Open Access

Gene Expression Profiling in Familial Adenomatous Polyposis Adenomas and Desmoid Disease

Hereditary Cancer in Clinical Practice20075:79

https://doi.org/10.1186/1897-4287-5-2-79

Received: 9 February 2007

Accepted: 25 April 2007

Published: 15 June 2007

Abstract

Gene expression profiling is a powerful method by which alterations in gene expression can be interrogated in a single experiment. The disease familial adenomatous polyposis (FAP) is associated with germline mutations in the APC gene, which result in aberrant β-catenin control. The molecular mechanisms underlying colorectal cancer development in FAP are being characterised but limited information is available about other symptoms that occur in this disorder. Although extremely rare in the general population, desmoid tumours in approximately 10% of FAP patients. The aim of this study was to determine the similarities and differences in gene expression profiles in adenomas and compare them to those observed in desmoid tumours. Illumina whole genome gene expression BeadChips were used to measure gene expression in FAP adenomas and desmoid tumours. Similarities between gene expression profiles and mechanisms important in regulating formation of FAP adenomas and desmoid tumours were identified. This study furthers our understanding of the mechanisms underlying FAP and desmoid tumour formation.

Keywords

gene expression profiling FAP adenomos desmoid tumours

Introduction

Familial adenomatous polyposis (FAP) is a rare form of colorectal cancer caused by germline mutations in the adenomatous polyposis coli (APC) gene. Approximately 70–90% of FAP patients have identifiable germline mutations in APC [1, 2]. FAP is clinically characterized by the formation of hundreds to thousands of adenomas that carpet the entire colon and rectum [3]. Although initially benign the risk of malignant transformation increases with age such that, if left untreated, colorectal carcinoma usually develops before the age of 40 years [4].

Loss of APC results in dysregulation of the Wnt signalling pathway that leads to the constitutional activation of the transcription factor Tcf-4, which has been associated with adenoma formation [5]. Alterations in Wnt signalling cause stem cells to retain their ability to divide in the upper intestinal crypt, thereby forming monocryptal adenomas [6]. Eventually the adenomas may acquire metastatic potential, resulting in carcinoma development [7]. Not all adenomas will progress to malignant tumours; however, due to the abundance of adenomas carcinoma development is virtually assured [8].

Apart from the apparent loss of APC function, little is known about the molecular processes involved in adenoma initiation [6]. Similarly, the molecular events occurring during the transformation of adenomas into carcinomas are poorly understood, as are the mechanisms that underlie the development of extra-colonic disease in FAP.

It is well established that FAP patients are susceptible to benign extra-colonic tumours, including desmoid tumours [3]. Although rare in the general population, desmoids occur in approximately 10% of FAP patients and they are the second most common cause of death [9]. Desmoid tumours are poorly encapsulated and consist of spindle-shaped fibroblast cells with varying quantities of collagen [10]. Despite their apparent inability to metastasize, desmoid tumours can be extremely aggressive [11].

It has been speculated that desmoid formation is a result of an abnormal wound healing response [12]. Desmoids can affect surrounding viscera, causing potentially fatal complications [13]. FAP-associated desmoid tumours are usually associated with germline APC mutations [14], but somatic APC mutations have been detected in sporadic desmoid tumours [15].

Microarray technology has an enormous potential for applications in the endeavour to better understand tumours and their development [16]. The ability to detect expression levels of thousands of genes can identify particular genes that are either up- or down-regulated in different tumour types [17]. Tumours that are currently categorized by similar morphology, such as desmoid tumours, may be more usefully divided into subtypes according to their expression profiles [18]. Particular expression profiles in tumours may also be capable of predicting the clinical outcome in specific patients in the early stages of tumour development [18]. In colorectal cancer, gene expression profiles of adenomas and adenocarcinomas have been compared and subsets of genes expressed at common levels in both lesions have been identified as well as expression patterns that are unique to each [19]. Gene expression profiling has the potential to identify factors involved in the malignant transformation of adenomas, and may aid in the diagnosis of benign versus malignant disease.

Although genome-wide expression studies have been reported on FAP adenomas and desmoid tumours, the present one of the first to compare the two tissue types. The first aim of this study was to identify distinct gene expression profiles for colorectal and stomach FAP adenomas and desmoid tumours. The second aim was to determine the similarity between the gene expression profiles in FAP adenomas and desmoid tumours to identify mechanisms important in regulating formation of these lesions. To achieve this, mRNA from normal colon, FAP stomach and colon adenomas and desmoid tumours was measured using whole human genome expression BeadChips (Illumina). The findings of this study further our understanding of the mechanisms underlying FAP and desmoid tumour formation.

Materials and methods

FAP adenoma and tumour tissue and controls

Frozen adenoma tissue from 4 FAP patients was available for this study. Colorectal FAP adenoma A was from an individual aged 40 at the time of surgery. Genetic testing revealed a heterozygous A5465T change in the APC gene, causing a missense change from aspartic acid to valine at position 1822 in the amino acid sequence. The specimen obtained for this study was obtained as a result of a proctocolectomy. The pathology report indicated that over 100 tubulovillous adenomas were present in the original specimen, with no evidence of invasive tumour. Patients B, C and D harboured the same frameshift mutation, a 4 base pair deletion at position 3462–3465 of the APC gene. Patient B was diagnosed with FAP at the age of 11 years, patient C at 13 years of age, and patient D at the age of 37 years. One gastric adenoma was obtained from patient D, in addition to a colonic adenoma. Normal colon tissue from 7 healthy individuals with no history of FAP or desmoid disease was used as a mixed reference sample for this study.

Desmoid Disease Tissue

Desmoid tumour tissue from two individuals was available for this study. Patient A had FAP-associated desmoid disease. There was a family history of FAP, but no known history of desmoid disease. The individual harboured a 1 bp deletion in exon 15 of the APC gene resulting in a frameshift that introduced a premature stop codon at amino acid position 964. Patient B had a family history of FAP and desmoid disease. This patient harboured a 17 bp duplication in exon 15 of the APC gene, which introduced a premature stop codon at amino acid position 1969. A previously established fibroblast cell line from a healthy individual with no history of FAP or desmoid disease was used as a control for this study. The fibroblast cell line was cultured in 1× Complete DMEM media at 37°C (5% CO2).

RNA Extraction

2–3 mm2 pieces of fresh frozen FAP adenoma and desmoid tumour tissue were cut from the original sample and transferred immediately to 1 ml Trizol reagent (Invitrogen, USA). Similarly, approximately 1–10 × 106 control fibroblast cells were lysed in 1 ml Trizol reagent (Invitrogen, USA). RNA was extracted per manufacturer's instructions. The RNA pellet was washed with 75% ethanol, before being dissolved in 20 μl water.

