specific cell populations from 38 patients were profiled. Minimal common regions (MCR) of alterations were defined for each sample type, and metastatic samples displayed the most number of alterations. Clinically localized prostate cancer samples with high Gleason grade resembled metastatic samples with respect to the size of altered regions and number of affected genes. A total of 9 out of 13 MCRs in the putative precursor lesion, high-grade prostatic intraepithelial neoplasia (PIN), showed an overlap with prostate cancer cases (amplifications in 3q29, 5q31.3-q32, 6q27, and 8q24.3 and deletions in 6q22.31, 16p12.2, 17q21.2, and 17q21.31), whereas postatrophic hyperplasia (PAH) did not exhibit this overlap. Interestingly, prostate cancers that do not overexpress ETS family members (i.e., gene fusion–negative prostate cancers) harbor differential aberrations in 1q23, 6q16, 6q21, 10q23, and 10q24. Integrative analysis with matched mRNA profiles identified genetic alterations in several proposed candidate genes implicated in prostate cancer progression. [Cancer Res 2007;67(17):8229–39]
HOXA5 Acts Directly Downstream of Retinoic Acid Receptor β and Contributes to Retinoic Acid–Induced Apoptosis and Growth Inhibition
Authors: Chen Hexin , Zhang Huiping , Lee Jishin , Liang Xiaohui , Wu Xinyan , Zhu Tao , Lo Pang-kuo , Zhang Xiaokun , Sukumar Saraswati
Published: September 2007

The promise of retinoids as chemopreventive agents in breast cancer is based on the differentiation and apoptosis induced upon their binding to the retinoic acid (RA) receptor β (RARβ). We have previously shown that HOXA5 induces apoptosis in breast cancer cells. In this study, we investigated whether RA/RARβ and HOXA5 actions intersect to induce apoptosis and differentiation in breast cancer cells. We found that HOXA5 expression can be induced by RA only in RARβ-positive breast cancer cells. We have, for the first time, identified the RA response element in HOXA5, which was found to be located in the 3′ end of the gene. Chromatin immunoprecipitation assays showed that RARβ binds directly to this region in vivo. Overexpression of RARβ strongly enhances RA responsiveness, and knocking down RARβ expression abolishes RA-mediated induction of HOXA5 expression in breast cancer cells. In addition, there is coordinated loss of both HOXA5 and RARβ expression during neoplastic transformation and progression in the breast epithelial cell model, MCF10A. Knockdown of HOXA5 expression partially abrogates retinoid-induced apoptosis and promotes cell survival upon RA treatment. These results strongly suggest that HOXA5 acts directly downstream of RARβ and may contribute to retinoid-induced anticancer and chemopreventive effects. [Cancer Res 2007;67(17):8007–13]
Systemic Delivery of γ134.5-Deleted Herpes Simplex Virus-1 Selectively Targets and Treats Distant Human Xenograft Tumors That Express High MEK Activity
Authors: Veerapong Jula , Bickenbach Kai A. , Shao Michael Y. , Smith Kerrington D. , Posner Mitchell C. , Roizman Bernard , Weichselbaum Ralph R.
Published: September 2007

Δγ134.5 mutant herpes simplex type 1 viruses are under active clinical investigation as oncolytic therapy for cancer. Mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) activity has been shown to suppress protein kinase R and thereby confer oncolytic susceptibility to some human tumors by R3616, a virus deleted for both copies of γ134.5. We report that systemic delivery of R3616 can selectively target and destroy human xenograft tumors that overexpress MEK activity compared with tumors that express lower MEK activity. These results suggest systemic delivery of R3616 may be effective in the treatment of some human tumors. [Cancer Res 2007;67(17):8301–6]
Leveraging the Immune System during Chemotherapy: Moving Calreticulin to the Cell Surface Converts Apoptotic Death from “Silent” to Immunogenic
Authors: Obeid Michel , Panaretakis Theocharis , Tesniere Antoine , Joza Nick , Tufi Roberta , Apetoh Lionel , Ghiringhelli François , Zitvogel Laurence , Kroemer Guido
Published: September 2007

