bluuebunny/crossref_metadata_2025 huggingface dataset:
Data from OX40 Agonist Therapy Enhances CD8 Infiltration and Decreases Immune Suppression in the Tumor
Authors: Gough Michael J. , Ruby Carl E. , Redmond William L. , Dhungel Birat , Brown Alexis , Weinberg Andrew D.
Published: March 2023

Abstract
Acquisition of full T-cell effector function and memory differentiation requires appropriate costimulatory signals, including ligation of the costimulatory molecule OX40 (TNFRSF4, CD134). Tumors often grow despite the presence of tumor-specific T cells and establish an environment with weak costimulation and immune suppression. Administration of OX40 agonists has been shown to significantly increase the survival of tumor-bearing mice and was dependent on the presence of both CD4 and CD8 T cells during tumor-specific priming. To understand how OX40 agonists work in mice with established tumors, we developed a model to study changes in immune cell populations within the tumor environment. We show here that systemic administration of OX40 agonist antibodies increased the proportion of CD8 T cells at the tumor site in three different tumor models. The function of the CD8 T cells at the tumor site was also increased by administration of OX40 agonist antibody, and we observed an increase in the proportion of antigen-specific CD8 T cells within the tumor. Despite decreases in the proportion of T regulatory cells at the tumor site, T regulatory cell function in the spleen was unaffected by OX40 agonist antibody therapy. Interestingly, administration of OX40 agonist antibody caused significant changes in the tumor stroma, including decreased macrophages, myeloid-derived suppressor cells, and decreased expression of transforming growth factor-β. Thus, therapies targeting OX40 dramatically changed the tumor environment by enhancing the infiltration and function of CD8 T cells combined with diminished suppressive influences within the tumor. [Cancer Res 2008;68(13):5206–15]
Data from GSTP1 Promoter Haplotypes Affect DNA Methylation Levels and Promoter Activity in Breast Carcinomas
Authors: Rønneberg Jo Anders , Tost Jörg , Solvang Hiroko K. , Alnaes Grethe I.G. , Johansen Fredrik E. , Brendeford Elen M. , Yakhini Zohar , Gut Ivo G. , Lønning Per Eystein , Børresen-Dale Anne-Lise , Gabrielsen Odd S. , Kristensen Vessela N.
Published: March 2023

Abstract
The CpG island spanning the transcription start of the glutathione S-transferase P1 becomes methylated in a variety of human cancers including breast cancer. To study the effect of sequence variation on hypermethylation of the GSTP1 promoter, we analyzed the genetic and epigenetic variability in 90 tumors from patients with locally advanced breast cancer. High-resolution quantitative analysis revealed large variability in the DNA methylation levels. Lack of methylation was more often observed in the basal and normal-like estrogen receptor (ER)-negative tumors, and methylated GSTP1 was associated with better overall survival (P = 0.00063). Studies of the genetic variation identified 14 different haplotypes. The distribution of methylation levels of tumors homozygous for the most frequent haplotype was significantly different from other haplotype combinations (P = 0.011), the difference being more pronounced in ER-positive (P = 0.005) and progesterone receptor–positive (P = 0.008) tumors. Regression modeling identified the ER status and haplotype as the main determinants of DNA methylation variability. We identified a putative c-Myb response element (MRE) that was present in one of two minimal promoter haplotypes. In vitro analysis showed that c-Myb binds to the MRE, but binding was weakened by the two polymorphisms. Transient cotransfections in luminal-type and basal-like breast cancer cell lines confirmed cell-specific differential binding of c-Myb to the polymorphic sites, leading to a change in the expression from the GSTP1 promoter in vivo. GSTP1 expression was moderately but significantly (P = 0.01) reduced after siRNA-mediated knockdown of c-Myb. Our results indicate that haplotype structure of a promoter is important for the extent of DNA methylation. [Cancer Res 2008;68(14):5562–71]
Data from Enhanced Activation of Epidermal Growth Factor Receptor Caused by Tumor-Derived E-Cadherin Mutations
Authors: Bremm Anja , Walch Axel , Fuchs Margit , Mages Jörg , Duyster Justus , Keller Gisela , Hermannstädter Christine , Becker Karl-Friedrich , Rauser Sandra , Langer Rupert , von Weyhern Claus Hann , Höfler Heinz , Luber Birgit
Published: March 2023

Abstract
Mutations of the tumor suppressor E-cadherin and overexpression of the receptor tyrosine kinase epidermal growth factor receptor (EGFR) are among the most frequent genetic alterations associated with diffuse-type gastric carcinoma. Accumulating evidence suggests a functional relationship between E-cadherin and EGFR that regulates both proteins. We report that somatic mutation of E-cadherin is associated with increased activation of EGFR followed by enhanced recruitment of the downstream acting signaling components growth factor receptor binding protein 2 and Shc, and activation of Ras. Reduced complex formation of mutant E-cadherin — with an in frame deletion of exon 8 in the extracellular domain resulting in reduced adhesion and increased motility — with EGFR was observed compared with wild-type E-cadherin. We conclude that reduced binding of mutant E-cadherin to EGFR in a multicomponent complex or reduced stability of the complex may enhance EGFR surface motility, thereby facilitating EGFR dimerization and activation. Furthermore, reduced surface localization due to enhanced internalization of mutant E-cadherin compared with the wild-type protein was observed. The internalization of EGFR was decreased in response to epidermal growth factor stimulation in cells expressing mutant E-cadherin, suggesting that mutation of E-cadherin also influences the endocytosis of EGFR. Moreover, we show increased activation of EGFR in gastric carcinoma samples with mutant E-cadherin lacking exons 8 or 9. In summary, we describe activation of EGFR by mutant E-cadherin as a novel mechanism in tumor cells that explains the enhanced motility of tumor cells in the presence of an extracellular mutation of E-cadherin. [Cancer Res 2008;68(3):707–14]
Data from Clonal Selection in Malignant Transformation of Human Fibroblasts Transduced with Defined Cellular Oncogenes
Authors: Mahale Alka M. , Khan Zahid A.T. , Igarashi Makoto , Nanjangud Gouri J. , Qiao Rui Fang , Yao Shen , Lee Sam W. , Aaronson Stuart A.
Published: March 2023

