Candidate pathway analysis of surfactant proteins identifies CTSH and SFTA2 that influences lung cancer risk.

Pulmonary surfactant is a lipoprotein synthesized and secreted by alveolar type II cells in lung. We evaluated the associations between 200,139 single nucleotide polymorphisms (SNPs) of 40 surfactant-related genes and lung cancer risk using genotyped data from two independent lung cancer genome-wide association studies. Discovery data included 18,082 cases and 13,780 controls of European ancestry. Replication data included 1,914 cases and 3,065 controls of European descent. Using multivariate logistic regression, we found novel SNPs in surfactant-related genes CTSH [rs34577742 C > T, odds ratio (OR) = 0.90, 95% confidence interval (CI) = 0.89-0.93, P = 7.64 × 10-9] and SFTA2 (rs3095153 G > A, OR = 1.16, 95% CI = 1.10-1.21, P = 1.27 × 10-9) associated with overall lung cancer in the discovery data and validated in an independent replication data-CTSH (rs34577742 C > T, OR = 0.88, 95% CI = 0.80-0.96, P = 5.76 × 10-3) and SFTA2 (rs3095153 G > A, OR = 1.14, 95% CI = 1.01-1.28, P = 3.25 × 10-2). Among ever smokers, we found SNPs in CTSH (rs34577742 C > T, OR = 0.89, 95% CI = 0.85-0.92, P = 1.94 × 10-7) and SFTA2 (rs3095152 G > A, OR = 1.20, 95% CI = 1.14-1.27, P = 4.25 × 10-11) associated with overall lung cancer in the discovery data and validated in the replication data-CTSH (rs34577742 C > T, OR = 0.88, 95% CI = 0.79-0.97, P = 1.64 × 10-2) and SFTA2 (rs3095152 G > A, OR = 1.15, 95% CI = 1.01-1.30, P = 3.81 × 10-2). Subsequent transcriptome-wide association study using expression weights from a lung expression quantitative trait loci study revealed genes most strongly associated with lung cancer are CTSH (PTWAS = 2.44 × 10-4) and SFTA2 (PTWAS = 2.32 × 10-6).

Genome-wide interaction analysis identified low-frequency variants with sex disparity in lung cancer risk.

Differences by sex in lung cancer incidence and mortality have been reported which cannot be fully explained by sex differences in smoking behavior, implying existence of genetic and molecular basis for sex disparity in lung cancer development. However, the information about sex dimorphism in lung cancer risk is quite limited despite the great success in lung cancer association studies. By adopting a stringent two-stage analysis strategy, we performed a genome-wide gene-sex interaction analysis using genotypes from a lung cancer cohort including ~ 47 000 individuals with European ancestry. Three low-frequency variants (minor allele frequency < 0.05), rs17662871 [odds ratio (OR) = 0.71, P = 4.29×10-8); rs79942605 (OR = 2.17, P = 2.81×10-8) and rs208908 (OR = 0.70, P = 4.54×10-8) were identified with different risk effect of lung cancer between men and women. Further expression quantitative trait loci and functional annotation analysis suggested rs208908 affects lung cancer risk through differential regulation of Coxsackie virus and adenovirus receptor gene expression in lung tissues between men and women. Our study is one of the first studies to provide novel insights about the genetic and molecular basis for sex disparity in lung cancer development.

Identification of genetically predicted DNA methylation markers associated with non-small cell lung cancer risk among 34,964 cases and 448,579 controls.

BACKGROUND: Although the associations between genetic variations and lung cancer risk have been explored, the epigenetic consequences of DNA methylation in lung cancer development are largely unknown. Here, the genetically predicted DNA methylation markers associated with non-small cell lung cancer (NSCLC) risk by a two-stage case-control design were investigated. METHODS: The genetic prediction models for methylation levels based on genetic and methylation data of 1595 subjects from the Framingham Heart Study were established. The prediction models were applied to a fixed-effect meta-analysis of screening data sets with 27,120 NSCLC cases and 27,355 controls to identify the methylation markers, which were then replicated in independent data sets with 7844 lung cancer cases and 421,224 controls. Also performed was a multi-omics functional annotation for the identified CpGs by integrating genomics, epigenomics, and transcriptomics and investigation of the potential regulation pathways. RESULTS: Of the 29,894 CpG sites passing the quality control, 39 CpGs associated with NSCLC risk (Bonferroni-corrected p ≤ 1.67 × 10-6 ) were originally identified. Of these, 16 CpGs remained significant in the validation stage (Bonferroni-corrected p ≤ 1.28 × 10-3 ), including four novel CpGs. Multi-omics functional annotation showed nine of 16 CpGs were potentially functional biomarkers for NSCLC risk. Thirty-five genes within a 1-Mb window of 12 CpGs that might be involved in regulatory pathways of NSCLC risk were identified. CONCLUSIONS: Sixteen promising DNA methylation markers associated with NSCLC were identified. Changes of the methylation level at these CpGs might influence the development of NSCLC by regulating the expression of genes nearby. PLAIN LANGUAGE SUMMARY: The epigenetic consequences of DNA methylation in lung cancer development are still largely unknown. This study used summary data of large-scale genome-wide association studies to investigate the associations between genetically predicted levels of methylation biomarkers and non-small cell lung cancer risk at the first time. This study looked at how well larotrectinib worked in adult patients with sarcomas caused by TRK fusion proteins. These findings will provide a unique insight into the epigenetic susceptibility mechanisms of lung cancer.

