bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of self-contained breathing apparatus on ventricular function during strenuous exercise
Authors: Nelson Michael D. , Haykowsky Mark J. , Mayne Jonathan R. , Jones Richard L. , Petersen Stewart R.
Published: November 2008

The purpose of this study was to investigate left-ventricular function during strenuous exercise with the self-contained breathing apparatus (SCBA). With the use of two-dimensional echocardiography, images of the left ventricle (LV) were acquired during sustained exercise (3 × 10 min) under two conditions: 1) SCBA, or 2) low resistance breathing valve. Twenty healthy men volunteered for the study, and in each condition subjects wore fire protective equipment. Heart rate, systolic blood pressure, cavity areas during systole and diastole (ESCA and EDCA, respectively), esophageal pressure, ventilation rate, oxygen consumption, perceived physical, thermal and respiratory distress, and core temperature were measured at regular intervals. Urine specific gravity (<1.020 g/ml) and hematological variables were used to infer hydration status. All subjects began both trials in a euhydrated state. No differences were found between conditions for heart rate, systolic blood pressure, ventilation rate, oxygen consumption, perceived distress, or any hematological variables. Peak expiratory esophageal pressure was always higher ( P < 0.05), while EDCA and stroke area (SA) were significantly lower ( P < 0.05) with the SCBA. ESCA, end-systolic transmural pressure (ESTMP), and LV contractility (ESTMP/ESCA) were similar between conditions. Sustained exercise with fire protective equipment resulted in significant reductions in EDCA, ESCA, and SA from the start of exercise, which was associated with a 6.3 ± 0.8% reduction in plasma volume, an increase in core temperature (37.0 ± 0.4 to 38.8 ± 0.3°C), and a significant increase in heart rate (146.9 ± 2.1 to 181.7 ± 2.4 beats/min) throughout exercise. The results from this study support research by others showing that increased intrathoracic pressure reduces LV preload (EDCA); however, the novelty of the present study is that when venous return is compromised by sustained exercise and heat stress, SA cannot be maintained.
Lysosomal Cathepsin B Participates in the Podosome-Mediated Extracellular Matrix Degradation and Invasion via Secreted Lysosomes in v-Src Fibroblasts
Authors: Tu Chun , Ortega-Cava Cesar F. , Chen Gengsheng , Fernandes Norvin D. , Cavallo-Medved Dora , Sloane Bonnie F. , Band Vimla , Band Hamid
Published: November 2008

Podosomes mediate cell migration and invasion by coordinating the reorganization of actin cytoskeleton and focal matrix degradation. MMP and serine proteases have been found to function at podosomes. The lysosomal cysteine cathepsins, a third major class of matrix-degrading enzymes involved in tumor invasion and tissue remodeling, have yet to be linked to podosomes with the exception of cathepsin K in osteoclasts. Using inhibitors and shRNA-mediated depletion, we show that cathepsin B participates in podosomes-mediated focal matrix degradation and invasion in v-Src–transformed fibroblasts. We observed that lysosomal marker LAMP-1 localized at the center of podosome rosettes protruding into extracellular matrix using confocal microscopy. Time-lapse live-cell imaging revealed that lysosomal vesicles moved to and fused with podosomes. Disruption of lysosomal pH gradient with Bafilomycin A1, chloroquine, or ammonium chloride greatly enhanced the formation of podosomes and increased the matrix degradation. Live-cell imaging showed that actin structures, induced shortly after Bafilomycin A1 treatment, were closely associated with lysosomes. Overall, our results suggest that cathepsin B, delivered by lysosomal vesicles, is involved in the matrix degradtion of podosomes. [Cancer Res 2008;68(22):9147–56]
NOTCH Is Part of the Transcriptional Network Regulating Cell Growth and Survival in Mouse Plasmacytomas
Authors: Shin Dong-Mi , Shaffer Daniel J. , Wang Hongsheng , Roopenian Derry C. , Morse Herbert C.
Published: November 2008

Aside from Myc-activating translocations characteristic of plasmacytomas (PCT), little is known about genetic factors and signaling pathways responsible for the development of spontaneous B-cell lineage lymphomas of mice. Here, we characterized the transcriptional profiles of PCT, centroblastic diffuse large B-cell lymphomas (CBL), and high-grade splenic marginal zone B-cell lymphoma (MZL++) using high-throughput quantitative reverse transcription-PCR. Expression profiles of CBL and MZL++ were strikingly similar and quite unlike that of PCT. Among the genes expressed at significantly higher levels by PCT were a number involved in NOTCH signaling, a finding supported by gene set enrichment analyses of microarray data. To investigate the importance of this pathway, NOTCH signaling was blocked in PCT cell lines by treatment with a γ-secretase inhibitor (GSI) or transduction of a dominant-negative mutant of MAML1. These treatments resulted in reduced expression of NOTCH transcriptional targets in association with impaired proliferation and increased apoptosis. GSI treatment of transformed plasma cells in a primary PCT also induced apoptosis. These results integrate NOTCH activation with oncogenic signaling pathways downstream of translocated Myc in the pathogenesis of mouse PCT, two signaling pathways also implicated in development of human multiple myeloma and T-cell lymphoblastic lymphoma. [Cancer Res 2008;68(22):9202–11]
AKT1 Inhibits Homologous Recombination by Inducing Cytoplasmic Retention of BRCA1 and RAD51
Authors: Plo Isabelle , Laulier Corentin , Gauthier Laurent , Lebrun Fabienne , Calvo Fabien , Lopez Bernard S.
Published: November 2008