Abstract
Recent evidence has implied that disruption of a limited number of defined cellular pathways is necessary and sufficient for neoplastic conversion of a variety of normal human cell types in tissue culture. We show instead that malignancy in such models results from an iterative process of clonal selection in vitro and/or in vivo. Normal human fibroblasts underwent malignant transformation after transduction with telomerase, cyclin-dependent kinase 4, dominant-negative p53, and activated Ras or MEK. Furthermore, culture conditions favoring overgrowth resulted in clonal selection, which with added Ras or MEK oncogenes led to the emergence of tumorigenic clones. Such tumors showed variable degrees of malignancy with some even exhibiting metastasis. SV40 small t antigen (ST) has been reported to be necessary and sufficient to convert human fibroblasts with these pathway aberrations to a polyclonal tumor. However, we observed that clonal tumors emerged even with ST addition. Genomic instability was markedly increased by p53 and Rb pathway abrogation. Under the same conditions, fibroblasts with these alterations failed to induce tumors, implying that genomic instability may be necessary but not sufficient for malignant transformation. These findings indicate that the minimum number of events required for malignant transformation of human fibroblasts is greater than has been enumerated by such oncogene addition strategies and support a stochastic cancer progression model initiated by four defined cellular alterations. [Cancer Res 2008;68(5):1417–26]
Data from Inhibition of Endoplasmic Reticulum Stress–Induced Apoptosis of Melanoma Cells by the ARC Protein
Authors: Chen Li Hua , Jiang Chen Chen , Watts Ralph , Thorne Rick F. , Kiejda Kelly A. , Zhang Xu Dong , Hersey Peter
Published: March 2023

Abstract
We have shown previously that most melanoma cell lines are insensitive to endoplasmic reticulum (ER) stress–induced apoptosis, but resistance can be reversed through activation of caspase-4 by inhibition of the MEK/ERK pathway. We report in this study that apoptosis was induced by the ER stress inducer thapsigargin or tunicamycin via a caspase-8–mediated pathway in the melanoma cell line Me1007, although the MEK/ERK pathway was activated in this cell line. The high sensitivity of Me1007 to ER stress–induced apoptosis was associated with low expression levels of the apoptosis repressor with caspase recruitment domain (ARC) protein that was expressed at relatively high levels in the resistant melanoma cell lines. Transfection of cDNA encoding ARC into Me1007 cells inhibited both caspase-8 activation and apoptosis induced by thapsigargin or tunicamycin. In contrast, inhibition of ARC by small interfering RNA knockdown sensitized the resistant melanoma cell lines to ER stress–induced apoptosis, which was inhibitable by blockage of caspase-8 activation. Both exogenous and endogenous ARC seemed to predominantly locate to the cytoplasm and mitochondria and could be coimmunoprecipitated with caspase-8. Taken together, ER stress can potentially activate multiple apoptosis signaling pathways in melanoma cells in a context-dependent manner. Whereas the MEK/ERK signaling pathway plays an important role in inhibiting ER stress–induced caspase-4 activation, ARC seems to be critical in blocking activation of casapse-8 in melanoma cells subjected to ER stress. [Cancer Res 2008;68(3):834–42]
Data from Improved Cellular Pharmacokinetics and Pharmacodynamics Underlie the Wide Anticancer Activity of Sagopilone
Authors: Hoffmann Jens , Vitale Ilio , Buchmann Bernd , Galluzzi Lorenzo , Schwede Wolfgang , Senovilla Laura , Skuballa Werner , Vivet Sonia , Lichtner Rosemarie B. , Vicencio José M. , Panaretakis Theocharis , Siemeister Gerhard , Lage Hermann , Nanty Lisa , Hammer Stefanie , Mittelstaedt Kevin , Winsel Sebastian , Eschenbrenner Julia , Castedo Maria , Demarche Carine , Klar Ulrich , Kroemer Guido
Published: March 2023

Abstract
Sagopilone (ZK-EPO) is the first fully synthetic epothilone undergoing clinical trials for the treatment of human tumors. Here, we investigate the cellular pathways by which sagopilone blocks tumor cell proliferation and compare the intracellular pharmacokinetics and the in vivo pharmacodynamics of sagopilone with other microtubule-stabilizing (or tubulin-polymerizing) agents. Cellular uptake and fractionation/localization studies revealed that sagopilone enters cells more efficiently, associates more tightly with the cytoskeleton, and polymerizes tubulin more potently than paclitaxel. Moreover, in contrast to paclitaxel and other epothilones [such as the natural product epothilone B (patupilone) or its partially synthetic analogue ixabepilone], sagopilone is not a substrate of the P-glycoprotein efflux pumps. Microtubule stabilization by sagopilone caused mitotic arrest, followed by transient multinucleation and activation of the mitochondrial apoptotic pathway. Profiling of the proapoptotic signal transduction pathway induced by sagopilone with a panel of small interfering RNAs revealed that sagopilone acts similarly to paclitaxel. In HCT 116 colon carcinoma cells, sagopilone-induced apoptosis was partly antagonized by the knockdown of proapoptotic members of the Bcl-2 family, including Bax, Bak, and Puma, whereas knockdown of Bcl-2, Bcl-XL, or Chk1 sensitized cells to sagopilone-induced cell death. Related to its improved subcellular pharmacokinetics, however, sagopilone is more cytotoxic than other epothilones in a large panel of human cancer cell lines in vitro and in vivo. In particular, sagopilone is highly effective in reducing the growth of paclitaxel-resistant cancer cells. These results underline the processes behind the therapeutic efficacy of sagopilone, which is now evaluated in a broad phase II program. [Cancer Res 2008;68(13):5301–8]
Data from MicroRNA-Mediated Inhibition of Prostate-Derived Ets Factor Messenger RNA Translation Affects Prostate-Derived Ets Factor Regulatory Networks in Human Breast Cancer
Authors: Findlay Victoria J. , Turner David P. , Moussa Omar , Watson Dennis K.
Published: March 2023

