bluuebunny/crossref_metadata_2025 huggingface dataset:
Certification Schemes and the Impacts on Forests and Forestry
Authors: Auld Graeme , Gulbrandsen Lars H. , McDermott Constance L.
Published: August 2008

Certification schemes have emerged in recent years to become a significant and innovative venue for standard setting and governance in the environmental realm. This review examines these schemes in the forest sector where, arguably, their development is among the most advanced of the sustainability labeling initiatives. Beginning with the origins, history, and features of schemes, the review synthesizes and assesses what we know about the direct effects and broader consequences of forest certification. Bearing in mind underlying factors affecting producers' decisions to certify, direct effects are examined by describing the uptake of schemes, the improvements to management of audited forests, and the ameliorative potential of certification for landscape-level concerns such as deforestation and forest protection. In assessing broader consequences, we look beyond the instrument itself to detail positive and negative unintended consequences, spillover effects, and longer-term and slow-moving effects that flow from the emergence of the certification innovation.
Visualizing form and function in organotypic slices of the adult mouse parotid gland
Authors: Warner Jennifer D. , Peters Christian G. , Saunders Rudel , Won Jong Hak , Betzenhauser Matthew J. , Gunning William T. , Yule David I. , Giovannucci David R.
Published: August 2008

An organotypic slice preparation of the adult mouse parotid salivary gland amenable to a variety of optical assessments of fluid and protein secretion dynamics is described. The semi-intact preparation rendered without the use of enzymatic treatment permitted live-cell imaging and multiphoton analysis of cellular and supracellular signals. Toward this end we demonstrated that the parotid slice is a significant addition to the repertoire of tools available to investigators to probe exocrine structure and function since there is currently no cell culture system that fully recapitulates parotid acinar cell biology. Importantly, we show that a subpopulation of the acinar cells of parotid slices can be maintained in short-term culture and retain their morphology and function for up to 2 days. This in vitro model system is a significant step forward compared with enzymatically dispersed acini that rapidly lose their morphological and functional characteristics over several hours, and it was shown to be long enough for the expression and trafficking of exogenous protein following adenoviral infection. This system is compatible with a variety of genetic and physiological approaches used to study secretory function.
Perivascular nitric oxide and superoxide in neonatal cerebral hypoxia-ischemia
Authors: Fabian Roderic H. , Perez-Polo J. Regino , Kent Thomas A.
Published: August 2008

Decreased cerebral blood flow (CBF) has been observed following the resuscitation from neonatal hypoxic-ischemic injury, but its mechanism is not known. We address the hypothesis that reduced CBF is due to a change in nitric oxide (NO) and superoxide anion O2− balance secondary to endothelial NO synthase (eNOS) uncoupling with vascular injury. Wistar rats (7 day old) were subjected to cerebral hypoxia-ischemia by unilateral carotid occlusion under isoflurane anesthesia followed by hypoxia with hyperoxic or normoxic resuscitation. Expired CO2 was determined during the period of hyperoxic or normoxic resuscitation. Laser-Doppler flowmetry was used with isoflurane anesthesia to monitor CBF, and cerebral perivascular NO and O2− were determined using fluorescent dyes with fluorescence microscopy. The effect of tetrahydrobiopterin supplementation on each of these measurements and the effect of apocynin and Nω-nitro-l-arginine methyl ester (l-NAME) administration on NO and O2− were determined. As a result, CBF in the ischemic cortex declined following the onset of resuscitation with 100% O2 (hyperoxic resuscitation) but not room air (normoxic resuscitation). Expired CO2 was decreased at the onset of resuscitation, but recovery was the same in normoxic and hyperoxic resuscitated groups. Perivascular NO-induced fluorescence intensity declined, and O2−-induced fluorescence increased in the ischemic cortex after hyperoxic resuscitation up to 24 h postischemia. l-NAME treatment reduced O2− relative to the nonischemic cortex. Apocynin treatment increased NO and reduced O2− relative to the nonischemic cortex. The administration of tetrahydrobiopterin following the injury increased perivascular NO, reduced perivascular O2−, and increased CBF during hyperoxic resuscitation. These results demonstrate that reduced CBF follows hyperoxic resuscitation but not normoxic resuscitation after neonatal hypoxic-ischemic injury, accompanied by a reduction in perivascular production of NO and an increase in O2−. The finding that tetrahydrobiopterin, apocynin, and l-NAME normalized radical production suggests that the uncoupling of perivascular NOS, probably eNOS, due to acquired relative tetrahydrobiopterin deficiency occurs after neonatal hypoxic-ischemic brain injury. It appears that both NOS uncoupling and the activation of NADPH oxidase participate in the changes of reactive oxygen concentrations seen in cerebral hypoxic-ischemic injury.
Cytochrome P-450 2C9 signaling does not contribute to age-associated vascular endothelial dysfunction in humans
Authors: Donato Anthony J. , Eskurza Iratxe , Jablonski Kristen L. , Gano Lindsey B. , Pierce Gary L. , Seals Douglas R.
Published: August 2008