AKT1 is frequently up-regulated in sporadic breast cancer, whereas BRCA1 is frequently mutated in familial breast cancer. Because BRCA1 is involved in homologous recombination (HR), we addressed whether AKT1 also has an effect on this process. We showed that AKT1 repressed HR through cytoplasmic retention of BRCA1 and RAD51 proteins, resulting in a BRCA1-deficient–like phenotype. This process does not require direct BRCA1 phosphorylation by AKT1. The cytoplasmic retention of BRCA1 and RAD51 correlated with activated AKT1 in tumor cell lines and in biopsies from sporadic breast cancers. Under nonpathologic conditions, fibroblast growth factor, which activates AKT1 and stimulates proliferation in fibroblasts, impaired excessive HR without fully inhibiting it, promoting genome stability. Our study reveals that the regulation of BRCA1 and RAD51 is altered in a high frequency of sporadic breast cancers and highlights the role of extracellular AKT signaling-dependent regulation of HR and genome stability. [Cancer Res 2008;68(22):9404–12]
Type 2 Bias of T Cells Expanded from the Blood of Melanoma Patients Switched to Type 1 byIL-12p70mRNA–Transfected Dendritic Cells
Authors: Minkis Kira , Kavanagh Daniel G. , Alter Galit , Bogunovic Dusan , O'Neill David , Adams Sylvia , Pavlick Anna , Walker Bruce D. , Brockman Mark A. , Gandhi Rajesh T. , Bhardwaj Nina
Published: November 2008

Melanoma patients may exhibit a TH2-skewed cytokine profile within blood and tumor-infiltrating lymphocytes. Therapies that induce beneficial TH1-type tumor-specific immune responses, therefore, are highly desirable. Dendritic cells (DC) are widely used as immune adjuvants for cancer. Before their administration, DC are generally induced to mature with a cocktail of recombinant cytokines [interleukin (IL)-1β, tumor necrosis factor α, and IL-6] and prostaglandin E2 (PGE2), which is added to preserve the ability of DC to migrate to draining lymph nodes. However, PGE2 suppresses the production of IL-12p70, a cytokine essential for differentiation of TH1 responses. In this study, human DC were transfected with IL-12p70 mRNA and tested for their ability to alter the TH2 type bias manifested by blood T cells of patients with melanoma. Transfected DC secreted high levels of bioactive IL-12p70, as indicated by their capacity to enhance natural killer cell activity, skew TH1 responses in allogeneic mixed lymphocyte reactions through reduction of IL-4 and IL-5, and prime CD8+ T cells to the melanoma-associated antigen Melan A/MART-1. Furthermore, T-cell lines primed in vitro from the blood of melanoma patients showed strong type 2 skewing that was dramatically reversed by IL-12p70 transfection of autologous DC. Thus, IL-12p70 transfection of clinical DC preparations may enhance type 1 antitumor responses and may thereby contribute to effective immune-based therapy. [Cancer Res 2008;68(22):9441–50]
Involvement of the Leptin Receptor in the Immune Response in Intestinal Cancer
Authors: Aloulou Nijez , Bastuji-Garin Sylvie , Le Gouvello Sabine , Abolhassani Mohammad , Chaumette Marie Thérèse , Charachon Antoine , Leroy Karen , Sobhani Iradj
Published: November 2008

The incidence of colorectal cancers (CRC) may be influenced by environmental factors, including nutrition. The role of peptides regulating food intake in controlling the growth and recurrence of human tumors is controversial. Leptin, a cytokine-like peptide, regulates food intake. We investigated the expression of leptin and its receptor in 171 consecutive patients (78 female and 93 male; 71 years) with CRC. Leptin concentrations in the serum (ELISA) were determined before tumor removal. ObRb was characterized in tumors and normal homologous tissues and culture cells (HT29, HCT116, and HCT116 with a transferred chromosome 3) by using immunocytochemistry, immunohistochemistry, reverse transcription-PCR (RT-PCR), and Western blotting. Microsatellite instability (MSI) phenotype was characterized by immunohistochemistry and pentaplex PCR. mRNAs of cytokines and chemokines were quantified in tumors and in normal homologous tissues (RT-PCR) in 43 patients. Adequate statistical tests, including multivariate analysis adjusted for pathologic tumor-node-metastasis (pTNM), MSI-H, and ObRb phenotypes, were used. Higher expression of ObRb in tumors compared with the homologous normal mucosa, pTNM staging but not leptin serum level, was associated with patients' progression-free survival (PFS). Tumor ObRb phenotype and pTNM were independent predictive factors of PFS. ObRb was more strongly expressed in HCT116 cells than in HCT116-Ch3 cells as well as in MSI-H tumors than in microsatellite stability and potentially associated with efficient cytotoxic antitumoral response as assessed by immunohistochemistry and RT-PCR measurements. We suggest that leptin receptor expression in tumors is involved in adaptive immune response in sporadic colon and rectal tumors likely via MSI-H phenotype orientation. [Cancer Res 2008;68(22):9413–22]
Leptin Receptor–Related Immune Response in Colorectal Tumors: The Role of Colonocytes and Interleukin-8
Authors: Abolhassani Mohammad , Aloulou Nijez , Chaumette Marie Thérèse , Aparicio Thomas , Martin-Garcia Nadine , Mansour Hicham , Le Gouvello Sabine , Delchier Jean Charles , Sobhani Iradj
Published: November 2008

We have shown that ObRb, the leptin receptor, is overexpressed in colorectal cancer cells, and that this may influence the patients' outcome. We investigated colonocytes as leptin targets and characterized their pivotal role in antitumor immune response. Cytokine and chemokine mRNAs in HT29 cells were measured by targeted arrays. In vitro, normal colonocytes and human colon cancer cells (HT29, Caco-2, SW480, and HCT116) were used to investigate ObRb transduction system and cytokine releases. Animal colonocytes and CD8 splenocytes and human HT29, HCT116, and CD8+ cells from blood donors were used to investigate the lymphocyte response to the colonocytes when stimulated by leptin. Leptin-induced cytokine releases in the normal colonic mucosa and tumor growth and cytokine releases within tumors in vivo were measured in male rats and nude mice, respectively. Statistical analysis was done by Fisher's exact and Mann-Whitney U tests. Various cytokines and their receptors were produced in normal and tumoral colonocytes in response to leptin by increasing nuclear factor-κB activation. Interleukin-8 (IL-8) was the main cytokine produced in vitro. The levels of IL-8 and its receptor, CXCR1, were higher in tumors than in homologous normal mucosa. Systemic leptin enhanced the proinflammatory cytokines in normal colonocytes and in HT29 xenografted tumor colonocytes. Colonocyte-derived products after leptin treatment stimulated perforin and granzyme B expressions in normal CD8+ T cells in vitro. Leptin triggers an inflammatory response in tumor tissue by directly stimulating colonocytes, which can recruit T cytotoxic cells in the tumor microenvironment. [Cancer Res 2008;68(22):9423–32]
Hypoxia-Inducible Factor-2α Correlates to Distant Recurrence and Poor Outcome in Invasive Breast Cancer
Authors: Helczynska Karolina , Larsson Anna-Maria , Holmquist Mengelbier Linda , Bridges Esther , Fredlund Erik , Borgquist Signe , Landberg Göran , Påhlman Sven , Jirström Karin
Published: November 2008

