bluuebunny/crossref_metadata_2025 huggingface dataset:
Experimental Design and Analysis of Antibody Microarrays: Applying Methods from cDNA Arrays
Authors: Eckel-Passow Jeanette E. , Hoering Antje , Therneau Terry M. , Ghobrial Irene
Published: July 2017

Protein expression microarrays, also called antibody arrays, represent a new technology that allows the expression level of proteins to be assessed directly. As is also the case with gene expression microarrays, it is hoped that protein expression microarrays will aid in biomarker discovery, predicting disease outcomes and response to treatments, and detecting molecular mechanisms and/or pathways associated with a particular disease state. However, accurately achieving these aims is dependent upon suitable experimental designs, normalization procedures that eliminate systematic bias, and appropriate statistical analyses to assess differential expression or expose expression patterns. In the last five years, a large amount of research has been devoted to two-color cDNA arrays to improve experimental design, normalization and statistical analyses to assess differential expression and classification. These methods are directly applicable to two-color antibody arrays. The objective of this article is to discuss statistical methods that have been developed for cDNA arrays and describe how the methods can be directly applied to antibody arrays.
Constitutive Expression and Costimulatory Function of LIGHT/TNFSF14 on Human Melanoma Cells and Melanoma-Derived Microvesicles
Authors: Mortarini Roberta , Scarito Alessia , Nonaka Daisuke , Zanon Marina , Bersani Ilaria , Montaldi Elisabetta , Pennacchioli Elisabetta , Patuzzo Roberto , Santinami Mario , Anichini Andrea
Published: July 2017

Neoplastic cells are thought to have defective expression of costimulatory molecules. However, in this study, we show that human melanoma cells express LIGHT/TNFSF14, a ligand of herpesvirus entry mediator on T cells and of lymphotoxin β receptor on stromal cells. In vitro, melanoma cells stained for LIGHT in the intracellular compartment, with weak or negative cell surface expression. However, LIGHT was expressed on tumor-derived microvesicles released from melanoma cells. In vivo, LIGHT was found in metastatic lesions, and the extent of lymphotoxin β receptor expression on the stromal cells was significantly associated with a “brisk” T-cell infiltrate in the neoplastic tissue. In the lesions with a brisk T-cell infiltrate, stromal cells surrounding the tumor also stained for the T-cell attractant chemokine CCL21. The intratumoral T lymphocytes frequently expressed herpesvirus entry mediator and were characterized by a differentiated phenotype. Coculture of lymphocytes with LIGHT+ melanoma-derived microvesicles or even with LIGHT+ melanoma cells in the presence of interleukin-2 costimulated LIGHT-dependent CD3+CD8+ T-cell proliferation. However, lymphocyte coculture with LIGHT+ microvesicles in the presence of interleukin-2 was also associated with an apoptotic response as documented by increased binding of Annexin V by CD3+CD8+ T cells. These data suggest that LIGHT constitutively expressed in human melanoma cells and microvesicles may contribute to regulate T-cell responses to tumor cells.
Down-regulation of Mitochondrial F1F0-ATP Synthase in Human Colon Cancer Cells with Induced 5-Fluorouracil Resistance
Authors: Shin Young-Kyoung , Yoo Byong Chul , Chang Hee Jin , Jeon Eunkyung , Hong Sung-Hye , Jung Mi-Sun , Lim Soo-Jeong , Park Jae-Gahb
Published: July 2017

5-Fluorouracil (5-FU) is widely used for treatment of advanced colorectal cancer. However, it is common for such patients to develop resistance to 5-FU, and this drug resistance becomes a critical problem for chemotherapy. The mechanisms underlying this resistance are largely unknown. To screen for proteins possibly responsible for 5-FU resistance, cells resistant to 5-FU were derived from human colon cancer cell lines and two-dimensional gel electrophoresis–based comparative proteomics was done. Two-dimensional gel electrophoresis data showed there was lower expression of the α subunit of mitochondrial F1F0-ATP synthase (ATP synthase) in 5-FU–resistant cells compared with parent cells. Western blotting showed that expression of other ATP synthase complex subunits was also lower in 5-FU–resistant cell lines and that these resistant cells also showed decreased ATP synthase activity and reduced intracellular ATP content. The ATP synthase inhibitor, oligomycin A, strongly antagonized 5-FU–induced suppression of cell proliferation. When 5-FU sensitivity was compared with ATP synthase activity in six different human colon cancer cell lines, a positive correlation has been found. Furthermore, suppressed ATP synthase d-subunit expression by siRNA transfection increased cell viability in the presence of 5-FU. Bioenergetic dysfunction of mitochondria has been reported as a hallmark of many types of cancers (i.e., down-regulation of ATP synthase β-subunit expression in liver, kidney, colon, squamous oesophageal, and lung carcinomas, as well as in breast and gastric adenocarcinomas). Our findings show that ATP synthase down-regulation may not only be a bioenergetic signature of colorectal carcinomas but may also lead to cellular events responsible for 5-FU resistance.
Pak1 Phosphorylation of Snail, a Master Regulator of Epithelial-to-Mesenchyme Transition, Modulates Snail's Subcellular Localization and Functions
Authors: Yang Zhibo , Rayala Suresh , Nguyen Diep , Vadlamudi Ratna K. , Chen Shiuan , Kumar Rakesh
Published: July 2017

