bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic analysis of metabolic traits in an intercross between body weight-selected chicken lines
Authors: Ek Weronica , Strömstedt Lina , Wahlberg Per , Siegel Paul , Andersson Leif , Carlborg Örjan
Published: March 2010

A network of four interacting loci has been reported previously to influence growth in two lines of chickens divergently selected for body weight at 56 days of age. Located on chromosomes 3 ( Growth4), 4 ( Growth6), 7 ( Growth9), and 20 ( Growth12), they explained nearly half of the difference in body weight at selection age between the two lines. The original study reported effects on body weight and fat deposition, but no attempts were made to explore the effects of the network on other phenotypes measured in the F2 population. In this study we conducted further analyses to evaluate the specific effects of the four-locus network on other metabolic traits as well as refining results from the original study by including a larger number of genetic markers in the quantitative trait locus (QTL) regions. We confirm the previously described effect of the epistatic network on body weight and show that the network increases the total amount of muscle and fat as well as the weight of the internal organs. The network as a whole did not change the relative content of any studied organs or tissues in the body. There was, however, a significant interaction between the loci on chromosomes 3 and 7 that changed the relative proportion of abdominal fat and breast muscle in the chicken by increasing abdominal fat weight without a corresponding increase in muscle mass.
Association ofMAOA,5-HTT, andNETpromoter polymorphisms with gene expression and protein activity in human placentas
Authors: Zhang Huiping , Smith Graeme N. , Liu Xudong , Holden Jeanette J. A.
Published: March 2010

Monoamine oxidase A (MAOA) and the transporters for serotonin (5-HTT) and norepinephrine (NET) may play important roles in regulating maternal monoamine neurotransmitters transferred across the placenta to the fetus. We investigated whether promoter polymorphisms in MAOA (uVNTR), 5-HTT (5-HTTLPR), and NET (NETpPR AAGG4) could influence gene expression and protein activity in human placentas. Normal term human placentas ( n = 73) were collected, and placental MAOA, 5-HTT, and NET mRNA levels and protein activity were determined. The mRNA levels or protein activities were compared between different genotype groups. Placentas hemizygous (male fetus) or homozygous (female fetus) for MAOA uVNTR 4-repeat allele had significantly higher MAOA mRNA levels than those hemizygous or homozygous for the 3-repeat allele ( P = 0.001). However, no significant difference in MAOA enzyme activity was found for these two groups of genotypes ( P = 0.161). Placentas with the 5-HTTLPR short (S)-allele (S/S+S/L) had significantly lower 5-HTT mRNA levels and serotonin uptake rate than those homozygous for the long (L)-allele (L/L) (mRNA: P < 0.001; serotonin transporting activity: P < 0.001). Placentas homozygous for the NET AAGG4L4allele had significantly higher NET mRNA levels, as well as dopamine and norepinephrine uptake rates, than those with the S4/L4genotype (mRNA: P < 0.001; dopamine transporting activity: P = 0.012; norepinephrine transporting activity: P = 0.011). These findings suggest that the three promoter polymorphisms of MAOA, 5-HTT, and NET influence gene expression levels and protein activity of these genes in human placentas, potentially leading to different fetal levels of maternal monoamine neurotransmitters, which may have an impact on fetal neurodevelopment.
Proteomics Strategy for Identifying Candidate Bioactive Proteins in Complex Mixtures: Application to the Platelet Releasate
Authors: O'Connor Roisin , Cryan Lorna M. , Wynne Kieran , de Stefani Andreas , Fitzgerald Desmond , O'Brien Colm , Cagney Gerard
Published: March 2010

Proteomic approaches have proven powerful at identifying large numbers of proteins, but there are fewer reports of functional characterization of proteins in biological tissues. Here, we describe an experimental approach that fractionates proteins released from human platelets, linking bioassay activity to identity. We used consecutive orthogonal separation platforms to ensure sensitive detection: (a) ion-exchange of intact proteins, (b) SDS-PAGE separation of ion-exchange fractions and (c) HPLC separation of tryptic digests coupled to electrospray tandem mass spectrometry. Migration of THP-1 monocytes in response to complete or fractionated platelet releasate was assessed and located to just one of the forty-nine ion-exchange fractions. Over 300 proteins were identified in the releasate, with a wide range of annotated biophysical and biochemical properties, in particular platelet activation, adhesion, and wound healing. The presence of PEDF and involucrin, two proteins not previously reported in platelet releasate, was confirmed by western blotting. Proteins identified within the fraction with monocyte promigratory activity and not in other inactive fractions included vimentin, PEDF, and TIMP-1. We conclude that this analytical platform is effective for the characterization of complex bioactive samples.
FOXD3 Is a Mutant B-RAF–Regulated Inhibitor of G1-S Progression in Melanoma Cells
Authors: Abel Ethan V. , Aplin Andrew E.
Published: March 2010

The forkhead box transcription factor FOXD3 is a stemness factor that prevents the production of melanocyte progenitors from the developing neural crest; however, its role in human cancers is not known. Transformation of melanocytes gives rise to melanoma. In two thirds of melanomas, the serine/threonine kinase B-RAF is mutated to a constitutively active form. Here, we show that FOXD3 levels are upregulated following attenuation of B-RAF and mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK) signaling in mutant B-RAF harboring human melanoma cells. This effect was selective because FOXD3 was not upregulated following MEK inhibition in wild-type B-RAF melanoma cells and mutant B-RAF thyroid carcinoma cells. Ectopic FOXD3 expression potently inhibited melanoma cell growth without altering mutant B-RAF activation of ERK1/2. Inhibition of cell growth was due to a potent G1 cell cycle arrest and was associated with p53-dependent upregulation of p21Cip1. FOXD3-induced cell cycle arrest was prevented by p53 depletion and, to a lesser extent, p21Cip1 depletion. These studies show that FOXD3 is suppressed by B-RAF, uncover a novel role and mechanism for FOXD3 as a negative cell cycle regulator, and have implications for the repression of melanocytic lineage cells. Cancer Res; 70(7); 2891–900
Antitumor Activity of Hyaluronic Acid Synthesis Inhibitor 4-Methylumbelliferone in Prostate Cancer Cells
Authors: Lokeshwar Vinata B. , Lopez Luis E. , Munoz Daniel , Chi Andrew , Shirodkar Samir P. , Lokeshwar Soum D. , Escudero Diogo O. , Dhir Neetika , Altman Norman
Published: March 2010

