bluuebunny/crossref_metadata_2025 huggingface dataset:
Nitrosoureas Inhibit the Stathmin-Mediated Migration and Invasion of Malignant Glioma Cells
Authors: Liang Xing-Jie , Choi Yong , Sackett Dan L. , Park John K.
Published: July 2008

Malignant gliomas are the most common primary intrinsic brain tumors and are highly lethal. The widespread migration and invasion of neoplastic cells from the initial site of tumor formation into the surrounding brain render these lesions refractory to definitive surgical treatment. Stathmin, a microtubule-destabilizing protein that mediates cell cycle progression, can also regulate directed cell movement. Nitrosoureas, traditionally viewed as DNA alkylating agents, can also covalently modify proteins such as stathmin. We therefore sought to establish a role for stathmin in malignant glioma cell motility, migration, and invasion and determine the effects of nitrosoureas on these cell movement–related processes. Scratch wound–healing recovery, Boyden chamber migration, Matrigel invasion, and organotypic slice invasion assays were performed before and after the down-regulation of cellular stathmin levels and in the absence and presence of sublethal nitrosourea ([1-(2-chloroethyl)-3-cyclohexyl-l-nitrosourea]; CCNU) concentrations. We show that decreases in stathmin expression lead to significant decreases in malignant glioma cell motility, migration, and invasion. CCNU, at a concentration of 10 μmol/L, causes similar significant decreases, even in the absence of any effects on cell viability. The direct inhibition of stathmin by CCNU is likely a contributing factor. These findings suggest that the inhibition of stathmin expression and function may be useful in limiting the spread of malignant gliomas within the brain, and that nitrosoureas may have therapeutic benefits in addition to their antiproliferative effects. [Cancer Res 2008;68(13):5267–72]
15-Hydroxyprostaglandin Dehydrogenase is a Target of Hepatocyte Nuclear Factor 3β and a Tumor Suppressor in Lung Cancer
Authors: Huang Guosheng , Eisenberg Rosana , Yan Min , Monti Stefano , Lawrence Earl , Fu Pingfu , Walbroehl Jaclyn , Löwenberg Ester , Golub Todd , Merchan Jaime , Tenen Daniel G. , Markowitz Sanford D. , Halmos Balazs
Published: July 2008

The forkhead transcription factor hepatocyte nuclear factor 3β (HNF3β) is essential in foregut development and the regulation of lung-specific genes. HNF3β expression leads to growth arrest and apoptosis in lung cancer cells and HNF3β is a candidate tumor suppressor in lung cancer. In a transcriptional profiling study using a conditional cell line system, we now identify 15-PGDH as one of the major genes induced by HNF3β expression. 15-PGDH is a critical metabolic enzyme of proliferative prostaglandins, an antagonist to cyclooxygenase-2 and a tumor suppressor in colon cancer. We confirmed the regulation of 15-PGDH expression by HNF3β in a number of systems and showed direct binding of HNF3β to 15-PGDH promoter elements. Western blotting of lung cancer cell lines and immunohistochemical examination of human lung cancer tissues found loss of 15-PGDH expression in ∼65% of lung cancers. Further studies using in vitro cell-based assays and in vivo xenograft tumorigenesis assays showed a lack of in vitro but significant in vivo tumor suppressor activity of 15-PGDH via an antiangiogenic mechanism analogous to its role in colon cancer. In summary, we identify 15-PGDH as a direct downstream effector of HNF3β and show that 15-PGDH acts as a tumor suppressor in lung cancer. [Cancer Res 2008;68(13):5040–8]
Tumor-Induced Immune Suppression ofIn vivoEffector T-Cell Priming Is Mediated by the B7-H1/PD-1 Axis and Transforming Growth Factor β
Authors: Wei Shuang , Shreiner Andrew B. , Takeshita Nobuhiro , Chen Lieping , Zou Weiping , Chang Alfred E.
Published: July 2008

We have generated effector T cells from tumor-draining lymph nodes (TDLN) that are efficacious in adoptive immunotherapy. We now examine the effect of concomitant tumors on the generation of effector T cells. We inoculated methylcholanthrene (MCA) 205 in the flanks of normal mice and mice bearing MCA 205 lung metastases. TDLN cells from these mice were activated and expanded in vitro, and adoptively transferred to mice bearing lung metastases. Effector T cells generated from TDLN in mice with only flank tumor mediated potent antitumor activity. However, antitumor efficacy of the effector T cells generated from TDLN in mice with pre-existent lung tumor (cTDLN) was reduced. Phenotyping studies showed that dendritic cells in cTDLN expressed higher levels of B7-H1, whereas cTDLN T cells expressed higher levels of PD-1. The levels of IFNγ were reduced, and the levels of CD4+Foxp3+ regulatory T cells were increased in cTDLN versus TDLN. The in vitro activation of cTDLN was increased by blocking B7-H1 or transforming growth factor (TGF)-β. Importantly, we found a synergistic up-regulation of IFNγ with simultaneous blockade of B7-H1 and TGF-β that was much greater than observed with TDLN. In vitro activation of cTDLN with anti–B7-H1 and anti–TGF-β and in vivo administration of these antibodies after adoptive transfer resulted in the abrogation of the suppression associated with cTDLN. These results show a major role for the B7-H1/PD-1 axis and TGF-β as synergistic suppressive mechanisms in cTDLN. Our data have clinical relevance in the generation of effector T cells in the tumor-bearing host. [Cancer Res 2008;68(13):5432–7]
Myeloid-Derived Suppressor Cells Promote Cross-Tolerance in B-Cell Lymphoma by Expanding Regulatory T Cells
Authors: Serafini Paolo , Mgebroff Stephanie , Noonan Kimberly , Borrello Ivan
Published: July 2008

