bluuebunny/crossref_metadata_2025 huggingface dataset:
A Unique Mucin Immunoenhancing Peptide with Antitumor Properties
Authors: Herbert Lynn M. , Grosso Joseph F. , Dorsey Mantley , Fu Tihui , Keydar Iafa , Cejas Mabel A. , Wreschner Daniel H. , Smorodinski Nechama , Lopez Diana M.
Published: September 2005

Implantation of DA-3 mammary tumor cells into BALB/c mice results in tumor growth, metastatic lesions, and death. These cells were transfected with genes encoding for either the transmembrane (DA-3/TM) or secreted (DA-3/sec) form of human mucin 1 (MUC1). Although the gene for the secreted form lacks the transmembrane and cytoplasmic domains, the 5′ sequences of these mucins are identical; however, the gene for the secreted mucin isoform ends with a sequence encoding for a unique 11 amino acid peptide. The DA-3/TM or DA-3 cells transfected with the neomycin vector only (DA-3/neo) have the same in vivo growth characteristics as the parent cell line. In contrast, DA-3/sec cells fail to grow when implanted in immunocompetent BALB/c animals. DA-3/sec cells implanted in nude mice resulted in tumor development verifying the tumorigenic potential of these cells. Pre-exposure of BALB/c mice to DA-3/sec cells afforded protection against challenge with DA-3/TM or DA-3/neo mammary tumors and the unrelated tumors K7, an osteosarcoma, and RENCA, a renal cell carcinoma. Partial protection against subsequent tumor challenges was also achieved by substituting the 11 amino acid peptide found only in the secreted MUC1 isoform, for the live DA-3/sec cells. Notably, the efficacy of this peptide is not strain restricted because it also retarded the growth of Lewis lung carcinoma cells in C57 BL/6 mice. These findings reveal that a unique peptide present in the secreted MUC1 has immunoenhancing properties and may be a potential agent for use in immunotherapy.
Transforming Growth Factor-β Pathway Serves as a Primary Tumor Suppressor in CD8+ T Cell Tumorigenesis
Authors: Lucas Philip J. , McNeil Nicole , Hilgenfeld Eva , Choudhury Baishakhi , Kim Seong-Jin , Eckhaus Michael A. , Ried Thomas , Gress Ronald E.
Published: September 2005

Tumorigenesis in rodents, as well as in humans, has been shown to be a multistep process, with each step reflecting an altered gene product or gene regulatory process leading to autonomy of cell growth. Initial genetic mutations are often associated with dysfunctional growth regulation, as is demonstrated in several transgenic mouse models. These changes are often followed by alterations in tumor suppressor gene function, allowing unchecked cell cycle progression and, by genomic instability, additional genetic mutations responsible for tumor metastasis. Here we show that reduced transforming growth factor-β signaling in T lymphocytes leads to a rapid expansion of a CD8+ memory T-cell population and a subsequent transformation to leukemia/lymphoma as shown by multiple criteria, including peripheral blood cell counts histology, T-cell receptor monoclonality, and host transferability. Furthermore, spectral karyotype analysis of the tumors shows that the tumors have various chromosomal aberrations. These results suggest that reduced transforming growth factor-β signaling acts as a primary carcinogenic event, allowing uncontrolled proliferation with consequent accumulation of genetic defects and leukemic transformation.
Identification of Hypoxia-Regulated Proteins in Head and Neck Cancer by Proteomic and Tissue Array Profiling
Authors: Chen Yijun , Shi Gongyi , Xia Wei , Kong Christina , Zhao Shuchun , Gaw Allison F. , Chen Eunice Y. , Yang George P. , Giaccia Amato J. , Le Quynh-Thu , Koong Albert C.
Published: September 2005

Hypoxia within solid tumors decreases therapeutic efficacy, and identification of hypoxia markers may influence the choice of therapeutic modality. Here, we used a proteomic approach to identify hypoxia-regulated proteins and validated their use as endogenous indicators of tumor hypoxia. Using two-dimensional gel electrophoresis and PowerBlot (antibody-based array), we identified a group of 20 proteins that are increased ≥1.5-fold during hypoxia. The majority of these proteins such as IκB kinase β (IKKβ), MKK3b, highly expressed in cancer (HEC), density-regulated protein 1, P150glued, nuclear transport factor 2, binder of ARL 2, Paxillin, and transcription termination factor I have not been previously reported to be hypoxia inducible. The increase in these proteins under hypoxia was mediated through posttranscriptional mechanisms. We additionally characterized the role of IKKβ, a regulator of the nuclear factor-κB transcription factor, during hypoxia. We demonstrated that IKKβ mediates cell survival during hypoxia and is induced in a variety of squamous cell carcinoma cell lines. Furthermore, we showed that IKKβ expression from tumor specimens correlated with tumor oxygenation in patients with head and neck squamous cell carcinomas. These data suggest that IKKβ is a novel endogenous marker of tumor hypoxia and may represent a new target for anticancer therapy.
A Protective Role for Matrix Metalloproteinase-3 in Squamous Cell Carcinoma
Authors: McCawley Lisa J. , Crawford Howard C. , King Lloyd E. , Mudgett John , Matrisian Lynn M.
Published: September 2005