The total RNA was purified using a Qiagen RNeasy MiniElute Cleanup Kit as per manufacturer's instructions. The concentration of the purified total RNA samples was measured using a Quant-It RiboGreen RNA Assay Kit (Invitrogen, USA) and a fluorometer (Fluostar OPTIMA) as per manufacturer's instructions.

RNA amplification

To synthesise first and second strand cDNA and amplify biotinylated cRNA from the total RNA, an Illumina Totalprep RNA Amplification Kit was used as per manufacturer's instructions.

The purified cRNA samples were quantified to determine the volume required for the BeadChip hybridisation step via the Quant-iT RiboGreen RNA Assay Kit as described previously.

Illumina BeadChip Procedure

Hybridisation to the Illumina Sentrix 8 BeadChip was performed according to the manufacturer's instructions without modification. The Sentrix 8 BeadChips were read using an Illumina Beadarray reader (San Diego, CA, USA).

Data Analysis

Analysis and normalisation of expression data from the 24,000 transcripts was carried out using BeadStudio 2.0 (Illumina, San Diego, CA, USA). The t-test error model and cubic spline normalisation was used for all samples. A differential analysis was applied to all adenoma and tumour samples using the Illumina custom test of significance, utilising the mixed normal colon control as the reference group. GeneSpring 5.0 (Agilant, Santa Clara, CA, USA) used standard correlation and distance to create dendrograms (Experiment trees) to show relationships between gene expression profiles. A second dendrogram (Gene tree) was created for each gene list using standard correlation and distance to show relationships between the expression levels of genes across the groups.

Results

Gene expression data from over 23,000 genes on Illumina HumRef-8 BeadChips was analysed and normalised using Illumina BeadStudio 2.0 software. Cubic spline normalisation and the t-test error model were employed for all the FAP adenoma, normal colon and desmoid tumour samples. Correlation analyses identified the average R2 value of the duplicates for each sample as 0.950 ± 0.04. An average of each duplicate pair was then taken before additional analysis was carried out.

Differential gene expression analysis in FAP adenomas and healthy colon tissue

Differential analysis using the mixed normal colon control as the reference group was applied to all adenoma and tumour samples. Genes in each analysis were excluded if their fluorescence detection score was less than 0.99, and if their differential score was less than 13 (p > 0.05). From the genes that met the exclusion criteria, according to detection and differential scores, lists were generated for genes both up- and down-regulated more than 2-fold in the FAP adenoma samples compared to the mixed normal colon control. The genes commonly up- and down-regulated across all the FAP adenomas are shown in Tables 1 and 2 and genes that were commonly up- or down-regulated across the 4 colorectal FAP adenomas only are shown in Tables 3 and 4 respectively.
Table 1

Genes commonly up-regulated more than 2-fold in all FAP polyps compared to normal colon

Symbol

Gene Name

Transcription/Transcriptional Regulation

TBPL1

TBP-like 1

Other

 

ZCWCC2

Zinc finger, CW-type with coiled-coil domain 2

KIAA1324

Maba1

FLJ20366

Hypothetical protein FLJ20366

ATOH8

Atonal homolog 8 (Drosophila)

Table 2

Genes commonly down-regulated more than 2-fold in all FAP polyps compared to normal colon

Symbol

Gene Name

Cell Cycle Control

 

PPP3CB

Protein phosphatase 3 (formerly 2B), catalytic subunit, beta isoform (calcineurin A beta)

Transport

 

SLC20A1

Solute carrier family 20 (phosphate transporter), member 1

P2RX4

Purinergic receptor P2X, ligand-gated ion channel, 4, tv-2

Metabolism

 

PC

Pyruvate carboxylase, nuclear gene encoding mitochondrial protein, tv-2

PRSS3

Protease, serine, 3 (mesotrypsin)

ST6GALNAC6

CMP-NeuAC: (beta)-N-acetylgalactosaminide (alpha) 2,6-sialyltransferase member IV

Signal Transduction

 

IL2RG

Interleukin 2 receptor, gamma (severe combined immunodeficiency)

TJP3

Tight junction protein 3 (zona occludens 3)

Cell Adhesion

 

CDC42

Cell division cycle 42 (GTP binding protein, 25 kDa), tv-2

GSN

Gelsolin (amyloidosis, Finnish type), tv-2

TAGLN

Transgelin

Apoptosis

 

DAPK3

Death-associated protein kinase 3

Structural

 

KRT19

Keratin 19

TPM2

Tropomyosin 2 (beta)

Other

 

CTGF

Connective tissue growth factor

EPS8L2

EPS8-like 2

LRRC1

Leucine rich repeat containing 1

NS5ATP13TP2

NS5ATP13TP2 protein

PTPRR

Protein tyrosine phosphatase, receptor type, R, tv-2

RICH1

RhoGAP interacting with CIP4 homologs 1

SMTN

Smoothelin, tv-2

Table 3

Genes commonly up-regulated 2-fold or more in colorectal FAP polyps compared to normal colon

Symbol

Gene Name

Cell Cycle Control

 

CCNB2

Cyclin B2

CDKN3

Cyclin-dependent kinase inhibitor 3

AURKB

Aurora kinase B

Cell Cycle

 

HCAP-G

Chromosome condensation protein G

PRC1

Protein regulator of cytokinesis 1, tv-1

KIF2C

Kinesin family member 2C

CHC1

Chromosome condensation 1

SMC4L1

SMC4 structural maintenance of chromosome 4-like 1 (yeast)

Pfs2

DNA replication complex GINS protein PSF2

RNASEH2A

Ribonuclease H2, large subunit

Transcription/Transcriptional Regulation

FLJ20315

Hypothetical protein FLJ20315

TBPL1

TBP-like 1

LOC89958

Hypothetical protein LOC89958

HMGN1

High-mobility group nucleosome binding domain 1

ZNF22

Zinc finger protein 22 (KOX 15)

PTTG1

Pituitary tumour-transforming 1

NFE2L3

Nuclear factor (erythroid-derived 2)-like 3

SOX9

SRY (sex determining region Y)-box 9 (campomelic dysplasia, autosomal sex-reversal)

Transport

 

SLC12A2

Solute carrier family 12 (sodium/potassium/chloride transporters) member 2

CLCA1

Chloride channel, calcium activated, family member 1

LCN2

Lipocalin 2 (oncogene 24p3)

Metabolism

 

SORD

Sorbitol dehydrogenase

TPRT

Trans-prenyltransferase

QTRT1

Queuine tRNA-ribosyltransferase 1 (tRNA-guanine transglycosylase)