Tumor-induced T-cell tolerance is a major mechanism that facilitates tumor progression and limits the efficacy of immune therapeutic interventions. Regulatory T cells (Treg) play a central role in the induction of tolerance to tumor antigens, yet the precise mechanisms regulating its induction in vivo remain to be elucidated. Using the A20 B-cell lymphoma model, here we identify myeloid-derived suppressor cells (MDSC) as the tolerogenic antigen presenting cells capable of antigen uptake and presentation to tumor-specific Tregs. MDSC-mediated Treg induction requires arginase but is transforming growth factor-β independent. In vitro and in vivo inhibition of MDSC function, respectively, with NOHA or sildenafil abrogates Treg proliferation and tumor-induced tolerance in antigen-specific T cells. These findings establish a role for MDSCs in antigen-specific tolerance induction through preferential antigen uptake mediating the recruitment and expansion of Tregs. Furthermore, therapeutic interventions, such as in vivo phosphodiesterase 5–inhibition, which effectively abrogate the immunosuppressive role of MDSCs and reduce Treg numbers, may play a critical role in delaying and/or reversing tolerance induction. [Cancer Res 2008;68(13):5439–49]
RETRACTED: Methylation Mediated Silencing of MicroRNA-1 Gene and Its Role in Hepatocellular Carcinogenesis
Authors: Datta Jharna , Kutay Huban , Nasser Mohd W. , Nuovo Gerard J. , Wang Bo , Majumder Sarmila , Liu Chang-Gong , Volinia Stefano , Croce Carlo M. , Schmittgen Thomas D. , Ghoshal Kalpana , Jacob Samson T.
Published: July 2008

MicroRNAs (miR) are a class of small (∼21 nucleotide) noncoding RNAs that, in general, negatively regulate gene expression. Some miRs harboring CGIs undergo methylation-mediated silencing, a characteristic of many tumor suppressor genes. To identify such miRs in liver cancer, the miRNA expression profile was analyzed in hepatocellular carcinoma (HCC) cell lines treated with 5-azacytidine (DNA hypomethylating agent) and/or trichostatin A (histone deacetylase inhibitor). The results showed that these epigenetic drugs differentially regulate expression of a few miRs, particularly miR-1-1, in HCC cells. The CGI spanning exon 1 and intron 1 of miR-1-1 was methylated in HCC cell lines and in primary human HCCs but not in matching liver tissues. The miR-1-1 gene was hypomethylated and activated in DNMT1−/− HCT 116 cells but not in DNMT3B null cells, indicating a key role for DNMT1 in its methylation. miR-1 expression was also markedly reduced in primary human hepatocellular carcinomas compared with matching normal liver tissues. Ectopic expression of miR-1 in HCC cells inhibited cell growth and reduced replication potential and clonogenic survival. The expression of FoxP1 and MET harboring three and two miR-1 cognate sites, respectively, in their respective 3′-untranslated regions, was markedly reduced by ectopic miR-1. Up-regulation of several miR-1 targets including FoxP1, MET, and HDAC4 in primary human HCCs and down-regulation of their expression in 5-AzaC–treated HCC cells suggest their role in hepatocarcinogenesis. The inhibition of cell cycle progression and induction of apoptosis after re-expression of miR-1 are some of the mechanisms by which DNA hypomethylating agents suppress hepatocarcinoma cell growth. [Cancer Res 2008;68(13):5049–58]
Dietary Energy Restriction Modulates the Activity of AMP-Activated Protein Kinase, Akt, and Mammalian Target of Rapamycin in Mammary Carcinomas, Mammary Gland, and Liver
Authors: Jiang Weiqin , Zhu Zongjian , Thompson Henry J.
Published: July 2008

Dietary energy restriction (DER) inhibits mammary carcinogenesis, yet mechanisms accounting for its protective activity have not been fully elucidated. In this study, we tested the hypothesis that DER exerts effects on intracellular energy sensing pathways, resulting in alterations of phosphorylated proteins that play a key role in the regulation of cancer. Experiments were conducted using the 1-methyl-1-nitrosourea–induced mammary cancer model in which rats were 0%, 20%, or 40% energy restricted during the postinitiation stage of carcinogenesis. Parallel experiments were done in non–carcinogen-treated rats in which effects of DER at 0%, 5%, 10%, 20%, or 40% in liver were investigated. In a DER dose–dependent manner, levels of Thr172 phosphorylated AMP-activated protein kinase (AMPK) increased in mammary carcinomas with a concomitant increase in phosphorylated acetyl-CoA-carboxylase, a direct target of AMPK, the phosphorylation of which is regarded as an indicator of AMPK activity. Levels of phosphorylated mammalian target of rapamycin (mTOR) decreased with increasing DER, and down-regulation of mTOR activity was verified by a decrease in the phosphorylation state of two mTOR targets, 70-kDa ribosomal protein S6 kinase (p70S6K) and eukaryote initiation factor 4E binding protein 1 (4E-BP1). Coincident with changes in mTOR phosphorylation, levels of activated protein kinase B (Akt) were also reduced. Similar patterns were observed in mammary glands and livers of non–carcinogen-treated rats. This work identifies components of intracellular energy sensing pathways, specifically mTOR, its principal upstream regulators, AMPK and Akt, and its downstream targets, p70S6K and 4E-BP1, as candidate molecules on which to center mechanistic studies of DER. [Cancer Res 2008;68(13):5492–9]
Single-Cell Transcription Site Activation Predicts Chemotherapy Response in Human Colorectal Tumors
Authors: Pezo Rossanna C. , Gandhi Saumil J. , Shirley L. Andrew , Pestell Richard G. , Augenlicht Leonard H. , Singer Robert H.
Published: July 2008