Elevated expression of matrix metalloproteinase-3 (MMP-3/stromelysin-1) is associated with a variety of tumor types, although its in vivo functional role remains unclear. In human and murine squamous cell carcinoma (SCC), MMP-3 is expressed in the stromal compartment at all of the stages of tumor progression and is expressed by the malignant epithelial cells in late-stage, highly invasive tumors. To elucidate whether MMP-3 plays a causal role during SCC, wild-type and MMP-3 null mice were subjected to chemical carcinogenesis procedures by topical application of either the complete carcinogen 1-methyl-3-nitro-1-nitroso-guanidine or two-stage initiation and promotion with 7,12-dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol-13-acetate. Contrasting with our expectations, tumors originating on MMP-3 null mice had enhanced initial tumor growth rates as compared with control animals, although there was no difference in tumor onset or incidence. This elevated rate in growth was coupled with an elevated proliferative index and a reduced vasculature density but with no significant effect on apoptosis. Tumors from MMP-3 null mice had a prevalence of undifferentiated spindle tumors as compared with controls, which was concomitant with a higher percentage of MMP-3 null mice evidencing surface lung metastases. Tumor progression in MMP-3 null mice was inversely associated with leukocyte infiltration, in which an overall reduction in tumor-associated macrophages and neutrophils was evident. We propose that MMP-3 is expressed as a protective response and plays an important role in host defense during SCC tumorigenesis.
Genetic signatures of High- and Low-Risk Aberrant Crypt Foci in a Mouse Model of Sporadic Colon Cancer
Authors: Nambiar Prashant R. , Nakanishi Masako , Gupta Rishi , Cheung Evelyn , Firouzi Ali , Ma Xiao-Jun , Flynn Christopher , Dong Mei , Guda Kishore , Levine Joel , Raja Rajiv , Achenie Luke , Rosenberg Daniel W.
Published: September 2005

To determine whether cancer risk is related to histopathological features of preneoplastic aberrant crypt foci (ACF), gene expression analysis was performed on ACF from two mouse strains with differing tumor sensitivity to the colonotropic carcinogen, azoxymethane. ACF from sensitive A/J mice were considered at high risk, whereas ACF from resistant AKR/J mice were considered at low risk for tumorigenesis. A/J and AKR/J mice received weekly injections of azoxymethane (10 mg/kg body weight), and frozen colon sections were prepared 6 weeks later. Immunohistochemistry was performed using biomarkers associated with colon cancer, including adenomatous polyposis coli, β-catenin, p53, c-myc, cyclin D1, and proliferating cell nuclear antigen. Hyperplastic ACF, dysplastic ACF, microadenomas, adjacent normal-appearing epithelium, and vehicle-treated colons were laser captured, and RNA was linearly amplified (LCM-LA) and subjected to cDNA microarray-based expression analysis. Patterns of gene expression were identified using adaptive centroid algorithm. ACF from low- and high-risk colons were not discriminated by immunohistochemistry, with the exception of membrane staining of β-catenin. To develop genetic signatures that predict cancer risk, LCM-LA RNA from ACF was hybridized to cDNA arrays. Of 4896 interrogated genes, 220 clustered into six broad clusters. A total of 226 and 202 genes was consistently altered in lesions from A/J and AKR/J mice, respectively. Although many alterations were common to both strains, expression profiles stratified high- and low- risk lesions. These data demonstrate that ACF with distinct tumorigenic potential have distinguishing molecular features. In addition to providing insight into colon cancer promotion, our data identify potential biomarkers for determining colon cancer risk in humans.
Breast Cancer Cells Can Evade Apoptosis-Mediated Selective Killing by a Novel Small Molecule Inhibitor of Bcl-2
Authors: Real Pedro Jose , Cao Yeyu , Wang Renxiao , Nikolovska-Coleska Zaneta , Sanz-Ortiz Jaime , Wang Shaomeng , Fernandez-Luna Jose Luis
Published: September 2005

Proteins of the Bcl-2 family are key regulators of caspase activation and apoptosis. Some members of this family, notably Bcl-2 and Bcl-xL, are overexpressed in cancer cells, which have been associated with chemoresistance. We have designed and synthesized a small molecule inhibitor of Bcl-2, named YC137, and studied its role in cancer cells. In vitro studies showed that YC137 inhibits the binding of the Bid BH3 peptide to Bcl-2, thus disrupting an interaction essential for the antiapoptotic activity of Bcl-2. This inhibitor induces apoptosis of hematopoietic progenitors overexpressing Bcl-2 but not Bcl-xL and breast cancer cells that express high levels of Bcl-2. On the contrary, a variety of normal primary cells, including CD34+ progenitors, myoblasts, and peripheral blood mononuclear cells, do not respond to the inhibitor. A breast cancer cell line resistant to YC137 was generated. Analysis of resistant cells revealed a reduced expression of Bcl-2, which correlated with low activation of signal transducer and activator of transcription-3 (Stat3) and reduced expression of the human epidermal growth factor receptor-2 (HER2). Of note, YC137-resistant cells were more sensitive to apoptosis induced by chemotherapy. Because HER2 has not been linked previously to the Stat3-Bcl-2 transcriptional pathway, we additionally confirmed that specific blockade of HER2 in breast cancer cells resulted in down-regulation of Stat3 activity and reduced levels of Bcl-2. Consistently, HER2 blockade led to YC137 resistance. These data provide evidence for the selective killing of tumor cells by YC137 and represent the first example of in vitro selection of cancer cells refractory to a Bcl-2 inhibitor.
The Centrosomal Kinase Nek2 Displays Elevated Levels of Protein Expression in Human Breast Cancer
Authors: Hayward Daniel G. , Clarke Robert B. , Faragher Alison J. , Pillai Meenu R. , Hagan Iain M. , Fry Andrew M.
Published: September 2005