The process of epithelial-mesenchymal transition plays a pivotal role in the conversion of early stage tumors into invasive malignancies, and has been shown to be regulated by the zinc finger phosphoprotein, Snail; however, no upstream signaling kinases have been shown to modulate Snail functions. Since the invasiveness of breast cancer cells is also influenced by p21-activated kinase 1 (Pak1) signaling, we investigated Pak1's potential mechanistic role in the regulation of Snail functions. We found for the first time that Pak1 promotes transcription repression activity of Snail from E-cadherin, occludin, and aromatase promoters. Pak1 regulates the repressor activity of Snail by phosphorylating on Ser246. Pak1 phosphorylation of Snail supports Snail's accumulation in the nucleus as well as its repressor functions. A Ser246Ala substitution in Snail or Pak1 knockdown by short interference RNA blocked Pak1-mediated Snail phosphorylation, leading to increased cytoplasmic accumulation of Snail and attenuation of Snail repressor activity in breast cancer cells. The regulation of phosphorylation and function of Snail by Pak1 represents a novel mechanism by which a signaling kinase might contribute to the process of epithelial-mesenchymal transition.
Role of SRC-1 in the Promotion of Prostate Cancer Cell Growth and Tumor Progression
Authors: Agoulnik Irina U. , Vaid Ajula , Bingman William E. , Erdeme Halime , Frolov Anna , Smith Carolyn L. , Ayala Gustavo , Ittmann Michael M. , Weigel Nancy L.
Published: July 2017

Prostate cancer is initially androgen dependent and there is evidence that androgen receptor continues to play a role in androgen-independent prostate cancer. Androgen receptor activity depends both on the level of androgens and on the level of coactivators that interact with androgen receptor. Our goal was to evaluate the role of the androgen receptor coactivator SRC-1 in prostate cancer progression. Using tissue arrays to measure SRC-1 protein levels, we found that increased SRC-1 expression in clinically localized, androgen-dependent cancer is associated with clinical and pathologic variables of increased tumor aggressiveness. Interestingly, there was variable expression of SRC-1 in normal prostate tissue which correlated with the staining intensity of the corresponding cancer tissue. To test the contribution of SRC-1, we examined its role in androgen-dependent LNCaP and androgen-independent C4-2 prostate cancer cell lines. Using small interfering RNA to reduce expression of androgen receptor, we found that androgen receptor was required both for cell growth and for basal expression of prostate-specific antigen in the androgen-independent C4-2 cell line. Thus, although the cells can grow in an androgen-depleted medium, they remained androgen receptor dependent. Reduction of SRC-1 expression significantly reduced growth and altered androgen receptor target gene regulation in both LNCaP and C4-2 cell lines whereas it had no effect on the growth of the androgen receptor–negative PC-3 and DU145 prostate cancer cell lines. Although the requirement for androgens and androgen receptor in the development of prostate cancer is well established, our study implicates enhanced androgen receptor activity through elevated expression of SRC-1 in the development of more aggressive disease in men with prostate cancer.
Mechanisms of Enhanced Radiation Response following Epidermal Growth Factor Receptor Signaling Inhibition by Erlotinib (Tarceva)
Authors: Chinnaiyan Prakash , Huang Shyhmin , Vallabhaneni Geetha , Armstrong Eric , Varambally Sooryanarayana , Tomlins Scott A. , Chinnaiyan Arul M. , Harari Paul M.
Published: July 2017

Erlotinib (Tarceva) is an orally available HER1 (epidermal growth factor receptor, EGFR) tyrosine kinase inhibitor advancing through clinical trials for the treatment of a range of human malignancies. In this study, we examine the capacity of erlotinib to modulate radiation response and investigate specific mechanisms underlying these interactions in human tumor cell lines and xenografts. The impact of erlotinib on cell cycle kinetics was analyzed using flow cytometry, and the impact on apoptosis was evaluated via fluorescein-labeled pan-caspase inhibition and poly(ADP-ribose) polymerase cleavage. Radiation-induced EGFR autophosphorylation and Rad51 expression were examined by Western blot analysis. Radiation survival was analyzed using a clonogenic assay and assessment of in vivo tumor growth was done using a mouse xenograft model system. Microarray studies were carried out using 20 K human cDNA microarray and select genes were validated using quantitative reverse transcription-PCR (RT-PCR). Independently, erlotinib and radiation induce accumulation of tumor cells in G1 and G2-M phase, respectively, with a reduction of cells in S phase. When combined with radiation, erlotinib promotes a further reduction in S-phase fraction. Erlotinib enhances the induction of apoptosis, inhibits EGFR autophosphorylation and Rad51 expression following radiation exposure, and promotes an increase in radiosensitivity. Tumor xenograft studies confirm that systemic administration of erlotinib results in profound tumor growth inhibition when combined with radiation. cDNA microarray analysis assessing genes differentially regulated by erlotinib following radiation exposure identifies a diverse set of genes deriving from several functional classes. Validation is confirmed for several specific genes that may influence radiosensitization by erlotinib including Egr-1, CXCL1, and IL-1β. These results identify the capacity of erlotinib to enhance radiation response at several levels, including cell cycle arrest, apoptosis induction, accelerated cellular repopulation, and DNA damage repair. Preliminary microarray data suggests additional mechanisms underlying the complex interaction between EGFR signaling and radiation response. These data suggest that the erlotinib/radiation combination represents a strategy worthy of further examination in clinical trials.
Activin A Mediates Growth Inhibition and Cell Cycle Arrest through Smads in Human Breast Cancer Cells
Authors: Burdette Joanna E. , Jeruss Jacqueline S. , Kurley Sarah J. , Lee Eun Jig , Woodruff Teresa K.
Published: July 2017

