bluuebunny/crossref_metadata_2025 huggingface dataset:
Association of Breast Cancer DNA Methylation Profiles with Hormone Receptor Status and Response to Tamoxifen
Authors: Widschwendter Martin , Siegmund Kimberly D. , Müller Hannes M. , Fiegl Heidi , Marth Christian , Müller-Holzner Elisabeth , Jones Peter A. , Laird Peter W.
Published: September 2005

We have generated DNA methylation profiles of 148 human breast tumors and found significant differences in hormone receptor (HR) status between clusters of DNA methylation profiles. Of 35 DNA methylation markers analyzed, the ESR1 gene, encoding estrogen receptor α, proved to be the best predictor of progesterone receptor status, whereas methylation of the PGR gene, encoding progesterone receptor, was the best predictor of estrogen receptor status. ESR1 methylation outperformed HR status as a predictor of clinical response in patients treated with the antiestrogen tamoxifen, whereas promoter methylation of the CYP1B1 gene, encoding a tamoxifen- and estradiol-metabolizing cytochrome P450, predicted response differentially in tamoxifen-treated and nontamoxifen-treated patients. High levels of promoter methylation of the ARHI gene, encoding a RAS-related small G-protein, were strongly predictive of good survival in patients who had not received tamoxifen therapy. Our results reveal an as yet unrecognized degree of interaction between DNA methylation and HR biology in breast cancer cells and suggest potentially clinically useful novel DNA methylation predictors of response to hormonal and non-hormonal breast cancer therapy.
Integrin α5β1 Promotes Survival of Growth-Arrested Breast Cancer Cells
Authors: Korah Reju , Boots Monika , Wieder Robert
Published: September 2005

The mechanisms of long-term survival of occult breast cancer cells in the bone marrow microenvironment are not known. Using selected bone marrow stromal components with demonstrated roles in promoting growth arrest and survival of breast cancer cells, we reconstituted an in vitro model for dormancy of breast cancer cells in bone marrow. According to this model, basic fibroblast growth factor, a mammary differentiation factor abundant in the bone marrow stroma, induces growth arrest of relatively well-differentiated breast cancer cells, induces a spread appearance, and restricts their survival to fibronectin by up-regulating integrin α5β1. Most of the basic fibroblast growth factor-arrested cells fail to establish optimal ligation to fibronectin and undergo cell death. Cells that do attach to fibronectin, another major constituent of the bone marrow microenvironment, stay alive and growth-arrested for many weeks. Although capable of adhering to other stromal proteins collagen and laminin, dormant cells do not gain a survival advantage from these interactions. Using function-blocking peptides, we show a specific contribution of α5β1-fibronectin interaction in maintaining survival of growth-arrested cells, potentially by negatively modulating apoptotic response via signaling pathways. Blocking of phosphatidylinositol 3′-kinase and Akt inhibits survival of dormant clones, demonstrating this as one of those pathways. Experiments with human bone marrow stroma cocultures confirm the role of fibronectin ligation in maintaining survival of dormant clones.
A Mouse Mammary Tumor Virus-Like Long Terminal Repeat Superantigen in Human Breast Cancer
Authors: Wang Yue , Jiang Jian-Dong , Xu Dongping , Li Yan , Qu Chunfeng , Holland James F. , Pogo Beatriz G-T.
Published: September 2005

We previously reported a 660-bp mouse mammary tumor virus (MMTV)-like env gene sequence in approximately 38% of human breast cancer DNA, but not in normal breasts or other tumors. This MMTV-like env gene sequence was expressed in 66% of the env gene-positive human breast cancers. An entire proviral structure was identified in human breast cancer DNA with high homology to MMTV and low homology to known human endogenous retrovirus. MMTV-like long terminal repeat (LTR) sequences were also detected in 41.5% of human breast cancers. They contain hormone-responsive elements, TEF-1 family elements, and the open reading frame for the superantigen (SAg). We have now amplified and sequenced MMTV-like sag sequences from 10 human breast cancers, and we found that they are highly homologous to those of MMTV. However, deletions and insertions at the COOH-terminal of sag were observed. The immune function of the human MMTV-like LTR SAg was also investigated. The sag gene was cloned and expressed in a human B-cell line (Ramos). T-cell proliferation and cytokine releasing assays were performed after cocultivation of T cells with irradiated Ramos SAg-expressing cells. The results indicate that expression of the human SAg stimulates T-cell activation in vitro, as the mouse SAg does. Because the T-cell responses in vitro are considered similar to those in vivo, these results suggest that the human LTR SAg might also play a role in human breast carcinogenesis.
Overexpression, Amplification, and Androgen Regulation of TPD52 in Prostate Cancer
Authors: Rubin Mark A. , Varambally Sooryanarayana , Beroukhim Rameen , Tomlins Scott A. , Rhodes Daniel R. , Paris Pamela L. , Hofer Matthias D. , Storz-Schweizer Martina , Kuefer Rainer , Fletcher Jonathan A. , Hsi Bae-Li , Byrne Jennifier A. , Pienta Kenneth J. , Collins Colin , Sellers William R. , Chinnaiyan Arul M.
Published: September 2005

Gains in the long arm of chromosome 8 (8q) are believed to be associated with poor outcome and the development of hormone-refractory prostate cancer. Based on a meta-analysis of gene expression microarray data from multiple prostate cancer studies (D. R. Rhodes et al., Cancer Res 2002;62:4427–33), a candidate oncogene, Tumor Protein D52 (TPD52), was identified in the 8q21 amplicon. TPD52 is a coiled-coil motif-bearing protein, potentially involved in vesicle trafficking. Both mRNA and protein levels of TPD52 were highly elevated in prostate cancer tissues. Array comparative genomic hybridization and amplification analysis using single nucleotide polymorphism arrays demonstrated increased DNA copy number in the region encompassing TPD52. Fluorescence in situ hybridization on tissue microarrays confirmed TPD52 amplification in prostate cancer epithelia. Furthermore, our studies suggest that TPD52 protein levels may be regulated by androgens, consistent with the presence of androgen response elements in the upstream promoter of TPD52. In summary, these findings suggest that dysregulation of TPD52 by genomic amplification and androgen induction may play a role in prostate cancer progression.
The Nuclear Factor κB Subunits RelA/p65 and c-Rel Potentiate but Are Not Required for Ras-Induced Cellular Transformation
Authors: Hanson Julie L. , Hawke Noel A. , Kashatus David , Baldwin Albert S.
Published: September 2005