In contrast to prior belief, tumor cell apoptosis is not necessarily silent but can be immunogenic. By tracing how anthracyclines and γ-irradiation trigger immunogenic cell deaths, we found that they were causally connected to the exposure of calreticulin on the tumor cell surface, before apoptosis in the tumor cell itself occurred. Furthermore, we showed that calreticulin exposure was necessary and sufficient to increase proimmunogenic killing by other chemotherapies. Our findings suggest that calreticulin could serve as a biomarker to predict therapy-associated immune responses, and that tactics to expose calreticulin might improve the clinical efficacy of many cancer therapies. [Cancer Res 2007;67(17):7941–4]
Chromosomal Instability and Supernumerary Centrosomes Represent Precursor Defects in a Mouse Model of T-Cell Lymphoma
Authors: van de Wetering Christopher I. , Knudson C. Michael
Published: September 2007

A hallmark of carcinogenesis is resistance to cell death. However, recent studies indicate that Bax expression increased apoptosis and promoted oncogenesis. In this study, we hypothesized that Bax promotes tumor formation by increasing chromosomal instability (CIN). Consistent with this hypothesis, spectral karyotype analysis (SKY) of lymphomas derived from Lck-Bax38/1 mice were consistently aneuploid. To determine if CIN precedes tumor formation, quantitative cytogenetic analysis, SKY analysis, and quantitative centrosome staining were done on thymocytes from young premalignant mice. Between 6 and 10 weeks of age, thymi from Bax-expressing mice (either p53+/+ or p53−/−) had an increased percentage of aneuploid cells as well as an increase in cells with supernumerary centrosomes. For 3- to 6-week-old mice, Bax expression increased aneuploidy and supernumerary centrosomes in p53−/− mice but not in p53+/+ animals. Importantly, both aneuploidy and supernumerary centrosomes were attenuated by Bcl-2. Remarkably, SKY analysis showed multiple independent aneuploid populations in the p53−/− Bax-expressing mice between 3 and 6 weeks of age. These results indicate that oligoclonal aneuploidy and supernumerary centrosomes are early hallmarks of Bax-induced lymphoma formation and support a novel link between the Bcl-2 family and CIN. The data provide an attractive model for the paradoxical effects of the Bcl-2 family on carcinogenesis that have been observed in multiple studies of both humans and mice. [Cancer Res 2007;67(17):8081–8]
A Chemical Screen Identifies Anisomycin as an Anoikis Sensitizer That Functions by Decreasing FLIP Protein Synthesis
Authors: Mawji Imtiaz A. , Simpson Craig D. , Gronda Marcela , Williams Moyo A. , Hurren Rose , Henderson Clare J. , Datti Alessandro , Wrana Jeffrey L. , Schimmer Aaron D.
Published: September 2007

Malignant epithelial cells with metastatic potential resist apoptosis that normally occurs upon loss of anchorage from the extracellular matrix, a process termed “anoikis.” Resistance to anoikis enables malignant cells to survive in an anchorage-independent manner, which leads to the formation of distant metastases. To understand the regulation of anoikis, we designed, automated, and conducted a high-throughput chemical screen for anoikis sensitizers. PPC-1 anoikis–resistant prostate cancer cells were seeded in hydrogel-coated ultralow binding plates for suspension conditions and standard tissue culture plates to promote adhesion. After seeding, cells were treated with aliquots from a library of previously characterized small molecules, and viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt, assay. From this chemical screen, we identified anisomycin that induced apoptosis in suspension conditions, but was not toxic to these cells grown under adherent conditions. Anisomycin sensitized cells to anoikis by decreasing levels of the caspase-8 inhibitor FLIP and subsequently activating the death receptor pathway of caspase activation. Although anisomycin activated c-Jun-NH2-kinase and p38, these kinases were not functionally important for the effect of anisomycin on anoikis and FLIP. Rather, anisomycin decreased FLIP and sensitized cells to anoikis by inhibiting its protein synthesis. Finally, we showed that anisomycin decreased distal tumor formation in a mouse model of prostate cancer metastases. Thus, a novel chemical screen identified anisomycin as an anoikis sensitizer that acts by decreasing FLIP protein synthesis. Our results suggest that FLIP is a suppressor of anoikis and inhibiting FLIP protein synthesis may be a useful antimetastatic strategy. [Cancer Res 2007;67(17):8307–15]
Radiosensitization and Modulation of p44/42 Mitogen-Activated Protein Kinase by 2-Methoxyestradiol in Prostate Cancer Models
Authors: Casarez Eli V. , Dunlap-Brown Marya E. , Conaway Mark R. , Amorino George P.
Published: September 2007