Aneuploidy and chromosome instability are common abnormalities in human cancer. Loss of control over mitotic progression, multipolar spindle formation, and cytokinesis defects are all likely to contribute to these phenotypes. Nek2 is a cell cycle-regulated protein kinase with maximal activity at the onset of mitosis that localizes to the centrosome. Functional studies have implicated Nek2 in regulation of centrosome separation and spindle formation. Here, we present the first study of the protein expression levels of the Nek2 kinase in human cancer cell lines and primary tumors. Nek2 protein is elevated 2- to 5-fold in cell lines derived from a range of human tumors including those of cervical, ovarian, breast, prostate, and leukemic origin. Most importantly, by immunohistochemistry, we find that Nek2 protein is significantly up-regulated in preinvasive in situ ductal carcinomas of the breast as well as in invasive breast carcinomas. Finally, by ectopic expression of Nek2A in immortalized HBL100 breast epithelial cells, we show that increased Nek2 protein leads to accumulation of multinucleated cells with supernumerary centrosomes. These data highlight the Nek2 kinase as novel potential target for chemotherapeutic intervention in breast cancer.
Enhanced Redundancy in Akt and Mitogen-activated Protein Kinase-induced Survival of MalignantversusNormal Prostate Epithelial Cells
Authors: Uzgare Aarti R. , Isaacs John T.
Published: September 2005

Activation of the downstream akt and mitogen-activated protein kinases is associated with development and progression of prostate cancer to the lethal androgen-independent state. However, the causal role of these downstream kinases in androgen-independent prostate cancers is unknown. In this study, activation and requirements of akt and mitogen-activated protein kinase (erk, p38, and jnk) signaling for the survival and proliferation of five malignant human cell lines encompassing the spectrum of androgen-independent prostate cancers was compared with the activation and requirements in normal prostate epithelial cells. Using Western blotting with phospho-antibodies, we detected differential activation in exponentially growing, growth factor-deprived, and restimulated cultures of malignant versus normal cells. The inhibition of erk, p38, jnk, and akt with U0126, SB203580, SP600125, and Akt inhibitor, respectively, document that normal cells require simultaneous erk and jnk signaling for survival, plus akt signaling for proliferation. In malignant cells, however, only jnk inhibition as monotherapy produces a consistent apoptotic response, although the combinatorial inhibition of jnk, erk, p38 plus akt results in statistically enhanced apoptosis. These results demonstrate that prostate cancer progression to a lethal androgen-independent state involves the acquisition of an enhanced redundancy in downstream survival signaling.
Mechanistic and Predictive Profiling of 5-Fluorouracil Resistance in Human Cancer Cells
Authors: Wang Weiguang , Cassidy James , O’Brien Vincent , Ryan Kevin M. , Collie-Duguid Elaina
Published: September 2005

Gene expression was analyzed in five pairs of 5-fluorouracil (5-FU) resistant and parental cancer cell lines on DNA microarrays. In unsupervised analysis, a prediction rule was built from the expression profiles of 29 genes, and 5-FU sensitivity class was predicted with 100% accuracy and high predictive strength. In supervised analysis of key 5-FU pathways, expression of 91 genes was associated with 5-FU sensitivity phenotype and segregated samples accordingly in hierarchical analysis. Key genes involved in 5-FU activation were significantly down-regulated (thymidine kinase, 2.9-fold; orotate phosphoribosyltransferase, 2.3-fold; uridine monophosphate kinase, 3.2-fold; pyrimidine nucleoside phosphorylase 3.6-fold) in resistant cells. Overexpression of thymidylate synthase and its adjacent gene, c-Yes, was detected in the resistant cell lines. The mRNA and protein overexpression of nuclear factor κB (NFκB) p65 and related antiapoptotic c-Flip gene was detected in resistant cells. The 5-FU-resistant cell lines also showed high NFκB DNA-binding activity. Cotransfection of NFκB p50 and p65 cDNA induced 5-FU resistance in MCF-7 cells. Both NFκB- and 5-FU-induced resistant cell lines manifested reduced expression of genes governing G1-S and S-phase transition. Expression of genes involved in DNA replication was also down-regulated in resistant cell lines. These findings were highly consistent with the slower growth rate, higher proportion of G1, and lower proportion of S-phase cells in the resistant cell lines. This phenotype may protect resistant cells from cell death induced by incorporation of 5-FU into DNA chains, by allowing time to repair 5-FU-induced damage. Our findings may provide novel targets for tackling 5-FU resistance.
Blockade of Paclitaxel-Induced Thymidine Phosphorylase Expression Can Accelerate Apoptosis in Human Prostate Cancer Cells
Authors: Kikuno Nobuyuki , Moriyama-Gonda Nobuko , Yoshino Tateki , Yoneda Tatsuaki , Urakami Shinji , Terashima Masaharu , Yoshida Manabu , Kishi Hirofumi , Shigeno Kazushi , Shiina Hiroaki , Igawa Mikio
Published: September 2005

Recently, survival benefit by chemotherapy using paclitaxel (PTX) and the induction of thymidine phosphorylase (TP) by PTX have been reported in several solid tumors. On the other hand, TP confers antiapoptotic effect on tumor cells through inhibition of caspase-8 activation in vitro. On the basis of these previous observations, we hypothesized that (a) TP can be induced after PTX treatment in human prostate cancer (PC) and (b) blockade of PTX-induced TP expression can enhance the apoptotic processes in human PC cells. PTX was used to find TP expression in all eight hormone-refractory PC cases after chemotherapy; however, cleaved caspase-8 was not expressed after chemotherapy in the six hormone-refractory PC cases with strong TP expression. In PC cell lines (PC-3, DU 145, and LNCaP), TP expression after PTX treatment was clearly up-regulated in a dose-dependent manner. Cell viability of PC cell lines treated with PTX and TP antisense was significantly reduced in a time-dependent and dose-dependent manner compared with the PTX treatment alone. Likewise, apoptotic index of PC cells treated with PTX and TP antisense was significantly increased in comparison with PTX alone. After complete blockade of PTX-induced TP translation by TP antisense transfection, cleaved form of caspase-3 and poly(ADP-ribose) polymerase was increased, and this exaggeration of apoptosis also ran parallel with caspase-8 activation in a PTX dose-dependent manner. However, in PC cell lines treated with TP antisense alone, neither caspase-3 nor poly(ADP-ribose) polymerase was cleaved despite caspase-8 activation. These results indicate that PTX-induced TP up-regulation is associated with decreased caspase-8 activation. This study is the first report showing that blockade of PTX-induced TP expression could exaggerate the processing of apoptosis in PC cells treated with PTX. Our results provide preclinical evidence that TP could be a new molecular target for enhancing the potency of PTX-mediated apoptosis in PC cells.
Hepatocyte Growth Factor/c-Met Signaling Promotes the Progression of Experimental Human Neuroblastomas
Authors: Hecht Monica , Papoutsi Maria , Tran Hoa Dinh , Wilting Joerg , Schweigerer Lothar
Published: September 2005