Candidate gene and pathway approaches, and unbiased gene expression profiling, have identified marker signatures predictive of tumor phenotypes, such as drug sensitivity and invasive or metastatic potential. However, application of such information to evaluation of tumors in the clinic is limited by cell heterogeneity in the tumor. We have developed a novel method of fluorescence in situ hybridization (FISH) that can detect transcriptional activation of individual genes at their site in single cells in the interphase nucleus. A major obstacle in the treatment of colorectal cancer is relative insensitivity to the chemotherapeutic agent 5-Fluorouracil (5-FU). Here, we have developed a sensitive approach to predict relative sensitivity of colorectal cancer cells to 5-FU, using FISH with probes targeted to nascent mRNAs to measure the number of individual cells with active transcription sites for a panel of candidate genes. These results reveal that the transcriptional status of four key genes, thymidylate synthase (TYMS), MORF-related gene X (MRGX), Bcl2-antagonist/killer (BAK), and ATPase, Cu2+ transporting β polypeptide (ATP7B), can accurately predict response to 5-FU. As proof of principle, we show that this transcriptional profile is predictive of response to 5-FU in a small number of patient colon tumor tissues. This approach provides a novel ability to identify and characterize unique minor cell populations in the tumor that may exhibit relative resistance to chemotherapy. [Cancer Res 2008;68(13):4977–82]
Transporter-Mediated Protection against Thiopurine-Induced Hematopoietic Toxicity
Authors: Krishnamurthy Partha , Schwab Matthias , Takenaka Kazumasa , Nachagari Deepa , Morgan Jessica , Leslie Mark , Du Weinan , Boyd Kelli , Cheok Meyling , Nakauchi Hiromitsu , Marzolini Catia , Kim Richard B. , Poonkuzhali Balasubramanian , Schuetz Erin , Evans William , Relling Mary , Schuetz John D.
Published: July 2008

Thiopurines are effective immunosuppressants and anticancer agents, but intracellular accumulation of their active metabolites (6-thioguanine nucleotides, 6-TGN) causes dose-limiting hematopoietic toxicity. Thiopurine S-methyltransferase deficiency is known to exacerbate thiopurine toxicity. However, many patients are highly sensitive to thiopurines for unknown reasons. We show that multidrug-resistance protein 4 (Mrp4) is abundant in myeloid progenitors and tested the role of the Mrp4, an ATP transporter of monophosphorylated nucleosides, in this unexplained thiopurine sensitivity. Mrp4-deficient mice experienced Mrp4 gene dosage-dependent toxicity caused by accumulation of 6-TGNs in their myelopoietic cells. Therefore, Mrp4 protects against thiopurine-induced hematopoietic toxicity by actively exporting thiopurine nucleotides. We then identified a single-nucleotide polymorphism (SNP) in human MRP4 (rs3765534) that dramatically reduces MRP4 function by impairing its cell membrane localization. This SNP is common (>18%) in the Japanese population and indicates that the increased sensitivity of some Japanese patients to thiopurines may reflect the greater frequency of this MRP4 SNP. [Cancer Res 2008;68(13):4983–9]
Tumor-Induced Suppressor of Cytokine Signaling 3 Inhibits Toll-like Receptor 3 Signaling in Dendritic Cells via Binding to Tyrosine Kinase 2
Authors: Zeng Bin , Li Haijie , Liu Yu , Zhang Zhuohan , Zhang Yuan , Yang Rongcun
Published: July 2008

The suppressor of cytokine signaling (SOCS) family of negative regulatory proteins is up-regulated in response to several cytokines and pathogen-associated molecular patterns (PAMP) and suppresses cellular signaling responses by binding receptor phosphotyrosine residues. Exposure of bone marrow–derived dendritic cells (BMDC) to 1D8 cells, a murine model of ovarian carcinoma, suppresses their ability to express CD40 and stimulate antigen-specific responses in response to PAMPs and, in particular, to polyinosinic acid:poly-CMP (polyI:C) with the up-regulated SOCS3 transcript and protein levels. The ectopic expression of SOCS3 in both the macrophage cell line RAW264.7 and BMDCs decreased signaling in response to both polyI:C and IFNα. Further, knockdown of SOCS3 transcripts significantly enhanced the responses of RAW264.7 and BMDCs to both polyI:C and IFNα. Immunoprecipitation and pull-down studies show that SOCS3 binds to the IFNα receptor tyrosine kinase 2 (TYK2). Because polyI:C triggers autocrine IFNα signaling, binding of SOCS3 to TYK2 may thereby suppress the activation of BMDCs by polyI:C and IFNα. Thus, elevated levels of SOCS3 in tumor-associated DCs may potentially resist the signals induced by Toll-like receptor 3 ligands and type I IFN to decrease DC activation via binding with IFNα receptor TYK2. [Cancer Res 2008;68(13):5397–404]
Curcumin Decreases Specificity Protein Expression in Bladder Cancer Cells
Authors: Chadalapaka Gayathri , Jutooru Indira , Chintharlapalli Sudhakar , Papineni Sabitha , Smith Roger , Li Xiangrong , Safe Stephen
Published: July 2008

Curcumin is the active component of tumeric, and this polyphenolic compound has been extensively investigated as an anticancer drug that modulates multiple pathways and genes. In this study, 10 to 25 μmol/L curcumin inhibited 253JB-V and KU7 bladder cancer cell growth, and this was accompanied by induction of apoptosis and decreased expression of the proapoptotic protein survivin and the angiogenic proteins vascular endothelial growth factor (VEGF) and VEGF receptor 1 (VEGFR1). Because expression of survivin, VEGF, and VEGFR1 are dependent on specificity protein (Sp) transcription factors, we also investigated the effects of curcumin on Sp protein expression as an underlying mechanism for the apoptotic and antiangiogenic activity of this compound. The results show that curcumin induced proteasome-dependent down-regulation of Sp1, Sp3, and Sp4 in 253JB-V and KU7 cells. Moreover, using RNA interference with small inhibitory RNAs for Sp1, Sp3, and Sp4, we observed that curcumin-dependent inhibition of nuclear factor κB (NF-κB)–dependent genes, such as bcl-2, survivin, and cyclin D1, was also due, in part, to loss of Sp proteins. Curcumin also decreased bladder tumor growth in athymic nude mice bearing KU7 cells as xenografts and this was accompanied by decreased Sp1, Sp3, and Sp4 protein levels in tumors. These results show for the first time that one of the underlying mechanisms of action of curcumin as a cancer chemotherapeutic agent is due, in part, to decreased expression of Sp transcription factors in bladder cancer cells. [Cancer Res 2008;68(13):5345–54]
Curcumin Blocks RON Tyrosine Kinase–Mediated Invasion of Breast Carcinoma Cells
Authors: Narasimhan Madhusudhanan , Ammanamanchi Sudhakar
Published: July 2008