Differential regulation as well as target gene specificity of the two hypoxia-inducible factor (HIF)-α subunits HIF-1α and HIF-2α in various tumors and cell lines have been suggested. In breast cancer, the prognostic significance of HIF-1α is not clear-cut and that of HIF-2α is largely unknown. Using IHC analyses of HIF-1α, HIF-2α, and vascular endothelial growth factor (VEGF) expression in a tissue microarray of invasive breast cancer specimens from 512 patients, we investigated the expression patterns of the 2 HIF-α subunits in relation to established clinicopathologic variables, VEGF expression, and survival. HIF-1α and HIF-2α protein levels and their effect on survival were additionally analyzed in a second cohort of 179 patients. To evaluate the individual role of each subunit in the hypoxic response and induction of VEGF, HIF-α protein and HIF-α and VEGF mRNA levels were further studied in cultured breast cancer cells after hypoxic induction and/or knockdown of HIF-α subunits by siRNA by Western blot and Quantitative Real-Time PCR techniques. We showed that although HIF-1α and HIF-2α protein levels in breast cancer specimens were not interrelated, high levels of both HIF-1α and HIF-2α associated to high VEGF expression. HIF-2α expression was an independent prognostic factor associated to reduced recurrence-free and breast cancer–specific survival, whereas HIF-1α did not exhibit these correlations. In cultured cells, acute hypoxia induced both HIF-proteins. At prolonged hypoxia, HIF-2α remained accumulated, whereas HIF-1α protein levels decreased, in agreement with the oxygen level and time-dependent induction of HIFs recently reported in neuroblastoma. [Cancer Res 2008;68(22):9212–20]
Posttranscriptional Regulation of the Breast Cancer Susceptibility Gene BRCA1 by the RNA Binding Protein HuR
Authors: Saunus Jodi M. , French Juliet D. , Edwards Stacey L. , Beveridge Dianne J. , Hatchell Esme C. , Wagner Sarah A. , Stein Sandra R. , Davidson Andrew , Simpson Kaylene J. , Francis Glenn D. , Leedman Peter J. , Brown Melissa A.
Published: November 2008

BRCA1 is a breast cancer susceptibility gene that is down-regulated in a significant proportion of sporadic breast cancers. BRCA1 is posttranscriptionally regulated by RNA-binding proteins, the identities of which are unknown. HuR is an RNA binding protein implicated in posttranscriptional regulation of many genes and is overexpressed in sporadic breast cancer. To investigate the possibility that these two molecules are functionally linked in breast cancer, we performed bioinformatic analysis of the BRCA1 3′ untranslated region (UTR), RNA-protein assays with the HuR protein and the BRCA1 3′UTR, and immunohistochemical analysis of a cohort of breast tumors using antibodies against BRCA1 and HuR. Here, we describe the identification of two predicted HuR-binding sites in the BRCA1 3′UTR, one of which binds specifically to HuR. We also show that this interaction is disrupted by single nucleotide substitutions in the BRCA1 3′UTR and that endogenous HuR protein associates with BRCA1 transcripts in T47D and MCF7 breast cancer cells. Expression of ectopic HuR results in a significant decrease in BRCA1 protein expression and also BRCA1 3′UTR activity. Immunohistochemical analysis revealed that although BRCA1 and HuR expression were associated with some clinicopathologic features of the tumors, there was no statistically significant correlation between BRCA1 and HuR protein expression. These results identify the first posttranscriptional protein regulator of BRCA1 and have implications for understanding BRCA1 regulation in human breast cancer. [Cancer Res 2008;68(22):9469–78]
Bone Morphogenetic Protein-7 Inhibits Telomerase Activity, Telomere Maintenance, and Cervical Tumor Growth
Authors: Cassar Lucy , Li He , Pinto Alexander Ruvantha , Nicholls Craig , Bayne Sharyn , Liu Jun-Ping
Published: November 2008

Telomere maintenance is critical in tumor cell immortalization. Here, we report that the cytokine bone morphogenetic protein-7 (BMP7) inhibits telomerase activity that is required for telomere maintenance in cervical cancer cells. Application of human recombinant BMP7 triggers a repression of the human telomerase reverse transcriptase (hTERT) gene, shortening of telomeres, and hTERT repression–dependent cervical cancer cell death. Continuous treatment of mouse xenograft tumors with BMP7, or silencing the hTERT gene, results in sustained inhibition of telomerase activity, shortening of telomeres, and tumor growth arrest. Overexpression of hTERT lengthens telomeres and blocks BMP7-induced tumor growth arrest. Thus, BMP7 negatively regulates telomere maintenance, inducing cervical tumor growth arrest by a mechanism of inducing hTERT gene repression. [Cancer Res 2008;68(22):9157–66]
Melanoma-Specific Memory T Cells Are Functionally Active inRetTransgenic Mice without Macroscopic Tumors
Authors: Umansky Viktor , Abschuetz Oliver , Osen Wolfram , Ramacher Marcel , Zhao Fang , Kato Masashi , Schadendorf Dirk
Published: November 2008

