Transcript Poster - University of British Columbia

Effect of Inhaled Budesonide in Smokers with Bronchial Dysplasia
BC Cancer Research Centre
675 West 10th Avenue
Raj
1,2
Chari ,
Kim
1
Lonergan ,
Luc
4
Girard ,
4
Minna ,
4
Gazdar ,
3
Yao ,
3
You ,
2
Ng ,
1,2
Lam ,
John
Adi
Ruisheng
Ming
Raymond
Wan
1,2
1,2
Calum MacAulay , Stephen Lam
[1]British Columbia Cancer Agency, Vancouver, BC, Canada, [2]University of British Columbia, Vancouver, BC, Canada,
[3]Washington University in St. Louis, St. Louis, MO, USA, [4]UT Southwestern Medical Centre4, 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 U133A 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, CYP1B1 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 N01-CN85188, & NCI SPORE CA 70907 & NIH 1P01 CA096964
Effect of Active Smoking
Effect of Budesonide
Comparison of Affymetrix U133A and SAGE
Description of Dataset
Specimens
Higher in Current Smokers
34
SAGE
11
9
A
cDN
Affymetrix
U133A
data was obtained from the
B.
Nla III
digestion
paper
by
Spira
et
al. [Proc Natl Acad Sci U S A.
C.
Linker
ligation
2004;101(27):10143-8]
and
“Tagging”
SAGE
libraries were generated from human
enzyme
digestion
bronchial epithelial
cells obtained by bronchial
Ligation
D.
brushings
during
Ditag bronchoscopy of heavy
Amplification
smokers
GTAC
AAAAA
TTTTT
AAAAA
TTTTT
GTAC
AAAAA
TTTTT
GTAC
A
AAT
AAT
TT T A
AA
TT A
AA
TTT
TG
CA
C
GTA
T
AA
TT
AA
TT
TGC
CAT
G A
TG
CATAC
G
C
GTATG
A
CAT C
G
GTA
C
A
TTAAAA
AA TT
TTTAAAT
TT
AA
CATG
GTAC
AAA
TTTTT
CATG
GTAC
CATG
GTAC
CATG
GTAC
CATG
GTAC
CATG
GTAC
CATG
GTAC
Nla III digestion
SAGE Library Construction
AA
AAA TT
TTT
Serial
Analysis of Gene Expression (SAGE)
E.
CATG
GTAC
G
CAT
Ligation
TGC
CAT
G A
G
CAT
C
GTA
SAGEF.deduces expression
profile based on
Cloning, sequencing and
bioinformatics analysis
the relative abundance
of sequence tags.
Platform
Affymetrix U133A array was used to
profile the samples obtained from the 10
subjects in the six month study
Affymetrix U133A 2.0 array was used for
the 7 subjects in the one month study
C
GTA
RNA sample A
Gene tags
Gene tags
SAGE is not limited to genes spotted on the array
SAGE produces quantitative, discrete (digital) data
values
~150,000 tags enumerated per library
Objectives
Filtering of data
To determine the effect of active tobacco smoking on gene
expression profiles in human bronchial epithelial cells retrieved by
bronchial brushing during bronchoscopy
To compare the gene expression profiles obtained by Affymetrix
U133A array with Serial Analysis of Gene Expression (SAGE)
To characterize the effects of inhaled budesonide on the gene
expression profiles of bronchial cells from current versus former
smokers with bronchial dysplasia
To identify genes associated with regression of bronchial dysplasia
after treatment with inhaled budesonide
Hypotheses
SAGE identifies differentially expressed genes not captured by
previous microarray analysis
Current and former smokers respond differently to inhaled
budesonide
Regression of bronchial dysplasia after budesonide is
associated with different gene expression profiles versus nonresponders
Evaluation Criteria
RNA sample B
Copies
Chemopreventive agents for lung cancer may exhibit a differential
effect in current versus former or never smokers
Previous microarray studies showed different gene expression profiles
between current and former smokers
Budesonide 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]
Expression profile
Affymetrix data filtered as explained by Spira et al.
