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GENES, CHROMOSOMES & CANCER 49:270281 (2010)

Polymorphisms in the Transforming Growth Factor


Beta 1 Pathway in Relation to Colorectal Cancer
Progression
Asta Forsti,1,2* Xuchen Li,1 Kerstin Wagner,1 Bjorn Tavelin,3 Kerstin Enquist,4 Richard Palmqvist,5 Andrea Altieri,1
Goran Hallmans,4 Kari Hemminki,1,2 and Per Lenner3
1
Department of Molecular Genetic Epidemiology,German Cancer Research Center,Heidelberg,Germany
2
Center for Primary Health Care Research,Clinical Research Center,Lund University,Malm, Sweden
3
Department of Oncology,Norrlands University Hospital,Ume, Sweden
4
Department of Public Health and Clinical Medicine/Nutritional Research,Ume University,Ume, Sweden
5
Department of Medical Biosciences,Ume University,Ume, Sweden

Transforming growth factor b1 (TGFB1) acts as a growth inhibitor of normal colonic epithelial cells, however, as a tumor
promoter of colorectal cancer (CRC) cells. To explore the association between genetic polymorphisms in the TGFB1 path-
way and CRC susceptibility and clinical outcome, we carried out a casecontrol study on a Swedish population of 308
CRC cases and 585 age- and gender-matched controls. The cases were sampled prospectively and had up to 16 years fol-
low-up, making the study material particularly suitable for survival analysis. On the basis of their reported or predicted
functional effect, nine single-nucleotide polymorphisms (TGFB1: Leu10Pro; TGFBR1: 9A/6A and IVS7G24A; FURIN: C-
229T; THBS1: T42C; LTBP1L: C-256G; LTBP4: T-893G and Thr750Ala; BAMBI: T-779A) were selected for genotyping. We
evaluated the associations between genotypes and CRC and Dukes stage. Survival probabilities were compared between
different subgroups. The observed statistically signicant associations included a decreased CRC risk for TGFBR1
IVS7G24A minor allele carriers (odds ratio (OR): 0.72, 95% condence interval (CI): 0.530.97), less aggressive tumors
with Dukes stage AB for carriers of LTBP4 Thr750Ala and BAMBI T-779A minor alleles (OR: 0.58, 95%CI: 0.360.93 and
OR: 0.51, 95%CI: 0.290.89, respectively) and worse survival for FURIN C-229T heterozygotes (hazard ratio: 1.63, 95%CI:
1.082.46). As this is the rst study about the inuence of the polymorphisms in the TGFB1 pathway on CRC progression,
further studies in large independent cohorts are warranted. VC 2009 Wiley-Liss, Inc.

INTRODUCTION sague et al., 2000; Broderick et al., 2007; Xu and


The transforming growth factor beta (TGFB) Pasche, 2007).
pathway is an important modulator of several bio- TGFB1 is secreted in a biologically inactive
logical processes, including cell proliferation, dif- form and as part of a large latent complex (LLC)
ferentiation, migration, and apoptosis (Massague that is targeted to the extracellular matrix (ECM)
et al., 2000). The signaling pathway of TGFB1, (Annes et al., 2003). The TGFB1 LLC comprises
the most abundant form of the TGFB isoforms the mature TGFB1, the latency-associated pro-
(Xu and Pasche, 2007), plays an important role in tein (LAP), and the latent transforming growth
carcinogenesis, having both tumor-suppressing factor beta binding protein (LTBP) (Annes et al.,
and -promoting activities (Akhurst and Derynck, 2003). LTBPs are required for correct folding,
2001). TGFB1 is a potent inhibitor of normal co- efcient secretion, and targeting of TGFB1 to
lonic epithelial cells acting as a tumor suppressor.
However, TGFB1 promotes survival, invasion, Supported by: EU, Grant number: LSHC-CT-2004-503465;
and metastasis of colorectal cancer (CRC) cells, Wallenberg Consortium North, Sweden.
thereby acting as an oncogene (Akhurst and Der- *Correspondence to: Asta Forsti, Department of Molecular
Genetic Epidemiology, German Cancer Research Center (DKFZ),
ynck, 2001). In CRC, several mutations affecting Im Neuenheimer Feld 580, Heidelberg 69120, Germany.
the TGFB1 signaling pathway have been identi- E-mail: a.foersti@dkfz.de
ed, including somatic mutations in the TGFBR2, Received 24 July 2009; Accepted 3 November 2009
DOI 10.1002/gcc.20738
SMAD2, and SMAD4 genes and low-penetrance Published online 8 December 2009 in
alleles in the SMAD7 and TGFBR1 genes (Mas- Wiley InterScience (www.interscience.wiley.com).

