Search

Background: Diesel exhaust is a complex mixture, including polycyclic aromatic hydrocarbons (PAH) and nitrated PAHs (nitro-PAH), many of which are potent mutagens and possible bladder carcinogens. To explore the association between diesel exposure and bladder carcinogenesis, we examined the relationship between exposure and somatic mutations and mutational signatures in bladder tumors. Methods: Targeted sequencing was conducted in bladder tumors from the New England Bladder Cancer Study. Using data on 797 cases and 1,418 controls, two-stage polytomous logistic regression was used to evaluate etiologic heterogeneity between bladder cancer subtypes and quantitative, lifetime estimates of respirable elemental carbon (REC), a surrogate for diesel exposure. Poisson regression was used to evaluate associations between REC and mutational signatures. Results: We observed significant heterogeneity in the diesel-bladder cancer risk relationship, with a strong positive association among cases with high-grade, nonmuscle invasive TP53-mutated tumors compared with controls [ORTop Tertile vs.Unexposed, 4.8; 95% confidence interval (CI), 2.2–10.5; Ptrend Conclusions: The relationship between diesel exhaust and bladder cancer was heterogeneous based on the presence of TP53 mutations in tumors, further supporting the link between PAH exposure and TP53 mutations in carcinogenesis. Future studies that can identify nitro-PAH signatures in exposed tumors are warranted to add human data supporting the link between diesel and bladder cancer. Impact: This study provides additional insight into the etiology and possible mechanisms related to diesel exhaust-induced bladder cancer.

REC and nitro-PAH signatures in New England

Genes on panel

Cosine similarities for all signatures

Case-case associations between REC and TP53 mutation, NEBCS

specific TP53 mutations and ever diesel

Background:Diesel exhaust is a complex mixture, including polycyclic aromatic hydrocarbons (PAH) and nitrated PAHs (nitro-PAH), many of which are potent mutagens and possible bladder carcinogens. To explore the association between diesel exposure and bladder carcinogenesis, we examined the relationship between exposure and somatic mutations and mutational signatures in bladder tumors.Methods:Targeted sequencing was conducted in bladder tumors from the New England Bladder Cancer Study. Using data on 797 cases and 1,418 controls, two-stage polytomous logistic regression was used to evaluate etiologic heterogeneity between bladder cancer subtypes and quantitative, lifetime estimates of respirable elemental carbon (REC), a surrogate for diesel exposure. Poisson regression was used to evaluate associations between REC and mutational signatures.Results:We observed significant heterogeneity in the diesel-bladder cancer risk relationship, with a strong positive association among cases with high-grade, nonmuscle invasive TP53-mutated tumors compared with controls [ORTop Tertile vs.Unexposed, 4.8; 95% confidence interval (CI), 2.2–10.5; Ptrend Pheterogeneity = 0.002]. In muscle-invasive tumors, we observed a positive association between diesel exposure and the nitro-PAH signatures of 1,6-dintropyrene (RR, 1.93; 95% CI, 1.28–2.92) and 3-nitrobenzoic acid (RR, 1.97; 95% CI, 1.33–2.92).Conclusions:The relationship between diesel exhaust and bladder cancer was heterogeneous based on the presence of TP53 mutations in tumors, further supporting the link between PAH exposure and TP53 mutations in carcinogenesis. Future studies that can identify nitro-PAH signatures in exposed tumors are warranted to add human data supporting the link between diesel and bladder cancer.Impact:This study provides additional insight into the etiology and possible mechanisms related to diesel exhaust-induced bladder cancer.

