GENES, CHROMOSOMES & CANCER 49:688–698 (2010)

ADAM28: A Potential Oncogene Involved in

Asbestos-Related Lung Adenocarcinomas
Casey M. Wright,1,2* Jill E. Larsen,1,2 Nicholas K. Hayward,3 Maria U. Martins,2 Maxine E. Tan,2
Morgan R. Davidson,1,2 Santiyagu M. Savarimuthu,1,2 Rebecca E. McLachlan,2
Linda H. Passmore,2 Morgan N. Windsor,4 Belinda E. Clarke,5 Edwina E. Duhig,5
Ian A. Yang,1,2 Rayleen V. Bowman,1,2 and Kwun M. Fong1,2
1
School of Medicine,The University of Queensland,QLD, Australia
2
Department of Thoracic Medicine,The Prince Charles Hospital,QLD, Australia
3
Oncogenomics Laboratory,Queensland Institute of Medical Research,QLD, Australia
4
Department of Thoracic Surgery,The Prince Charles Hospital,QLD, Australia
5
Department of Anatomical Pathology,The Prince Charles Hospital,QLD, Australia

Asbestos-related lung cancer accounts for 4–12% of all lung cancers worldwide. Since putative mechanisms of carcinogen-
esis differ between asbestos and tobacco induced lung cancers, tumors induced by the two agents may be genetically dis-
tinct. To identify gene expression biomarkers associated with asbestos-related lung tumorigenicity we performed gene
expression array analysis on tumors of 36 patients with primary lung adenocarcinoma, comparing 12 patients with lung
asbestos body counts above levels associated with urban dwelling (ARLC-AC: asbestos-related lung cancer-adenocarci-
noma) with 24 patients with no asbestos bodies (NARLC-AC: non-asbestos related lung cancer-adenocarcinoma). Genes
differentially expressed between ARLC-AC and NARLC-AC were identified on fold change and P value, and then priori-
tized using gene ontology. Candidates included ZNRF3, ADAM28, PPP1CA, IRF6, RAB3D, and PRDX1. Expression of these
six genes was technically and biologically replicated by qRT-PCR in the training set and biologically validated in three inde-
pendent test sets. ADAM28, encoding a disintegrin and metalloproteinase domain protein that interacts with integrins,
was consistently upregulated in ARLC across all four datasets. Further studies are being designed to investigate the possi-
ble role of this gene in asbestos lung tumorigenicity, its potential utility as a marker of asbestos related lung cancer for
purposes of causal attribution, and its potential as a treatment target for lung cancers arising in asbestos exposed
persons. VC 2010 Wiley-Liss, Inc.

INTRODUCTION Many asbestos-related lung cancers may result

Lung cancer is a leading cause of death globally
with some suggesting that 25% of lung cancers are
not attributable to tobacco exposure (Sun et al.,
2007). The strength of this association and the prev-
alence of tobacco exposure have made quantifying
the contribution of occupational and environmental
factors, including asbestos exposure and air pollu-
tion, difficult. Presently, the contribution of asbes-
tos to lung cancer risk is unknown although global
estimates predict that 4–12% of lung cancer cases
may be due to asbestos (Lee, et al., 1998; Hender-
son et al., 2004a). Attribution of asbestos involve-
ment in people exposed to both tobacco and
asbestos is difficult because of uncertainties sur-
rounding biological interactions between the two
carcinogens, lack of unique histopathological differ-
ences and lack of other biomarkers capable of dis-
tinguishing between asbestos-related lung cancer
(ARLC) and nonasbestos related lung cancer
(NARLC).
from the combined effect of asbestos and carcino-
gens in tobacco smoke, with the possibility of a
synergistic relationship first proposed in an early
study of United States insulation workers (Doll,
1955). Since then many have argued whether this
relationship is best described by an additive or
Additional Supporting Information may be found in the online
version of this article.
Supported by: NHMRC and Queensland Smart State project
grants, NHMRC Practitioner Fellowship (KF), NHMRC Senior
Principal Research Fellowship (NH), NHMRC Career
Development Award (IY), NHMRC Biomedical Scholarship (SS,
CW), Cancer Council Queensland Senior Research Fellowship
(KF), Queensland Clinical Research Fellowship (KF, IY), The
Prince Charles Hospital Foundation, Dust Diseases Board (New
South Wales, Australia).
*Correspondence to: Casey M. Wright, Room 2, Level 1 Clini-
cal Sciences Building, The Prince Charles Hospital, Rode Road,
Chermside, QLD 4032, Australia. E-mail: c.wright@uq.edu.au
Received 26 November 2009; Accepted 22 March 2010
DOI 10.1002/gcc.20779
Published online 29 April 2010 in
Wiley InterScience (www.interscience.wiley.com).