4-Methylumbelliferone (4-MU) is a hyaluronic acid (HA) synthesis inhibitor with anticancer properties; the mechanism of its anticancer effects is unknown. We evaluated the effects of 4-MU on prostate cancer cells. 4-MU inhibited proliferation, motility, and invasion of DU145, PC3-ML, LNCaP, C4-2B, and/or LAPC-4 cells. At IC50 for HA synthesis (0.4 mmol/L), 4-MU induced >3-fold apoptosis in prostate cancer cells, which could be prevented by the addition of HA. 4-MU induced caspase-8, caspase-9, and caspase-3 activation, PARP cleavage, upregulation of Fas-L, Fas, FADD and DR4, and downregulation of bcl-2, phosphorylated bad, bcl-XL, phosphorylated Akt, phosphorylated IKB, phosphorylated ErbB2, and phosphorylated epidermal growth factor receptor. At IC50, 4-MU also caused >90% inhibition of NF-κB reporter activity, which was prevented partially by the addition of HA. With the exception of caveolin-1, HA reversed the 4-MU–induced downregulation of HA receptors (CD44 and RHAMM), matrix-degrading enzymes (MMP-2 and MMP-9), interleukin-8, and chemokine receptors (CXCR1, CXCR4, and CXCR7) at the protein and mRNA levels. Expression of myristoylated-Akt rescued 4-MU–induced apoptosis and inhibition of cell growth and interleukin-8, RHAMM, HAS2, CD44, and MMP-9 expression. Oral administration of 4-MU significantly decreased PC3-ML tumor growth (>3-fold) when treatment was started either on the day of tumor cell injection or after the tumors became palpable, without organ toxicity, changes in serum chemistry, or body weight. Tumors from 4-MU–treated animals showed reduced microvessel density (∼3-fold) and HA expression but increased terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling–positive cells and expression of apoptosis-related molecules. Therefore, the anticancer effects of 4-MU, an orally bioavailable and relatively nontoxic agent, are primarily mediated by inhibition of HA signaling. Cancer Res; 70(7); 2613–23
NF-κB–Inhibited Acute Myeloid Leukemia Cells Are Rescued from Apoptosis by Heme Oxygenase-1 Induction
Authors: Rushworth Stuart A. , Bowles Kristian M. , Raninga Prahlad , MacEwan David J.
Published: March 2010

Despite high basal NF-κB activity in acute myeloid leukemia (AML) cells, inhibiting NF-κB in these cells has little or no effect on inducing apoptosis. We previously showed that heme oxygenase-1 (HO-1) underlies this resistance of AML to tumor necrosis factor–induced apoptosis. Here, we describe a mechanism by which HO-1 is a silent antiapoptotic factor only revealed when NF-κB is inhibited, thus providing a secondary antiapoptotic mechanism to ensure AML cell survival and chemoresistance. We show that inhibition of NF-κB increased HO-1 expression in primary AML cells compared with that of nonmalignant cells. In addition, we observed this suppressed HO-1 level in AML cells compared with CD34+ nonmalignant control cells. Using chromatin immunoprecipitation assay and small interfering RNA knockdown, we showed that the NF-κB subunits p50 and p65 control this suppression of HO-1 in AML cells. Finally, we showed that inhibition of HO-1 and NF-κB in combination significantly induced apoptosis in AML cells but not in noncancerous control cells. Thus, NF-κB inhibition combined with HO-1 inhibition potentially provides a novel therapeutic approach to treat chemotherapy-resistant forms of AML. Cancer Res; 70(7); 2973–83
Single-Nucleotide Polymorphisms Inside MicroRNA Target Sites Influence Tumor Susceptibility
Authors: Nicoloso Milena S. , Sun Hao , Spizzo Riccardo , Kim Hyunsoo , Wickramasinghe Priyankara , Shimizu Masayoshi , Wojcik Sylwia E. , Ferdin Jana , Kunej Tanja , Xiao Lianchun , Manoukian Siranoush , Secreto Giorgio , Ravagnani Fernando , Wang Xuemei , Radice Paolo , Croce Carlo M. , Davuluri Ramana V. , Calin George A.
Published: March 2010