Ever diesel and nitro-PAH signatures adjusted for smoking

study characteristics

Purpose:Exome- and whole-genome sequencing of muscle-invasive bladder cancer has revealed important insights into the molecular landscape; however, there are few studies of non–muscle-invasive bladder cancer with detailed risk factor information.Experimental Design:We examined the relationship between smoking and other bladder cancer risk factors and somatic mutations and mutational signatures in bladder tumors. Targeted sequencing of frequently mutated genes in bladder cancer was conducted in 322 formalin-fixed paraffin-embedded bladder tumors from a population-based case–control study. Logistic regression was used to calculate odds ratios (OR) and 95% confidence intervals (CI), evaluating mutations and risk factors. We used SignatureEstimation to extract four known single base substitution mutational signatures and Poisson regression to calculate risk ratios (RR) and 95% CIs, evaluating signatures and risk factors.Results:Non-silent KDM6A mutations were more common in females than males (OR = 1.83; 95% CI, 1.05–3.19). There was striking heterogeneity in the relationship between smoking status and established single base substitution signatures: current smoking status was associated with greater ERCC2-Signature mutations compared with former (P = 0.024) and never smoking (RR = 1.40; 95% CI, 1.09–1.80; P = 0.008), former smoking was associated with greater APOBEC-Signature13 mutations (P = 0.05), and never smoking was associated with greater APOBEC-Signature2 mutations (RR = 1.54; 95% CI, 1.17–2.01; P = 0.002). There was evidence that smoking duration (the component most strongly associated with bladder cancer risk) was associated with ERCC2-Signature mutations and APOBEC-Signature13 mutations among current (Ptrend = 0.005) and former smokers (P = 0.0004), respectively.Conclusions:These data quantify the contribution of bladder cancer risk factors to mutational burden and suggest different signature enrichments among never, former, and current smokers.

Purpose: Exome- and whole-genome sequencing of muscle-invasive bladder cancer has revealed important insights into the molecular landscape; however, there are few studies of non–muscle-invasive bladder cancer with detailed risk factor information. Experimental Design: We examined the relationship between smoking and other bladder cancer risk factors and somatic mutations and mutational signatures in bladder tumors. Targeted sequencing of frequently mutated genes in bladder cancer was conducted in 322 formalin-fixed paraffin-embedded bladder tumors from a population-based case–control study. Logistic regression was used to calculate odds ratios (OR) and 95% confidence intervals (CI), evaluating mutations and risk factors. We used SignatureEstimation to extract four known single base substitution mutational signatures and Poisson regression to calculate risk ratios (RR) and 95% CIs, evaluating signatures and risk factors. Results: Non-silent KDM6A mutations were more common in females than males (OR = 1.83; 95% CI, 1.05–3.19). There was striking heterogeneity in the relationship between smoking status and established single base substitution signatures: current smoking status was associated with greater ERCC2-Signature mutations compared with former (P = 0.024) and never smoking (RR = 1.40; 95% CI, 1.09–1.80; P = 0.008), former smoking was associated with greater APOBEC-Signature13 mutations (P = 0.05), and never smoking was associated with greater APOBEC-Signature2 mutations (RR = 1.54; 95% CI, 1.17–2.01; P = 0.002). There was evidence that smoking duration (the component most strongly associated with bladder cancer risk) was associated with ERCC2-Signature mutations and APOBEC-Signature13 mutations among current (Ptrend = 0.005) and former smokers (P = 0.0004), respectively. Conclusions: These data quantify the contribution of bladder cancer risk factors to mutational burden and suggest different signature enrichments among never, former, and current smokers.

Type III IFNs are important mediators of antiviral immunity. IFN-λ4 is a unique type III IFN because it is produced only in individuals who carry a dG allele of a genetic variant rs368234815-dG/TT. Counterintuitively, those individuals who can produce IFN-λ4, an antiviral cytokine, are also less likely to clear hepatitis C virus infection. In this study, we searched for unique functional properties of IFN-λ4 that might explain its negative effect on hepatitis C virus clearance. We used fresh primary human hepatocytes (PHHs) treated with recombinant type III IFNs or infected with Sendai virus to model acute viral infection and subsequently validated our findings in HepG2 cell line models. Endogenous IFN-λ4 protein was detectable only in Sendai virus–infected PHHs from individuals with the dG allele, where it was poorly secreted but highly functional, even at concentrations < 50 pg/ml. IFN-λ4 acted faster than other type III IFNs in inducing antiviral genes, as well as negative regulators of the IFN response, such as USP18 and SOCS1. Transient treatment of PHHs with IFN-λ4, but not IFN-λ3, caused a strong and sustained induction of SOCS1 and refractoriness to further stimulation with IFN-λ3. Our results suggest unique functional properties of IFN-λ4 that can be important in viral clearance and other clinical conditions.

