Gene expression profiling of colorectal adenomas and early invasive carcinomas by cDNA array analysis

It is generally accepted that most colorectal carcinomas arise in pre-existing adenomas. Morphologically, colorectal adenomas can be divided into two groups, protruded type and flat type. The aim of this study was to clarify relevant alterations of gene expression associated with the early stage of colorectal carcinogenesis. Using cDNA array, we analysed the expression profiles of 550 cancer-related genes in 36 colorectal adenomas (18 flat-type and 18 protruded-type adenomas) and 14 early invasive carcinomas. Among the 550 genes, we chose 32 genes the average expression levels of which were at least three-fold up- or downregulated in tumour tissues compared with levels in matched normal tissues. A total of 13 and 19 genes were identified as up- and downregulated genes in tumour tissues, respectively. Among the upregulated genes, the average expression levels of E1AF, bone morphogenic protein (BMP)-4, insulin-like growth factor (IGF)-2, inducible nitric oxide synthase (iNOS), tissue inhibitors of metalloproteinase (TIMP)-1, Smad4, and nm23 in tumour tissues were over five times higher than those in matched normal tissues. Colorectal adenomas and early invasive carcinomas were divided into two major clusters by clustering analysis. Moreover, flat- and protruded-type adenomas were divided into two major clusters by clustering analysis. The expression profiles obtained by the cDNA array clearly indicate that colorectal adenomas and early invasive carcinomas have specific expression profiles. Likewise, the gene expression profiles of flat- and protruded-type adenomas are different. These results indicate that molecular classification of early colorectal tumours by a cDNA array is feasible.

malignancies in the world. Although alternative pathways exist, it is generally accepted that most colorectal carcinomas arise in pre-existing adenomas (Jass et al, 2002). Despite a large number of studies, little is known about molecular alterations associated with the heterogeneity of colorectal carcinomas.
Morphologically, early colorectal tumors can be divided into two groups, protruded type and flat type. Recently, flat-type colorectal tumours have been reported not only in Japan (Sakashita et al, 2000) but also in Western countries (Olschwang et al, 1998). Previous studies showed that flat-type colorectal tumours tended to reach deeper layers earlier and to show higher rates of lymphatic invasion and lymph node metastasis than did protruded-type tumours (Kuramoto and Oohara, 1989;Mueller et al, 1998). Moreover, it has been thought that some flat-type cancers correspond to de novo cancers, which contain no observable adenomatous component and may develop through a distinct genetic pathway (Yashiro et al, 2001). Thus, it would be interesting to examine gene expression profiles of colorectal adenomas and early invasive colorectal carcinomas, because comparison of these two groups of tumours will provide information about genes that play an important role during progression from adenoma to carcinoma.
DNA array technology enables measure of the mRNA expression levels of thousands of genes in a single assay. Mainly advanced cancer has been analysed in gene expression profiling-based studies on colorectal cancer (Alon et al, 1999;Backert et al, 1999;Hegde et al, 2001;Kitahara et al, 2001;Notterman et al, 2001;Takemasa et al, 2001;Agrawal et al, 2002;Birkenkamp-Demtroder et al, 2002;Lin et al, 2002;Zou et al, 2002;Frederiksen et al, 2003;Muro et al, 2003;Tureci et al, 2003;Williams et al, 2003;Bertucci et al, 2004). Only a small number of adenoma and early invasive cancer tissues have been analysed in previous studies, and the issue of flat-and protruded-type adenoma tissues has not been directly addressed (Notterman et al, 2001;Agrawal et al, 2002;Lin et al, 2002;Williams et al, 2003).
In this study, we therefore applied cDNA array technology to analyse the gene expression profiles of 36 colorectal adenomas (18 flat-type and 18 protruded-type adenomas) and 14 early invasive carcinomas. This is the first study showing molecular classification of early colorectal tumours by a cDNA array analysis. treatment. These tumour samples consisted of 36 colorectal adenomas and 14 early invasive carcinomas (pT1 in the TNM classification of the Union International Contre Cancer). Normal epithelial tissue samples and tumour tissue samples were carefully macrodissected by expert pathologists. In case of early invasive carcinomas, tumour tissue samples were taken from the macroscopically visible deepest invading part of the tumour after surgical resection. Each tissue specimen was divided into two pieces after resection. For total RNA extraction, one sample was immediately frozen in liquid nitrogen at the time of surgery and stored at À801C until extraction. The other sample was processed for pathological examination using haematoxylin and eosin staining for the evaluation of the tumour cell content. Only specimens containing more than 80% tumour cells were used for analysis (Horiuchi et al, 2003). The histopathological features of the carcinoma specimens were classified according to the TNM classification system. Locations of the colorectal tumours were divided into proximal colon (caecum and ascending and transverse colon) and distal colon (descending and sigmoid colon and rectum). Macroscopic types were divided into protruded type (height of tumour X3 mm) and flat type (height of tumour o3 mm). It was difficult to divide early invasive carcinoma into protruded type or flat type because colorectal tumours become thick when they have invaded the submucosal layer. Therefore, macroscopic type was classified in only colorectal adenomas. The clinicopathological characteristics of colorectal tumours are shown in Table 1. Informed consent was obtained from each subject, and the institutional review committee approved this study.