Extensive data indicate that oncoproteins, such as oncogenic H-Ras, initiate signal transduction cascades that ultimately lead to the activation of specific transcription factors. We and others have previously demonstrated that Ras activates the inherent transcriptional activation function of the transcription factor nuclear factor κB (NF-κB). Supportive of the importance of NF-κB in transformation, Ras-induced cellular transformation can be suppressed by expression of IκBα, an inhibitor of NF-κB, or by dominant-negative forms of the upstream activator IκB kinase (IKK). However, conclusive evidence for a requirement for NF-κB subunits in oncogenic transformation has not been reported. Furthermore, there is little understanding of the gene targets controlled by NF-κB that might support oncogenic conversion. The data presented here demonstrate that, although both p65 and c-Rel enhance the frequency of Ras-induced cellular transformation, these NF-κB subunits are not essential for Ras to transform spontaneously immortalized murine fibroblasts. Microarray analysis identified a set of genes induced by Ras that is dependent on NF-κB for their expression and that likely play contributory roles in promoting Ras-induced oncogenic transformation.
P27kip1 Down-Regulation Is Associated with Trastuzumab Resistance in Breast Cancer Cells
Authors: Nahta Rita , Takahashi Takeshi , Ueno Naoto T. , Hung Mien-Chie , Esteva Francisco J.
Published: September 2005

Trastuzumab (Herceptin) is a recombinant humanized monoclonal antibody directed against HER-2. The objective response rate to trastuzumab monotherapy is 12–34% for a median duration of 9 months, by which point most patients become resistant to treatment. We created two trastuzumab-resistant (TR) pools from the SKBR3 HER-2-overexpressing breast cancer cell line to study the mechanisms by which breast cancer cells escape trastuzumab-mediated growth inhibition. Both pools maintained her-2 gene amplification and protein overexpression. Resistant cells demonstrated a higher S-phase fraction by flow cytometry and a faster doubling time of 24–36 h compared with 72 h for parental cells. The cyclin-dependent kinase inhibitor p27kip1 was decreased in TR cells, and cyclin-dependent kinase 2 activity was increased. Importantly, exogenous addition of p27kip1 increased trastuzumab sensitivity. Additionally, resistant cells displayed heightened sensitivity to the proteasome inhibitor MG132, which induced p27kip1 expression. Thus, we propose that trastuzumab resistance may be associated with decreased p27kip1 levels and may be susceptible to treatments that induce p27kip1 expression.
Induction of Centrosome Amplification and Chromosome Instability in Human Bladder Cancer Cells by p53 Mutation and Cyclin E Overexpression
Authors: Kawamura Kenji , Izumi Hideki , Ma Zhiyong , Ikeda Ryosuke , Moriyama Manabu , Tanaka Tatsuro , Nojima Takayuki , Levin Linda S. , Fujikawa-Yamamoto Kohzaburo , Suzuki Koji , Fukasawa Kenji
Published: September 2005

Centrosome amplification frequently occurs in human cancers and is a major cause of chromosome instability (CIN). In mouse cells, centrosome amplification can be readily induced by loss or mutational inactivation of p53. In human cells, however, silencing of endogenous p53 alone does not induce centrosome amplification or CIN, although high degrees of correlation between p53 mutation and CIN/centrosome amplification in human cancer can be detected, suggesting the presence of additional regulatory mechanism(s) in human cells that ensures the numeral integrity of centrosomes and genomic integrity. Cyclin E, a regulatory subunit for CDK2 that plays a key role in centrosome duplication, frequently is overexpressed in human cancers. We found that cyclin E overexpression, together with loss of p53, efficiently induces centrosome amplification and CIN in human bladder cancer cells but not by either cyclin E overexpression or loss of p53 alone. We extended these findings to bladder cancer specimens and found that centrosome amplification is strongly correlated with concomitant occurrence of cyclin E overexpression and p53 inactivation but not with either cyclin E overexpression or p53 inactivation alone. Because cyclin E expression is strictly controlled in human cells compared with mouse cells, our findings suggest that this stringent regulation of cyclin E expression plays an additional role underlying numeral homeostasis of centrosomes in human cells and that deregulation of cyclin E expression, together with inactivation of p53, results in centrosome amplification.
Adenovirus Vector-Mediatedin VivoGene Transfer of OX40 Ligand to Tumor Cells Enhances Antitumor Immunity of Tumor-Bearing Hosts
Authors: Andarini Sita , Kikuchi Toshiaki , Nukiwa Mio , Pradono Prasenohadi , Suzuki Takuji , Ohkouchi Shinya , Inoue Akira , Maemondo Makoto , Ishii Naoto , Saijo Yasuo , Sugamura Kazuo , Nukiwa Toshihiro
Published: September 2005