We have recently shown that macrophage-stimulating protein (MSP) promotes the invasion of recepteur d'origine nantais (RON), a tyrosine kinase receptor–positive MDA-MB-231, MDA-MB-468 breast cancer cells, and also identified the regulatory elements required for RON gene expression. In this report, we have analyzed the efficacy of a chemopreventive agent, curcumin, in blocking RON tyrosine kinase–mediated invasion of breast cancer cells. Reverse transcription-PCR and Western analysis indicated the down-regulation of the RON message and protein, respectively, in MDA-MB-231 and MDA-MB-468 cells. Significantly, curcumin-mediated inhibition of RON expression resulted in the blockade of RON ligand, MSP-induced invasion of breast cancer cells. We have identified two putative nuclear factor-κB p65 subunit binding sites on the RON promoter. Using chromatin immunoprecipitation analysis and site-directed mutagenesis of the RON promoter, we have confirmed the binding of p65 to the RON promoter. Our data show that curcumin reduces RON expression by affecting p65 protein expression and transcriptional activity. Treatment of MDA-MB-231 cells with pyrrolidine dithiocarbamate, an inhibitor of p65, or small interfering RNA knockdown of p65, blocked RON gene expression and MSP-mediated invasion of MDA-MB-231 cells. This is the first report showing the regulation of human RON gene expression by nuclear factor-κB and suggests a potential therapeutic role for curcumin in blocking RON tyrosine kinase–mediated invasion of carcinoma cells. [Cancer Res 2008;68(13):5185–92]
Increasing Melanoma Cell Death Using Inhibitors of Protein Disulfide Isomerases to Abrogate Survival Responses to Endoplasmic Reticulum Stress
Authors: Lovat Penny E. , Corazzari Marco , Armstrong Jane L. , Martin Shaun , Pagliarini Vittoria , Hill David , Brown Anna M. , Piacentini Mauro , Birch-Machin Mark A. , Redfern Christopher P.F.
Published: July 2008

Exploiting vulnerabilities in the intracellular signaling pathways of tumor cells is a key strategy for the development of new drugs. The activation of cellular stress responses mediated by the endoplasmic reticulum (ER) allows cancer cells to survive outside their normal environment. Many proteins that protect cells against ER stress are active as protein disulfide isomerases (PDI) and the aim of this study was to test the hypothesis that apoptosis in response to ER stress can be increased by inhibiting PDI activity. We show that the novel chemotherapeutic drugs fenretinide and velcade induce ER stress–mediated apoptosis in melanoma cells. Both stress response and apoptosis were enhanced by the PDI inhibitor bacitracin. Overexpression of the main cellular PDI, procollagen-proline, 2-oxoglutarate-4-dioxygenase β subunit (P4HB), resulted in increased PDI activity and abrogated the apoptosis-enhancing effect of bacitracin. In contrast, overexpression of a mutant P4HB lacking PDI activity did not increase cellular PDI activity or block the effects of bacitracin. These results show that inhibition of PDI activity increases apoptosis in response to agents which induce ER stress and suggest that the development of potent, small-molecule PDI inhibitors has significant potential as a powerful tool for enhancing the efficacy of chemotherapy in melanoma. [Cancer Res 2008;68(13):5363–8]
Improved Therapeutic Results by Pretargeted Radioimmunotherapy of Non–Hodgkin's Lymphoma with a New Recombinant, Trivalent, Anti-CD20, Bispecific Antibody
Authors: Sharkey Robert M. , Karacay Habibe , Litwin Samuel , Rossi Edmund A. , McBride William J. , Chang Chien-Hsing , Goldenberg David M.
Published: July 2008

We examined whether a pretargeting method using a new recombinant anti-CD20 bispecific antibody (bsMAb) followed by 90Y-1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid (90Y-DOTA)-peptide could reduce hematologic toxicity yet improve therapeutic responses compared with conventional 90Y-anti-CD20 IgG and a chemically conjugated bsMAb. TF4, a humanized, tri-Fab bsMAb with two Fabs binding CD20 and one Fab binding histamine-succinyl-glycine (HSG), developed by the dock and lock (DNL) method, was tested in nude mice with Ramos B-cell lymphomas. Optimal pretargeting required a 29-h interval between TF4 and 90Y-DOTA-HSG, and 20-fold more moles of TF4. TF4 cleared more rapidly from the blood than anti-CD20 IgG, with early processing in the liver, spleen, and kidney. At 24 h, TF4 improved tumor uptake of 111In-HSG-peptide 2.6-fold [13% versus 5% injected dose per gram (ID/g)] and enhanced tumor to blood ratios >45-fold (770 versus 17), compared with an anti-CD20 Fab × anti-HSG Fab chemical conjugate, and by 1.6-fold (9.0% versus 5.6% ID/g) and 1,600-fold (522 versus 0.32), respectively, compared with radiolabeled anti-CD20 IgG. A severe (≥90%) and prolonged reduction of WBCs was observed at the maximum dose of 90Y-anti-CD20 IgG, whereas pretargeting resulted in a ≤60% transient drop. TF4 pretargeting resulted in highly significant improvement in survival, curing 33% to 90% of the animals, even at relatively low doses, whereas most tumors progressed quickly without cures with 90Y-anti-CD20 IgG. These results indicate an improved therapeutic index with pretargeted radioimmunotherapy (RAIT) using a DNL-constructed tri-Fab, bsMAb, compared with conventional therapy with directly radiolabeled antibody or with a chemically conjugated bsMAb. These encouraging results prompt testing these constructs for pretargeting RAIT in patients. [Cancer Res 2008;68(13):5282–90]
Haploinsufficiency of the Maspin Tumor Suppressor Gene Leads to Hyperplastic Lesions in Prostate
Authors: Shao Long-jiang , Shi Heidi Y. , Ayala Gustavo , Rowley David , Zhang Ming
Published: July 2008

