bluuebunny/crossref_metadata_2025 huggingface dataset:
Studies of mucus in mouse stomach, small intestine, and colon. II. Gastrointestinal mucus proteome reveals Muc2 and Muc5ac accompanied by a set of core proteins
Authors: Rodríguez-Piñeiro Ana M. , Bergström Joakim H. , Ermund Anna , Gustafsson Jenny K. , Schütte André , Johansson Malin E. V. , Hansson Gunnar C.
Published: July 2013

The mucus that protects the surface of the gastrointestinal tract is rich in specialized O-glycoproteins called mucins, but little is known about other mucus proteins or their variability along the gastrointestinal tract. To ensure that only mucus was analyzed, we combined collection from explant tissues mounted in perfusion chambers, liquid sample preparation, single-shot mass spectrometry, and specific bioinformatics tools, to characterize the proteome of the murine mucus from stomach to distal colon. With our approach, we identified ∼1,300 proteins in the mucus. We found no differences in the protein composition or abundance between sexes, but there were clear differences in mucus along the tract. Noticeably, mucus from duodenum showed similarities to the stomach, probably reflecting the normal distal transport. Qualitatively, there were, however, fewer differences than might had been anticipated, suggesting a relatively stable core proteome (∼80% of the total proteins identified). Quantitatively, we found significant differences (∼40% of the proteins) that could reflect mucus specialization throughout the gastrointestinal tract. Hierarchical clustering pinpointed a number of such proteins that correlated with Muc2 (e.g., Clca1, Zg16, Klk1). This study provides a deeper knowledge of the gastrointestinal mucus proteome that will be important in further understanding this poorly studied mucosal protection system.
Morning blood pressure surge is associated with arterial stiffness and sympathetic baroreflex sensitivity in hypertensive seniors
Authors: Okada Yoshiyuki , Galbreath M. Melyn , Shibata Shigeki , Jarvis Sara S. , Bivens Tiffany B. , Vongpatanasin Wanpen , Levine Benjamin D. , Fu Qi
Published: July 2013

Morning blood pressure (BP) surge is considered to be an independent risk factor for cardiovascular diseases. We tested the hypothesis that increased large-artery stiffness and impaired sympathetic baroreflex sensitivity (BRS) contribute to augmented morning surge in elderly hypertensive subjects. Morning surge was assessed as morning systolic BP averaged for 2 h just after waking up minus minimal sleeping systolic BP by using ambulatory BP monitoring (ABPM) in 40 untreated hypertensive [68 ± 1 (SE) yr] and 30 normotensive (68 ± 1 yr) subjects. Beat-by-beat finger BP and muscle sympathetic nerve activity (MSNA) were recorded in the supine position and at 60° upright tilt. We assessed arterial stiffness with carotid-to-femoral pulse wave velocity (cfPWV) and sympathetic BRS during spontaneous breathing. Awake and asleep ABPM-BPs and morning surge were higher in hypertensive than normotensive subjects (all P < 0.001). cfPWV was higher ( P = 0.002) and sympathetic BRS was lower ( P = 0.096) in hypertensive than normotensive subjects. Hypertensive subjects with morning surge ≥35 mmHg (median value) had higher cfPWV (11.9 ± 0.5 vs. 9.9 ± 0.4 m/s, P = 0.002) and lower sympathetic BRS (supine: −2.71 ± 0.25 vs. −3.73 ± 0.29, P = 0.011; upright: −2.62 ± 0.22 vs. −3.51 ± 0.35 bursts·100 beats−1·mmHg−1, P = 0.052) than those with morning surge <35 mmHg. MSNA indices were similar between groups (all P > 0.05), while upright total peripheral resistance was higher in hypertensive subjects with greater morning surge than those with lesser morning surge ( P = 0.050). Morning surge was correlated positively with cfPWV ( r = 0.59, P < 0.001) and negatively with sympathetic BRS ( r = 0.51, P < 0.001) in hypertensive subjects only. Thus, morning BP surge is associated with arterial stiffness and sympathetic BRS, as well as vasoreactivity during orthostasis in hypertensive seniors.
Cardiac overexpression of Mammalian enabled (Mena) exacerbates heart failure in mice
Authors: Belmonte Stephen L. , Ram Rashmi , Mickelsen Deanne M. , Gertler Frank B. , Blaxall Burns C.
Published: July 2013

Mammalian enabled (Mena) is a key regulator of cytoskeletal actin dynamics, which has been implicated in heart failure (HF). We have previously demonstrated that cardiac Mena deletion produced cardiac dysfunction with conduction abnormalities and hypertrophy. Moreover, elevated Mena expression correlates with HF in human and animal models, yet the precise role of Mena in cardiac pathophysiology is unclear. In these studies, we evaluated mice with cardiac myocyte-specific Mena overexpression (TTA/TgTetMena) comparable to that observed in cardiac pathology. We found that the hearts of TTA/TgTetMena mice were functionally and morphologically comparable to wild-type littermates, except for mildly increased heart mass in the transgenic mice. Interestingly, TTA/TgTetMena mice were particularly susceptible to cardiac injury, as these animals experienced pronounced decreases in ejection fraction and fractional shortening as well as heart dilatation and hypertrophy after transverse aortic constriction (TAC). By “turning off” Mena overexpression in TTA/TgTetMena mice either immediately prior to or immediately after TAC surgery, we discovered that normalizing Mena levels eliminated cardiac hypertrophy in TTA/TgTetMena animals but did not preclude post-TAC cardiac functional deterioration. These findings indicate that hearts with increased levels of Mena fare worse when subjected to cardiac injury and suggest that Mena contributes to HF pathophysiology.
Influence of spontaneously occurring bursts of muscle sympathetic nerve activity on conduit artery diameter
Authors: Fairfax Seth T. , Padilla Jaume , Vianna Lauro C. , Holwerda Seth H. , Davis Michael J. , Fadel Paul J.
Published: July 2013

