Citations

Abstract Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-186.

Abstract We have previously reported that the protease activity of uPA delays tumor progression in a syngeneic model of breast cancer, and that the inhibitory effects are due to the protease activity of uPA (Merchan et. al, JNCI, 2006: 7; 98(11)). We also reported that changes in the uPA/PAI-1 balance favoring the proteolytic activity of uPA are associated with delayed tumor growth and reduction in lung metastases in vivo (Jing, et al, AACR 2009 meeting, #1124). The aim of this study was to extend the above findings in other in vivo syngeneic cancer models and to characterize the changes in gene expression mediated by uPA overexpression. We generated stably uPA overexpressing murine renal cancer (RENCA) and murine colon cancer (MC-38) cell lines using lentiviral vectors carrying m-uPA and selecting high uPA expressing clones for in vivo studies. uPA protein expression and in vitro growth was similar between the uPA overexpressing and control cancer cells. Similar with our findings in the 4T1 model, mice harboring RENCA tumors overexpressing uPA had reduced tumor growth than controls. Conversely, mice carrying uPA-overexpressing MC-38 tumors had significantly enhanced tumor growth compared to controls. To understand the causes for the opposite in vivo effects of uPA overexpressing RENCA and MC-38 tumors, we first compared uPA's proteolytic activity vs. protein concentration between both cell lines. To our surprise, the uPA proteolytic activity of (uPA overexpressing) MC-38 conditioned medium (CM) was significantly lower (> 15-fold) than that of stable RENCA cell lines even though uPA protein levels were comparable. To assess if the differences in uPA proteolytic activity between MC38 and RENCA transfectants was due to differential endogenous PAI-1 expression, we measured PAI-1 levels in MC38 and RENCA transfectants CM (ELISA). We found that PAI-1 expression in MC 38 was significantly higher (30-fold) than RENCA cells. The above data strongly suggest that in MC-38 tumors, uPA overexpression promoted tumor growth in part due to high endogenous PAI-1 levels. On the other hand, in the RENCA model, uPA overexpression (and low PAI-1 expression) was associated with significantly enhanced proteolytic activity and delayed tumor growth. Gene expression arrays of early murine mammary tumors overexpressing active uPA, showed significant tumor down regulation of several members of the MMP family, which may explain the in vivo antitumor effects. The above data suggest that the pro-or anti tumorigenic/metastatic effects of uPA overexpression depend on its proteolytic activity, and indicate that the status of endogenous tumor PAI-1 may regulate uPA's effects on tumor progression and metastases. The above data may help reconcile contradictory experimental and clinical findings on the role of proteases and protease inhibitors on tumor progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 516.

AbstractGenome‐wide association studies (GWAS) are being conducted at an unprecedented rate in population‐based cohorts and have increased our understanding of the pathophysiology of complex disease. Regardless of context, the practical utility of this information will ultimately depend upon the quality of the original data. Quality control (QC) procedures for GWAS are computationally intensive, operationally challenging, and constantly evolving. Here we enumerate some of the challenges in QC of GWAS data and describe the approaches that the electronic MEdical Records and Genomics (eMERGE) network is using for quality assurance in GWAS data, thereby minimizing potential bias and error in GWAS results. We discuss common issues associated with QC of GWAS data, including data file formats, software packages for data manipulation and analysis, sex chromosome anomalies, sample identity, sample relatedness, population substructure, batch effects, and marker quality. We propose best practices and discuss areas of ongoing and future research. Curr. Protoc. Hum. Genet. 68:1.19.1‐1.19.18 © 2011 by John Wiley & Sons, Inc.

Abstract The contribution of emphysema to lung cancer risk has been recognized, but the effect size needs to be further defined. In this study, 565 primary lung cancer cases were enrolled though a prospective lung cancer cohort at Mayo Clinic, and 450 controls were smokers participating in a lung cancer screening study in the same institution using spiral computed tomography (CT). Cases and controls were frequency matched on age, gender, race, smoking status, and residential region. CT imaging using standard protocol at the time of lung cancer diagnosis (case) or during the study (control) was assessed for emphysema by visual scoring CT analysis as a percentage of lung tissue destroyed. The clinical definition of emphysema was the diagnosis recorded in the medical documentation. Using multiple logistic regression models, emphysema (≥5% on CT) was found to be associated with a 3.8-fold increased lung cancer risk in Caucasians, with higher risk in subgroups of younger (<65 years old, OR = 4.64), heavy smokers (≥40 pack-years, OR = 4.46), and small-cell lung cancer (OR = 5.62). When using >0% or ≥10% emphysema on CT, lung cancer risk was 2.79-fold or 3.33-fold higher than controls. Compared with CT evaluation (using criterion ≥5%), the sensitivity, specificity, positive and negative predictive values, and the accuracy of the clinical diagnosis for emphysema in controls were 19%, 98%, 73%, 84%, and 83%, respectively. These results imply that an accurate evaluation of emphysema could help reliably identify individuals at greater risk of lung cancer among smokers. Cancer Prev Res; 4(1); 43–50. ©2010 AACR.