Oxidative stress impairs endothelium-dependent dilation (EDD) with aging in healthy sedentary adults. Increased cytochrome P-450 2C9 (CYP 2C9) signaling can contribute to oxidative stress-mediated suppression of EDD, but its role in aging is unknown. We hypothesized that inhibition of CYP 2C9 signaling with sulfaphenazole would improve EDD in older, but not young, healthy sedentary adults. At baseline, increases in forearm blood flow (FBF; venous occlusion plethysmography) in response to brachial artery infusions of ACh (1, 2, 4, and 8 μg·100 ml forearm volume−1·min−1), an endothelium-dependent dilator, were smaller in older [ n = 14, 63 ± 1 (SE) yr] than in young ( n = 11, 23 ± 2 yr) adults ( P < 0.05), with a reduction in peak FBF of 32% (11.8 ± 1.7 vs. 17.3 ± 2.3 ml·100 ml tissue−1·min−1). Infusion of sulfaphenazole at doses that block CYP 2C9 signaling in humans did not affect the FBF responses to ACh in the older (peak FBF = 13.0 ± 4.3 ml·100 ml tissue−1·min−1, P = 0.41) or the young (peak FBF = 17.1 ± 1.9 ml·100 ml tissue−1·min−1, P = 0.55) adults. Coadministration of the nitric oxide inhibitor l-NMMA and sulfaphenazole decreased the FBF response to ACh in young and older subjects ( P < 0.05); the effect was smaller in the older subjects, but group differences in EDD remained ( P < 0.05). Endothelium-independent dilation assessed with sodium nitroprusside was not different in the young and older subjects. These results provide the first support for the concept that increased CYP 2C9 signaling does not contribute to impairments in EDD with aging in healthy adults.
Vascular Endothelial Growth Factor Reduces Tamoxifen Efficacy and Promotes Metastatic Colonization and Desmoplasia in Breast Tumors
Authors: Qu Zhican , Van Ginkel Sabrina , Roy Anshu M. , Westbrook Louise , Nasrin Mubina , Maxuitenko Yulia , Frost Andra R. , Carey Delicia , Wang Wenquan , Li Rongbao , Grizzle William E. , Thottassery Jaideep V. , Kern Francis G.
Published: August 2008

Clinical studies have shown that decreased tamoxifen effectiveness correlates with elevated levels of vascular endothelial growth factor (VEGF)-A165 in biopsy samples of breast cancers. To investigate the mechanisms underlying tamoxifen resistance and metastasis, we engineered the estrogen receptor (ER)–positive MCF-7 human breast cancer cell line to express VEGF to clinically relevant levels in a doxycycline-regulated manner. Induction of VEGF expression in orthotopically implanted xenografts that were initially tamoxifen responsive and noninvasive resulted in tamoxifen-resistant tumor growth and metastasis to the lungs. Lung metastases were also observed in a VEGF-dependent manner following tail vein injection of tumor cells. At both primary and metastatic sites, VEGF-overexpressing tumors exhibited extensive fibroblastic stromal content, a clinical feature called desmoplasia. VEGF-induced metastatic colonies were surrounded by densely packed stromal cells before detectable angiogenesis, suggesting that VEGF is involved in the initiation of desmoplasia. Because expression of VEGF receptors R1 and R2 was undetectable in these tumor cells, the observed VEGF effects on reduction of tamoxifen efficacy and metastatic colonization are most likely mediated by paracrine signaling that enhances tumor/stromal cell interactions and increases the level of desmoplasia. This study reveals new roles for VEGF in breast cancer progression and suggests that combination of antiestrogens and VEGF inhibitors may prolong tamoxifen sensitivity and prevent metastasis in patients with ER-positive tumors. [Cancer Res 2008;68(15):6232–40]
Diagnostic and Prognostic MicroRNAs in Stage II Colon Cancer
Authors: Schepeler Troels , Reinert Jørgen T. , Ostenfeld Marie S. , Christensen Lise L. , Silahtaroglu Asli N. , Dyrskjøt Lars , Wiuf Carsten , Sørensen Frank J. , Kruhøffer Mogens , Laurberg Søren , Kauppinen Sakari , Ørntoft Torben F. , Andersen Claus L.
Published: August 2008

MicroRNAs (miRNA) are a class of small noncoding RNAs with important posttranscriptional regulatory functions. Recent data suggest that miRNAs are aberrantly expressed in many human cancers and that they may play significant roles in carcinogenesis. Here, we used microarrays to profile the expression of 315 human miRNAs in 10 normal mucosa samples and 49 stage II colon cancers differing with regard to microsatellite status and recurrence of disease. Several miRNAs were differentially expressed between normal tissue and tumor microsatellite subtypes, with miR-145 showing the lowest expression in cancer relative to normal tissue. Microsatellite status for the majority of cancers could be correctly predicted based on miRNA expression profiles. Furthermore, a biomarker based on miRNA expression profiles could predict recurrence of disease with an overall performance accuracy of 81%, indicating a potential role of miRNAs in determining tumor aggressiveness. The expression levels of miR-320 and miR-498, both included in the predictive biomarker, correlated with the probability of recurrence-free survival by multivariate analysis. We successfully verified the expression of selected miRNAs using real-time reverse transcription-PCR assays for mature miRNAs, whereas in situ hybridization was used to detect the accumulation of miR-145 and miR-320 in normal epithelial cells and adenocarcinoma cells. Functional studies showed that miR-145 potently suppressed growth of three different colon carcinoma cell lines. In conclusion, our results suggest that perturbed expression of numerous miRNAs in colon cancer may have a functional effect on tumor cell behavior, and, furthermore, that some miRNAs with prognostic potential could be of clinical importance. [Cancer Res 2008;68(15):6416–24]
RUNX3 Methylation Reveals that Bladder Tumors Are Older in Patients with a History of Smoking
Authors: Wolff Erika M. , Liang Gangning , Cortez Connie C. , Tsai Yvonne C. , Castelao J. Esteban , Cortessis Victoria K. , Tsao-Wei Denice D. , Groshen Susan , Jones Peter A.
Published: August 2008