Abstract
Prostate-derived Ets factor (PDEF) is an ETS transcription factor expressed in normal tissues with high epithelial cell content and noninvasive breast cancer cells. A putative tumor suppressor PDEF protein expression is often lost during progression to a more invasive phenotype. Interestingly, PDEF mRNA has been found to be retained or even overexpressed in the absence of protein; however, the mechanisms for this remain to be elucidated. This study identifies two microRNAs (miRNA) that directly act on and repress PDEF mRNA translation, leading to the loss of PDEF protein expression and the gain of phenotypes associated with invasive cells. In addition, we show that these miRNAs are elevated in human breast tumor samples. Together, these data describe a mechanism of regulation that explains, for the first time, the lack of correlation between PDEF mRNA and protein levels, providing insight into the underexplored role of posttranscriptional regulation and how this contributes to dysregulated protein expression in cancer. These observations have critical implications for therapeutically targeting miRNAs that contribute to cancer progression. [Cancer Res 2008;68(20):8499–506]
Data from Cell Type Variation in Responses to Antimitotic Drugs that Target Microtubules and Kinesin-5
Authors: Shi Jue , Orth James D. , Mitchison Tim
Published: March 2023

Abstract
To improve cancer chemotherapy, we need to understand the mechanisms that determine drug sensitivity in cancer and normal cells. Here, we investigate this question across a panel of 11 cell lines at a phenotypic and molecular level for three antimitotic drugs: paclitaxel, nocodazole, and an inhibitor of kinesin-5 (also known as KSP, Eg5, Kif11). Using automated microscopy with markers for mitosis and apoptosis (high content screening), we find that the mitotic arrest response shows relatively little variation between cell types, whereas the tendency to undergo apoptosis shows large variation. We found no correlation between levels of mitotic arrest and apoptosis. Apoptosis depended on entry into mitosis and occurred both from within mitosis and after exit. Response to the three drugs strongly correlated, although paclitaxel caused more apoptosis in some cell lines at similar levels of mitotic arrest. Molecular investigations showed that sensitivity to apoptosis correlated with loss of an antiapoptotic protein, XIAP, during the drug response, but not its preresponse levels, and to some extent also correlated with activation of the p38 and c-Jun NH2 kinase pathways. We conclude that variation in sensitivity to antimitotic drugs in drug-naive cell lines is governed more by differences in apoptotic signaling than by differences in mitotic spindle or spindle assembly checkpoint proteins and that antimitotics with different mechanisms trigger very similar, but not identical, responses. [Cancer Res 2008;68(9):3269–76]
Data from Targeting Heat Shock Protein 90 Overrides the Resistance of Lung Cancer Cells by Blocking Radiation-Induced Stabilization of Hypoxia-Inducible Factor-1α
Authors: Kim Woo-Young , Oh Seung Hyun , Woo Jong-Kyu , Hong Waun Ki , Lee Ho-Young
Published: March 2023

Abstract
Hypoxia-inducible factor-1 (HIF-1) has been suggested to play a major role in tumor radioresistance. However, the mechanisms through which irradiation regulates HIF-1α expression remain unclear. The purpose of this study was to investigate the mechanisms that mediate HIF-1 activation and thus radioresistance. Here, we show that irradiation induces survival and angiogenic activity in a subset of radioresistant lung cancer cell lines by elevating HIF-1α protein expression. Radiation induced HIF-1α protein expression mainly through two distinct pathways, including an increase in de novo protein synthesis via activation of phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) and stabilization of HIF-1α protein via augmenting the interaction between heat shock protein 90 (Hsp90) and HIF-1α protein. Whereas the PI3K/Akt/mTOR pathway was activated by irradiation in all the lung cancer cells examined, the Hsp90-HIF-1α interaction was enhanced in the resistant cells only. Inhibition of Hsp90 function by 17-allylamino-17-demethoxygeldanamycin or deguelin, a novel natural inhibitor of Hsp90, suppressed increases in HIF-1α/Hsp90 interaction and HIF-1α expression in radioresistant cells. Furthermore, combined treatment of radiation with deguelin significantly decreased the survival and angiogenic potential of radioresistant lung cancer cells in vitro. We finally determined in vivo that systemic administration of deguelin resulted in profound inhibition of tumor growth and angiogenesis when combined with radiation. These results provide a strong rationale to target Hsp90 as a means to block radiation-induced HIF-1α and thus to circumvent radioresistance in lung cancer cells. [Cancer Res 2009;69(4):1624–32]
Data from Carcinogen-Induced Gene Promoter Hypermethylation Is Mediated by DNMT1 and Causal for Transformation of Immortalized Bronchial Epithelial Cells
Authors: Damiani Leah A. , Yingling Christin M. , Leng Shuguang , Romo Paul E. , Nakamura Jun , Belinsky Steven A.
Published: March 2023