Large increases in muscle sympathetic nerve activity (MSNA) can decrease the diameter of a conduit artery even in the presence of elevated blood pressure, suggesting that MSNA acts to regulate conduit artery tone. Whether this influence can be extrapolated to spontaneously occurring MSNA bursts has not been examined. Therefore, we tested the hypothesis that MSNA bursts decrease conduit artery diameter on a beat-by-beat basis during rest. Conduit artery responses were assessed in the brachial (BA), common femoral (CFA) and popliteal (PA) arteries to account for regional differences in vascular function. In 20 young men, MSNA, mean arterial pressure (MAP), conduit artery diameter, and shear rate (SR) were continuously measured during 20-min periods of supine rest. Spike-triggered averaging was used to characterize beat-by-beat changes in each variable for 15 cardiac cycles following all MSNA bursts, and a peak response was calculated. Diameter increased to a similar peak among the BA (+0.14 ± 0.02%), CFA (+0.17 ± 0.03%), and PA (+0.18 ± 0.03%) following MSNA bursts (all P < 0.05 vs. control). The diameter rise was positively associated with an increase in MAP in relation to increasing amplitude and consecutive numbers of MSNA bursts ( P < 0.05). Such relationships were similar between arteries. SR changes following MSNA bursts were heterogeneous between arteries and did not appear to systematically alter diameter responses. Thus, in contrast to our hypothesis, spontaneously occurring MSNA bursts do not directly influence conduit arteries with local vasoconstriction or changes in shear, but rather induce a systemic pressor response that appears to passively increase conduit artery diameter.
Activation of melanocortin receptors in the intermediolateral cell column of the upper thoracic cord elicits tachycardia in the rat
Authors: Iwasa Masamitsu , Kawabe Kazumi , Sapru Hreday N.
Published: July 2013

Melanocortin receptors (MCRs) are present in the intermediolateral cell column of the spinal cord (IML). We tested the hypothesis that activation of MCRs in the IML elicits cardioacceleratory responses and the source of melanocortins in the IML may be the melanocortin-containing neurons in the hypothalamic arcuate nucleus (ARCN). Experiments were done in urethane-anesthetized, artificially ventilated adult male Wistar rats. Microinjections (50 nl) of α-melanocyte stimulating hormone (α-MSH) (0.4–2 mM) and adrenocorticotropic hormone (ACTH) (0.5–2 mM) into the right IML elicited increases in heart rate (HR). These tachycardic responses were blocked by microinjections of melanocortin receptor 4 (MC4R) antagonists [SHU9119 (0.25 mM) or agouti-related protein (AGRP, 0.1 mM)] into the right IML. Stimulation of right ARCN by microinjections (30 nl) of N-methyl-d-aspartic acid (NMDA, 10 mM) elicited increases in HR. Blockade of MC4Rs in the ipsilateral IML at T1–T3 using SHU9119 (0.25 mM) attenuated the tachycardic responses elicited by subsequent microinjections of NMDA into the ipsilateral ARCN. ARCN neurons retrogradely labeled by microinjections of Fluoro-Gold into the right IML showed immunoreactivity for proopiomelanocortin (POMC), α-MSH, and ACTH. Fibers immunoreactive for POMC, α-MSH, and ACTH were present in the IML at T1-T3. These results indicated that activation of MC4Rs in the right IML elicited tachycardia and one of the sources of melanocortins in the IML is the ARCN. Melanocortin levels are elevated in stress and ARCN neurons are activated during stress. Our results allude to the possibility that cardiac effects of stress may be mediated via melanocortin containing ARCN neurons that project to the IML.
Human-induced pluripotent stem cell-derived cardiomyocytes exhibit temporal changes in phenotype
Authors: Ivashchenko Christine Y. , Pipes Gordon C. , Lozinskaya Irina M. , Lin Zuojun , Xiaoping Xu , Needle Saul , Grygielko Eugene T. , Hu Erding , Toomey John R. , Lepore John J. , Willette Robert N.
Published: July 2013

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) have been recently derived and are used for basic research, cardiotoxicity assessment, and phenotypic screening. However, the hiPS-CM phenotype is dependent on their derivation, age, and culture conditions, and there is disagreement as to what constitutes a functional hiPS-CM. The aim of the present study is to characterize the temporal changes in hiPS-CM phenotype by examining five determinants of cardiomyocyte function: gene expression, ion channel functionality, calcium cycling, metabolic activity, and responsiveness to cardioactive compounds. Based on both gene expression and electrophysiological properties, at day 30 of differentiation, hiPS-CMs are immature cells that, with time in culture, progressively develop a more mature phenotype without signs of dedifferentiation. This phenotype is characterized by adult-like gene expression patterns, action potentials exhibiting ventricular atrial and nodal properties, coordinated calcium cycling and beating, suggesting the formation of a functional syncytium. Pharmacological responses to pathological (endothelin-1), physiological (IGF-1), and autonomic (isoproterenol) stimuli similar to those characteristic of isolated adult cardiac myocytes are present in maturing hiPS-CMs. In addition, thyroid hormone treatment of hiPS-CMs attenuated the fetal gene expression in favor of a more adult-like pattern. Overall, hiPS-CMs progressively acquire functionality when maintained in culture for a prolonged period of time. The description of this evolving phenotype helps to identify optimal use of hiPS-CMs for a range of research applications.
Transdifferentiation of alveolar epithelial type II to type I cells is controlled by opposing TGF-β and BMP signaling
Authors: Zhao Lan , Yee Min , O'Reilly Michael A.
Published: July 2013

