bone morphogenic protein 3 inactivation is an early and frequent event in colorectal cancer...

RESEARCH ARTICLES

Bone Morphogenic Protein 3 InactivationIs an Early and Frequent Event in ColorectalCancer Development

Kim Loh,1 June A. Chia,1 Sonia Greco,1 Sarah-Jane Cozzi,1 Ron L. Buttenshaw,1 Catherine E. Bond,1 Lisa A. Simms,1

Tanya Pike,1 Joanne P. Young,2 Jeremy R. Jass,3 Kevin J. Spring,1 Barbara A. Leggett,1 and Vicki L. J. Whitehall1*

1Conjoint Gastroenterology Laboratory,Royal Brisbane andWomen’s Hospital Research Foundation Clinical Research Centre andQueensland Institute of Medical Research,Brisbane 4029,Australia2Familial Cancer Laboratory,Queensland Institute of Medical Research,Brisbane 4029,Australia3Departmentof Cellular Pathology,St Mark’s Hospital,HarrowHA13UJ,UK

Bone morphogenic proteins (BMPs) are members of the TGFB growth factor superfamily with well-described functions in

bone formation. Although disrupted BMP signalling in tumor development has more recently been investigated, a role for

BMP3 in colorectal cancer (CRC) has remained largely unexplored. The aim of this study was to investigate BMP3 disruption

in CRCs in relation to both the traditional and serrated pathways of tumor progression. BMP3 was down-regulated as assessed

by real-time PCR in 50 of 56 primary tumors (89%). Bisulfite sequencing of the putative promoter revealed extensive hyper-

methylation in the cell line HT29, in which expression could be restored by treatment with a methyltransferase inhibitor. Aber-

rant hypermethylation was observed in 33/60 (55%) tumors and was highly correlated with microsatellite instability (P < 0.01),

the CpG Island Methylator Phenotype (P < 0.01), BRAF oncogene mutation (P < 0.01), and proximal location (P < 0.001).

Methylation was also frequently observed in serrated and traditional adenomatous polyps (22/29, 76%). Re-introduction of

BMP3 into cell lines revealed marked growth suppression supporting the functional relevance of this alteration in colorectal tu-

mor development. This study provides molecular and functional data supporting the importance of BMP3 silencing as an early

and frequent event in colorectal tumors progressing via the serrated and traditional pathways. VVC 2008 Wiley-Liss, Inc.

INTRODUCTION

Bone morphogenic protein 3 (BMP3) is a mem-

ber of the transforming growth factor beta (TGFB)

superfamily of cytokines, which includes BMPs,

activins, and TGFB isoforms. These growth factors

act by binding to type II surface receptors

(including BMPRII, ACTRII, and TGFBRII),

which then recruit and subsequently phosphoryl-

ate type I receptors. This series of events activates

a signalling cascade via phosphorylation of recep-

tor-mediated SMADs, which ultimately results in

the transcriptional regulation of SMAD4 target

genes. Receptor specificity of BMP3 has not been

defined, but it has been suggested that BMPs may

also signal via SMAD-independent pathways to

achieve growth suppression (Beck et al., 2006). In

vitro studies have suggested a role for BMP3 as a

negative growth regulator in bone marrow progeni-

tor cells through inhibition of DNA synthesis and

proliferation (Amedee et al., 1994). There have

been no recent studies to define the function of

the BMP3 ligand in tumorigenesis and a potential

role in the SMAD signalling pathway has not been

demonstrated.

BMP family members were initially studied due

to their function in development, particularly bone

formation, however more recent evidence suggests

an important role in tumorigenesis. De-regulated

expression in colorectal cancer (CRC) has been

reported for family members including BMP4which is over-expressed (Nosho et al., 2005) and

BMP2, BMP3, and BMP5 which are down-regu-

lated (Hardwick et al., 2004; Koehler et al., 2004;

Koinuma et al., 2005). Aberrant promoter hyper-

methylation has been reported as a mechanism of

down-regulated expression for some family mem-

bers including BMP3B and BMP6 in lung cancer

(Kraunz et al., 2005). Perhaps the most compelling

*Correspondence to: Vicki L. J. Whitehall, The Queensland Insti-tute of Medical Research, 300 Herston Road, Herston QLD 4029,Australia. E-mail: [email protected]

Supported by: The National Health and Medical Research Coun-cil of Australia, Grant number: 290203; Queensland Cancer Fund(145), Queensland Health Pathology and Scientific Services, TheRoyal Brisbane and Women’s Hospital Research Foundation andThe Walter Paulson Tumor Bank.

Received 2 October 2007; Accepted 30 January 2008

DOI 10.1002/gcc.20552

Published online 29 February 2008 inWiley InterScience (www.interscience.wiley.com).

VVC 2008 Wiley-Liss, Inc.

GENES, CHROMOSOMES & CANCER 47:449–460 (2008)

evidence for the importance of disrupted BMP sig-

nalling in the early stages of CRC development was

the finding of germline mutation of BMPR1A in ju-

venile polyposis syndrome kindreds (Howe et al.,

2001; Zhou et al., 2001). A conditional Bmpr1a2/2murine model also developed intestinal polyposis

and provided evidence for a link between BMP and

Wnt signalling via PI3K-AKT (He et al., 2004).