OX40 ligand (OX40L), the ligand for OX40 on activated CD4+ T cells, has adjuvant properties for establishing effective T-cell immunity, a potent effector arm of the immune system against cancer. The hypothesis of this study is that in vivo genetic engineering of tumor cells to express OX40L will stimulate tumor-specific T cells by the OX40L-OX40 engagement, leading to an induction of systemic antitumor immunity. To investigate this hypothesis, s.c. established tumors of three different mouse cancer cells (B16 melanoma, H-2b; Lewis lung carcinoma, H-2b; and Colon-26 colon adenocarcinoma, H-2d) were treated with intratumoral injection of a recombinant adenovirus vector expressing mouse OX40L (AdOX40L). In all tumor models tested, treatment of tumor-bearing mice with AdOX40L induced a significant suppression of tumor growth along with survival advantages in the treated mice. The in vivo AdOX40L modification of tumors evoked tumor-specific cytotoxic T lymphocytes in the treated host correlated with in vivo priming of T helper 1 immune responses in a tumor-specific manner. Consistent with the finding, the antitumor effect provided by intratumoral injection of AdOX40L was completely abrogated in a CD4+ T cell-deficient or CD8+ T cell-deficient condition. In addition, ex vivo AdOX40L-transduced B16 cells also elicited B16-specific cytotoxic T lymphocyte responses, and significantly suppressed the B16 tumor growth in the immunization-challenge experiment. All of these results support the concept that genetic modification of tumor cells with a recombinant OX40L adenovirus vector may be of benefit in cancer immunotherapy protocols.
Cannabinoids Inhibit the Vascular Endothelial Growth Factor Pathway in Gliomas
Authors: Blázquez Cristina , González-Feria Luis , Álvarez Luis , Haro Amador , Casanova M. Llanos , Guzmán Manuel
Published: September 2005

Cannabinoids inhibit tumor angiogenesis in mice, but the mechanism of their antiangiogenic action is still unknown. Because the vascular endothelial growth factor (VEGF) pathway plays a critical role in tumor angiogenesis, here we studied whether cannabinoids affect it. As a first approach, cDNA array analysis showed that cannabinoid administration to mice bearing s.c. gliomas lowered the expression of various VEGF pathway-related genes. The use of other methods (ELISA, Western blotting, and confocal microscopy) provided additional evidence that cannabinoids depressed the VEGF pathway by decreasing the production of VEGF and the activation of VEGF receptor (VEGFR)-2, the most prominent VEGF receptor, in cultured glioma cells and in mouse gliomas. Cannabinoid-induced inhibition of VEGF production and VEGFR-2 activation was abrogated both in vitro and in vivo by pharmacological blockade of ceramide biosynthesis. These changes in the VEGF pathway were paralleled by changes in tumor size. Moreover, intratumoral administration of the cannabinoid Δ9-tetrahydrocannabinol to two patients with glioblastoma multiforme (grade IV astrocytoma) decreased VEGF levels and VEGFR-2 activation in the tumors. Because blockade of the VEGF pathway constitutes one of the most promising antitumoral approaches currently available, the present findings provide a novel pharmacological target for cannabinoid-based therapies.
DNA Polymerase ζ Regulates Cisplatin Cytotoxicity, Mutagenicity, and The Rate of Development of Cisplatin Resistance
Authors: Wu Fang , Lin Xinjian , Okuda Tsuyoshi , Howell Stephen B.
Published: September 2005

DNA polymerase ζ participates in translesional bypass replication. Here we show that reduced expression of the catalytic subunit hREV3 renders human fibroblasts more sensitive to the cytotoxic effect of cisplatin, reduces their sensitivity to the ability of cisplatin exposure to generate drug resistant variants in the surviving population, and reduces the rate of emergence of resistance to cisplatin at the population level. Reduction of REV3 mRNA did not alter the rate of cisplatin adduct removal but did impair both spontaneous and cisplatin-induced extrachromosomal homologous recombination and attenuated bypass replication as reflected by reduced ability to express luciferase from a platinated plasmid. Cisplatin induced a concentration- and time-dependent increase in hREV3 mRNA. The results indicate that, following formation of cisplatin adducts in DNA, REV3 mRNA levels increase, and polymerase ζ functions to promote both cell survival and the generation of drug-resistant variants in the surviving population. We conclude that when cisplatin adducts are present in the DNA, polymerase ζ is an important contributor to cisplatin-induced genomic instability and the subsequent emergence of resistance to this chemotherapeutic agent.
Inhibition of Breast Cancer Metastasis by Selective Synthetic Polypeptide against CXCR4
Authors: Liang Zhongxing , Wu Tao , Lou Hong , Yu Xiwen , Taichman Russell S. , Lau Stephen K. , Nie Shuming , Umbreit Jay , Shim Hyunsuk
Published: September 2005

Metastasis shares many similarities with leukocyte trafficking. Among those chemokine receptors thought to be involved in hemopoietic cell homing, stromal cell-derived factor-1 and its receptor CXC chemokine receptor-4 (CXCR4) have received considerable attention. Like hemopoietic cell homing, levels of stromal cell-derived factor-1 are high at sites of breast cancer metastasis including lymph node, lung, liver, and the marrow. Moreover, CXCR4 expression is low in normal breast tissues and high in malignant tumors, suggesting that a blockade of CXCR4 might limit tumor metastasis. We therefore investigated the role of a synthetic antagonist 14-mer peptide (TN14003) in inhibiting metastasis in an animal model. Not only was TN14003 effective in limiting metastasis of breast cancer by inhibiting migration, but it may also prove useful as a diagnostic tool to identify CXCR4 receptor-positive tumor cells in culture and tumors in paraffin-embedded clinical samples.
Activated Signal Transducer and Activator of Transcription (STAT) 3
Authors: Silver Debra L. , Naora Honami , Liu Jinsong , Cheng Wenjun , Montell Denise J.
Published: September 2005

Constitutive activation of the Janus-activated kinase/signal transducer and activator of transcription (STAT) pathway promotes the proliferation and survival of cancer cells in culture and is associated with various cancers, including those of the ovary. We found that constitutively activated STAT3 levels correlated with aggressive clinical behavior of ovarian carcinoma specimens. Furthermore, inhibition of STAT3 reduced the motility of ovarian cancer cells in vitro. Surprisingly, we found that activated STAT3 localized not only to nuclei but also to focal adhesions in these cells. Activated STAT3 coimmunoprecipitated with phosphorylated paxillin and focal adhesion kinase and required paxillin and Src for its localization to focal adhesions. These results suggest that Janus-activated kinase/STAT signaling may contribute to ovarian cancer cell invasiveness.
A Novel Synthetic Inhibitor of CDC25 Phosphatases
Authors: Brezak Marie-Christine , Quaranta Muriel , Mondésert Odile , Galcera Marie-Odile , Lavergne Olivier , Alby Frédéric , Cazales Martine , Baldin Véronique , Thurieau Christophe , Harnett Jeremiath , Lanco Christophe , Kasprzyk Philip G. , Prevost Gregoire P. , Ducommun Bernard
Published: September 2005