V
C 2009 Wiley-Liss, Inc.
TGFB1 PATHWAY AND COLORECTAL CANCER PROGRESSION 271
TABLE 1. Characteristics of the CRC Cases inhibits TGFB1 signaling by forming a hetero-
at the Time of Diagnosisa dimer with TGFBR2.
Variable Single-nucleotide polymorphisms (SNPs),
which are located within the promoter or the cod-
Mean age  SD (range) 61.9  7.2 (3577)
Gender (n, %)
ing sequence, may regulate the expression of the
Female 174 (56.5) genes in the TGFB1 signaling pathway or affect
Male 134 (43.5) the function of the coded proteins respectively.
Total 308 (100) Thus, genotyping of these SNPs could provide a
Dukes stage (n, %) simple method to predict the risk and the pro-
A 60 (19.5)
B 99 (32.1)
gression of cancer. On the basis of the reported
C 80 (26.0) associations with cancer, putative functional
D 57 (18.5) effects of the SNPs and linkage disequilibrium
Missing 12 (3.9) (LD) between the SNPs, we selected nine poly-
Tumor location (n, %) morphisms (Leu10Pro in TGFB1; 9A/6A polyala-
Colon 178 (57.8)
Rectum 106 (34.4)
nine polymorphism and IVS7G24A in TGFBR1;
Not specied 24 (7.8) T42C in THBS1; C-229T in FURIN; C-256G in
LTBP1L; T-893G and Thr750Ala in LTBP4; T-
SD, standard deviation; n, number of patients.
a
The controls were matched with the corresponding case by gender,
779A in BAMBI) in the TGFB1 pathway for gen-
age at baseline (6 months), and time of sampling (2 months). otyping in a Swedish incident CRC series. The
They had to be alive at the time of diagnosis of the corresponding relationships between these polymorphisms and
case and without any previous cancer diagnosis, except carcinoma
in situ of cervix uteri.
CRC susceptibility, progression, and prognosis
were evaluated.

the ECM. The human genome has four LTBP MATERIALS AND METHODS
genes (LTBP1-4), among which LTBP1, LTBP3,
and LTBP4 are able to bind mature TGFB1- Blood Samples
LAP complex. These three proteins are also The study was performed on genomic DNA
expressed in human colon and rectum (Saharinen from 308 Swedish CRC cases together with 585
et al., 1999). In addition, disruption of the gene controls, matched with age and gender. The cases
encoding LTBP4 has been shown to cause CRC and controls were drawn from the population-
in mice (Sterner-Kock et al., 2002). Proteolytic based Vasterbotten intervention project and the
processing by FURIN convertase on the Arg-His- mammary screening project, which contain blood
Arg-Arg site of the pro-TGFB1 (Dubois et al., samples collected between October 1987 and
1995), and interaction between the amino acid April 2002 from an ethnically homogenous popu-
sequence Lys-Arg-Phe-Lys of thrombospondin-1 lation living in a geographically dened region in
(THBS1) and LAP (Murphy-Ullrich and Pocza- North Sweden (Kaaks et al., 2002; Pukkala et al.,
tek, 2000) are essential steps in the formation of 2007). Prospective cases were identied from the
the biologically active TGFB1. cohorts by record linkage to the regional cancer
In the TGFB1 signaling, TGFB1 binds to the registry. The controls were selected from the
cell surface receptor transforming growth factor same cohort as the corresponding case. They
beta receptor 2 (TGFBR2), which results in their were matched by gender, age at baseline (6
binding to and phosphorylation of transforming months) and the time of sampling (2 months).
growth factor beta receptor 1 (TGFBR1). Subse- The controls had to be alive at the time of diag-
quently, SMADs are phosphorylated by activated nosis of the corresponding case and without any
TGFBR1 and translocated into the nucleus, previous cancer diagnosis, except of carcinoma in
where they regulate transcription of target genes situ of cervix uteri. The female versus male distri-
(Massague et al., 2000; Akhurst and Derynck, bution among the cases was 56.5% versus 43.5%
2001; Xu and Pasche, 2007). TGFB1 signaling and among the controls 56.1% versus 43.9%. The
can be inhibited by the pseudo receptor BAMBI mean age at sampling of the cases was 56.8 years
(Onichtchouk et al., 1999). BAMBI is a trans- (range: 30.173.4) and of the controls 56.8 years
membrane protein that lacks an intracellular do- (range: 30.073.8). The blood samples were
main but has sequence similarity to the stored at 80 C until the time of sample selection
extracellular domain of TGFBR1 and thereby it and DNA isolation for genotyping analyses. The