Single-nucleotide polymorphisms (SNP) associated with polygenetic disorders, such as breast cancer (BC), can create, destroy, or modify microRNA (miRNA) binding sites; however, the extent to which SNPs interfere with miRNA gene regulation and affect cancer susceptibility remains largely unknown. We hypothesize that disruption of miRNA target binding by SNPs is a widespread mechanism relevant to cancer susceptibility. To test this, we analyzed SNPs known to be associated with BC risk, in silico and in vitro, for their ability to modify miRNA binding sites and miRNA gene regulation and referred to these as target SNPs. We identified rs1982073-TGFB1 and rs1799782-XRCC1 as target SNPs, whose alleles could modulate gene expression by differential interaction with miR-187 and miR-138, respectively. Genome-wide bioinformatics analysis predicted ∼64% of transcribed SNPs as target SNPs that can modify (increase/decrease) the binding energy of putative miRNA::mRNA duplexes by >90%. To assess whether target SNPs are implicated in BC susceptibility, we conducted a case-control population study and observed that germline occurrence of rs799917-BRCA1 and rs334348-TGFR1 significantly varies among populations with different risks of developing BC. Luciferase activity of target SNPs, allelic variants, and protein levels in cancer cell lines with different genotypes showed differential regulation of target genes following overexpression of the two interacting miRNAs (miR-638 and miR-628-5p). Therefore, we propose that transcribed target SNPs alter miRNA gene regulation and, consequently, protein expression, contributing to the likelihood of cancer susceptibility, by a novel mechanism of subtle gene regulation. Cancer Res; 70(7); 2789–98
Smac Mimetic Increases Chemotherapy Response and Improves Survival in Mice with Pancreatic Cancer
Authors: Dineen Sean P. , Roland Christina L. , Greer Rachel , Carbon Juliet G. , Toombs Jason E. , Gupta Puja , Bardeesy Nabeel , Sun Haizhou , Williams Noelle , Minna John D. , Brekken Rolf A.
Published: March 2010

Failure of chemotherapy in the treatment of pancreatic cancer is often due to resistance to therapy-induced apoptosis. A major mechanism for such resistance is the expression and activity of inhibitors of apoptosis proteins (IAP). Smac (second mitochondria–derived activator of caspase) is a mitochondrial protein that inhibits IAPs. We show that JP1201, a Smac mimetic, is a potent enhancer of chemotherapy in robust mouse models of pancreatic cancer. Combination of JP1201 with gemcitabine reduced primary and metastatic tumor burden in orthotopic xenograft and syngenic tumor models, induced regression of established tumors, and prolonged survival in xenograft and transgenic models of pancreatic cancer. The effect of JP1201 was phenocopied by XIAP small interfering RNA in vitro and correlated with elevated levels of tumor necrosis factor α protein in vivo. The continued development of JP1201 and other strategies designed to enhance therapy-induced apoptosis in pancreatic cancer is warranted. Cancer Res; 70(7); 2852–61
FoxM1B Regulates NEDD4-1 Expression, Leading to Cellular Transformation and Full Malignant Phenotype in Immortalized Human Astrocytes
Authors: Dai Bingbing , Pieper Russell O. , Li Dawei , Wei Ping , Liu Mingguang , Woo Shiao Y. , Aldape Kenneth D. , Sawaya Raymond , Xie Keping , Huang Suyun
Published: March 2010

Our recent studies have shown that the FoxM1B transcription factor is overexpressed in human glioma tissues and that the level of its expression correlates directly with glioma grade. However, whether FoxM1B plays a role in the early development of glioma (i.e., in transformation) is unknown. In this study, we found that the FoxM1B molecule causes cellular transformation and tumor formation in normal human astrocytes (NHA) immortalized by p53 and pRB inhibition. Moreover, brain tumors that arose from intracranial injection of FoxM1B-expressing immortalized NHAs displayed glioblastoma multiforme (GBM) phenotypes, suggesting that FoxM1B overexpression in immortalized NHAs not only transforms the cells but also leads to GBM formation. Mechanistically, our results showed that overexpression of FoxM1B upregulated NEDD4-1, an E3 ligase that mediates the degradation and downregulation of phosphatase and tensin homologue (PTEN) in multiple cell lines. Decreased PTEN in turn resulted in the hyperactivation of Akt, which led to phosphorylation and cytoplasmic retention of FoxO3a. Blocking Akt activation with phosphoinositide 3-kinase/Akt inhibitors inhibited the FoxM1B-induced transformation of immortalized NHAs. Furthermore, overexpression of FoxM1B in immortalized NHAs increased the expression of survivin, cyclin D1, and cyclin E, which are important molecules for tumor growth. Collectively, these results indicate that overexpression of FoxM1B, in cooperation with p53 and pRB inhibition in NHA cells, promotes astrocyte transformation and GBM formation through multiple mechanisms. Cancer Res; 70(7); 2951–61
Dual Regulation by Apurinic/Apyrimidinic Endonuclease-1 Inhibits Gastric Epithelial Cell Apoptosis during Helicobacter pylori Infection
Authors: Chattopadhyay Ranajoy , Bhattacharyya Asima , Crowe Sheila E.
Published: March 2010

Human apurinic/apyrimidinic endonuclease-1 (APE-1), a key enzyme involved in repair of oxidative DNA base damage, is an important transcriptional coregulator. We previously reported that Helicobacter pylori infection induces apoptosis and increases APE-1 expression in human gastric epithelial cells (GEC). Although both the DNA repair activity and the acetylation-mediated transcriptional regulation of APE-1 are required to prevent cell death, the mechanisms of APE-1–mediated inhibition of infection-induced apoptosis are unclear. Here, we show that short hairpin RNA–mediated stable suppression of APE-1 results in increased apoptosis in GEC after H. pylori infection. We show that programmed cell death involves both the caspase-9–mediated mitochondrial pathway and the caspase-8–dependent extrinsic pathway by measuring different markers for both the pathways. Overexpression of wild-type APE-1 in APE-1–suppressed GEC reduced apoptosis after infection; however, overexpression of the DNA repair mutant or the nonacetylable mutant of APE-1 alone was unable to reduce apoptosis, suggesting that both DNA repair and acetylation functions of APE-1 modulate programmed cell death. We show for the first time that the DNA repair activity of APE-1 inhibits the mitochondrial pathway, whereas the acetylation function inhibits the extrinsic pathway during H. pylori infection. Thus, our findings establish that the two different functions of APE-1 differentially regulate the intrinsic and the extrinsic pathway of H. pylori–mediated GEC apoptosis. As proapoptotic and antiapoptotic mechanisms determine the development and progression of gastritis, gastric ulceration, and gastric cancer, this dual regulatory role of APE-1 represents one of the important molecular strategies by H. pylori to sustain chronic infection. Cancer Res; 70(7); 2799–808
New Strategies in Estrogen Receptor–Positive Breast Cancer
Authors: Johnston Stephen R.D.
Published: March 2010