CDC25 dual-specificity phosphatases are essential regulators that dephosphorylate and activate cyclin-dependent kinase/cyclin complexes at key transitions of the cell cycle. CDC25 activity is currently considered to be an interesting target for the development of new antiproliferative agents. Here we report the identification of a new CDC25 inhibitor and the characterization of its effects at the molecular and cellular levels, and in animal models.

BN82002 inhibits the phosphatase activity of recombinant human CDC25A, B, and C in vitro. It impairs the proliferation of tumoral cell lines and increases cyclin-dependent kinase 1 inhibitory tyrosine phosphorylation. In synchronized HeLa cells, BN82002 delays cell cycle progression at G1-S, in S phase and at the G2-M transition. In contrast, BN82002 arrests U2OS cell cycle mostly in the G1 phase. Selectivity of this inhibitor is demonstrated: (a) by the reversion of the mitotic-inducing effect observed in HeLa cells upon CDC25B overexpression; and (b) by the partial reversion of cell cycle arrest in U2OS expressing CDC25. We also show that BN82002 reduces growth rate of human tumor xenografts in athymic nude mice.

BN82002 is a original CDC25 inhibitor that is active both in cell and animal models. This greatly reinforces the interest in CDC25 as an anticancer target.
Bryostatin-1 Enhances the Maturation and Antigen-Presenting Ability of Murine and Human Dendritic Cells
Authors: Do Yoonkyung , Hegde Venkatesh L. , Nagarkatti Prakash S. , Nagarkatti Mitzi
Published: September 2005

In this study, we investigated the effect of bryostatin-1 (Bryo-1), an antineoplastic agent, on dendritic cell (DC) maturation, activation, and functions. Murine bone marrow-derived DCs on culture with Bryo-1 alone, Bryo-1 + calcium ionophore (CI), but not CI alone exhibited morphologic changes characteristic of mature DCs and expressed increased levels of CD40, CD80, and CD86. Moreover, Bryo-1 + CI–treated DCs exhibited enhanced antigen-presenting ability to naive and antigen-specific T cells and alloreactive T cells. Bryo-1 + CI–mediated activation of DCs involved protein kinase C (PKC), especially PKC-α, -δ, and -ι, and addition of PKC inhibitors impaired their ability to activate T cells. Bryo-1 + CI treatment of DCs did not activate mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase, p38 MAPK, or stress-activated protein kinase/c-Jun NH2-terminal kinase pathways. Finally, treatment of DCs with Bryo-1 alone and Bryo-1 + CI, but not CI alone, induced nuclear translocation of nuclear factor κB as studied by confocal microscopy. DCs generated from human peripheral blood monocytes or from human cord blood CD34+ hematopoietic stem cells, when cultured with Bryo-1 + CI, also showed maturation and increased T-cell stimulatory activity. Bryo-1 + CI was more potent in inducing maturation and activation of DCs when compared with other agents such as tumor necrosis factor α, lipopolysaccharide, or phorbol 12-myristate 13acetate + CI. Collectively, the current study shows for the first time that Bryo-1 alone or in combination with CI may promote the maturation of DCs and therefore may be useful in development of DC-based cancer immunotherapy.
Fluoxetine Inhibits Multidrug Resistance Extrusion Pumps and Enhances Responses to Chemotherapy in Syngeneic and in Human Xenograft Mouse Tumor Models
Authors: Peer Dan , Dekel Yaron , Melikhov Dina , Margalit Rimona
Published: September 2005

Multidrug resistance (MDR) operated by extrusion pumps such as P-glycoprotein and multidrug-resistance-associated-proteins, is a major reason for poor responses and failures in cancer chemotherapy. MDR modulators (chemosensitizers) were found among drugs approved for noncancer indications and their derivatives. Yet toxicity, adverse effects, and poor solubility at doses required for MDR reversal prevent their clinical application. Among newly designed chemosensitizers, some still suffer from toxicity and adverse effects, whereas others progressed to clinical trials. Diversities among tumors and among MDR pumps indicate a need for several clinically approved MDR modulators. Here we report for the first time that fluoxetine (Prozac), the well-known antidepressant, is a highly effective chemosensitizer. In vitro, fluoxetine enhanced (10- to 100-fold) cytotoxicity of anticancer drugs (doxorubicin, mitomycin C, vinblastine, and paclitaxel) in drug-resistant but not in drug-sensitive cells (5 and 3 lines, respectively). Fluoxetine increased drug accumulation within MDR-cells and inhibited drug efflux from those cells. In vivo, fluoxetine enhanced doxorubicin accumulation within tumors (12-fold) with unaltered pharmacokinetics. In four resistant mouse tumor models of both syngeneic and human xenograft, combination treatment of fluoxetine and doxorubicin generated substantial (P < 0.001) improvements in tumor responses and in survivals (2- to 3-fold). Moreover, fluoxetine reversed MDR at doses that are well below its human safety limits, free of the severe dose-related toxicity, adverse effects, and poor solubility that are obstacles to other chemosensitizers. This low-dose range, together with the findings reported here, indicate that fluoxetine has a high potential to join the arsenal of MDR reversal agents that may reach the clinic.
A Mouse Skin Multistage Carcinogenesis Model Reflects the Aberrant DNA Methylation Patterns of Human Tumors
Authors: Fraga Mario F. , Herranz Michel , Espada Jesús , Ballestar Esteban , Paz Maria F. , Ropero Santiago , Erkek Emel , Bozdogan Onder , Peinado Héctor , Niveleau Alain , Mao Jian-Hua , Balmain Alan , Cano Amparo , Esteller Manel
Published: September 2005