V
C 2010 Wiley-Liss, Inc.
 ADAM28, A POSSIBLE ONCOGENE IN ARLC-AC
multiplicative model (Vainio and Boffetta 1994;
Liddell 2001; Lee, 2001). More recent reviews
tend to support a cumulative exposure model for
lung cancer as a consequence of past asbestos ex-
posure (Churg, 1993; Egilman and Reinert, 1996;
Billings and Howard, 2000; Hodgson and Darn-
ton, 2000).
Asbestos fibers have been shown to accumulate
near mitotic spindles, and it has been postulated
that asbestos interferes directly with chromo-
somes (Hesterberg and Barrett, 1985; Dopp et al.,
1995). In vitro studies using DNA repair deficient
Cell lines have reported time and dose depend-
ent increases in DNA double-strand breaks after
asbestos exposure (Okayasu et al., 1999), and
others have suggested that asbestos can physically
shear DNA (Libbus et al., 1989). Fiber size,
shape, and diameter appear to be important varia-
bles in asbestos-cell interactions, with long
straight needle-like fibers showing a high interac-
tion rate associated with altered cellular morphol-
ogy (Cardinali et al., 2005). Several pathways
have been implicated in asbestos-induced lung
injury with cellular functions, including oxidative
stress response, inflammation, DNA damage
response, mitochondrial activity, and apoptosis
thought to be triggered by asbestos fibers
(Nymark et al., 2008). Free radicals that trigger
oxidative stress responses may originate from
direct chemical interactions with the fiber surface
or from macrophages activated during inflamma-
tory processes (Dusinska et al., 2004). It has been
postulated that generation of excessive amounts
of reactive oxygen species (ROS) and reactive
nitrogen species (RNS) result in DNA damage
and chromosomal aberrations (Dusinska et al.,
2004; Topinka et al., 2004).
Microarray technology has shown that gene
expression profiles differ between closely related
tumor types (Dyrskjot et al., 2003; Komuro et al.,
2005). Furthermore, gene expression based classi-
fiers predicting recurrence, prognosis, survival,
and treatment response have been developed for
a many types of cancer (Schramm et al., 2005;
Larsen et al., 2006; Raponi et al., 2006). The aim
of this study was to investigate whether tumor
gene expression profiles differ between individu-
als with evidence of prior asbestos exposure
(measured by pulmonary asbestos lung fiber
counts) and those without. We sought to identify
biomarkers of asbestos carcinogenicity in lung
adenocarcinomas that could be technically and
biologically validated test and in independent
datasets.
689
MATERIALS AND METHODS
Sample Collection and Subject Selection
The study population described previously
(Larsen et al., 2007) consisted of 36 cases of pri-
mary lung adenocarcinoma. Lung asbestos fiber
count was measured using the bleach digestion
technique described by (Roggli, 1992) and
(Wright, et al., 2008). Given that urban dwellers
without occupational asbestos exposure have
fewer than 20 asbestos bodies/gram wet weight
(AB/gww) lung tissue (Roggli et al., 1982), we
classified subjects with asbestos fiber burdens >
¼ 20 AB/gww as ‘‘moderately exposed’’ (n ¼ 12)
and subjects with 0 AB/gww as ‘‘nonexposed’’
(n ¼ 24). Subjects were clinically characterized
for tumor phenotype (age, gender, tobacco expo-
sure, stage, lobe location, and differentiation)
(Table 1). Subjects gave informed written con-
sent for tissue resection and this study was
approved by The Prince Charles Hospital Human
Research Ethics Committee.
RNA Extraction and Gene Expression Analysis
Total RNA was extracted from 20–30 mg of
fresh frozen lung tumor tissue using the TRI-
ZOLTM reagent (Invitrogen, California), treated
with DNaseI (Ambion, Texas) to remove
genomic contamination and reverse transcribed to
cDNA using a two-step reaction. RNA quality
was assessed with an Agilent 2100 Bioanalyzer
(Agilent Technologies, California) and quantified
by Nanodrop (Thermo Scientific, Delaware).
RNA quality for the independent test set was
assessed by gel electrophoresis and examination
of A260/A280 (<1.8 excluded) and A260/A230 (<0.7
excluded) ratios.
Microarray experiments were performed
according to MIAME guidelines as described pre-
viously (Larsen et al., 2007). Total RNA from
each tumor sample was hybridized with Human
Universal Reference RNA (Stratagene, California)
to 22K Human Oligo microarrays printed by the
British Columbia Gene Array Facility using the
Operon Human Genome Oligo set v2.0 contain-
ing 21,329 70-mer probes, representing
14,200
known transcripts. Arrays were scanned using an
Affymetrix GMS418 confocal scanner (Affyme-
trix, California) and probe quality assessed using
Imagene V5.1 (BioDiscovery, California). All data
were subject to filtering and Lowess normalization
procedures using AVADIS bioinformatics software
(Version 4.3, Strand Genomics, Bangalore, India).
Genes, Chromosomes & Cancer DOI 10.1002/gcc
690 WRIGHT ET AL.