PAICS

Phosphoribosylaminoimidazole carboxylase, Phosphoribosylaminoimidazole succinocarboxamide synthetase

DPH2L2

DPH2-like 2 (S. cerevisiae), tv-1

ALOX5

Arachidonate 5-lipoxygenase

IARS

Isoleucine-tRNA synthetase, tv-short

BRIX

BRIX

TK1

Thymidine kinase 1, soluble

Oncogenesis

 

EPHB2

EphB2 (EPHB2), tv-1

BCL11A

B-cell CLL/lymphoma 11A (zinc finger protein) tv-1

MAP17

Membrane-associated protein 17

GDF15

Growth differentiation factor 15

Signalling

 

RACGAP1

Rac GTPase activating protein 1

mRNA Processing

 

LSM5

LSM5 homolog, U6 small nuclear RNA associated (S. cerevisiae)

THOC3

THO complex 3

Cell Adhesion

 

C20orf42

Chromosome 20 open reading frame 42

Translation

 

UK114

Translational inhibitor protein p14.5

Other

 

ZCWCC2

Zinc finger, CW-type with coiled-coil domain 2

KIAA1324

Maba1

FLJ10514

Hypothetical protein FLJ10514

ENC1

Ectodermal-neural cortex (with BTB-like domain)

PTTG2

Pituitary tumour-transforming 2

C21orf59

Chromosome 21 open reading frame 59

WDR12

WD repeat domain 12

LXN

Latexin protein

Other

 

KIAA1892

KIAA1892

KIAA1797

KIAA1797

GLCE

Glucuronyl C5-epimerase

KIAA0101

KIAA0101 gene product

RRP46

Exosome component Rrp46

S100P

S100 calcium binding protein P

PRDX4

Peroxiredoxin 4

FLJ20366

Hypothetical protein FLJ20366

F12

Coagulation factor XII (Hageman factor)

IGFBP2

Insulin-like growth factor binding protein 2 (36 kD)

GW112

Differentially expressed in hematopoietic lineages

C10orf3

Chromosome 10 open reading frame 3

ATOH8

Atonal homolog 8 (Drosophila)

MFN1

Mitofusin 1, nuclear gene encoding mitochondrial protein, tv-2

QPCT

Glutaminyl-peptide cyclotransferase (glutaminyl cyclase)

UBE2S

Ubiquitin-conjugating enzyme E2S

Table 4

Genes commonly down-regulated 2-fold or more in colorectal FAP polyps compared to normal colon

Symbol

Gene Name

Cell Cycle Control

 

FOSB

FBJ murine osteosarcoma viral oncogene homolog B

PPP3CB

Protein phosphatase 3, catalytic subunit, beta isoform (calcineurn A beta)

Cell Cycle

 

MXI1

MAX interacting protein 1, tv-2

CABLES1

Cdk5 and Abl enzyme substrate 1

PMP22

Peripheral myelin protein 22, tv-3

DTR

Diphtheria toxin receptor (heparin-binding epidermal growth factor-like growth factor)

Transcription/Transcriptional Regulation

HLX1

H2.0-like homeo box 1 (Drosophila)

NKX2–3

NK2 transcription factor related, locus 3 (Drosophila)

SOX18

SRY (sex determining region Y)-box 18

FNBP1

Formin-binding protein 1

COL4A1

Collagen, type IV, alpha 1

SIRT6

Sirtuin (silent mating type information regulation 2 homolog) 6 (S. cerevisiae)

SIRT7

Sirtuin (silent mating type information regulation 2 homolog) 7 (S. cerevisiae)

AIM1L

Absent in melanoma 1-like

C19orf21

Chromosome 19 open reading frame 21

Transport

 

FBXO32

F-box only protein 32, tv-2

KCNMA1

Potassium large conductance calcium-activated channel, subfamily M, alpha member 1

MYADM

Myeloid-associated differentiation marker

AQP8

Aquaporin 8

SLC17A4

Solute carrier family 17 (sodium phosphate), member 4

SLCO2A1

Solute carrier organic anion transporter family, member 2A1

SGK

Serum/glucocorticoid regulated kinase

P2RX4

Purinergic receptor P2X, ligand-gated ion channel, 4, tv-2

SLC20A1

Solute carrier family 20 (phosphate transporter), member 1

VAMP5

Vesicle-associated membrane protein 5 (myobrevin)

Metabolism

 

MGC4171

Hypothetical protein MGC4171

LIPH

Lipase, member H

KIAA0992

Palladin

KIAA0828

KIAA0828 protein

SULT1A2

Sulfotransferase family, cytosolic, 1A, phenol-preferring, member 2, tv-1

UPP1

Uridine phosphorylase 1, tv-1

BTNL3

Butyrophilin-like 3, tv-2

KIAA0934

KIAA0934 protein

AK1

Adenylate kinase 1

DPYSL3

Dihydropyrimidinase-like 3

PLCD1

Phospholipase C, delta 1

CA4

Carbonic anhydrase IV

SVIL

Supervillin, tv-1

PC

Pyruvate carboxylase, nuclear gene encoding mitochondrial protein, tv-2

TMPRSS2

Transmembrane protease, serine 2

PRSS3

Protease, serine, 3 (mesotrypsin)

PCK1

Phosphoenolpyruvate carboxykinase 1 (soluble)

ST6GALNAC6

CMP-NeuAC: (beta)-N-acetylgalactosaminide (alpha)2,6-sialyltransferase member IV

RARRES2

Retinoic acid receptor responder (tazarotene induced) 2

Tumour Suppression

 

PPAP2A

Phosphatidic acid phosphatase type 2A, tv-1

Signalling

 

RGL1

Ral guanine nucleotide dissociation stimulator-like 1

EFNA1

Ephrin-A1, tv-1

SDCBP2

Syndecan binding protein (syntenin) 2, tv-2

GUCA2A

Guanylate cyclase activator 2A (guanylin)

BSG

Basigin (OK blood group), tv-4

TRIF

TIR domain containing adaptor inducing interferon-beta

ILK

Integrin-linked kinase

TJP3

Tight junction protein 3 (zona occludens 3)

PRKCD

Protein kinase C, delta

ITPKA

Inositol 1,4,5-trisposphate 3-kinase A

IL2RG

Interleukin 2 receptor, gamma (severe combined immunodeficiency)

LNK

Lymphocyte adaptor protein

Cell Adhesion

 

PC-LKC

Protocadherin LKC

DCN

Decorin, tv-E

FLNA

Filamin A, alpha (actin binding protein 280)