Whereas accepted models of tumorigenesis exist for genetic lesions, the timing of epigenetic alterations in cancer is not clearly understood. We have analyzed the profile of aberrations in DNA methylation occurring in cells lines and primary tumors of one of the best-characterized mouse carcinogenesis systems, the multistage skin cancer progression model. Initial analysis using high-performance capillary electrophoresis and immunolocalization revealed a loss of genomic 5-methylcytosine associated with the degree of tumor aggressiveness. Paradoxically, this occurs in the context of a growing number of hypermethylated CpG islands of tumor suppressor genes at the most malignant stages of carcinogenesis. We have observed this last phenomenon using two approaches, a candidate gene approach, studying genes with well-known methylation-associated silencing in human tumors, and a mouse cDNA microarray expression analysis after treatment with DNA demethylating drugs. The transition from epithelial to spindle cell morphology is particularly associated with major epigenetic alterations, such as E-cadherin methylation, demethylation of the Snail promoter, and a decrease of the global DNA methylation. Analysis of data obtained from the cDNA microarray strategy led to the identification of new genes that undergo methylation-associated silencing and have growth-inhibitory effects, such as the insulin-like growth factor binding protein-3. Most importantly, all of the above genes were also hypermethylated in human cancer cell lines and primary tumors, underlining the value of the mouse skin carcinogenesis model for the study of aberrant DNA methylation events in cancer cells.
Mechanism of the Pharmacokinetic Interaction between Methotrexate and Benzimidazoles
Authors: Breedveld Pauline , Zelcer Noam , Pluim Dick , Sönmezer Özgür , Tibben Matthijs M. , Beijnen Jos H. , Schinkel Alfred H. , van Tellingen Olaf , Borst Piet , Schellens Jan H. M.
Published: September 2005

The antifolate drug methotrexate (MTX) is transported by breast cancer resistance protein (BCRP; ABCG2) and multidrug resistance-associated protein1–4 (MRP1–4; ABCC1–4). In cancer patients, coadministration of benzimidazoles and MTX can result in profound MTX-induced toxicity coinciding with an increase in the serum concentrations of MTX and its main metabolite 7-hydroxymethotrexate. We hypothesized that benzimidazoles interfere with the clearance of MTX and/or 7-hydroxymethotrexate by inhibition of the ATP-binding cassette drug transporters BCRP and/or MRP2, two transporters known to transport MTX and located in apical membranes of epithelia involved in drug disposition. First, we investigated the mechanism of interaction between benzimidazoles (pantoprazole and omeprazole) and MTX in vitro in membrane vesicles from Sf9 cells infected with a baculovirus containing human BCRP or human MRP2 cDNA. In Sf9-BCRP vesicles, pantoprazole and omeprazole inhibited MTX transport (IC50 13 μm and 36 μm, respectively). In Sf9-MRP2 vesicles, pantoprazole did not inhibit MTX transport and at high concentrations (1 mm), it even stimulated MTX transport 1.6-fold. Secondly, we studied the transport of pantoprazole in MDCKII monolayers transfected with mouse Bcrp1 or human MRP2. Pantoprazole was actively transported by Bcrp1 but not by MRP2. Finally, the mechanism of the interaction was studied in vivo using Bcrp1−/− mice and wild-type mice. Both in wild-type mice pretreated with pantoprazole to inhibit Bcrp1 and in Bcrp1−/− mice that lack Bcrp1, the clearance of i.v. MTX was decreased significantly 1.8- to 1.9-fold compared with the clearance of i.v. MTX in wild-type mice. The conclusion is as follows: benzimidazoles differentially affect transport of MTX mediated by BCRP and MRP2. Competition for BCRP may explain the clinical interaction between MTX and benzimidazoles.
From the Cyclooxygenase-2 Inhibitor Celecoxib to a Novel Class of 3-Phosphoinositide-Dependent Protein Kinase-1 Inhibitors
Authors: Zhu Jiuxiang , Huang Jui-Wen , Tseng Ping-Hui , Yang Ya-Ting , Fowble Joseph , Shiau Chung-Wai , Shaw Yeng-Jeng , Kulp Samuel K. , Chen Ching-Shih
Published: September 2005

The blockade of Akt activation through the inhibition of 3-phosphoinositide-dependent kinase-1 (PDK-1) represents a major signaling mechanism whereby celecoxib mediates apoptosis. Celecoxib, however, is a weak PDK-1 inhibitor (IC50, 48 μm), requiring at least 30 μm to exhibit discernable effects on the growth of tumor cells in vitro. Here, we report the structure-based optimization of celecoxib to develop PDK-1 inhibitors with greater potency in enzyme inhibition and growth inhibition. Kinetics of PDK-1 inhibition by celecoxib with respect to ATP suggest that celecoxib derivatives inhibit PDK-1 by competing with ATP for binding, a mechanism reminiscent to that of many kinase inhibitors. Structure-activity analysis together with molecular modeling was used to generate compounds that were tested for their potency in inhibiting PDK-1 kinase activity and in inducing apoptosis in PC-3 prostate cancer cells. Docking of potent compounds into the ATP-binding site of PDK-1 was performed for lead optimization, leading to two compounds, OSU-03012 and OSU-03013, with IC50 values in PDK-1 inhibition and apoptosis induction in the low μm range. Exposure of PC-3 cells to these agents led to Akt dephosphorylation and inhibition of p70 S6 kinase activity. Moreover, overexpression of constitutively active forms of PDK-1 and Akt partially protected OSU-03012-induced apoptosis. Screening in a panel of 60 cell lines and more extensive testing in PC-3 cells indicated that the mean concentration for total growth inhibition was ∼3 μm for both agents. Considering the conserved role of PDK-1/Akt signaling in promoting tumorigenesis, these celecoxib analogs are of translational relevance for cancer prevention and therapy.
Reduced Infiltration of Class A Scavenger Receptor Positive Antigen-Presenting Cells Is Associated with Prostate Cancer Progression
Authors: Yang Guang , Addai Josephine , Tian Wei-hua , Frolov Anna , Wheeler Thomas M. , Thompson Timothy C.
Published: September 2005