The transforming growth factor-β (TGF-β) superfamily of growth factors is responsible for a variety of physiologic actions, including cell cycle regulation. Activin is a member of the TGF-β superfamily that inhibits the proliferation of breast cancer cells. Activin functions by interacting with its type I and type II receptors to induce phosphorylation of intracellular signaling molecules known as Smads. Smads regulate transcription of many genes in a cell- and tissue-specific manner. In this study, the role of activin A in growth regulation of breast cancer cells was investigated. Activin stimulated the Smad-responsive promoter, p3TP, 2-fold over control in T47D breast cancer cells. Activin inhibited cellular proliferation of T47D breast cancer cells after 72 hours, an effect that could be abrogated by incubation with the activin type I receptor inhibitor, SB431542. Activin arrested T47D cells in the G0-G1 cell cycle phase. Smad2 and Smad3 were phosphorylated in response to activin and accumulated in the nucleus of treated T47D cells. Infection of T47D cells with adenoviral Smad3 resulted in cell cycle arrest and activation of p3TP-luciferase, whereas a adenoviral dominant-negative Smad3 blocked activin-mediated cell cycle arrest and gene transcription. Activin maintained expression of p21 and p27 cyclin-dependent kinase inhibitors involved in cell cycle control, enhanced expression of p15, reduced cyclin A expression, and reduced phosphorylation of the retinoblastoma (Rb) protein. Smad3 overexpression recapitulated activin-induced p15 expression and repression of cyclin A and Rb phosphorylation. These data indicate that activin A inhibits breast cancer cellular proliferation and activates Smads responsible for initiating cell cycle arrest.
Synergistic Augmentation of Rapamycin-Induced Autophagy in Malignant Glioma Cells by Phosphatidylinositol 3-Kinase/Protein Kinase B Inhibitors
Authors: Takeuchi Hayato , Kondo Yasuko , Fujiwara Keishi , Kanzawa Takao , Aoki Hiroshi , Mills Gordon B. , Kondo Seiji
Published: July 2017

The mammalian target of rapamycin (mTOR) is a downstream effector of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway and a central modulator of cell proliferation in malignant gliomas. Therefore, the targeting of mTOR signaling is considered a promising therapy for malignant gliomas. However, the mechanisms underlying the cytotoxic effects of a selective mTOR inhibitor, rapamycin, on malignant glioma cells are poorly understood. The purpose of this study was thus to elucidate how rapamycin exerts its cytotoxic effects on malignant glioma cells. We showed that rapamycin induced autophagy but not apoptosis in rapamycin-sensitive malignant glioma U87-MG and T98G cells by inhibiting the function of mTOR. In contrast, in rapamycin-resistant U373-MG cells, the inhibitory effect of rapamycin was minor, although the phosphorylation of p70S6 kinase, a molecule downstream of mTOR, was remarkably inhibited. Interestingly, a PI3K inhibitor, LY294002, and an Akt inhibitor, UCN-01 (7-hydroxystaurosporine), both synergistically sensitized U87-MG and T98G cells as well as U373-MG cells to rapamycin by stimulating the induction of autophagy. Enforced expression of active Akt in tumor cells suppressed the combined effects of LY294002 or UCN-01, whereas dominant-negative Akt expression was sufficient to increase the sensitivity of tumor cells to rapamycin. These results indicate that rapamycin exerts its antitumor effect on malignant glioma cells by inducing autophagy and suggest that in malignant glioma cells a disruption of the PI3K/Akt signaling pathway could greatly enhance the effectiveness of mTOR inhibitors.
Androgen Receptor–Dependent PSA Expression in Androgen-Independent Prostate Cancer Cells Does Not Involve Androgen Receptor Occupancy of the PSA Locus
Authors: Jia Li , Coetzee Gerhard A.
Published: July 2017

It is widely suspected that androgen-independent prostate cancer growth depends on androgen receptor signaling via ill-defined mechanisms. Prostate-specific antigen (PSA) expression is often used to measure androgen receptor activity in cells and prostate cancer progression in patients. In the present study, we have compared androgen receptor activity using PSA and human male germ cell–associated kinase (hMAK), as read-outs in androgen-dependent LNCaP and androgen-independent C4-2B cells. As expected, very little PSA and hMAK expression were detected in LNCaP cells in the absence of androgens, whereas substantial expression of PSA was observed only in C4-2B cells under the same conditions. The addition of dihydrotestosterone to the culture medium increased the expression of both genes in both cell types. Comprehensive chromatin immunoprecipitation analysis of the entire PSA locus and an androgen-response element in hMAK unexpectedly revealed that androgen receptor was not occupying any site in the absence of dihydrotestosterone in either cell type. In line with the expression data, and in the absence of dihydrotestosterone, histone acetylation and RNA polymerase II occupancy was substantial at the PSA locus in C4-2B but not in LNCaP cells. In the presence of dihydrotestosterone, androgen receptor was found to occupy mainly the enhancer region of PSA in both cell types, accompanied with increases in histone acetylation and RNA polymerase II occupancy. Although the androgen receptor was not directly involved in the androgen-independent expression of PSA in C4-2B cells, small interfering RNA knock-down of androgen receptor significantly reduced PSA expression in both the presence and absence of dihydrotestosterone. In contrast, hMAK expression was decreased only in the presence of dihydrotestosterone after androgen receptor knock-down. We conclude that androgen-independent expression of PSA in C4-2B cells does not rely on the direct occupancy of the androgen receptor at the PSA locus, but is nevertheless affected indirectly via unknown androgen receptor–dependent mechanism(s) that influence the expression from some but not all androgen receptor target genes.
Valosin-Containing Protein Phosphorylation at Ser784 in Response to DNA Damage
Authors: Livingstone Mark , Ruan Hong , Weiner Jessica , Clauser Karl R. , Strack Peter , Jin Shengfang , Williams Amy , Greulich Heidi , Gardner James , Venere Monica , Mochan Tamara A. , DiTullio Richard A. , Moravcevic Katarina , Gorgoulis Vassilis G. , Burkhardt Anne , Halazonetis Thanos D.
Published: July 2017