Abstract
A better understanding of key molecular changes during transformation of lung epithelial cells could affect strategies to reduce mortality from lung cancer. This study uses an in vitro model to identify key molecular changes that drive cell transformation and the likely clonal outgrowth of preneoplastic lung epithelial cells that occurs in the chronic smoker. Here, we show differences in transformation efficiency associated with DNA repair capacity for two hTERT/cyclin-dependent kinase 4, immortalized bronchial epithelial cell lines after low-dose treatment with the carcinogens methylnitrosourea, benzo(a)pyrene-diolepoxide 1, or both for 12 weeks. Levels of cytosine-DNA methyltransferase 1 (DNMT1) protein increased significantly during carcinogen exposure and were associated with the detection of promoter hypermethylation of 5 to 10 genes in each transformed cell line. Multiple members of the cadherin gene family were commonly methylated during transformation. Stable knockdown of DNMT1 reversed transformation and gene silencing. Moreover, stable knockdown of DNMT1 protein before carcinogen treatment prevented transformation and methylation of cadherin genes. These studies provide a mechanistic link between increased DNMT1 protein, de novo methylation of tumor suppressor genes, and reduced DNA repair capacity that together seem causal for transformation of lung epithelial cells. This finding supports the development of demethylation strategies for primary prevention of lung cancer in smokers. [Cancer Res 2008;68(21):9005–14]
Data from Maintenance of Constitutive IκB Kinase Activity by Glycogen Synthase Kinase-3α/β in Pancreatic Cancer
Authors: Wilson Willie , Baldwin Albert S.
Published: March 2023

Abstract
Constitutive nuclear factor κB (NF-κB) activation is among the many deregulated signaling pathways that are proposed to drive pancreatic cancer cell growth and survival. Recent reports suggest that glycogen synthase kinase-3β (GSK-3β) plays a key role in maintaining basal NF-κB target gene expression and cell survival in pancreatic cancer cell lines. However, the mechanism by which GSK-3β facilitates constitutive NF-κB signaling in pancreatic cancer remains unclear. In this report, we analyze the contributions of both GSK-3 isoforms (GSK-3α and GSK-3β) in regulating NF-κB activation and cell proliferation in pancreatic cancer cell lines (Panc-1 and MiaPaCa-2). We show that GSK-3 isoforms are differentially required to maintain basal NF-κB DNA binding activity, transcriptional activity, and cell proliferation in Panc-1 and MiaPaCa-2 cells. Our data also indicate that IκB kinase (IKK) subunits are not equally required to regulate pancreatic cancer–associated NF-κB activity and cell growth. Importantly, we provide the first evidence that GSK-3 maintains constitutive NF-κB signaling in pancreatic cancer by regulating IKK activity. These data provide new insight into GSK-3–dependent NF-κB regulation and further establish GSK-3 and IKK as potential therapeutic targets for pancreatic cancer. [Cancer Res 2008;68(19):8156–63]
Data from ArgBP2-Dependent Signaling Regulates Pancreatic Cell Migration, Adhesion, and Tumorigenicity
Authors: Taieb David , Roignot Julie , André Frédéric , Garcia Stéphane , Masson Bénédicte , Pierres Anne , Iovanna Juan-Lucio , Soubeyran Philippe
Published: March 2023

Abstract
The poor prognosis of pancreatic cancer is due to rapid locoregional invasion, the early development of metastases, and the limited efficacy of current therapies. To date, none of the identified oncogenes and suppressors involved in this disease have led to efficient treatments. Here, we describe that the scaffold protein ArgBP2 is repressed during oncogenic transformation of the pancreas. We could show, using a pancreatic cancer cell line model, that this repression of ArgBP2 participates in the progression of this disease. Interestingly, in vitro analyses revealed that the antitumoral potential of ArgBP2 is linked to the control of cell adhesion and migration rather than to the regulation of cell proliferation or sensitivity to apoptosis. Moreover, we could detail part of the molecular mechanism responsible by identifying new ArgBP2-interacting proteins, and show that this function is partly achieved by the control of a WAVE/PTP-PEST/c-Abl signaling complex. These findings point to a new mechanism of pancreatic cancer progression leading to invasion and metastasis and suggest that the ArgBP2 signaling pathway could represent a new target for cancer therapy. [Cancer Res 2008;68(12):4588–96]
Data from Antimyeloma Activity of the Orally Bioavailable Dual Phosphatidylinositol 3-Kinase/Mammalian Target of Rapamycin Inhibitor NVP-BEZ235
Authors: McMillin Douglas W. , Ooi Melissa , Delmore Jake , Negri Joseph , Hayden Patrick , Mitsiades Nicolas , Jakubikova Jana , Maira Sauveur-Michel , Garcia-Echeverria Carlos , Schlossman Robert , Munshi Nikhil C. , Richardson Paul G. , Anderson Kenneth C. , Mitsiades Constantine S.
Published: March 2023

Abstract
The phosphatidylinositol 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) pathway mediates proliferation, survival, and drug resistance in multiple myeloma (MM) cells. Here, we tested the anti-MM activity of NVP-BEZ235 (BEZ235), which inhibits PI3K/Akt/mTOR signaling at the levels of PI3K and mTOR. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric survival assays showed that MM cell lines exhibited dose- and time-dependent decreased viability after exposure to BEZ235 (IC50, 25–800 nmol/L for 48 hours). MM cells highly sensitive (IC50, <25 nmol/L) to BEZ235 (e.g., MM.1S, MM.1R, Dox40, and KMS-12-PE) included both lines sensitive and resistant to conventional (dexamethasone, cytotoxic chemotherapeutics) agents. Pharmacologically relevant BEZ235 concentrations (25–400 nmol/L) induced rapid commitment to and induction of MM.1S and OPM-2 cell death. Furthermore, normal donor peripheral blood mononuclear cells were less sensitive (IC50, >800 nmol/L) than the majority of MM cell lines tested, suggesting a favorable therapeutic index. In addition, BEZ235 was able to target MM cells in the presence of exogenous interleukin-6, insulin-like growth factor-1, stromal cells, or osteoclasts, which are known to protect against various anti-MM agents. Molecular profiling revealed that BEZ235 treatment decreased the amplitude of transcriptional signatures previously associated with myc, ribosome, and proteasome function, as well as high-risk MM and undifferentiated human embryonic stem cells. In vivo xenograft studies revealed significant reduction in tumor burden (P = 0.011) and survival (P = 0.028) in BEZ235-treated human MM tumor-bearing mice. Combinations of BEZ235 with conventional (e.g., dexamethasone and doxorubicin) or novel (e.g., bortezomib) anti-MM agents showed lack of antagonism. These results indicate that BEZ235 merits clinical testing, alone and in combination with other agents, in MM. [Cancer Res 2009;69(14):5835–42]
Data from Magnetic Resonance Imaging Defines Cervicovaginal Anatomy, Cancer, and VEGF Trap Antiangiogenic Efficacy in Estrogen-Treated K14-HPV16 Transgenic Mice
Authors: Garbow Joel R. , Santeford Andrea C. , Anderson Jeff R. , Engelbach John A. , Arbeit Jeffrey M.
Published: March 2023