Polymorphic tandem DNA repeats activate the human telomerase reverse transcriptase gene.

Multiple independent sequence variants of the hTERT locus have been associated with telomere length and cancer risks in genome-wide association studies. Here, we identified an intronic variable number tandem repeat, VNTR2-1, as an enhancer-like element, which activated hTERT transcription in a cell in a chromatin-dependent manner. VNTR2-1, consisting of 42-bp repeats with an array of enhancer boxes, cooperated with the proximal promoter in the regulation of hTERT transcription by basic helix-loop-helix transcription factors and maintained hTERT expression during embryonic stem-cell differentiation. Genomic deletion of VNTR2-1 in MelJuSo melanoma cells markedly reduced hTERT transcription, leading to telomere shortening, cellular senescence, and impairment of xenograft tumor growth. Interestingly, VNTR2-1 lengths varied widely in human populations; hTERT alleles with shorter VNTR2-1 were underrepresented in African American centenarians, indicating its role in human aging. Therefore, this polymorphic element is likely a missing link in the telomerase regulatory network and a molecular basis for genetic diversities of telomere homeostasis and age-related disease susceptibilities.

Genome-wide association meta-analysis identifies pleiotropic risk loci for aerodigestive squamous cell cancers.

Squamous cell carcinomas (SqCC) of the aerodigestive tract have similar etiological risk factors. Although genetic risk variants for individual cancers have been identified, an agnostic, genome-wide search for shared genetic susceptibility has not been performed. To identify novel and pleotropic SqCC risk variants, we performed a meta-analysis of GWAS data on lung SqCC (LuSqCC), oro/pharyngeal SqCC (OSqCC), laryngeal SqCC (LaSqCC) and esophageal SqCC (ESqCC) cancers, totaling 13,887 cases and 61,961 controls of European ancestry. We identified one novel genome-wide significant (Pmeta<5x10-8) aerodigestive SqCC susceptibility loci in the 2q33.1 region (rs56321285, TMEM273). Additionally, three previously unknown loci reached suggestive significance (Pmeta<5x10-7): 1q32.1 (rs12133735, near MDM4), 5q31.2 (rs13181561, TMEM173) and 19p13.11 (rs61494113, ABHD8). Multiple previously identified loci for aerodigestive SqCC also showed evidence of pleiotropy in at least another SqCC site, these include: 4q23 (ADH1B), 6p21.33 (STK19), 6p21.32 (HLA-DQB1), 9p21.33 (CDKN2B-AS1) and 13q13.1(BRCA2). Gene-based association and gene set enrichment identified a set of 48 SqCC-related genes rel to DNA damage and epigenetic regulation pathways. Our study highlights the importance of cross-cancer analyses to identify pleiotropic risk loci of histology-related cancers arising at distinct anatomical sites.

Hydrocodone, Oxycodone, and Morphine Metabolism and Drug-Drug Interactions.

Awareness of drug interactions involving opioids is critical for patient treatment as they are common therapeutics used in numerous care settings, including both chronic and disease-related pain. Not only do opioids have narrow therapeutic indexes and are extensively used, but they have the potential to cause severe toxicity. Opioids are the classical pain treatment for patients who suffer from moderate to severe pain. More importantly, opioids are often prescribed in combination with multiple other drugs, especially in patient populations who typically are prescribed a large drug regimen. This review focuses on the current knowledge of common opioid drug-drug interactions (DDIs), focusing specifically on hydrocodone, oxycodone, and morphine DDIs. The DDIs covered in this review include pharmacokinetic DDI arising from enzyme inhibition or induction, primarily due to inhibition of cytochrome p450 enzymes (CYPs). However, opioids such as morphine are metabolized by uridine-5'-diphosphoglucuronosyltransferases (UGTs), principally UGT2B7, and glucuronidation is another important pathway for opioid-drug interactions. This review also covers several pharmacodynamic DDI studies as well as the basics of CYP and UGT metabolism, including detailed opioid metabolism and the potential involvement of metabolizing enzyme gene variation in DDI. Based upon the current literature, further studies are needed to fully investigate and describe the DDI potential with opioids in pain and related disease settings to improve clinical outcomes for patients. SIGNIFICANCE STATEMENT: A review of the literature focusing on drug-drug interactions involving opioids is important because they can be toxic and potentially lethal, occurring through pharmacodynamic interactions as well as pharmacokinetic interactions occurring through inhibition or induction of drug metabolism.