We previously reported that bone marrows of breast cancer patients contained tumor antigen–specific CD8+ T cells with central or effector memory phenotype. Using a recently developed ret transgenic mouse melanoma model, we now show that bone marrows and tumors of transgenic mice contain high frequencies of CD8+ T cells specific for the melanoma antigen tyrosinase-related protein 2 and showing mostly effector memory phenotype. Moreover, increased numbers of bone marrow tyrosinase-related protein-2–specific effector memory CD8+ T cells are also detected in transgenic animals older than 20 weeks with disseminated melanoma cells in the bone marrow and lymph nodes but showing no visible skin tumors and no further melanoma progression. After a short-term coincubation with dendritic cells generated from the bone marrow and pulsed with melanoma lysates, bone marrow memory T cells from mice without macroscopic melanomas produced IFN-γ in vitro and exerted antitumor activity in vivo after adoptive transfer into melanoma-bearing mice. Our data indicate that functionally active bone marrow–derived melanoma-specific memory T cells are detectable at the phase of microscopic tumor load, suggesting that thereby they could control disseminated melanoma cells. [Cancer Res 2008;68(22):9451–8]
OSU-03012, a Novel Celecoxib Derivative, Induces Reactive Oxygen Species–Related Autophagy in Hepatocellular Carcinoma
Authors: Gao Ming , Yeh Pei Yen , Lu Yen-Shen , Hsu Chih-Hung , Chen Kuen-Feng , Lee Wei-Chung , Feng Wen-Chi , Chen Ching-Shih , Kuo Min-Liang , Cheng Ann-Lii
Published: November 2008

Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of cancer death worldwide. Systemic treatments for HCC have been largely unsuccessful. OSU-03012 is a derivative of celecoxib with anticancer activity. The mechanism of action is presumably 3-phosphoinositide–dependent kinase 1 (PDK1) inhibition. This study investigated the potential of OSU-03012 as a treatment for HCC. OSU-03012 inhibited cell growth of Huh7, Hep3B, and HepG2 cells with IC50 below 1 μmol/L. In Huh7 cells, OSU-03012 did not suppress PDK1 or AKT activity. Terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling assay and flow cytometry analysis indicated that OSU-03012 did not induce cellular apoptosis. Instead, morphologic studies by light and electron microscopy, as well as special biological staining with monodansylcadaverine, acridine orange, and microtubule-associated protein 1 light chain 3, revealed OSU-03012–induced autophagy of Huh7 cells. This OSU-03012–induced autophagy was inhibited by 3-methyladenine. Moreover, reactive oxygen species (ROS) accumulation was detected after OSU-03012 treatment. Blocking ROS accumulation with ROS scavengers inhibited autophagy formation, indicating that ROS accumulation and subsequent autophagy formation might be a major mechanism of action of OSU-03012. Daily oral treatment of BALB/c nude mice with OSU-03012 suppressed the growth of Huh7 tumor xenografts. Electron microscopic observation indicated that OSU-03012 induced autophagy in vivo. Together, our results show that OSU-03012 induces autophagic cell death but not apoptosis in HCC and that the autophagy-inducing activity is at least partially related to ROS accumulation. [Cancer Res 2008;68(22):9348–57]
Membrane Type 1 Matrix Metalloproteinase–Mediated Stromal Syndecan-1 Shedding Stimulates Breast Carcinoma Cell Proliferation
Authors: Su Gui , Blaine Stacy A. , Qiao Dianhua , Friedl Andreas
Published: November 2008

Mounting evidence implicates stromal fibroblasts in breast carcinoma progression. We have recently shown in three-dimensional coculture experiments that human mammary fibroblasts stimulate the proliferation of T47D breast carcinoma cells and that this activity requires the shedding of the heparan sulfate proteoglycan syndecan-1 (Sdc1) from the fibroblast surface. The goal of this project was to determine the mechanism of Sdc1 ectodomain shedding. The broad spectrum matrix metalloproteinase (MMP) inhibitor GM6001 specifically blocked Sdc1-mediated carcinoma cell growth stimulation, pointing toward MMPs as critical enzymes involved in Sdc1 shedding. MMP-2 and membrane type 1 MMP (MT1-MMP) were the predominant MMPs expressed by the mammary fibroblasts. Fibroblast-dependent carcinoma cell growth stimulation in three-dimensional coculture was abolished by MT1-MMP expression silencing with small interfering RNA and restored either by adding recombinant MT1-MMP catalytic domain or by expressing a secreted form of Sdc1 in the fibroblasts. These findings are consistent with a model where fibroblast-derived MT1-MMP cleaves Sdc1 at the fibroblast surface, leading to paracrine growth stimulation of carcinoma cells by Sdc1 ectodomain. The relevance of MT1-MMP in paracrine interactions was further supported by coculture experiments with T47D cells and primary fibroblasts isolated from human breast carcinomas or matched normal breast tissue. Carcinoma-associated fibroblasts stimulated T47D cell proliferation significantly more than normal fibroblasts in three-dimensional coculture. Function-blocking anti–MT1-MMP antibody significantly inhibited the T47D cell growth stimulation in coculture with primary fibroblasts. In summary, these results ascribe a novel role to fibroblast-derived MT1-MMP in stromal-epithelial signaling in breast carcinomas. [Cancer Res 2008;68(22):9558–65]
Garlic Constituent Diallyl Trisulfide Prevents Development of Poorly Differentiated Prostate Cancer and Pulmonary Metastasis Multiplicity in TRAMP Mice
Authors: Singh Shivendra V. , Powolny Anna A. , Stan Silvia D. , Xiao Dong , Arlotti Julie A. , Warin Renaud , Hahm Eun-Ryeong , Marynowski Stanley W. , Bommareddy Ajay , Potter Douglas M. , Dhir Rajiv
Published: November 2008