The response of eukaryotic cells to DNA damage includes the activation of phosphatidylinositol-3 kinase–related kinases (PIKK), such as ATM, ATR, and DNA-dependent protein kinase (DNA-PK). These three kinases have very similar substrate specificities in vitro, but in vivo, their substrates overlap only partially. Several in vivo substrates of ATM and ATR have been identified and almost all of them are involved in DNA damage–induced cell cycle arrest and/or apoptosis. In contrast, few in vivo substrates of DNA-PK have been identified. These include histone H2AX and DNA-PK itself. We identify here valosin-containing protein (VCP) as a novel substrate of DNA-PK and other PIKK family members. VCP is phosphorylated at Ser784 within its COOH terminus, a region previously shown to target VCP to specific intracellular compartments. Furthermore, VCP phosphorylated at Ser784 accumulated at sites of DNA double-strand breaks (DSBs). VCP is a protein chaperone that unfolds and translocates proteins. Its phosphorylation in response to DNA damage and its recruitment to sites of DNA DSBs could indicate a role of VCP in DNA repair.
Improved Effector Functions of a Therapeutic Monoclonal Lewis Y-Specific Antibody by Glycoform Engineering
Authors: Schuster Manfred , Umana Pablo , Ferrara Claudia , Brünker Peter , Gerdes Christian , Waxenecker Günter , Wiederkum Susanne , Schwager Cornelia , Loibner Hans , Himmler Gottfried , Mudde Geert C.
Published: July 2017

The aim of the present study was to produce glycosylation variants of the therapeutic Lewis Y-specific humanized IgG1 antibody IGN311 to enhance cell-killing effector function. This was achieved via genetic engineering of the glycosylation machinery of the antibody-producing host. Antibody genes were transiently cotransfected with acetyl-glycosaminyltransferase-III genes into human embryonic kidney-EBV nuclear antigen cells. A control wild-type antibody, IGN311wt, was expressed in the same host using identical expression vectors, but without cotransfection of genes for acetyl-glycosaminyltransferase-III expression. Both expression products were purified to homogeneity and characterized. The glycoengineered expression product (IGN312-Glyco-I) showed a remarkably homogenous N-linked glycosylation pattern consisting of one major hybrid-type, nonfucosylated and agalactosylated form carrying a bisecting GlcNAc-group. Wild-type expression product (IGN311wt) on the other hand was glycosylated by a multitude of different core-fucosylated complex-type structures of variable degrees of galactosylation. Target affinity of the glycoengineered antibody as well as heavy and light chain assembly were not affected by acetyl-glycosaminyltransferase-III expression. In vitro experiments showed a ∼10-fold increase of antibody-dependent cellular cytotoxicity of the glycoengineered antibody using different Lewis Y-positive target cancer cell lines (SK-BR-3, SK-BR-5, OVCAR-3, and Kato-III). Complement-mediated cytotoxicity of IGN312-Glyco-I was 0.4-fold reduced using SK-BR-5 as target cell line. The reduction of complement activation could be prevented and even converted into a slight increase of activity by using a different molecular-biological approach directing the glycosylation towards increased levels of complex N-linked oligosaccharides of bisected, nonfucosylated type, as a result of cotransfection of mannosidase II together with acetyl-glycosaminyltransferase-III.
Membrane-Type 1 Matrix Metalloproteinase Expression Is Regulated by Zonula Occludens-1 in Human Breast Cancer Cells
Authors: Polette Myriam , Gilles Christine , Nawrocki-Raby Béatrice , Lohi Jouko , Hunziker Walter , Foidart Jean-Michel , Birembaut Philippe
Published: July 2017

The acquisition of a migratory/invasive phenotype by tumor cells is characterized by the loss of cell-cell adhesion contacts and the expression of degradative properties. In this study, we examined the effect of the disorganization of occludin/zonula occludens (ZO)-1 tight junction (TJ) complexes on the expression of membrane-type 1 matrix metalloproteinase (MT1-MMP). We first compared the expression of MT1-MMP and the localization of occludin/ZO-1 complexes in breast tumor cell lines displaying various degrees of invasiveness. We showed that the expression of MT1-MMP in invasive breast tumor cell lines correlates with the absence of occludin and with a cytoplasmic localization of ZO-1. In contrast, noninvasive cell lines displayed a membrane staining for both ZO-1 and occludin and did not express MT1-MMP. In vivo, cytoplasmic ZO-1 and MT1-MMP could be detected in invasive tumor clusters of human breast carcinomas. We then used RNA interference strategy to inhibit ZO-1 expression in invasive BT549 cells and to evaluate the effect of ZO-1 down-regulation on MT1-MMP expression. We observed that ZO-1 small interfering RNA transfection down-regulates MT1-MMP mRNAs and proteins and subsequently decreases the ability of tumor cells to invade a reconstituted basement membrane in a Boyden chamber assay. Inversely, transfection of expression vectors encoding wild-type ZO-1 or the NH2-terminal fragment of ZO-1 comprising the PSD95/DLG/ZO-1 domains in BT549 activated a human MT1-MMP promoter luciferase reporter construct and increased cell invasiveness. Such transfections concomitantly activated the β-catenin/TCF/LEF pathway. Our results therefore show that ZO-1, besides its structural role in TJ assembly, can intervene in signaling events promoting tumor cell invasion.
High Nuclear S100A6 (Calcyclin) Is Significantly Associated with Poor Survival in Pancreatic Cancer Patients
Authors: Vimalachandran Dale , Greenhalf William , Thompson Christopher , Lüttges Jutta , Prime Wendy , Campbell Fiona , Dodson Andrew , Watson Richard , Crnogorac-Jurcevic Tatjana , Lemoine Nicholas , Neoptolemos John , Costello Eithne
Published: July 2017