Maspin is a key tumor suppressor gene in prostate and breast cancers with diverse biological functions. However, how maspin regulates prostate tumor progression is not fully understood. In this study, we have used maspin heterozygous knockout mice to determine the effect of maspin haploinsufficiency on prostate development and tumor progression. We report that loss of one copy of maspin gene in Mp+/− heterozygous knockout mice leads to the development of prostate hyperplastic lesions, and this effect was mediated through decreased level of cyclin-dependent kinase inhibitors p21 and p27. Prostate hyperplastic lesions in Mp+/− mice also induced stromal reaction, which occurred in both aged prostate tissues and in neonatal prostates during early ductal morphogenesis. We showed that maspin was also expressed in prostate smooth muscle cells (PSMC), and recombinant maspin increased PSMC cell adhesion but inhibited cell proliferation. We also observed a defective interaction between epithelial cells and basement membrane in the prostate of Mp+/− mice, which was accompanied with a changed pattern of matrix deposition and a loss of epithelial cell polarity. Therefore, we have identified a novel property of maspin, which involves the control of the proliferation in prostate epithelial and smooth muscle cells. This is the first report that a partial loss of maspin caused an early developmental defect of the prostate and prostate hyperplastic lesions in mouse. [Cancer Res 2008;68(13):5143–51]
miR-206 Expression Is Down-regulated in Estrogen Receptor α–Positive Human Breast Cancer
Authors: Kondo Naoto , Toyama Tatsuya , Sugiura Hiroshi , Fujii Yoshitaka , Yamashita Hiroko
Published: July 2008

Expression levels of estrogen receptor (ER) α govern estrogen-dependent growth, response to endocrine therapy, and prognosis in ERα-positive breast cancer. Multiple mechanisms involved in altering ERα gene expression in breast cancer have been identified, including ERα gene amplification as well as transcriptional silencing by DNA methylation of CpG islands within the ERα promoter and mutations within the open reading frame of ERα. However, expression levels of ERα in breast cancer tissues differ widely among patients, and frequently change during disease progression and in response to systemic therapies. Recent evidence has shown that microRNA mutations or misexpression correlate with various human cancers, and miR-206 is reported to decrease endogenous ERα mRNA and protein levels in human MCF-7 breast cancer cells via two specific target sites within the 3′-untranslated region of the human ERα transcript. In this study, we show for the first time that miR-206 expression is markedly decreased in ERα-positive human breast cancer tissues assayed by quantitative reverse transcription-PCR analysis. Moreover, we observe that miR-206 expression is inversely correlated with ERα but not ERβ mRNA expression in breast cancer tissues. Transfection experiments revealed that introduction of miR-206 into estrogen-dependent MCF-7 breast cancer cells inhibits cell growth in a dose- and time-dependent manner. Our results suggest that miR-206 could be a novel candidate for endocrine therapy that targets only ERα in breast cancer. [Cancer Res 2008;68(13):5004–8]
Serotonin Regulates Macrophage-Mediated Angiogenesis in a Mouse Model of Colon Cancer Allografts
Authors: Nocito Antonio , Dahm Felix , Jochum Wolfram , Jang Jae Hwi , Georgiev Panco , Bader Michael , Graf Rolf , Clavien Pierre-Alain
Published: July 2008

Serotonin, a neurotransmitter with numerous functions in the central nervous system (CNS), is emerging as an important signaling molecule in biological processes outside of the CNS. Recent advances have implicated serotonin as a regulator of inflammation, proliferation, regeneration, and repair. The role of serotonin in tumor biology in vivo has not been elucidated. Using a genetic model of serotonin deficiency (Tph1−/−) in mice, we show serotonin to be crucial for the growth of s.c. colon cancer allografts in vivo. Serotonin does not enhance tumor cell proliferation but acts as a regulator of angiogenesis by reducing the expression of matrix metalloproteinase 12 (MMP-12) in tumor-infiltrating macrophages, entailing lower levels of angiostatin—an endogenous inhibitor of angiogenesis. Accordingly, serotonin deficiency causes slower growth of s.c. tumors by reducing vascularity, thus increasing hypoxia and spontaneous necrosis. The biological relevance of these effects is underscored by the reconstitution of serotonin synthesis in Tph1−/− mice, which restores allograft phenotype in all aspects. In conclusion, we show how serotonin regulates angiogenesis in s.c. colon cancer allografts by influencing MMP-12 expression in tumor-infiltrating macrophages, thereby affecting the production of circulating angiostatin. [Cancer Res 2008;68(13):5152–8]
NO Signaling Confers Cytoprotectivity through the Survivin Network in Ovarian Carcinomas
Authors: Engels Knut , Knauer Shirley K. , Loibl Sibylle , Fetz Verena , Harter Philipp , Schweitzer Andrea , Fisseler-Eckhoff Annette , Kommoss Friedrich , Hanker Lars , Nekljudova Valentina , Hermanns Iris , Kleinert Hartmut , Mann Wolf , du Bois Andreas , Stauber Roland H.
Published: July 2008