Neuroblastoma is the most frequent solid childhood malignancy. Despite aggressive therapy, mortality is high due to rapid tumor progression to advanced stages. The molecules and mechanisms underlying poor prognosis are not well understood. Here, we report that cultured human neuroblastoma cells express the hepatocyte growth factor (HGF) and its receptor c-Met. Binding of HGF to c-Met triggers receptor autophosphorylation, indicating functional relevance of this interaction. HGF activates several downstream effectors of c-Met such as the mitogen-activated protein kinases extracellular signal-regulated kinase 1/extracellular signal-regulated kinase 2 and phospholipase C-γ, whereas signal transducer and activator of transcription 3 is constitutively activated in neuroblastoma cells expressing c-Met. In addition, HGF is able to stimulate expression and proteolytic activity of matrix metalloproteinase-2 and tissue-type plasminogen activator in neuroblastoma cells, thereby promoting degradation of extracellular matrix components. We show that HGF stimulates invasion of neuroblastoma cells in vitro and in vivo, and it promotes the formation of angiogenic neuroblastomas in vivo. These processes can be blocked by specific inhibitors of the mitogen-activated protein kinase cascade, by inhibitors of phospholipase C-γ, and also by the expression of a dominant negative signal transducer and activator of transcription 3 mutant. Our data provide the first evidence that the HGF/c-Met pathway is essential for invasiveness and malignant progression of human neuroblastomas. They further suggest that specific inhibitors of this pathway may be suitable as therapeutic agents to improve clinical outcome of neuroblastomas.
An Epidermal Growth Factor Receptor Intron 1 Polymorphism Mediates Response to Epidermal Growth Factor Receptor Inhibitors
Authors: Amador Maria L. , Oppenheimer Darin , Perea Sofia , Maitra Anirban , Cusati George , Iacobuzio-Donahue Christi , Baker Sharyn D. , Ashfaq Raheela , Takimoto Chris , Forastiere Arlene , Hidalgo Manuel
Published: September 2005

This study tested the hypothesis that the number of CA single sequence repeat (CA-SSR) in the intron 1 of the epidermal growth factor receptor (egfr) gene, which affects transcription efficiency of the gene, is associated with the response to EGFR inhibitors. To this end, we determined the number of CA dinucleotides in the intron 1 of the egfr gene in a panel of 12 head and neck cancer cell lines that lack egfr gene amplification and measured the expression of EGFR (mRNA and protein), as well as response to EGFR inhibition. Cells with lower number of CA dinucleotides in the CA-SSR had higher expression of the EGFR gene and protein and were more sensitive to the inhibitory effects of erlotinib, a small molecule inhibitor of the EGFR tyrosine-kinase. Phenotypic modification by silencing EGFR mRNA expression in a susceptible cell line induced resistance to the drug. The number of CA dinucleotide was equivalent in genomic and tumor DNA obtained from 30 patients with head and neck cancer. In a clinical study in colorectal cancer, subjects with lower number of CA dinucleotide frequently developed skin toxicity, a feature that is related to the antitumor activity of this class of drugs. These results suggest that polymorphic variations in the intron 1 of the egfr gene is associated with response to EGFR inhibitors and may provide an explanation as to why the development of skin toxicity is associated with a favorable outcome in patients treated with these agents.
Cleavage of Lumican by Membrane-Type Matrix Metalloproteinase-1 Abrogates This Proteoglycan-Mediated Suppression of Tumor Cell Colony Formation in Soft Agar
Authors: Li Yingyi , Aoki Takanori , Mori Yuya , Ahmad Munirah , Miyamori Hisashi , Takino Takahisa , Sato Hiroshi
Published: September 2005

The small leucine-rich proteoglycan lumican was identified from a human placenta cDNA library by the expression cloning method as a gene product that interacts with membrane-type matrix metalloproteinase-1 (MT1-MMP). Coexpression of MT1-MMP with lumican in HEK293T cells reduced the concentration of lumican secreted into culture medium, and this reduction was abolished by addition of the MMP inhibitor BB94. Lumican protein from bovine cornea and recombinant lumican core protein fused to glutathione S-transferase was shown to be cleaved at multiple sites by recombinant MT1-MMP. Transient expression of lumican in HEK293 cells induced expression of tumor suppressor gene product p21/Waf-1, which was abrogated by the coexpression of MT1-MMP concomitant with a reduction in lumican concentration in culture medium. Stable expression of lumican in HeLa cells induced expression of p21 and reduction of colony formation in soft agar, which were both abolished by the expression of MT1-MMP. HT1080 fibrosarcoma cells stably transfected with the lumican cDNA (HT1080/Lum), which express endogenous MT1-MMP, secreted moderate levels of lumican; however, treatment of HT1080/Lum cells with BB94 resulted in accumulation of lumican in culture medium. The expression levels of p21 in HT1080/Lum were proportional to the concentration of secreted lumican and showed reverse corelation with colony formation in soft agar. These results suggest that MT1-MMP abrogates lumican-mediated suppression of tumor cell colony formation in soft agar by degrading this proteoglycan, which down-regulates it through the induction of p21.
Cyclooxygenase-1 Deletion Enhances Apoptosis but Does Not Protect Against Ultraviolet Light-Induced Tumors
Authors: Pentland Alice P. , Scott Glynis , VanBuskirk JoAnne , Tanck Carol , LaRossa Gina , Brouxhon Sabine
Published: September 2005