MSN

Moesin

SORBS1

Sorbin and SH3 domain containing 1

TAGLN

Transgelin

CDC42

Cell division cycle 42 (GTP binding protein, 25 kDa), tv-2

COL4A2

Collagen, type IV, alpha 2

DBN1

Drebin 1, tv-1

GSN

Gelsolin (amyloidosis, Finnish type), tv-2

ACTG2

Actin, gamma 2, smooth muscle, enteric

ACTA2

Actin, alpha 2, smooth muscle, aorta

CGN

Cingulin

Apoptosis

 

RIPK3

Receptor-interacting serine-threonine kinase 3

FOSL2

FOS-like antigen 2

DAPK3

Death-associated protein kinase 3

LGALS1

Lectin, galactoside-binding, soluble, 1 (galactin 1)

GADD45B

Growth arrest and DNA-damage-inducible, beta

Structural

 

CLDN5

Claudin 5 (transmembrane protein deleted in velocardiofacial syndrome)

KRT19

Keratin 19

TPM2

Tropomyosin 2 (beta)

Other

 

DUSP5

Dual specificity phosphatase 5

CLIPR-59

CLIP-170-related protein

PTPRR

Protein tyrosine phosphatase, receptor type, R, tv-2

SMTN

Smoothelin, tv-2

CEACAM1

Carcinoembryonic antigen-related cell adhesion molecule 1 (biliary glycoprotein)

EPS8L2

EPS8-like 2

RICH1

RhoGAP interacting with CIP4 homologs 1

PDZK2

PDZ domain containing 2

CHKL

Choline kinase-like, tv-1

DIP13B

DIP13 beta

NS5ATP13TP2

NS5ATP13TP2 protein

M-RIP

Myosin phosphatase-Rho interacting protein

MTMR9

Myotubularin related protein 9

LRRC1

Leucine rich repeat containing 1

CTGF

Connective tissue growth factor

DSCR1L1

Down syndrome critical region gene 1-like 1

TU12B1-TY

TU12B1-TY protein

MYH11

Myosin, heavy polypeptide 11, smooth muscle, tv-SM1

FLJ23471

MICAL-like 2, tv-2

DKFZP434B044

Hypothetical protein DKFZp434B044

MUCDHL

Mucin and cadherin-like, tv-2

MMP28

Matrix metalloproteinase 28, tv-1

TRIM15

Tripartite motif-containing 15, tv-1

COL6A2

Collagen, type VI, alpha 2, tv-2C2

SELM

Selenoprotein SelM

ZAK

Sterile alpha motif and leucine zipper containing kinase AZK

SMTN

Smoothelin, tv-3

TNXB

Tenascin XB, tv-XB-S

EPS8L1

EPS8-like 1, tv-3

FLJ10350

Hypothetical protein FLJ10350

DKFZP762C186

Tangerin

TBC1D1

TBC1 (tre-2/USP6, BUB2, cdc16) domain family, member 1

KIAA1145

KIAA1145 protein

PKIG

Protein kinase (cAMP-dependent, catalytic) inhibitor gamma, tv-2

PKIB

Protein kinase (cAMP-dependent, catalytic) inhibitor beta, tv-3

IGSF9

Immunoglobulin superfamily, member 9

LOC90313

Hypothetical protein BC004507

FLJ22582

Hypothetical protein FLJ22582

KIAA0063

KIAA0063 gene product

FSTL1

Follistatin-like 1

PRNP

Prion protein (Creutzfeld-Jakob disease, Gerstmann-Strausler-Scheinker syndrome, fatal familial insomnia), tv-2

ANKRD25

Ankyrin repeat domain 25

STOM

Stomatin, tv-2

FLJ46603

FLJ46603 protein

RAIN

Ras-interacting protein

DHRS9

Dehydrogenase/reductase (SDR family) member 9, tv-1

LIMS2

LIM and senescent cell antigen-like domains 2

ARHGEF18

Rho/rac guanine nucleotide exchange factor (GEF) 18

KIAA0285

KIAA0285 gene product

PDLIM7

PDZ and LIM domain 7 (enigma), tv-1

CXX1

CAAX box 1

MGP

Matrix GIa protein

PTPRH

Protein tyrosine phosphatase, receptor type, H

SPARC

Secreted protein, acidic, cysteine-rich (osteonectin)

FLJ90022

Hypothetical protein FLJ90022

SERPING1

Serine (or cysteine) proteinase inhibitor, clade G (C1 inhibitor), member 1, (angioedema, hereditary)

CSRP1

Cysteine and glycine-rich protein 1

KIAA0513

KIAA0513 gene product

OAS1

2',5'-oligoadenylate synthetase 1, 40/46 kDa

Cluster analysis was performed using GeneSpring 5.0 software in order to further characterise the similarity across the FAP samples and to determine if there was differential gene expression compared to healthy colon tissue. The stomach FAP duplicates display profiles slightly distinct from the other FAP adenomas. The normal colon duplicate profiles are unique to all other profiles (Figure 1).
Figure 1

Cluster analysis of FAP polyps and mixed normal colon. The columns represent the gene expression profiles of each sample. Green – low expression level, yellow – medium expression level, red – high expression level. The relationships between each sample are shown by the upper dendrogram. The colouring in the upper dendrogram represents the sample type: green (left) – normal colon; blue – colorectal FAP polyps; yellow – stomach FAP. 1 – Normal Colon Duplicate; 2 – Normal Colon Duplicate; 3 – Colorectal FAP Polyp A Duplicate; 4 – Colorectal FAP Polyp A Duplicate; 5 – Colorectal FAP Polyp D Duplicate; 6 – Colorectal FAP Polyp D Duplicate; 7 – Colorectal FAP Polyp B Duplicate; 8 – Colorectal FAP Polyp B Duplicate; 9 – Colorectal FAP Polyp C Duplicate; 10 – Colorectal FAP Polyp C Duplicate; 11 – Stomach FAP Polyp D Duplicate; 12 – Stomach FAP Polyp D Duplicate.