cDNA array analysis
Total RNA was extracted from specimens using the acid guanidinum thiocyanate -phenol -chloroform extraction method and treated with DNase I. Biotin-labelled cDNA targets were made from 2.5 mg of total RNA using Gene Navigator cDNA amplification system (Toyobo, Osaka, Japan), including random 9-mer, biotin-16-dUTP, and ReverTraAceTM reverse transcriptase. Free biotin-16-dUTP in the reaction was removed by ethanol precipitation. Gene Navigator cDNA array filter (human cancer, Toyobo) consisted of 550 cancer-related genes and 11 housekeeping genes in duplicate. A complete list is available on the Internet (http:// www.toyobo.co.jp). Hybridisation was performed overnight at 681C in PerfectHybTM (Toyobo). Filters were washed three times in 2 Â SSC/0.1% SDS at 681C for 10 min each, followed by three washes in 0.1 Â SSC/0.1% SDS at 681C for 10 min each. Specific signals on the filters were detected by the chemiluminescence detection kit (Imaging highTM, Toyobo). CDP-Star was used as the chemiluminescent substrate. Quantitative assessment of the signals on the filters was performed by scanning on a Fluor-S MultiImager System (Bio-Rad, Richmond, CA, USA) followed by image analysis using ImaGene software (BioDiscovery, Los Angeles, CA, USA). The data were analysed with normalisation to glyceraldehyde-3phosphate dehydrogenase (GAPDH) expression. The average of three experiments was calculated.

Statistical analysis
Expression of each target gene was assessed for associations with clinicopathological characteristics using Mann -Whitney U-test for average tumour expressions.
Among the 13 genes the average expression levels of which were at least three-fold upregulated in tumour tissues compared with levels in matched normal tissues, the average expression levels of E1AF, BMP-4, IGF-2, iNOS, TIMP-1, Smad4, and nm23 genes in tumour tissues were over five times higher than those in matched normal tissues (Table 2). Semiquantitative reverse transcriptasepolymerase chain reaction (RT -PCR) analysis of these differentially expressed genes gave results consistent with those by a cDNA array analysis (Figure 1).

DISCUSSION
Using a cDNA array, we analysed the gene expression profiles of 36 colorectal adenoma and 14 early invasive carcinoma tissues to clarify characteristic changes associated with the early stage Table 2 Average expression levels of E1AF, BMP4, IGF-2, iNOS, TIMP-1, Smad4, and nm23 genes in tumour tissues were over five times higher than those in the matched normal tissues  Flat type Protruded type Figure 2 A two-dimensional hierarchical clustering of 32 genes across 50 colorectal tumours. The colour in each well represents relative expression of each gene (vertical axis) in each paired sample (horizontal axis); red, increased in tumour tissues; green, decreased in the tumour tissues. In the sample axis, early invasive carcinomas and adenomas were separated into two different trunks. In the gene axis, 32 genes were clustered in different branches according to their similarity; the shorter the branches, the greater the similarity. In adenomas, subclusters of flat type and protruded type were selected for further analysis (see Figure 3).
of colorectal carcinogenesis. The reason why we chose early invasive carcinoma is that it represents the early stage of colorectal carcinoma. Among the 550 genes the expression profiles of which were analysed, we chose 32 genes the average expression levels of which were at least three-fold up-or downregulated in 50 tumour tissues compared with levels in 50 matched normal tissues. Among the 13 upregulated genes, the expression levels of E1AF, BMP-4, IGF-2, iNOS, TIMP-1, Smad4, and nm23 genes were over five-times higher than those in matched normal tissues.