Inhibition or deletion of cyclooxygenase (COX)-2 has been demonstrated to protect against squamous cell cancer in many studies. Although much effort has focused on COX-2 inhibition, recent work indicates that COX-1 deletion may be nearly as protective. In this study, we used SKH-1 hairless mice in which COX-1 was selectively deleted to examine the role of COX-1 in photocarcinogenesis. After UV exposure, 40–60% less prostaglandin E2 was detected in COX-1−/− animals compared with wild-type (WT) controls. A 4-fold induction of keratinocyte apoptosis was observed in knockouts relative to WT animals, as documented by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling and caspase-3 staining. Proliferation was not significantly different in COX-1+/+, COX-1+/−, and COX-1−/− animals. When susceptibility to UV-induced tumor formation was studied, tumor number, average tumor size, and time of tumor onset in COX-1−/− animals were identical to WT controls. Thus, enhanced apoptosis did not alter UV-induced skin carcinogenesis, suggesting other effects are key to nonsteroidal anti-inflammatory drug chemoprevention. These results contrast sharply with data obtained using the classic 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate cancer model in which a prominent protective effect of COX-1−/− is present. The lack of protection observed here confirms cancer mechanisms are distinct in UV- and tumor promotor-induced cancer models and indicates that chemoprevention strategies must specifically address cancer causes to be effective.
Overexpression of Hyperactive Integrin-Linked Kinase Leads to Increased Cellular Radiosensitivity
Authors: Cordes Nils
Published: September 2005

Integrin-linked kinase (ILK), bound to the cytoplasmic tails of integrin β1, β2, and β3, is thought to signal through AKT and glycogen synthase kinase-3β (GSK-3β) for survival and proliferation regulation. To determine the role of ILK in the cellular radiation response, stably transfected A549 lung cancer cells overexpressing either wild-type (ILK-wk) or hyperactive ILK (ILK-hk) were studied for survival, signaling, proliferation, and examined in immunofluorescence and adhesion assays. Strong radiosensitization was observed in ILK-hk in contrast to ILK-wk mutants and empty vector controls. ILK small interfering RNA transfections showed radioresistance similar to irradiation on fibronectin. AKT, GSK-3β-cyclin D1, mitogen-activated protein kinase kinase 1/2-mitogen-activated protein kinase, and c-Jun NH2-terminal kinase signaling was dysregulated in irradiated ILK-hk mutants. Immunofluorescence stainings of ILK-hk cells indicated disturbed ILK and paxillin membrane localization with concomitant decrease in focal adhesions. Profound ILK-hk-dependent changes in morphology were characterized by spindle-like cell shape, cell size reduction, increased cell protrusions, strong formation of membranous f-actin rings, and significantly reduced adhesion to matrix proteins. Additionally, ILK-wk and ILK-hk overexpression impaired β1-integrin clustering and protein Tyr-phosphorylation. Taken together, the data provide evidence that ILK signaling modulates the cellular radiation response involving diverse signaling pathways and through changes in f-actin-based processes such as focal adhesion formation, cell adhesion, and spreading. Identification of ILK and its signaling partners as potential targets for tumor radiosensitization might promote innovative anticancer strategies by providing insight into the mechanism of cell adhesion-mediated radioresistance, oncogenic transformation, and tumor growth and spread.
Functional Attributes of Mucosal Immunity in Cervical Intraepithelial Neoplasia and Effects of HIV Infection
Authors: Kobayashi Akiko , Greenblatt Ruth M. , Anastos Kathryn , Minkoff Howard , Massad Leslie S. , Young Mary , Levine Alexandra M. , Darragh Teresa M. , Weinberg Vivian , Smith-McCune Karen K.
Published: September 2005

The role of mucosal immunity in human papillomavirus (HPV)-related cervical diseases is poorly understood. To characterize the local immune microenvironment in cervical intraepithelial neoplasia (CIN) 2/3 and determine the effects of HIV infection, we compared samples from three groups: normal cervix, CIN 2/3 from immunocompetent women (HIV− CIN 2/3), and CIN 2/3 from HIV seropositive women (HIV+ CIN 2/3). CIN 2/3 lesions contained increased numbers of immune cells from both the acquired and innate arms of the immune response in stroma [CD4+ and CD8+ T cells, macrophages, mast cells, B cells, neutrophils, and natural killer (NK) cells] and dysplastic epithelium (CD4+ T cells, macrophages, and NK cells). Immune cells in CIN 2/3 expressed activation markers, as measured by interleukin-2 receptor (IL-2R) and transcription factor T bet. Interferon-γ production was significantly up-regulated in CIN lesions and was expressed by CD4+ and CD8+ T cells and NK cells, indicating the activation of immune cells. Abundant presence of transforming growth factor-β+ CD25+ cells in the infiltrates associated with CIN lesions, and of immature CD1a+ dendritic cells expressing IL-10 and transforming growth factor-β, indicate that CIN is associated with an influx of immune cells that produce a mixture of proinflammatory and regulatory cytokines. In HIV+ CIN, immune cell densities (CD4+ T cells, macrophages, neutrophils, and NK cells) and expression of interferon-γ were significantly decreased compared with HIV− CIN. Regulatory cytokines were also down-regulated in this group. Therefore, both pro- and anti-inflammatory responses present in CIN 2/3 lesions are suppressed in HIV-seropositive women.
The Role of Defective Mismatch Repair in Small Bowel Adenocarcinoma in Celiac Disease
Authors: Potter D. Dean , Murray Joseph A. , Donohue John H. , Burgart Lawrence J. , Nagorney David M. , van Heerden Jon A. , Plevak Matthew F. , Zinsmeister Alan R. , Thibodeau Stephen N.
Published: September 2005