CYP2C19 Plays a Major Role in the Hepatic N-Oxidation of Cotinine.

The primary mode of metabolism of nicotine is via the formation of cotinine by the enzyme CYP2A6. Cotinine undergoes further CYP2A6-mediated metabolism by hydroxylation to 3-hydroxycotinine and norcotinine, but can also form cotinine-N-glucuronide and cotinine-N-oxide (COX). The goal of this study was to investigate the enzymes that catalyze COX formation and determine whether genetic variation in these enzymes may affect this pathway. Specific inhibitors of major hepatic cytochrome P450 (P450) enzymes were used in cotinine-N-oxidation reactions using pooled human liver microsomes (HLMs). COX formation was monitored by ultrahigh-pressure liquid chromatography-tandem mass spectrometry and enzyme kinetic analysis was performed using microsomes from P450-overexpressing human embryonic kidney 293 (HEK293) cell lines. Genotype-phenotype analysis was performed in a panel of 113 human liver specimens. Inhibition of COX formation was only observed in HLMs when using inhibitors of CYP2A6, CYP2B6, CYP2C19, CYP2E1, and CYP3A4. Microsomes from cells overexpressing CYP2A6 or CYP2C19 exhibited similar N-oxidation activity against cotinine, with maximum reaction rate over Michaelis constant values (intrinsic clearance) of 4.4 and 4.2 nL/min/mg, respectively. CYP2B6-, CYP2E1-, and CYP3A4-overexpressing microsomes were also active in COX formation. Significant associations (P < 0.05) were observed between COX formation and genetic variants in CYP2C19 (*2 and *17 alleles) in HLMs. These results demonstrate that genetic variants in CYP2C19 are associated with decreased COX formation, potentially affecting the relative levels of cotinine in the plasma or urine of smokers and ultimately affecting recommended smoking cessation therapies. SIGNIFICANCE STATEMENT: This study is the first to elucidate the enzymes responsible for cotinine-N-oxide formation and genetic variants that affect this biological pathway. Genetic variants in CYP2C19 have the potential to modify nicotine metabolic ratio in smokers and could affect pharmacotherapeutic decisions for smoking cessation treatments.

Inhibition of Nicotine Metabolism by Cannabidiol (CBD) and 7-Hydroxycannabidiol (7-OH-CBD).

Cannabis-based products have experienced notable increases in co-usage alongside tobacco products. Several cannabinoids exhibit inhibition of a number of cytochrome P450 (CYP) and UDP glucuronosyltransferase (UGT) enzymes, but few studies have examined their inhibition of enzymes involved in nicotine metabolism. The goal of the present study was to examine potential drug-drug interactions occurring in the nicotine metabolism pathway perpetrated by cannabidiol (CBD) and its active metabolite, 7-hydroxy-CBD (7-OH-CBD). The inhibitory effects of CBD and 7-OH-CBD were tested in microsomes from HEK293 cells overexpressing individual metabolizing enzymes and from human liver tissue. Assays with overexpressing microsomes demonstrated that CBD and 7-OH-CBD inhibited CYP-mediated nicotine metabolism. Binding-corrected IC50,u values for CBD inhibition of nicotine metabolism to cotinine and nornicotine, and cotinine metabolism to trans-3'-hydroxycotinine (3HC), were 0.27 ± 0.060, 0.23 ± 0.14, and 0.21 ± 0.14 µM, respectively, for CYP2A6; and 0.26 ± 0.17 and 0.029 ± 0.0050 µM for cotinine and nornicotine formation, respectively, for CYP2B6. 7-OH-CBD IC50,u values were 0.45 ± 0.18, 0.16 ± 0.08, and 0.78 ± 0.23 µM for cotinine, nornicotine, and 3HC formation, respectively, for CYP2A6, and 1.2 ± 0.44 and 0.11 ± 0.030 µM for cotinine and nornicotine formation, respectively, for CYP2B6. Similar IC50,u values were observed in HLM. Inhibition (IC50,u = 0.37 ± 0.06 µM) of 3HC to 3HC-glucuronide formation by UGT1A9 was demonstrated by CBD. Significant inhibition of nicotine metabolism pathways by CBD and 7-OH-CBD suggests that cannabinoids may inhibit nicotine metabolism, potentially impacting tobacco addiction and cessation.