OBJECTIVE The islet in type 2 diabetes is characterized by β-cell apoptosis, β-cell endoplasmic reticulum stress, and islet amyloid deposits derived from islet amyloid polypeptide (IAPP). Toxic oligomers of IAPP form intracellularly in β-cells in humans with type 2 diabetes, suggesting impaired clearance of misfolded proteins. In this study, we investigated whether human-IAPP (h-IAPP) disrupts the endoplasmic reticulum–associated degradation/ubiquitin/proteasome system. RESEARCH DESIGN AND METHODS We used pancreatic tissue from humans with and without type 2 diabetes, isolated islets from h-IAPP transgenic rats, isolated human islets, and INS 832/13 cells transduced with adenoviruses expressing either h-IAPP or a comparable expression of rodent-IAPP. Immunofluorescence and Western blotting were used to detect polyubiquitinated proteins and ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) protein levels. Proteasome activity was measured in isolated rat and human islets. UCH-L1 was knocked down by small-interfering RNA in INS 832/13 cells and apoptosis was evaluated. RESULTS We report accumulation of polyubiquinated proteins and UCH-L1 deficiency in β-cells of humans with type 2 diabetes. These findings were reproduced by expression of oligomeric h-IAPP but not soluble rat-IAPP. Downregulation of UCH-L1 expression and activity to reproduce that caused by h-IAPP in β-cells induced endoplasmic reticulum stress leading to apoptosis. CONCLUSIONS Our results indicate that defective protein degradation in β-cells in type 2 diabetes can, at least in part, be attributed to misfolded h-IAPP leading to UCH-L1 deficiency, which in turn further compromises β-cell viability.

Abstract The low efficiency of reprogramming and genomic integration of virus vectors obscure the potential application of induced pluripotent stem (iPS) cells; therefore, identification of chemicals and cooperative factors that may improve the generation of iPS cells will be of great value. Moreover, the cellular mechanisms that limit the reprogramming efficiency need to be investigated. Through screening a chemical library, we found that two chemicals reported to upregulate E-cadherin considerably increase the reprogramming efficiency. Further study of the process indicated that E-cadherin is upregulated during reprogramming and the established iPS cells possess E-cadherin-mediated cell–cell contact, morphologically indistinguishable from embryonic stem (ES) cells. Our experiments also demonstrate that overexpression of E-cadherin significantly enhances reprogramming efficiency, whereas knockdown of endogenous E-cadherin reduces the efficiency. Consistently, abrogation of cell–cell contact by the inhibitory peptide or the neutralizing antibody against the extracellular domain of E-cadherin compromises iPS cell generation. Further mechanistic study reveals that adhesive binding activity of E-cadherin is required. Our results highlight the critical role of E-cadherin-mediated cell–cell contact in reprogramming and suggest new routes for more efficient iPS cell generation.

Abstract Background: Multiple recent genome-wide studies of single nucleotide polymorphisms (SNP) reported associations between candidate chromosome loci and lung cancer susceptibility. We evaluated five of the top candidate SNPs (rs402710, rs2736100, rs4324798, rs16969968, and rs8034191) for their effects on lung cancer risk and overall survival. Methods: Over 1,700 cases and 2,200 controls were included in this study. Seven independent, complementary case-control data sets were tested for risk assessment encompassing cigarette smokers and never smokers, using unrelated controls and unaffected full-sibling controls. Five patient groups were tested for survival prediction stratified by smoking status, histology subtype, and treatment. Results: After considering a history of chronic obstructive pulmonary disease as a risk factor altering lung cancer risk and comparing to sibling controls, none of the five SNPs remained significant. However, the variant rs4324798 was significant in predicting overall survival (hazard ratio, 0.46; 95% confidence interval, 0.30-0.73; P = 0.001) in small cell lung cancer. Conclusions: None of the five candidate SNPs in lung cancer risk can be confirmed in our study. The previously reported association could be explained by disparity in tobacco smoke exposure and chronic obstructive pulmonary disease history between cases and controls. Instead, we found rs4324798 to be an independent predictor in small cell lung cancer survival, warranting further elucidation of the underlying mechanisms.Cancer Epidemiol Biomarkers Prev; 19(1); 240–4