Exposure to tobacco smoke is associated with increased DNA methylation at certain genes in both lung and bladder tumors. We sought to identify interactions in bladder cancer between DNA methylation and a history of smoking, along with any possible effect of aging. We measured DNA methylation in 342 transitional cell carcinoma tumors at BCL2, PTGS2 (COX2), DAPK, CDH1 (ECAD), EDNRB, RASSF1A, RUNX3, TERT, and TIMP3. The prevalence of methylation at RUNX3, a polycomb target gene, increased as a function of age at diagnosis (P = 0.031) and a history of smoking (P = 0.015). RUNX3 methylation also preceded methylation at the other eight genes (P < 0.001). It has been proposed that DNA methylation patterns constitute a “molecular clock” and can be used to determine the “age” of normal tissues (i.e., the number of times the cells have divided). Because RUNX3 methylation increases with age, is not present in normal urothelium, and occurs early in tumorigenesis, it can be used for the first time as a molecular clock to determine the age of a bladder tumor. Doing so reveals that tumors from smokers are “older” than tumors from nonsmokers (P = 0.009) due to tumors in smokers either initiating earlier or undergoing more rapid cell divisions. Because RUNX3 methylation is acquired early on in tumorigenesis, then its detection in biopsy or urine specimens could provide a marker to screen cigarette smokers long before any symptoms of bladder cancer are present. [Cancer Res 2008;68(15):6208–14]
An Integrative Genomic and Proteomic Analysis of PIK3CA, PTEN, and AKT Mutations in Breast Cancer
Authors: Stemke-Hale Katherine , Gonzalez-Angulo Ana Maria , Lluch Ana , Neve Richard M. , Kuo Wen-Lin , Davies Michael , Carey Mark , Hu Zhi , Guan Yinghui , Sahin Aysegul , Symmans W. Fraser , Pusztai Lajos , Nolden Laura K. , Horlings Hugo , Berns Katrien , Hung Mien-Chie , van de Vijver Marc J. , Valero Vicente , Gray Joe W. , Bernards René , Mills Gordon B. , Hennessy Bryan T.
Published: August 2008

Phosphatidylinositol 3-kinase (PI3K)/AKT pathway aberrations are common in cancer. By applying mass spectroscopy–based sequencing and reverse-phase protein arrays to 547 human breast cancers and 41 cell lines, we determined the subtype specificity and signaling effects of PIK3CA, AKT, and PTEN mutations and the effects of PIK3CA mutations on responsiveness to PI3K inhibition in vitro and on outcome after adjuvant tamoxifen. PIK3CA mutations were more common in hormone receptor–positive (34.5%) and HER2-positive (22.7%) than in basal-like tumors (8.3%). AKT1 (1.4%) and PTEN (2.3%) mutations were restricted to hormone receptor–positive cancers. Unlike AKT1 mutations that were absent from cell lines, PIK3CA (39%) and PTEN (20%) mutations were more common in cell lines than tumors, suggesting a selection for these but not AKT1 mutations during adaptation to culture. PIK3CA mutations did not have a significant effect on outcome after adjuvant tamoxifen therapy in 157 hormone receptor–positive breast cancer patients. PIK3CA mutations, in comparison with PTEN loss and AKT1 mutations, were associated with significantly less and inconsistent activation of AKT and of downstream PI3K/AKT signaling in tumors and cell lines. PTEN loss and PIK3CA mutation were frequently concordant, suggesting different contributions to pathophysiology. PTEN loss rendered cells significantly more sensitive to growth inhibition by the PI3K inhibitor LY294002 than did PIK3CA mutations. Thus, PI3K pathway aberrations likely play a distinct role in the pathogenesis of different breast cancer subtypes. The specific aberration present may have implications for the selection of PI3K-targeted therapies in hormone receptor–positive breast cancer. [Cancer Res 2008;68(15):6084–91]
RhoA-Dependent Regulation of Cell Migration by the Tumor Suppressor hSNF5/INI1
Authors: Caramel Julie , Quignon Frédérique , Delattre Olivier
Published: August 2008

Malignant rhabdoid tumors (MRT) are extremely aggressive pediatric tumors caused by the inactivation of the hSNF5/INI1 tumor suppressor gene, which encodes a core member of the SWI/SNF chromatin remodeling complex. Roles for hSNF5/INI1 in cell cycle and differentiation have been documented. Based on the observation that MRTs are highly invasive, we investigated a role for hSNF5/INI1 in cell migration. MRT cell lines exhibit high migration properties that are dramatically reduced upon hSNF5/INI1 expression. This effect is associated with the disorganization of the actin stress fiber network and is mediated by the inhibition of the activity of the small GTPase RhoA, through a nuclear, SWI/SNF-dependent transcriptional mechanism. We further show that the knockdown of hSNF5/INI1 in epithelial 293T or MCF7 cells results in increased cell size, loss of cell-cell adhesions, and enhanced migration, associated with an increased RhoA activity. Finally, we show that the SNF5 homology domain is required for hSNF5/INI1-mediated inhibition of migration, and that a missense mutation (S284L) associated with cancer is sufficient to impair hSNF5/INI1 function in migration. We conclude that the inhibition of migration is another crucial tumor suppressor function of hSNF5/INI1, in addition to its previously described functions in proliferation and differentiation, and that its loss-of-function in MRTs may account for the high invasiveness and metastatic potential of these tumors. [Cancer Res 2008;68(15):6154–61]
Variants in Inflammation Genes and the Risk of Biliary Tract Cancers and Stones: A Population-Based Study in China
Authors: Hsing Ann W. , Sakoda Lori C. , Rashid Asif , Andreotti Gabriella , Chen Jinbo , Wang Bin-Shen , Shen Ming-Chang , Chen Bingshu E. , Rosenberg Philip S. , Zhang Mingdong , Niwa Shelley , Chu Lisa , Welch Robert , Yeager Meredith , Fraumeni Joseph F. , Gao Yu-Tang , Chanock Stephen J.
Published: August 2008