Inhibition of the aromatase enzyme by exemestane cysteine conjugates.

Exemestane (EXE) is an aromatase inhibitor used to treat hormone-dependent breast cancer. EXE is extensively metabolized, with unchanged EXE and its active metabolite 17-dihydroexemestane (17-DHE) accounting for 17 and 12%, respectively, of total plasma EXE in vivo The major circulating EXE metabolites are the cysteine conjugates of EXE and 17-DHE, and the 17-DHE glucuronide, which together account for 70% of total plasma EXE in vivo The goal of the present study was to examine the inhibition potential of major metabolites of EXE through inhibition assays using aromatase-overexpressing cells and pooled ovarian tissues. Estrone formation was used as a measure of aromatase activity and was detected and quantified using UPLC-MS. EXE-cys, 17ß-DHE, and 17ß-DHE-cys all exhibited inhibition of estrone formation at both 1 µM and 10 µM concentrations, with 17ß-DHE and EXE-cys showing significant inhibition of estrone formation (63% each) at 10 µM. In contrast, 17ß-DHE-Gluc displayed minimal inhibition (5-8%) at both concentrations. In ovarian tissue, EXE-cys and 17ß-DHE showed similar patterns of inhibition, with 49% and 47% inhibition, respectively, at 10 µM. The IC50 value for EXE-cys (16 {plus minus} 10 µM) was similar to 17ß-DHE (9.2 {plus minus} 2.7 µM) and higher than EXE (1.3 {plus minus} 0.28 µM), and all three compounds showed time-dependent inhibition with IC50 shifts of 13 {plus minus} 10, 5.0 {plus minus} 2.5 and 36 {plus minus} 12-fold, respectively. Given its high circulating levels in patients taking EXE, these results suggest that EXE-cys may contribute to the pharmacologic effect of EXE in vivo Significance Statement The current study is the first to examine the major phase II metabolites of EXE (EXE-cys, 17ß-DHE-cys, and 17ß-DHE-Gluc) for inhibition potential against the target enzyme, aromatase (CYP19A1). EXE-cys was found to significantly inhibit aromatase in a time dependent manner. Given its high circulating levels in patients taking EXE, this phase II metabolite may play an important role in reducing circulating estrogen levels in vivo.

Integration of multiomic annotation data to prioritize and characterize inflammation and immune-related risk variants in squamous cell lung cancer.

Clinical trial results have recently demonstrated that inhibiting inflammation by targeting the interleukin-1ß pathway can offer a significant reduction in lung cancer incidence and mortality, highlighting a pressing and unmet need to understand the benefits of inflammation-focused lung cancer therapies at the genetic level. While numerous genome-wide association studies (GWAS) have explored the genetic etiology of lung cancer, there remains a large gap between the type of information that may be gleaned from an association study and the depth of understanding necessary to explain and drive translational findings. Thus, in this study we jointly model and integrate extensive multiomics data sources, utilizing a total of 40 genome-wide functional annotations that augment previously published results from the International Lung Cancer Consortium (ILCCO) GWAS, to prioritize and characterize single nucleotide polymorphisms (SNPs) that increase risk of squamous cell lung cancer through the inflammatory and immune responses. Our work bridges the gap between correlative analysis and translational follow-up research, refining GWAS association measures in an interpretable and systematic manner. In particular, reanalysis of the ILCCO data highlights the impact of highly associated SNPs from nuclear factor-κB signaling pathway genes as well as major histocompatibility complex mediated variation in immune responses. One consequence of prioritizing likely functional SNPs is the pruning of variants that might be selected for follow-up work by over an order of magnitude, from potentially tens of thousands to hundreds. The strategies we introduce provide informative and interpretable approaches for incorporating extensive genome-wide annotation data in analysis of genetic association studies.

Genetic variants in CYP2B6 and HSD17B12 associated with risk of squamous cell carcinoma of the head and neck.

