Effect of Inhaled Budesonide in Smokers with Bronchial

Effect of Inhaled Budesonide in Smokers with Bronchial Dysplasia BC Cancer Research Centre 675 West 10 th Avenue Raj 1, 2 Chari , Kim 1 Lonergan , Luc 4 Girard , 4 Minna , 4 Gazdar , 3 Yao , 3 You , 2 Ng , John Adi Ruisheng Ming Raymond Wan 1, 2 Calum Mac. Aulay , Stephen Lam [1]British Columbia Cancer Agency, Vancouver, BC, Canada, [2]University of British Columbia, Vancouver, BC, Canada, [3]Washington University in St. Louis, MO, USA, [4]UT Southwestern Medical Centre 4, Dallas, TX, USA Abstract Methods Background: Budesonide, an inhaled corticosteroid used in the treatment of asthma, has been shown to be an effective chemopreventive agent in an animal model of adenocarcinoma [Carcinogenesis 1997 Oct 18(10): 2015 -7]. In humans, although inhaled budesonide for 6 months was not effective in regression of bronchial dysplasia, a significantly higher rate of resolution of CT detected small lung nodules was observed, some of which may represent pre-neoplastic lesions in the peripheral lung [Clinical Cancer Research 2004 Oct 1 10(19): 6502 -11]. Despite being used as a drug for treatment of asthma and COPD for over two decades, the in-vivo effects of inhaled steroids on gene expression profiles of bronchial epithelial cells in smokers are still poorly understood. Objective: The objective of this study is to characterize the effects of inhaled budesonide on the gene expression profiles of bronchial cells from current and former smokers with bronchial dysplasia. Methods: Bronchial cells were obtained before and after six months of treatment with budesonide 800 mcg BID by inhalation. After two rounds of linear amplification of the extracted RNA, the gene expression profiles were analyzed using the Affymetrix U 133 A microarray chip. Results: Using Principal Component Analysis, the effect of active smoking was found to be stronger than the effect of budesonide. In current smokers, more Phase 1 enzyme genes were upregulated compared with former smokers. However, Phase 2 enzyme genes were up-regulated in former smokers but down-regulated in current smokers. Specifically, CYP 1 B 1 was shown to have a two-fold increase in current smokers after Budesonide treatment and approximately a 1. 5 -fold decrease in former smokers after treatment. In a separate analysis, it was found that genes upregulated in current smokers had the tendency to be down-regulated in former smokers after treatment. As well, potential genes have been identified which correlate with the response to Budesonide treatment. Conclusions: A differential effect of Budesonide on gene expression profile was found between current and former smokers. Furthermore, we have characterized potential genes which correlate with the level of response. Targeting genes and pathways that correlate with patient response may be beneficial in screening of new chemopreventive agents. Supported by NIH contract N 01 -CN 85188, & NCI SPORE CA 70907 & NIH 1 P 01 CA 096964 Effect of Active Smoking Effect of Budesonide Comparison of Affymetrix U 133 A and SAGE Description of Dataset Specimens Higher in Current Smokers Objectives ØTo determine the effect of active tobacco smoking on gene expression profiles in human bronchial epithelial cells retrieved by bronchial brushing during bronchoscopy ØTo compare the gene expression profiles obtained by Affymetrix U 133 A array with Serial Analysis of Gene Expression (SAGE) ØTo characterize the effects of inhaled budesonide on the gene expression profiles of bronchial cells from current versus former smokers with bronchial dysplasia ØTo identify genes associated with regression of bronchial dysplasia after treatment with inhaled budesonide Hypotheses ØSAGE identifies differentially expressed genes not captured by previous microarray analysis ØCurrent and former smokers respond differently to inhaled budesonide ØRegression of bronchial dysplasia after budesonide is associated with different gene expression profiles versus nonresponders Vancouver BC, V 5 Z 1 L 3 ph: 604 -675 -8000 ext. 7703 email: rchari@bccrc. ca Results Current Smokers Affymetrix U 133 A 18 34 SAGE 11 9 ØAffymetrix U 133 A data was obtained from the paper by Spira et al. [Proc Natl Acad Sci U S A. 2004; 101(27): 10143 -8] ØSAGE libraries were generated from human bronchial epithelial cells obtained by bronchial brushings during bronchoscopy of heavy smokers Serial Analysis of Gene Expression (SAGE) SAGE deduces expression profile based on the relative abundance of sequence tags. ØSAGE is not limited to genes spotted on the array ØSAGE produces quantitative, discrete (digital) data values Ø~150, 000 tags enumerated per library Filtering of data ØAffymetrix data filtered as explained by Spira et al. [Proc Natl Acad Sci U S A. 2004; 101(27): 10143 -8] ØData for SAGE tags was retained only if raw tag counts were greater than 1 in at least one library Statistical Methods ØA combination of the student’s t-test, fold-change and permutation test were used in the analysis ØDifferentially expressed genes were selected at a significance level of p ≤ 0. 001 ØT-test and fold-change are commonly used in the literature ØPermutation test Øsuitable test for a small sample size Ønon-parametric test Øperforms well in identifying differentially expressed genes Human bronchial epithelial cells were obtained in 10 smokers with bronchial dysplasia by bronchial brushings before and after daily treatment with 1600 µg of inhaled budesonide/placebo for six months (Clin Cancer Res 2004; 10: 65026511). Specimens were also obtained from 7 subjects before and after one month treatment with budesonide Platform ØAffymetrix U 133 A array was used to profile the samples obtained from the 10 subjects in the six month study ØAffymetrix U 133 A 2. 