Endocrine therapy has led to a significant improvement in outcomes for women with estrogen receptor–positive (ER+) breast cancer. Current questions in the adjuvant setting include the optimal duration of endocrine therapy, and the accurate molecular prediction of endocrine responsiveness using gene array–based assays compared with ER expression itself. In advanced disease, novel selective estrogen receptor antagonists (SERM) have failed to make an impact, although the pure ER antagonist fulvestrant may have a role, albeit optimal dose and sequence remain unclear. Overcoming de novo or acquired endocrine resistance remains critical to enhancing further the benefit of existing endocrine therapies. Recent progress has been made in understanding the molecular biology associated with acquired endocrine resistance, including adaptive “cross-talk” between ER and peptide growth factor receptor pathways such as epidermal growth factor receptor (EGFR)/human epidermal growth factor receptor 2 (HER2). Future strategies that are being evaluated include combining endocrine therapy with inhibitors of growth factor receptors or downstream signaling pathways, to treat or prevent critical resistance pathways that become operative in ER+ tumors. Preclinical experiments have provided great promise for this approach, although clinical data remain mixed. Enriching trial recruitment by molecular profiling of different ER+ subtypes will become increasingly important to maximize additional benefit that new agents may bring to current endocrine therapies for breast cancer. Clin Cancer Res; 16(7); 1979–87. ©2010 AACR.
Effect of Low-Dose Tamoxifen on Steroid Receptor Coactivator 3/Amplified in Breast Cancer 1 in Normal and Malignant Human Breast Tissue
Authors: Haugan Moi Line L. , Hauglid Flågeng Marianne , Gandini Sara , Guerrieri-Gonzaga Aliana , Bonanni Bernardo , Lazzeroni Matteo , Gjerde Jennifer , Lien Ernst A. , De Censi Andrea , Mellgren Gunnar
Published: March 2010

Purpose: Nuclear receptor coactivator expression and activity may partly explain the complex agonist/antagonist effects of tamoxifen at clinical level. In a preoperative trial, dose reduction from 20 to 1 mg tamoxifen was associated with retained antiproliferative effect on breast cancer. Here, we assessed the gene expression of the steroid receptor coactivators SRC-1, SRC-2/transcription intermediary factor 2, and SRC-3/amplified in breast cancer 1 (AIB1) and the growth factor receptor HER-2/neu under three tamoxifen dose regimens.

Experimental Design: Surgical specimens from estrogen receptor–positive breast cancer and adjacent normal breast tissue from 64 patients treated 4 weeks preoperatively with 20, 5, or 1 mg/d tamoxifen and 28 nontreated breast cancer controls were analyzed for coactivator and HER-2/neu mRNA expression using real-time reverse transcription-PCR. The gene expression levels were related to immunohistochemical expression of Ki67, serum levels of insulin-like growth factor I and sex hormone binding globulin, other prognostic factors, and clinical outcome.

Results: The coactivators and HER-2/neu mRNA levels were higher in malignant compared with normal tissue (P < 0.001). Tamoxifen significantly increased the expression of coactivators in normal and malignant tissue irrespective of dose, especially for SRC-3/AIB1 (P < 0.001 tamoxifen-treated versus nontreated subjects). SRC-3/AIB1 and HER-2/neu mRNA levels were positively correlated (P = 0.016), but the coactivators could not explain the variability of Ki67, insulin-like growth factor I, and sex hormone binding. Although not significant, SRC-3/AIB1 tended to be higher in subjects with poor clinical outcome and unfavorable prognostic factors.

Conclusions: Increased coactivator mRNA levels seem to be an early response to tamoxifen without dose-response relationship in the 1- to 20-mg range. Clinical and molecular effects of low-dose tamoxifen should be further explored. Clin Cancer Res; 16(7); 2176–86. ©2010 AACR.
A Phase II Study of PD-0325901, an Oral MEK Inhibitor, in Previously Treated Patients with Advanced Non–Small Cell Lung Cancer
Authors: Haura Eric B. , Ricart Alejandro D. , Larson Timothy G. , Stella Philip J. , Bazhenova Lyudmila , Miller Vincent A. , Cohen Roger B. , Eisenberg Peter D. , Selaru Paulina , Wilner Keith D. , Gadgeel Shirish M.
Published: March 2010

Purpose: To evaluate the efficacy of mitogen-activated protein kinase/extracellular signal-related kinase kinase inhibitor PD-0325901 in advanced non–small cell lung cancer patients who had experienced treatment failure after, or were refractory to, standard systemic therapy.

Experimental Design: This open-label, phase II study initially evaluated 15 mg PD-0325901 twice daily administered intermittently (3 weeks on/1 week off; schedule A). As this schedule was not well tolerated, a second schedule was introduced as follows: 5 days on/2 days off for 3 weeks, followed by 1 week off (schedule B). The primary end point was objective response.