Recent studies have reported elevated levels of S100A6 in pancreatic ductal adenocarcinoma cells. Here, we describe a detailed analysis of S100A6 expression in benign (n = 32), malignant (n = 60), and premalignant pancreatic ductal cells [96 pancreatic intraepithelial neoplasias (PanIN) from 46 patients]. S100A6 staining was more intense in malignant cells than in benign cells (P = 0.0001). In malignant cells, staining was higher in the nucleus than in the cytoplasm (P = 0.003). Univariate analysis revealed a significant decrease in survival time for patients with high levels of nuclear (P = 0.01) but not cytoplasmic (P = 0.20) S100A6. No evidence was found for an association between nuclear S100A6 expression and other variables, including gender, age at surgery, tumor size or grade, nodal metastases, resection margin, vascular invasion, perineural invasion, p53 or Smad4 levels (both linked to survival in previous studies), or the p65 subunit of nuclear factor-κB (a potential regulator of S100A6). Although nodal metastases and resection margin involvement were also associated with poor survival (P = 0.06 in both cases), multivariate analysis suggests that nuclear S100A6 is a significant independent indicator of survival (P = 0.003). Whereas PanIN 1a lesions showed a general absence of S100A6 staining, there was a progressive increase in the proportion of positively stained PanINs with increasing PanIN grade. In particular, we observed an increase in the frequency and intensity of nuclear staining. Our results suggest that up-regulation of S100A6 is an early event in pancreatic cancer development and that elevated levels of nuclear S100A6 may affect clinical outcome.
Influence of Mast Cells on Structural and Functional Manifestations of Radiation-Induced Heart Disease
Authors: Boerma Marjan , Wang Junru , Wondergem Jan , Joseph Jacob , Qiu Xiaohua , Kennedy Richard H. , Hauer-Jensen Martin
Published: July 2017

Radiation-induced heart disease (RIHD), characterized by accelerated atherosclerosis and adverse tissue remodeling, is a serious sequelae after radiotherapy of thoracic and chest wall tumors. Adverse cardiac remodeling in RIHD and other cardiac disorders is frequently accompanied by mast cell hyperplasia, suggesting that mast cells may affect the development of cardiac fibrosis. This study used a mast cell–deficient rat model to define the role of mast cells in RIHD.

Mast cell–deficient rats (Ws/Ws) and mast cell–competent littermate controls (+/+) were exposed to 18 Gy localized single-dose irradiation of the heart. Six months after irradiation, cardiac function was examined by echocardiography and Langendorff-perfused isolated heart preparation, whereas structural changes were assessed using quantitative histology and immunohistochemical analysis.

Mast cell–deficient rats exhibited more severe postradiation changes than mast cell–competent littermates. Hence, mast cell–deficient rats exhibited a greater upward/leftward shift in the left ventricular (LV) diastolic pressure-volume relationship (P = 0.001), a greater reduction in in vivo LV diastolic area (from 0.50 ± 0.024 cm in age-matched controls to 0.24 ± 0.032 cm after irradiation; P = 0.006), and a greater increase in LV posterior wall thickness (from 0.13 ± 0.003 cm in age-matched controls to 0.15 ± 0.003 cm after irradiation; P = 0.04). Structural analysis revealed more pronounced postradiation accumulation of interstitial collagen III but less myocardial degeneration in hearts from mast cell–deficient rats.

These data show that the absence of mast cells accelerates the development of functional changes in the irradiated heart, particularly diastolic dysfunction, and suggest that, in contrast to what has been the prevailing assumption, the role of mast cells in RIHD is predominantly protective.
Big Mitogen-Activated Protein Kinase 1/Extracellular Signal-Regulated Kinase 5 Signaling Pathway Is Essential for Tumor-Associated Angiogenesis
Authors: Hayashi Masaaki , Fearns Colleen , Eliceiri Brian , Yang Young , Lee Jiing-Dwan
Published: July 2017

Although big mitogen-activated protein kinase 1 (BMK1) has been shown to be critical for embryonic angiogenesis, the role of BMK1 in tumor-associated neovascularization is poorly understood. Exogenous tumors were established in BMK1+/+, BMK1flox/+, or BMK1flox/flox mice carrying the Mx1-Cre transgene. Induced deletion of host BMK1 gene significantly reduced the volumes of B16F10 and LL/2 tumor xenografts in BMK1flox/flox mice by 63% and 72%, respectively. Examining the tumors in these induced BMK1-knockout animals showed a significant decrease in vascular density. Localized reexpression of BMK1 in BMK1-knockout mice by administration of adenovirus encoding BMK1 restored tumor growth and angiogenesis to the levels observed in wild-type mice. These observations were further supported by in vivo Matrigel plug assays in which vascular endothelial growth factor– and basic fibroblast growth factor–induced neovacularization was impaired by removing BMK1. Through screening with the Pepchip microarray, we discovered that in BMK1-knockout endothelial cells, phosphorylation of ribosomal protein S6 (rpS6) at Ser235/236 was mostly abrogated, and this BMK1-dependent phosphorylation required the activity of p90 ribosomal S6 kinase (RSK). Immunofluorescent analysis of tumor vasculature from BMK1-knockout and control animals revealed a strong correlation between the presence of BMK1 and the phosphorylation of rpS6 in tumor-associated endothelial cells of blood vessels. As both RSK and rpS6 are known to be important for cell proliferation and survival, which are critical endothelial cell functions during neovascularization, these findings suggest that the BMK1 pathway is crucial for tumor-associated angiogenesis through its role in the regulation of the RSK-rpS6 signaling module.
Prevention of Colon Cancer by Low Doses of Celecoxib, a Cyclooxygenase Inhibitor, Administered in Diet Rich in ω-3 Polyunsaturated Fatty Acids
Authors: Reddy Bandaru S. , Patlolla Jagan M. , Simi Barbara , Wang S.H. , Rao Chinthalapally V.
Published: July 2017