Sporadic CRC can be divided into clinically rele-

vant subgroups based on gene expression profiles,

which reflect different pathways of tumor progres-

sion. Two major pathways of colorectal tumorigen-

esis include the ‘traditional’ adenoma-carcinoma

pathway and the ‘serrated’ pathway, the latter

being associated with serrated lesions including

hyperplastic polyps (HPs) and the more recently

described sessile serrated adenoma (SSA), which

are larger than HPs and occur more frequently in

the proximal colon. Whilst the traditional pathway

is characterized by chromosomal instability and tar-

get gene mutation (Fearon and Vogelstein, 1990),

serrated pathway lesions usually exhibit extensive

aberrant promoter hypermethylation (CpG Island

Methylator Phenotype, CIMP), microsatellite insta-

bility (MSI), and activating mutation of the BRAFoncogene (Toyota et al., 1999; Park et al., 2003; Kam-

bara et al., 2004). KRAS mutation, which like BRAFmutation upregulates the MAPK pathway, rarely

occurs in CIMP1 tumors displaying a high level of

MSI (MSI-H), but frequently in CIMP1microsatel-

lite instability low (MSI-L) (Jass et al., 1999; White-

hall et al., 2002) and microsatellite stable cancers

(MSS) (van Rijnsoever et al., 2002).

We identified BMP3 as a candidate tumor sup-

pressor gene in CRC using suppressive subtractive

hybridization (Diatchenko et al., 1996) and vali-

dated its down-regulation in a panel of CRCs

stratified for MSI, CIMP, and the presence of

BRAF or KRAS mutation. Methylation, loss of het-

erozygosity (LOH) and mutation were assessed as

possible mechanisms for down-regulation and cell

growth in vitro was assessed following re-introduc-

tion of BMP3 into cell lines. A variety of precursor

polyps were studied to examine the temporal con-

text and developmental pathway specificity of

BMP3 disruption. The data presented herein pro-

vide evidence that BMP3 is a frequent and early tar-

get in both the traditional and serrated pathways.

MATERIALS ANDMETHODS

Patient Samples and CRC Cell Lines

All CRC and matched normal mucosa samples

were taken from surgical resection specimens col-

lected at the Royal Brisbane and Women’s Hospital

in Australia between 1993 and 2003. Tumors were

defined as microsatellite instability high (MSI-H)

if >30% markers were positive, MSI-low (MSI-L)

if <30% and microsatellite stable (MSS) if no

markers were mutated as previously described

(Whitehall et al., 2002). The cohort was enriched

for MSI (21 MSI-H, 16 MSI-L, 23 MSS) but was

otherwise unselected. A further 7 MSI-H tumors

were included from patients with hereditary non-

polyposis colorectal cancer (HNPCC) for compari-

son. The majority of tumors had previously been

characterised for KRAS mutation and CIMP

(Whitehall et al., 2002). Twenty-nine precancerous

polyps had previously been classified histologically

(JRJ) and characterised for BRAF and KRAS muta-

tion (Spring et al., 2006). Purified colonic epithelial

cells were isolated from surrounding mucosa as

previously described (Whitehead et al., 1987). The

study protocol was approved by the Royal Brisbane

and Women’s Hospital Human Research Ethics

Committee and the Bancroft Human Research

Ethics Committee of the Queensland Institute of

Medical Research. Written, informed consent was

obtained from all study patients. All cell lines were

routinely maintained in RPMI1640 media under

standard culture conditions.

BMP3 Expression Analysis

Total RNA was extracted using RNeasy Midi

Preps (QIAGEN Inc, Valencia, CA) and cDNAwas

synthesised using random hexamers and SUPER-

SCRIPT III (Invitrogen, Carlsbad, CA). A 152bp

cDNA product was amplified for 30 cycles at 618Cannealing (BMP3RTF: 50-TGGATTGAACCTCG

GAATTGC-30 and BMP3RTR: 50-GCTTCAAA

GACTTTGGC ATGG-30). Cell line expression

was assessed by RT-PCR and compared with the

18S housekeeping gene (18SF: 50-AAACGGCTA

CCACATCCAAG-30 and 18SR: 50-CCTCCAATG

GATCCTCGTTA-30). Relative expression levels

of BMP3 in primary tumors and matched normal

mucosa were quantified using semi-quantitative

real time PCR and normalised to b-actin (b-actinF:50-TCATGAAGTGTGACGTGGACATC-30 and

b-actinR: 50-CAGGAGGAG CAATGATCTTGA

TCT-30). The PCR was performed in duplicate

on a RotorGene3000 (Corbett Research PTY

LTD, Sydney, NSW, Australia) machine using

SYBR Green PCR master mix (Invitrogen). The

relative expression of BMP3 to b-actin (R value)

was calculated using the Pfaffl method (Pfaffl

2001).

Genes, Chromosomes & Cancer DOI 10.1002/gcc

450 LOH ET AL.

Methylation Analysis

Putative CpG islands in the 50UTR of BMP3were identified using EMBOSS CpGPlot (Larsen

et al., 1992) and CpG Island Searcher (Takai and

Jones 2002). Genomic DNA was bisulfite-modified

using the CpGenome Bisulfite Modification Kit

(S7820, Chemicon International, Temecula, CA).