Abstract
Noninvasive detection of dysplasia provides a potential platform for monitoring the efficacy of chemopreventive therapy of premalignancy, imaging the tissue compartments comprising dysplasia: epithelium, microvasculature, and stromal inflammatory cells. Here, using respiratory-gated magnetic resonance imaging (MRI), the anatomy of premalignant and malignant stages of cervical carcinogenesis in estrogen-treated K14-HPV16 transgenic mice was noninvasively defined. Dynamic contrast enhanced (DCE)-MRI was used to quantify leakage across premalignant dysplastic microvasculature. Vascular permeability as measured by DCE-MRI, Ktrans, was similar in transgenic (0.053 ± 0.020 min−1; n = 32 mice) and nontransgenic (0.056 ± 0.029 min−1; n = 17 mice) animals despite a 2-fold increase in microvascular area in the former compared with the latter. DCE-MRI did detect a significant decrease in vascular permeability accompanying diminution of dysplastic microvasculature by the antiangiogenic agent, vascular endothelial growth factor Trap (Ktrans = 0.052 ± 0.013 min−1 pretreatment; n = 6 mice versus Ktrans = 0.019 ± 0.008 min−1 post-treatment; n = 5 mice). Thus, we determined that the threshold of microvessel leakage associated with cervical dysplasia was <17 kDa and highlighted the potential of DCE-MRI to noninvasively monitor the efficacy of antiangiogenic drugs or chemoprevention regimens targeting the vasculature in premalignant cervical dysplasia. [Cancer Res 2009;69(20):7945–52]
Data from Genomic Profiling of MicroRNAs in Bladder Cancer: miR-129 Is Associated with Poor Outcome and Promotes Cell Death In vitro
Authors: Dyrskjøt Lars , Ostenfeld Marie S. , Bramsen Jesper B. , Silahtaroglu Asli N. , Lamy Philippe , Ramanathan Ramshanker , Fristrup Niels , Jensen Jens L. , Andersen Claus L. , Zieger Karsten , Kauppinen Sakari , Ulhøi Benedicte P. , Kjems Jørgen , Borre Michael , Ørntoft Torben F.
Published: March 2023

Abstract
microRNAs (miRNA) are involved in cancer development and progression, acting as tumor suppressors or oncogenes. Here, we profiled the expression of 290 unique human miRNAs in 11 normal and 106 bladder tumor samples using spotted locked nucleic acid–based oligonucleotide microarrays. We identified several differentially expressed miRNAs between normal urothelium and cancer and between the different disease stages. miR-145 was found to be the most down-regulated in cancer compared with normal, and miR-21 was the most up-regulated in cancer. Furthermore, we identified miRNAs that significantly correlated to the presence of concomitant carcinoma in situ. We identified several miRNAs with prognostic potential for predicting disease progression (e.g., miR-129, miR-133b, and miR-518c*). We localized the expression of miR-145, miR-21, and miR-129 to urothelium by in situ hybridization. We then focused on miR-129 that exerted significant growth inhibition and induced cell death upon transfection with a miR-129 precursor in bladder carcinoma cell lines T24 and SW780 cells. Microarray analysis of T24 cells after transfection showed significant miR-129 target down-regulation (P = 0.0002) and pathway analysis indicated that targets were involved in cell death processes. By analyzing gene expression data from clinical tumor samples, we identified significant expression changes of target mRNA molecules related to the miRNA expression. Using luciferase assays, we documented a direct link between miR-129 and the two putative targets GALNT1 and SOX4. The findings reported here indicate that several miRNAs are differentially regulated in bladder cancer and may form a basis for clinical development of new biomarkers for bladder cancer. [Cancer Res 2009;69(11):4851–60]
Data from E-Cadherin Deficiency Initiates Gastric Signet-Ring Cell Carcinoma in Mice and Man
Authors: Humar Bostjan , Blair Vanessa , Charlton Amanda , More Helen , Martin Iain , Guilford Parry
Published: March 2023