Identification of agents that are nontoxic but can delay onset and/or progression of prostate cancer, which is the second leading cause of cancer-related deaths among men in the United States, is highly desirable. We now show that p.o. gavage of garlic constituent diallyl trisulfide (DATS; 1 and 2 mg/day, thrice/week for 13 weeks beginning at age 8 weeks) significantly inhibits progression to poorly differentiated prostate carcinoma and pulmonary metastasis multiplicity in transgenic adenocarcinoma of mouse prostate (TRAMP) mice without any side effects. There was a trend of a decrease in average wet weights of the urogenital tract and prostate gland in 1 and 2 mg DATS–treated mice compared with controls (∼25–46% decrease in DATS-treated mice compared with controls). The incidence and the area of the dorsolateral prostate occupied by the poorly differentiated carcinoma were significantly lower in both 1 and 2 mg DATS–treated mice compared with control mice. In addition, DATS administration resulted in a statistically significant decrease in pulmonary metastasis multiplicity compared with controls (P = 0.002). The dorsolateral prostate from DATS-treated TRAMP mice exhibited decreased cellular proliferation in association with induction of cyclinB1 and securin protein levels, and suppression of the expression of neuroendocrine marker synaptophysin. However, DATS administration did not have any appreciable effect on apoptosis induction, angiogenesis, or natural killer and dendritic cell function. In conclusion, the results of the present study show, for the first time, that DATS administration prevents progression to invasive carcinoma and lung metastasis in TRAMP mice. [Cancer Res 2008;68(22):9503–11]
A Human Antibody–Drug Conjugate Targeting EphA2 Inhibits Tumor GrowthIn vivo
Authors: Jackson Dowdy , Gooya John , Mao Shenlan , Kinneer Krista , Xu Linda , Camara Margarita , Fazenbaker Christine , Fleming Ryan , Swamynathan Sudha , Meyer Damon , Senter Peter D. , Gao Changshou , Wu Herren , Kinch Michael , Coats Steven , Kiener Peter A. , Tice David A.
Published: November 2008

The EphA2 receptor tyrosine kinase is selectively expressed on the surface of many different human tumors. We have previously shown that tumor cells can be targeted by EphA2 monoclonal antibodies and that these antibodies function, in part, by inducing EphA2 internalization and degradation. In this report, we describe the isolation and characterization of a fully human monoclonal antibody (1C1) that selectively binds both the human and rodent EphA2 receptor. After cell binding, the antibody induces rapid tyrosine phosphorylation, internalization, and degradation of the EphA2 receptor. Because monoclonal antibodies that selectively bind tumor cells and internalize provide a vehicle for targeted delivery of cytotoxics, 1C1 was conjugated to the microtubule inhibitor monomethylauristatin phenylalanine using a stable maleimidocaproyl linker. The anti-EphA2 antibody-drug conjugate [1C1–maleimidocaproyl-MMAF (mcMMAF)] stimulated the activation of caspase-3/caspase-7 and the death of EphA2-expressing cells with IC50 values as low as 3 ng/mL. Similarly, the conjugate induced degradation of the EphA2 receptor and inhibited tumor growth in vivo. Administration of 1C1-mcMMAF at doses as low as 1 mg/kg once weekly resulted in significant growth inhibition of EphA2-expressing tumors without any observable adverse effects in mouse xenograft and rat syngeneic tumor models. Our data support the use of an antibody-drug conjugate approach to selectively target and inhibit the growth of EphA2-expressing tumors. [Cancer Res 2008;68(22):9367–74]
Small Interfering RNA Molecules Targeting Endothelin-Converting Enzyme-1 Inhibit Endothelin-1 Synthesis and the Invasive Phenotype of Ovarian Carcinoma Cells
Authors: Rayhman Oleg , Klipper Eyal , Muller Laurent , Davidson Ben , Reich Reuven , Meidan Rina
Published: November 2008

Endothelin-1 (ET-1) has been implicated in the progression of various cancers, including ovarian carcinoma. We found that the ovarian carcinoma cell lines ES2 and OVCAR3 and tumors from different anatomic sites expressed ET-1 system members [ET receptor A and ET-converting enzyme-1 (ECE-1)]. However, only ECE-1 was significantly higher in the solid tumors compared with effusions. We therefore investigated the effect of RNA interference-induced knockdown of ECE-1, the key enzyme in ET-1 production, on these two ovarian carcinoma cell lines. Small interfering RNA (siRNA) targeting of ECE-1 markedly reduced ECE-1 mRNA and protein levels, which subsequently led to 80% to 90% inhibition of ET-1 peptide secretion by the cells. ECE-1 silencing also profoundly affected the behavior of tumor cells compared with cells treated with scrambled siRNA. Silenced cells exhibited (a) reduced ET-1–dependent p44/42 mitogen-activated protein kinase phosphorylation; (b) decreased invasiveness and matrix metalloproteinase-2 activity; (c) improved adhesion to basal lamina proteins, laminin-1, and collagen IV; and (d) increased E-cadherin, an epithelial adhesion molecule, and reduced N-cadherin expression, a mesenchymal marker. Altered cell adherence is one of the hallmarks of the transformed phenotype, often characterized by the loss of the epithelial features and the gain of a mesenchymal phenotype. ECE-1 ablation did not, however, alter viable ovarian carcinoma cell numbers. Addition of exogenous ET-1 reversed the effects cited above. Taken together, these data indicate that siRNA is an effective tool for manipulating ECE-1 expression, ET-1 biosynthesis, and invasiveness of ovarian carcinoma. ECE-1 silencing may therefore develop into a promising novel anticancer therapy. [Cancer Res 2008;68(22):9265–73]
A Novel Mechanism of Late Gene Silencing Drives SV40 Transformation of Human Mesothelial Cells
Authors: Carbone Michele , Pannuti Antonio , Zhang Lei , Testa Joseph R. , Bocchetta Maurizio
Published: November 2008