Alveolar epithelial type II (ATII) cells are essential for maintaining normal lung homeostasis because they produce surfactant, express innate immune proteins, and can function as progenitors for alveolar epithelial type I (ATI) cells. Although autocrine production of transforming growth factor (TGF)-β1 has been shown to promote the transdifferentiation of primary rat ATII to ATI cells in vitro, mechanisms controlling this process still remain poorly defined. Here, evidence is provided that Tgf-β1, - 2, - 3 mRNA and phosphorylated SMAD2 and SMAD3 significantly increase as primary cultures of mouse ATII cells transdifferentiate to ATI cells. Concomitantly, bone morphogenetic protein ( Bmp)-2 and -4 mRNA, and phosphorylated SMAD1/5/8 expression decrease. Exogenously supplied recombinant human TGF-β1 inhibited BMP signaling and enhanced transdifferentiation by promoting the loss of ATII cell-specific gene expression and weakly stimulating ATI cell-specific gene expression. On the other hand, exogenously supplied recombinant human BMP-4 inhibited TGF-β signaling and delayed transdifferentiation by inhibiting the gain in ATI cell-specific gene expression and weakly delaying the loss of ATII cell-specific gene expression. In mouse lung epithelial (MLE15) cells, small-interfering RNA (siRNA) knockdown of TGF-β receptor type-1 enhanced basal expression of ATII genes while siRNA RNA knockdown of BMP receptors type-1a and -1b enhanced basal expression of ATI genes. Together, these results suggest that the rate of ATII cell transdifferentiation is controlled by the opposing actions of BMP and TGF-β signaling that switch during the process of transdifferentiation.
DNA methylation in nasal epithelial cells from smokers: identification of ULBP3-related effects
Authors: Rager Julia E. , Bauer Rebecca N. , Müller Loretta L. , Smeester Lisa , Carson Johnny L. , Brighton Luisa E. , Fry Rebecca C. , Jaspers Ilona
Published: July 2013

We previously demonstrated that, in nasal epithelial cells (NECs) from smokers, methylation of an antiviral gene was associated with impaired antiviral defense responses. To expand these findings and better understand biological mechanisms underlying cigarette smoke (CS)-induced modifications of host defense responses, we aimed to compare DNA methylation of genes that may play a role in antiviral response. We used a two-tiered analytical approach, where we first implemented a genome-wide strategy. NECs from smokers differed in the methylation levels of 390 genes, the majority (84%) of which showed decreased methylation in smokers. Secondly, we generated an a priori set of 161 antiviral response-related genes, of which five were differentially methylated in NEC from smokers ( CCL2, FDPS, GSK3B, SOCS3, and ULBP3). Assessing these genes at the systems biology level revealed a protein interaction network associated with CS-induced epigenetic modifications involving SOCS3 and ULBP3 signaling, among others. Subsequent confirmation studies focused on SOCS3 and ULBP3, which were hypomethylated and hypermethylated, respectively. Expression of SOCS3 was increased, whereas ULBP3 expression was decreased in NECs from smokers. Addition of the demethylating agent 5-Aza-2-deoxycytidine enhanced ULBP3 expression in NECs from smokers. Furthermore, infection of differentiated NECs with influenza virus resulted in significantly lower levels of ULBP3 in cells from smokers. Taken together, our findings show that genomic DNA methylation profiles are altered in NECs from smokers and that these changes are associated with decreased antiviral host defense responses, indicating that epigenenic dysregulation of genes such as SOCS3 and ULBP3 likely impacts immune responses in the epithelium.
Cooperation and Antagonism among Cancer Genes: The Renal Cancer Paradigm
Authors: Peña-Llopis Samuel , Christie Alana , Xie Xian-Jin , Brugarolas James
Published: July 2013

It is poorly understood how driver mutations in cancer genes work together to promote tumor development. Renal cell carcinoma (RCC) offers a unique opportunity to study complex relationships among cancer genes. The four most commonly mutated genes in RCC of clear-cell type (the most common type) are two-hit tumor suppressor genes, and they cluster in a 43-Mb region on chromosome 3p that is deleted in approximately 90% of tumors: VHL (mutated in ∼80%), PBRM1 (∼50%), BAP1 (∼15%), and SETD2 (∼15%). Meta-analyses that we conducted show that mutations in PBRM1 and SETD2 co-occur in tumors at a frequency higher than expected by chance alone, indicating that these mutations may cooperate in tumorigenesis. In contrast, consistent with our previous results, mutations in PBRM1 and BAP1 tend to be mutually exclusive. Mutation exclusivity analyses (often confounded by lack of statistical power) raise the possibility of functional redundancy. However, mutation exclusivity may indicate negative genetic interactions, as proposed herein for PBRM1 and BAP1, and mutations in these genes define RCC with different pathologic features, gene expression profiles, and outcomes. Negative genetic interactions among cancer genes point toward broader context dependencies of cancer gene action beyond tissue dependencies. An enhanced understanding of cancer gene dependencies may help to unravel vulnerabilities that can be exploited therapeutically. Cancer Res; 73(14); 4173–9. ©2013 AACR.
Lessons from Functional Analysis of Genome-Wide Association Studies
Authors: Sur Inderpreet , Tuupanen Sari , Whitington Thomas , Aaltonen Lauri A. , Taipale Jussi
Published: July 2013

Most cancer-associated single-nucleotide polymorphisms (SNP) identified using genome-wide association studies are located outside of protein-coding regions, and their significance and mode of action have been a source of continuing debate. One proposed mechanism of action of the SNPs is that they would affect the activity of enhancer elements regulating critical target genes. In this review, we summarize recent results that substantiate this model. These studies have identified a cancer-specific enhancer element at the 8q24 gene desert that controls the expression of the MYC oncogene. We further discuss implications of the observed difference between normal growth control and cancer for drug development, and the inherent features of genome-wide association studies that may specifically lead to identification of disease-specific regulatory elements. Cancer Res; 73(14); 4180–4. ©2013 AACR.
C1GALT1 Enhances Proliferation of Hepatocellular Carcinoma Cells via Modulating MET Glycosylation and Dimerization
Authors: Wu Yao-Ming , Liu Chiung-Hui , Huang Miao-Juei , Lai Hong-Shiee , Lee Po-Huang , Hu Rey-Heng , Huang Min-Chuan
Published: July 2013