Results: All patients had received prior systemic therapy (median of two regimens, including epidermal growth factor receptor inhibitors in 26%). Of 13 patients treated on schedule A, three discontinued due to adverse events (blurred vision, fatigue, and hallucinations, respectively). Twenty-one patients received schedule B. Main toxicities included diarrhea, fatigue, rash, vomiting, nausea, and reversible visual disturbances. Hematologic toxicity consisted mainly of mild-to-moderate anemia, without neutropenia. Chemistry abnormalities were rare. Mean (coefficient of variation) PD-0325901 trough plasma concentrations were 100 ng/mL (52%) and 173 ng/mL (73%) for schedules A and B, respectively, above the minimum target concentration established in preclinical studies (16.5 ng/mL). There were no objective responses. Seven patients had stable disease. Median (95% confidence interval) progression-free survival was 1.8 months (1.5-1.9) and overall survival was 7.8 months (4.5-13.9).

Conclusions: PD-0325901 did not meet its primary efficacy end point. Future studies should focus on PD-0325901 schedule, rational combination strategies, and enrichment of patient selection based on mode of action. Clin Cancer Res; 16(8); 2450–7. ©2010 AACR.
Lack of Correlation between Epidermal Growth Factor Receptor Status and Response to Panitumumab Monotherapy in Metastatic Colorectal Cancer
Authors: Hecht J. Randolph , Mitchell Edith , Neubauer Marcus A. , Burris Howard A. , Swanson Paul , Lopez Timothy , Buchanan Glenn , Reiner Maureen , Gansert Jennifer , Berlin Jordan
Published: March 2010

Purpose: Panitumumab, a fully human anti–epidermal growth factor receptor (EGFR) monoclonal antibody, is approved as monotherapy for the treatment of metastatic colorectal cancer. We evaluated the association of tumor EGFR expression levels with outcomes in patients with chemorefractory metastatic colorectal cancer.

Experimental Design: Two phase II, multicenter, single-arm, open-label studies enrolled chemorefractory patients with tumors expressing low/negative (1-9%/<1%; Low/Negative EGFR study) or high (≥10%; High EGFR study) levels of EGFR. Patients received panitumumab 6 mg/kg every two weeks until disease progression or intolerance. End points included objective response rate (per response evaluation criteria in solid tumors), progression-free survival (PFS), overall survival (OS), and safety. Exploratory analyses by tumor KRAS status were carried out.

Results: A total of 203 patients (Low/Negative EGFR) and 185 patients (High EGFR) enrolled in the studies. The overall response rate was 5.7% [95% confidence interval (95% CI), 2.6-10.5] in patients with low/negative EGFR and 4.2% (95% CI, 1.6-9.0) in patients with high EGFR; the response rate at week 16 was 4% in both studies (all partial responses). Median PFS times were 8.1 weeks (95% CI, 7.1-12.6), 8.1 weeks (95% CI, 7.4-11.1), and 7.3 weeks (95% CI, 7.1-7.6) in patients with negative, low, and high levels of EGFR expression, respectively. PFS and OS were longer in patients with wild-type KRAS than those with mutant KRAS. As expected, most adverse events were skin related.

Conclusions: These studies confirm previous reports that tumor EGFR expression levels are not associated with efficacy with an anti-EGFR antibody and that anti-EGFR antibody therapy should be limited to those patients whose tumors express wild-type KRAS. Clin Cancer Res; 16(7); 2205–13. ©2010 AACR.
Outcome of Photodynamic Therapy Using NPe6 for Bronchogenic Carcinomas in Central Airways >1.0 cm in Diameter
Authors: Usuda Jitsuo , Ichinose Shuji , Ishizumi Taichirou , Hayashi Hiroki , Ohtani Keishi , Maehara Sachio , Ono Shoutarou , Honda Hidetoshi , Kajiwara Naohiro , Uchida Osamu , Tsutsui Hidemitsu , Ohira Tatsuo , Kato Harubumi , Ikeda Norihiko
Published: March 2010

Purpose: Most centrally located early lung cancers (CLELC) <1.0 cm in diameter do not invade beyond the bronchial cartilage, and photodynamic therapy (PDT) with Photofrin is currently recommended as a treatment option for such lesions. NPe6 is a second-generation photosensitizer, and because it has a longer absorption band (664 nm) than Photofrin (630 nm), we hypothesized that NPe6-PDT would exert a strong antitumor effect against cancer lesions >1.0 cm in diameter, which are assumed to involve extracartilaginous invasion and to be unsuitable for treatment with Photofrin-PDT.

Experimental Design: Between June 2004 and December 2008, 75 patients (91 lesions) with CLELC underwent NPe6-PDT after the extent of their tumors had been assessed by fluorescence bronchoscopy for photodynamic diagnosis and tumor depth had been assessed by optical coherence tomography.

Results: Seventy cancer lesions ≤1.0 cm in diameter and 21 lesions >1.0 cm in diameter were identified, and the complete response rate was 94.0% (66 of 70) and 90.4% (19 of 21), respectively. After the mass of large tumors and deeply invasive tumors had been reduced by electrocautery, NPe6-PDT was capable of destroying the residual cancer lesions.

Conclusion: NPe6-PDT has a strong antitumor effect against CLELCs >1.0 cm in diameter that have invaded beyond the bronchial cartilage, thereby enabling the destruction of residual cancer lesions after mass reduction of large nodular- or polypoid-type lung cancers by electrocautery. The PDT guidelines for lung cancers should therefore be revised because use of NPe6-PDT will enable expansion of the clinical indications for PDT. Clin Cancer Res; 16(7); 2198–204. ©2010 AACR.
A Bispecific Enediyne-Energized Fusion Protein Containing Ligand-Based and Antibody-Based Oligopeptides against Epidermal Growth Factor Receptor and Human Epidermal Growth Factor Receptor 2 Shows Potent Antitumor Activity
Authors: Guo Xiao-fang , Zhu Xiao-fei , Shang Yue , Zhang Sheng-hua , Zhen Yong-su
Published: March 2010

Purpose: The cooverexpression of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) observed in many human tumors and their synergistic interaction in the transformation of cells make these receptors important targets for the development of new targeted therapeutics. Targeting of EGFR and HER2 simultaneously has been pursued as a strategy with which to potentially increase efficiency and selectivity in therapy of certain cancers. This study was set to construct a bispecific energized fusion protein (Ec-LDP-Hr-AE) consisting of two oligopeptides against EGFR and HER2, and lidamycin, and investigate its antitumor efficacy.