Background: Second to cigarette smoking, occupational exposures are a leading risk factor for the development of bladder cancer. Occupational exposure to diesel exhaust in particular has been linked to increased bladder cancer risk in several epidemiologic studies. To provide insight into possible mechanisms that influence this association, we examined the relationship between diesel exhaust and somatic mutations in bladder tumors. Methods: Targeted sequencing of frequently mutated genes in bladder cancer was conducted in formalin-fixed paraffin-embedded bladder tumors from a population-based case-control study in New England. Lifetime occupational histories combined with exposure-oriented questions were used to estimate cumulative exposure to respirable elemental carbon (REC), a primary index for diesel exhaust. REC exposure was modeled both continuously (per 100 μg/m3-yrs) and categorically (by tertiles). Two-stage polytomous logistic regression was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs) to evaluate etiologic heterogeneity by tumor subtypes using data on 797 cases and 1,418 controls. Results: We observed significant heterogeneity in the relationship between diesel exhaust and risk of bladder cancer by TP53 mutation status (p-heterogeneity=0.013). When comparing patients with TP53+ tumors to controls, there was a strong association with increasing cumulative REC exposure (ORTertile 1 vs. Unexposed=1.4, 95% CI=0.7, 2.8; ORTertile 2 vs. Unexposed=1.3, 95% CI=0.6, 2.7; ORTertile 3 vs. Unexposed=2.8, 95% CI=1.4, 5.7; p-trend=0.003), while there was no apparent association between cumulative REC exposure and bladder cancer among patients with wild-type TP53 tumors (p-trend=0.603). Subtype analyses that incorporate clinical features (stage: non-muscle invasive/muscle-invasive and grade: low-grade/high-grade) with TP53 mutation, also indicate etiologic heterogeneity in the relationship between diesel exhaust and bladder cancer (p-het=0.002), with the strongest association observed between increasing cumulative REC and risk of bladder cancer among cases with non-muscle invasive, high-grade tumors (including carcinoma in situ) with TP53 mutations (OR per 100 μg/m3-yrs =1.14, 95% CI=1.00, 1.29). Conclusions: Our results suggest that diesel engine exhaust may influence bladder cancer risk by impacting the cell cycle, especially among patients with high-risk non-muscle invasive tumors that are more likely to progress to invasive disease. More work will be needed to understand the impact of diesel-generated oxidative stress on tumor suppressor genes like TP53 and cell cycle regulation in the development of bladder cancer. Citation Format: Nicole Gonzalez, Nina Rao, Michael Dean, Ludmila Prokunina-Olsson, Dalsu Baris, Molly Schwenn, Alison Johnson, Nathaniel Rothman, Debra Silverman, Stella Koutros. Diesel engine exhaust is associated with TP53 mutations and high-risk non-muscle invasive bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 861.

Interferon lambda 4 (IFN-λ4) is a novel type-III interferon that can be generated only in individuals carrying a ΔG frame-shift allele of an exonic genetic variant (rs368234815-ΔG/TT). The rs368234815-ΔG allele is strongly associated with decreased clearance of hepatitis C virus (HCV) infection. Here, we further explored the biological function of IFN-λ4 expressed in human hepatic cells-a hepatoma cell line HepG2 and fresh primary human hepatocytes (PHHs). We performed live confocal imaging, cell death and proliferation assays, mRNA expression profiling, protein detection, and antibody blocking assays using transient and inducible stable in vitro systems. Not only did we observe significant intracellular retention of IFN-λ4 but also detected secreted IFN-λ4 in the culture media of expressing cells. Secreted IFN-λ4 induced strong activation of the interferon-stimulated genes (ISGs) in IFN-λ4-expressing and surrounding cells in transwell assays. Specifically, in PHHs, secreted IFN-λ4 induced expression of the CXCL10 transcript and a corresponding pro-inflammatory chemokine, IP-10. In IFN-λ4-expressing HepG2 cells, we also observed decreased proliferation and increased cell death. All IFN-λ4-induced phenotypes--activation of ISGs, decreased proliferation, and increased cell death--could be inhibited by an anti-IFN-λ4-specific antibody. Our study offers new insights into biology of IFN-λ4 and its possible role in HCV clearance.