To evaluate the role of chronic inflammation in the development of gallstones and biliary tract cancer, we examined the risk associated with 62 single nucleotide polymorphisms (SNPs), in 22 inflammation-related genes, in a population-based case-control study conducted in Shanghai, China, where the incidence of biliary tract cancer has been increasing in recent decades. The study included 411 cases with biliary tract cancer (237 gallbladder, 127 extrahepatic bile duct, and 47 ampulla of Vater), 895 with biliary stones, and 786 controls randomly selected from the population. Unconditional logistic regression was used to calculate odds ratios and 95% confidence intervals for the association of individual SNPs and haplotypes with biliary stones and biliary tract cancer. Of the 62 SNPs examined, 14 were related to the risk of biliary cancer and stones. Specifically, variants in the IL8, IL8RB, RNASEL, and NOS2 genes were associated with biliary stones, whereas VEGF variants were associated with gallbladder cancer. Of the 10 genes with multiple SNPs from which we inferred haplotypes, only one IL8RB haplotype, consisting of 3 SNPs (rs2230054, rs1126579, and rs1126580), was associated with the risk of bile duct cancer (P = 0.003) and biliary stones (P = 0.02), relative to the most frequent haplotype. In summary, common variants in genes that influence inflammatory responses may predispose to gallstones and biliary tract cancer, suggesting the need for future studies into the immunologic and inflammatory pathways that contribute to biliary diseases, including cancer. [Cancer Res 2008;68(15):6442–52]
Effect of Ablation or Inhibition of Stromal Matrix Metalloproteinase-9 on Lung Metastasis in a Breast Cancer Model Is Dependent on Genetic Background
Authors: Martin Michelle D. , Carter Kathy J. , Jean-Philippe Sharon R. , Chang Mayland , Mobashery Shahriar , Thiolloy Sophie , Lynch Conor C. , Matrisian Lynn M. , Fingleton Barbara
Published: August 2008

Matrix metalloproteinases (MMP) are a family of enzymes with a myriad of functions. Lately, we have come to realize that broad-spectrum inhibition of these enzymes, as was tried unsuccessfully in multiple phase III trials in cancer patients, is likely unwise given the protumorigenic and antitumorigenic functions of various family members. Here, we used the multistage mammary tumor model MMTV-PyVT to investigate roles for either MMP7 or MMP9 in tumor progression. We found no effect of genetic ablation of MMP7 or MMP9 on the multifocal tumors that developed in the mammary glands. Lack of MMP7 also had no effect on the development of lung metastases, suggesting that MMP7 is irrelevant in this model. In contrast, MMP9 deficiency was associated with an 80% decrease in lung tumor burden. The predominant cellular source of MMP9 was myeloid cells, with neutrophils being the largest contributor in tumor-bearing lungs. Experimental metastasis assays corroborated the role of host-derived MMP9 in lung metastasis and also facilitated determination of a time frame most relevant for the MMP9-mediated effect. The lung tumors from MMP9-deficient mice showed decreased angiogenesis. Surprisingly, the antimetastatic outcome of MMP9 ablation seemed to be dependent on strain. Only mice that had genetic background derived from C57BL/6 showed reduced metastasis, whereas mice fully of the FVB/N background showed no significant effect. These strain-specific responses were also observed in a study using a highly selective pharmacologic inhibitor of MMP9 and thus suggest that responses to MMP inhibition are controlled by genetic differences. [Cancer Res 2008;68(15):6251–9]
The HER4/4ICD Estrogen Receptor Coactivator and BH3-Only Protein Is an Effector of Tamoxifen-Induced Apoptosis
Authors: Naresh Anjali , Thor Ann D. , Edgerton Susan M. , Torkko Kathleen C. , Kumar Rakesh , Jones Frank E.
Published: August 2008

Greater than 40% of breast cancer patients treated with tamoxifen exhibit de novo or acquired tumor resistance. Recent clinical evidence indicates that loss of expression of HER4 is an independent marker for tamoxifen resistance. In direct corroboration with clinical observations, suppression of HER4 expression in the tamoxifen-sensitive MCF-7 and T47D breast tumor cell lines resulted in resistance to tamoxifen-induced apoptosis. Furthermore, HER4 expression was lost in three independent MCF-7 models of acquired tamoxifen resistance. The HER4 intracellular domain (4ICD) is an independently signaling nuclear protein that functions as a potent ERα coactivator. In addition, mitochondrial 4ICD functions as a proapoptotic BH3-only protein. Tamoxifen disrupts an estrogen-driven interaction between ERα and 4ICD while promoting mitochondrial accumulation of the 4ICD BH3-only protein. BCL-2 inhibition of tamoxifen-induced apoptosis and tamoxifen activation of BAK, independent of BAX, further supports a role for 4ICD during tamoxifen-induced apoptosis. Finally, reintroduction of HER4, but not HER4 with a mutated BH3 domain, restores tamoxifen sensitivity to tamoxifen-resistant TamR cells in a xenograft model. Clinically, breast cancer patients with tumor expression of nuclear 4ICD responded to tamoxifen therapy with no clinical failures reported after 14 years of follow-up, whereas 20% of patients lacking nuclear 4ICD expression succumbed to their disease within 10 years of diagnosis. Our identification of the HER4/4ICD BH3-only protein as a critical mediator of tamoxifen action provides a clinically important role for 4ICD in human cancer and reveals a potential tumor marker to predict patient response to tamoxifen therapy. [Cancer Res 2008;68(15):6387–95]
Down-regulation of Myeloid Cell Leukemia-1 through Inhibiting Erk/Pin 1 Pathway by Sorafenib Facilitates Chemosensitization in Breast Cancer
Authors: Ding Qingqing , Huo Longfei , Yang Jer-Yen , Xia Weiya , Wei Yongkun , Liao Yong , Chang Chun-Ju , Yang Yan , Lai Chien-Chen , Lee Dung-Fang , Yen Chia-Jui , Chen Yun-Ju Rita , Hsu Jung-Mao , Kuo Hsu-Ping , Lin Chun-Yi , Tsai Fuu-Jen , Li Long-Yuan , Tsai Chang-Hai , Hung Mien-Chie
Published: August 2008