E1AF (human PEA3/ETV4) is an ets family transcriptional factor. We recently reported that E1AF plays a key role in the progression of colorectal carcinoma (Horiuchi et al, 2003). Thus, our results of cDNA array analysis extend roles of E1AF in the late stage to early stage of colorectal carcinogenesis. BMP-4 is a member of the TGF-b superfamily of growth factors. It has been reported that BMP-4 is overexpressed and secreted by human colon cancer cells with mutant APC genes (Kim et al, 2002). Our results suggest that BMP-4 overexpression plays an important role in the early stage of colorectal carcinogenesis. iNOS has been −3.0 1:1 3.0 Flat type Protruded type Figure 3 A two-dimensional hierarchical clustering of 32 genes across 36 colorectal adenomas. The colour in each well represents relative expression of each gene (vertical axis) in each paired sample (horizontal axis); red, increased in adenoma tissues; green, decreased in adenoma tissues. In the sample axis, flat-and protruded-type adenomas were separated into two different trunks.
reported to play a crucial role in cancer development by promoting angiogenesis (Jenkins et al, 1995). Our results are consistent with those of previous studies showing an important role of iNOS in the early stage of colorectal carcinogenesis (Xu et al, 2003). Interestingly, nitric oxide (NO), generated by iNOS, reportedly augments the synergistic interaction between E1AF and its transcription coactivator CBP/p300, resulting in the facilitation of induction of tumour-related genes, such as COX-2 (Liu et al, 2004). Several lines of evidence suggest that IGF-2 plays an important role in the progression of colorectal tumours (Lambert et al, 1990). Moreover, expression of IGF-2 protein has been reported to be associated with advanced tumour stage and poor survival (Kawamoto et al, 1998;Peters et al, 2003). It has also been suggested that IGF-2 plays a role in the development of liver metastasis from colorectal cancer (Kawamoto et al, 1999). Thus, our results extend roles of IGF-2 in the late stage to early stage of colorectal carcinogenesis.
Among the four known tissue inhibitors of metalloproteinases (TIMPs), TIMP-1 has functions apart from its protease inhibitory action. Several investigators have reported that TIMP-1 has growth-promoting properties and might also stimulate tumour growth by inhibiting apoptosis (Holten-Andersen et al, 2002). Overexpression of TIMP-1 has been reported in colorectal cancer tissues (Murashige et al, 1996). Holten-Andersen et al (2005) recently reported that TIMP-1 mRNA was detected in all of 24 cases of colorectal cancer tissues by in situ hybridisation, but it was detected in only two of seven adenoma tissues. In the current study, we also found that the average expression levels of TIMP-1 were significantly higher in the early invasive carcinoma group than in the adenoma group. Besides, levels of TIMP-1 in blood were significantly elevated in colorectal cancer patients compared to healthy donors, and high plasma TIMP-1 levels were associated with short survival of colorectal cancer patients (Holten-Andersen et al, 2005). Therefore, TIMP-1 appears to be a novel marker for detection of early colorectal cancer and for prognostic stratification of colorectal cancer patients.
Smad4 is an intracellular transmitter of TGF-b signals and its tumour suppressor function is presumed to reside in its capacity to mediate TGF-b-induced growth inhibition. However, there is accumulating evidence that this hypothesis may be too simple (Muller et al, 2002). Although functional inactivation of Smad4 in colorectal cancer frequently occurs at late stages when tumours acquire invasive and metastatic capabilities, the roles of TGF-b signals in carcinogenesis are complex and also comprise tumourpromoting functions in colorectal carcinogenesis (Muller et al, 2002;Reinacher-Schick et al, 2004). Nevertheless, the reason for the overexpression of Smad4 in early colorectal tumour tissues remains unknown. It may be induced to inhibit tumour growth by some compensatory mechanisms. Further analysis is needed to clarify this issue.
The nm23 gene was first identified as a gene the expression level of which was reduced in highly metastatic rodent tumours relative to poorly metastatic tumour cells (Steeg et al, 1988). The transfection of nm23 cDNA into various cancer cell lines resulted in suppression of the metastatic potential of motility, invasion, or colonisation (Suzuki et al, 2004). However, Bertucci et al (2004) recently reported over-and downexpression of nm23 in colorectal cancer tissues and in those with poor prognosis, respectively. The reason why nm23 gene was highly expressed in tumour tissues in the current study may be due to the fact that all tumour samples We identified 22 genes the expression levels of which differed significantly in colorectal adenomas and early invasive carcinomas. Colorectal adenomas and early invasive carcinomas were divided into two major clusters by clustering analysis. This result is consistent with that of a recent study showing that nine colorectal adenomas were separated from 11 differentiated colorectal carcinomas by using oligonucleotide arrays (Lin et al, 2002). The expression profiles obtained by our cDNA array demonstrated that colorectal adenomas and early invasive carcinomas have specific expression profiles. Among the seven upregulated genes the expression levels of which were over five times higher than those in the matched normal tissues, the expression levels of IGF-2, E1AF, iNOS, nm23, Smad4, and TIMP-1 genes were significantly higher in the early invasive carcinoma group than in the adenoma group. These results suggest that these genes play an important role in the progression from adenoma to carcinoma.