Abstract
The importance of loss of the cell-cell adhesion molecule E-cadherin (encoded by CDH1) to tumor progression is well established. However, CDH1 germ-line mutations predispose to the cancer susceptibility syndrome hereditary diffuse gastric cancer (HDGC), suggesting a role for E-cadherin in tumor initiation. The earliest indications of cancer in the stomachs of CDH1 mutation carriers are microscopic foci of intramucosal signet-ring cell carcinoma (SRCC; designated “eHDGC”). Here, we used N-methyl-N-nitrosourea (MNU) to promote gastric carcinogenesis in wild-type (wt) and cdh1+/− mice. MNU induced a variety of gastric tumors; however, intramucosal SRCC developed with an 11 times higher incidence in cdh1+/− mice compared with wt mice. The murine SRCC resembled the human eHDGCs in that they were hypoproliferative, lacked nuclear β-catenin accumulation, and had reduced membrane localization of E-cadherin and its interacting junctional proteins. The down-regulation of E-cadherin in the murine SRCCs confirmed the importance of the second CDH1 hit to the initiation of diffuse gastric cancer. CDH1 promoter hypermethylation has been proposed to be a major second hit in advanced HDGC; however, its contribution to eHDGC was unknown. We thus examined a series of human eHDGC and detected CDH1 promoter methylation in 50% of foci. Promoter methylation was accompanied by reduced wt CDH1 mRNA levels in the foci and had a monoclonal pattern, consistent with an epigenetic initiation of disease. Together, these findings provide compelling evidence for a deficiency in cell-to-cell adhesion being sufficient to initiate diffuse gastric cancer in the absence of hyperproliferation and β-catenin activation. [Cancer Res 2009;69(5):2050–6]
Data from Hyperactivation of the Insulin-like Growth Factor Receptor I Signaling Pathway Is an Essential Event for Cisplatin Resistance of Ovarian Cancer Cells
Authors: Eckstein Niels , Servan Kati , Hildebrandt Barbara , Pölitz Anne , Jonquières Georg von , Wolf-Kümmeth Sybille , Napierski Inge , Hamacher Alexandra , Kassack Matthias U. , Budczies Jan , Beier Manfred , Dietel Manfred , Royer-Pokora Brigitte , Denkert Carsten , Royer Hans-Dieter
Published: March 2023

Abstract
Platinum plays a central role in the therapy of ovarian cancer, and the emergence of platinum resistance is a major obstacle for clinical management of the disease. We treated A2780 ovarian cancer cells by weekly cycles of cisplatin over a period of 6 months and unveiled that enhanced insulin-like growth factor I receptor (IGF-IR) expression and autocrine IGF-I are associated with hyperactivation of the IGF-IR and phosphatidylinositol-3-OH kinase (PI3K) pathways in cisplatin-resistant cells. IGF-IR expression levels increased during treatment cycles and correlated with cisplatin resistance. Purified IGF-I induced cisplatin resistance in diverse ovarian cancer cell lines, and small molecule inhibitors proved that IGF-IR and PI3K are essential for cisplatin resistance. Similar results were obtained with BG-1 ovarian cancer cells. Cytogenetic and array comparative genomic hybridization analyses revealed selection and de novo formation of chromosomal alterations during resistance development. An analysis of gene expression profiles of primary ovarian carcinomas identified the regulatory subunit PIK3R2 of PI3-kinase as a significant negative prognosis factor for ovarian cancer. We conclude that targeting the IGF-IR and the PI3K pathways is a promising new strategy to treat cisplatin-resistant ovarian carcinomas. [Cancer Res 2009;69(7):2996–3003]
Data from The Role of the Mouse Y Chromosome on Susceptibility to Testicular Germ Cell Tumors
Authors: Anderson Philip D. , Lam Man-Yee , Poirier Christophe , Bishop Colin E. , Nadeau Joseph H.
Published: March 2023

Abstract
Testicular germ cell tumors (TGCT) are sex limited, occurring only in males with a Y chromosome. Recently, the gr/gr deletion on the human Y chromosome was associated with increased risk of TGCTs. In addition, the presence of Y chromosome sequences is associated with TGCTs in cases of gonadal dysgenesis. TGCTs in strain 129 males recapitulate many aspects of testicular cancer in human infants and can be used to evaluate the role of the Y chromosome in TGCT risk. We used chromosome substitution strains and a sex-reversing mutant to test the role of the Y chromosome on TGCT susceptibility. Our results show that a Y-linked gene that does not differ among the tested strains is essential for tumorigenesis. [Cancer Res 2009;69(8):3614–8]
Data from p27Kip1 Stabilization Is Essential for the Maintenance of Cell Cycle Arrest in Response to DNA Damage
Authors: Cuadrado Myriam , Gutierrez-Martinez Paula , Swat Aneta , Nebreda Angel R. , Fernandez-Capetillo Oscar
Published: March 2023

Abstract
One of the current models of cancer proposes that oncogenes activate a DNA damage response (DDR), which would limit the growth of the tumor in its earliest stages. In this context, and in contrast to studies focused on the acute responses to a one-time genotoxic insult, understanding how cells respond to a persistent source of DNA damage might become critical for future studies in the field. We here report the discovery of a novel damage-responsive pathway, which involves p27Kip1 and retinoblastoma tumor suppressors and is only implemented after a persistent exposure to clastogens. In agreement with its late activation, we show that this pathway is critical for the maintenance, but not the initiation, of the cell cycle arrest triggered by DNA damage. Interestingly, this late response is independent of the canonical ataxia telangiectasia mutated–dependent and ataxia telangiectasia mutated and Rad3-related–dependent DDR but downstream of p38 mitogen-activated protein kinase. Our results might help to reconcile the oncogene-induced DNA damage model with the clinical evidence that points to non-DDR members as the most important tumor suppressors in human cancer. [Cancer Res 2009;69(22):8726–32]
Data from GPR109A Is a G-protein–Coupled Receptor for the Bacterial Fermentation Product Butyrate and Functions as a Tumor Suppressor in Colon
Authors: Thangaraju Muthusamy , Cresci Gail A. , Liu Kebin , Ananth Sudha , Gnanaprakasam Jaya P. , Browning Darren D. , Mellinger John D. , Smith Sylvia B. , Digby Gregory J. , Lambert Nevin A. , Prasad Puttur D. , Ganapathy Vadivel
Published: March 2023