Bisulfite sequencing was performed for three

patients and matched normal mucosa samples as

well as the cell line HT29. Overlapping primer

pairs (BMP3cpgF1: 50-TAGTTTGGTGTAAGTT

AAGAG-30; BMP3cpgR1: 50-ACCTAACA AATA

AACTCTTCC-30 and BMP3cpgF2: 50-GGAGG

GAAGGTATAGATAGATT-30; BMP3cpgR2: 50-ATCACCTAACACA ACTTTA-30) were utilized

to amplify a 786 bp fragment of the BMP3 50UTR

and exon1. PCR was performed for 38 cycles at an

annealing temperature of 56.58C (product 1) or

548C (product 2). The PCR products were purified

(QIAquick PCR purification kit, QIAGEN) and

cloned into pGEM-T (Promega Corp, Madison,

WI). Clones were automatically sequenced using

T7 and SP6 primers using Big Dye Terminator

v3.1 polymerase (Applied Biosystems, Foster City,

CA). HT29 was also treated with the methylation

inhibitor 5-Aza-20-deoxycytidine (5-Aza-dC) to

assess the relationship between methylation and

BMP3 gene expression. Exponentially growing

cells were treated with 0.0, 2.5, 5.0, 7.5, or 10.0 lM5-Aza-dC then harvested 4 days later. For this

experiment, BMP3 expression scores were normal-

ised to the 18S housekeeper gene using standard

curve analysis.

Methylation-specific PCR (MSP) was performed

for 60 normal and tumor pairs, 7 HNPCC samples,

29 polyps and 12 cell lines. Primers specific to

unmethylated DNA (BMP3-UF: 50-GGAGTTTA

ATTTTTGGTTTTGTTGTT-30 and BMP3-UR:

50-ATC AACTCCCAACATCACTACA-30) or

methylated DNA (BMP3-MF: 50-GTTTAA

TTTTCGGTTTCGTCGTCGT-30 and BMP3-MR: 50-GTCGACTCCCGACGTCGC TACG-30)amplified 73 bp and 70 bp products, respectively.

Each PCR was performed for 30 cycles at 648Cannealing. DNA for archival polyps differed in

extraction using Chelex-100 (Coombs et al., 1999)

and bisulfite modification using the MethylEasy

DNA Bisulfite Modification Kit (Human Genetic

Signatures PTY LTD, Sydney, NSW, Australia).

Loss of Heterozygosity Analysis

Chromosome band 4q21 LOH was assessed

using D4S2964, AFM318zc9 and D4S2922, whereD4S2964 is the most proximal marker and BMP3 is

located between AFM318zc9 and D4S2922 (most

distal marker). Each reaction was performed for 35

cycles at 568C annealing. PCR products were sepa-

rated through 5% denaturing polyacrylamide gels.

All samples showing two distinct allelic bands in

normal mucosa DNA were considered informative.

Results were examined by three independent

observers and LOH was scored as positive when a

clear reduction in signal intensity was observed in

one of the alleles in the tumor DNA compared

with the same allele in the normal DNA of the

same patient.

Mutation Analysis

Mutations were assessed by direct sequencing

and high resolution melt analysis (HRM) in 5 MSI-

H, 6 MSI-L, 9 MSS cancers and 12 cell lines.

Assessment of 20 primary CRCs was considered

sufficient to determine if mutation is a common

mechanism of gene silencing for BMP3. Primer

sequences and amplification conditions are shown

in Table 1. HRM profiles were assessed on a Rotor-

Gene6000 (Corbett Research) and products were

purified using a GF-1 Nucleic Acid Extraction Kit

(Vivantis). About 3–10 ng was sequenced using

BigDye3.1 Terminator Cycle Sequencing premix

TABLE 1. BMP3 Mutation Screening Primers

Primer Product size (bp) Annealing temp. (8C) No. of cycles

Exon1F 50-TCCTTGCGCCTTCGGAGTGTC-30 415 64 30Exon1R 50-ACCGCGGGAAGGGAGTCTCA-30

Exon2aF 50-TGTCATATAGTGAAGTAATGGTCTTGTT-30 357 64 35Exon2aR 50-GAGTGATATCTTTAGACAGCCAGGAC-30

Exon2bF 50-TCAACTCCTTGGCCATCTGTCAGT-30 434 64 30Exon2bR 50-TGTAAGGCTTTCTCTCCTCCCACA-30

Exon2cF 50-CTCTGCAGAACAACGAGCTTCCT-30 408 64 35Exon2cR 50-ACTTAGTCAATGTGTCTTTCTACTAATGG-30

Exon3F 50-TAATGAGGACTGAGGAGTGGAAACG-30 331 64 35Exon3R 50-AAAAACAGGAAGAAGTCCATAAAAATAA-30


451BMP3 IN COLORECTAL CANCER

(Applied Biosystems). If novel variants were

detected, the HRM analysis was extended to

include the remaining 40 tumor and all normal

samples from the initial cohort. The BRAF V600E

mutation was also analysed in all tumor samples

using an allelic discrimination assay as previously

described (Kambara et al., 2004).