TABLE 1. Demographics for the TPCH Training and Independent Test Set

Training set Test set

ARLC NARLC ARLC NARLC
(>20 AB/gww) (0 AB/gww) P-value (>20 AB/gww) (0 AB/gww) P-value
N 12 24 28 30
Age (Years, Mean  SD) 69.8 (6.9) 64.4 (8.7) 7.0E-2 67.8 (9.0) 67.7 (8.5) 9.57E-1
Sex
Male 9 19 7.77E-1 19 20 3.89E-1
Female 3 5 9 10
Smoking history
Ever 11 24 20 26
Never 1 0 7 3
Unknown – - 1 1
Pack years (Mean  SD) 35.8 (35) 55.3 (23.05) 5.2E-2 38.7 (31.8) 47.4 (31.0) 2.97E-1
TNM tumour stage
(IA and IB) 5 20 13 11
(IIA and IIB) 3 4 4 5
(IIIA and IIIB) 4 0 2 5
(IV) 3 1
Unclassifiablea 6.0E-3 6 8 6.64E-1
Self-reported asbestos exposure
Yes 12 3 7 2
No 0 15 10 21
Unknown 0 6 7 2
Missing – – 4 5
Asbestos fibre count (Mean  SD) 107 (89) 0 (0) <1.0E-4 84.4 (95) 0 (0) <1.0E-4
ARLC, Asbestos-related lung cancer; NARLC, Non-asbestos related lung cancer; AB/gww, asbestos bodies/gram wet weight; SD, standard devia-
tion; AB, asbestos bodies.
a
unclassifiable, nodes or presence of metastases not able to be assessed.

Of the 21,329 probes on the chip 20,597 passed fil-
tering criteria and were used in expression analy-
sis. All expression data has been deposited in the
National Center for Biotechnology Information
(NCBI) Gene Expression Omnibus (GEO Acces-
sion GSE20875).
Bioinformatics and Statistical Analysis
Candidate genes were identified by meeting
criteria of (a) statistical significance (P < 0.001)
and (b) magnitude of expression change (fold
change >1.5). Ontologies were obtained by Gene
Ontology and Pathway Analysis (P < 0.05). Selec-
tion of genes at P < 0.001 (by student t test)
allowed control of the false discovery rate, limit-
ing the likelihood of selecting false positives.
Hierarchical clustering analysis was performed on
differentially expressed genes at the P < 0.001
level using the Pearson absolute distance measure
with complete linkage to identify whether dis-
tinct gene expression profiles existed between
ARLC-AC and NARLC-AC. We also used three
independent datasets to test the validity of candi-
date genes in asbestos-related tumors. Statistical
analyses for microarray experiments were per-
formed using BRB ArrayTools Version 3.5; devel-
oped by Dr Richard Simon and Amy Peng Lam,
http://linus.nci.hih.gov/
brb/tool.htm. Group com-
parisons, correlations, and associations were per-
formed using v2 tests and two-tailed t tests where
appropriate using SPSS statistical software (Ver-
sion 13, SPSS Inc Chicago, Illinois). A P value less
than 0.05 (two-tailed) was considered statistically
significant.
Gene Ontology and Pathway Analysis
Over-represented gene ontologies were deter-
mined using the Expression Analysis Systematic
Explorer (EASE) Online Interface: Chromosome,
GO Biological Process, GO Molecular function,
GO Cellular component, and Kyoto Encyclopedia
of Genes and Genomes (KEGG) pathway (Ver-
sion 1.21, http://david.niaid.nih.gov/david/ease.htm).
Genes were selected from differential gene ex-
pression analysis at the P < 0.05 level (Fisher
exact test) and compared with all probes showing
large variations on the microarray. Relaxation of
P value stringency allowed the identification of
significant pathways using a larger number of
genes to allow visualization of all biologically