[Proc Natl Acad Sci U S A. 2004;101(27):10143-8]
Data for SAGE tags was retained only if raw tag
counts were greater than 1 in at least one library
Statistical Methods
A combination of the student’s t-test, fold-change
and permutation test were used in the analysis
Differentially expressed genes were selected at a
significance level of p ≤ 0.001
T-test and fold-change are commonly used in the
literature
Permutation test
suitable test for a small sample size
non-parametric test
performs well in identifying differentially
expressed genes
Identify genes that show similar
expression differences before and after
treatment in 4 of 5 subjects
Fold-change of ≥ 1.5 (log2 fold of
0.585) between baseline and six months
Remove genes if similar expression
pattern seen in the placebo arm
Findings validated in the dataset of the
one-month study
30
25
20
Affymetrix U133A
15
SAGE
10
5
20
15
Affymetrix U133A
SAGE
10
5
0
0
All 3 Criteria (↑ Former)
All 3 Criteria (↑Current)
Comparison 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
Using the 3 criteria
In current smokers, SAGE data shows 30 tags (mapping to 27
unique genes) and Affymetrix U133A gives 7 probes (mapping 5
unique genes) that are higher compared to former smokers
In former smokers, SAGE data suggests 22 tags (mapping to
18 unique genes) and Affymetrix U133A has none that are higher
relative current smokers
Examples of Differentially Expressed Genes - Phase
1& 2 Toxin Exposure Genes
1800
Current Smoker
1600
(x104)
Former Smoker
1400
1200
1000
800
600
400
200
CYP1B1
Response characterized as complete
response (CR), partial response (PR) and
progressive disease (PD)
CR 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
Identify genes that have a ≥ 1.5 fold
change in complete responders only
Must also exhibit a gradient of
expression between CR, PR and PD.
Smoking Status and Response to
Inhaled Budesonide
 Identify genes which have ≥ 1.5 fold
change exclusively in all former smokers
but not in all current smokers and viceversa
 Remove genes if similar expression
pattern seen in placebo arm
Example of genes with increased (left) and decreased expression (right) in
complete responders. Among the up-regulated genes are: caveolin 2 (CAV2),
hydroxyprostaglandin dehyrogenase 15-(NAD) (HPGD), TU3A protein (TU3A)
and the down-regulated genes are: toll-like receptor 3 (TLR3), TIR domain
containing adaptor inducing interferon-beta (TRIF), mitogen-activated protein
kinase kinase 6 (MAP2K6).
HPGD has been recently shown to mediate anti-inflammatory effects in lung
adenocarcinoma cell lines [Arch Biochem Biophys. 2005;435(1):50-5]
TRIF and TLR3 potentially implicate down-regulation of the Toll-like receptor
pathway
Subset of Genes Differentially Expressed Between Current and
Former Smokers
2000
0
Correlation of Gene Expression
with Clinical Response
Genes Correlating with Clinical Response
25
Number of probes or tags
18
G.
email: [email protected]
Higher in Former Smokers
35
Tags Per Million
Affymetrix U133A
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
Number of probes or tags
Platform
Current
Smokers
G
CATAC
GT
Background
ph:604-675-8000 ext.7703
Results
Former
Smokers
A.
Vancouver BC, V5Z 1L3
CYP1A1
GST2
GPX2
Increased in Current Smokers,
Decreased in Former Smokers
Increased in Former Smokers,
Decreased in Current Smokers
MYO6 - Myosin VI
TM7SF4 - transmembrane 7 superfamily
member 4
MAP4K5 - mitogen-activated protein
kinase kinase kinase kinase 5
CORO1A - coronin, actin binding protein,
1A
TPD52 - tumor protein D52
NMT2 - N-myristoyltransferase 2
CYP1B1 - cytochrome P450, family 1,
subfamily B, polypeptide 1
PLA2 - phospholipase A2, group IVB
(cytosolic)
CYP/(GST+GPX)
Phase 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
1CR, 1PR
Current (n = 5)
3
1CR, 1PR, 1PD
Conclusions
Effect of Budesonide Independent of Response
and Smoking Status
(Below) Sample of genes up-regulated and down-regulated after
treatment in at least 4 of 5 subjects
Also exhibit same pattern in at least 5 of 7 subjects in validation
dataset (U133A 2.0)
Down-Regulated
Up-Regulated
TRBC1 - T cell receptor beta constant 1
TOP2A - topoisomerase (DNA) II alpha 170kDa
CCL5 - chemokine (C-C motif) ligand 5
HPGD - hydroxyprostaglandin dehydrogenase
15-(NAD)
Analysis of SAGE data affords more discovery of significant genes as compared
to the Affymetrix U133A platform
Budesonide exhibits anti-inflammatory effects as evidenced by increased HPGD,
furthermore this gene is increased even higher in complete responders.
Expression changes of some of the genes appeared as soon as one month after
budesonide exposure, while others do not appear until six months
Inhaled 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 N01-CN85188, & NCI SPORE CA 70907 & NIH 1P01 CA096964
& Genome Canada/Genome BC