The class A macrophage scavenger receptor (SR-A) is expressed in antigen presenting cells and is involved in host immune responses. Germ-line mutation of this gene has been associated with increased risk of human prostate cancer. However, there is little known about its expression in normal or neoplastic human prostate tissues. Double immunofluorescent labeling with monoclonal antibodies to SR-A and specific macrophage and dendritic cell markers was used to identify cells expressing SR-A in human prostate tissues. SR-A immunohistochemical staining was performed on paraffin sections of normal prostate, prostatic intraepithelial neoplasia (PIN) lesions, and prostate cancers from radical prostatectomy specimens. SR-A was expressed in a subset of macrophages and dendritic cells that infiltrated prostatic tissues. The majority of SR-A-positive cells coexpressed CD68, and a relatively low percentage expressed S100 protein. The number of SR-A-positive cells was significantly increased in PIN as compared with normal prostatic tissue (P = 0.0176). In contrast, the number of SR-A-positive cells decreased with tumor progression. A lower SR-A-positive cell density was associated with higher clinical stage (ρ = −0.26; P = 0.0234). Inverse associations were also found between SR-A density and positive lymph nodes (ρ = −0.23; P = 0.0437), tumor size (ρ = −0.31; P = 0.0100) and preoperative PSA levels (ρ = −0.32; P = 0.0057). SR-A density is a significant predictor of disease-free survival after surgery univariately (P = 0.0003), as well as multivariately, adjusted for known clinical and pathological markers including preoperative prostate-specific antigen, clinical stage, Gleason score, surgical margin, extraprostatic extension, and seminal vesicle invasion, as well as lymph node metastasis (P = 0.0021). The preferential accumulation of SR-A-positive cells in PIN suggests a role for SR-A in the APC response to early malignancy. A reduction in the number of SR-A-positive cells demarcates tumor progression as indicated by clinical and pathological correlations. Our results additionally indicate that systematic measurement of SR-A density is a strong prognostic marker for clinical outcome after surgery.
Hypermethylation of a Small CpGuanine-Rich Region Correlates with Loss of Activator Protein-2α Expression during Progression of Breast Cancer
Authors: Douglas Donna B. , Akiyama Yoshimitsu , Carraway Hetty , Belinsky Steven A. , Esteller Manel , Gabrielson Edward , Weitzman Sigmund , Williams Trevor , Herman James G. , Baylin Stephen B.
Published: September 2005

The transcription factor activator protein-2α (AP-2α) has recently been implicated as a tumor suppressor protein that can be lost during tumor progression and that exhibits growth-inhibitory properties when overexpressed in cancer cell lines. We now demonstrate that hypermethylation of a discrete 5′ region within a promoter CpG island of the gene is associated in breast cancer with the loss of AP-2α expression. Multiple CpG sites within the island become hypermethylated during breast cancer evolution. However, only hypermethylation of the most CpG-rich region, a small, ∼300-bp area at the 3′ end of exon 1, fully distinguishes neoplastic from normal breast tissue and correlates with transcriptional silencing. In cell culture, silenced AP-2α, associated with exon 1 hypermethylation, is re-expressed by 5-aza-2′deoxycytidine resulting in the restoration of a functional DNA sequence-specific binding protein. In vivo, as detected by a very sensitive nested PCR approach, methylation of the discrete AP-2α exon 1 region does not occur in normal breast epithelium and occurs in only 3 (16%) of 19 ductal carcinoma in situ (DCIS) lesions, but is present in 12 (75%) of 16 invasive breast tumors (P < 0.001; DCIS versus invasive cancers). Tumors unmethylated for this region expressed AP-2α protein throughout, whereas tumors with hypermethylation showed large areas of loss. Our studies then determine that hypermethylation of a small region of a CpG island correlates with silencing of AP-2α in breast cancer and suggest that inactivation of this gene could be a factor in, and a useful marker for, the progression of DCIS lesions.
Targeting Endogenous Transforming Growth Factor β Receptor Signaling in SMAD4-Deficient Human Pancreatic Carcinoma Cells Inhibits Their Invasive Phenotype
Authors: Subramanian Gayathri , Schwarz Roderich E. , Higgins Linda , McEnroe Glenn , Chakravarty Sarvajit , Dugar Sundeep , Reiss Michael
Published: September 2005

Transforming growth factor-β (TGF-β) suppresses tumor formation by blocking cell cycle progression and maintaining tissue homeostasis. In pancreatic carcinomas, this tumor suppressive activity is often lost by inactivation of the TGF-β-signaling mediator, Smad4. We found that human pancreatic carcinoma cell lines that have undergone deletion of MADH4 constitutively expressed high endogenous levels of phosphorylated receptor-associated Smad proteins (pR-Smad2 and pR-Smad3), whereas Smad4-positive lines did not. These elevated pR-Smad levels could not be attributed to a decreased dephosphorylation rate nor to increased expression of TGF-β type I (TβR-I) or type II (TβR-II) receptors. Although minimal amounts of free bioactive TGF-β1 and TGF-β2 were detected in conditioned medium, treatment with a pan-specific (but not a TGF-β3 specific) TGF-β-neutralizing antibody and with anti-αVβ6 integrin antibody decreased steady-state pSmad2 levels and activation of a TGF-β-inducible reporter gene in neighboring cells, respectively. Thus, activation of TGF-β at the cell surface was responsible for the increased autocrine endogenous and paracrine signaling. Blocking TβR-I activity using a selective kinase inhibitor (SD-093) strongly decreased the in vitro motility and invasiveness of the pancreatic carcinoma cells without affecting their growth characteristics, morphology, or the subcellular distribution of E-cadherin and F-actin. Moreover, exogenous TGF-β strongly stimulated in vitro invasiveness of BxPC-3 cells, an effect that could also be blocked by SD-093. Thus, the motile and invasive properties of Smad4-deficient pancreatic cancer cells are at least partly driven by activation of endogenous TGF-β signaling. Therefore, targeting the TβR-I kinase represents a potentially powerful novel therapeutic approach for the treatment of this disease.
Galectin-1(L11A) Predicted from a Computed Galectin-1 Farnesyl-Binding Pocket Selectively Inhibits Ras-GTP
Authors: Rotblat Barak , Niv Hagit , André Sabine , Kaltner Herbert , Gabius Hans-Joachim , Kloog Yoel
Published: September 2005