Genes, Chromosomes & Cancer DOI 10.1002/gcc


272 FORSTI ET AL.

samples were randomly divided on the 96-well from a BMP-responsive element, Bre7 (Sekiya
plates. The laboratory personnel were kept et al., 2004b) and which was in partial LD with the
blinded to the casecontrol status throughout the A-191C SNP (D0 1.0; r2 0.42). The LTBP1S
study. Clinical data for the CRC cases were and LTBP3 genes were not considered for
retrieved from the registry managed by the further analyses as no SNPs with putative func-
Northern Sweden Collaborative Group for Colo- tional effect in the screened promoter regions were
rectal Diseases (Table 1). Information about the observed.
date of death was collected from the Swedish
population register, with a follow-up until Sep-
tember 2006. The data of cause of death were
not available. The study was approved by the PCR and Sequencing
ethical committee of Karolinska Institute Syd and The polymerase chain reaction (PCR) was
Umea University. used to amplify the selected DNA regions.
Amplication was performed with 5 ng of
genomic DNA in a 10 ll reaction volume using
SNP Selection 1 PCR buffer, 1.5 mM of MgCl2, 0.11 lM of
The selected genes were screened for SNPs of dNTP-Mixture (Invitrogen, Paisley, United King-
interest based on the following criteria: (1) SNPs dom), 0.15 lM of each primer (MWG Biotech
known to be associated with cancer in the litera- AG, Ebersberg, Germany), and 0.3 U Platinum-
ture, (2) SNPs with putative effects on gene tran- Taq DNA polymerase (Invitrogen). The PCR
scription using the TESS and TFSEARCH was programmed as initial denaturation at 94 C
prediction tools (http://www.cbil.upenn.edu/tess/ for 1 min, followed by three cycles of 94 C for 45
and http://www.cbrc.jp/research/db/TFSEARCH. sec, an optimized annealing temperature T1 for
html, respectively) or on protein function because 45 sec and extension at 72 C for 45 sec. Addi-
of change of coding sequence using PolyPhen tional 32 cycles were performed consisting of
prediction tool (http://coot.embl.de/PolyPhen/), denaturation at 94 C for 30 sec, T11 C for 30
and (3) LD between the SNPs based on both our sec and extension at 72 C for 30 sec, followed by
screening results and the data of the International a nal extension at 72 C for 5 min. PCR primer
HapMap Project (http://www.hapmap.org/index. sequences and the optimized annealing tempera-
html.en) (Haploview; Barrett et al., 2005). Nine tures are available upon request. The PCR prod-
polymorphisms (Leu10Pro in TGFB1; 9A/6A poly- ucts were visualized on 2% TAE agarose gels
alanine polymorphism and IVS7G24A in containing 0.01% ethidium bromide and puried
TGFBR1; T42C in THBS1; C-229T in FURIN; by ExoSAP-ITV (USB Amersham, Uppsala, Swe-
R

C-256G in LTBP1L; T-893G and Thr750Ala in den) for 30 min at 37 C followed by 15 min at
LTBP4; T-779A in BAMBI) were selected for geno- 85 C.
typing (Table 2). The SNPs from the TGFB1, The sequencing reaction was carried out using
TGFBR1, FURIN, and LTBP1L genes were the BigDyeV Terminator v. 3.1 Cycle Sequencing
R

selected based on their reported functionality or Kit (Applied Biosystems, Foster City, USA) in a
association with cancer (Ayoubi et al., 1994; Pasche 10 ll volume (1.75 ll 5 sequencing buffer, 1 ll
et al., 1999; Dunning et al., 2003; Densem et al., primer [10 lM], 0.5 ll Big Dye, and 0.52 ll
2004; Hannah et al., 2004; Sparks et al., 2004; Bian PCR product depending on intensity of the DNA
et al., 2005; Zhang, 2005; Chen et al., 2006b; Higa- band on the agarose gel). The sequencing reac-
shi et al., 2006; Berndt et al., 2007; Daley et al., tion was performed using forward and reverse
2007; Skoglund et al., 2007; Xu and Pasche, 2007; primer separately according to the following pro-
Saltzman et al., 2008). The SNPs from the THBS1, gram: 96 C for 1 min, 27 cycles of 96 C for 16
LTBP4, and BAMBI genes were selected for geno- sec, 54 C for 5 sec, and 60 C for 4 min.
typing based on their putative effects on gene tran- The PCR and sequencing reactions were per-
scription, protein function, and/or their high LD formed in GeneAmp PCR System 9700 thermo-
with other putatively functional SNPs. In the cyclers (Applied Biosystems) and MJ Research
BAMBI gene, we observed a novel SNP, A-191C, PTC-225 gradient cyclers (Biorad, Hercules,
which is the rst nucleotide 50 to a SMAD-binding USA). The sequencing reaction products were
element (Sekiya et al., 2004b). However, no Taq- analyzed on an ABI PRISM 3100 genetic ana-
man assay could be designed for this SNP and we lyzer (Applied Biosystem), and the primary
genotyped T-779A, which is located 77 bp away sequence data were aligned and analyzed using

Genes, Chromosomes & Cancer DOI 10.1002/gcc


TABLE 2. Veried SNPs in the Screened Regions of the TGFB1 Pathway Genes, Their Reported Association with Cancer, Putative Effect on Gene Expression or on Protein Function and Linkage
Disequilibrium (LD) Between the SNPs Screened in our Study