Altered glycosylation is a hallmark of cancer. The core 1 β1,3-galactosyltransferase (C1GALT1) controls the formation of mucin-type O-glycans, far overlooked and underestimated in cancer. Here, we report that C1GALT1 mRNA and protein are frequently overexpressed in hepatocellular carcinoma tumors compared with nontumor liver tissues, where it correlates with advanced tumor stage, metastasis, and poor survival. Enforced expression of C1GALT1 was sufficient to enhance cell proliferation, whereas RNA interference–mediated silencing of C1GALT1 was sufficient to suppress cell proliferation in vitro and in vivo. Notably, C1GALT1 attenuation also suppressed hepatocyte growth factor (HGF)–mediated phosphorylation of the MET kinase in hepatocellular carcinoma cells, whereas enforced expression of C1GALT1 enhanced MET phosphorylation. MET blockade with PHA665752 inhibited C1GALT1-enhanced cell viability. In support of these results, we found that the expression level of phospho-MET and C1GALT1 were associated in primary hepatocellular carcinoma tissues. Mechanistic investigations showed that MET was decorated with O-glycans, as revealed by binding to Vicia villosa agglutinin and peanut agglutinin. Moreover, C1GALT1 modified the O-glycosylation of MET, enhancing its HGF-induced dimerization and activation. Together, our results indicate that C1GALT1 overexpression in hepatocellular carcinoma activates HGF signaling via modulation of MET O-glycosylation and dimerization, providing new insights into how O-glycosylation drives hepatocellular carcinoma pathogenesis. Cancer Res; 73(17); 5580–90. ©2013 AACR.
Distinct FAK Activities Determine Progenitor and Mammary Stem Cell Characteristics
Authors: Luo Ming , Zhao Xiaofeng , Chen Song , Liu Suling , Wicha Max S. , Guan Jun-Lin
Published: July 2013

Mammary stem cells (MaSC) and progenitor cells are important for mammary gland development and maintenance and may give rise to mammary cancer stem cells (MaCSC). Yet, there remains limited understanding of how these cells contribute to tumorigenesis. Here, we show that conditional deletion of focal adhesion kinase (FAK) in embryonic mammary epithelial cells (MaEC) decreases luminal progenitors and basal MaSCs, reducing their colony-forming and regenerative potentials in a cell-autonomous manner. Loss of FAK kinase activity in MaECs specifically impaired luminal progenitor proliferation and alveologenesis, whereas a kinase-independent activity of FAK supported ductal invasion and basal MaSC activity. Deficiency in luminal progenitors suppressed tumorigenesis and MaCSC formation in a mouse model of breast cancer. In contrast with the general inhibitory effect of FAK attenuation, inhibitors of FAK kinase preferentially inhibited proliferation and tumorsphere formation of luminal progenitor-like, but not MaSC-like, human breast cancer cells. Our findings establish distinct kinase-dependent and -independent activities of FAK that differentially regulate luminal progenitors and basal MaSCs. We suggest that targeting these distinct functions may tailor therapeutic strategies to address breast cancer heterogeneity more effectively. Cancer Res; 73(17); 5591–602. ©2013 AACR.
Germline Missense Variants in the BTNL2 Gene Are Associated with Prostate Cancer Susceptibility
Authors: FitzGerald Liesel M. , Kumar Akash , Boyle Evan A. , Zhang Yuzheng , McIntosh Laura M. , Kolb Suzanne , Stott-Miller Marni , Smith Tiffany , Karyadi Danielle M. , Ostrander Elaine A. , Hsu Li , Shendure Jay , Stanford Janet L.
Published: July 2013

Background: Rare, inherited mutations account for 5% to 10% of all prostate cancer cases. However, to date, few causative mutations have been identified.

Methods: To identify rare mutations for prostate cancer, we conducted whole-exome sequencing (WES) in multiple kindreds (n = 91) from 19 hereditary prostate cancer (HPC) families characterized by aggressive or early-onset phenotypes. Candidate variants (n = 130) identified through family- and bioinformatics-based filtering of WES data were then genotyped in an independent set of 270 HPC families (n = 819 prostate cancer cases; n = 496 unaffected relatives) for replication. Two variants with supportive evidence were subsequently genotyped in a population-based case–control study (n = 1,155 incident prostate cancer cases; n = 1,060 age-matched controls) for further confirmation. All participants were men of European ancestry.

Results: The strongest evidence was for two germline missense variants in the butyrophilin-like 2 (BTNL2) gene (rs41441651, p.Asp336Asn and rs28362675, p.Gly454Cys) that segregated with affection status in two of the WES families. In the independent set of 270 HPC families, 1.5% (rs41441651; P = 0.0032) and 1.2% (rs28362675; P = 0.0070) of affected men, but no unaffected men, carried a variant. Both variants were associated with elevated prostate cancer risk in the population-based study (rs41441651: OR, 2.7; 95% CI, 1.27–5.87; P = 0.010; rs28362675: OR, 2.5; 95% CI, 1.16–5.46; P = 0.019).

Conclusions: Results indicate that rare BTNL2 variants play a role in susceptibility to both familial and sporadic prostate cancer.

Impact: Results implicate BTNL2 as a novel prostate cancer susceptibility gene. Cancer Epidemiol Biomarkers Prev; 22(9); 1520–8. ©2013 AACR.
Long-Term Statin Use and Risk of Ductal and Lobular Breast Cancer among Women 55 to 74 Years of Age
Authors: McDougall Jean A. , Malone Kathleen E. , Daling Janet R. , Cushing-Haugen Kara L. , Porter Peggy L. , Li Christopher I.
Published: July 2013

Background: Mechanistic studies largely support the chemopreventive potential of statins. However, results of epidemiologic studies investigating statin use and breast cancer risk have been inconsistent and lacked the ability to evaluate long-term statin use.