Colony Formation Assay

A mammalian expression construct

(pcDNA3.1::BMP3) was designed to examine the

effect of re-introducing BMP3 into null-expressing

CRC cell lines. A nested PCR was performed to

amplify the coding sequence (BMP3-NestedF

50-CCAGCTGGTTT GGAGTTCAAC-30 and

BMP3-NestedR 50-ATTGAATTAAGCATTCAAA

TGAG-30; and internal BMP3-Kpn1-Koz-F 50-CGGGGTACCGCCATGGCTGGGGCGAGCAGG

CTGCTCTTTCT-30 and BMP3-EcoR1-R 50-GACAGTAGAGTCTTGCGCTTGCAGATAAG

AATTCCG-30). In addition to the EcoR1/Kpn1cloning sites, a Kozak sequence was engineered

into the forward primer to enhance translation ini-

tiation. One microgram of either pcDNA3.1(1)

(Invitrogen) or pcDNA3.1::BMP3 was transfected

with FuGENE6 (Roche) at a ratio of 3:1. Eight cell

lines (HT29, HCT116, Lim1215, SW48, SW480,

LS174T, RKO, and DLD1) were transfected in

triplicate at an initial density of approximately

50%. Cells were allowed to recover for 48 h before

applying selective media at a final concentration of

700 ng/lL G418 (GibcoBRL) for 10–14 days, after

which time no untransfected control cells were sur-

viving and transfected colonies were stained with

0.25% crystal violet/80% methanol.

Statistical Analysis

Data was analyzed using GraphPad Prism Ver-

sion 4 (Motulsky, 2003). Expression data was ana-

lysed based on empirical fold differences initially

using Kruskal–Wallis (nonparametric) analysis fol-

lowed by a Dunn’s multiple comparison post-test.

Differences between individual groups were fur-

ther probed using the Wilcoxon test (nonpara-

metric) for paired samples. P-values � 0.05 were

considered statistically significant.

RESULTS

BMP3 Expression in Primary Colorectal

Cancers and Cell Lines

BMP3 was expressed in all normal colonic mu-

cosa and purified crypts, but not in any of eleven

CRC cell lines (HT29, LIM1863, SW480, LOVO,

RKO, DLD1, SW48, HCT116, LS174T,

LIM1215, and LISP1). RT-PCR results for a nor-

mal mucosa sample and three cell lines are shown

in Figure 1. BMP3 was down-regulated in the ma-

jority of primary, sporadic CRCs examined by

qRT-PCR, irrespective of MSI-status (20/21 MSI-

H, 13/15 MSI-L and 17/20 MSS) (Fig. 1C). When

tumors were compared with matched normal mu-

cosa from the same patient, the mean fold down-

regulation was 984 for MSI-H (P < 0.0001), 673 for

MSI-L (P < 0.0001) and 949 for MSS cancers (P <0.0006) (Wilcoxon matched pairs nonparametric

t-test, two tailed). Only two tumors showed up-

regulated expression as compared to matched nor-

mal mucosa, with one MSI-H tumor showing 5-

fold up-regulation and one MSS showing 46-fold

up-regulation. A further 2 MSI-L and 2 MSS

tumors showed no significant deregulation (P <1.5-fold). No significant difference in normalised

expression scores were observed between purified

crypts and whole normal tissue extractions.

BMP3 Promoter Hypermethylation

A putative CpG island was identified bioinfor-

matically using two independent algorithms. Bisul-

fite sequencing of 71 CpG sites in this region

revealed extensive hypermethylation in two CRCs

(T1, T2) and HT29, but not in a third CRC (T3)

or 3 matched normal mucosa samples (N1, N2,

N3) (Fig. 2). In the two MSI-H tumors, which

showed 942 (T1) and 3111 (T2) fold down-regula-

tion of BMP3 expression, the majority of clones

were heavily methylated. The remaining largely

unmethylated clones most likely originated from

stromal cells. Interestingly, 1/10 clones from N2

showed 17/71 (24%) sites methylated, which may

represent a methylator field effect in the normal

mucosa. Comparatively little methylation was seen

in the MSI-L tumor (T3) where BMP3 was down-

regulated only 8 fold. HT29 was heavily methyl-

ated in 12/12 clones and transcript expression

could be restored by treatment with 5-Aza-dC

(Fig. 3A). This strongly supported promoter hyper-

methylation as a mechanism for BMP3 silencing in

HT29.

The frequency of BMP3 hypermethylation was

assessed by methylation specific PCR (MSP) in a

larger cohort of 60 sporadic CRCs, 7 HNPCC, 29

polyps and 12 cell lines. The MSP assay target

region was located immediately 50 of the transla-

tion start site (Fig. 2). It was considered conserva-

tive to place the primers in this region of dense

methylation as the region closer to the transcrip-


452 LOH ET AL.

tional start site had comparatively fewer methyla-

tion events, which would have resulted in a less

sensitive assay. Figure 3B shows a tumor positive

for methylation (Patient A), negative for methyla-

tion (Patient B), the fully methylated cell line

HT29 and the partially methylated cell line

SW480. Methylation occurred with decreasing fre-

quency in relation to MSI-status, with 17/21 (81%)

MSI-H, 7/16 (44%) MSI-L and 9/23 (39%) MSS

cancers being methylated. The frequency of meth-

ylation was significantly higher in MSI-H tumors

compared to MSI-L (P < 0.025) or MSS (P <0.01). Only a single HNPCC tumor (1/7, 14%)

showed methylation, suggesting a strong associa-

tion with sporadic rather than familial MSI-H sta-

tus (P < 0.01). Aberrant BMP3 promoter hyper-

methylation was highly tumor-specific with not a

single normal mucosa sample (of the 67 tested)

showing methylation by MSP (P < 0.001). The

majority of methylated cancers (28/33, 85%)

showed significant down-regulation of BMP3expression. Furthermore, the single MSS tumour

(S17) with BMP3 up-regulated 46-fold was not

methylated, supporting the hypothesis that the

region targeted by the MSP primers is important

for gene silencing.