Differential gene expression analysis in desmoid tumours and control fibroblasts

The average expression in the desmoid tumours was compared to the control fibroblast cell line and significantly altered expression identified by differential gene expression analysis. Genes in each analysis were excluded if their fluorescence detection score was less than 0.99, and if their differential score was less than 13 (p > 0.05). Genes with differential expression and up- or down-regulated more than 2-fold in the desmoid tumour samples compared to the normal fibroblast cell line were compiled into lists (Tables 5 and 6).
Table 5

Genes commonly up-regulated 2-fold or more in desmoid tumours compared to normal fibroblast cells

Symbol

Gene Name

Cell Cycle Control

 

PTN

Pleiotrophin (heparin binding growth factor 8, neurite growth-promoting factor 1)

GAS7

Growth arrest-specific 7, tv-b

CDKN1C

Cyclin-dependent kinase inhibitor 1C (p57, Kip2)

TGFB3

Transforming growth factor, beta 3

Cell Cycle

 

NEK3

NIMA (never in mitosis gene a)-related kinase 3, tv-2

Transcription/Transcriptional Regulation

BHLHB2

Basic helix-loop-helix domain containing, class B, 2

COL4A1

Collagen, type IV, alpha 1

COL4A2

Collagen, type IV, alpha 2

DNAJB2

DnaJ (Hsp40) homolog, subfamily B, member 2

ELF2

E74-like factor 2 (ets domain transcription factor), tv-1

EVI1

Ecotropic viral integration site 1

FKBP1A

FK506 binding protein 1A, 12 kDa, tv-12A

FLJ10404

Hypothetical protein FLJ10404

HDAC8

Histone deacetylase 8

JUN

v-jun sarcoma virus 17 oncogene homolog (avian)

KIF2C

Kinesin family member C2

NUCKS

Nuclear ubiquitous casein kinase and cyclin-dependent kinase substrate

PBX2

Pre-B-cell leukemia transcription factor 2

PPIE

Peptidylprolyl isomerase E (cyclophilin E), tv-2

PRR3

Proline-rich polypeptide 3

TEAD2

TEA domain family member 2

TLE2

Transducin-like enhancer of split 2 (E(sp1)) homolog, Drosophila

TLE4

Transducin-like enhancer of split 4 (E(sp1)) homolog, Drosophila

ZNF22

Zinc finger protein 22 (KOX15)

ZNF254

Zinc finger protein 254

TDRD3

Tudor domain containing 3

ZNF300

Zinc finger protein 300

MEF2C

MADS box transcription enhancer factor 2, polypeptide C (myocyte enhancer factor 2C)

NAB1

NGFI-A binding protein 1 (EGR1 binding protein 1)

Hes4

bHLH factor Hes4

C19orf13

Chromosome 19 open reading frame 13

ARNT

Aryl hydrocarbon receptor nuclear translocator, tv-2

ZNF266

Zinc finger protein 266

ZNF26

Zinc finger protein 26 (KOX 20)

MGC51082

Hypothetical protein MGC51082

TGIF2

TGFB-induced factor 2 (TALE family homeobox)

MYST3

MYST histone acetyltransferase (monocytic leukemia) 3

M96

Likely ortholog of mouse metal response element binding transcription factor 2

BAZ2B

Bromodomain adjacent to zinc finger domain, 2B

Transport

 

NXT1

NTF2-like export factor 1

ABCA1

ATP-binding cassette, sub-family A, member 1

SLC25A29

Solute carrier family 25, member 29

SLC16A9

Solute carrier family 16 (monocarboxylic acid transporters), member 9

PSCD1

Pleckstrin homology, Sec7 and coiled-coil domains 1(cytohesin 1), tv-2

AQP1

Aquaporin 1(Channel-forming integral protein, 28 kDa) tv-1

SCNN1D

Sodium channel, nonvoltage-gated, delta

SLC22A17

Solute carrier family 22 (organic cation transporter), member 17, tv-2

Metabolism

 

SULT1A1

Sulfotransferase family, cytosolic, 1A, phenol-preferring, member 1, tv-1

CH25H

Cholesterol 25-hydroxylase

QTRTD1

Queuine tRNA-ribosyltransferase domain containing 1

FLJ23749

Hypothetical protein FLJ23749

FLJ10706

Hypothetical protein FLJ10706

USP52

Ubiquitin specific protease 52

RARRES2

Retinoic acid receptor responder (tazarotene induced) 2

ADAM19

A distintegrin and metalloproteinase domain 19 (meltrin beta), tv-2

AUTS2

Autism susceptibility candidate 2

GALNT3

UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3 (GalNAc-T3)

KIAA0140

KIAA0140

ODC-p

Ornithine decarboxylase-like

PCSK5

Proprotein convertase subtilisin/kexin type 5

Oncogenesis

 

AKAP13

A kinase (PRKA) anchor protein 13, tv-3

MGP

Matrix Gla protein

EWSR1

Ewing sarcoma breakpoint region 1, tv-EWS-b

SFRP4

Secreted frizzled-related protein 4

SRPUL

Sushi-repeat protein

Signalling

 

GABBR1

Gamma-aminobutyric acid (GABA) B receptor, 1, tv-2

CAPS

Calcyphosine, tv-2

NET1

Neuroepithelial cell transforming gene 1

PRKCH

Protein kinase C, eta

PPP2R2B

Protein phosphatase 2 (formerly 2A), regulatory subunit B (PR52), beta isoform, tv-4

RGS16

Regulator of G-protein signalling 16

PTHR1

Parathyroid hormone receptor 1

TMPEI

Transmembrane, prostate androgen induced RNA, tv-4

ARHU

Ras homolog gene family, member U

CHN1

Chimerin (chimaerin) 1

EFNB3

Ephrin-B3

GFRA2

GDNF family receptor alpha 2

GNB4

Guanine nucleotide binding protein (G protein), beta polypeptide 4

IL11RA

Interleukin 11 receptor, alpha, tv-1

ITPKB

Inositol 1,4,5-trisphosphate 3-kinase B

KIF13B

Kinesin family member 13B

MAP4K1

Mitogen-activated protein kinase kinase kinase kinase 1

MLP

MARCKS-like protein

PDGFRL

Platelet-derived growth factor receptor-like

PRKCABP

Protein kinase C, alpha binding protein

RASD1

RAS, dexamethasone-induced 1

TNFAIP6

Tumour necrosis factor, alpha-induced protein 6

Cell Adhesion

 

COL7A1

Collagen, type VII, alpha 1 (epidermolysis bullosa, dystrophic, dominant and recessive)

ISLR

Immunoglobulin superfamily containing leucine-rich repeat, tv-1

Apoptosis

 

PPP1R13B

Protein phosphatase 1, regulatory (inhibitor) subunit 13B

AXUD1

AXIN1 up-regulated 1

CASP10

Caspase 10, apoptosis-related cysteine protease, tv-B

MX1

Myxovirus (influenza virus) resistance 1, interferon-inducible protein p78 (mouse)

PCBP4

Poly(rC) binding protein 4, tv-4

TNFRSF19

Tumour necrosis factor receptor superfamily, member 19, tv-2

TNFRSF25

Tumour necrosis factor receptor superfamily, member 25, tv-7

Tumourigenesis

 