Gene Location SNP reference ID Variationa MAF in our screen MAF in Caucasians Association with cancer Putative effect LD (our study)
TGFB1 Exon1 rs1800470 Leu10Pro [T/C] 0.34 0.270.41 Associated with Located in the signal NA
(Dunning et al., 2003; colorectal adenoma, peptide sequence, affects
Sparks et al., 2004; adenomatous and TGFB1 secretion and
Berndt et al., 2007) hyperplastic polyps, serum level (Dunning
adenocarcinomas et al., 2003; Berndt
(Sparks et al., 2004; et al., 2007)
Berndt et al., 2007;
Saltzman et al., 2008)
TGFBR1 Exon1 rs11466445 [9Ala/6Ala] 0.11 0.05-0.12 Associated with Located in the signal NA
(Skoglund et al., 2007) different cancers, peptide sequence,
especially with colorectal effect through its
cancer (Pasche et al., secondary signaling
1999; Xu and effects (Xu and Pasche,
Pasche, 2007) 2007); affects cell
migration and invasion
(Rosman et al., 2008)
Intron7 rs334354 IVS7G124A 0.21 0.22b Associated with Effect through its LD NA
invasive breast cancer, with other SNPs;
lung cancer, kidney captures 32 SNPs
and bladder carcinomas out of 35 common
(Zhang 2005; Chen et al., SNPs in the gene regionb
2006b)
THBS1 Promoter rs2169830 [T-1224C] 0.35 Not reported Not reported No predicted effect High LD between the
SNPs in the promoter
and the 50 UTR
(D0 1.0; r 1.0)
Promoter rs2664139 [T-697C] 0.34 Not reported Not reported No predicted effect
50 UTR rs1478605 [G5A] 0.33 0.22b Not reported No predicted effect
50 UTR rs1478604 [T142C] 0.29 0.22b Not reported Located within a putative
TF binding site for E2F-1c
Exon13 rs2228262 Asn700Ser [A/G] 0.05 0.08b Not reported via inuce on Ca2 binding Not in LD with the
afnity, affects proper protein SNPs in promoter
function (Hannah et al., 2004) and 50 UTR
(D0 1.0; r  0.14)
FURIN Promoter rs4932178 [C-229T] 0.35 0.41b Not reported Located within a putative TF NA
binding site for SP1 (Ayoubi
et al., 1994; Densem
et al., 2004)c
LTBP1L Promoter Novel [T-1879C] 0.14 Not reported Not reported No predicted effect Not in LD with the
SNPs in promoter
(Continued)

(Continued)
TABLE 2. Veried SNPs in the Screened Regions of the TGFB1 Pathway Genes, Their Reported Association with Cancer, Putative Effect on Gene Expression or on Protein Function and Linkage
Disequilibrium (LD) Between the SNPs Screened in our Study (Continued)
Gene Location SNP reference ID Variationa MAF in our screen MAF in Caucasians Association with cancer Putative effect LD (our study)
Promoter Novel [C-256G] 0.27 Not reported Associated with Located in a minimal High LD with C-34A
clinical outcome of promoter (Koski (D0 1.0; r2 1.0)
ovarian cancer in et al., 1999)
the Japanese
population
(Higashi et al., 2006)
Promoter Novel [C-228T] 0.23 Not reported Not reported Located in a minimal Not in LD with the
promoter (Koski et al., 1999) SNPs in promoter
Promoter Novel [C-85G] 0.15 Not reported Not reported Located in a minimal Not in LD with the
promoter (Koski et al., 1999) SNPs in promoter
Promoter Novel [C-34A] 0.30 Not reported Associated with Located in a minimal High LD with C-256G
clinical outcome of promoter (Koski et al., 1999) (D0 1.0; r2 1.0)
ovarian cancer in the
Japanese population
(Higashi et al., 2006)
Promoter Novel [C-30T] 0.18 Not reported Not reported Located in a minimal promoter Not in LD with
(Koski et al., 1999) the SNPs in promoter
LTBP1S Promoter Novel [C-994G] 0.19 Not reported Not reported No predicted effect Not in LD with the
SNPs in promoter
Promoter Novel [A-935G] 0.04 Not reported Not reported No predicted effect Not in LD with the
SNPs in promoter
Promoter rs12712338 [G-922A] 0.42 Not reported Not reported No predicted effect Not in LD with the
SNPs in promoter
Promoter rs12620839 [C-220T] 0.12 0.07b Not reported No predicted effect Not in LD with the
SNPs in promoter
Promoter Novel [G-112A] 0.22 Not reported Not reported No predicted effect Not in LD with the
SNPs in promoter
LTBP3 Promoter No SNPs
LTBP4 Promoter rs11673027 [T-893G] 0.15 Not reported Not reported Located within a putative Not in LD with the
TF binding site for C/EBPc,d other promoter SNP
or the nsSNPs
Promoter Novel [C-201A] 0.13 Not reported Not reported No predicted effect Not in LD with the
other promoter SNP
or the nsSNPs
Exon5 rs2303729 Val157Ile [G/A] 0.40 0.45b Not reported AA exchange, unknown effecte High LD with the
other nsSNPs
(D0 > 0.90; r > 0.59 )
Exon17 rs1131620 Thr750Ala [A/G] 0.43 0.42b Not reported AA exchange, benign effecte High LD with the other
nsSNPs (D0 > 0.91;
r > 0.77)
(Continued)