BMP3 promoter hypermethylation was highly

correlated with other molecular and clinical fea-

tures which have previously been associated with

the serrated neoplastic pathway including CIMP

(P < 0.01), BRAF mutation (P < 0.01) and proxi-

mal location in the colon (P < 0.001) (Table 2). A

significant association was observed between

BMP3 methylation and mutation of either BRAF or

KRAS, with 26/33 (79%) of methylated tumors hav-

ing one or the other mutation, compared with 13/

27 (48%) of unmethylated tumors having mutation

of either oncogene (P < 0.025). MSP results were

suggestive of biallelic methylation in 6/11 cell

lines, including the two with a BRAF mutation

(Table 3). Despite repeated attempts no band

could be produced using either primer pair for

LISP1 which may suggest biallelic deletion of the

BMP3 locus in this cell line. The majority of polyps

assessed showed detectable methylation (Table 2),

including 100% (5/5) SSAs which are considered

the precursors to MSI-H / CIMP1 tumors. Five of

the eight HPs and all the SSAs also had a BRAF

Figure 1. Expression of BMP3 is shown for a normal colonic mucosa sample (A) but was absent in thecell lines HT29, HCT116, and SW480 (B) as demonstrated by RT-PCR. Normalized qRT-PCR expressionscores are shown normal and tumor specimens stratified by MSI (C).



mutation. Interestingly, 5/8 (62.5%) TAs and 7/8

(87.5%) TVAs were also methylated, suggesting a

wider involvement in early lesion formation. None

of the TAs or TVAs had a BRAF mutation. BMP3methylation was not associated with polyp size, an-

atomical location or total number of polyps in the

bowel.

Loss of Heterozygosity Analysis

LOH of the BMP3 locus was examined using 3

polymorphic DNA markers on chromosome band

4q21 (Table 4). Scoring was performed in a blinded

fashion by three independent observers and 100%

concordance was obtained. Overall, 21/38 (55%) of

cases were informative. Two tumors in which

BMP3 was down-regulated but not methylated had

LOH of all three markers (S2 and L15). Overall, 7/

31 (23%) tumors with at least one informative

marker showed LOH and corresponding down-reg-

ulation of BMP3, with four also being methylated,

suggesting biallelic inactivation. Neither of the

two samples (S17 and L2) which had shown up-

regulation of BMP3 mRNA expression had either

methylation or LOH. Taken together, methylation

or LOH may account for decreased BMP3 expres-

sion in 17/21 (81%) MSI-H, 8/15 (53%) MSI-L and

10/23 (43%) MSS cancers.

Mutation Analysis

The three BMP3 coding exons were directly

sequenced to scan for somatic mutations in 5 MSI-

H, 6 MSI-L, 9 MSS tumors, and 12 cell lines

(Table 5). All variants detected by direct sequenc-

ing were also observed by high resolution melt

(HRM) analysis. The most common variant was

exon 2 G895A (Arg192Gln) which was identified in

5/23 (22%) MSI-H, 2/16 (12.5%) MSI-L and 4/22

Figure 2. Genomic structure, 50 CpG island and bisulfite sequencingmap for BMP3. Exons are indicated by boxes, with the coding regionshaded in black (A). The transcription start sites (TSS) is indicated by astar and translation start site indicated by an arrow. The identified CpGisland is depicted in (B) and the region selected for bisulfite sequencingis indicated. CpG sites which were bisulfite sequenced (C) are indicatedby circles, with filled in circles indicating methylation. Each row ofcircles reflects a unique clone. The CpG sites chosen for MSP are indi-cated by arrows and the region indicated by a dotted box.

Figure 3. Treatment with the methylation inhibitor 5-aza-deoxycytidine resulted in BMP3 transcript re-expression in HT29 a concentration-dependent manner (A). Methylation specific PCR (B) indicated specificBMP3 methylation in the tumor of patient A whilst the tumor of patient B was unmethylated. The cell lineHT29 showed putative biallelic methylation whilst putative monoallelic methylation was observed forSW480.