Polycyclic aromatic hydrocarbons (PAH) and tobacco-specific nitrosamines (TSNA) metabolism-related genes play an important role in the development of cancers. We assessed the associations of genetic variants in genes involved in the metabolism of PAHs and TSNA with risk of squamous cell carcinoma of the head and neck (SCCHN) in European populations using two published genome-wide association study datasets. In the single-locus analysis, we identified two SNPs (rs145533669 and rs35246205) in CYP2B6 to be associated with risk of SCCHN (P = 1.57 × 10-4 and .004, respectively), two SNPs (EPHX1 rs117522494 and CYP2B6 rs145533669) to be associated with risk of oropharyngeal cancer (P = .001 and .004, respectively), and one SNP (rs4359199 in HSD17B12) to be associated with risk of oral cancer (P = .006). A significant interaction effect was found between rs4359199 and drinking status on risks of SCCHN and oropharyngeal cancer (P < .05). eQTL and sQTL analyzes revealed that two SNPs (CYP2B6 rs35246205 and HSD17B12 rs4359199) were correlated with alternative splicing or mRNA expression levels of the corresponding genes in liver cells (P < .05 for both). In silico functional annotation suggested that these two SNPs may regulate mRNA expression by affecting the binding of transcription factors. Results from phenome-wide association studies presented significant associations between these genes and risks of other cancers, smoking behavior and alcohol dependence (P < .05). Thus, our study provided some insight into the underlying genetic mechanism of head and neck cancer, which warrants future functional validation.

Epigenetics in drug disposition & drug therapy: symposium report of the 24th North American meeting of the International Society for the Study of Xenobiotics (ISSX).

The 24th North American International Society for the Study of Xenobiotics (ISSX) meeting, held virtually from September 13 to 17, 2021, embraced the theme of "Broadening Our Horizons." This reinforces a key mission of ISSX: striving to share innovative science related to drug discovery and development. Session speakers and the ISSX New Investigators Group, which supports the scientific and professional development of student and early career ISSX members, elected to highlight the scientific content presented during the captivating session titled, "Epigenetics in Drug Disposition & Drug Therapy." The impact genetic variation has on drug response is well established; however, this session underscored the importance of investigating the role of epigenetics in drug disposition and drug discovery. Session speakers, Drs. Ning, McClay, and Lazarus, detailed mechanisms by which epigenetic players including long non-coding RNA (lncRNAs), microRNA (miRNAs), DNA methylation, and histone acetylation can alter the expression of genes involved in pharmacokinetics, pharmacodynamics, and toxicity. Dr. Ning detailed current knowledge about miRNAs and lncRNAs and the mechanisms by which they can affect the expression of drug metabolizing enzymes (DMEs) and nuclear receptors. Dr. Lazarus discussed the potential role of miRNAs on UDP-glucuronosyltransferase (UGT) expression and activity. Dr. McClay provided evidence that aging alters methylation and acetylation of DMEs in the liver, affecting gene expression and activity. These topics, compiled by the symposium organizers, presenters, and the ISSX New Investigators Group, are herein discussed, along with exciting future perspectives for epigenetics in drug disposition and drug discovery research.

Influence of Glutathione-S-Transferase A1*B Allele on the Metabolism of the Aromatase Inhibitor, Exemestane, in Human Liver Cytosols and in Patients Treated With Exemestane.

Exemestane (EXE) is used to treat postmenopausal women diagnosed with estrogen receptor positive (ER+) breast cancer. A major mode of metabolism of EXE and its active metabolite, 17ß-dihydroexemestane, is via glutathionylation by glutathione-S-transferase (GST) enzymes. The goal of the present study was to investigate the effects of genetic variation in EXE-metabolizing GST enzymes on overall EXE metabolism. Ex vivo assays examining human liver cytosols from 75 subjects revealed the GSTA1 *B*B genotype was associated with significant decreases in S-(androsta-1,4-diene-3,17-dion-6α-ylmethyl)-L-glutathione (P = 0.034) and S-(androsta-1,4-diene-17ß-ol-3-on-6α-ylmethyl)-L-gutathione (P = 0.014) formation. In the plasma of 68 ER+ breast cancer patients treated with EXE, the GSTA1 *B*B genotype was associated with significant decreases in both EXE-cysteine (cys) (29%, P = 0.0056) and 17ß-DHE-cys (34%, P = 0.032) as compared with patients with the GSTA1*A*A genotype, with significant decreases in EXE-cys (Ptrend = 0.0067) and 17ß-DHE-cys (Ptrend = 0.028) observed in patients with increasing numbers of the GSTA1*B allele. A near-significant (Ptrend = 0.060) trend was also observed for urinary EXE-cys levels from the same patients. In contrast, plasma and urinary 17ß-DHE-Gluc levels were significantly increased (36%, P = 0.00097 and 52%, P = 0.0089; respectively) in patients with the GSTA1 *B*B genotype. No significant correlations were observed between the GSTM1 null genotype and EXE metabolite levels. These data suggest that the GSTA1*B allele is associated with interindividual differences in EXE metabolism and may play a role in interindividual variability in overall response to EXE. SIGNIFICANCE STATEMENT: The present study is the first comprehensive pharmacogenomic investigation examining the role of genetic variability in GST enzymes on exemestane metabolism. The GSTA1 *B*B genotype was found to contribute to interindividual differences in the metabolism of EXE both ex vivo and in clinical samples from patients taking EXE for the treatment of ER+ breast cancer. Since GSTA1 is a major hepatic phase II metabolizing enzyme in EXE metabolism, the GSTA1*B allele may be an important biomarker for treatment outcomes and toxicities.