Suppression of the late gene expression, usually by integration of the viral DNA into the host genome, is a critical step in DNA tumor virus carcinogenesis. SV40 induces high rates of transformation in infected primary human mesothelial cells in tissue culture, leading to the formation of immortal cell lines (SV40-transformed human mesothelial cell lines, S-HML). The studies described here were designed to elucidate the unusual susceptibility of primary human mesothelial cells to SV40 carcinogenesis. We found that S-HML contained wild-type, mostly episomal SV40 DNA. In these cells, the early genes that code for the viral oncogenes are expressed; at the same time, the synthesis of the late genes, capsid proteins, is suppressed and S-HML are not lysed. Late gene suppression is achieved through the production of antisense RNA molecules. These antisense RNA molecules originate in the early region of the SV40 circular chromosome and proceed in antisense orientation into the late gene region, leading to the formation of highly unstable double-strand RNA, which is rapidly degraded. Our results reveal a novel biological mechanism responsible for the suppression of late viral gene products, an important step in viral carcinogenesis in humans. [Cancer Res 2008;68(22):9488–96]
Hypoxia-Mediated Induction of the Polyamine System Provides Opportunities for Tumor Growth Inhibition by Combined Targeting of Vascular Endothelial Growth Factor and Ornithine Decarboxylase
Authors: Svensson Katrin J. , Welch Johanna E. , Kucharzewska Paulina , Bengtson Per , Bjurberg Maria , Påhlman Sven , Ten Dam Gerdy B. , Persson Lo , Belting Mattias
Published: November 2008

Hypoxia is a hallmark of solid tumors, which may offer opportunities for targeted therapies of cancer; however, the mechanisms that link hypoxia to malignant transformation and tumor progression are not fully understood. Here, we show that up-regulation of the polyamine system promotes cancer cell survival during hypoxic stress. Hypoxia was found to induce polyamine transport and the key enzyme of polyamine biosynthesis, ornithine decarboxylase (ODC), in a variety of cancer cell lines. Increased ODC protein expression was shown in hypoxic, GLUT-1–expressing regions of tumor spheroids and experimental tumors, as well as in clinical tumor specimens. Hypoxic induction of the polyamine system was dependent on antizyme inhibitor (i.e., a key positive regulator of ODC and polyamine transport), as shown by RNA interference experiments. Interestingly, depletion of the polyamines during hypoxia resulted in increased apoptosis, which indicates an essential role of the polyamines in cancer cell adaptation to hypoxic stress. These results were supported by experiments in an in vivo glioma tumor model, showing significantly enhanced antitumor effects of the antiangiogenic, humanized anti–vascular endothelial growth factor (VEGF) antibody bevacizumab when used in combination with the well-established, irreversible inhibitor of ODC, α-difluoromethylornithine. Our results provide important insights into the hypoxic stress response in malignant cells and implicate combined targeting of VEGF and ODC as an alternative strategy to treat cancer disease. [Cancer Res 2008;68(22):9291–301]
Transforming Growth Factor β: Tumor Suppressor or Promoter? Are Host Immune Cells the Answer?
Authors: Yang Li , Moses Harold L.
Published: November 2008

Therapies targeting transforming growth factor β (TGFβ) signaling using neutralizing antibodies and small molecular inhibitors are in multiple clinical trails. However, TGFβ is known to work as both a tumor suppressor and a tumor promoter, and current knowledge does not provide sufficient information on what factors mediate this switch in function and when this switch occurs. Recent advances in multiple disciplines suggest that immune cells from the tumor host may provide the answer. [Cancer Res 2008;68(22):9107–11]
Role of a Novel Splice Variant of Mitotic Arrest Deficient 1 (MAD1), MAD1β, in Mitotic Checkpoint Control in Liver Cancer
Authors: Sze Karen Man-Fong , Ching Yick-Pang , Jin Dong-Yan , Ng Irene Oi-Lin
Published: November 2008

Loss of mitotic checkpoint contributes to chromosomal instability, leading to carcinogenesis. In this study, we identified a novel splicing variant of mitotic arrest deficient 1 (MAD1), designated MAD1β, and investigated its role in mitotic checkpoint control in hepatocellular carcinoma (HCC). The expression levels of human MAD1β were examined in hepatoma cell lines and human HCC samples. The functional roles of MAD1β in relation to the mitotic checkpoint control, chromosomal instability, and binding with MAD2 were assessed in hepatoma cell lines. On sequencing, MAD1β was found to have deletion of exon 4. It was expressed at both mRNA and protein levels in the nine hepatoma cell lines tested and was overexpressed in 12 of 50 (24%) human HCCs. MAD1β localized in the cytoplasm, whereas MAD1α was found in the nucleus. This cytoplasmic localization of MAD1β was due to the absence of a nuclear localization signal in MAD1α. In addition, MAD1β was found to physically interact with MAD2 and sequester it in the cytoplasm. Furthermore, expression of MAD1β induced mitotic checkpoint impairment, chromosome bridge formation, and aberrant chromosome numbers via binding with MAD2. Our data suggest that the novel splicing variant MAD1β may have functions different from those of MAD1α and may play opposing roles to MAD1α in mitotic checkpoint control in hepatocarcinogenesis. [Cancer Res 2008;68(22):9194–201]
Human Adrenomedullin Up-regulates Interleukin-13 Receptor α2 Chain in Prostate CancerIn vitroandIn vivo: A Novel Approach to Sensitize Prostate Cancer to Anticancer Therapy
Authors: Joshi Bharat H. , Leland Pamela , Calvo Alfonso , Green Jeffrey E. , Puri Raj K.
Published: November 2008