Genes, Chromosomes & Cancer DOI 10.1002/gcc

 ADAM28, A POSSIBLE ONCOGENE IN ARLC-AC
relevant ontologies. Additional functional and
pathway analyses were investigated using Ingenu-
ity Pathway Analysis software V7 (Ingenuity Sys-
tems, California).
Technical Replication
mRNA expression levels from the microarray
analysis were validated using qRT-PCR in the
training set. RNA was reverse transcribed to
cDNA using a two-step reaction. First strand syn-
thesis was primed by combination of random hex-
amers (100 ng/ll) and Oligo (dT)15 primers (200
ng/ll) (Promega, NSW, Australia) in a reaction
with 10 mM dNTPs. All qRT-PCR reactions
were performed using SYBR green chemistry
(Applied Biosystems, Warrington, United King-
dom). Forward and reverse primers for candidate
genes were designed using Primer Express V1.5
(Applied Biosystems) and sequences are shown in
the Supporting Information Table 3. Intron-span-
ning amplicons were as close as possible to the
microarray probe position. Three housekeeping
genes were amplified, with the geometric mean
of the housekeepers’ expression across individual
samples used for relative quantification. All sam-
ples were run in triplicate on a RotorGene-6000
(Corbett Research, Sydney, Australia). Genes
were deemed technically replicated if the direc-
tion of expression was consistent of at least 1.2-
fold magnitude.
Biological Validation
Biological validation of technically replicated
candidate genes was performed in three inde-
pendent test sets: (1) qRT-PCR mRNA quantifi-
cation on an independent test set of 58 primary
adenocarcinomas from our tumor bank with the
same as the training set; (2) 28 lung tumor sam-
ples studied by microarray (data obtained from
the authors) (Wikman et al., 2007), for which
prior asbestos exposure was assessed by a combi-
nation of pulmonary asbestos fiber burden and
occupational history; and (3) cell line data
described by Nymark et al., (2007) (GEO6013:
GDS2604, http://www.ncbi.nlm.nih.gov/sites/entrez?
db¼geo). These data (GEO6013:GDS2604) were
derived from a time course experiment measuring
gene expression changes in three commercial cell
lines (A549–human lung adenocarcinoma cell
line, BEAS-2B–nontumorigenic SV40 immortil-
ised human bronchial epithelial cell line and
Met5A-SV40 immortilised pleural mesothelial cell
691
line) purchased from the American Type Culture
Centre (ATCC, Rockville) and exposed to crodi-
colite asbestos (obtained from the International
Union Against Cancer, Johannesburg, South
Africa) for 1, 6, 24, 48, and 168 hr (Nymark et al.,
2007). Three cell lines RNA was extracted for
each time point and expression measured using
Affymetrix gene expression arrays.
RESULTS
Subject Characteristics
Mean and frequency distributions for age, gen-
der, and smoking history for the training and test
sets are shown in Table 1. There were no statisti-
cally significant differences in age and gender
between ARLC-AC and NARLC-AC and smok-
ing pack years was of borderline significance (P ¼
0.052) (Table 1). In the independent test set
there were no significant differences in age, gen-
der or smoking history (Table 1).
Microarray Analysis
A supervised analysis of 36 lung cancers based
on an univariate parametric t test, identified 270
probes differentially expressed between ARLC-
AC (n ¼ 12) and NARLC-AC (n ¼ 24) with 200
expected by chance (P < 0.01) and 89 probes at
P < 0.001 (20 expected by chance; most were
over-expressed in ARLC-AC tumors). The differ-
ence in mean expression between the two groups
was less than 2-fold for the majority of these
probes. Supervised hierarchical clustering using
Pearson’s absolute distance measure with com-
plete linkage, showed that the 89 probes signifi-
cant at the P < 0.001 level were able to clearly
distinguish between ARLC-AC and NARLC-AC
(Fig. 1). To rank the strongest candidate genes,
we selected genes with a differential mean
expression of 1.5-fold, reducing our list to 20
genes (Table 2). Most of these candidates
included genes of unknown function. In this anal-
ysis we focused on six genes with known func-
tion, a disintegrin and metalloproteinase domain
28 (ADAM28), protein phosphatase 1 catalytic
subunit alpha isoform (PPP1CA), member RAS
oncogene family (RAB3D), IFN regulatory factor
6 (IRF6), zinc ring finger 3 (ZNRF3), and peroxir-
edoxin 1 (PRDX1), and technically replicated
their expression levels by RT-PCR in the train-
ing set.
Genes, Chromosomes & Cancer DOI 10.1002/gcc
692 WRIGHT ET AL.
Figure 1. Heat map depicting the genomic profiles of asbestos-
related (blue) and non-asbestos related (yellow) lung adenocarcino-
mas. These genes were selected on the basis of statistical significance
(P < 0.001) and magnitude of gene expression change (>1.5-fold).
Genes are represented on the Y axis and tumor samples represented
on the X axis. Green areas represent genes that are under-expressed
in the tumor sample while red areas represent genes over expressed
in the tumor sample. The color bar represents the level of gene
expression across samples.
Technical Replication
The results of qRT-PCR for these six genes is
shown in Figure 2. If the qRT-PCR results
showed that the direction of expression change
between ARLC and NARLC was the same as
that from the microarray analysis, the gene was
considered technically validated. Four of the six
candidates were thus replicated in the training
set; PRDX1 (Microarray (MA) fold change
(ARLC/NARLC) 1.73; qRT-PCR fold change
(ARLC/NARLC) 1.39), ADAM28 (MA 2.26; qRT-
PCR 1.65), PPP1CA (MA 1.53; qRT-PCR 1.73)
and ZNRF3 (MA 1.99; qRT-PCR 1.13). All genes
were upregulated in ARLC with consistent mag-
nitude of fold change.
Biological Validation in Independent Test Sets
To biologically validate differential expression
of the four candidate genes, we performed qRT-
PCR analysis in an independent test set of lung
adenocarcinomas selected from our lung tumor
bank. Inclusion criteria for the test set (ARLC-
AC n ¼ 28, NARLC-AC n ¼ 30) was as for the
training set (Table 1). Genes were judged to be
biologically validated if the direction of expres-
sion (ratio of mean expression between ARLC
and NARLC) was the same as in the original
training set. In this cohort, upregulation of
PRDX1 (qRT-PCR training set (ARLC-AC/
NARLC-AC) FC ¼ 3.18) and ADAM28 (qRT-
PCR FC ¼ 1.25) (Supporting Information Table
1) were biologically validated.
Genes, Chromosomes & Cancer DOI 10.1002/gcc
We further tested two other independent data-
sets from phenotypically relevant studies (Wik-
man, et al., 2007) and Nymark et al., (2007)
(GDS2604). The Wikman data (kindly supplied
by the author) were from 28 lung cancer cases, of
whom 14 had been heavily exposed to asbestos
(as determined by occupational questionnaire and
pulmonary asbestos fiber burden) and 14 had no
evidence of prior asbestos exposure. The age and
gender matched tumors consisted of 11 adenocar-
cinomas (AC), 8 squamous cell carcinomas (SCC),
5 large cell carcinomas, 2 small cell lung cancers,
and 2 adenosquamous carcinomas. RNA was iso-
lated from tumor tissue and hybridized to Affy-
metrix HG-U133A gene expression microarrays.
Data confirmed up-regulation of ADAM28 (FC
1.38), RAB3D (FC 1.25), and IRF6 (FC 1.43), but
not PRDX1 (FC 1.00) or PPP1CA (FC 0.99) (Sup-
porting Information Table 1).
Analysis of the Nymark dataset (GDS2604)
demonstrated consistent upregulation of ADAM28
gene expression (Fig. 2). Furthermore, PRDX1
also showed a trend for increased expression in
A549 (7 days FC 1.19) and Met5A cell lines [2
days (a) FC 1.30; 2 days (b) FC 1.07) but not
BEAS-2B (2 days FC 1.07]. Conversely, no con-
sistent up-regulation for ZNRF3, PPP1CA,
RAB3D or IRF6 was apparent. (Supporting Infor-
mation Table 1 and 2). Figure 2 highlights the
gene expression changes observed across all data-
sets for the candidate genes.
Gene Ontology and Pathway Analysis of Genes
Dysregulated in ARLC
To identify biological processes over-expressed
in ARLC versus NARLC tumors, EASE V2
(http://david.niaid.nih.gov/david/ease.htm) was
used. This program compared genes identified at
P < 0.05 to the filtered gene list. Biological proc-
esses over-represented in ARLC included apop-
topic programming, release of cytoplasmic
sequestered NF-kappaB, T-cell differentiation
and activation, cellular defense response, immune
response, NF-kappaB-nucleus import, and regula-
tion of NF-kappaB nucleus import. Molecular
functions, including oxidoreductase activity, ser-
ine/threonine receptor kinase activity, and protein
kinase C activity, were also found to be over-rep-
resented. A full list of over-represented biologi-
cal, molecular functions, and cellular processes is
presented in Supporting Information Table 4.
Ingenuity Pathway Analysis (Ingenuity) was
also used to identify over-represented gene
 ADAM28, A POSSIBLE ONCOGENE IN ARLC-AC 693
TABLE 2. Microarray Identified Differentially Expressed Genes Between ARLC and NARLC Training Set