Celiac disease is associated with an increased risk of small bowel adenocarcinoma. The aims of this study were to investigate the molecular basis, assess outcomes, and identify clinicopathologic characteristics of small bowel adenocarcinoma in celiac disease. Retrospective case control cohort study of all celiac disease patients treated at our institution for small bowel adenocarcinoma and matched control patients with sporadic small bowel adenocarcinoma from July 1960 to November 2002. Mismatch repair (MMR) status was accessed by testing tissue for microsatellite instability (MSI) and for hMLH1 and hMSH2 protein expression. Over a 40-year time period, 18 patients with small bowel adenocarcinoma and celiac disease were treated at the Mayo Clinic. One celiac disease patient was excluded. High-frequency MSI (MSI-H) was identified in 8 of 11 (73%) and 2 of 22 (9%) available small bowel adenocarcinoma specimens in the celiac disease and control groups, respectively. In the celiac disease group, MSI-H was associated with loss of hMLH1 and hMSH2 in 6 and 1 specimens, respectively. Loss of hMLH1 occurred in both control tumors. Stage was associated with celiac disease status (P = 0.018), and 78% of controls were stage III or IV compared with 47% of celiac disease patients. Overall, survival was better (P = 0.025) in the celiac disease group compared with stage-matched controls. Celiac disease patients with small bowel adenocarcinoma had a high incidence defective MMR (73%) compared with controls and had better survival compared with stage-matched controls. In addition, celiac disease patients presented more frequently with early-stage small bowel adenocarcinoma. The better survival and earlier presentation of small bowel adenocarcinoma in celiac disease appears to be biologically associated with defective MMR.
Activation of Phosphatidylinositol 3-Kinase and Extracellular Signal-Regulated Kinase Is Required for Glial Cell Line-Derived Neurotrophic Factor-Induced Migration and Invasion of Pancreatic Carcinoma Cells
Authors: Veit Christine , Genze Felicitas , Menke Andre , Hoeffert Silke , Gress Thomas M. , Gierschik Peter , Giehl Klaudia
Published: September 2005

Pancreatic carcinoma cells exhibit a pronounced tendency to invade along and into intra- and extrapancreatic nerves, even at early stages of the disease. The neurotrophic factor glial cell line-derived neurotrophic factor (GDNF) has been shown to promote pancreatic cancer cell invasion. Here, we demonstrate that pancreatic carcinoma cell lines, such as PANC-1, expressed the RET and GDNF family receptor α receptor components for GDNF and that primary pancreatic tumor samples, derived from carcinomas with regional lymph node metastasis, exhibited marked expression of the mRNA encoding the RET51 isoform. Moreover, GDNF was an efficacious and potent chemoattractant for pancreatic carcinoma cells as examined in in vitro and in vivo model systems. Treatment of PANC-1 cells with GDNF resulted in activation of the monomeric GTPases N-Ras, Rac1, and RhoA, in activation of the mitogen-activated protein kinases extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) and in activation of the phosphatidylinositol 3-kinase/Akt pathway. Both inhibition of the Ras-Raf-MEK (mitogen-activated protein/ERK kinase)-ERK cascade by either stable expression of dominant-negative H-Ras(N17) or addition of the MEK1 inhibitor PD98059 as well as inhibition of the phosphatidylinositol 3-kinase pathway by LY294002 prevented GDNF-induced migration and invasion of PANC-1 cells. These results demonstrate that pancreatic tumor cell migration and possibly perineural invasion in response to GDNF is critically controlled by activation of the Ras-Raf-MEK-ERK and the phosphatidylinositol 3-kinase pathway.
α2HS-glycoprotein, an Antagonist of Transforming Growth Factor β
In vivo
, Inhibits Intestinal Tumor Progression
Authors: Swallow Carol J. , Partridge Emily A. , Macmillan Jennifer C. , Tajirian Tania , DiGuglielmo Gianni M. , Hay Kazy , Szweras Melanie , Jahnen-Dechent Willi , Wrana Jeff L. , Redston Mark , Gallinger Steven , Dennis James W.
Published: September 2005

Transforming growth factor (TGF)-β1 is associated with tumor progression and resistance to chemotherapy in established cancers, as well as host immune suppression. Here, we show that the serum glycoprotein α2-HS-glycoprotein (AHSG) blocks TGF-β1 binding to cell surface receptors, suppresses TGF-β signal transduction, and inhibits TGF-β-induced epithelial-mesenchymal transition, suggesting that AHSG may play a role in tumor progression. In 66 consecutive sporadic human colorectal cancer specimens, we observed a 3-fold depletion of ASHG in tumor compared with normal tissue, whereas levels of other abundant plasma proteins, albumin and transferrin, were equivalent. Using the Multiple intestinal neoplasia/+ (Min/+) mouse model of intestinal tumorigenesis, we found twice as many intestinal polyps overall, twice as many large polyps (>3 mm diameter), and more progression to invasive adenocarcinoma in Min/+ Ahsg−/− mice than in littermates expressing Ahsg. Phosphorylated Smad2 was more abundant in the intestinal mucosa and tumors of Min/+ mice lacking Ahsg, demonstrating increased TGF-β signaling in vivo. Furthermore, TGF-β-mediated suppression of immune cell function was exaggerated in Ahsg−/− animals, as shown by inhibition of macrophage activation and reduction in 12-O-tetradecanoylphorbol 13-acetate–induced cutaneous inflammation. Reconstitution of Ahsg−/− mice with bovine Ahsg suppressed endogenous TGF-β-dependent signaling to wild-type levels, suggesting that therapeutic enhancement of AHSG levels may benefit patients whose tumors are driven by TGF-β.
Retinoic Acid Increases the Expression of p53 and Proapoptotic Caspases and Sensitizes Keratinocytes to Apoptosis
Authors: Mrass Paul , Rendl Michael , Mildner Michael , Gruber Florian , Lengauer Barbara , Ballaun Claudia , Eckhart Leopold , Tschachler Erwin
Published: September 2005