Drug-Drug Interaction Between Orally Administered Hydrocodone-Acetaminophen and Inhalation of Cannabis Smoke: A Case Report.

Objective: To determine if a 2-day protocol measuring pharmacokinetic and pharmacodynamic characteristics can demonstrate drug-drug interactions when smoked cannabis is added to orally administered hydrocodone/acetaminophen combination products. Case Summary: A 51-year-old non-Hispanic white male with chronic pain diagnoses participated in a 2-day pilot protocol. The participant attended two 7-hour in-lab days where he received 10 blood draws each day and completed self-administered pain and anxiety surveys. For both days, the participant took his prescribed dose of hydrocodone/acetaminophen (1/2 tablet of 7.5 mg/325 mg combination product) with the addition of 1 smoked pre-rolled marijuana cigarette (labeled as 0.5 g; 22.17% Δ9-tetrahydrocannabinol; 0.12% cannabidiol) on Day 2. Blood specimens were analyzed using mass spectrometry to quantify the difference of plasma hydrocodone levels between Day 1 and Day 2. Results: Compared to Day 1, lower levels of pain and anxiety were reported during Day 2 with the addition of cannabis to oral hydrocodone/acetaminophen. Day 2 pharmacokinetic analysis also revealed more rapid absorption and overall lower levels of hydrocodone in plasma. Discussion: Lower hydrocodone plasma levels in Day 2 may indicate cannabis's effect on metabolism and reduce the risk of opioid toxicity. The quicker absorption rate of hydrocodone could explain lower pain and anxiety scores reported on the second day. Conclusion and Relevance: A 2-day protocol was able to capture differences across time in pharmacokinetic and pharmacodynamic measurements. Larger studies can be designed to better characterize the potential drug-drug interaction of cannabis and opioids.

Causal relationships between body mass index, smoking and lung cancer: Univariable and multivariable Mendelian randomization.

At the time of cancer diagnosis, body mass index (BMI) is inversely correlated with lung cancer risk, which may reflect reverse causality and confounding due to smoking behavior. We used two-sample univariable and multivariable Mendelian randomization (MR) to estimate causal relationships of BMI and smoking behaviors on lung cancer and histological subtypes based on an aggregated genome-wide association studies (GWASs) analysis of lung cancer in 29 266 cases and 56 450 controls. We observed a positive causal effect for high BMI on occurrence of small-cell lung cancer (odds ratio (OR) = 1.60, 95% confidence interval (CI) = 1.24-2.06, P = 2.70 × 10-4 ). After adjustment of smoking behaviors using multivariable Mendelian randomization (MVMR), a direct causal effect on small cell lung cancer (ORMVMR = 1.28, 95% CI = 1.06-1.55, PMVMR = .011), and an inverse effect on lung adenocarcinoma (ORMVMR = 0.86, 95% CI = 0.77-0.96, PMVMR = .008) were observed. A weak increased risk of lung squamous cell carcinoma was observed for higher BMI in univariable Mendelian randomization (UVMR) analysis (ORUVMR = 1.19, 95% CI = 1.01-1.40, PUVMR = .036), but this effect disappeared after adjustment of smoking (ORMVMR = 1.02, 95% CI = 0.90-1.16, PMVMR = .746). These results highlight the histology-specific impact of BMI on lung carcinogenesis and imply mediator role of smoking behaviors in the association between BMI and lung cancer.

Characterization of Cytosolic Glutathione S-Transferases Involved in the Metabolism of the Aromatase Inhibitor, Exemestane.