454 LOH ET AL.

(18%) MSS cancers and matched normal samples

(total 11/60, 18%) and 3/12 (25%) cell lines. The

SNP was reported to be at a lower frequency in the

normal population (NCBI rs3733549, 12.9%) but

this did not reach statistical significance. The silent

exon 2 SNP G1184A (Lys288Lys) was detected in

RKO but 0/20 primary tumor samples. A novel var-

iant of unknown functional significance was

detected in a 97-year-old patient with an MSI-H

tumor, as well as his matched normal mucosa

TABLE 2. Molecular and Clinical Correlations with BMP3 Methylation

n BMP3 Methylated BMP3 Unmethylated P-value

Age (Mean6 s.d.) 80.0 6 11.9 76.8 6 15.6Gender

Female 33 20 (61%) 13 (39%)Male 27 13 (48%) 14 (52%)

LocationProximal 29 23 (79%) 6 (21%) <0.001Distal 20 6 (30%) 14 (70%)

ACPS StageA 8 4 (50%) 4 (50%)B 31 19 (61%) 12 (39%)C 10 6 (60%) 4 (40%)D 9 3 (33%) 6 (66%)

MSI StatusMSI-H 21 17 (81%) 4 (19%) <0.025MSI-Low 16 7 (44%) 9 (56%)MSS 23 9 (39%) 14 (61%)

CIMPCIMP1 22 17 (77%) 5 (23%) <0.01CIMP2 26 8 (31%) 18 (69%)

KRASWild-type 44 25 (57%) 19 (43%)Mutant 16 8 (50%) 8 (50%)

BRAFWild-type 37 15 (41%) 22 (59%) <0.01Mutant 23 18 (78%) 5 (22%)

PolypsHP 8 7 (87.5%) 1 (12.5%)SSA 5 5 (100%) 0TA 8 5 (62.5%) 3 (37.5%)TVA 8 7 (87.5%) 1 (12.5%)

HP, hyperplastic polyp; SSA, sessile serrated adenoma; TA, tubular adenoma; TVA, tubulovillous adenoma.

TABLE 3. BMP3 Alterations in Colorectal Cell Lines

Cell line MSI-status BRAFa K-ras BMP3 Methb mRNAcAvg % pcDNA3.1

colonies

Coding sequence variants

Nt changed AA changed

HT29 MSS Mut wt 1ve 2ve 64.7 In1599T STOPLIM1863 MSS wt wt 1ve 2ve — - -SW480 MSS wt Mut Partial 2ve 34.9 G426A Val36MetLoVo MSI-H wt Mut 1ve 2ve — G895A Arg192GlnRKO MSI-H Mut wt 1ve 2ve 76.6 G1184A Lys288LysDLD-1 MSI-H wt Mut 1ve 2ve 49.8 - -SW48 MSI-H wt wt 1ve 2ve 243.3 - -HCT116 MSI-H wt Mut Partial 2ve 32.2 - -LS174T MSI-H wt Mut 2ve 2ve 19.7 G895A; A1562G Arg192Gln; Ser414SerLIM1215 MSI-H wt Mut 2ve 2ve 33.6 G895A Arg192GlnLISP-1 MSI-H wt Mut — 2ve — - -

awt, wild type; Mut, mutant.b1ve, positive;2ve, negative, —, not done.c2ve, no expression detected.d-, no mutation detected; Nt, nucleotide; AA, amino acid.



(T1168G, Ile283Thr, Fig. 4). Four novel variants

were found in cell lines only (Table 5). Of note is a

1 bp insertion in exon 3 of HT29 which results in a

premature stop codon.

Colony Formation Assay

Eight cell lines were transfected with either

pcDNA3.1 or with pcDNA3.1::BMP3 containing

the full length BMP3 coding sequence. Re-intro-

duction of BMP3 resulted in significant suppres-

sion of colony formation in all cell lines examined,

except SW48 in which greater than 2-fold increase

in colony numbers were seen (Fig. 5). This result

was reproduced in two independent experiments.

DISCUSSION

This is the first study to comprehensively

examine BMP3 in a well characterised series of col-

orectal tumors and precursor lesions. We have

demonstrated significant transcript repression in

the majority of colorectal tumors and cell lines and

shown that promoter hypermethylation is a major

mechanism for this down-regulation. Methylation

was highly correlated with other molecular deter-

TABLE 4. BMP3 Loss of Heterozygosity, Methylation, and Expressiona

Patient D4S2964b AFM318zc9b D4S2922b Methylationc BMP3 down-regulationd

S1 * * - U YesS2 l l l U YesS3 - - - U YesS4 - - - M YesS5 * * - U YesS6 * * x M YesS7 l l - M YesS8 l - - M YesS9 l - * M YesS10 - * x M YesS11 * - - M YesS12 * - l M YesS13 - - - U YesS14 - x x U YesS15 * * * U YesS16 * * - U YesS17 * * * U NoS18 * - - U YesS19 * - - U YesS20 * - - U YesS22 x x - U YesS23 - - - M -S24 * * - U YesL1 - * - U YesL2 * * * U YesL3 * * * M YesL4 * - - M YesL5 - * x M YesL6 * * l M YesL7 * * x M YesL9 - - - M YesL10 * * - M YesL12 - * - U YesL13 l X x U YesL14 * * - U YesL15 l l l U YesL16 * * * U YesL17 * * * U YesInformative Markers 27/37 (73%) 22/35 (63%) 11/32 (34%)LOH 6/27 (22%) 3/22 (14%) 4/11 (36%)

aBMP3 is located between AFM318zc9 and D4S2922.b*, normal; l, LOH; - ,noninformative; x, no product.cU, unmethylated; M, methylated.dyes, BMP3 transcript down-regulated; no, BMP3 not down-regulated; -, no detectable change in BMP3 expression.