Retinoids influence growth and differentiation of keratinocytes (KCs) and are widely used for the management of skin diseases and for prevention of nonmelanoma skin cancer (NMSC) in predisposed patients. Here we investigated the effect of all-trans-retinoic acid (ATRA) on KC apoptosis. When KCs were cultured in confluent monolayers for several days, they acquired resistance against UVB-induced apoptosis. In contrast, when the cells were treated with 1 μmol/L ATRA for 6 days and subsequently irradiated with different doses of UVB, they underwent massive apoptosis as assessed by morphology, expression of activated caspase-3, and DNA fragmentation. The same effect was observed when doxorubicin was used instead of UVB. Analysis by real-time PCR and Western blot revealed that ATRA treatment strongly increased the mRNA and protein expression of p53 and caspase-3, -6, -7, and -9, which are key regulators of apoptosis. UVB irradiation of ATRA-treated cells but not of control cells led to the accumulation of p53 protein and of its target gene Noxa. Inhibition of p53 and caspases with α-pifithrin and z-Val-Ala-Asp-fluoromethyl ketone, respectively, blocked UVB- and doxorubicin-induced apoptosis in ATRA-treated KCs. Analogous to the observed ATRA effects in monolayer cultures, in vitro-generated organotypic skin cultures reacted with up-regulation of p53 and proapoptotic caspases and displayed increased sensitivity to UVB-induced apoptosis. The ability of retinoic acid to regulate the expression of proapoptotic genes and to sensitize KCs to apoptosis may play a role in their prevention of NMSC in transplant patients and patients with DNA-repair deficiencies.
Transactivation of the
EGR1
Gene Contributes to Mutant p53 Gain of Function
Authors: Weisz Lilach , Zalcenstein Amir , Stambolsky Perry , Cohen Yehudit , Goldfinger Naomi , Oren Moshe , Rotter Varda
Published: September 2005

Tumor-associated mutants of the p53 tumor suppressor protein exert biological activities compatible with an oncogenic gain of function. To explore the underlying molecular mechanism, we performed microarray analysis, comparing p53-null cells to mutant p53-expressing cells. One of the genes up-regulated in the presence of mutant p53 was EGR1, a transcription factor implicated in growth control, apoptosis, and cancer. EGR1 induction by various types of stress is markedly augmented in cells expressing mutant p53. Moreover, chromatin immunoprecipitation analysis indicates that mutant p53 is physically associated with the EGR1 promoter. Functional assays indicate that induction of EGR1 by mutant p53 contributes to enhanced transformed properties and resistance to apoptosis. We propose that EGR1 is a significant contributor to mutant p53 gain of function.
Signaling and Transcriptional Changes Critical for Transformation of Human Cells by Simian Virus 40 Small Tumor Antigen or Protein Phosphatase 2A B56γ Knockdown
Authors: Moreno Carlos S. , Ramachandran Sumathi , Ashby Danita G. , Laycock Noelani , Plattner Courtney A. , Chen Wen , Hahn William C. , Pallas David C.
Published: September 2005

One set of genes sufficient for transformation of primary human cells uses the combination of Ha-Ras-V12, the telomerase catalytic subunit hTERT, SV40 large tumor antigen (LT), and SV40 small tumor antigen (ST). Whereas SV40 LT inactivates the retinoblastoma protein and p53, the contribution of ST is poorly understood. The essential helper function of ST requires a functional interaction with protein phosphatase 2A (PP2A). Here we have identified changes in gene expression induced by ST and show that ST mediates these changes through both PP2A-dependent and PP2A-independent mechanisms. Knockdown of PP2A B56γ subunit can substitute for ST expression to fully transform cells expressing LT, hTERT, and Ras-V12. We also identify those genes affected similarly in two cell lines that have been fully transformed from a common parental line by two alternative mechanisms, namely ST expression or PP2A B56γ subunit knockdown. ST altered expression of genes involved in proliferation, apoptosis, integrin signaling, development, immune responses, and transcriptional regulation. ST reduced surface expression of MHC class I molecules, consistent with a need for SV40 to evade immune detection. ST expression enabled cell cycle progression in reduced serum and src phosphorylation in anchorage-independent media, whereas B56γ knockdown required normal serum levels for these phenotypes. Inhibitors of integrin and src signaling prevented anchorage-independent growth of transformed cells, suggesting that integrin and src activation are key ST-mediated events in transformation. Our data support a model in which ST promotes survival through constitutive integrin signaling, src phosphorylation, and nuclear factor κB activation, while inhibiting cell-cell adhesion pathways.
Development of an Autocrine Neuregulin Signaling Loop with Malignant Transformation of Human Breast Epithelial Cells
Authors: Li Qunfang , Ahmed Sharlin , Loeb Jeffrey A.
Published: September 2005