Experimental Design: In vitro experiments measured the binding and internalization of bispecific Ec-LDP-Hr fusion protein. The potency of energized fusion proteins was also done in which the bispecific Ec-LDP-Hr-AE was compared with lidamycin (LDM) and its monospecific counterparts, Ec-LDP-AE and LDP-Hr-AE. In vivo, Ec-LDP-Hr-AE was given i.v. to nude mice bearing human ovarian carcinoma SK-OV-3 xenografts.

Results: Binding and internalization studies showed that bispecific fusion protein Ec-LDP-Hr bound to carcinoma cells specifically and then were internalized into the cytoplasm. Bispecific Ec-LDP-Hr-AE was more potent and selective in its cytotoxicity against different carcinoma cell lines than corresponding momospecific agents and LDM in vitro. In addition, Ec-LDP-Hr-AE significantly inhibited the growth of SK-OV-3 xenografts in nude mouse model. In vivo imaging study showed that FITC-labeled Ec-LDP-Hr was targeted and accumulated in the tumors.

Conclusion: A ligand-based and an antibody-based oligopeptide fused to the enediyne antibiotic LDM created a new bispecific fusion protein with low molecular weight and more potent in vitro and in vivo antitumor activity (than momospecific fusion proteins). Clin Cancer Res; 16(7); 2085–94. ©2010 AACR.
Decreased Selenium-Binding Protein 1 in Esophageal Adenocarcinoma Results from Posttranscriptional and Epigenetic Regulation and Affects Chemosensitivity
Authors: Silvers Amy L. , Lin Lin , Bass Adam J. , Chen Guoan , Wang Zhuwen , Thomas Dafydd G. , Lin Jules , Giordano Thomas J. , Orringer Mark B. , Beer David G. , Chang Andrew C.
Published: March 2010

Purpose: The chemopreventive effects of selenium have been extensively examined, but its role in cancer development or as a chemotherapeutic agent has only recently been explored. Because selenium-binding protein 1 (SELENBP1, SBP1, hSP56) has been shown to bind selenium covalently and selenium deficiency has been associated with esophageal adenocarcinoma (EAC), we examined its role in EAC development and its potential effect on chemosensitivity in the presence of selenium.

Experimental Design: SELENBP1 expression level and copy number variation were determined by oligonucleotide microarrays, real-time reverse transcription-PCR, tissue microarrays, immunoblotting, and single-nucleotide polymorphism arrays. Bisulfite sequencing and sequence analysis of reverse transcription-PCR–amplified products explored epigenetic and posttranscriptional regulation of SELENBP1 expression, respectively. WST-1 cell proliferation assays, senescence-associated β-galactosidase staining, immunoblotting, and flow cytometry were done to evaluate the biological significance of SELENBP1 overexpression in selenium-supplemented EAC cells.

Results: SELENBP1 expression decreased significantly in Barrett's esophagus to adenocarcinoma progression. Both epigenetic and posttranscriptional mechanisms seemed to modulate SELENBP1 expression. Stable overexpression of SELENBP1 in methylseleninic acid–supplemented Flo-1 cells resulted in enhanced apoptosis, increased cellular senescence, and enhanced cisplatin cytotoxicity. Although inorganic sodium selenite similarly enhanced cisplatin cytotoxicity, these two forms of selenium elicited different cellular responses.

Conclusions: SELENBP1 expression may be an important predictor of response to chemoprevention or chemosensitization with certain forms of selenium in esophageal tissues. Clin Cancer Res; 16(7); 2009–21. ©2010 AACR.
Subtype-Specific FBXW7 Mutation and MYCN Copy Number Gain in Wilms' Tumor
Authors: Williams Richard D. , Al-Saadi Reem , Chagtai Tasnim , Popov Sergey , Messahel Boo , Sebire Neil , Gessler Manfred , Wegert Jenny , Graf Norbert , Leuschner Ivo , Hubank Mike , Jones Chris , Vujanic Gordan , Pritchard-Jones Kathy ,   Children's Cancer and Leukaemia Group,   SIOP Wilms' Tumour Biology Group
Published: March 2010

Purpose: Wilms' tumor (WT), the most common pediatric renal malignancy, is associated with mutations in several well-characterized genes, most notably WT1, CTNNB1, WTX, and TP53. However, the majority of cases do not harbor mutations in these genes. We hypothesized that additional drivers of tumor behavior would be contained within areas of consistent genomic copy number change, especially those associated with the WT risk groups defined by the International Society of Paediatric Oncology (SIOP).

Experimental Design: We analyzed high-resolution (Affymetrix 250K single nucleotide polymorphism array) genomic copy number profiles of over 100 tumors from selected risk groups treated under the SIOP protocols, further characterizing genes of interest by sequencing, Multiplex Ligation–dependent Probe Amplification, or fluorescence in situ hybridization.

Results: We identified FBXW7, an E3 ubiquitin ligase component, as a novel Wilms' tumor gene, mutated or deleted in ∼4% of tumors examined. Strikingly, 3 of 14 (21%) of tumors with epithelial type histology after neoadjuvant chemotherapy had FBXW7 aberrations, whereas a fourth WT patient had germline mutations in both FBXW7 and WT1. We also showed that MYCN copy number gain, detected in 9 of 104 (8.7%) of cases, is relatively common in WT and significantly more so in tumors of the high risk diffuse anaplastic subtype (6 of 19, 32%).