BARD1

BRCA1 associated RING domain 1

LOH11CR2A

Loss of heterozygosity, 11, chromosomal region 2, gene A

Immune Response

 

HLA-DPA1

Major histocompatibility complex, class II, DP alpha 1

C1R

Complement component 1, r subcomponent

CXCL14

Chemokine (C-X-C motif) ligand 14

IFI27

Interferon, alpha-inducible protein 27, tv-α

MX2

Myxovirus (influenza virus) resistance 2 (mouse)

RNA Processing

 

DHX8

DEAH (Asp-Glu-Ala-His) box polypeptide 8

HNRPA1

Heterogeneous nuclear ribonucleoprotein A1, tv-1

SFRS11

Splicing factor, arginine/serine-rich 11

Structural

 

ACTL6

Actin-like 6

FBLN1

Fibulin 1 (FBLN1), tv-C

FBLN1

Fibulin 1 (FBLN1), tv-D

SMTN

Smoothelin, tv-2

Other

 

MT1H

Metallothionein 1H

C12orf14

Chromosome 12 open reading frame 14

PELI1

Pellino homolog 1 (Drosophila)

IFI44

Interferon-induced protein 44

C10orf6

Chromosome 10 open reading frame 6

C2orf11

Chromosome 2 open reading frame 11

FLJ31951

Hypothetical protein FLJ31951

ISYNA1

Myo-inositol 1-phosphate synthase A1

FLJ31614

Hypothetical protein FLJ31614

AD031

AD031 protein

CASC3

Cancer susceptibility candidate 3

GBA2

Glucosidase, beta (bile acid) 2

CGI-85

CGI-85 protein, tv-2

C14orf80

Chromosome 14 open reading frame 80

ACAS2L

Acetyl-Coenzyme A synthetase 2 (AMP forming)-like, nuclear gene encoding mitochondrial protein

DTX3

Deltex 3 homolog (Drosophila)

FLJ23059

Hypothetical protein FLJ23059

PIK3R1

Phosphoinositide-3-kinase, regulatory subunit, polypeptide 1 (p85 alpha), tv-2

KIAA1223

KIAA1223

STARD9

START domain containing 9

LOC375786

Hypothetical gene supported by AL713796

SR140

U2-associated SR140 protein

MIDN

Midnolin

SEC31L2

SEC31-like 2 (S. cerevisiae), tv-1

FLJ12178

Hypothetical protein FLJ12178

LOC157567

Hypothetical protein LOC157567

FLJ25005

FLJ25005 protein

WARP

von Willebrand factor A domain-related protein, tv-1

KIAA1036

KIAA1036

LOC374969

Hypothetical protein LOC374969

LOC155435

Hypothetical protein LOC155435

MGC9913

Hypothetical protein MGC9913

CASKIN2

CASK interacting protein 2

CFDP1

Craniofacial development protein 1

SPAG5

Sperm associated antigen 5

MMP23B

Matrix metalloproteinase 23B

AKAP8L

A kinase (PRKA) anchor protein 8-like

FLJ11029

Hypothetical protein FLJ11029

DDIT4

DNA-damage-inducible tv-4

APCDD1

Adenomatous Polyposis Coli down-regulated 1

CDW92

CDW92 antigen

Table 6

Genes commonly down-regulated 2-fold or more in desmoid tumours compared to normal fibroblast cells

Symbol

Gene Name

Cell Cycle

 

GRN

Granulin

QSCN6

Quiescin Q6

STAT1

Signal transducer and activator of transcription 1,91 kDa, tv-α

STAT1

Signal transducer and activator of transcription 1,91 kDa, tv-β

TIMP1

Tissue inhibitor of metalloproteinase 1 (erythroid potentiating activity, collagenase inhibitor)

Transcription/Transcriptional Regulation

HIST1H2BK

Histone 1, H2bk

LOXL1

Lysyl oxidase-like 1

MSC

Musculin (activated B-cell factor-1)

PRRX1

Paired related homeobox 1, tv-pmx-1b

ZDHHC14

Zinc finger, DHHC domain containing 14

Transport

 

GLRB

Glycine receptor, beta

PCOLCE2

Procollagen C-endopeptidase enhancer 2

SCAMP3

Secretory carrier membrane protein 3, tv-1

SLC31A2

Solute carrier family 31 (copper transporters), member 2

Metabolism

 

AK1

Adenylate kinase 1

AKR1C3

Aldo-keto reductase family 1, member C3 (3-alpha hydroxysteroid dehydrogenase, type II)

C1RL

Complement component 1, r subcomponent-like

COMT

Catechol-O-methyltransferase, tv-MB-COMT

CTSL

Cathepsin L, tv-2

GCLM

Glutamate-cysteine ligase, modifier subunit

GNPDA2

Glucosamine-6-phosphate deaminase 2

IDH1

Isocitrate dehydrogenase 1 (NADP+), soluble

NQO1

NAD(P)H dehydrogenase, quinone 1

PTGIS

Prostaglandin I2 (prostacyclin) synthase

SMPDL3A

Sphingomyelin phosphodiesterase, acid-like 3A

SPPL2A

Putative intramembrane cleaving protease

STS

Steroid sulfatase (microsomal), arylsulfatase C, isozyme S

UBE2G1

Ubiquitin-conjugating enzyme E2G 1 (UBC7 homolog, C. elegans), tv-1

UCHL1

Ubiquitin carboxyl-terminal esterase L1 (ubiquitin thiolesterase)

Tumour Suppression

 

MADH3

MAD, mothers against decapentaplegic homolog 3 (Drosophila)

Signalling

 

DEPDC6

DEP domain containing 6

DIRAS1

DIRAS family, GTP-binding RAS-like 1

PDGFRA

Platelet-derived growth factor receptor, alpha polypeptide

PENK

Proenkephalin

SARA2

SAR1a gene homolog 2 (S. cerevisiae)

SNTB1

Syntrophin, beta 1 (dystrophin-associated protein A1, 59 kDa, basic component 1)

DKFZp564I1922

Adlican

mRNA Processing

 

CSTF1

Cleavage stimulation factor, 3' pre-RNA, subunit 1, 50 kDa

Cell Adhesion

 

CNTNAP1

Contactin-associated protein 1

THBS2

Thrombospondin 2

ZYX

Zyxin

Apoptosis

 

C20orf97

Chromosome 20 open reading frame 97

DAPK1

Death-associated protein kinase 1

MAPK1

Mitogen-activated protein kinase 1, tv-1

Structural

 

KRT18

Keratin 18, tv-1

TUBG1

Tubulin, gamma 1

Immune Response

 

ANKRD15

Ankyrin repeat domain 15, tv-1

DPP4

Dipeptidylpeptidase 4 (CD26, adenosine deaminase complexing protein 2)