(Continued)
TABLE 2. Veried SNPs in the Screened Regions of the TGFB1 Pathway Genes, Their Reported Association with Cancer, Putative Effect on Gene Expression or on Protein Function and Linkage
Disequilibrium (LD) Between the SNPs Screened in our Study (Continued)
Gene Location SNP reference ID Variationa MAF in our screen MAF in Caucasians Association with cancer Putative effect LD (our study)
Exon18 rs1051303 Thr783Ala [A/G] 0.41 0.42b Not reported AA exchange, benign effecte High LD with the other
nsSNPs (D0 > 0.91;
r > 0.77)
Exon26 rs11668767 Arg1104Cys [C/T] 0.35 0.31b Not reported AA exchange, unknown High LD with the other
effecte nsSNPs (D0 > 0.90;
r > 0.59)
BAMBI Promoter rs610820 [T-779A] 0.49 0.53b Not reported 77bp away from Bre7 LD with the SNPs in
binding site promoter and intron
(Sekiya et al., 2004b) 1 (D0 1; r > 0.42)
Promoter Novel [A-191C] 0.34 Not reported Not reported 1bp away from SBE LD with the SNPs in
binding site (Sekiya promoter and
et al., 2004b) intron 1
Intron1 Rs675558 [IVS1G1404A] 0.41 0.38b Not reported No predicted effect; several LD with the SNPs
b-catenin response in promoter
elements
located in intron 1
(Sekiya et al., 2004a)
NA: not analyzed; TF: transcription factor; AA: amino acid; MAF: minor allele frequency.
SNPs selected for further analyses are shown in bold.
a
For nonsynonymous SNPs: amino acid change; for promoter SNPs: position relative to transcription start site; for intronic SNPs: location within the intron.
b
HapMap: http://www.hapmap.org/index.html.en.
c
TESS: http://www.cbil.upenn.edu/tess/.
d
TFSEARCH: http://www.cbrc.jp/research/db/TFSEARCH.html.
e
PolyPhen: http://coot.embl.de/PolyPhen.
276 FORSTI ET AL.

TABLE 3. Association of the Genotypes in the TGFB1 Pathway Genes with CRC Susceptibility

Gene Genotype Controls (%) Cases (%) OR (95%CI)a


TGFB1 rs1800470 (Leu10Pro)
TT 251 (44.9) 139 (47.3) 1.00 (reference)
TC 238 (42.6) 125 (42.5) 0.95 (0.701.28)
CC 70 (12.5) 30 (10.2) 0.77 (0.481.25)
TGFBR1 rs11466445 (9A/6A)
9A/9A 435 (78.0) 218 (74.4) 1.00 (reference)
9A/6A 115 (20.6) 69 (23.5) 1.20 (0.851.68)
6A/6A 8 (1.4) 6 (2.0) 1.50 (0.514.37)
TGFBR1 rs334354 (IVS7G24A)
GG 382 (65.7) 220 (72.8) 1.00 (reference)
GA 179 (30.8) 68 (22.5) 0.66 (0.480.91)
AA 20 (3.4) 14 (4.6) 1.22 (0.602.46)
GAAA 199 (34.3) 82 (27.2) 0.72 (0.530.97)
FURIN rs4932178 (C-229T)
CC 220 (38.8) 104 (34.8) 1.00 (reference)
CT 264 (46.0) 155 (51.8) 1.24 (0.911.69)
TT 90 (15.7) 40 (13.4) 0.94 (0.601.46)
THBS1 rs1478604 (T42C)
TT 316 (54.3) 160 (52.1) 1.00 (reference)
TC 222 (38.1) 124 (40.4) 1.10 (0.831.48)
CC 44 (7.6) 23 (7.5) 1.03 (0.601.77)
LTBP1L C-256G
CC 389 (68.0) 209 (69.4) 1.00 (reference)
CG 165 (28.8) 80 (26.6) 0.90 (0.661.24)
GG 18 (3.1) 12 (4.0) 1.24 (0.592.63)
LTBP4 rs11673027 (T-893G)
TT 466 (80.2) 258 (84.6) 1.00 (reference)
TG 107 (18.4) 44 (14.4) 0.74 (0.511.09)
GG 8 (1.4) 3 (1.0) 0.68 (0.182.58)
LTBP4 rs1131620 (Thr750Ala)
AA 196 (34.1) 107 (35.0) 1.00 (reference)
AG 285 (49.7) 138 (45.1) 0.89 (0.651.21)
GG 93 (16.2) 61 (19.9) 1.20 (0.801.79)
BAMBI rs610820 (T-779A)
TT 155 (27.0) 67 (22.0) 1.00 (reference)
TA 292 (50.8) 162 (53.3) 1.28 (0.911.81)
AA 128 (22.3) 75 (24.7) 1.36 (0.912.03)
a
The odds ratios (ORs) were considered statistically signicant when the 95% condence intervals (CIs) did not overlap 1.00, indicated in bold.

DNASTAR Lasergene 5.0 software (DNASTAR, 50 C for 2 min, 95 C for 10 min, and 3545
Madison, WI). cycles with 92 C for 15 sec and 60 C for 1 min in
the same PCR machines as in sequencing. The
samples were read and analyzed on an ABI
GENOTYPING PRISM 7900HT sequence detection system
(Applied Biosystems) using the SDS 1.2 software
Taqman (Applied Biosystems).
Allelic discrimination-based Taqman assays
(Applied Biosystems) were used for genotyping
the SNPs in TGFB1, TGFBR1, THBS1, LTBP4, Restriction Fragment Length Polymorphism
and BAMBI (assay IDs: C_1413390_20, As it was not possible to design TaqMan assays
C_3100547_20, C_2936796_10, C_8714829_10, for the FURIN C-229T and the LTBP1L C-
and C_1453131_10, respectively). The reaction 256G SNPs, these two SNPs were genotyped
was performed in 5 ll using 450 nM of each using restriction fragment length polymorphism
primer, 100 nM of each probe, and 2.5 ll Taq- (RFLP) method. The SNP in FURIN is located
Man Universal 2 PCR Master Mix (Applied within a HinfI restriction site. The 228 bp long
Biosystems) per reaction. PCR was performed at PCR product of FURIN was incubated with 4 U