Despite considerable success in the treatment of epithelial ovarian cancer (EOC), therapy resistance counteracts improvement of long-term survival. The dual role of survivin as an apoptosis inhibitor and mitotic regulator has been associated with disease outcome. However, the molecular mechanisms involved in the deregulated expression in EOC of survivin need further investigation. Here, we show that high amounts of the nitric oxide (NO) donors, S-nitroso-N-acetyl-penicillamine (SNAP) and sodium nitroprusside (SNP) or strong overexpression of the inducible nitric oxide synthase (iNOS) suppressed survivin levels via the p38MAPK pathway and triggered apoptosis in ovarian cancer cell lines (OCC). Importantly, low NO concentrations conferred resistance against carboplatin/paclitaxel-induced apoptosis. Cytoprotection was mediated by survivin because we observed its up-regulation subsequent to low SNAP/SNP doses or ectopic expression of low amounts of iNOS. Also, RNAi-mediated depletion of survivin blocked the antiapoptotic effects of NO signaling. Induction of survivin involves activation of the phosphatidylinositol-3-kinase (PI3K)/Akt pathway, which was antagonized by the PI3K-inhibitor, LY294002. Interestingly, application of the iNOS-inhibitor 1400W together with RNAi-mediated survivin down-regulation cooperatively enhanced drug-induced apoptosis in OCCs. The iNOS/survivin interdependencies seem to be also of clinical relevance because immunohistochemistry revealed that low iNOS levels correlate with survivin expression (P < 0.01) in carboplatin/paclitaxel-treated EOC patients with minimal postoperative residual tumor (n = 54). Also, iNOS and survivin expression were associated with increased risk for disease progression. Our study uncovers a novel molecular mechanism of how NO signaling may contribute to therapy resistance in EOC by modulating survivin expression. Pharmacogenetic iNOS/survivin-targeting strategies may hence be pursued to complement current treatment modalities in EOC. [Cancer Res 2008;68(13):5159–65]
The Warburg Effect in Leukemia-Stroma Cocultures Is Mediated by Mitochondrial Uncoupling Associated with Uncoupling Protein 2 Activation
Authors: Samudio Ismael , Fiegl Michael , McQueen Teresa , Clise-Dwyer Karen , Andreeff Michael
Published: July 2008

In 1956, Otto Warburg proposed that the origin of cancer cells was closely linked to a permanent respiratory defect that bypassed the Pasteur effect (i.e., the inhibition of anaerobic fermentation by oxygen). Since then, permanent defects in oxygen consumption that could explain the dependence of cancer cells on aerobic glycolysis have not been identified. Here, we show that under normoxic conditions exposure of leukemia cells to bone marrow–derived mesenchymal stromal cells (MSC) promotes accumulation of lactate in the culture medium and reduces mitochondrial membrane potential (ΔΨM) in both cell types. Notably, the consumption of glucose was not altered in cocultures, suggesting that the accumulation of lactate was the result of reduced pyruvate metabolism. Interestingly, the decrease in ΔΨM was mediated by mitochondrial uncoupling in leukemia cells and was accompanied by increased expression of uncoupling protein 2 (UCP2). HL60 cells fail to increase UCP2 expression, are not uncoupled after coculture, and do not exhibit increased aerobic glycolysis, whereas small interfering RNA–mediated suppression of UCP2 in OCI-AML3 cells reversed mitochondrial uncoupling and aerobic glycolysis elicited by MSC. Taken together, these data suggest that microenvironment activation of highly conserved mammalian UCPs may facilitate the Warburg effect in the absence of permanent respiratory impairment. [Cancer Res 2008;68(13):5198–205]
Splicing of a Novel Androgen Receptor Exon Generates a Constitutively Active Androgen Receptor that Mediates Prostate Cancer Therapy Resistance
Authors: Dehm Scott M. , Schmidt Lucy J. , Heemers Hannelore V. , Vessella Robert L. , Tindall Donald J.
Published: July 2008

The standard systemic treatment for prostate cancer (PCa) is androgen ablation, which causes tumor regression by inhibiting activity of the androgen receptor (AR). Invariably, PCa recurs with a fatal androgen-refractory phenotype. Importantly, the growth of androgen-refractory PCa remains dependent on the AR through various mechanisms of aberrant AR activation. Here, we studied the 22Rv1 PCa cell line, which was derived from a CWR22 xenograft that relapsed during androgen ablation. Three AR isoforms are expressed in 22Rv1 cells: a full-length version with duplicated exon 3 and two truncated versions lacking the COOH terminal domain (CTD). We found that CTD-truncated AR isoforms are encoded by mRNAs that have a novel exon 2b at their 3′ end. Functionally, these AR isoforms are constitutively active and promote the expression of endogenous AR-dependent genes, as well as the proliferation of 22Rv1 cells in a ligand-independent manner. AR mRNAs containing exon 2b and their protein products are expressed in commonly studied PCa cell lines. Moreover, exon 2b–derived species are enriched in xenograft-based models of therapy-resistant PCa. Together, our data describe a simple and effective mechanism by which PCa cells can synthesize a constitutively active AR and thus circumvent androgen ablation. [Cancer Res 2008;68(13):5469–77]
Synthesis and Anticancer Properties of Water-Soluble Zinc Ionophores
Authors: Magda Darren , Lecane Philip , Wang Zhong , Hu Weilin , Thiemann Patricia , Ma Xuan , Dranchak Patricia K. , Wang Xiaoming , Lynch Vincent , Wei Wenhao , Csokai Viktor , Hacia Joseph G. , Sessler Jonathan L.
Published: July 2008

Several water-solubilized versions of the zinc ionophore 1-hydroxypyridine-2-thione (ZnHPT), synthesized as part of the present study, have been found both to increase the intracellular concentrations of free zinc and to produce an antiproliferative activity in exponential phase A549 human lung cancer cultures. Gene expression profiles of A549 cultures treated with one of these water-soluble zinc ionophores, PCI-5002, reveal the activation of stress response pathways under the control of metal-responsive transcription factor 1 (MTF-1), hypoxia-inducible transcription factor 1 (HIF-1), and heat shock transcription factors. Additional oxidative stress response and apoptotic pathways were activated in cultures grown in zinc-supplemented media. We also show that these water-soluble zinc ionophores can be given to mice at 100 μmol/kg (300 μmol/m2) with no observable toxicity and inhibit the growth of A549 lung and PC3 prostate cancer cells grown in xenograft models. Gene expression profiles of tumor specimens harvested from mice 4 h after treatment confirmed the in vivo activation of MTF-1–responsive genes. Overall, we propose that water-solubilized zinc ionophores represent a potential new class of anticancer agents. [Cancer Res 2008;68(13):5318–25]
Pim Kinases Promote Cell Cycle Progression by Phosphorylating and Down-regulating p27Kip1 at the Transcriptional and Posttranscriptional Levels
Authors: Morishita Daisuke , Katayama Ryohei , Sekimizu Kazuhisa , Tsuruo Takashi , Fujita Naoya
Published: July 2008