2-Methoxyestradiol (2ME2) is an endogenous estradiol metabolite that inhibits microtubule polymerization, tumor growth, and angiogenesis. Because prostate cancer is often treated with radiotherapy, and 2ME2 has shown efficacy as a single agent against human prostate carcinoma, we evaluated 2ME2 as a potential radiosensitizer in prostate cancer models. A dose-dependent decrease in mitogen-activated protein kinase phosphorylation was observed in human PC3 prostate cancer cells treated with 2ME2 for 18 h. This decrease correlated with in vitro radiosensitization measured by clonogenic assays, and these effects were blocked by the expression of constitutively active MEK. Male nude mice with subcutaneous PC3 xenografts in the hind leg were treated with 2ME2 (75 mg/kg) p.o. for 5 days, and 2 Gy radiation fractions were delivered each day at 4 h after drug treatment. A statistically significant super-additive effect between radiation and 2ME2 was observed in this subcutaneous model, using analysis of within-animal slopes. A PC-3M orthotopic model was also used, with bioluminescence imaging as an end point. PC-3M cells stably expressing the luciferase gene were surgically implanted into the prostates of male nude mice. Mice were given oral doses of 2ME2 (75 mg/kg), with radiation fractions (3 Gy) delivered 4 h later. Mice were then imaged weekly for 4 to 5 weeks with a Xenogen system. A significant super-additive effect was also observed in the orthotopic model. These data show that 2ME2 is an effective radiosensitizing agent against human prostate cancer xenografts, and that the mechanism may involve a decrease in mitogen-activated protein kinase phosphorylation by 2ME2. [Cancer Res 2007;67(17):8316–24]
Mistaken Identity of Widely Used Esophageal Adenocarcinoma Cell Line TE-7
Authors: Boonstra Jurjen J. , van der Velden Albertina W. , Beerens Erwin C.W. , van Marion Ronald , Morita-Fujimura Yuiko , Matsui Yasuhisa , Nishihira Tetsuro , Tselepis Chris , Hainaut Pierre , Lowe Anson W. , Beverloo Berna H. , van Dekken Herman , Tilanus Hugo W. , Dinjens Winand N.M.
Published: September 2007

Cancer of the esophagus is the seventh leading cause of cancer death worldwide. Esophageal carcinoma cell lines are useful models to study the biological and genetic alterations in these tumors. An important prerequisite of cell line research is the authenticity of the used cell lines because the mistaken identity of a cell line may lead to invalid conclusions. Estimates indicate that up to 36% of the cell lines are of a different origin or species than supposed. The TE series, established in late 1970s and early 1980s by Nishihira et al. in Japan, is one of the first esophageal cancer cell line series that was used throughout the world. Fourteen TE cell lines were derived from human esophageal squamous cell carcinomas and one, TE-7, was derived from a primary esophageal adenocarcinoma. In numerous studies, this TE-7 cell line was used as a model for esophageal adenocarcinoma because it is one of the few esophageal adenocarcinoma cell lines existing. We investigated the authenticity of the esophageal adenocarcinoma cell line TE-7 by xenografting, short tandem repeat profiling, mutation analyses, and array-comparative genomic hybridization and showed that cell line TE-7 shared the same genotype as the esophageal squamous cell carcinoma cell lines TE-2, TE-3, TE-12, and TE-13. In addition, for more than a decade, independent TE-7 cultures from Japan, United States, United Kingdom, France, and the Netherlands had the same genotype. Examination of the TE-7 cell line xenograft revealed the histology of a squamous cell carcinoma. We conclude that the TE-7 cell line, used in several laboratories throughout the world, is not an adenocarcinoma, but a squamous cell carcinoma cell line. Furthermore, the cell lines TE-2, TE-3, TE-7, TE-12, and TE-13 should be regarded as one single squamous cell carcinoma cell line. [Cancer Res 2007;67(17):7996–8001]
Requirement for Rac1 in a K-ras–Induced Lung Cancer in the Mouse
Authors: Kissil Joseph L. , Walmsley Marita J. , Hanlon Linda , Haigis Kevin M. , Bender Kim Carla F. , Sweet-Cordero Alejandro , Eckman Matthew S. , Tuveson David A. , Capobianco Anthony J. , Tybulewicz Victor L.J. , Jacks Tyler
Published: September 2007