Methods: We used data from a population-based case–control study of breast cancer conducted in the Seattle–Puget Sound region to investigate the relationship between long-term statin use and breast cancer risk. Nine hundred sixteen invasive ductal carcinoma (IDC) and 1,068 invasive lobular carcinoma (ILC) cases in patients 55 to 74 years of age diagnosed between 2000 and 2008 were compared with 902 control women. All participants were interviewed in-person and data on hypercholesterolemia and all episodes of lipid-lowering medication use were collected through a structured questionnaire. We assessed the relationship between statin use and IDC and ILC risk using polytomous logistic regression.

Results: Current users of statins for 10 years or longer had a 1.83-fold increased risk of IDC [95% confidence interval (CI): 1.14–2.93] and a 1.97-fold increased risk of ILC (95% CI: 1.25–3.12) compared with never users of statins. Among women diagnosed with hypercholesterolemia, current users of statins for 10 years or longer had more than double the risk of both IDC (OR: 2.04, 95% CI: 1.17–3.57) and ILC (OR: 2.43, 95% CI: 1.40–4.21) compared with never users.

Conclusion: In this contemporary population-based case–control study, long-term use of statins was associated with increased risks of both IDC and ILC.

Impact: Additional studies with similarly high frequencies of statin use for various durations are needed to confirm this novel finding. Cancer Epidemiol Biomarkers Prev; 22(9); 1529–37. ©2013 AACR.
Cutaneous Markers of Photo-Damage and Risk of Basal Cell Carcinoma of the Skin: A Meta-Analysis
Authors: Khalesi Mohammad , Whiteman David C. , Doi Suhail A.R. , Clark Justin , Kimlin Michael G. , Neale Rachel E.
Published: July 2013

Epidemiologic research has shown that cutaneous markers of photo-damage are associated with risk of basal cell carcinoma (BCC). However, there has been no previous attempt to calculate pooled risk estimates. We conducted a systematic review and meta-analysis after extracting relevant studies published up to January 2013 from five electronic databases. Eligible studies were those that permitted quantitative assessment of the association between histologically confirmed BCC and actinic keratoses, solar elastosis, solar lentigines, or telangiectasia. Seven eligible studies were identified and summary odds ratios (ORs) were calculated using both random and quality effects models. Having more than ten actinic keratoses was most strongly associated with BCC, conferring up to a fivefold increase in risk (OR: 4.97; 95% CI: 3.26–7.58). Other factors, including solar elastosis, solar lentigines, and telangiectasia had weaker but positive associations with BCC with ORs around 1.5. Markers of chronic photo-damage are positively associated with BCC. The presence of actinic keratoses was the most strongly associated with BCC of the markers examined. This work highlights the relatively modest association between markers of chronic ultraviolet exposure and BCC. Cancer Epidemiol Biomarkers Prev; 22(9); 1483–9. ©2013 AACR.
Frequent Antibiotic Use and Second Breast Cancer Events
Authors: Wirtz Heidi S. , Buist Diana S.M. , Gralow Julie R. , Barlow William E. , Gray Shelly , Chubak Jessica , Yu Onchee , Bowles Erin J.A. , Fujii Monica , Boudreau Denise M.
Published: July 2013

Background: Antibiotic use may be associated with higher breast cancer risk and breast cancer mortality, but no study has evaluated the relation between antibiotic use and second breast cancer events (SBCE).

Methods: We conducted a retrospective cohort study among women ≥18 years, diagnosed with incident stage I/II breast cancer during 1990–2008. Antibiotic use and covariates were obtained from health plan administrative databases and medical record review. Frequent antibiotic use was defined as ≥4 antibiotic dispensings in any moving 12-month period after diagnosis. Our outcome was SBCE defined as recurrence or second primary breast cancer. We used multivariable Cox proportional hazards models to estimate HR and 95% confidence intervals (CI), accounting for competing risks.

Results: A total of 4,216 women were followed for a median of 6.7 years. Forty percent were frequent antibiotic users and 558 (13%) had an SBCE. Results are suggestive of a modest increased risk of SBCE (HR, 1.15; 95% CI, 0.88–1.50) among frequent antibiotic users compared with nonusers. Any potential increased risk was not supported when we evaluated recent use and past use. We observed no dose–response trends for SBCE with increasing duration of antibiotic use nor did we find evidence for altered SBCE risk in the antibiotic classes studied.

Conclusions: Frequent antibiotic use may be associated with modestly elevated risk of SBCEs, but the association was not significant.

Impact: Additional investigation by antibiotic class and underlying indication are important next steps given the high prevalence of frequent antibiotic use and growing number of breast cancer survivors. Cancer Epidemiol Biomarkers Prev; 22(9); 1588–99. ©2013 AACR.
Molecular Pathways: MERTK Signaling in Cancer
Authors: Cummings Christopher T. , DeRyckere Deborah , Earp H. Shelton , Graham Douglas K.
Published: July 2013

MERTK is a receptor tyrosine kinase of the TAM (Tyro3, Axl, MERTK) family, with a defined spectrum of normal expression. However, MERTK is overexpressed or ectopically expressed in a wide variety of cancers, including leukemia, non–small cell lung cancer, glioblastoma, melanoma, prostate cancer, breast cancer, colon cancer, gastric cancer, pituitary adenomas, and rhabdomyosarcomas, potentially resulting in the activation of several canonical oncogenic signaling pathways. These include the mitogen-activated protein kinase and phosphoinositide 3-kinase pathways, as well as regulation of signal transducer and activator of transcription family members, migration-associated proteins including the focal adhesion kinase and myosin light chain 2, and prosurvival proteins such as survivin and Bcl-2. Each has been implicated in MERTK physiologic and oncogenic functions. In neoplastic cells, these signaling events result in functional phenotypes such as decreased apoptosis, increased migration, chemoresistance, increased colony formation, and increased tumor formation in murine models. Conversely, MERTK inhibition by genetic or pharmacologic means can reverse these pro-oncogenic phenotypes. Multiple therapeutic approaches to MERTK inhibition are currently in development, including ligand “traps”, a monoclonal antibody, and small-molecule tyrosine kinase inhibitors. Clin Cancer Res; 19(19); 5275–80. ©2013 AACR.
Regulation of Cell Proliferation and Migration by Keratin19-Induced Nuclear Import of Early Growth Response-1 in Breast Cancer Cells
Authors: Ju Ji-hyun , Yang Wonseok , Lee Kyung-min , Oh Sunhwa , Nam KeeSoo , Shim Sarah , Shin Soon Young , Gye Myung Chan , Chu In-Sun , Shin Incheol
Published: July 2013