Ras biological activity necessitates membrane anchorage that depends on the Ras farnesyl moiety and is strengthened by Ras/galectin-1 interactions. We identified a hydrophobic pocket in galectin-1, analogous to the Cdc42 geranylgeranyl-binding cavity in RhoGDI, possessing homologous isoprenoid-binding residues, including the critical L11, whose RhoGDI L77 homologue changes dramatically on Cdc42 binding. By substituting L11A, we obtained a dominant interfering galectin-1 that possessed normal carbohydrate-binding capacity but inhibited H-Ras GTP-loading and extracellular signal-regulated kinase activation, dislodged H-Ras(G12V) from the cell membrane, and attenuated H-Ras(G12V) fibroblast transformation and PC12-cell neurite outgrowth. Thus, independently of carbohydrate binding, galectin-1 cooperates with Ras, whereas galectin-1(L11A) inhibits it.
Smad-Binding Defective Mutant of Transforming Growth Factor β Type I Receptor Enhances Tumorigenesis but Suppresses Metastasis of Breast Cancer Cell Lines
Authors: Tian Fang , Byfield Stacey DaCosta , Parks W. Tony , Stuelten Christina H. , Nemani Deepa , Zhang Ying E. , Roberts Anita B.
Published: September 2005

The role of transforming growth factor β (TGF-β) in carcinogenesis is complex, with tumor suppressor and pro-oncogenic activities depending on the particular tumor cell and its stage in malignant progression. We previously have demonstrated in breast cancer cell lines that Smad2/3 signaling played a dominant role in mediating tumor suppressor effects on well-differentiated breast cancer cell lines grown as xenografts and prometastatic effects on a more invasive, metastatic cell line. Our present data based on selective interference with activation of endogenous Smad2 and Smad3 by stable expression of a mutant form of the TGF-β type I receptor (RImL45) unable to bind Smad2/3 but with a functional kinase again show that reduction in Smad2/3 signaling by expression of RImL45 enhanced the malignancy of xenografted tumors of the well-differentiated MCF10A-derived tumor cell line MCF10CA1h, resulting in formation of larger tumors with a higher proliferative index and more malignant histologic features. In contrast, expression of RImL45 in the more aggressive MCF10CA1a cell line strongly suppressed formation of lung metastases following tail vein injection. These results suggest a causal, dominant role for the endogenous Smad2/3 signaling pathway in the tumor suppressor and prometastatic activities of TGF-β in these cells. Using an in vitro assay, we further show that non-Smad signaling pathways, including p38 and c-Jun NH2-terminal kinase, cooperate with TGF-β/Smads in enhancing migration of metastatic MCF10CA1a cells, but that, although necessary for migration, these other pathways are not sufficient for metastasis.
The Increased Expression of Peroxisome Proliferator-Activated Receptor-γ1 in Human Breast Cancer Is Mediated by Selective Promoter Usage
Authors: Wang Xin , Southard R. Chase , Kilgore Michael W.
Published: September 2005

Peroxisome proliferator-activated receptor-γ1 (PPARγ1) is transactivated by a wide range of ligands in normal human mammary epithelial and breast cancer cells. Although transactivation of PPARγ mediates the expression of genes that are markers of differentiation, its overexpression in cancers of the breast, thyroid, colon, and lung suggests its dysregulation may play a role in oncogenesis, cancer progression, or both. We report the overexpression of PPARγ is caused by the use of a tumor-specific promoter in breast cancer cells that is distinct from the promoter used in normal epithelia. Thus, the increase in PPARγ expression seen in breast cancer cells results from promoter recruitment, providing new insights into the expression and actions of PPARγ in breast cancer.
Spermine Acts as a Negative Regulator of Macrophage Differentiation in Human Myeloid Leukemia Cells
Authors: Gavin Igor M. , Glesne David , Zhao Yong , Kubera Cathryn , Huberman Eliezer
Published: September 2005

The role of putrescine, spermidine and spermine in phorbol 12-myristate-13-acetate (PMA)-induced macrophage differentiation was examined in human HL-60 and U-937 myeloid leukemia cells. Unlike other polyamines, spermine affected this differentiation by acting as a negative regulator. This negative regulation was established by showing that the PMA-induced macrophage phenotype, but not PMA-associated replication arrest, was abrogated (a) by replenishing the PMA-evoked decrease in cellular spermine levels with this polyamine from an exogenous source and (b) by blocking PMA-induced expression of the polyamine catabolic enzyme N1-spermidine/spermine acetyltransferase (SSAT) with antisense oligonucleotides in the presence of low substrate level. The PMA-evoked reduction in cellular spermine appears to result from an increase in the activity of SSAT and a decrease in the activity of ornithine decarboxylase, the polyamine biosynthetic enzyme. To a degree, these changes are due to corresponding changes in the expression of the genes that code for these enzymes. When cell differentiation is initiated, SSAT expression is increased after PMA-evoked activation of protein kinase C-β. The present studies raise the possibility that agents able to reduce spermine levels in patients’ myeloid leukemia cells may enhance the activity of differentiation therapy drugs for this type of leukemia.
Synergistic Activation of Innate Immunity by Double-Stranded RNA and CpG DNA Promotes Enhanced Antitumor Activity
Authors: Whitmore Mark M. , DeVeer Michael J. , Edling Andrea , Oates Rhonda K. , Simons Brenna , Lindner Daniel , Williams Bryan R. G.
Published: September 2005