Conclusions: Because MYCN is itself a target of FBXW7-mediated ubiquitination and degradation, these results suggest that a common pathway is dysregulated by different mechanisms in various WT subtypes. Emerging therapies that target MYCN, which is amplified in several other pediatric cancers, may therefore be of value in high risk Wilms' tumor. Clin Cancer Res; 16(7); 2036–45. ©2010 AACR.
Osteoclast-Derived Matrix Metalloproteinase-9 Directly Affects Angiogenesis in the Prostate Tumor–Bone Microenvironment
Authors: Bruni-Cardoso Alexandre , Johnson Lindsay C. , Vessella Robert L. , Peterson Todd E. , Lynch Conor C.
Published: March 2010

In human prostate to bone metastases and in a novel rodent model that recapitulates prostate tumor–induced osteolytic and osteogenic responses, we found that osteoclasts are a major source of the proteinase, matrix metalloproteinase (MMP)-9. Because MMPs are important mediators of tumor-host communication, we tested the effect of host-derived MMP-9 on prostate tumor progression in the bone. To this end, immunocompromised mice that were wild-type or null for MMP-9 received transplants of osteolytic/osteogenic-inducing prostate adenocarcinoma tumor tissue to the calvaria. Surprisingly, we found that that host MMP-9 significantly contributed to prostate tumor growth without affecting prostate tumor–induced osteolytic or osteogenic change as determined by microcomputed tomography, microsingle-photon emission computed tomography, and histomorphometry. Subsequent studies aimed at delineating the mechanism of MMP-9 action on tumor growth focused on angiogenesis because MMP-9 and osteoclasts have been implicated in this process. We observed (a) significantly fewer and smaller blood vessels in the MMP-9 null group by CD-31 immunohistochemistry; (b) MMP-9 null osteoclasts had significantly lower levels of bioavailable vascular endothelial growth factor-A164; and (c) using an aorta sprouting assay, conditioned media derived from wild-type osteoclasts was significantly more angiogenic than conditioned media derived from MMP-9 null osteoclasts. In conclusion, these studies show that osteoclast-derived MMP-9 affects prostate tumor growth in the bone microenvironment by contributing to angiogenesis without altering prostate tumor–induced osteolytic or osteogenic changes. Mol Cancer Res; 8(4); 459–70. ©2010 AACR.
Sulindac Metabolites Induce Proteosomal and Lysosomal Degradation of the Epidermal Growth Factor Receptor
Authors: Pangburn Heather A. , Ahnen Dennis J. , Rice Pamela L.
Published: March 2010

The epidermal growth factor receptor (EGFR) is a member of the ErbB family of receptor tyrosine kinases. In response to ligand, EGFR is internalized and degraded by the ubiquitin-proteasome/lysosome pathway. We previously reported that metabolites of the nonsteroidal anti-inflammatory drug sulindac downregulate the expression of EGFR and inhibit basal and EGF-induced EGFR signaling through extracellular signal–regulated kinase 1/2. We now have evaluated the mechanisms of sulindac metabolite–induced downregulation of EGFR. EGF-induced downregulation of EGFR occurs within 10 minutes and lasts for 24 hours. By contrast, downregulation of EGFR by sulindac sulfide and sulindac sulfone was first evident at 4 and 24 hours, respectively, with maximal downregulation at 72 hours. Pretreatment with either the lysosomal inhibitor chloroquine or the proteosomal inhibitor MG132 blocked sulindac metabolite–induced downregulation of EGFR. Sulindac metabolites also increased the ubiquitination of EGFR. Whereas sulindac metabolites inhibited phosphorylation of EGFR pY1068, they increased phosphorylation of EGFR pY1045, the docking site where c-Cbl binds, thereby enabling receptor ubiquitination and degradation. Immunofluorescence analysis of EGF and EGFR distribution confirmed the biochemical observations that sulindac metabolites alter EGFR localization and EGFR internalization in a manner similar to that seen with EGF treatment. Expression of ErbB family members HER2 and HER3 was also downregulated by sulindac metabolites. We conclude that downregulation of EGFR expression by sulindac metabolites is mediated via lysosomal and proteosomal degradation that may be due to drug-induced phosphorylation at pY1045 with resultant ubiquitination of EGFR. Thus, sulindac metabolite–induced downregulation of EGFR seems to be mediated through mechanism(s) similar, at least in part, to those involved in EGF-induced downregulation of EGFR. Cancer Prev Res; 3(4); 560–72. ©2010 AACR.
Serum Oxidized Protein and Prostate Cancer Risk within the Prostate Cancer Prevention Trial
Authors: Hoque Ashraful , Ambrosone Christine B. , Till Cathee , Goodman Phyllis J. , Tangen Cathy , Kristal Alan , Lucia Scott , Wang Qiao , Kappil Maya , Thompson Ian , Hsing Ann W. , Parnes Howard , Santella Regina M.
Published: March 2010

To evaluate the role of oxidative stress in prostate cancer risk, we analyzed serum levels of protein carbonyl groups in 1,808 prostate cancer cases and 1,805 controls, nested in the Prostate Cancer Prevention Trial, a randomized, placebo-controlled trial that found finasteride decreased prostate cancer risk. There were no significant differences in protein carbonyl levels in baseline samples between those later diagnosed with prostate cancer and those without at the end of study biopsy. Adjusted odds ratios and 95% confidence intervals (95% CI) for the 4th quartile of protein carbonyl level for the combined, placebo, and finasteride arms were 1.03 (95% CI, 0.85-1.24), 0.88 (95% CI, 0.69-1.12), and 1.27 (95% CI, 0.94-1.71), respectively. There were no significant associations between carbonyl level and risk when analyzing high-grade and low-grade disease separately, nor did finasteride affect protein oxidation levels. The results of this large nested case-control study do not support the hypothesis that oxidative stress, at least as measured by protein carbonyl level, plays a role in prostate cancer. Cancer Prev Res; 3(4); 478–83. ©2010 AACR.
Vaccine Prevention of Cancer: Can Endogenous Antigens Be Targeted?
Authors: Weiner Louis M. , Surana Rishi , Murray Joseph
Published: March 2010