Purpose: Keratin19 (KRT19) is the smallest known type I intermediate filament and is used as a marker for reverse transcriptase PCR–mediated detection of disseminated tumors. In this study, we investigated the functional analysis of KRT19 in human breast cancer.

Experimental Design: Using a short hairpin RNA system, we silenced KRT19 in breast cancer cells. KRT19 silencing was verified by Western blot analysis and immunocytochemistry. We further examined the effect of KRT19 silencing on breast cancer cells by cell proliferation, migration, invasion, colony formation assay, cell-cycle analysis, immunocytochemistry, immunohistochemistry, and mouse xenograft assay.

Results: Silencing of KRT19 resulted in increased cell proliferation, migration, invasion, and survival. These effects were mediated by upregulation of Akt signaling as a result of reduced PTEN mRNA expression. Silencing of KRT19 decreased the nuclear import of early growth response-1 (Egr1), a transcriptional factor for PTEN transcription, through reduced association between Egr1 and importin-7. We also confirmed that silencing of KRT19 increased tumor formation in a xenograft model.

Conclusions: KRT19 is a potential tumor suppressor that negatively regulates Akt signaling through modulation of Egr1 nuclear localization. Clin Cancer Res; 19(16); 4335–46. ©2013 AACR.
Relationships between LDH-A, Lactate, and Metastases in 4T1 Breast Tumors
Authors: Rizwan Asif , Serganova Inna , Khanin Raya , Karabeber Hazem , Ni Xiaohui , Thakur Sunitha , Zakian Kristen L. , Blasberg Ronald , Koutcher Jason A.
Published: July 2013

Purpose: To investigate the relationship between lactate dehydrogenase A (LDH-A) expression, lactate concentration, cell metabolism, and metastases in murine 4T1 breast tumors.

Experimental Design: Inhibition of LDH-A expression and protein levels were achieved in a metastatic breast cancer cell line (4T1) using short hairpin RNA (shRNA) technology. The relationship between tumor LDH-A protein levels and lactate concentration (measured by magnetic resonance spectroscopic imaging, MRSI) and metastases was assessed.

Results: LDH-A knockdown cells (KD9) showed a significant reduction in LDH-A protein and LDH activity, less acid production, decreased transwell migration and invasion, lower proliferation, reduced glucose consumption and glycolysis, and increase in oxygen consumption, reactive oxygen species (ROS), and cellular ATP levels, compared with control (NC) cells cultured in 25 mmol/L glucose. In vivo studies showed lower lactate levels in KD9, KD5, and KD317 tumors than in NC or 4T1 wild-type tumors (P < 0.01), and a linear relationship between tumor LDH-A protein expression and lactate concentration. Metastases were delayed and primary tumor growth rate decreased.

Conclusions: We show for the first time that LDH-A knockdown inhibited the formation of metastases, and was accompanied by in vivo changes in tumor cell metabolism. Lactate MRSI can be used as a surrogate to monitor targeted inhibition of LDH-A in a preclinical setting and provides a noninvasive imaging strategy to monitor LDH-A–targeted therapy. This imaging strategy can be translated to the clinic to identify and monitor patients who are at high risk of developing metastatic disease. Clin Cancer Res; 19(18); 5158–69. ©2013 AACR.
BRAF/NRAS Wild-Type Melanomas Have a High Mutation Load Correlating with Histologic and Molecular Signatures of UV Damage
Authors: Mar Victoria J. , Wong Stephen Q. , Li Jason , Scolyer Richard A. , McLean Catriona , Papenfuss Anthony T. , Tothill Richard W. , Kakavand Hojabr , Mann Graham J. , Thompson John F. , Behren Andreas , Cebon Jonathan S. , Wolfe Rory , Kelly John W. , Dobrovic Alexander , McArthur Grant A.
Published: July 2013

Purpose: The mutation load in melanoma is generally high compared with other tumor types due to extensive UV damage. Translation of exome sequencing data into clinically relevant information is therefore challenging. This study sought to characterize mutations identified in primary cutaneous melanomas and correlate these with clinicopathologic features.

Experimental Design: DNA was extracted from 34 fresh-frozen primary cutaneous melanomas and matched peripheral blood. Tumor histopathology was reviewed by two dermatopathologists. Exome sequencing was conducted and mutation rates were correlated with age, sex, tumor site, and histopathologic variables. Differences in mutations between categories of solar elastosis, pigmentation, and BRAF/NRAS mutational status were investigated.

Results: The average mutation rate was 12 per megabase, similar to published results in metastases. The average mutation rate in severely sun damaged (SSD) skin was 21 per Mb compared with 3.8 per Mb in non-SSD skin (P = 0.001). BRAF/NRAS wild-type (WT) tumors had a higher average mutation rate compared with BRAF/NRAS–mutant tumors (27 vs. 5.6 mutations per Mb; P = 0.0001). Tandem CC>TT/GG>AA mutations comprised 70% of all dinucleotide substitutions and were more common in tumors arising in SSD skin (P = 0.0008) and in BRAF/NRAS WT tumors (P = 0.0007). Targetable and potentially targetable mutations in WT tumors, including NF1, KIT, and NOTCH1, were spread over various signaling pathways.