Gene Fold
Probe ID Genbank symbol Gene description change Chromosome Map P-value
H200000836 NM_014045 LRP10 DKFZP564C1940 protein 1.806 14 14q11.2 8.36E-04
H200002032 NM_006147 IRF6 Interferon regulatory factor 6 1.671 1 1q32.3-q41 7.35E-04
H200006838 AL133117 THOC-003 Homo sapiens mRNA; cDNA 1.929 X Xq25-q26.3 8.81E-05
DKFZp586L1121 (from
clone DKFZp586L1121)
H200007710 AL050050 DKFZp566D133 DKFZp566D133 protein 1.669 9 14q13.2 2.87E-05
H200007800 NM_004160 PYY Peptide YY 1.512 17 17q21.1 2.24E-04
H200008192 NM_014265 ADAM28 A disintegrin and metallopro- 1.943 8 8p21.2 7.43E-04
teinase domain 28
H200008312 U92980 DT1P1A10 Hypothetical protein 1.559 X Xp11.22 1.35E-04
DT1P1A10
H200008482 NM_002574 PRDX1 Peroxiredoxin 1 1.593 1 1p34.1 1.04E-04
H200011070 NM_006419 SCYB13 Small inducible cytokine B sub- 1.569 4 4q21 3.66E-04
family (Cys-X-Cys motif),
member 13 (B-cell
chemoattractant)
H200013546 NM_022842 FLJ22969 Hypothetical protein FLJ22969 1.684 3 3p21.31 1.13E-05
H200014023 NM_018354 C20orf46 Chromosome 20 open reading 1.707 20 20p13 8.94E-05
frame 46
H200014241 NM_002708 PPP1CA Protein phosphatase 1, cata- 1.705 11 11q13 2.01E-05
lytic subunit, alpha isoform
H200016011 U80113 IGHV4 Human immunoglobulin heavy 1.796 14 14q32.33 7.43E-04
chain variable region (V4–
31) gene, partial cds
H200016279 NM_004283 RAB3D RAB3D, member RAS onco- 1.787 19 19p13.2 6.10E-05
gene family
H200017809 Z00008 IGKV1D-8 Immunoglobulin kappa variable 1.583 2 2 8.36E-04
1D-8
H200018915 AB051436 KIAA1133 Novel C3HC4 type Zinc fin- 1.811 22 22q12.1 8.52E-05
ger (ring finger)
H200019430 AK022017 0 Homo sapiens cDNA 1.602 3 3q13.2 1.75E-05
FLJ11955 fis, clone
HEMBB1000890
H200019567 AK024482 ZBT20 Homo sapiens mRNA for 1.579 7 7p22.2 1.74E-04
FLJ00076 protein, partial
cds
H200019644 NM_052875 MGC10485 Hypothetical protein 1.614 11 11q25 1.05E-03
MGC10485
H200020055 BC011360 LRP16 Homo sapiens, clone 1.771 11 11q11 2.32E-05
IMAGE:4177360, mRNA
H200020854 AK055590 Pseudogene Homo sapiens cDNA 1.829 0 1q32 4.13E-04
FLJ31028 fis, clone
HLUNG2000570, weakly
similar to 40S RIBOSOMAL
PROTEIN S10
The top 20 genes were selected on the basis of high magnitude (>1.5-fold) and statistical significance (P < 0.001). From this list, six high priority
candidates were selected for technical replication and biological validation.