Genes, Chromosomes & Cancer DOI 10.1002/gcc


TGFB1 PATHWAY AND COLORECTAL CANCER PROGRESSION 277
of HinfI (MBI Fermentas, St Leon-Rot, Ger- 1998). The KaplanMeier method was used to
many) for 3 hr at 37 C. Digestion with HinfI gen- estimate the survival probabilities, and the log-
erated 228 bp fragment for the CC genotype, rank test was used to test differences between
228, 150, and 78 bp fragments for the CT geno- subgroups. We used death by any cause as an
type and 150 and 78 bp fragments for the TT end point. Multivariate Cox proportional hazard
genotype. With the same experimental conditions model adjusted for age, gender, and Dukes stage
as for HinfI, MvaI (MBI Fermentas) was used to was used to estimate hazard ratios (HRs) for over-
digest the PCR product of LTBP1L. Digestion all survival. Statistical analyses were performed
with MvaI generated 431, 100, and 78 bp frag- using SPSS (Version 14).
ments for the CC genotype, 431, 269, 162, 100,
and 78 bp fragments for the CG genotype, and
RESULTS
269, 162, 100, and 78 bp fragments for the GG
genotype. Association with CRC Susceptibility
The genotype distribution of all of the geno-
Fluorescent Fragment Analysis typed polymorphisms in the controls followed
HWE (P > 0.05, data not shown). No gender-
Fluorescent fragment analysis was employed to
specic differences in the genotype distribution
analyze the polyalanine polymorphism in the
were observed (data not shown). As shown in Ta-
TGFBR1 gene, which involves a deletion of three
ble 3, none of the SNPs conferred an increased
alanines (6A) from a 9-alanine (9A) stretch. The
risk of CRC. However, a signicant negative
forward primer was 50 labeled with a uorescent
association was observed between the A allele
6-FAM dye. The PCR was performed as
carrier status of the TGFBR1 IVS7G24A SNP
described earlier. The PCR product was diluted
and CRC risk (OR: 0.72, 95% CI: 0.530.97).
to 1:10 and 1 ll of the dilution was added to 10
The results did not differ by tumor location, co-
ll HIDI-Formamide (Applied Biosystems) mixed
lon versus rectum (data not shown).
with 0.3 ll ROX350 size standard (Applied Bio-
systems). The reaction was denatured for 5 min
at 95 C with additional 5 min on ice and loaded Association with Dukes Stage
onto the ABI PRISM 3100 Genetic analyzer As shown in Table 4, the genotype distribution
(Applied Biosystems). The analyses were per- of the SNPs LTBP4 Thr750Ala and BAMBI
formed with the GeneScan software version 3.7 T-779A differed between the patients with
and the GeneMapper software version 3.0 Dukes stage AB and CD tumors. The
(Applied Biosystems). Genotyping of about 5% of patients with Dukes stage CD tumors were
the samples were randomly repeated with con- less likely to be carriers of the minor G allele of
cordant results. the LTBP4 Thr750Ala SNP than the patients
with Dukes stage AB tumors (OR: 0.58, 95%
CI: 0.360.93). Similarly, the TA and AA geno-
Statistical Analyses
types of the BAMBI T-779A SNP were less com-
The observed genotype frequencies in the con- mon among the patients with Dukes stage CD
trols were tested for HardyWeinberg equilib- tumors than in the patients with Dukes stage
rium (HWE), and the difference between the AB tumors (OR: 0.51, 95% CI: 0.290.89).
observed and expected frequencies was tested for
signicance using the v2 test. Statistical signi-
cance for the differences in the genotype fre- Association with Survival
quencies between the CRC cases and the The carriers of the rare TGFBR1 6A/6A geno-
controls was determined using the v2 test. Associ- type seemed to have worse survival compared to
ation between genotypes and CRC and Dukes the carriers of the other genotypes (Fig. 1A),
stage were estimated by odds ratios (ORs) and although the differences did not reach statistical
95% condence intervals (CIs), using conditional signicance (HR: 1.64, 95% CI: 0.604.51). Strati-
logistic regression. The power of the study (a cation of the cases by the FURIN genotype
0.05) was calculated using the power and sample- showed a worse survival for the CT genotype car-
size calculation software PS version 2.1.31 (http:// riers than for the carriers of the CC genotypes
biostat.mc.vanderbilt.edu/twiki/bin/view/Main/ (Fig. 1B, HR: 1.63, CI: 1.082.46), however, not
PowerSampleSize) (Dupont and Plummer, for the homozygous TT genotype carriers (HR:

Genes, Chromosomes & Cancer DOI 10.1002/gcc


278 FORSTI ET AL.

TABLE 4. Association of the Genotypes in the TGFb1 Pathway Genes with Dukes Stage at the Time of Diagnosis