Epidemiologic and animal studies suggest that a high-fat diet containing mixed lipids promotes colorectal cancer, whereas fish oil lacks promoting effect. Although cyclooxygenase-2 (COX-2) inhibitors are effective chemopreventive agents against colon carcinogenesis, administration of high doses of these agents over time may induce side effects. Here, we compared the efficacy of moderately high and low doses of celecoxib administered in diets high in mixed lipids (HFML) or fish oil (HFFO) against azoxymethane-induced colon carcinogenesis in male F344 rats. One day after the last azoxymethane treatment (15 mg/kg body weight once weekly for 2 weeks), groups of rats were fed the HFML and HFFO diets containing 0, 250, 500, and 1,000 ppm celecoxib. Rats were killed 26 weeks later and colon tumors were subjected to histopathologic examination and analyzed for total COX and COX-2 synthetic activities and COX-2 expression. Rats fed the HFFO diet showed significantly lower colon tumor incidence and multiplicity compared with rats fed the HFML diet. Celecoxib at 250, 500, and 1,000 ppm in either diet significantly suppressed colon carcinogenesis. Inhibition of colon adenocarcinomas were more pronounced in animals given 250 ppm celecoxib in HFFO diet compared with 250 ppm celecoxib given in HFML diet, suggesting some synergism between ω-3 polyunsaturated fatty acids (PUFA) and celecoxib. Inhibition of colon tumors by celecoxib was associated with lower levels of COX-2 activity and expression in colon tumors. These studies support the use of low doses of celecoxib in ω-3 PUFA–rich diet as a promising approach for clinical trials.
Accumulation of p53 and Reductions in XIAP Abundance Promote the Apoptosis of Prostate Cancer Cells
Authors: Mohapatra Subhra , Chu Baoky , Zhao Xiuhua , Pledger W.J.
Published: July 2017

Toward the goal of developing effective treatments for prostate cancers, we examined the effects of cyclin-dependent kinase inhibitors on the survival of prostate cancer cells. We show that roscovitine, R-roscovitine, and CGP74514A (collectively referred to as CKIs) induce the apoptosis of LNCaP and LNCaP-Rf cells, both of which express wild-type p53. Apoptosis required caspase-9 and caspase-3 activity, and cytochrome c accumulated in the cytosol of CKI-treated cells. Amounts of p53 increased substantially in CKI-treated cells, whereas amounts of the endogenous caspase inhibitor XIAP decreased. CKIs did not appreciably induce the apoptosis of LNCaP cells treated with pifithrin-α, which prevents p53 accumulation, or of prostate cancer cells that lack p53 function (PC3 and DU145). Ectopic expression of p53 in PC3 cells for 44 hours did not reduce XIAP abundance or induce apoptosis. However, p53-expressing PC3 cells readily apoptosed when exposed to CKIs or when depleted of XIAP by RNA interference. These findings show that CKIs induce the mitochondria-mediated apoptosis of prostate cancer cells by a dual mechanism: p53 accumulation and XIAP depletion. They suggest that these events in combination may prove useful in the treatment of advanced prostate cancers.
Therapeutic Targeting of the Hedgehog-GLI Pathway in Prostate Cancer
Authors: Sanchez Pilar , Clement Virginie , Ruiz i Altaba Ariel
Published: July 2017

The Hedgehog-GLI signaling pathway is important in animal development and tumorigenesis. Recent findings indicate that the growth and survival of human prostate cancer cells rely upon sustained signaling from the Hedgehog-GLI pathway. These findings have prompted a novel rational strategy for therapeutic treatment of prostate tumors, including metastatic tumors.
RUNX3, A Novel Tumor Suppressor, Is Frequently Inactivated in Gastric Cancer by Protein Mislocalization
Authors: Ito Kosei , Liu Qiang , Salto-Tellez Manuel , Yano Takashi , Tada Kotaro , Ida Hiroshi , Huang Canhua , Shah Nilesh , Inoue Masafumi , Rajnakova Andrea , Hiong Kum Chew , Peh Bee Keow , Han Hwan Chour , Ito Tomoko , Teh Ming , Yeoh Khay Guan , Ito Yoshiaki
Published: July 2017

Loss of RUNX3 expression is suggested to be causally related to gastric cancer as 45% to 60% of gastric cancers do not express RUNX3 mainly due to hypermethylation of the RUNX3 promoter. Here, we examined for other defects in the properties of RUNX3 in gastric cancers that express RUNX3. Ninety-seven gastric cancer tumor specimens and 21 gastric cancer cell lines were examined by immunohistochemistry using novel anti-RUNX3 monoclonal antibodies. In normal gastric mucosa, RUNX3 was expressed most strongly in the nuclei of chief cells as well as in surface epithelial cells. In chief cells, a significant portion of the protein was also found in the cytoplasm. RUNX3 was not detectable in 43 of 97 (44%) cases of gastric cancers tested and a further 38% showed exclusive cytoplasmic localization, whereas only 18% showed nuclear localization. Evidence is presented suggesting that transforming growth factor-β is an inducer of nuclear translocation of RUNX3, and RUNX3 in the cytoplasm of cancer cells is inactive as a tumor suppressor. RUNX3 was found to be inactive in 82% of gastric cancers through either gene silencing or protein mislocalization to the cytoplasm. In addition to the deregulation of mechanisms controlling gene expression, there would also seem to be at least one other mechanism controlling nuclear translocation of RUNX3 that is impaired frequently in gastric cancer.
JunB Induced by Constitutive CD30–Extracellular Signal-Regulated Kinase 1/2 Mitogen-Activated Protein Kinase Signaling Activates the CD30 Promoter in Anaplastic Large Cell Lymphoma and Reed-Sternberg Cells of Hodgkin Lymphoma
Authors: Watanabe Mariko , Sasaki Masataka , Itoh Kinji , Higashihara Masaaki , Umezawa Kazuo , Kadin Marshall E. , Abraham Lawrence J. , Watanabe Toshiki , Horie Ryouichi
Published: July 2017