456 LOH ET AL.

minants of the serrated pathway of neoplasia,

including MSI, CIMP and BRAF mutation, and

was common in serrated precursor polyps. Despite

these strong associations, BMP3 was also methyl-

ated in a subset of tubular adenomas and silenced

in the majority of non-MSI-H tumors, suggesting a

wider involvement in tumorigenesis. The mecha-

nism to explain down-regulation of BMP3 in the

majority of CRCs could not be explained by meth-

ylation, mutation or deletion analysis and other

potential mechanisms are discussed. Functional

data supporting a role for BMP3 in cancer was

obtained by re-introduction into null expressing

cell lines which resulted in marked growth sup-

pression. Taken together, these data support the

hypothesis that disruption of BMP signalling is an

early, perhaps rate-limiting step in colorectal tu-

morigenesis.

Figure 4. The T1168G mutation detected by high resolution melt analysis. The mutation is shown asrepresented by the normalised graph (A), difference plot (B) and direct sequencing (C).

TABLE 5. BMP3 Sequence Variants in Primary Tumors and Cell Lines

ExonNt changeobserved

Celllines

Tumors(frequency)

Information from NCBI-SNP

Ref SNP ID Frequenca Amino acid change

1 G426A 1/12 (0.08) 0/20 SNP? Unknown Val36Met2b G895A 3/12 (0.25) 6/20 (0.3), 5/40 (0.1),

Total: 11/60 (0.2)rs3733549 G/A (0.129), G/G

(0.871), A/A (0.0)Arg192Gln (Benign)

G1184A 1/12 (0.08) 0/20, 0/40, Total: 0/60 rs17005033 G/A (0.0), G/G(1.0), A/A (0.0)

Lys288Lys

T1168G 0/12 1/20 (0.05), 0/40,Total: 1/60 (0.02)

SNP? Unknown Ile283Ser

3 In1599T, Frame-shift 1/12 (0.08) 0/20 Mutation? Unknown Glu434TerA1562G 1/12 (0.08) 0/20 SNP? Unknown Ser414Ser

aBased on information from NCBI-SNP, Caucasian population genotypes for rs3733549 or European for rs1700503.bThe cohort for exon 2 was extended to include a further 40 patients.



Koinuma and colleagues identified BMP3 as a

methylated target in 8/8 tumors with concurrent

MLH1 methylation, as well as 4/8 tumors without

MLH1 methylated (Koinuma et al., 2005). This is

consistent with our data from a larger series of

tumors where we observed methylation in 81% of

21 MSI-H cancers (all with MLH1 methylation

detected by MSP, data not shown) compared to

just under half of the remaining tumors where

MLH1 was not methylated (44% MSI-L, 39%

MSS). The absence of methylation in 6/7 HNPCC

specimens was consistent with the hypothesis that

that BMP3 methylation is associated with CIMP

rather than MSI. The majority of polyps assayed

by MSP (83%) were methylated, including 7/8

HPs and 5/5 SSAs. This is consistent with a role for

disrupted BMP signalling in the serrated pathway

of neoplasia. Tubular adenomas are the accepted

precursor lesions for the traditional pathway of tu-

morigenesis. 5/8 TAs showed methylation, suggest-

ing BMP3 silencing may also be important in early

lesions of the traditional pathway. BMP3 was also

methylated in the majority of tubulovillous adeno-

mas. These are generally considered to be more

advanced lesions and show an increased frequency

of methylation events (Rashid et al., 2001). In com-

bination with the data from primary tumors, we

propose that BMP3 silencing via promoter hyper-

methylation plays an early role in the development

of most serrated pathway tumors as well as a subset

of tumors arising via the traditional pathway.

Importantly, BMP3 was also commonly down-regu-

lated in the absence of promoter hypermethyl-

ation. Potential mechanisms to explain the

decreased expression observed in these cancers

will be explored below.

Kraunz and colleagues demonstrated an associa-

tion between concurrent epigenetic silencing of

BMP3b and BMP6 with KRAS mutation in lung

cancer, suggesting a synergy between dampening

of the growth suppressing ability of the BMP sig-

nalling pathway and activation of the growth pro-

moting MAPK signalling pathway (Kraunz et al.,

2005). A similar correlation was observed in this

data series with BMP3 methylation being strongly

associated with BRAF mutation (P < 0.01), which

also activates MAPK signaling. Further experimen-

tation is necessary to test if this potential growth

advantage is the result of targeting both the BMP

and MAPK pathways, or the combination of CIMP

and MAPK pathway activation which has also been

demonstrated (Kambara et al., 2004; Yang et al.,

2004). SW48 was the only cell line used for the col-

ony formation assay which was wild type for both

BRAF and KRAS. Although the MAPK pathway

may have been activated at another point, it is

nevertheless interesting to note that this is also the

only cell line where growth suppression was not

observed, compared to the others with definite tar-

geting of the MAPK pathway.

We propose that biallelic promoter hypermethyl-

ation likely accounts for the dramatically reduced

expression of BMP3 in over half of the tumors

examined in this study. The remaining colon can-

cers studied were not methylated, despite 26 of

these 27 showing significantly down-regulated

expression. Gene deletion through LOH may have

Figure 5. Colony formation assay indicating growth inhibition in 7/8cell lines. Representative wells are shown for cell lines DLD1, LS174T,Lim1215, and SW48 (A) for empty vector (pcDNA3.1) and BMP3(pcDNA3.1::BMP3) transfected cells, whilst colony numbers are repre-sented as a percentage of empty vector controls in B.