Exemestane (EXE) is a hormonal therapy used to treat estrogen receptor-positive breast cancer by inhibiting the final step of estrogen biosynthesis catalyzed by the enzyme aromatase. Cysteine conjugates of EXE and its active metabolite 17ß-dihydro-EXE (DHE) are the major metabolites found in both the urine and plasma of patients taking EXE. The initial step in cysteine conjugate formation is glutathione conjugation catalyzed by the glutathione S-transferase (GST) family of enzymes. The goal of the present study was to identify cytosolic hepatic GSTs active in the GST-mediated metabolism of EXE and 17ß-DHE. Twelve recombinant cytosolic hepatic GSTs were screened for their activity against EXE and 17ß-DHE, and glutathionylated EXE and 17ß-DHE conjugates were detected by ultra-performance liquid chromatography tandem mass spectrometry. GST α (GSTA) isoform 1, GST µ (GSTM) isoform 3 and isoform 1 were active against EXE, whereas only GSTA1 exhibited activity against 17ß-DHE. GSTM1 exhibited the highest affinity against EXE with a Michaelis-Menten constant (KM) value that was 3.8- and 7.1-fold lower than that observed for GSTA1 and GSTM3, respectively. Of the three GSTs, GSTM3 exhibited the highest intrinsic clearance against EXE (intrinsic clearance = 0.14 nl·min-1·mg-1). The KM values observed for human liver cytosol against EXE (46 µM) and 17ß-DHE (77 µM) were similar to those observed for recombinant GSTA1 (53 and 30 µM, respectively). Western blot analysis revealed that GSTA1 and GSTM1 composed 4.3% and 0.57%, respectively, of total protein in human liver cytosol; GSTM3 was not detected. These data suggest that GSTA1 is the major hepatic cytosolic enzyme involved in the clearance of EXE and its major active metabolite, 17ß-DHE. SIGNIFICANCE STATEMENT: Most previous studies related to the metabolism of the aromatase inhibitor exemestane (EXE) have focused mainly on phase I metabolic pathways and the glucuronidation phase II metabolic pathway. However, recent studies have indicated that glutathionylation is the major metabolic pathway for EXE. The present study is the first to characterize hepatic glutathione S-transferase (GST) activity against EXE and 17ß-dihydro-EXE and to identify GST α 1 and GST µ 1 as the major cytosolic GSTs involved in the hepatic metabolism of EXE.

Cannabinoid Metabolites as Inhibitors of Major Hepatic CYP450 Enzymes, with Implications for Cannabis-Drug Interactions.

The legalization of cannabis in many parts of the United States and other countries has led to a need for a more comprehensive understanding of cannabis constituents and their potential for drug-drug interactions. Although (-)-trans-Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabinol (CBN) are the most abundant cannabinoids present in cannabis, THC metabolites are found in plasma at higher concentrations and for a longer duration than that of the parent cannabinoids. To understand the potential for drug-drug interactions, the inhibition potential of major cannabinoids and their metabolites on major hepatic cytochrome P450 (P450) enzymes was examined. In vitro assays with P450-overexpressing cell microsomes demonstrated that the major THC metabolites 11-hydroxy-Δ9-tetra-hydrocannabinol and 11-nor-9-carboxy-Δ9-THC-glucuronide competitively inhibited several major P450 enzymes, including CYP2B6, CYP2C9, and CYP2D6 (apparent Ki,u values = 0.086 ± 0.066 µM and 0.90 ± 0.54 µM, 0.057 ± 0.044 µM and 2.1 ± 0.81 µM, 0.15 ± 0.067 µM and 2.3 ± 0.54 µM, respectively). 11-Nor-9-carboxy-Δ9- tetrahydrocannabinol exhibited no inhibitory activity against any CYP450 tested. THC competitively inhibited CYP1A2, CYP2B6, CYP2C9, and CYP2D6; CBD competitively inhibited CYP3A4, CYP2B6, CYP2C9, CYP2D6, and CYP2E1; and CBN competitively inhibited CYP2B6, CYP2C9, and CYP2E1. THC and CBD showed mixed-type inhibition for CYP2C19 and CYP1A2, respectively. These data suggest that cannabinoids and major THC metabolites are able to inhibit the activities of multiple P450 enzymes, and basic static modeling of these data suggest the possibility of pharmacokinetic interactions between these cannabinoids and xenobiotics extensively metabolized by CYP2B6, CYP2C9, and CYP2D6. SIGNIFICANCE STATEMENT: Major cannabinoids and their metabolites found in the plasma of cannabis users inhibit several P450 enzymes, including CYP2B6, CYP2C9, and CYP2D6. This study is the first to show the inhibition potential of the most abundant plasma cannabinoid metabolite, THC-COO-Gluc, and suggests that circulating metabolites of cannabinoids play an essential role in CYP450 enzyme inhibition as well as drug-drug interactions.

Inhibition of UDP-Glucuronosyltransferase Enzymes by Major Cannabinoids and Their Metabolites.