ontologies and networks. The top five networks
significantly over-represented (P < 0.05),
included (1) inflammatory disease, cellular move-
ment, nervous system development and function,
(2) antigen presentation, cell-mediated immune
response, humoral immune response, (3) cellular
growth and proliferation, post-translational modi-
fication, lipid metabolism, (4) DNA replication,
recombination and repair, nucleic acid metabo-
lism, small molecule biochemistry, and (5) lipid
metabolism, small molecule biochemistry, and
molecular transport. Significantly over-repre-
sented molecular and cellular functions, included
cellular growth and proliferation, cell morphology,
energy production, molecular transport, and
nucleic acid metabolism. Figure 3 illustrates all
functions and pathways over-represented in
ARLC-AC.

Genes, Chromosomes & Cancer DOI 10.1002/gcc

694 WRIGHT ET AL.

Figure 2. Bar graph depicting gene expression changes for ZNRF3,
PPP1CA, PRDX1, RAB3D, ADAM28 and IRF6 in TPCH Microarray data,
TPCH qRT-PCR (Training and Testing sets), Nymark Microarray Data
and Wikman cell line gene expression data (A549, BEAS-2B and
Met5A). Fold changes are the mean of ARLC-AC/mean NARLC-AC
group. For the Wikman cell line data, ratios were calculated by divid-
ing the asbestos-treated/control for each cell line and time point.
TPCH denotes The Prince Charles Hospital. ADAM28 demonstrated
consistent up-regulation of gene expression across all datasets.

Figure 3. Ingenuity Pathway Analysis of differentially expressed genes identified for ARLC-AC at P <
0.05. Bar graph highlights significantly over-represented functions in asbestos-related lung adenocarcinoma.
[Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

DISCUSSION role in asbestos induced carcinogenicity (PRDX1,

We studied 36 lung cancers stratified by lung PPP1CA, ADAM28, ZNRF3, RAB3D, and IRF6).
asbestos fiber count with expression arrays and Technical replication and biological validation fil-
identified six candidate genes with a plausible tered down to one gene, ADAM28, which was