Abstract Anti-Smith (Sm) D autoantibodies are specific for systemic lupus erythematosus. In this investigation, the influence of HLA-D genes on immune responses to SmD was investigated. Mice with HLA-DR3, HLA-DR4, HLA-DQ0601, HLA-DQ0604, or HLA-DQ8 transgenes were immunized with recombinant SmD1, and their Ab responses were analyzed. Analysis by ELISA showed that all strains responded well to SmD. However, when synthetic SmD peptides were used as substrate, DR3 mice had the highest Ab response followed by DQ8, DQ0604, DQ0601, and DR4. A similar trend was observed in Western blot analysis using WEHI 7.1 cell lysate as the substrate, with the exception that DR4 mice did not generate detectable amounts of Abs. Only sera from DR3 and DQ0604 mice immunoprecipitated A-ribonucleoprotein (RNP), SmB, and SmD. Intermolecular epitope spreading to A-RNP and SmB was evident in DR3 and DQ0604 mice, as sera depleted of anti-SmD Abs were reactive with these proteins. DR3 mice also generated an immune response to C-RNP. Anti-nuclear Abs were detected in the majority of the DR3 mice, whereas moderate reactivities were seen in DQ0604 and DQ8 mice. Interestingly, only DR3 mice mounted an anti-dsDNA Ab response. Approximately half of the anti-dsDNA Abs were cross-reactive with SmD. Ab responses correlated with the strength of the T cell responses. Thus, HLA-DR3 appears to be the dominant HLA-D gene that determines the magnitude and quality of the anti-SmD immune response. In addition, our findings provide insights into the origin of the anti-dsDNA Abs often detected in patients with systemic lupus erythematosus.

Abstract Background: Exposure to secondhand smoke during adulthood has detrimental health effects, including increased lung cancer risk. Compared with adults, children may be more susceptible to secondhand smoke. This susceptibility may be exacerbated by alterations in inherited genetic variants of innate immunity genes. We hypothesized a positive association between childhood secondhand smoke exposure and lung cancer risk that would be modified by genetic polymorphisms in the mannose binding lectin-2 (MBL2) gene resulting in well-known functional changes in innate immunity. Methods: Childhood secondhand smoke exposure and lung cancer risk was assessed among men and women in the ongoing National Cancer Institute-Maryland Lung Cancer (NCI-MD) study, which included 624 cases and 348 controls. Secondhand smoke history was collected via in-person interviews. DNA was used for genotyping the MBL2 gene. To replicate, we used an independent case-control study from Mayo Clinic consisting of 461 never smokers, made up of 172 cases and 289 controls. All statistical tests were two-sided. Results: In the NCI-MD study, secondhand smoke exposure during childhood was associated with increased lung cancer risk among never smokers [odds ratio (OR), 2.25; 95% confidence interval (95% CI), 1.04-4.90]. This was confirmed in the Mayo study (OR, 1.47; 95% CI, 1.00-2.15). A functional MBL2 haplotype associated with high circulating levels of MBL and increased MBL2 activity was associated with increased lung cancer risk among those exposed to childhood secondhand smoke in both the NCI-MD and Mayo studies (OR, 2.52; 95% CI, 1.13-5.60, and OR, 2.78; 95% CI, 1.18-3.85, respectively). Conclusions: Secondhand smoke exposure during childhood is associated with increased lung cancer risk among never smokers, particularly among those possessing a haplotype corresponding to a known overactive complement pathway of the innate immune system. (Cancer Epidemiol Biomarkers Prev 2009;18(12):3375–83)

Mutation in leucine-rich repeat kinase-2 ( LRRK2 ) is the most common cause of late-onset Parkinson's disease (PD). Although most cases of PD are sporadic, some are inherited, including those caused by LRRK2 mutations. Because these mutations may be associated with a toxic gain of function, controlling the expression of LRRK2 may decrease its cytotoxicity. Here we show that the carboxyl terminus of HSP70-interacting protein (CHIP) binds, ubiquitinates, and promotes the ubiquitin proteasomal degradation of LRRK2. Overexpression of CHIP protects against and knockdown of CHIP exacerbates toxicity mediated by mutant LRRK2. Moreover, HSP90 forms a complex with LRRK2, and inhibition of HSP90 chaperone activity by 17AAG leads to proteasomal degradation of LRRK2, resulting in increased cell viability. Thus, increasing CHIP E3 ligase activity and blocking HSP90 chaperone activity can prevent the deleterious effects of LRRK2. These findings point to potential treatment options for LRRK2-associated PD.