0 array was used for the 7 subjects in the one month study Evaluation Criteria ØIdentify genes that show similar expression differences before and after treatment in 4 of 5 subjects ØFold-change of ≥ 1. 5 (log 2 fold of 0. 585) between baseline and six months ØRemove genes if similar expression pattern seen in the placebo arm ØFindings validated in the dataset of the one-month study Examples of Differentially Expressed Genes - Phase 1& 2 Toxin Exposure Genes Current Smoker 1600 (x 104) Former Smoker 1400 1200 1000 800 600 400 200 0 CYP 1 B 1 Correlation of Gene Expression with Clinical Response ØResponse characterized as complete response (CR), partial response (PR) and progressive disease (PD) ØCR is defined as complete regression of all dysplastic lesions to hyperplasia or normal, PR is regression of some but not all dysplastic lesions; PD is appearance of new lesions or progression of dysplastic lesions by ≥ 2 grades after treatment ØIdentify genes that have a ≥ 1. 5 fold change in complete responders only ØMust also exhibit a gradient of expression between CR, PR and PD. Smoking Status and Response to Inhaled Budesonide Ø Identify genes which have ≥ 1. 5 fold change exclusively in all former smokers but not in all current smokers and viceversa Ø Remove genes if similar expression pattern seen in placebo arm ØExample of genes with increased (left) and decreased expression (right) in complete responders. Among the up-regulated genes are: caveolin 2 (CAV 2), hydroxyprostaglandin dehyrogenase 15 -(NAD) (HPGD), TU 3 A protein (TU 3 A) and the down-regulated genes are: toll-like receptor 3 (TLR 3), TIR domain containing adaptor inducing interferon-beta (TRIF), mitogen-activated protein kinase 6 (MAP 2 K 6). ØHPGD has been recently shown to mediate anti-inflammatory effects in lung adenocarcinoma cell lines [Arch Biochem Biophys. 2005; 435(1): 50 -5] ØTRIF and TLR 3 potentially implicate down-regulation of the Toll-like receptor pathway Subset of Genes Differentially Expressed Between Current and Former Smokers 2000 1800 Genes Correlating with Clinical Response Higher in Former Smokers ØComparison of the number of probes or tags that are differentially expressed based on t-value, permutation test score, fold-change and all 3 criteria at significance level of p ≤ 0. 001 ØUsing the 3 criteria ØIn current smokers, SAGE data shows 30 tags (mapping to 27 unique genes) and Affymetrix U 133 A gives 7 probes (mapping 5 unique genes) that are higher compared to former smokers ØIn former smokers, SAGE data suggests 22 tags (mapping to 18 unique genes) and Affymetrix U 133 A has none that are higher relative current smokers Tags Per Million Platform Former Smokers Background ØChemopreventive agents for lung cancer may exhibit a differential effect in current versus former or never smokers ØPrevious microarray studies showed different gene expression profiles between current and former smokers ØBudesonide is one of the most effective chemopreventive agents in animal models of lung cancer but was found to be ineffective in regression of bronchial dysplasia in smokers [Clinical Cancer Research 2004 Oct 1 10(19): 6502 -11] 1, 2 Lam , CYP 1 A 1 GST 2 GPX 2 Increased in Current Smokers, Decreased in Former Smokers Increased in Former Smokers, Decreased in Current Smokers MYO 6 - Myosin VI TM 7 SF 4 - transmembrane 7 superfamily member 4 MAP 4 K 5 - mitogen-activated protein kinase 5 CORO 1 A - coronin, actin binding protein, 1 A TPD 52 - tumor protein D 52 NMT 2 - N-myristoyltransferase 2 CYP 1 B 1 - cytochrome P 450, family 1, subfamily B, polypeptide 1 PLA 2 - phospholipase A 2, group IVB (cytosolic) CYP/(GST+GPX) ØPhase I enzymes and cell cycle control genes was observed in current smokers compared to former smokers Budesonide Associated Expression Changes Subject Background and Response Smoking status # on active arm Clinical Response* Former (n = 5) 2 1 CR, 1 PR Current (n = 5) 3 1 CR, 1 PD Conclusions Effect of Budesonide Independent of Response and Smoking Status Ø(Below) Sample of genes up-regulated and down-regulated after treatment in at least 4 of 5 subjects ØAlso exhibit same pattern in at least 5 of 7 subjects in validation dataset (U 133 A 2. 0) Down-Regulated Up-Regulated TRBC 1 - T cell receptor beta constant 1 TOP 2 A - topoisomerase (DNA) II alpha 170 k. Da CCL 5 - chemokine (C-C motif) ligand 5 HPGD - hydroxyprostaglandin dehydrogenase 15 -(NAD) ØAnalysis of SAGE data affords more discovery of significant genes as compared to the Affymetrix U 133 A platform ØBudesonide exhibits anti-inflammatory effects as evidenced by increased HPGD, furthermore this gene is increased even higher in complete responders. ØExpression changes of some of the genes appeared as soon as one month after budesonide exposure, while others do not appear until six months ØInhaled budesonide has a differential effect in current versus smokers. Such differences such as effects on Phase I & II enzymes gene expression may account for differences in response between current and former smokers Acknowledgements Supported by NIH contract N 01 -CN 85188, & NCI SPORE CA 70907 & NIH 1 P 01 CA 096964 & Genome Canada/Genome BC