MR1

Major histocompatibility complex, class I-related

Other

 

ANGPTL2

Angiopoietin-like 2

ANTXR2

Anthrax toxin receptor 2

BCKDHB

Branched chain keto acid dehydrogenase E1, beta polypeptide (maple syrup urine disease), nuclear gene

 

encoding mitochondrial protein, tv-2

BZRP

Benzodiazapine receptor (peripheral), tv-PBR-S

C11orf17

Chromosome 11 open reading frame 17, tv-2

C6orf32

Chromosome 6 open reading frame 32

C9orf88

Chromosome 9 open reading frame 88

CDC42EP2

CDC42 effector protein (Rho GTPase binding) 2

CRLF1

Cytokine receptor-like factor 1

DIRC2

Disrupted in renal carcinoma 2

EDEM1

ER degradation enhancer, mannosidase alpha-like 1

FLJ20073

FLJ20073 protein

FLJ20272

Hypothetical protein FLJ20272

FLJ22582

Hypothetical protein FLJ22582

HOM-TES-103

HOM-TES-103 tumour antigen-like, tv-3

HSPC157

HSPC157 protein

KIAA0196

KIAA0196 gene product

LOC196463

Hypothetical protein LOC196463

LOC221091

Similar to hypothetical protein

LOC286343

Hypothetical protein LOC286343

LOC387908

Similar to Ferritin heavy chain (Ferritin H subunit)

LOC57168

Similar to aspartate beta hydroxylase (ASPH)

LRRFIP2

Leucine rich repeat (in FLII) interacting protein 2

LYPLA1

Lysophospholipase I

MGC12992

Hypothetical protein MGC12992

MGST1

Microsomal glutathione S-transferase 1, tv-1a

MOCOS

Molybdenum cofactor sulfurase

NNT

Nicotinamide nucleotide transhydrogenase

PKM2

Pyruvate kinase, muscle, tv-1

PPAP2B

Phosphatidic acid phosphatase type 2B, tv-2

PSFL

Anterior pharynx defective 1B-like

PTX3

Pentaxin-related gene, rapidly induced by IL-1 beta

S100A4

S100 calcium binding protein A4 (calcium protein, calvasculin, metastasin, murine placental homolog), tv-2

SLIT3

Slit homolog 3 (Drosophila)

SMP1

Small membrane protein 1

TRIM4

Tripartite motif-containing 4, tv-β

UNQ564

UNQ564

ZC3HAV1

Zinc finger CCCH type, antiviral 1, tv-2

To reveal any correlation between the expression profiles of desmoid tumours and FAP adenomas, the data from each group were compared. In the upper dendrogram (Figure 2) it can be seen that all the FAP adenomas cluster in the same group. The desmoid tumours and the normal fibroblast cell line clustered in an entirely different group to the FAP samples. The FAP adenomas and the normal colon have distinct gene profiles compared to the desmoid tumours and the normal fibroblasts. Within the FAP adenomas, the stomach adenoma and the normal colon have slightly different gene profiles compared to the colorectal adenomas.
Figure 2

Cluster analysis of FAP polyps, normal colon, desmoid tumours and normal fibroblasts. The columns represent the gene expression profiles of each sample. Green – low expression level, yellow – medium expression level, red -high expression level. The relationships between each sample are shown by the upper dendrogram. The colouring in the upper dendrogram represents the sample type: green (left) – normal colon; blue – colorectal FAP polyps; orange – stomach FAP polyp; green (right) – desmoid tumours; purple – fibroblast cell line. 1 – Normal Colon Duplicate; 2 – Normal Colon Duplicate; 3 – Colorectal FAP Polyp A Duplicate; 4 – Colorectal FAP Polyp A Duplicate; 5 – Colorectal FAP Polyp D Duplicate; 6 – Colorectal FAP Polyp D Duplicate; 7 – Colorectal FAP Polyp B Duplicate; 8 – Colorectal FAP Polyp B Duplicate; 9 – Colorectal FAP Polyp C Duplicate; 10 – Colorectal FAP Polyp C Duplicate; 11 – Stomach FAP Polyp D Duplicate; 12 – Stomach FAP Polyp D Duplicate; 13 – Desmoid Tumour A Duplicate; 14 – Desmoid Tumour A Duplicate; 15 – Desmoid Tumour C Duplicate; 16 – Desmoid Tumour C Duplicate; 17 – Fibroblast Cell Line Duplicate; 18 – Fibroblast Cell Line Duplicate.

Discussion

In this study, 24 K Illumina HumRef-8 BeadArrays were used to compare gene expression of FAP adenomas, desmoid tumours and normal fibroblasts. To date there have been a number of small scale gene expression studies on FAP adenoma tissue, the vast majority of which have employed immunohistochemistry (IHC). Most of these studies have been performed on individual genes that include E-cadhein, α-, β- and -catenin, COX-1, COX-2, and c-myc [2025]. In addition, one study used semi-quantitative RT-PCR to study GKLF [26]. The only report examining global gene expression in human FAP adenoma tissue identified 84 differentially expressed genes in adenomas compared to normal colon tissue [27].

In this study, the gene expression profiles obtained from the FAP adenomas indicate that colorectal adenomas are similar but distinctly different to the stomach adenomas. There were a large number of commonly expressed genes identified across the colorectal FAP adenomas, but when the differentially expressed genes from the stomach adenoma were included in the analysis the number of commonly expressed genes decreased dramatically. The genes that were differentially expressed in the four colonic adenomas and one stomach adenoma were investigated more closely in an attempt to identify common genetic features in FAP. From this analysis genes involved in the cell cycle, transcription and metabolism were the most frequently up-regulated. The most frequently down-regulated genes included those involved in metabolism, cell adhesion, signal transduction, transcription and transport. Since adenomas develop due to a breakdown in the fidelity of the Wnt signalling pathway it was not surprising to observe the over-expression of genes involved in cell cycle progression.

Altered Expression of Wnt/β-catenin Target Genes in Colorectal FAP Adenomas

It has been long established that deregulation of the Wnt signalling pathway due to APC mutations plays a major role in the progression of FAP [5]. The Wnt/β-catenin signalling pathway is involved in the control of expression of Sox9, PTTG1 and EphB2, all of which were found to be up-regulated by more than 2-fold in all the colorectal FAP adenomas compared to the normal colon.

PTTG1 is regulated by a TCF binding sequence in its promoter region [28]. The normal function of PTTG1 is to regulate chromosome segregation during cell division [29]. Over-expression of PTTG1 has been reported frequently in various types of cancer, including colorectal, and has been associated with angiogenesis [3032]. The role of PTTG1 in angiogenesis is thought to be a result of its part in mediating the secretion of the basic fibroblast growth factor into the extracellular matrix, which promotes proliferation and migration of colorectal cancer cells [30, 31].