This perspective on the report by Beatty et al. in this issue of the journal (beginning on page 438) discusses the prevention of cancer through vaccination strategies that target antigens associated with tumor promotion and progression. Such approaches were first developed for treating cancer. We address cancer vaccination in the context of a mouse model of inflammatory bowel disease expressing MUC1, an epithelial mucin aberrantly expressed during chronic inflammation and in colorectal carcinogenesis, and in a broader context that includes the potential of targeting the tumor microenvironment for immunoprevention in humans. Obstacles in developing effective cancer vaccines, including antigen selection, immunoediting, and tumor-mediated immunosuppression, are also discussed. Cancer Prev Res; 3(4); 410–5. ©2010 AACR.
Estrogen Actions on Neuroendocrine Glia
Authors: Micevych Paul , Bondar Galyna , Kuo John
Published: March 2010

Astrocytes are the most abundant cells in the central nervous system (CNS). It appears that astrocytes are as diverse as neurons, having different phenotypes in various regions throughout the brain and participating in intercellular communication that involves signaling to neurons. It is not surprising then that astrocytes in the hypothalamus have an active role in the CNS regulation of reproduction. In addition to the traditional mechanism involving ensheathment of neurons and processes, astrocytes may have a critical role in regulating estrogen-positive feedback. Work in our laboratory has focused on the relationship between circulating estradiol and progesterone synthesized de novo in the brain. We have demonstrated that circulating estradiol stimulates the synthesis of progesterone in adult hypothalamic astrocytes, and this neuroprogesterone is critical for initiating the LH surge. Estradiol cell signaling is initiated at the cell membrane and involves the transactivation of metabotropic glutamate receptor type 1a (mGluR1a) leading to the release of intracellular stores of calcium. We used surface biotinylation to demonstrate that estrogen receptor-α (ERα) is present in the cell membrane and has an extracellular portion. Like other membrane receptors, ERα is inserted into the membrane and removed via internalization after agonist stimulation. This trafficking is directly regulated by estradiol, which rapidly and transiently increases the levels of membrane ERα, and upon activation, increases internalization that finally leads to ERα degradation. This autoregulation temporally limits membrane-initiated estradiol cell signaling. Thus, neuroprogesterone, the necessary signal for the LH surge, is released when circulating levels of estradiol peak on proestrus and activate progesterone receptors whose expression has been induced by the gradual rise of estradiol during follicular development.
Approaches for Evaluating Rare Polymorphisms in Genetic Association Studies
Authors: Li Qizhai , Zhang Hong , Yu Kai
Published: March 2010

Most current genetic association studies, including genome-wide association studies, look for the single nucleotide polymorphisms (SNPs) with a relatively large minor allele frequency (MAF) (e.g. >5%) in the search for genetic loci underlying the susceptibility for complex diseases. The strategy of focusing on common SNPs in genetic association studies is very effective under the common-disease-common-variant (CDCV) hypothesis, which claims that common diseases are caused by common variants that have relatively small to moderate effects. Although the CDCV hypothesis has become the dogma guiding the conduct of association studies over the past decade, growing evidence from recent empirical data and simulations suggests that the causal genetic polymorphisms, including SNPs and copy number variants (CNVs), for common diseases have a wide spectrum of MAFs, ranging from rare to common. Unlike the analysis for common genetic variants, statistical approaches for the analysis of rare variants receive very little attention. Methods developed for common variants usually rely on their asymptotic properties, which can be inaccurate for the study of the rare variants with limited sample size. Although Fisher’s exact test can be used for such a scenario, it is usually conservative and thus its usefulness is diminished to some extent. Here we propose two novel approaches for the analysis of rare genetic variants. Simulation studies and two real examples demonstrate the advantages of the proposed methods over the existing methods.
Molecular Characterization of α-Thalassemia in Adana, Turkey: A Single Center Study
Authors: Guvenc Birol , Yildiz Sule Menziletoglu , Tekinturhan Ferda , Dincer Suleyman , Akyuzluer Inci , Okten Secaatin , Erkman Hakan
Published: March 2010

Background/Aim: α-Thalassemia (α-thal) is a widespread genetic disorder throughout the world caused primarily by reduced synthesis of the α-globin chains, and it has been found at a high incidence in Turkey. Our aim in this study was to determine the frequency and molecular properties of α-thal in Adana, Turkey. Material and Methods: A total of 3,000 individuals comprising premarital couples or patients with anemia were screened between 2007 and 2008. Hematological parameters were analyzed using an automatic cell counter, and to detect the carriers of hemoglobin variants, high-performance liquid chromatography was used. Molecular screening of the α-globin gene was carried out by an Alpha-Globin StripAssay® which is based on multiplex PCR for specific amplification. Results: We have identified 225 cases with α-thal and found that the prevalence of α-thal is 7.5% in this area. In molecular analyses, the α-thal gene mutations α3.7, α4.2, --MED, --20.5, αPA-2α, αααanti-3.7, and αPA-1α were detected. Conclusion: Our results showed that the α-thal mutations represent a great heterogeneity and that the -α3.7 deletion has the highest frequency in Adana.