Conclusion: Melanomas arising in SSD skin have higher mutation loads and contain a spectrum of molecular subtypes compared with BRAF- and NRAS-mutant tumors indicating multigene screening approaches and combination therapies may be required for management of these patients. Clin Cancer Res; 19(17); 4589–98. ©2013 AACR.
PARP-1 Regulates Resistance of Pancreatic Cancer to TRAIL Therapy
Authors: Yuan Kaiyu , Sun Yong , Zhou Tong , McDonald Jay , Chen Yabing
Published: July 2013

Purpose: Activating extrinsic apoptotic pathways targeting death receptors (DR) using agonistic antibodies or TNF-related apoptosis-inducing ligand (TRAIL) is promising for cancer therapy. However, most pancreatic cancers are resistant to TRAIL therapy. The present studies aimed to identify combination therapies that enhance the efficacy of TRAIL therapy and to investigate the underlying mechanisms.

Experimental Design: A xenograft model in nude mice was used to determine pancreatic cancer tumorigenesis and therapeutic efficacy of TRA-8, a monoclonal agonistic antibody for DR5. Pancreatic cancer cells were used to characterize mechanisms underlying PARP-1 regulation of TRA-8–induced apoptosis in vitro.

Results: PARP-1 was found highly expressed in the TRA-8–resistant PANC-1 and Suit-2 cells, compared with TRA-8–sensitive BxPc-3 and MiaPaca-2. Inhibition of PARP-1 with a pharmacologic inhibitor sensitized PANC-1 and Suit2 cells to TRA-8–induced apoptosis in a dose-dependent manner. Furthermore, siRNAs specifically knocking down PARP-1 markedly enhanced TRA-8–induced apoptosis in vitro and augmented the efficacy of TRA-8 therapy on tumorigenesis in vivo. PARP-1 knockdown increased TRA-8–induced activation of caspase-8 in the death-induced signaling complex (DISC). Immunoprecipitation with DR5 antibody identified the recruitment of PARP-1 and PARP-1–mediated protein poly-ADP-ribosylation (pADPr) modification in the DR5-associated DISC. Further characterization revealed that PARP-1–mediated pADPr modification of caspase-8 inhibited caspase-8 activation, which may contribute to its function in regulating TRA-8 resistance.

Conclusions: Our studies provide molecular insights into a novel function of PARP-1 in regulating the extrinsic apoptosis machinery and also support interventions combining PARP-1 inhibitors with DR agonists for pancreatic cancer therapy. Clin Cancer Res; 19(17); 4750–9. ©2013 AACR.
Clinical, Pathologic, and Biologic Features Associated with BRAF Mutations in Non–Small Cell Lung Cancer
Authors: Cardarella Stephanie , Ogino Atsuko , Nishino Mizuki , Butaney Mohit , Shen Jeanne , Lydon Christine , Yeap Beow Y. , Sholl Lynette M. , Johnson Bruce E. , Jänne Pasi A.
Published: July 2013

Purpose: BRAF mutations are found in a subset of non–small cell lung cancers (NSCLC). We examined the clinical characteristics and treatment outcomes of patients with NSCLC harboring BRAF mutations.

Experimental Design: Using DNA sequencing, we successfully screened 883 patients with NSCLC for BRAF mutations between July 1, 2009 and July 16, 2012. Baseline characteristics and treatment outcomes were compared between patients with and without BRAF mutations. Wild-type controls consisted of patients with NSCLC without a somatic alteration in BRAF, KRAS, EGFR, and ALK. In vitro studies assessed the biologic properties of selected non-V600E BRAF mutations identified from patients with NSCLC.

Results: Of 883 tumors screened, 36 (4%) harbored BRAF mutations (V600E, 18; non-V600E, 18) and 257 were wild-type for BRAF, EGFR, KRAS, and ALK negative. Twenty-nine of 36 patients with BRAF mutations were smokers. There were no distinguishing clinical features between BRAF-mutant and wild-type patients. Patients with advanced NSCLC with BRAF mutations and wild-type tumors showed similar response rates and progression-free survival (PFS) to platinum-based combination chemotherapy and no difference in overall survival. Within the BRAF cohort, patients with V600E-mutated tumors had a shorter PFS to platinum-based chemotherapy compared with those with non-V600E mutations, although this did not reach statistical significance (4.1 vs. 8.9 months; P = 0.297). We identified five BRAF mutations not previously reported in NSCLC; two of five were associated with increased BRAF kinase activity.

Conclusions: BRAF mutations occur in 4% of NSCLCs and half are non-V600E. Prospective trials are ongoing to validate BRAF as a therapeutic target in NSCLC. Clin Cancer Res; 19(16); 4532–40. ©2013 AACR.
Phase II Study of Bevacizumab in Combination with Sorafenib in Recurrent Glioblastoma (N0776): A North Central Cancer Treatment Group Trial
Authors: Galanis Evanthia , Anderson S. Keith , Lafky Jackie M. , Uhm Joon H. , Giannini Caterina , Kumar Shaji K. , Kimlinger Teresa K. , Northfelt Donald W. , Flynn Patrick J. , Jaeckle Kurt A. , Kaufmann Timothy J. , Buckner Jan C.
Published: July 2013

Purpose: We hypothesized that vertical blockade of VEGF signaling by combining bevacizumab with sorafenib in patients with recurrent glioblastoma would result in a synergistic therapeutic effect. We also investigated whether VEGF, VEGFR2 and hypoxia-inducible factor-1α single-nucleotide polymorphisms (SNP), circulating biomarkers of angiogenesis, and MRI markers such as apparent diffusion coefficient (ADC) are correlated with treatment efficacy and/or toxicity.

Experimental Design: Patients received bevacizumab (5 mg/kg every 2 weeks) with sorafenib (200 mg twice a day, weekly, days 1–5; group A). Due to toxicity, the starting sorafenib dose was subsequently modified to 200 mg every day (group B).