Double-stranded RNA (dsRNA) and unmethylated CpG sequences in DNA are pathogen-associated molecular patterns of viruses and bacteria that activate innate immunity. To examine whether dsRNA and CpG DNA could combine to provide enhanced stimulation of innate immune cells, murine macrophages were stimulated with poly-rI:rC (pIC), a dsRNA analog, and CpG-containing oligodeoxynucleotides (CpG-ODN). Combined treatments demonstrated synergy in nitric oxide, interleukin (IL)-12, tumor necrosis factor α, and IL-6 production. Studies using neutralizing antibodies for type I interferons (IFNs), IFN-α and IFN-β, indicated that nitric oxide synthase synergism is mediated by paracrine/autocrine effects of IFN-β. In contrast, enhanced cytokine production occurred independent of type I IFN and was maintained in macrophages from IFN-α/β receptor knockout mice. Cotransfection of human Toll-like receptors 3 and 9 (receptors for dsRNA and CpG DNA, respectively) into 293T cells supported synergistic activation of an IL-8 promoter reporter construct by pIC, indicating interaction of the signaling pathways in driving the synergy response. In vivo stimulation of mice with pIC and CpG-ODN demonstrated synergy for serum IL-6 and IL-12p40 levels that correlated with an enhanced antitumor effect against established B16-F10 experimental pulmonary metastases. Treatment of tumor-bearing mice with pIC and CpG-ODN in combination resulted in enhanced nitric oxide synthase expression in lung tissue and enhanced up-regulation of class I major histocompatibility complex on splenic dendritic cells relative to treatments with either agent alone. In conclusion, the combined detection of viral pathogen-associated molecular patterns, i.e., dsRNA and CpG DNA, may mimic definitive viral recognition, resulting in an enhanced innate immune response that could be used for tumor vaccination or immunotherapy.
Therapeutic Effectiveness of Recombinant Cancer Vaccines Is Associated with a Prevalent T-Cell Receptor α Usage by Melanoma-specific CD8+ T Lymphocytes
Authors: De Palma Raffaele , Marigo Ilaria , Del Galdo Francesco , De Santo Carmela , Serafini Paolo , Cingarlini Sara , Tüting Thomas , Lenz Julia , Basso Giuseppe , Milan Gabriella , Zanovello Paola , Bronte Vincenzo
Published: September 2005

Definition of immune variables that correlate with the antitumor activity of cancer vaccines is critical for monitoring immunotherapy protocols. To define surrogate end points predictive of the therapeutic efficacy of recombinant vaccines based on melanoma antigen tyrosinase-related protein (TRP)-2, we evaluated several properties of antigen-specific CD8+ T lymphocytes in single mice undergoing either prophylactic or therapeutic immunization. Predictive markers for the efficacy of genetic vaccination were identified in the prophylactic model used. Interestingly, the number of tetramer+ CD8+ T lymphocytes expanded in vitro after a single cycle of stimulation with the immunodominant TRP-2 peptide was of the highest predictive value. In the therapeutic model, no variable examined at a single mouse level predicted the long-term therapeutic effect. Mice that survived did not show the highest expansion of antigen-specific lymphocytes or the more functionally active effectors, ex vivo or after in vitro culture with the peptide antigen. Successful therapy correlated strictly with the skewing of the T-cell receptor repertoire of tetramer-sorted, TRP-2–specific CD8+ T lymphocytes, which showed a preferential α chain usage with a common CDR3 region.
Integrin Fibronectin Receptors in Matrix Metalloproteinase-1–Dependent Invasion by Breast Cancer and Mammary Epithelial Cells
Authors: Jia Yifeng , Zeng Zhao-Zhu , Markwart Sonja M. , Rockwood Korrene F. , Ignatoski Kathleen M. Woods , Ethier Stephen P. , Livant Donna L.
Published: September 2005

Integrins contribute to progression in many cancers, including breast cancer. For example, the interaction of α5β1 with plasma fibronectin causes the constitutive invasiveness of human prostate cancer cells. Inhibition of this process reduces tumorigenesis and prevents metastasis and recurrence. In this study, naturally serum-free basement membranes were used as invasion substrates. Immunoassays were used to compare the roles of α5β1 and α4β1 fibronectin receptors in regulating matrix metalloproteinase (MMP)-1–dependent invasion by human breast cancer and mammary epithelial cells. We found that a peptide consisting of fibronectin PHSRN sequence, Ac-PHSRN-NH2, induces α5β1-mediated invasion of basement membranes in vitro by human breast cancer and mammary epithelial cells. PHSRN-induced invasion requires interstitial collagenase MMP-1 activity and is suppressed by an equimolar concentration of a peptide consisting of the LDV sequence of the fibronectin connecting segment, Ac-LHGPEILDVPST-NH2, in mammary epithelial cells, but not in breast cancer cells. This sequence interacts with α4β1, an integrin that is often down-regulated in breast cancer cells. Immunoblotting shows that the PHSRN peptide stimulates MMP-1 production by serum-free human breast cancer and mammary epithelial cells and that the LDV peptide represses PHSRN-stimulated MMP-1 production only in mammary epithelial cells. Furthermore, PHSRN stimulates MMP-1 activity in breast cancer cells and mammary epithelial cells with a time course that closely parallels invasion induction. Thus, down-regulation of surface α4β1 during oncogenic transformation may be crucial for establishment of the α5β1-induced, MMP-1–dependent invasive phenotype of breast cancer cells.