Bis(benzimidazolium)butane (BBIM-butane) system acts as a new guest molecule for dibenzo-24-crown-8 (DB24C8). This is the only [BBIM]DB24C8 pseudorotaxanes where two conformers coexist (detected by 1H NMR and structure established by DFT studies); major conformer and minor conformer. Low temperature 1H NMR (240, 253 K) was done for a representative [BBIM-butane]DB24C8 pseudorotaxane that could not be detected at room-temperature. DFT calculations for this [BBIM-butane]DB24C8 pseudorotaxane were done both with B3LYP and MPW1PW91 functionals. The MPW1PW91 optimized structures identified possible stable major and minor conformer with the strongest H-bonding. We have compared the characteristics of [BBIM]DB24C8 [2]pseudorotaxanes comprising the [BBIM-methane]DB24C8, [BBIM-ethane]DB24C8, [BBIM-propane]DB24C8 and [BBIM-butane]DB24C8 systems. The feasibility of formation of these pseudorotaxanes decreases considerably from [BBIM-methane]DB24C8 to the [BBIM-butane]DB24C8 system. The presence of these threaded complexes was initially screened based on 1H NMR and substantiated further by 2D NMR spectroscopy (NOESY and COSY), X-ray crystallography, HR-ESI-MS and DFT calculations.

Exome and whole-genome sequencing of muscle-invasive (MI) bladder cancer has revealed important insights into the molecular landscape; however, there are few studies of non-muscle invasive (NMI) bladder cancer with detailed information on bladder cancer risk factors. We used deep, targeted amplicon sequencing of the coding regions of 44 genes frequently mutated in bladder cancer (average 630x coverage for tumor and 350x for germline DNA) to characterize somatic mutations and the mutational spectrum in 307 formalin fixed paraffin embedded bladder tumors (n=260 NMI and n=47 MI) from a population-based case-control study with detailed information on smoking and other risk factors. Variants identified by mutation calling pipelines were validated by manual review of aligned sequences. Logistic regression was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs) to evaluate somatic mutations and risk factors. We used SignatureEstimation to extract the four known single base substitution mutational signatures (COSMIC: Sig1, Sig2, Sig13, ERCC2-Signature) and Poisson regression to calculate risk ratios (RRs) and 95%CIs to evaluate signatures and risk factors. The most frequently mutated gene in NMI tumors was FGFR3 (52% mutated) and in MI tumors was TP53 (51% mutated). FGFR3 (89%), STAG2 (81%), and PIK3CA (78%) mutations were significantly higher in Ta tumors compared to T1 or MI tumors. Mutations in histone/chromatin regulating genes (KDM6A, KMT2D, KMT2C, CREBBP, ARID1A, and EP300) were present at a high frequency in low grade Ta tumors. Non-silent KDM6Amutations were significantly more common in females compared to males (OR=1.79,95%CI:1.03-3.11); in females these mutations largely occurred in Ta tumors (86%). There was striking heterogeneity in the relationship between smoking status (current, former, never) and the established signatures: only current smoking was associated with greater ERCC2-Signature variants compared to both former smoking (p-value=0.015) and never smoking (p-value=0.023); only former smoking was associated with APOBEC-Sig13 variants (RR=1.83,95%CI: 1.10-3.04,p-value=0.019); and only never smoking was associated with the APOBEC-Sig2 variants (RR=1.52,95%CI: 1.15-2.01,p-value=0.003). Further, there was significant evidence that smoking duration, the component of smoking most strongly associated with bladder cancer risk, was associated with ERCC2-Signature variants and APOBEC-Sig13 variants among current (p-trend=0.013) and former smokers (p=0.002), respectively. Exploration of insertion/deletions revealed that both status and duration of smoking was significantly associated with presence of any 1bp deletions (p-trend=0.007); these were enriched for deletions in KDM6A (p=0.0003). In conclusion, these data quantify the contribution of bladder cancer risk factors to mutational burden in a population-based series of tumors and reveal distinct signature contributions among never, former and current smokers. Citation Format: Stella Koutros, Nina Rao, Lee E. Moore, Michael L. Nickerson, Donghyuk Lee, Bin Zhu, Larissa Pardo, Dalsu Baris, Molly Schwenn, Alison Johnson, Kristine Jones, Montserrat Garcia-Closas, Ludmila Prokunina-Olsson, Debra T. Silverman, Nathaniel Rothman, Michael Dean. Integrative, targeted deep sequencing of bladder tumors reveals novel associations between cancer gene mutations and mutational signatures with major risk factors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-051.