Interleukin-13 (IL-13) receptor α2 (IL-13Rα2), a high-affinity IL-13 binding subunit and a tumor antigen, is amplified in a variety of human tumor cell lines and tumors in vivo. By cDNA microarray, we have shown that gene transfer of human and rat adrenomedullin (AM) up-regulates IL-13Rα2 in a human prostate tumor cell line. Here, we show that IL-13Rα2 mRNA and protein are also up-regulated in PC-3 prostate tumor cells by recombinant AM (rAM) and human synthetic AM peptide in a dose-dependent manner in vitro and in vivo in mouse prostate tumor model. The 8- to 10-fold up-regulation of IL-13Rα2 by rAM or AM peptide in prostate tumor cells in vitro and in vivo increased their sensitivity to IL-13PE cytotoxin consisting of IL-13 and a truncated form of Pseudomonas exotoxin. Immunodeficient mice with established prostate tumors transfected with AM or treated with AM peptide showed reduction in tumor size by intratumoral administration of IL-13PE in a dose-dependent manner. At the highest dose (three 100 μg/kg/d every alternate day), >70% reduction of tumor size was observed compared with controls (P ≤ 0.01). These results indicate that two completely unrelated hormones (AM and IL-13) are closely related to each other and that we have identified a novel role of AM in sensitizing certain types of prostate tumors to IL-13R–directed therapeutic agent. [Cancer Res 2008;68(22):9311–7]
Antitumor Effect of SN-38–Releasing Polymeric Micelles, NK012, on Spontaneous Peritoneal Metastases from Orthotopic Gastric Cancer in Mice Compared with Irinotecan
Authors: Eguchi Nakajima Takako , Yanagihara Kazuyoshi , Takigahira Misato , Yasunaga Masahiro , Kato Ken , Hamaguchi Tetsuya , Yamada Yasuhide , Shimada Yasuhiro , Mihara Keichiro , Ochiya Takahiro , Matsumura Yasuhiro
Published: November 2008

7-Ethyl-10-hydroxy-camptothecin (SN-38), an active metabolite of irinotecan hydrochloride (CPT-11), has potent antitumor activity. Moreover, we have reported the strong antitumor activity of NK012 (i.e., SN-38–releasing polymeric micelles) against human cancer xenografts compared with CPT-11. Here, we investigated the advantages of NK012 over CPT-11 treatment in mouse models of gastric cancer with peritoneal dissemination. NK012 or CPT-11 was i.v. administered thrice every 4 days at their respective maximum tolerable doses (NK012, 30 mg/kg/day; CPT-11, 67 mg/kg/day) to mice receiving orthotopic transplants of gastric cancer cell lines (44As3Luc and 58As1mLuc) transfected with the luciferase gene (n = 5). Antitumor effect was evaluated using the photon counting technique. SN-38 concentration in gastric tumors and peritoneal nodules was examined by high-performance liquid chromatography (HPLC) 1, 24, and 72 hours after each drug injection. NK012 or CPT-11 distribution in these tumors was evaluated using a fluorescence microscope on the same schedule. In both models, the antitumor activity of NK012 was superior to that of CPT-11. High concentrations of SN-38 released from NK012 were detected in gastric tumors and peritoneal nodules up to 72 hours by HPLC. Only a slight conversion from CPT-11 to SN-38 was observed from 1 to 24 hours. Fluorescence originating from NK012 was detected up to 72 hours, whereas that from CPT-11 disappeared until 24 hours. NK012 also showed antitumor activity against peritoneal nodules. Thus, NK012 showing enhanced distribution with prolonged SN-38 release may be ideal for cancer treatment because the antitumor activity of SN-38 is time dependent. [Cancer Res 2008;68(22):9318–22]
Repression of 15-Hydroxyprostaglandin Dehydrogenase Involves Histone Deacetylase 2 and Snail in Colorectal Cancer
Authors: Backlund Michael G. , Mann Jason R. , Holla Vijaykumar R. , Shi Qiong , Daikoku Takikoku , Dey Sudhansu K. , DuBois Raymond N.
Published: November 2008

Prostaglandin E2 (PGE2) promotes cancer progression by modulating proliferation, apoptosis, angiogenesis, and the immune response. Enzymatic degradation of PGE2 involves the NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH). Recent reports have shown a marked diminution of 15-PGDH expression in colorectal carcinomas (CRC). We report here that treatment of CRC cells with histone deacetylase (HDAC) inhibitors, including sodium butyrate and valproic acid, induces 15-PGDH expression. Additionally, we show that pretreatment of CRC cells with HDAC inhibitors can block epidermal growth factor–mediated or Snail-mediated transcriptional repression of 15-PGDH. We show an interaction between Snail and HDAC2 and the binding of HDAC2 to the 15-PGDH promoter. In vivo, we observe increased Hdac2 expression in Apc-deficient mouse adenomas, which inversely correlated with loss of 15-Pgdh expression. Finally, in human colon cancers, elevated HDAC expression correlated with down-regulation of 15-PGDH. These data suggest that class I HDACs, specifically HDAC2, and the transcriptional repressor Snail play a central role in the suppression of 15-PGDH expression. These results also provide a cyclooxygenase-2–independent mechanism to explain increased PGE2 levels that contribute to progression of CRC. [Cancer Res 2008;68(22):9331–7]
Induction of Cytoplasmic Accumulation of p53: A Mechanism for Low Levels of Arsenic Exposure to Predispose Cells for Malignant Transformation
Authors: Huang Yelin , Zhang Jianglin , McHenry Kevin T. , Kim Mihee M. , Zeng Weiqi , Lopez-Pajares Vanessa , Dibble Christian C. , Mizgerd Joseph P. , Yuan Zhi-Min
Published: November 2008

Although epidemiologic studies have linked arsenic exposure to the development of human cancer, the mechanisms underlying the tumorigenic role of arsenic remain largely undefined. We report here that treatment of cells with sodium arsenite at the concentrations close to environmental exposure is associated with the up-regulation of Hdm2 and the accumulation of p53 in the cytoplasm. Through the mitogen-activated protein kinase pathway, arsenite stimulates the P2 promoter–mediated expression of Hdm2, which then promotes p53 nuclear export. As a consequence, the p53 response to genotoxic stress is compromised, as evidenced by the impaired p53 activation and apoptosis in response to UV irradiation or 5FU treatment. The ability of arsenite to impede p53 activation is further demonstrated by a significantly blunted p53-dependent tissue response to 5FU treatment when mice were fed with arsenite-containing water. Together, our data suggests that arsenic compounds predispose cells to malignant transformation by up-regulation of Hdm2 and subsequent p53 inactivation. [Cancer Res 2008;68(22):9131–6]
Genome-Wide Functional Synergy between Amplified and Mutated Genes in Human Breast Cancer
Authors: Nikolsky Yuri , Sviridov Evgeny , Yao Jun , Dosymbekov Damir , Ustyansky Vadim , Kaznacheev Valery , Dezso Zoltan , Mulvey Laura , Macconaill Laura E. , Winckler Wendy , Serebryiskaya Tatiana , Nikolskaya Tatiana , Polyak Kornelia
Published: November 2008