Neuregulin (NRG) is a heparin-binding factor that activates members of the epidermal growth factor family of tyrosine kinase receptors including erbB2 that is overexpressed in more aggressive types of breast cancer. The exact role that NRG plays in breast cancer is complicated by the fact that NRG has been shown to have both proliferative and antiproliferative effects, depending on the breast cancer cell line used. Using an isogenic series of breast epithelial cell lines (MCF10A) ranging from benign to malignant, we found that the actions of NRG changed from antiproliferative to proliferative as the cells progress to cancer. This correlated with a progressive inability of NRG to down-regulate a group of proliferation genes identified previously using cDNA microarrays. As the cells progress to malignancy, they expressed higher levels of erbB2 and lower levels of erbB3 and secreted high levels of NRG into the culture media, resulting in high basal levels of erbB receptor phosphorylation. Disruption of this autocrine signaling loop by blocking ligand-induced receptor activation inhibited cancer cell proliferation. These results demonstrate that the transition of MCF10A cells from normal to premalignant to malignant correlates with the development of a constitutively active autocrine NRG signaling loop that promotes cell proliferation and suggest that disrupting this autocrine loop may provide an important therapeutic measure to control breast cancer cell growth.
Reduction of Human Metastatic Breast Cancer Cell Aggressiveness on Introduction of Either Form A or B of the Progesterone Receptor and Then Treatment with Progestins
Authors: Sumida Tomoki , Itahana Yoko , Hamakawa Hiroyuki , Desprez Pierre-Yves
Published: September 2005

The sex steroid hormone progesterone (Pg) is critically involved in the development of the mammary gland, and it also is thought to play a role in breast cancer progression. However, the effect of Pg on malignant phenotypes is not fully understood in breast cancer. We previously reported that in Pg receptor (PR)–positive T47D breast cancer cells, Pg was able to counterbalance the stimulatory effect of estrogen or serum on proliferation and on expression level of Id-1, which generally stimulates cell proliferation and inhibits differentiation. Conversely, metastatic MDA-MB231 breast cancer cells lack PR and express high levels of Id-1 constitutively, and Pg showed no effect on Id expression, proliferation, and invasion in these cells. However, after introducing PR (either PR-A or PR-B) into MDA-MB231 cells, Pg inhibited the expression of Id-1 mRNA drastically. PR-transfected MDA-MB231 cells exhibited less proliferative activity after Pg treatment than parental or control MDA-MB231 cells, an effect which correlated well with reduction of Id-1 mRNA. This inhibitory effect on proliferation was accompanied by p21 up-regulation and c-myc down-regulation. Moreover, Pg-treated PR transfectants showed significant morphologic change, appearing more flattened and spread out than control ethanol-treated cells. Boyden chamber invasion assay revealed that PR-transfected MDA-MB231 cells also lost most of their invasive properties after Pg treatment. Zymographic analysis revealed that Pg drastically inhibited matrix metalloproteinase-9 (MMP-9) activity in cells transfected with either PR-A or PR-B. To determine whether Id-1 could act as a key mediator of the effects of Pg, we prepared cells transfected with Id-1 and PR. The morphologic change and p21 up-regulation still were observed after Pg treatment. However, c-myc down-regulation was not observed; the proliferative and invasive activities were mostly recovered; and MMP-9 down-regulation could not be detected anymore. From these observations, we conclude that either form of the PR is sufficient to reduce the malignant phenotypes on treatment with Pg and that Id-1 plays an important role as a mediator of the effects of Pg on breast cancer cell proliferation and invasion.
Prostaglandin E2 Enhances Pancreatic Cancer Invasiveness through an Ets-1-Dependent Induction of Matrix Metalloproteinase-2
Authors: Ito Hiromichi , Duxbury Mark , Benoit Eric , Clancy Thomas E. , Zinner Michael J. , Ashley Stanley W. , Whang Edward E.
Published: September 2005

Accumulating evidence suggests an important role for cyclooxygenase-2 (COX-2) in the pathogenesis of a wide range of malignancies. Here we tested the hypothesis that the COX-2 product prostaglandin E2 (PGE2) increases cellular invasive potential by inducing matrix metalloproteinase-2 (MMP-2) expression and activity through an extracellular signal-regulated kinase (ERK)/Ets-1-dependent mechanism in pancreatic cancer. PANC-1 and MIAPaCa-2 pancreatic cancer cells were treated with PGE2 or rofecoxib, a selective COX-2 inhibitor. MMP-2 expression and activity were assayed using Western blot analysis and zymography, respectively. MMP-2 promoter activity was analyzed with a luciferase-based assay. Ets-1 activity was analyzed using gel shift assay. Ets-1 expression was specifically silenced using RNA interference. Cellular invasive and migratory potentials were determined using a Boyden chamber assay with or without Matrigel, respectively. Exogenous PGE2 induced MMP-2 expression and activity and increased ERK1/2 phosphorylation, Ets-1 binding activity, and MMP-2 promoter activity. PGE2 also increased cellular migratory and invasive potentials. The mitogen-activated protein kinase kinase inhibitor PD98059 and Ets-1 silencing each abolished PGE2-induced increases in MMP-2 expression. PD98059 and Ets-1 silencing each abrogated the effect of PGE2 on cellular invasive potential but not on cellular migratory potential. Rofecoxib suppressed MMP-2 expression and activity, Ets-1 binding activity, MMP-2 promoter activity, and cellular migratory and invasive potentials. These results suggest that PGE2 mediates pancreatic cancer cellular invasiveness through an ERK/Ets-1-dependent induction of MMP-2 expression and activity. They also suggest that COX-2 inhibition may represent a strategy to inhibit invasive potential in pancreatic cancer.