IFN-λ4, a recently discovered type-III interferon (IFN), is genetically regulated, and has clinical significance because of its association with impaired clearance of hepatitis C virus (HCV) infection. Type-III IFNs play a critical role in the innate immune response to viral infections. Although IFN-λ4 induces a similar antiviral profile as other type-III IFNs, its specific contribution to the innate immune response is unclear. Delineating the functional properties of IFN-λ4 that distinguish it from other type-III IFNs may give insights into its unique role during HCV, and other infections. Here we compared antiviral responses induced by all four type-III IFNs in HepG2 cells. We show that IFN-λ4 attained 50% peak antiviral activity by 4 hours while other type-III IFNs were only at 20% of peak activity at this time. The rapid antiviral response induced by IFN-λ4 was followed by a sharp decline to less than 25% of peak activity within 24 hours, while activity of other type-III IFNs were still over 60% from peak. By comparing antiviral activity of IFN-λ4 and IFN-λ3 against Sendai virus infection in primary human hepatocytes, we show that IFN-λ4 had stronger antiviral activity than IFN-λ3 early in the course of infection, but comparable activity at later time points. We also show that despite its poor secretion, IFN-λ4 was significantly more potent than IFN-λ3 in inducible cell lines engineered to express either protein. In summary, we show that IFN-λ4 is more active during the early stages of viral infection compared to other type-III IFNs, which maybe a clinically important window for restricting viral infection in the host, and affecting disease outcome.

Sex tourism is the dark side of the global phenomenon of tourism. Every day we read about the benefits of tourism, its income and employment potential, its ability to bridge the gap between the rich and the poor, its potential to overcome uneven development in backward regions of the world. Above all, its contribution to the balance of payments and foreign exchange earnings for the decolonised and third world countries that must enter the capitalist mainstream if they are to make the transition to the twenty‐first century. Asks why sex tourism is being condoned and wonders why more voices are not raised in protest against its continuance.

The ability to generate interferon lambda 4 (IFNL4), the most recently discovered member of the type-III interferon family, has been associated with impaired clearance of hepatitis C virus (HCV). IFNL4 signals through an IFNL receptor complex that serves all type-III interferons and consists of receptors IFNLR1 and IL10R2. Signaling through IFNL receptor complex leads to activation of the JAK-STAT pathway and transcriptional activation of interferon-stimulated genes (ISG). Intracellular expression of IFNL4 also causes reduced proliferation and increased cell death in human hepatic cells but it remains unclear if this is a result of JAK-STAT signaling through IFNL receptor complex or through some other alternative mechanisms. To address this question, we used CRISPR-CAS9 system to knock-out IFNLR1 in a hepatoma HepG2 cells. We designed and tested six guide RNAs (gRNAs) for IFNLR1 exons. CAS9 protein and gRNAs that most efficiently induced mutations in IFNLR1 were introduced into HepG2 cells to generate stable cell lines. No obvious defects were observed in IFNLR1-KO-HepG2 cells, indicating that loss of IFNLR1 had no deleterious effects on cell viability. We selected three stable clones which had sequencing-confirmed inactivating IFNLR1 mutations and Western blot-confirmed lack of IFNLR1 protein expression. Compared to the parental HepG2 cells the IFNLR1-KO-HepG2 cells were deficient in the ability to induce the luciferase-tagged interferon-stimulated response element (ISRE-Luc) reporter after treatment with recombinant IFNL4 and IFNL3 proteins, which signal through the same receptor complex. At the same time, response to IFNa treatment was not affected because IFNa signals through its own receptor complex. These results indicate that, as other type-III interferons, IFNL4 induces ISRE signaling through IFNL receptor complex. INFLR1-KO-HepG2 cells provide a highly efficient model for studies of factors involved in signaling of type-III interferons, including IFNL4. Next, we will investigate alternative pathways involved in anti-proliferative and cell death-related phenotypes induced by IFNL4 in HepG2 cell. We will perform RNA-seq on parental and IFNLR1-KO-HepG2 cells treated with or without IFNL4, IFNL3 and IFNa. These results will contribute to a better understanding of IFNL4 function, which can be relevant for viral infections and cancer. Citation Format: Fang Wang, Olusegun O. Onabajo, Nina Rao, Ludmila Prokunina-Olsson. Exploration of alternative pathways mediated by IFNL4 and related to cell proliferation and death in a hepatoma cell line. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4419.

“Lord Jesus Christ, Son of God. have mercy on the cities, the islands and villages of our orhodox fatherland, as well as the holy monasteries which are scourged by the worldly touristic wave.“Grace us with a solution to this dramatic problem and protect our brethren who are sorely tried by the modernistic spirit of these contemporary western brethren”. (A new prayer recommended by the Greek Orthodox Church)