Genes, Chromosomes & Cancer DOI 10.1002/gcc

 ADAM28, A POSSIBLE ONCOGENE IN ARLC-AC
consistently upregulated in several different
ARLC cohorts. It was also dysregulated experi-
mentally in cell lines treated with asbestos.
ADAM28 (a disintegrin and metalloproteinase
domain protein) encodes two isoforms (mem-
brane-associated ADAM28m and secreted
ADAM28s) and is often upregulated in lung,
breast, and kidney cancers (Mitsui, et al., 2006;
Ohtsuka, et al., 2006; Roemer, et al., 2004). Ele-
vated expression of ADAM28 occurs in lung
tumors compared to non-cancerous tissue and in
primary tumors with lymph node metastases ver-
sus primary tumors without metastases (Ohtsuka
et al., 2006). ADAM28 may also be involved in
activation of EGFR, MAPK1, MAPK3, and release
of CD44, with a possible role in the in the prolif-
eration of lung cancer cell line NCI-H292 (Hart
S et al., 2004). In addition, ADAM28 has been
shown to interact with integrins and is involved
in the digestion of IGFBP3 through release of
IGF1, a potent inducer of cell proliferation
(Bridges et al., 2003; Mochizuki et al., 2004).
This digestion has been shown to be inhibited by
TIMP3 and TIMP4 (tissue inhibitors of metallo-
proteinases). While the short term exposure of
cell lines to asbestos experiments represent acute
challenges, which may not necessarily mimic the
effects of chronic exposures with long latency,
similar gene expression changes nevertheless sup-
port a role for ADAM28.
Protein expression studies conducted by Oht-
suka et al., (2006) and Mitsui et al., (2006) showed
strong expression of ADAM28 at the mRNA and
protein level in NSCLC and breast cancer, respec-
tively. Ohtsuka et al., demonstrated localization of
ADAM28 to squamous cell carcinoma and adeno-
carcinoma cells with immunoblotting, confirming
the presence of a 42 kDa protein band in tumor tis-
sue, absent in matching control noncarcinoma lung
(Ohtsuka et al., 2006). qRT-PCR analysis con-
firmed increased expression of ADAM28 (>9-fold
increase) in carcinoma versus control noncarci-
noma tissue, which correlated with tumor size
(>30 mm) and lymph node metastases. A similar
study performed by Mitsui et al., (2006) in breast
cancer showed localization of ADAM28 to the cyto-
plasm of carcinoma cells with immunoblotting
identifying a similar sized product (42kDa) present
in breast carcinoma but not control non-neoplastic
breast tissue. Approximately 65% of cases had
strong expression at the mRNA and protein level.
These two studies provide evidence that expres-
sion changes observed at the mRNA level corre-
spond to changes in protein.
695
The remaining candidate genes, (PRDX1,
IRF6, ZNRF3, PPP1CA, and RAB3)D were all up-
regulated in tumors from asbestos-exposed sub-
jects; although the data were not as strong as that
of ADAM28, these are not fully excluded as
potentially significant ARLC-AC genes. PRDX1
appears to be regulated by oxidative stress (Ishii
et al., 1993), and cigarette smoke, with high lev-
els expressed in most tissues (Graves et al.,
2009). Regulation by cigarette smoke is depend-
ent on expression of the transcription factor Nrf2
with homozygous knockdown of Nrf2 in mouse
liver, shown to decrease expression (Powis and
Montfort, 2001; Rangasamy, et al., 2004). Several
studies have proposed PRDX1 as a potential bio-
marker for lung cancer. Chang et al., (2001)
observed elevated levels of PRDX1 protein
expression in A549 cells compared with BEAS-
2B, while Kim et al., showed that the risk of
death increased with increased PRDX1 expression
in stage 1 NSCLC (P ¼ 0.036) (Kim et al., 2007;
Kim et al., 2008). Elevated PRDX1 expression
has also been observed in mesothelioma, another
asbestos-related cancer (Kinnula et al., 2002) con-
sistent with our hypothesis that PRDX1 levels are
increased by release of reactive oxygen species in
response to asbestos fiber. PPP1CA is a member
of the protein phosphatase family, and is essential
for cell division. Concordant increases in gene
copy number and expression have been observed
in oral squamous cell carcinomas (Hsu et al.,
2006). PPP1CA interacts with cancer related
genes, including TP53 (Ruiz et al., 2008) and
BRCA1 (Winter et al., 2007). In contrast, little is
known about the function of ZNRF3. Although
well studied in Van der Woude syndrome, little
is known about the role of IRF6 in cancer (Kondo
et al., 2002; Wang et al., 2003; de Medeiros et al.,
2008). In normal breast tissue, increased expres-
sion of IRF6 has been shown to induce mRNA
expression of the tumor suppressor gene Maspin
(Bailey and Hendrix, 2008). Studies suggest that
IRF6 is a key regulator of the cell cycle, promot-
ing progression to the G(0) state, and in cancer
allowing uncontrolled cell proliferation (Bailey
and Hendrix, 2008). RAB3D is a member of the
ras oncogene family with a role in vesicular traffic
control in eukaryotic cells. (Sehgal, et al., 1997)
identified this as a putative target in glioblastoma
multiforme, and found increased expression in
several different tumor types, including lung,
with potential oncogenic function.
Previous studies identified several regions of
copy number alteration in asbestos related
Genes, Chromosomes & Cancer DOI 10.1002/gcc
696 WRIGHT ET AL.
tumors, notably 2p16, 9q33.1, and 19p (Nymark
et al., 2006, 2009; Kettunen et al., 2009). In the