The UDP-glucuronosyltransferase (UGT) family of enzymes play a central role in the metabolism and detoxification of a wide range of endogenous and exogenous compounds. UGTs exhibit a high degree of structural similarity and display overlapping substrate specificity, often making estimations of potential drug-drug interactions difficult to fully elucidate. One such interaction yet to be examined may be occurring between UGTs and cannabinoids, as the legalization of recreational and medicinal cannabis and subsequent co-usage of cannabis and therapeutic drugs increases in the United States and internationally. In the present study, the inhibition potential of the major cannabinoids Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabinol (CBN), as well as their major metabolites, was determined in microsomes isolated from HEK293 cells overexpressing individual recombinant UGTs and in microsomes from human liver and kidney specimens. The highest inhibition was seen by CBD against the glucuronidation activity of UGTs 1A9, 2B4, 1A6, and 2B7, with binding-corrected IC50 values of 0.12 ± 0.020 µM, 0.22 ± 0.045 µM, 0.40 ± 0.10 µM, and 0.82 ± 0.15 µM, respectively. Strong inhibition of UGT1A9 was also demonstrated by THC and CBN, with binding-corrected IC50 values of 0.45 ± 0.12 µM and 0.51 ± 0.063 µM, respectively. Strong inhibition of UGT2B7 was also observed for THC and CBN; no or weak inhibition was observed with cannabinoid metabolites. This inhibition of UGT activity suggests that in addition to playing an important role in drug-drug interactions, cannabinoid exposure may have important implications in patients with impaired hepatic or kidney function. SIGNIFICANCE STATEMENT: Major cannabinoids found in the plasma of cannabis users inhibit several UDP-glucuronosyltransferase (UGT) enzymes, including UGT1A6, UGT1A9, UGT2B4, and UGT2B7. This study is the first to show the potential of cannabinoids and their metabolites to inhibit all the major kidney UGTs as well as the two most abundant UGTs present in liver. This study suggests that as all three major kidney UGTs are inhibited by cannabinoids, greater drug-drug interaction effects might be observed from co-use of cannabinods and therapeutics that are cleared renally.

Genome-wide association study of INDELs identified four novel susceptibility loci associated with lung cancer risk.

Genome-wide association studies (GWAS) have identified 45 susceptibility loci associated with lung cancer. Only less than SNPs, small insertions and deletions (INDELs) are the second most abundant genetic polymorphisms in the human genome. INDELs are highly associated with multiple human diseases, including lung cancer. However, limited studies with large-scale samples have been available to systematically evaluate the effects of INDELs on lung cancer risk. Here, we performed a large-scale meta-analysis to evaluate INDELs and their risk for lung cancer in 23,202 cases and 19,048 controls. Functional annotations were performed to further explore the potential function of lung cancer risk INDELs. Conditional analysis was used to clarify the relationship between INDELs and SNPs. Four new risk loci were identified in genome-wide INDEL analysis (1p13.2: rs5777156, Insertion, OR = 0.92, p = 9.10 × 10-8 ; 4q28.2: rs58404727, Deletion, OR = 1.19, p = 5.25 × 10-7 ; 12p13.31: rs71450133, Deletion, OR = 1.09, p = 8.83 × 10-7 ; and 14q22.3: rs34057993, Deletion, OR = 0.90, p = 7.64 × 10-8 ). The eQTL analysis and functional annotation suggested that INDELs might affect lung cancer susceptibility by regulating the expression of target genes. After conducting conditional analysis on potential causal SNPs, the INDELs in the new loci were still nominally significant. Our findings indicate that INDELs could be potentially functional genetic variants for lung cancer risk. Further functional experiments are needed to better understand INDEL mechanisms in carcinogenesis.

Transcriptome-wide association study reveals candidate causal genes for lung cancer.

We have recently completed the largest GWAS on lung cancer including 29,266 cases and 56,450 controls of European descent. The goal of our study has been to integrate the complete GWAS results with a large-scale expression quantitative trait loci (eQTL) mapping study in human lung tissues (n = 1,038) to identify candidate causal genes for lung cancer. We performed transcriptome-wide association study (TWAS) for lung cancer overall, by histology (adenocarcinoma, squamous cell carcinoma and small cell lung cancer) and smoking subgroups (never- and ever-smokers). We performed replication analysis using lung data from the Genotype-Tissue Expression (GTEx) project. DNA damage assays were performed in human lung fibroblasts for selected TWAS genes. As expected, the main TWAS signal for all histological subtypes and ever-smokers was on chromosome 15q25. The gene most strongly associated with lung cancer at this locus using the TWAS approach was IREB2 (pTWAS = 1.09E-99), where lower predicted expression increased lung cancer risk. A new lung adenocarcinoma susceptibility locus was revealed on 9p13.3 and associated with higher predicted expression of AQP3 (pTWAS = 3.72E-6). Among the 45 previously described lung cancer GWAS loci, we mapped candidate target gene for 17 of them. The association AQP3-adenocarcinoma on 9p13.3 was replicated using GTEx (pTWAS = 6.55E-5). Consistent with the effect of risk alleles on gene expression levels, IREB2 knockdown and AQP3 overproduction promote endogenous DNA damage. These findings indicate genes whose expression in lung tissue directly influences lung cancer risk.