Abstract
Short-chain fatty acids, generated in colon by bacterial fermentation of dietary fiber, protect against colorectal cancer and inflammatory bowel disease. Among these bacterial metabolites, butyrate is biologically most relevant. GPR109A is a G-protein–coupled receptor for nicotinate but recognizes butyrate with low affinity. Millimolar concentrations of butyrate are needed to activate the receptor. Although concentrations of butyrate in colonic lumen are sufficient to activate the receptor maximally, there have been no reports on the expression/function of GPR109A in this tissue. Here we show that GPR109A is expressed in the lumen-facing apical membrane of colonic and intestinal epithelial cells and that the receptor recognizes butyrate as a ligand. The expression of GPR109A is silenced in colon cancer in humans, in a mouse model of intestinal/colon cancer, and in colon cancer cell lines. The tumor-associated silencing of GPR109A involves DNA methylation directly or indirectly. Reexpression of GPR109A in colon cancer cells induces apoptosis, but only in the presence of its ligands butyrate and nicotinate. Butyrate is an inhibitor of histone deacetylases, but apoptosis induced by activation of GPR109A with its ligands in colon cancer cells does not involve inhibition of histone deacetylation. The primary changes in this apoptotic process include down-regulation of Bcl-2, Bcl-xL, and cyclin D1 and up-regulation of death receptor pathway. In addition, GPR109A/butyrate suppresses nuclear factor-κB activation in normal and cancer colon cell lines as well as in normal mouse colon. These studies show that GPR109A mediates the tumor-suppressive effects of the bacterial fermentation product butyrate in colon. [Cancer Res 2009;69(7):2826–32]
Data from RASSF1A Mediates p21Cip1/Waf1-Dependent Cell Cycle Arrest and Senescence through Modulation of the Raf-MEK-ERK Pathway and Inhibition of Akt
Authors: Thaler Sonja , Hähnel Patricia S. , Schad Arno , Dammann Reinhard , Schuler Martin
Published: March 2023

Abstract
Promoter hypermethylation preventing expression of the RAS association domain family 1 isoform A (RASSF1A) gene product is among the most abundant epigenetic deregulations in human cancer. Restoration of RASSF1A inhibits tumor cell growth in vitro and in murine xenograft models. Rassf1a-deficient mice feature increased spontaneous and carcinogen-induced tumor formation. Mechanistically, RASSF1A affects several cellular functions, such as microtubule dynamics, migration, proliferation, and apoptosis; however, its tumor-suppressive mechanism is incompletely understood. To study the functional consequences of RASSF1A expression in human cancer cells, we made use of a doxycycline-inducible expression system and a RASSF1A-deficient lung cancer cell line. We observed that RASSF1A induces cell cycle arrest in G1 phase and senescence in vitro and in tumors established in immunodeficient mice. RASSF1A-mediated growth inhibition was accompanied by the up-regulation of the cyclin-dependent kinase inhibitor p21Cip1/Waf1 and proceeded independently of p53, p14Arf, and p16Ink4a. Loss of p21Cip1/Waf1 or coexpression of the human papilloma virus 16 oncoprotein E7 was found to override RASSF1A-induced cell cycle arrest and senescence. Conditional RASSF1A affected mitogen-activated protein kinase and protein kinase B/Akt signaling to up-regulate p21Cip1/Waf1 and to facilitate its nuclear localization. In summary, RASSF1A can mediate cell cycle arrest and senescence in human cancer cells by p53-independent regulation of p21Cip1/Waf1. [Cancer Res 2009;69(5):1748–57]
Data from Inhibition of Azoxymethane-Induced Colorectal Cancer by CP-31398, a TP53 Modulator, Alone or in Combination with Low Doses of Celecoxib in Male F344 Rats
Authors: Rao Chinthalapally V. , Steele Vernon E. , Swamy Malisetty V. , Patlolla Jagan M.R. , Guruswamy Suresh , Kopelovich Levy
Published: March 2023

Abstract
Tumor suppressor p53 plays a major role in colorectal cancer development. The present study explores the effects of p53-modulating agent CP-31398 alone and combined with celecoxib on azoxymethane-induced aberrant crypt foci (ACF) and colon adenocarcinomas in F344 rats. Maximum tolerated doses were 400 and 3,000 ppm for CP-31398 and celecoxib, respectively. ACF and tumor efficacy endpoints were carried out on azoxymethane-treated 7-week-old rats (48 per group) fed the control AIN-76A diet. Two weeks after carcinogen treatment, rats were fed the diets containing 0, 150, or 300 ppm CP-31398, 300 ppm celecoxib, or 150 ppm CP-31398 plus 300 ppm celecoxib. ACF and colon adenocarcinomas were determined at 8 and 48 weeks after azoxymethane treatment, respectively. Dietary CP-31398 was shown to suppress mean colonic total ACF by 43% and multicrypt ACF by 63%; dietary CP-31398 at 150 and 300 ppm suppressed adenocarcinoma incidence by 30.4% (P < 0.02) and 44% (P < 0.005), respectively, and adenocarcinoma multiplicity by 51% (P < 0.005) and 65% (P < 0.0001), respectively. Dietary celecoxib suppressed colon adenocarcinoma incidence (60%; P < 0.0003) and multiplicity (70%; P < 0.0001). Importantly, combination of low-dose CP-31398 and celecoxib suppressed colon adenocarcinoma incidence by 78% and multiplicity by 90%. Rats that were fed the high-dose CP-31398 or a combination of low-dose CP-31398 and celecoxib showed considerable enhancement of p53 and p21WAF1/CIP expression, apoptosis, and reduced tumor cell proliferation in colonic tumors. These observations show, for the first time, that CP-31398 possesses significant dose-dependent chemopreventive activity in a well-established colon cancer model and that a combination of low-dose CP-31398 and celecoxib significantly enhanced colon cancer chemopreventive efficacy. [Cancer Res 2009;69(20):8175–82]
Data from 17β-Hydroxysteroid Dehydrogenase Type 12 in Human Breast Carcinoma: A Prognostic Factor via Potential Regulation of Fatty Acid Synthesis
Authors: Nagasaki Shuji , Suzuki Takashi , Miki Yasuhiro , Akahira Jun-ichi , Kitada Kunio , Ishida Takanori , Handa Hiroshi , Ohuchi Noriaki , Sasano Hironobu
Published: March 2023