Gene Genotype A B (%) C D (%) OR (95%CI)


TGFB1 rs1800470 (Leu10Pro)
TT 73 (46.2) 64 (47.1) 1.00 (reference)
TC 69 (43.7) 58 (42.6) 0.96 (0.591.56)
CC 16 (10.1) 14 (10.3) 1.00 (0.452.20)
TGFBR1 rs11466445 (9A/6A)
9A/9A 117 (74.1) 98 (72.6) 1.00 (reference)
9A/6A 38 (24.1) 33 (24.4) 1.04 (0.611.78)
6A/6A 3 (1.9) 4 (3.0) 1.59 (0.357.29)
TGFBR1 rs334354 (IVS7G24A)
GG 120 (73.6) 100 (71.9) 1.00 (reference)
GA 38 (23.3) 31 (22.3) 0.98 (0.571.69)
AA 5 (3.1) 8 (5.8) 1.92 (0.616.05)
FURIN rs4932178 (C-229T)
CC 55 (34.8) 49 (34.8) 1.00 (reference)
CT 75 (47.5) 80 (56.7) 1.20 (0.732.00)
TT 28 (17.7) 12 (8.5) 0.48 (0.221.05)
THBS1 rs1478604 (T42C)
TT 93 (56.0) 65 (46.1) 1.00 (reference)
TC 64 (38.6) 63 (44.7) 1.41 (0.882.26)
CC 9 (5.4) 13 (9.2) 2.07 (0.835.12)
LTBP1L C-256G
CC 113 (69.8) 96 (68.6) 1.00 (reference)
CG 44 (27.2) 37 (26.4) 0.99 (0.591.66)
GG 5 (3.1) 7 (5.0) 1.65 (0.515.36)
LTBP4 rs11673027 (T-893G)
TT 138 (84.1) 120 (85.1) 1.00 (reference)
TG 25 (15.2) 19 (13.5) 0.87 (0.461.67)
GG 1 (0.6) 2 (1.4) 2.30 (0.2125.68)
LTBP4 rs1131620 (Thr750Ala)
AA 48 (29.3) 59 (41.5) 1.00 (reference)
AG 78 (47.6) 60 (42.3) 0.63 (0.381.04)
GG 38 (23.2) 23 (16.2) 0.49 (0.260.94)
AGGG 116 (70.8) 83 (58.5) 0.58 (0.360.93)
BAMBI rs610820 (T-779A)
TT 26 (16.8) 39 (28.5) 1.00 (reference)
TA 94 (60.6) 62 (45.3) 0.44 (0.240.79)
AA 35 (22.6) 36 (26.3) 0.69 (0.351.35)
TAAA 129 (83.2) 98 (71.6) 0.51 (0.290.89)
a
The odds ratios (ORs) were considered statistically signicant when the 95% condence intervals (CIs) did not overlap 1.00, indicated in bold.

1.39, CI: 0.712.71). The carriers of the BAMBI TGFBR1; T42C in THBS1; C-229T in FURIN;
TT genotype had a statistically not signicantly C-256G in LTBP1L; T-893G and Thr750Ala in
decreased survival compared to the carriers of the LTBP4; T-779A in BAMBI) on the susceptibility
other genotypes (Fig. 1C, HR: 1.28, CI: 0.77 and clinical outcome of CRC.
2.15). None of the other SNPs had any effect on This study was particularly suitable for survival
survival (data not shown). analysis because all patients were sampled before
diagnosis of CRC, independently of their geno-
type (Pukkala et al., 2007). The follow-up time
DISCUSSION was up to 16 years after diagnosis, allowing analy-
In the normal intestinal epithelium TGFB1 sis of long-term survival. The proportion of
acts as a growth inhibitor, but in malignant cells women (56.5%) in our study was slightly higher
it may act as a tumor promoter. Therefore, any than in other studies about the effects of the
disturbances in the TGBF1 pathway may affect TGB1 pathway genes on the risk of CRC; how-
CRC susceptibility and progression. We investi- ever, we did not observe any gender-specic dif-
gated the effect of nine polymorphisms in ferences in genotype distribution in concordance
TGFB1 pathway genes (Leu10Pro in TGFB1; 9A/ with the previous studies (Pasche et al., 1999;
6A polyalanine polymorphism and IVS7G24A in Sparks et al., 2004; Berndt et al., 2007; Saltzman