The serine/threonine kinase Pim is known to promote cell cycle progression and to inhibit apoptosis leading to tumorigenesis. However, the precise mechanisms remain unclear. We show, herein, that all the Pim family members (Pim1, Pim2, and Pim3) bind to and directly phosphorylate the cyclin-dependent kinase inhibitor p27Kip1 at threonine-157 and threonine-198 residues in cells and in vitro. The Pim-mediated phosphorylation induced p27Kip1 binding to 14-3-3 protein, resulting in its nuclear export and proteasome-dependent degradation. Ectopic expression of Pim kinases overcome the G1 arrest mediated by wild-type p27Kip1 but not by phosphorylation-resistant T157A-p27Kip1 or T198A-p27Kip1. In addition to the posttranslational regulations, p27Kip1 promoter assay revealed that Pim kinases also had the ability to suppress p27Kip1 transcription. Pim-mediated phosphorylation and inactivation of forkhead transcription factors, FoxO1a and FoxO3a, was involved in the transcriptional repression of the p27Kip1 gene. In contrast, inhibition of Pim signaling by expressing the dominant-negative form of Pim1 increased nuclear p27Kip1 level and attenuated cell proliferation. Because the CDK inhibitor p27Kip1 plays a crucial role in tumor suppression by inhibiting abnormal cell cycle progression, Pim kinases promote cell cycle progression and tumorigenesis by down-regulating p27Kip1 expression at both transcriptional and posttranslational levels. [Cancer Res 2008;68(13):5076–85]
Frequent Detection of Merkel Cell Polyomavirus in Human Merkel Cell Carcinomas and Identification of a Unique Deletion in the VP1 Gene
Authors: Kassem Ahmad , Schöpflin Anja , Diaz Carlos , Weyers Wolfgang , Stickeler Elmar , Werner Martin , zur Hausen Axel
Published: July 2008

Merkel cell carcinoma (MCC) is a rare but very aggressive human malignancy of the elderly or immunosuppressed patients. Recently, the clonal integration of a new human polyoma virus, which was termed Merkel cell polyomavirus (MCPyV), has been reported in 8 of 10 MCC patients. In the present study, we studied the formalin-fixed and paraffin-embedded tissue specimens of 39 MCC for the presence of MCPyV by PCR. We applied four different primer sets directed against the large T antigen and the VP1 gene of MCPyV. We were able to detect MCPyV in 77% (n = 30) of MCC as confirmed by sequence analyses of the PCR products. Sequence analyses showed only minor nucleotide changes compared with the previously published MCC sequences. In addition, one patient revealed the amplification of two PCR products using PCR primers directed against the VP1 gene. Sequence analyses confirmed the presence of the expected 351-bp PCR product and in addition a second PCR product of 261 bp containing a unique 90-bp deletion in the VP1 gene, which will lead to a predicted loss of 28 amino acids. The unique 90-bp deletion within the VP1 gene possibly is a result of incomplete viral integration of MCPyV in the MCC. The presence of MCPyV in the majority of MCC tissue specimens in our study strongly underlines a possible role for MCPyV as an etiologic agent in the carcinogenesis of MCC. [Cancer Res 2008;68(13):5009–13]
Steroid Receptor Coactivator-3/AIB1 Promotes Cell Migration and Invasiveness through Focal Adhesion Turnover and Matrix Metalloproteinase Expression
Authors: Yan Jun , Erdem Halime , Li Rile , Cai Yi , Ayala Gustavo , Ittmann Michael , Yu-Lee Li-yuan , Tsai Sophia Y. , Tsai Ming-Jer
Published: July 2008

Steroid receptor coactivator-3 (SRC-3)/AIB1 is a member of the p160 nuclear receptor coactivator family involved in development and cell cycle progression. We previously showed that SRC-3/AIB1 is required for prostate cancer cell proliferation and survival. Here, we reported that the elevated SRC-3/AIB1 expression is significantly correlated with human prostate cancer seminal vesicle invasion and lymph node metastasis. Furthermore, SRC-3/AIB1 is associated with increased prostate cancer cell migration and invasion. SRC-3/AIB1 is required for focal adhesion turnover and focal adhesion kinase activation. In addition, SRC-3/AIB1 directly regulates transcription of matrix metalloproteinase (MMP)-2 and MMP-13 through its coactivation of AP-1 and PEA3. Taken together, these data suggest that SRC-3/AIB1 plays an essential role in prostate cancer cell invasion and metastasis. [Cancer Res 2008;68(13):5460–8]
Mechanisms by which E-Selectin Regulates Diapedesis of Colon Cancer Cells under Flow Conditions
Authors: Tremblay Pierre-Luc , Huot Jacques , Auger François A.
Published: July 2008

Diapedesis, the passage of circulating tumor cells across the endothelium, is a critical determinant in most cases of metastasis. Using a laminar flow chamber and a tissue-engineered blood vessel, we found that E-selectin is required not only for the initial adhesion and rolling of circulating HT-29 colon cancer cells on the endothelium but also for their subsequent diapedesis. These processes require both the intracellular and extracellular domains of E-selectin. We also identified three distinct mechanisms by which circulating cancer cells interact with E-selectin to initiate their diapedesis: formation of a mosaic between cancer cells and endothelial cells, paracellular diapedesis at the junction of three endothelial cells, and transcellular diapedesis. We also obtained evidence indicating that E-selectin–dependent paracellular extravasation is independent of intercellular adhesion molecule and vascular cell adhesion molecule and that it requires the activation of extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase downstream of E-selectin. This is supported by the observation that the adenoviral-mediated expression of the E-selectin mutant Y603F is associated with both an inhibition of ERK and paracellular extravasation. Our study is the first to clearly establish, under dynamic and shear stress conditions, how E-selectin regulates diapedesis of circulating cancer cells. These results provide new insights in understanding the metastatic process. [Cancer Res 2008;68(13):5167–76]
Activation of Notch Signaling in a Xenograft Model of Brain Metastasis
Authors: Nam Do-Hyun , Jeon Hye-Min , Kim Shiyeon , Kim Mi Hyun , Lee Young-Ju , Lee Min Su , Kim Hyunggee , Joo Kyeung Min , Lee Dong-Sup , Price Janet E. , Bang Sa Ik , Park Woong-Yang
Published: July 2008