Myeloid cell leukemia-1 (Mcl-1), a Bcl-2–like antiapoptotic protein, plays a role in cell immortalization and chemoresistance in a number of human malignancies. A peptidyl-prolyl cis/trans isomerase, Pin1 is involved in many cellular events, such as cell cycle progression, cell proliferation, and differentiation through isomerizing prophosphorylated substrates. It has been reported that down-regulation of Pin1 induces apoptosis, and that Erk phosphorylates and up-regulates Mcl-1; however, the underlying mechanisms for the two phenomena are not clear yet. Here, we showed that Pin 1 stabilizes Mcl-1, which is required for Mcl-1 posphorylation by Erk. First, we found expression of Mcl-1 and Pin1 were positively correlated and associated with poor survival in human breast cancer. We then showed that Erk could phosphorylate Mcl-1 at two consensus residues, Thr 92 and 163, which is required for the association of Mcl-1 and Pin1, resulting in stabilization of Mcl-1. Moreover, Pin1 is also required for the up-regulation of Mcl-1 by Erk activation. Based on this newly identified mechanism of Mcl-1 stabilization, two strategies were used to overcome Mcl-1–mediated chemoresistance: inhibiting Erk by Sorafenib, an approved clinical anticancer drug, or knocking down Pin1 by using a SiRNA technique. In conclusion, the current report not only unravels a novel mechanism to link Erk/Pin1 pathway and Mcl-1–mediated chemoresistance but also provides a plausible combination therapy, Taxol (Paclitaxel) plus Sorafenib, which was shown to be effective in killing breast cancer cells. [Cancer Res 2008;68(15):6109–17]
Targeted Overexpression of Vav3 Oncogene in Prostatic Epithelium Induces Nonbacterial Prostatitis and Prostate Cancer
Authors: Liu Yin , Mo Jun Qin , Hu Qiande , Boivin Gregory , Levin Linda , Lu Shan , Yang Dianer , Dong Zhongyun , Lu Shan
Published: August 2008

Our previous study revealed that Vav3 oncogene is overexpressed in human prostate cancer, activates androgen receptor (AR), and stimulates growth in prostate cancer cells. The purpose of this study is to further determine the potential role of Vav3 in prostate cancer development in genetically engineered mouse model. We generated Vav3 transgenic mice by targeted overexpression of a constitutive active Vav3 in the prostatic epithelium. We found that overexpression of Vav3 led to development of mouse prostatic intraepithelial neoplasia and prostate cancer at the age of as early as 3 months. The AR signaling axis and phosphatidylinositol 3-kinase-Akt signaling were elevated in the prostate glands of Vav3 transgenic mice. In addition to prostate cancer, Vav3 transgenic mice developed significant nonbacterial chronic prostatitis in the prostate gland with notable infiltration of lymphomononuclear cells (monocytes, lymphocytes, and plasma cells), which was associated with elevated incidence of prostate cancer. DNA microarray and signaling pathway analysis revealed that the top diseases and disorders were inflammatory diseases and cancer of the prostate gland in Vav3 transgenic mice. In vitro analysis showed that overexpression of Vav3 in prostate cancer cells enhanced nuclear factor-κB (NF-κB) activity, implicating an underlying mechanism of innate inflammatory response induced by elevated Vav3 activity. These data showed that Vav3 overexpression in the prostate epithelium enhanced both the AR signaling axis and NF-κB–mediated pathway, which potentially contributed to the development of nonbacterial prostatitis and prostate cancer. [Cancer Res 2008;68(15):6396–406]
Serum-Soluble B7x Is Elevated in Renal Cell Carcinoma Patients and Is Associated with Advanced Stage
Authors: Thompson R. Houston , Zang Xingxing , Lohse Christine M. , Leibovich Bradley C. , Slovin Susan F. , Reuter Victor E. , Cheville John C. , Blute Michael L. , Russo Paul , Kwon Eugene D. , Allison James P.
Published: August 2008

B7x is the newest member of the B7-CD28 family and is thought to dampen immune responses via negative costimulation. Tumor expression of B7x was recently described in renal cell carcinoma (RCC) and was associated with poor outcome. We developed an assay to detect serum-soluble B7x (sB7x) and investigated 101 patients with clear cell RCC who underwent nephrectomy between 2003 and 2007. For controls, we obtained serum from 101 sex-matched blood donors within the same age range. Following an ELISA for sB7x, detectable levels (>0.1 ng/mL) of sB7x were observed in 53 RCC patients compared with 18 controls (P < 0.001). Median (range) concentrations of sB7x for RCC patients and controls were 14.4 ng/mL (0.1–56.9) and 2.7 ng/mL (0.2–37.1), respectively. For RCC patients with detectable sB7x, median levels were significantly higher for patients with a tumor thrombus (19.2 versus 6.6 ng/mL; P = 0.007), positive lymph nodes (41.3 versus 10.3 ng/mL; P = 0.018), and distant metastases at nephrectomy (43.3 versus 8.5 ng/mL; P = 0.002) and tended to be higher in patients with high-grade tumors (18.8 versus 8.5; P = 0.090). Additionally, median sB7x levels for tumor-node-metastasis stage I to IV RCC were 6.6, 10.3, 14.5, and 43.3 ng/mL, respectively (P = 0.012). In this first evaluation of sB7x in RCC, we show that RCC patients are more likely to have detectable sB7x compared with controls and higher sB7x levels correlate with advanced tumor stage. These early results merit further investigation of this serum marker for potential diagnostic and prognostic purposes. [Cancer Res 2008;68(15):6054–8]
Topology of NGEP, a Prostate-Specific Cell:Cell Junction Protein Widely Expressed in Many Cancers of Different Grade Level
Authors: Das Sudipto , Hahn Yoonsoo , Walker Dawn A. , Nagata Satoshi , Willingham Mark C. , Peehl Donna M. , Bera Tapan K. , Lee Byungkook , Pastan Ira
Published: August 2008