Abstract
17β-Hydroxysteroid dehydrogenase type 12 (17β-HSD12) has been shown to be involved in elongation of very long chain fatty acid (VLCFA) as well as in biosynthesis of estradiol (E2). 17β-HSD12 expression was also reported in breast carcinomas but its functions have remained unknown. In this study, we examined the correlation between mRNA expression profiles determined by microarray analysis and tissue E2 concentrations obtained from 16 postmenopausal breast carcinoma cases. No significant correlations were detected between 17β-HSD12 expression and E2 concentration. We then immunolocalized this enzyme in 110 cases of invasive ductal carcinoma. 17β-HSD12 immunoreactivity in breast carcinoma cells was significantly associated with poor prognosis of the patients. We further examined the biological significance of 17β-HSD12 using cell-based studies. Small interfering RNA–mediated knockdown of 17β-HSD12 in SK-BR-3 (estrogen receptor–negative breast carcinoma cell line) resulted in significant growth inhibition, which was recovered by the addition of VLCFAs such as arachidonic acid. The status of 17β-HSD12 immunoreactivity was also correlated with adverse clinical outcome in cyclooxygenase 2 (COX2)–positive breast cancer patients but not in COX2-negative patients. Therefore, these findings indicated that 17β-HSD12 was not necessarily related to intratumoral E2 biosynthesis, at least in human breast carcinoma, but was rather correlated with production of VLCFAs such as arachidonic acid, which may subsequently be metabolized to prostaglandins by COX2 and result in tumor progression of the patients. [Cancer Res 2009;69(4):1392–9]
Data from Cancer-Specific Transgene Expression Mediated by Systemic Injection of Nanoparticles
Authors: Chisholm Edward J. , Vassaux Georges , Martin-Duque Pilar , Chevre Raphael , Lambert Olivier , Pitard Bruno , Merron Andrew , Weeks Mark , Burnet Jerome , Peerlinck Inge , Dai Ming-Shen , Alusi Ghassan , Mather Stephen J. , Bolton Katherine , Uchegbu Ijeoma F. , Schatzlein Andreas G. , Baril Patrick
Published: March 2023

Abstract
The lack of safe and efficient systemic gene delivery vectors has largely reduced the potential of gene therapy in the clinic. Previously, we have reported that polypropylenimine dendrimer PPIG3/DNA nanoparticles are capable of tumor transfection upon systemic administration in tumor-bearing mice. To be safely applicable in the clinic, it is crucial to investigate the colloidal stability of nanoparticles and to monitor the exact biodistribution of gene transfer in the whole body of the live subject. Our biophysical characterization shows that dendrimers, when complexed with DNA, are capable of forming spontaneously in solution a supramolecular assembly that possesses all the features required to diffuse in experimental tumors through the enhanced permeability and retention effect. We show that these nanoparticles are of sizes ranging from 33 to 286 nm depending on the DNA concentration, with a colloidal stable and well-organized fingerprint-like structure in which DNA molecules are condensed with an even periodicity of 2.8 nm. Whole-body nuclear imaging using small-animal nano-single-photon emission computed tomography/computer tomography scanner and the human Na/I symporter (NIS) as reporter gene shows unique and highly specific tumor targeting with no detection of gene transfer in any of the other tissues of tumor-bearing mice. Tumor-selective transgene expression was confirmed by quantitative reverse transcription-PCR at autopsy of scanned animals, whereas genomic PCR showed that the tumor sites are the predominant sites of nanoparticle accumulation. Considering that NIS imaging of transgene expression has been recently validated in humans, our data highlight the potential of these nanoparticles as a new formulation for cancer gene therapy. [Cancer Res 2009;69(6):2655–62]
Data from MMP13, Birc2 (cIAP1), and Birc3 (cIAP2), Amplified on Chromosome 9, Collaborate with p53 Deficiency in Mouse Osteosarcoma Progression
Authors: Ma Ou , Cai Wei-Wen , Zender Lars , Dayaram Tajhal , Shen Jianhe , Herron Alan J. , Lowe Scott W. , Man Tsz-Kwong , Lau Ching C. , Donehower Lawrence A.
Published: March 2023

Abstract
Osteosarcoma is the primary malignant cancer of bone and particularly affects adolescents and young adults, causing debilitation and sometimes death. As a model for human osteosarcoma, we have been studying p53+/− mice, which develop osteosarcoma at high frequency. To discover genes that cooperate with p53 deficiency in osteosarcoma formation, we have integrated array comparative genomic hybridization, microarray expression analyses in mouse and human osteosarcomas, and functional assays. In this study, we found seven frequent regions of copy number gain and loss in the mouse p53+/− osteosarcomas but have focused on a recurrent amplification event on mouse chromosome 9A1. This amplicon is syntenic with a similar chromosome 11q22 amplicon identified in several human tumor types. Three genes on this amplicon, the matrix metalloproteinase gene MMP13 and the antiapoptotic genes Birc2 (cIAP1) and Birc3 (cIAP2), show elevated expression in mouse and human osteosarcomas. We developed a functional assay using clonal osteosarcoma cell lines transduced with lentiviral short hairpin RNA vectors to show that down-regulation of MMP13, Birc2, or Birc3 resulted in reduced tumor growth when transplanted into immunodeficient recipient mice. These experiments revealed that high MMP13 expression enhances osteosarcoma cell survival and that Birc2 and Birc3 also enhance cell survival but only in osteosarcoma cells with the chromosome 9A1 amplicon. We conclude that the antiapoptotic genes Birc2 and Birc3 are potential oncogenic drivers in the chromosome 9A1 amplicon. [Cancer Res 2009;69(6):2559–67