A single cancer cell contains large numbers of genetic alterations that in combination create the malignant phenotype. However, whether amplified and mutated genes form functional and physical interaction networks that could explain the selection for cells with combined alterations is unknown. To investigate this issue, we characterized copy number alterations in 191 breast tumors using dense single nucleotide polymorphism arrays and identified 1,747 genes with copy number gain organized into 30 amplicons. Amplicons were distributed unequally throughout the genome. Each amplicon had distinct enrichment pattern in pathways, networks, and molecular functions, but genes within individual amplicons did not form coherent functional units. Genes in amplicons included all major tumorigenic pathways and were highly enriched in breast cancer–causative genes. In contrast, 1,188 genes with somatic mutations in breast cancer were distributed randomly over the genome, did not represent a functionally cohesive gene set, and were relatively less enriched in breast cancer marker genes. Mutated and gained genes did not show statistically significant overlap but were highly synergistic in populating key tumorigenic pathways including transforming growth factor β, WNT, fibroblast growth factor, and PIP3 signaling. In general, mutated genes were more frequently upstream of gained genes in transcription regulation signaling than vice versa, suggesting that mutated genes are mainly regulators, whereas gained genes are mostly regulated. ESR1 was the major transcription factor regulating amplified but not mutated genes. Our results support the hypothesis that multiple genetic events, including copy number gains and somatic mutations, are necessary for establishing the malignant cell phenotype. [Cancer Res 2008;68(22):9532–40]
Prediction of Recurrence-Free Survival in Postoperative Non–Small Cell Lung Cancer Patients by Using an Integrated Model of Clinical Information and Gene Expression
Authors: Lee Eung-Sirk , Son Dae-Soon , Kim Sung-Hyun , Lee Jinseon , Jo Jisuk , Han Joungho , Kim Heesue , Lee Hyun Joo , Choi Hye Young , Jung Youngja , Park Miyeon , Lim Yu Sung , Kim Kwhanmien , Shim Young Mog , Kim Byung Chul , Lee Kyusang , Huh Nam , Ko Christopher , Park Kyunghee , Lee Jae Won , Choi Yong Soo , Kim Jhingook
Published: November 2008

Purpose: One of the main challenges of lung cancer research is identifying patients at high risk for recurrence after surgical resection. Simple, accurate, and reproducible methods of evaluating individual risks of recurrence are needed.

Experimental Design: Based on a combined analysis of time-to-recurrence data, censoring information, and microarray data from a set of 138 patients, we selected statistically significant genes thought to be predictive of disease recurrence. The number of genes was further reduced by eliminating those whose expression levels were not reproducible by real-time quantitative PCR. Within these variables, a recurrence prediction model was constructed using Cox proportional hazard regression and validated via two independent cohorts (n = 56 and n = 59).

Results: After performing a log-rank test of the microarray data and successively selecting genes based on real-time quantitative PCR analysis, the most significant 18 genes had P values of <0.05. After subsequent stepwise variable selection based on gene expression information and clinical variables, the recurrence prediction model consisted of six genes (CALB1, MMP7, SLC1A7, GSTA1, CCL19, and IFI44). Two pathologic variables, pStage and cellular differentiation, were developed. Validation by two independent cohorts confirmed that the proposed model is significantly accurate (P = 0.0314 and 0.0305, respectively). The predicted median recurrence-free survival times for each patient correlated well with the actual data.

Conclusions: We have developed an accurate, technically simple, and reproducible method for predicting individual recurrence risks. This model would potentially be useful in developing customized strategies for managing lung cancer.
Role of the Erythropoietin Receptor in ETV6/RUNX1-Positive Acute Lymphoblastic Leukemia
Authors: Inthal Andrea , Krapf Gerd , Beck Dominik , Joas Ruth , Kauer Max O. , Orel Lukas , Fuka Gerhard , Mann Georg , Panzer-Grümayer E. Renate
Published: November 2008

Purpose: We explored the mechanisms leading to the distinct overexpression of EPOR as well as the effects of EPO signaling on ETV6/RUNX1-positive acute lymphoblastic leukemias.

Experimental Design: ETV6/RUNX1-expressing model cell lines and leukemic cells were used for real-time PCR of EPOR expression. Proliferation, viability, and apoptosis were analyzed on cells exposed to EPO, prednisone, or inhibitors of EPOR pathways by [3H]thymidine incorporation, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and Annexin V/propidium iodide staining. Western blot analysis was done to detect activation of signaling proteins. Serum EPO levels and sequences of the EPOR (n = 53) as well as hemoglobin levels were taken from children with acute lymphoblastic leukemia enrolled in Austrian protocols.

Results: We show here that ectopic expression of ETV6/RUNX1 induced EPOR up-regulation. Anemia, however, did not appear to influence EPOR expression on leukemic cells, although children with ETV6/RUNX1-positive leukemias had a lower median hemoglobin than controls. Exposure to EPO increased proliferation and survival of ETV6/RUNX1-positive leukemias in vitro, whereas blocking its binding site did not alter cell survival. The latter was not caused by activating mutations in the EPOR but might be triggered by constitutive activation of phosphatidylinositol 3-kinase/Akt, the major signaling pathway of EPOR in these cells. Moreover, prednisone-induced apoptosis was attenuated in the presence of EPO in this genetic subgroup.

Conclusions: Our data suggest that ETV6/RUNX1 leads to EPOR up-regulation and that activation by EPO might be of relevance to the biology of this leukemia subtype. Further studies are, however, needed to assess the clinical implications of its apoptosis-modulating properties.