Purpose: The potential of metastasis can be predicted from clinical features like tumor size, histologic grade, and gene expression patterns. We examined the whole-genome transcriptomic profile of a xenograft model of breast cancer to understand the characteristics of brain metastasis.

Experimental Design: Variants of the MDA-MB-435 cell were established from experimental brain metastases. The LvBr2 variant was isolated from lesions in a mouse injected in the left ventricle of the heart, and these cells were used for two cycles of injection into the internal carotid artery and selection of brain lesions, resulting in the Br4 variant. To characterize the different metastatic variants, we examined the gene expression profile of MDA-MB-435, LvBr2, and Br4 cells using microarrays.

Results: We could identify 2,016 differentially expressed genes in Br4 by using the F test. Various metastasis-related genes and a number of genes related to angiogenesis, migration, tumorigenesis, and cell cycle were differentially expressed by the Br4 cells. Notably, the Notch signaling pathway was activated in Br4, with increased Jag2 mRNA, activated Notch intracellular domain, and Notch intracellular domain/CLS promoter-luciferase activity. Br4 cells were more migratory and invasive than MDA-MB-435 cells in collagen and Matrigel Transwell assays, and the migration and invasion of Br4 cells were significantly inhibited by inactivation of Notch signaling using DAPT, a γ-secretase inhibitor, and RNA interference–mediated knockdown of Jagged 2 and Notch1.

Conclusions: Taken together, these results suggest that we have isolated variants of a human cancer cell line with enhanced brain metastatic properties, and the activation of Notch signaling might play a crucial role in brain metastasis.
Molecular Risk Stratification of Medulloblastoma Patients Based on Immunohistochemical Analysis of MYC, LDHB, and CCNB1 Expression
Authors: de Haas Talitha , Hasselt Nancy , Troost Dirk , Caron Huib , Popovic Mara , Zadravec-Zaletel Lorna , Grajkowska Wieslawa , Perek Marta , Osterheld Maria-Chiara , Ellison David , Baas Frank , Versteeg Rogier , Kool Marcel
Published: July 2008

Purpose: Medulloblastoma is the most common malignant embryonal brain tumor in children. The current clinical risk stratification to select treatment modalities is not optimal because it does not identify the standard-risk patients with resistant disease or the unknown number of high-risk patients who might be overtreated with current protocols. The aim of this study is to improve the risk stratification of medulloblastoma patients by using the expression of multiple prognostic markers in combination with current clinical parameters.

Experimental Design: Candidate prognostic markers were selected from literature or from medulloblastoma expression data. Selected genes were immunohistochemically analyzed for their prognostic value using medulloblastoma tissue arrays containing 124 well-characterized patient samples.

Results: Protein expression analyses showed that the combined expression of three genes was able to predict survival in medulloblastoma patients. Low MYC expression identified medulloblastoma patients with a very good outcome. In contrast, concomitant expression of LDHB and CCNB1 characterized patients with a very poor outcome. Multivariate analyses showed that both expression of MYC and the LDHB/CCNB1 gene signature were strong prognostic markers independent of the clinical parameters metastasis and residual disease. Combined analysis of clinical and molecular markers enabled greater resolution of disease risk than clinical factors alone.

Conclusions: A molecular risk stratification model for medulloblastoma patients is proposed based on the signature of MYC, LDHB, and CCNB1 expression. Combined with clinical variables, the model may provide a more accurate basis for targeting therapy in children with this disease.
Genetic Changes of Wnt Pathway Genes Are Common Events in Metaplastic Carcinomas of the Breast
Authors: Hayes Michael J. , Thomas Dafydd , Emmons Agnieszka , Giordano Thomas J. , Kleer Celina G.
Published: July 2008

Purpose: Metaplastic carcinomas are distinct invasive breast carcinomas with aberrant nonglandular differentiation, which may be spindle, squamous, or chondroid. The limited effective treatments result from the lack of knowledge of its molecular etiology. Given the role of the Wnt pathway in cell fate and in the development of breast cancer, we hypothesized that defects in this pathway may contribute to the development of metaplastic carcinomas.

Design: In 36 primary metaplastic carcinomas, we comprehensively determined the prevalence of and mechanism underlying β-catenin and Wnt pathway deregulation using immunohistochemistry for β-catenin expression and localization and mutational analysis for CTNNB1 (encoding β-catenin), APC, WISP3, AXIN1, and AXIN2 genes. By immunohistochemistry, normal β-catenin was seen as membrane staining, and it was aberrant when >5% of tumor cells had nuclear or cytoplasmic accumulation or reduced membrane staining.

Results: By immunohistochemistry, aberrant β-catenin was present in 33 of 36 (92%) cases, revealing deregulation of the Wnt pathway. CTNNB1 missense mutations were detected in 7 of 27 (25.9%) tumors available for mutation analyses. All mutations affected the NH2-terminal domain of β-catenin, presumably rendering the mutant protein resistant to degradation. Two of 27 (7.4%) tumors had mutations of APC, and 5 (18.5%) carried a frame shift mutation of WISP3. No AXIN1 or AXIN2 mutations were found.

Conclusions: Activation of the Wnt signaling pathway is common in this specific subtype of breast carcinoma. The discovery of CTNNB1, APC, and WISP3 mutations may result in new treatments for patients with metaplastic carcinomas of the breast.