New gene expressed in prostate (NGEP) is a prostate-specific polytopic membrane protein found at high concentrations at cell:cell contact regions. To determine if NGEP is a useful target for antibody-based therapy of prostate cancer, we performed an immunohistochemical analysis of 126 human prostate carcinoma samples using polyclonal anti-NGEP sera and found that 91% of the cancers express NGEP protein. To elucidate the topology of NGEP and guide the development of monoclonal antibodies (mAb) reacting with the extracellular regions of NGEP, a hemagglutinin epitope tag was inserted at several positions within the NGEP sequence. The tagged proteins were expressed in 293T cells and locations of the tags were determined by immunofluorescence in intact or permeabilized cells. The results indicate that NGEP contains eight transmembrane domains with both the NH2 and COOH termini of NGEP located inside the cell. We produced mAb to three regions that are predicted to be intracellular based on the epitope tag data (amino acids 1-352, 441-501, and 868-933), and as predicted, the mAb only detected the protein in permeabilized cells. NGEP is a glycoprotein with predicted glycosylation sites at N809 and N824. When these residues were converted to glutamine, glycosylation was abolished, confirming that the residues are extracellular. Our findings on the expression and the orientation of the NGEP protein serve as an important framework for the development of mAb targeting the extracellular regions of NGEP that could be used for prostate cancer immunotherapy. [Cancer Res 2008;68(15):6306–12]
17β-Estradiol Mobilizes Bone Marrow–Derived Endothelial Progenitor Cells to Tumors
Authors: Suriano Robert , Chaudhuri Devyani , Johnson Raja Singh , Lambers Erin , Ashok Badithe T. , Kishore Raj , Tiwari Raj K.
Published: August 2008

Neovascularization is critical for tumor growth and development. The cellular mediators for this process are yet to be defined. We discovered that bone marrow–derived endothelial progenitor cells (BM-EPC), having the phenotype (CD133+, CD34+, VEGFR-2+), initiate neovascularization in response to TG1-1 mammary cells implanted in the inguinal mammary gland of Tie-2 GFP transgenic mice. The fluorescence tag allowed for tracing the migration of green fluorescent protein–tagged endothelial progenitor cells to tumor tissues. We discovered that 17-β estradiol supplementation of ovariectomized mice significantly enhanced BM-EPC–induced neovascularization and secretion of angiogenic factors within the tumor microenvironment. Cell-based system analyses showed that estrogen-stimulated BM-EPCs secreted paracrine factors which enhanced TG1-1 cell proliferation and migration. Furthermore, TG1-1 cell medium supplemented with estrogen-induced BM-EPC mediated tubulogenesis, which was an experimental in vivo representation of the neovasculature. Our data provide evidence of BM-EPC mammary tumor cell interactions and identify a novel cellular mediator of tumor progression that can be exploited clinically. [Cancer Res 2008;68(15):6038–42]
Methadone, Commonly Used as Maintenance Medication for Outpatient Treatment of Opioid Dependence, Kills Leukemia Cells and Overcomes Chemoresistance
Authors: Friesen Claudia , Roscher Mareike , Alt Andreas , Miltner Erich
Published: August 2008

The therapeutic opioid drug methadone (d,l-methadone hydrochloride) is the most commonly used maintenance medication for outpatient treatment of opioid dependence. In our study, we found that methadone is also a potent inducer of cell death in leukemia cells and we clarified the unknown mechanism of methadone-induced cell killing in leukemia cells. Methadone inhibited proliferation in leukemia cells and induced cell death through apoptosis induction and activated apoptosis pathways through the activation of caspase-9 and caspase-3, down-regulation of Bcl-xL and X chromosome–linked inhibitor of apoptosis, and cleavage of poly(ADP-ribose) polymerase. In addition, methadone induced cell death not only in anticancer drug–sensitive and apoptosis-sensitive leukemia cells but also in doxorubicin-resistant, multidrug-resistant, and apoptosis-resistant leukemia cells, which anticancer drugs commonly used in conventional therapies of leukemias failed to kill. Depending on caspase activation, methadone overcomes doxorubicin resistance, multidrug resistance, and apoptosis resistance in leukemia cells through activation of mitochondria. In contrast to leukemia cells, nonleukemic peripheral blood lymphocytes survived after methadone treatment. These findings show that methadone kills leukemia cells and breaks chemoresistance and apoptosis resistance. Our results suggest that methadone is a promising therapeutic approach not only for patients with opioid dependence but also for patients with leukemias and provide the foundation for new strategies using methadone as an additional anticancer drug in leukemia therapy, especially when conventional therapies are less effective. [Cancer Res 2008;68(15):6059–64]
Imaging of Urokinase-Type Plasminogen Activator Receptor Expression Using a 64Cu-Labeled Linear Peptide Antagonist by microPET
Authors: Li Zi-Bo , Niu Gang , Wang Hui , He Lina , Yang Lily , Ploug Michael , Chen Xiaoyuan
Published: August 2008

Purpose: Malignant tumors are capable of degrading the surrounding extracellular matrix, resulting in local invasion or metastasis. Urokinase-type plasminogen activator (uPA) and its cell surface receptor (uPAR) are central molecules in one of the major protease systems involved in extracellular matrix degradation. Noninvasive imaging of this receptor in vivo with radiolabeled peptides that specifically target uPAR may therefore be useful to decipher the potential invasiveness of malignant lesions.

Experimental Design: In this study, we developed a 64Cu-labeled uPAR-binding peptide for positron emission tomography (PET) imaging. A linear, high-affinity uPAR-binding peptide antagonist AE105 was conjugated with 1,4,7,10-tetraazadodecane-N,N′,N″,N‴-tetraacetic acid (DOTA) and labeled with 64Cu for microPET imaging of mice bearing U87MG human glioblastoma (uPAR positive) and MDA-MB-435 human breast cancer (uPAR negative).