Results: Fifty-four patients were enrolled: 19 patients in group A and 35 in group B. Objective response rate was 18.5% with median duration of 6.7 months (range 0.5–24.1 months). Six-month progression-free survival (PFS6) was 20.4% (11/54), and median overall survival (OS) was 5.6 months [95% confidence interval (CI), 4.7–8.2]; outcome was similar between the two dose groups. We identified SNPs in the VEGF and VEGFR2 promoter regions, which were associated with PFS6 (P < 0.022). Among molecular markers of angiogenesis, a higher log2 baseline level of stromal cell–derived factor-1 was associated with PFS6 success (P = 0.04). Circulating endothelial cells decreased during treatment with subsequent increase at disease progression (P = 0.022). Imaging analysis showed a trend associating ADC-L with poor outcome.

Conclusions: The bevacizumab/sorafenib combination did not improve outcome of patients with recurrent glioblastoma versus historic bevacizumab-treated controls. Biologic markers of response and resistance to bevacizumab in gliomas were identified which merit prospective validation. Clin Cancer Res; 19(17); 4816–23. ©2013 AACR.
Phase II Randomized Trial Comparing High-Dose IFN-α2b with Temozolomide Plus Cisplatin as Systemic Adjuvant Therapy for Resected Mucosal Melanoma
Authors: Lian Bin , Si Lu , Cui Chuanliang , Chi Zhihong , Sheng Xinan , Mao Lili , Li Siming , Kong Yan , Tang Bixia , Guo Jun
Published: July 2013

Purpose: Mucosal melanoma is rare and associated with extremely poor prognosis. However, standard adjuvant therapy for mucosal melanoma has not been established. We conducted a randomized phase II clinical trial in patients with resected mucosal melanoma to compare the efficacy and safety of high-dose IFN-α2b (HDI) and temozolomide-based chemotherapy as adjuvant therapy.

Experimental Design: Patients with mucosal melanoma in stage II/III after surgery were randomized into three groups: observation group (group A, surgery alone), HDI group (group B, treated with 15 × 106 U/m2/d IFN-α2b, followed by 9 × 106 U IFN-α2b), and temozolomide (200 mg/m2/d) plus cisplatin (75 mg/m2) group (group C). The endpoints were relapse-free survival (RFS), overall survival (OS), and toxicities.

Results: One hundred and eighty-nine patients were enrolled and finally analyzed. With a median follow-up of 26.8 months, the median RFS was 5.4, 9.4, and 20.8 months for group A, B, and C, respectively. Estimated median OS for group A, B, and C was 21.2, 40.4, and 48.7 months, respectively. Patients treated with temozolomide plus cisplatin showed significant improvements in RFS (P < 0.001) and OS (P < 0.01) than those treated with either HDI or surgery alone. Toxicities were generally mild to moderate.

Conclusion: Both temozolomide-based chemotherapy and HDI are effective and safe as adjuvant therapies for resected mucosal melanoma as compared with observation alone. However, HDI tends to be less effective than temozolomide-based chemotherapy for patients with resected mucosal melanoma in respect to RFS. The temozolomide plus cisplatin regimen might be a better choice for patients with resected mucosal melanoma. Clin Cancer Res; 19(16); 4488–98. ©2013 AACR.
Memantine Protects Rats Treated with Intrathecal Methotrexate from Developing Spatial Memory Deficits
Authors: Cole Peter D. , Vijayanathan Veena , Ali Nafeeza F. , Wagshul Mark E. , Tanenbaum Eric J. , Price Jeremy , Dalal Vidhi , Gulinello Maria E.
Published: July 2013

Purpose: To test whether memantine can prevent methotrexate-induced cognitive deficits in a preclinical model.

Experimental Design: After noting that methotrexate exposure induces prolonged elevations of the glutamate analog homocysteic acid (HCA) within cerebrospinal fluid, we tested whether intrathecal injection of HCA would produce memory deficits similar to those observed after intrathecal methotrexate. We then tested whether memantine, an antagonist of the N-methyl-d-aspartate (NMDA) subclass of glutamate receptors, could protect animals treated with clinically relevant doses of intrathecal methotrexate against developing memory deficits. Finally, we asked whether memantine affected this pathway beyond inhibiting the NMDA receptor by altering expression of the NMDA receptor or affecting concentrations of HCA or glutamate within the central nervous system.

Results: Four intrathecal doses of methotrexate induced deficits in spatial memory, persisting at least one month following the final injection. Intrathecal HCA was sufficient to reproduce this deficit. Concurrent administration of memantine during the period of methotrexate exposure was protective, decreasing the incidence of methotrexate-induced spatial memory deficits from 56% to 20% (P < 0.05). Memantine neither altered expression of NMDA receptors within the hippocampus nor blunted the methotrexate-induced increases in glutamate or HCA.

Conclusions: Excitotoxic glutamate analogs including HCA contribute to cognitive deficits observed after intrathecal methotrexate. Memantine, an NMDA receptor antagonist, reduces the incidence of cognitive deficits in rats treated with intrathecal methotrexate, and may therefore benefit patients with cancer receiving similar treatment. Clin Cancer Res; 19(16); 4446–54. ©2013 AACR.
Accelerated Approval and Breakthrough Therapy Designation: Oncology Drug Development on Speed?
Authors: Yao James C. , Meric-Bernstam Funda , Lee J. Jack , Eckhardt S. Gail
Published: July 2013

Recent advances in biotechnology have led to discoveries resulting in major improvements in the therapy of refractory malignancies, although most advanced cancers remain incurable. Thus, there is global consensus around the need to streamline the drug approval process for effective agents. Accelerated Approval and Breakthrough Therapy Designation hold the promise of making new treatments available sooner through the use of smaller studies using intermediate endpoints. Here, we consider the inherent limitations of smaller studies and discuss the strategies for hastening oncology drug development while maintaining high-efficacy standards. Clin Cancer Res; 19(16); 4305–8. ©2013 AACR.