458 LOH ET AL.

contributed to down-regulation in up to seven

cases whilst no definitely causative mutations were

seen in any of the primary tumors examined.

Future studies of larger patient cohorts will be nec-

essary to further investigate the potential role of

rare mutations in the BMP3 coding and promoter

regions. Putative single nucleotide polymorphisms

of unknown functional significance were also iden-

tified and future large, population-based studies

will be necessary to determine if these germline

variants may contribute to disease susceptibility.

Heritable variations in gene expression have

recently been associated with single nucleotide

polymorphisms (Dixon et al., 2007; Stranger et al.,

2007). Such variants may alter a gene’s propensity

for silencing, for example, by methylation. An in-

herent predisposition or resistance to methylation

induced gene silencing has recently been proposed

in the context of long range coordinate gene silenc-

ing (Frigola et al., 2006; Hitchins et al., 2007). This

describes the existence of a ‘repressed genomic

neighbourhood’ whereby all genes within a large

genomic region will be silenced as a result of his-

tone modification. Only a subset of these genes

will be silenced by methylation and it is intriguing

to speculate that the propensity for methylation

will be determined by primary structure of the pro-

moter and modified based on sequence variation.

Proof of principle was recently demonstrated for

theMGMT gene whereby a promoter sequence var-

iant modulated susceptibility to promoter hyper-

methylation (Ogino et al., 2007).

Other mechanisms to explain the frequent

downregulation of the BMP3 expression in CRCs

may include mutational targeting of the BMP3promoter or transcription regulatory proteins, in-

creased transcript degradation as well as upstream

disruption of BMP3 signalling proteins. Gene tran-

scription may be reduced due to BMP3 promoter

mutations, disruption of necessary transcription

factors or upstream signalling elements. The

BMP3 transcript may also be a direct target of rapid

degradation mediated by a miRNA which is aber-

rantly up-regulated in CRC. Although this has yet

to be experimentally validated, a number of miR-

NAs predicted to target BMP3 are listed in the

miRBase Target database (http://microrna.sanger.

ac.uk). There is currently no published data on

BMP3-interacting proteins to provide clues as to a

possible signalling pathway. It may be postulated

that BMP3 will signal in the TGFB pathway based

on sequence homology to TGFB family members,

however there is currently no published experi-

mental evidence to support this hypothesis. Fur-

ther functional studies to elucidate BMP3 receptor

specificity or binding partners will be critical to fur-

ther explore its role in colorectal tumorigenesis.

In 7/8 CRC cell lines examined in this study, re-

expression of BMP3 resulted in significant inhibi-

tion of colony formation. Similarly, treatment of

human bone marrow osteoprogenitor cells with

BMP3 protein inhibited proliferation (Amedee

et al., 1994), and in vitro administration of BMP6

inhibited growth of prostate cancer cells (Haudens-

child et al., 2004). BMP5, 6, and 7 have been

shown to inhibit growth and induce apoptosis in

myeloma cells (Ro et al., 2004), whilst BMP2 pro-

motes apoptosis and differentiation whilst inhibi-

ting proliferation in vitro (Hardwick et al., 2004).

The SMAD pathway is disrupted in a number of

the cell lines examined (Ijichi et al., 2001), how-

ever it remains to be demonstrated if BMP3 plays

a role in this pathway or whether it exerts a tumour

suppressive effect via SMAD-independent mecha-

nisms, as has previously been suggested (Beck

et al., 2006). In vitro experiments to study apopto-

sis, proliferation and migration are now necessary

to further establish a role for BMP3 in colorectal

tumorigenesis. It is also possible that other BMP

family members may compensate for the down-

regulation of BMP3. Identification of binding part-

ners and receptor specificity for BMP3 will be nec-

essary before further exploring this hypothesis.

Inhibition of BMP signalling has also been

linked to early polyp development in vivo. Inhibi-

tion of intestinal BMP signalling via villin-driventransgenic expression of the BMP inhibitor noggin

leads to de novo crypt formation and polyp devel-

opment in mice (Haramis et al., 2004). In vivo

delivery of BMP4 suppressed glioblastoma growth

in mice by reducing proliferation (Piccirillo et al.,

2006). A Bmp32/2 mouse has been developed

which showed increased trabecular bone density,

however this model was not intestine-specific and

was only examined at 6 weeks of age (Bahamonde

and Lyons, 2001). An intestine-specific BMP32/2model would now be useful to aid in the elucida-

tion of the role of this gene in colorectal polyp and/

or tumor development.

In summary, this study provides compelling evi-

dence for the importance of BMP3 inactivation in

early polyp formation and colorectal tumor devel-

opment. The frequent observation of BMP3 meth-

ylation in colorectal polyps and cancers, but not in

normal mucosa samples, suggests this may be an

attractive target for the future development of mo-

lecular blood and/or stool screening tests for the

early detection of lesions with neoplastic potential.



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460 LOH ET AL.

bone morphogenic protein 3 inactivation is an early and frequent event in colorectal cancer...

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