current study only one of our candidates was
found in a region previously associated with copy
number loss (RAB3D, 19p13.2) suggesting that
the altered expression of candidates identified in
this study are not necessarily driven by copy
number alterations. A recent review by Nymark
et al., (2008), provides an excellent overview of
the many molecular and genetic changes in
asbestos-related lung cancer. In a comparison of
matched normal and lung tumor tissue, Ruosaari
et al., (2008) have identified several functional
pathways significantly different between asbestos-
exposed and nonexposed lung cancer patients
including ion transport, NF-jB signaling, DNA
repair, and spliceosome and nucleosome com-
plexes. Furthermore, many pathways downregu-
lated in asbestos-exposed subjects were related to
protein ubiquination, involved in multiple cell
processes, including cell cycling, apoptosis, and
DNA repair. Consistent with previous investiga-
tions, we also found over-representation of NF-
jB related gene ontologies.
A limitation of this study is the potential for
Type I errors. Primary selection by P value
allows control of the false discovery rate (Benja-
mini and Hochberg, 1995). By combining this
with secondary selection of those genes whose
expression differed between the groups by great-
est magnitude, our candidate identification pro-
cess incorporated essential elements of statistical
and biological significance (Chen et al., 2007). To
focus down to a manageable number of candi-
dates, we prioritized on biological functions that
seemed most relevant to differences in tumor
biology between ARLC-AC and NARLC-AC.
Nonetheless, some genes excluded on ontological
groups could nevertheless be relevant.
Another potential limitation is the relative size
of the training set (N ¼ 36). Increasing the sam-
ple size of this study would lend greater power to
our analysis and perhaps avoid overfitting to the
particular tumors within the sample; however,
microarray technology is limited by cost and tech-
nical requirements. A larger sample size may
have permitted further lowering of the false dis-
covery rate such that the differentially expressed
gene list contained fewer falsely declared genes.
To our knowledge only one public dataset of
lung cancers with annotated asbestos exposure or
lung fiber burden information is available (Wik-
man et al., 2007,) and this is limited by its rela-
tively small sample numbers, mixed histology
Genes, Chromosomes & Cancer DOI 10.1002/gcc
types, and methods of prior asbestos exposure
assessment. While we investigated differences in
the gene expression profiles of moderately
exposed (> ¼ 20 AB/gww) and nonexposed pri-
mary lung ACs, the Wikman group compared
subjects with high occupational exposures and
nonexposed subjects with lung cancer patients of
mixed histologies. This and most other asbestos
exposed cohorts who develop lung cancer have
also smoked tobacco. Thus comparative studies
are effectively comparing tumor expression pro-
files of asbestos and tobacco exposed subjects
with tobacco only exposed subjects. Whether
ADAM28 upregulation would also occur in
tobacco treated cells is not known. It is possible
that combined exposure to both asbestos and
tobacco increases the expression of ADAM28
more so, than exposure to either carcinogen
alone. Further functional experiments are
required to demonstrate whether tobacco, asbes-
tos or tobacco plus asbestos induce the greatest
change in ADAM28 expression. Finally, to our
knowledge there are no publicly available data-
sets investigating ADAM28 expression in lung ep-
ithelium or preneoplasia from ARLC-AC and
NARLC-AC making it difficult to investigate
whether alteration of ADAM28 expression is an
early event in ARLC-AC. Further studies may be
required to determine whether ADAM28 expres-
sion is altered in early preneoplastic lesions.
In conclusion, we have identified ADAM28 as a
potential asbestos-related cancer biomarker with
consistent upregulation of ADAM28 across in-
house and independent datasets. ADAM28 has
previously been implicated as an oncogene in
NSCLC (Ohtsuka, et al., 2006) with a role in cell
proliferation and metastases (Okada, 2007). Gene
expression changes observed for ADAM28 could
be due to asbestos exposure independent of
tobacco smoke exposure but additional experi-
ments are needed to confirm this. Further func-
tional studies, including transfection experiments
and immunohistochemistry (IHC) are required to
confirm that protein expression parallels mRNA
expression, although the lack of a commercial
antibody verified for use in formalin-fixed paraf-
fin-embedded tissue and relatively modest fold
change differences pose a challenge for accurate
quantification of protein staining by IHC.
ACKNOWLEDGMENTS
The authors acknowledge Harriet Wikman
(from The Finnish Institute of Occupational
 ADAM28, A POSSIBLE ONCOGENE IN ARLC-AC
Health) who provided the raw data files for the
independent microarray dataset of 28 lung tumors
with/without prior asbestos exposure. The authors
thank MW and the other thoracic surgeons at
TPCH for assisting with sample collection, LP
and the research nurses at TPCH for collecting
patient consents and finally the patients for pro-
viding lung samples, without whom we could not
conduct this study.
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