Results: Surface plasmon resonance measurements show that AE105 with DOTA conjugated at the α-amino group (DOTA-AE105) has high affinity toward uPAR. microPET imaging reveals a rapid and high accumulation of 64Cu-DOTA-AE105 in uPAR-positive U87MG tumors (10.8 ± 1.5%ID/g at 4.5 hours, n = 3) but not in uPAR-negative MDA-MB-435 tumors (1.2 ± 0.6%ID/g at 4.5 hours, n = 3). Specificity of this peptide-based imaging of uPAR was validated by further control experiments. First, a nonbinding variant of AE105 carrying a single amino acid replacement (Trp→Glu) does not target U87MG tumors in vivo. Second, targeting of U87MG tumors by 64Cu-DOTA-AE105 is specifically inhibited by a nonlabeled antagonist.

Conclusion: The successful demonstration of the ability of a 64Cu labeled uPAR-specific probe to visualize uPAR expression in vivo may allow clinical translation of this class of radiopharmaceuticals for uPAR-positive cancer detection and patient stratification for uPA/uPAR system-based cancer therapy.
T-Cell Responses to Survivin in Cancer Patients Undergoing Radiation Therapy
Authors: Schaue Dörthe , Comin-Anduix Begonya , Ribas Antoni , Zhang Li , Goodglick Lee , Sayre James W. , Debucquoy Annelies , Haustermans Karin , McBride William H.
Published: August 2008

Purpose: The goal of this study was to determine if radiation therapy (RT) of human cancer enhances or diminishes tumor-specific T-cell reactivity. This is important if immunotherapy is to be harnessed to improve the outcome of cancer radiotherapy.

Experimental Design: Lymphocytes were isolated from colorectal cancer (CRC) patients before, during, and after presurgical chemoradiotherapy. Similar samples were taken from prostate cancer patients receiving standard RT. The level of CD8+ T cells capable of binding tetramers for the tumor-associated antigen survivin, which is overexpressed in both cancer types, was enumerated in HLA-A*0201 patient samples. CD4+, CD25high, Foxp3+ cells were also enumerated to evaluate therapy-induced changes in Tregulatory cells. For CRC patients, most of whom were enrolled in a clinical trial, pathologic response data were available, as well as biopsy and resection specimens, which were stained for cytoplasmic and intranuclear survivin.

Results: Survivin-specific CD8+ T lymphocytes were detected in the peripheral blood of CRC and prostate cancer patients and increased after therapy in some, but not all, patients. Increases were more common in CRC patients whose tumor was downstaged after chemoradiotherapy. Biopsy specimens from this cohort generally had higher nuclear to cytoplasmic survivin expression. Tregulatory cells generally increased in the circulation following therapy but only in CRC patients.

Conclusion: This study indicates that RT may increase the likelihood of some cancer patients responding to immunotherapy and lays a basis for future investigations aimed at combining radiation and immunotherapy.
Tumor Cell Gene Expression Changes Following Short-term In vivo Exposure to Single Agent Chemotherapeutics are Related to Survival in Multiple Myeloma
Authors: Burington Bart , Barlogie Bart , Zhan Fenghuang , Crowley John , Shaughnessy John D.
Published: August 2008

Changes in global gene expression patterns in tumor cells following in vivo therapy may vary by treatment and provide added or synergistic prognostic power over pretherapy gene expression profiles (GEP). This molecular readout of drug-cell interaction may also point to mechanisms of action/resistance. In newly diagnosed patients with multiple myeloma (MM), microarray data were obtained on tumor cells prior to and 48 hours after in vivo treatment using dexamethasone (n = 45) or thalidomide (n = 42); in the case of relapsed MM, microarray data were obtained prior to (n = 36) and after (n = 19) lenalidomide administration. Dexamethasone and thalidomide induced both common and unique GEP changes in tumor cells. Combined baseline and 48-hour changes in GEP in a subset of genes, many related to oxidative stress and cytoskeletal dynamics, were predictive of outcome in newly diagnosed MM patients receiving tandem transplants. Thalidomide-altered genes also changed following lenalidomide exposure and predicted event-free and overall survival in relapsed patients receiving lenalidomide as a single agent. Combined with baseline molecular features, changes in GEP following short-term single-agent exposure may help guide treatment decisions for patients with MM. Genes whose drug-altered expression were found to be related to survival may point to molecular switches related to response and/or resistance to different classes of drugs.
Pattern of Serum Autoantibodies Allows Accurate Distinction between a Tumor and Pathologies of the Same Organ
Authors: Ludwig Nicole , Keller Andreas , Comtesse Nicole , Rheinheimer Stefanie , Pallasch Christian , Fischer Ulrike , Fassbender Klaus , Steudel Wolf Ingo , Lenhof Hans-Peter , Meese Eckart
Published: August 2008

Purpose: Recent studies impressively showed the diagnostic potential of seroreactivity patterns for different tumor types, offering the prospect for low-cost screening of numerous tumor types simultaneously. One of the major challenges toward this goal is to prove that seroreactivity profiles do not only allow for identifying a tumor but also allow for distinguishing tumors from other pathologies of the same organ.

Experimental Design: We chose glioma as a model system and tested 325 sera (88 glioma, 95 intracranial tumors, 60 other brain pathologies, and 82 healthy controls) for seroreactivity on a panel of 35 antigens.

Results: We were able to discriminate between glioma and all other sera with cross-validated specificity of 86.1%, sensitivity of 85.2%, and accuracy of 85.8%. We obtained comparably good results for the separation of glioma versus nontumor brain pathologies and glioma versus other intracranial tumors.