The Sox9 gene encodes a transcription factor that is required for chondrogenesis and male gonad development [32], which is under the control of the Wnt signalling pathway [33]. The expression of the Sox9 gene in the intestine is dependent on the activity of the β-catenin/TCF-4 complex, although it is unknown whether this complex interacts directly with the Sox9 promoter or through another of its targets [33].

The EphB2 gene encodes the Eph receptor B, which has been shown to be a target of the Wnt signalling pathway [34]. There is evidence to suggest that normal patterning in the epithelium of the intestinal crypts is coordinated by EphB2 and its ligand, ephrin B [34]. Over-expression of EphB2 is often found in colorectal cancers, but there is confusion about its role in tumourigenesis. Many studies on other tumours have reported EphB2 over-expression as a marker of poor prognosis, but recent studies in colorectal cancer have suggested otherwise [35, 36].

Altered Expression of Cell Cycle-Related Genes in Colorectal FAP Adenomas

A number of genes found to be commonly up-regulated in the adenomas used in this study have previously been reported as being over-expressed in various types of cancers. These genes include the cell cycle-related genes Chromosome condensation protein G (HCAP-G), Protein regulator of cytokinesis 1 (PRC1), SMC4 structural maintenance of chromosome 4-like 1 (SMC4L1) and Cyclin B2 (CCNB2) [3739]. Although these genes are associated with tumour development none have been thoroughly characterized in FAP to date.

Altered Gene Expression in Desmoid Tumours

A limited number of gene expression studies have been performed on desmoid tumours, primarily due to the difficulties in obtaining tissue. Two reports have studied gene expression in desmoid disease using 6.8 K, 19 K and 33 K Affymetrix microarrays [40, 41]. Skubitz and Skubitz (2004) [40] reported that ADAM12, WISP-1, Sox-11 and fibroblast activation protein-a are uniquely expressed in desmoids. Denys et al. (2004) identified 69 differentially expressed genes in desmoid tumour tissue compared to normal fibroblasts, before focusing on the down-regulation of IGFBP-6 [41].

A number of genes that were identified as being differentially expressed in desmoid tumours in this study have been reported previously. The over-expressed genes include transforming growth factor β3 (TGFβ3), a distintegrin and metalloproteinase domain 19 (ADAM19), chimerin 1 (CHN1), and ephrin-B3 (EFNB3) [40, 41]. The under-expressed genes include quiescin Q6 (QSCN6), prostaglandin I2 synthase (PTGIS), proenkephalin (PENK), keratin 18 (KRT18), cytokine receptor-like factor 1 (CRLF1), pentaxin-related gene (PTX3) and endoglin (ENG) [41].

Ephrin-B3, a Wnt Target Overexpressed in Desmoid Tumours

The known Wnt/β-catenin target gene ephrin-B3 [42] has been found in this study to be up-regulated more than 2-fold in desmoid tumours compared to normal fibroblasts. The ephrins are ligands for the EPH receptor family, whose normal function is to organize cell patterning in the intestinal crypts [34]. In addition, more recent observations suggest that ephrins are tumour suppressors, although the mechanism by which this is affected remains to be clarified [3, 43, 44]. Further investigation into the precise role of ephrin-B3 is required before any conclusions can be made regarding its role in desmoid disease.

Wound Healing-Associated Genes Differentially Expressed in Desmoid Tumours

Two genes, transforming growth factor β-3 (TGFβ3) and pleiotrophin (PTN), were found to be differentially expressed in desmoid tumours. Both genes are associated with wound healing and could potentially explain the growth advantage of desmoid tumours [45].

TGFβ3 is a multifunctional protein, having roles in cell proliferation and differentiation during embryogenesis and wound healing [46]. Pleiotrophin has been reported to be strongly expressed in many human cancers, and is thought to promote malignant transformation and angiogenesis [47]. It is also frequently found to be up-regulated during the wound healing process [48].

In this study, three genes associated with negative regulation of the wound response have been identified as being under-expressed in desmoid tumours. The three genes are: signal transducer and activator of transcription 1 (STAT1), mothers against decapentaplegic homolog 3 (MADH3 or Smad3) and mothers against decapentaplegic homolog 6 (MADH6 or Smad6). STAT1 enhances transcription in response to interferon-, an action which has been shown to inhibit the wound healing response by preventing phosphorylation of Smad2 and Smad3 [49]. This in turn inhibits the action of TGFβ on the wound response [50]. The role of Smad3 in the wound response is not entirely understood; however, the absence of Smad3 causes an accelerated healing response, even though its over-expression has also been shown to promote healing [51, 52]. Smad6 is a known inhibitor of TGFβ, and has shown to be down-regulated in keloids [53].

The abundance of wound response-related genes found to be deregulated in the desmoid tumours in this study adds to the notion that desmoid formation is an abnormal wound response. The finding of over-expressed genes involved in fibroblast proliferation and migration could explain the abnormal proliferation and local invasiveness of desmoid tumours. The down-regulation of angiogenesis-associated genes could account for the poor vascularisation of desmoids.

The limiting factor in this study of desmoid tumours is the small number of desmoids available. In order to reach more conclusions regarding the exact molecular nature of desmoids and their growth mechanisms, a much larger sample size would be required.

Comparison of FAP Adenoma and Desmoid Tumour Molecular Profiles

It has long been recognized that desmoid tumours occur with a much higher frequency in FAP patients than in the general population. The apparent role of aberrant Wnt signalling in both diseases could indicate a molecular similarity between the two. Although Wnt target genes were identified as being up-regulated in both tumour types in this study, the specific genes were different in the two groups. The finding of different Wnt targets could be attributed to the use of different control groups for the FAP adenomas and desmoid tumours. Nevertheless, the molecular profiles obtained using cluster analysis clearly demonstrated that FAP adenomas and desmoid tumours display distinctly different gene expression profiles.

Declarations

Acknowledgements

This work was supported in part by funds from the NBN Childhood Cancer Research Group, the University of Newcastle, the Clive and Vera Ramaciotti Centre for Gene Function Analysis, and the Hunter Medical Research Institute (HMRI).

Authors’ Affiliations

(1)
School of Biomedical Sciences, University of Newcastle
(2)
Hunter Medical Research Institute, New Lambton Heights
(3)
Molecular Genetics, HAPS, New Lambton Heights
(4)
Hunter Medical Research Institute, Level 3 John Hunter Hospital

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