High expression of CD30 and JunB is characteristic of tumor cells in anaplastic large cell lymphoma (ALCL) and Hodgkin lymphoma (HL). Possible interactions of CD30 and JunB were examined in this study. We found that the CD30 promoter in tumor cells of both nucleophosmin (NPM)-anaplastic lymphoma kinase (ALK)–positive and NPM-ALK-negative ALCL and HL is regulated by a constitutively active CD30–extracellular signal-regulated kinase (ERK) 1/2 mitogen-activated protein kinase (MAPK). Phosphorylation of ERK1/2 MAPK was confirmed in nuclei of tumor cells in both ALCL and HL. CD30-ERK1/2 MAPK signals induce JunB expression, which maintains high activity of the CD30 promoter. JunB induction seems to be largely independent of nuclear factor κB in ALCL and HL. These results show a common mechanism of CD30 overexpression in ALCL and HL, although the outcome of CD30 signaling differs between NPM-ALK-positive ALCL and NPM-ALK-negative ALCL, cutaneous ALCL, and HL as we recently reported.
Targeting of Urokinase Plasminogen Activator Receptor in Human Pancreatic Carcinoma Cells Inhibits c-Met– and Insulin-like Growth Factor-I Receptor–Mediated Migration and Invasion and Orthotopic Tumor Growth in Mice
Authors: Bauer Todd W. , Liu Wenbiao , Fan Fan , Camp Ernest R. , Yang Anthony , Somcio Ray J. , Bucana Corazon D. , Callahan Jennifer , Parry Graham C. , Evans Douglas B. , Boyd Douglas D. , Mazar Andrew P. , Ellis Lee M.
Published: July 2017

Pancreatic carcinomas express high levels of urokinase-type plasminogen activator (uPA) and its receptor (uPAR), both of which mediate cell migration and invasion. We investigated the hypotheses that (a) insulin-like growth factor-I (IGF-I)– and hepatocyte growth factor (HGF)–mediated migration and invasion of human pancreatic carcinoma cells require uPA and uPAR function and (b) inhibition of uPAR inhibits tumor growth, retroperitoneal invasion, and hepatic metastasis of human pancreatic carcinomas in mice. Using transwell assays, we investigated the effect of IGF-I and HGF on L3.6pl migration and invasion. We measured the induction of uPA and uPAR following treatment of cells with IGF-I and HGF using immunoprecipitation and Western blot analysis. The importance of uPA and uPAR on L3.6pl cell migration and invasion was studied by inhibiting their activities with amiloride and antibodies before cytokine treatment. In an orthotopic mouse model of human pancreatic carcinoma, we evaluated the effect of anti-uPAR monoclonal antibodies with and without gemcitabine on primary tumor growth, retroperitoneal invasion, and hepatic metastasis. IGF-I and HGF mediated cell migration and invasion in L3.6pl cells. In addition, IGF-I and HGF induced uPA and uPAR expression in L3.6pl cells. In vitro, blockade of uPA and uPAR activity inhibited IGF-I– and HGF-mediated cell migration and invasion. Treatment of mice with anti-uPAR monoclonal antibody significantly decreased pancreatic tumor growth and hepatic metastasis and completely inhibited retroperitoneal invasion. Our study shows the importance of the uPA/uPAR system in pancreatic carcinoma cell migration and invasion. These findings suggest that uPAR is a potential target for therapy in patients with pancreatic cancer.
Loss of Epigenetic Control of Synuclein-γ Gene as a Molecular Indicator of Metastasis in a Wide Range of Human Cancers
Authors: Liu Haiyan , Liu Wei , Wu Yinwei , Zhou Yue , Xue Rong , Luo Chan , Wang Lan , Zhao Wei , Jiang Jian-Dong , Liu Jingwen
Published: July 2017

Metastasis is a major contributing factor to poor prognosis in cancer. Reliable and sensitive biomarkers that indicate the development of metastasis of primary tumors would be of great clinical use. In this study, we show that the neuronal protein synuclein-γ (SNCG) is abnormally expressed in a high percentage (67.5%) of tumor tissues of diversified cancer types, including liver, esophagus, colon, gastric, lung, prostate, cervical, and breast cancer, but rarely expressed in tumor-matched nonneoplastic adjacent tissues (0.6%). Expressions of SNCG protein in different cancer types all display stage-specific patterns of very low expression in stage I and high expression in stages II to IV. Importantly, we observe a strong association between SNCG protein expression in primary tumors with distant metastasis in patients regardless of the cancer type (60.6%, P < 0.001). By performing genomic sequencing and methylation-specific PCR assays, we identify an inclusive demethylation of CpG sites within the CpG island of SNCG gene in every tumor sample (100%) across all cancer types, illustrating a universal loss of the epigenetic control of SNCG gene expression in tumors and further demonstrating that the demethylation of SNCG CpG island is primarily responsible for the aberrant expression of SNCG protein in cancerous tissues. These new findings strongly suggest that reactivation of SNCG gene expression by DNA demethylation is a common critical contributing factor to malignant progression of many solid tumors and its expression in primary carcinomas is an effective molecular indicator of distant metastasis. Our studies also suggest that the methylation status of SNCG gene can be used as a sensitive molecular tool in early detections of tumorigenesis.
Prostate Cancer Cells Promote Osteoblastic Bone Metastases through Wnts
Authors: Hall Christopher L. , Bafico Anna , Dai Jinlu , Aaronson Stuart A. , Keller Evan T.
Published: July 2017