Genes, Chromosomes & Cancer DOI 10.1002/gcc


TGFB1 PATHWAY AND COLORECTAL CANCER PROGRESSION 279
the SNPs would be expected to be somewhat
stronger than the ones in the GWASs. For SNPs
with a risk genotype frequency of 1020% in
controls, we had a power of 80% to detect an
OR of 1.8 and 1.6, respectively, at a signicance
level 0.05. Although some statistically signicant
associations were observed between the studied
SNPs and the risk of CRC, Dukes stage and sur-
vival, none of the SNPs were associated with
more than one of the studied end points, thus
decreasing the usefulness of the ndings in the
clinics.
In earlier studies, both positive and negative
associations have been reported between the
Leu10Pro polymorphism in TGFB1 and colorectal
adenomas, adenomatous and hyperplastic polyps
and adenocarcinomas (Sparks et al., 2004; Berndt
et al., 2007; Saltzman et al., 2008). In one study,
the association was stronger in patients with rec-
tal adenomas than in patients with distal adeno-
mas (Berndt et al., 2007). The SNP is located in
the signal peptide sequence and it has been
shown to affect TGFB1 secretion and serum
level (Dunning et al., 2003; Berndt et al., 2007).
In our current investigation, we did not nd any
effect of the TGFB1 Leu10Pro on CRC suscepti-
bility or clinical outcome.
The 6A allele in the signal peptide sequence
of the TGFBR1 gene has been suggested to be a
susceptibility allele for CRC (Pasche et al., 1999;
Bian et al., 2005; Xu and Pasche, 2007), although
more recent studies, including a meta-analysis
with 2,627 CRC cases and 3,387 controls, have
excluded the association between the TGFBR1
6A allele and risk of CRC (Castillejo et al., 2009;
Skoglund et al., 2007, 2009). In our study, the 6A
allele was more common in the patients with
CRC (13.8%) than in the control population
(11.7%). It was also more common in cases with
Figure 1. Overall survival after diagnosis of CRC according to the
Dukes stage CD tumors (15.2%) than in the
patients genotypes of the SNPs (A) rs114664459, TGFBR1 9A/6A cases with Dukes stage AB tumors (13.9%).
(9A/9A vs. 9A/6A P 0.46, 9A/9A vs. 6A/6A P 0.27, 9A/6A vs.
6A/6A P 0.39), (B) rs4932178, FURIN C-229T (CC vs. CT, P
Also, survival of the 6A/6A genotype carriers
0.04, CC vs. TT P 0.85, CT vs. TT P 0.21), and (C) rs610820, seemed to be worse compared to 9A/9A carriers.
BAMBI T-779A (TT vs. TA P 0.08, TT vs. AA P 0.40, TA vs. AA
P 0.55).
The rareness of the 6A/6A genotype may have
contributed to the fact, that none of the results
were statistically signicant. However, as this is
et al., 2008; Castillejo et al., 2009). With our sam- the rst report about the effect of the 9A/6A
ple size of 308 patients with CRC and 585 con- polymorphism on clinical outcome of CRC, fur-
trols, we had a limited power to detect the ther studies are warranted.
relatively low ORs observed for common SNPs in The IVS7G24A SNP in the TGFBR1 gene
the recent genome-wide association studies has been reported to associate with invasive
(GWASs) (Easton and Eeles, 2008; Houlston breast cancer, lung cancer, kidney, and bladder
et al., 2008). However, as the SNPs were selected carcinomas (Zhang, 2005; Chen et al., 2006b), but
based on functional considerations, the effect of not with familial CRC in a recent Swedish study

Genes, Chromosomes & Cancer DOI 10.1002/gcc


280 FORSTI ET AL.

(Skoglund Lundin et al., 2009). In our study, sion and in that way affect TGFB1 signaling in
based on prospective cases from North Sweden, CRC cells.
the A allele of IVS7G24A in TGFBR1 had a sig- In summary, although the polymorphisms in
nicant protective effect on the susceptibility to our study were selected based on their reported
CRC. An explanation for this apparent discrep- association with cancer, their putative function
ancy between these studies may be the selection and their LD with other SNPs, only a few of
criteria of the cases. Although we selected pro- them had an effect on CRC susceptibility,
spective cases from two population-based cohorts, Dukes stage or survival. However, as this is the
Skoglund Lundin et al. (2009) recruited the cases rst study about the inuence of the SNPs in the
from families diagnosed and counseled as familial TGFB1 pathway on clinical outcome of CRC and
CRC. Alternatively, the results may be due to as TGFB1 signaling plays a dual role during
chance, because both studies were relatively CRC development and progression, the results
small, our study consisting of 308 cases and the should be validated in larger independent stud-
one by Skoglund Lundin et al. (2009) of 262 ies, preferable with prospectively collected study
cases. According to the HapMap, the IVS7G materials like in the present study. These materi-
24A SNP captures 32 of 35 common SNPs als are particularly suitable for survival analysis
(MAF  10%) with r2 > 0.8 in the TGFBR1 because all patients are sampled before diagnosis
gene. Thus, its effect could be through its LD of CRC, independently of their genotype.
with any of the SNPs. Five of the SNPs are
located in the 30 UTR, making them candidates ACKNOWLEDGMENTS
for functional variants due to their possible effect
We thank Asa Agren (Department of Public
on post-transcriptional gene regulation (Chen
Health and Clinical Medicine/Nutritional
et al., 2006a).
Research, Umea University, Sweden) for her ef-
No studies about the effect of the SNPs in
ciency and skill in keeping track of samples and
FURIN, THBS1, LTBP4, and BAMBI on CRC
data. The Northern Sweden Collaborative Group
susceptibility or clinical outcome have been pub-
for Colorectal Diseases is appreciated for provid-
lished thus far. In this study, the carriers of the
ing the clinical data.
CT genotype of FURIN C-229T had a worse sur-
vival than the carriers of the CC genotype (CT
vs. CC, P 0.04). However, no effect on survival
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