Conclusion: Our study provides first evidence that seroreactivity patterns allow for an accurate discrimination between a tumor and pathologies of the same organ even between different tumor types of the same organ.
OPG Is Regulated by β-Catenin and Mediates Resistance to TRAIL-Induced Apoptosis in Colon Cancer
Authors: De Toni Enrico N. , Thieme Susanne E. , Herbst Andreas , Behrens Andrea , Stieber Petra , Jung Andreas , Blum Helmut , Göke Burkhard , Kolligs Frank T.
Published: August 2008

Purpose: Resistance to apoptosis is a hallmark of cancer and correlates with aggressiveness of tumors and poor prognosis. The Wnt/β-catenin pathway plays a pivotal role in the genesis of colorectal cancer by mechanisms not fully elucidated yet. Previous studies have linked regulation of osteoprotegerin (OPG) in bone to Wnt/β-catenin signaling. As OPG also serves as a decoy receptor for tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), we hypothesized that OPG might play a role in mediating resistance to apoptosis in colorectal cancer cells.

Experimental Design: Expression analysis and functional studies in human colorectal cancer cell lines and determination of expression in primary tumors and sera from patients with colorectal cancer.

Results: We found production of OPG in colorectal cancer cells to be regulated by β-catenin/Tcf-4. Addition of exogenous OPG to colorectal cancer cells caused resistance to TRAIL. Similarly, accumulation of OPG in medium of cultivated cells caused resistance to TRAIL, and this could be reverted by removal of OPG. Furthermore, OPG levels were significantly increased in serum of patients with advanced disease.

Conclusions: We conclude that the Wnt/β-catenin pathway contributes to carcinogenesis and cancer cell survival by driving expression of OPG. Expression of the survival factor OPG might provide colorectal cancer cells with an essential growth advantage and contribute to cell invasion and metastasis. Inhibition of OPG expression might offer a new therapeutic approach for the treatment of patients with colorectal tumors overexpressing OPG and make these tumors sensitive to TRAIL-induced apoptosis.
The Targeted Immunocytokine L19-IL2 Efficiently Inhibits the Growth of Orthotopic Pancreatic Cancer
Authors: Wagner Karola , Schulz Petra , Scholz Arne , Wiedenmann Bertram , Menrad Andreas
Published: August 2008

Purpose: Effective control of pancreatic cancer has been hampered primarily by the lack of tumor specificity of current treatment modalities. The highly specific antibody-mediated delivery of therapeutic agents to the tumor microenvironment might overcome this problem. We therefore investigated the therapeutic efficacy of the targeted immunocytokine L19-Interleukin-2 (L19-IL2), consisting of the human single-chain Fv antibody L19, which is highly specific for the extradomain B (ED-B) of fibronectin, and the human cytokine IL-2, in pancreatic cancer.

Experimental Design: Therapeutic effects of L19-IL-2, IL-2, and gemcitabine on tumor growth and metastasis were evaluated in orthotopic mouse models for pancreatic cancer. Immunohistochemistry was done to define ED-B expression, tumor necrosis, apoptosis, proliferation, and invasion of macrophages and natural killer (NK) cells. NK cells were depleted by i.v. injection of an anti-asialo-GM-1 antibody.

Results: ED-B is selectively expressed in human pancreatic cancer and in primary tumors and metastases of the mouse models. L19-IL-2 therapy was clearly superior to untargeted IL-2 or gemcitabine and inhibited tumor growth and metastasis with remarkable long-term tumor control. Therapeutic effects were associated with the induction of extensive tumor necrosis and inhibition of tumor cell proliferation. Immunohistochemistry revealed an increase of macrophages and NK cells in the tumor tissue, suggesting immune-mediated mechanisms. The functional relevance of NK cells for the therapeutic effect of the targeted immunocytokine L19-IL-2 was confirmed by NK cell depletion, which completely abolished its antitumor efficacy.

Conclusions: These preclinical results strongly encourage the initiation of clinical studies using L19-IL-2 in pancreatic cancer.
Epithelial-Mesenchymal Transition in Cervical Cancer: Correlation with Tumor Progression, Epidermal Growth Factor Receptor Overexpression, and Snail Up-Regulation
Authors: Lee Mei-Yi , Chou Cheng-Yang , Tang Ming-Jer , Shen Meng-Ru
Published: August 2008

Purpose: Acquisition of epithelial-mesenchymal transition (EMT) by primary carcinoma cells is associated with disrupted epithelial integrity, local invasion, and ultimately metastasis. Little is known about the existence and function of EMT in cervical cancer. This study aims to investigate the regulation of EMT in cervical squamous cell carcinoma.

Experimental Design: We investigated the molecular events of EMT in surgical specimens, which present the progression of cervical carcinoma. Two cervical cancer cell lines and the primary culture of normal cervical epithelia were used to study the regulatory mechanisms of EMT.

Results: The chronic epidermal growth factor (EGF) treatment induces the elongation of cell shape, increases cell scattering, and enhances cell invasion. EGF treatment down-regulates E-cadherin and up-regulates vimentin in cervical cancer cells. These characteristics are consistent with the morphologic changes, molecular events, and functional significance of EMT. EGF receptor (EGFR) signaling inactivates glycogen synthase kinase-3β, which results in the nuclear accumulation of up-regulated Snail and then leads to EMT program. α5β1 integrin signaling and extracellular matrix fibronectin can modulate EGF-induced EMT. Importantly, the immunofluorescent stainings of surgical specimens indicate that cervical carcinoma progression is accompanied by EGFR overexpression, which is in parallel with decreased E-cadherin and increased vimentin. Up-regulation and nuclear accumulation of Snail correlate with EMT program in tumor tissues.

Conclusion: EGF cooperates with α5β1 integrin signaling to induce EMT in cervical cancer cells via up-regulated Snail. Blockade of EGFR activity or expression may provide a potential target for the treatment of cervical cancer progression.