Prostate cancer produces painful osteoblastic bone metastases. Although prostate cancer cells produce numerous osteogenic factors, to date, none have been shown to mediate osteoblastic bone metastases in an in vivo model of prostate cancer. Wnts are a large family of proteins that promote bone growth. Wnt activity is antagonized by endogenous proteins including dickkopf-1 (DKK-1). We explored if prostate cancer cells mediate osteoblastic activity through Wnts using DKK-1 as a tool to modify Wnt activity. A variety of Wnt mRNAs were found to be expressed in prostate cancer cell lines and Wnt mRNA expression was increased in primary prostate cancer compared with nonneoplastic prostate tissue. In addition to expressing Wnts, PC-3 prostate cancer cells expressed the Wnt inhibitor DKK-1. To determine if DKK-1 masked Wnt-mediated osteoblastic activity in osteolytic PC-3 cells, the cells were stably transfected with DKK-1 short hairpin RNA. Decreasing DKK-1 enabled PC-3 cells to induce osteoblastic activity, including alkaline phosphatase production and mineralization, in murine bone marrow stromal cells indicating that DKK-1 blocked Wnt-mediated osteoblastic activity in PC-3 cells. Another prostate cancer cell line, C4-2B, induces mixed osteoblastic/osteolytic lesions. To determine if Wnts contribute to C4-2B's ability to induce mixed osteoblastic/osteolytic lesions, C4-2B cells were stably transfected with either empty vector or DKK-1 expression vector to block Wnt activity. The cells were then injected in the tibiae of mice and allowed to grow for 12 weeks. Blocking Wnt activity converted the C4-2B cells to a highly osteolytic tumor. Taken together, these data show that Wnts contribute to the mechanism through which prostate cancer induces osteoblastic activity.
Multiple Microalterations Detected at High Frequency in Oral Cancer
Authors: Baldwin Corisande , Garnis Cathie , Zhang Lewei , Rosin Miriam P. , Lam Wan L.
Published: July 2017

The development of array comparative genomic hybridization (array CGH) at tiling-path resolution has enabled the detection of gene-sized segmental DNA copy number gains and losses. Here, we present the first application of whole genome tiling-path array CGH to archival clinical specimens for the detailed analysis of oral squamous cell carcinomas (OSCC). We describe the genomes of 20 OSCCs as well as a selection of matched normal DNA in unprecedented detail. Examination of their whole genome profiles enabled the identification of alterations ranging in size from whole-arm, segmental, to gene size alterations. Tiling-path resolution enabled the detection of many more alterations within each tumor than previously reported, many of which include narrow alterations found to be frequent events among the 20 OSCCs. We report the presence of several novel frequent submegabase alterations, such as the 0.58 Mb gain at 5p15.2 containing triple functional domain (TRIO), detected in 45% of cases. We also report the first coamplification of two gene clusters, by fine-mapping the precise base pair boundaries of the high-level amplification at 11q22.2-22.3 containing both matrix metalloproteinase and baculoviral IAP repeat-containing protein 2 (BIRC) gene clusters. These results show the large improvement in detection sensitivity and resolution compared with genome interval marker arrays and the utility of tiling resolution array CGH for the detection of both submegabase and single copy gains and losses in cancer gene discovery.
EGFR Tyrosine Kinase Domain Mutations Are Detected in Histologically Normal Respiratory Epithelium in Lung Cancer Patients
Authors: Tang Ximing , Shigematsu Hisayuki , Bekele B. Nebiyou , Roth Jack A. , Minna John D. , Hong Waun Ki , Gazdar Adi F. , Wistuba Ignacio I.
Published: July 2017

To determine whether EGFR tyrosine kinase domain mutations are early events in the pathogenesis of lung adenocarcinomas, we tested for the presence of EGFR mutations in histologically normal bronchial and bronchiolar epithelia from lung adenocarcinomas bearing the common EGFR mutations. DNA was extracted from microdissected tissue obtained from 21 tumors with known EGFR mutations, 16 tumors without mutation, and 90 sites of normal bronchial and bronchiolar epithelium from the same surgical specimens. With the use of PCR and direct DNA sequencing, EGFR mutations identical to the tumors were detected in the normal respiratory epithelium in 9 of 21 (43%) patients with EGFR mutant adenocarcinomas but none in patients without mutation in the tumors. The finding of mutations being more frequent in normal epithelium within tumor (43%) than in adjacent sites (24%) suggests a localized field effect phenomenon. Our findings indicate that mutation of the tyrosine kinase domain of EGFR is an early event in the pathogenesis of lung adenocarcinomas, and suggest EGFR mutations as an early detection marker and chemoprevention target.
Methods of Academic Course Planning for Cancer Biology PhD Students to Enhance Knowledge of Clinical Oncology
Authors: Mattes Malcolm D. , Swart Elizabeth , Markwell Steven M. , Wen Sijin , Vona-Davis Linda C.
Published: July 2017

Little is known about how clinical oncology concepts are taught to PhD students or the most effective methods of doing so. In this study, electronic surveys were sent to faculty and students at PhD training programs, assessing their institution's methods of clinical oncology education and their perspective on optimal approaches to clinical oncology education. Only 40.0% of students reported any clinical oncology component to their institution's training, and only 26.5% had a clinician on their graduate advisory committee. Forty-three percent of students believed that they had a good understanding for translating basic science research into clinical practice, and 77.2% of all participants believed dual degree MD/PhD students were superior to PhD students in this regard. Lectures on clinical oncology research topics were the most valuable type of experience for all participants and were also the most common type of experience utilized. Working with a clinician to develop a clinical trial with correlative endpoints was also highly valued, but was only utilized by approximately 10% of programs. Faculty rated the value of nearly all types of clinical oncology exposure significantly lower than did students. Inclusion of the approaches identified in this study is likely to enhance PhD training in oncology-related disciplines. Cancer Res; 77(18); 4741–4. ©2017 AACR.