On the other hand, GAK was the most downregulated gene in the early invasive carcinoma group relative to the adenoma group. GAK is a serine/threonine kinase that shows high homology outside its kinase domain with auxilin. Like auxilin, GAK has been shown to be a cofactor for uncoating clathrin vesicles in vitro. Zhang et al (2004) reported that downregulation of GAK by small hairpin RNA increased the levels of epidermal growth factor (EGF) receptor expression and tyrosine kinase activity, resulting in a large increase in the levels of activated extracellular signalregulated kinase 5 and Akt. Moreover, downregulation of GAK has been reported to result in outgrowth of monkey kidney CV1P cells in soft agar, raising the possibility that loss of GAK function may promote tumorigenesis. Thus, our results suggest that downregulation of GAK plays an important role in the progression from colorectal adenoma to carcinoma.
The adenoma -carcinoma sequence (ACS) is widely accepted as a pathogenesis of colorectal carcinoma. A multistep genetic model for colorectal carcinogenesis based on the ACS has been proposed (Vogelstein et al, 1988). In the ACS sequence, mutations in the K-ras gene and various tumour suppressor genes, such as APC and p53, are known to accumulate during the progression from normal to malignant tissue. Although coexistence of all three mutations has been reported to be a rare occurrence (Smith et al, 2002), the majority of sporadic colorectal carcinomas are still thought to develop and progress through this pathway. It has been thought that de novo cancers develop from normal colonic mucosa directly. However, critical genetic abnormality is not known. Most protruded-type colorectal cancers have adenomatous elements in the periphery when found at an early stage, suggesting that these cancers have arisen from pre-existing adenomas. On the other hand, adenomatous components are not detectable microscopically in some flat-type cancers, suggesting that flat-type cancers correspond to de novo cancer (Yashiro et al, 2001). The reason why we could not detect any changes in the expression of APC, p53, and K-ras genes may be due to the fact that mutations of these genes do not necessarily result in alterations of mRNA expression levels.
In the current study, flat-and protruded-type adenomas were divided into two major clusters by clustering analysis. We identified 27 genes the expression levels of which differed significantly in flat-and protruded-type adenomas. The expression levels of 16 genes (PMS1, nm23, p21, FAK, Smad4, c-jun, ECGF-1, Erk1, GAK, GSTP1, IGF-2, Laminin b-3, MMP-15, Mucin3, Rho GDIb, and TIMP-1) were significantly higher in the flat-type group than in the protruded-type group. On the other hand, the expression levels of 11 genes (HLA-DQ, Cdc42, Egr-2, Eph, Galectin-1, gp130, GST-II, MDR1, p120, Ras-GAP, and Rho 8) were significantly lower in the flat-type group than in the protruded-type group. Among the 18 genes the expression levels of which were significantly different in the early invasive carcinoma group and the flat-type adenoma group, the expression levels of eight genes (p21, MUC-2, Erk1, Mucin 3, aN-catenin, PMS1, Lamin b3, and GAK) and 10 genes (Rho GDIb, c-jun, Cdc42, iNOS, Galectin-1, TIMP-1, GSTP1, ECGF1, nm23, and Smad4) were significantly higher and lower, respectively, in the flat-type adenoma group than in the early invasive cancer group. On the other hand, among the 28 genes the expression levels of which were significantly different in the early invasive carcinoma group and the protrudedtype adenoma group, the expression levels of 13 genes (GAK, MUC-2, PMS1, aN-catenin, Cdc42, Eph, gp130, GST-II, Rho 8, Ras-GAP, p120, MDR1, and Egr-2) and 15 genes (iNOS, FAK, MMP-15, E1AF, IGF-2, Laminin b3, Smad4, TIMP1, nm23, Rho GDIb, GSTP1, c-jun, ECGF1, Erk1, and Mucin 3) were significantly higher and lower, respectively, in the protruded-type adenoma group than in the early invasive cancer group. These results suggest that flatand protruded-type adenomas have specific expression profiles and that genes that play a crucial role in the progression from each type of adenoma to carcinoma are different.
In conclusion, the expression profiles obtained by the cDNA array clearly indicate that colorectal adenomas and early invasive carcinomas have specific expression profiles. Likewise, the gene expression profiles of flat-and protruded-type adenomas are different. These results indicate that molecular classification of early colorectal tumours by a cDNA array is feasible.