Given the prevalence of Ras mutations in human cancer, it is critical to understand the effector pathways downstream of oncogenic Ras leading to transformation. To directly assess the requirement for Rac1 in K-ras–induced tumorigenesis, we employed a model of lung cancer in which an oncogenic allele of K-ras could be activated by Cre-mediated recombination in the presence or absence of conditional deletion of Rac1. We show that Rac1 function is required for tumorigenesis in this model. Furthermore, although Rac1 deletion alone was compatible with cell viability and proliferation, when combined with K-ras activation in primary epithelial cells, loss of Rac1 caused a profound reduction in proliferation. These data show a specific requirement for Rac1 function in cells expressing oncogenic K-ras. [Cancer Res 2007;67(17):8089–94]
Subdural Haematomas and Physical Abuse in the First Two Years of Life
Authors: Trenchs Victoria , Curcoy Ana Isabel , Navarro Ramón , Pou Jordi
Published: September 2007

Objective: To analyze our institution’s work-up for patients with a diagnosis of subdural haematoma (SDH) in order to determine how many of them are secondary to child abuse, as well as to examine their final functional outcome. Methods: Retrospective review of children under 2 years of age diagnosed as having SDH between 1995 and 2005. Results: A total of 35 cases were identified. Fifteen patients that had underlying conditions that predispose them to bleed were excluded. Among the remaining 20 patients, seizures and head trauma were the main causes for consultation. All patients had a coagulation study and a head computed tomography carried out, 11 of these had a magnetic resonance imaging and 1 had a post-mortem examination. Bilateral SDHs in different stages of evolution was the most common pattern of intracranial haemorrhage. Fourteen infants had a skeletal survey, 4 had a bone scintigraphy and 19 had an ophthalmoscopic examination. Fractures were diagnosed in 7 patients and retinal haemorrhages in 11. The final diagnoses were: 10 shaken baby syndromes, 4 idiopathic SDH, 3 strokes, 2 coagulopathies and 1 accidental head injury. Upon follow-up, 1 patient had died and 9 had sustained permanent disabilities. Conclusions: Cases of infantile SDH are usually thoroughly investigated. In spite of this, sometimes it is not possible to determine the SDH aetiology. Nonetheless, shaken baby syndrome remains the most frequent cause of SDH in infants, and it carries a poor prognosis.
Surgical Treatment of 13 Pediatric Patients with Dandy-Walker Syndrome
Authors: Yüceer Nurullah , Mertol Tansu , Arda Nuri
Published: September 2007

We present our experience with the treatment of 13 patients with Dandy-Walker syndrome. The common presenting symptom and associated central nervous system anomaly were enlargement of head and occipital encephalocele, respectively. Eleven out of 13 patients were treated surgically after stabilization of systemic medical status. Two patients could not be operated because of poor medical condition. In 6 patients with an opened passage between posterior fossa cyst and lateral ventricle, cystoperitoneal shunt system with medium pressure valve was the treatment of choice. In 5 patients with no relation between cyst and ventricle, cystoperitoneal and ventriculoperitoneal shunting with ‘Y’ connectors were applied separately. Another patient with a shunt infection was treated by shunt system renewal combined with parenteral antibiotics. One patient died 7 months after the operation due to recurrent meningitis.