Significant overexpression of oligophrenin-1 in colorectal tumors detected by cDNA microarray analysis

Cancer Letters 172 (2001) 67±73

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Signi®cant overexpression of oligophrenin-1 in colorectal tumors detected by cDNA microarray analysis NõÂdia Alice Pinheiro a,b, OtaÂvia Luisa Caballero b, Fernando Soares c, Luis F.L. Reis b, Andrew John George Simpson b,* b

a Department of Biochemistry, Instituto de QuõÂmica, University of SaÄo Paulo, SaÄo Paulo, Brazil Laboratory of Cancer Genetics, Ludwig Institute for Cancer Research, Rua Professor Antonio Prudente, 109 ± 4th Floor, 01509010, SaÄo Paulo Branch, SaÄo Paulo, Brazil c Hospital do CaÃncer A.C. Camargo, SaÄo Paulo, Brazil

Received 20 March 2001; received in revised form 25 April 2001; accepted 6 June 2001

Abstract The human oligophrenin-1 gene is ubiquitously expressed at low levels and expressed at high levels in the developing neuroepithelium of the neural tube. Mutations in this gene have been related to the X-linked mental retardation. Using cDNA microarrays, we found evidence that oligophrenin-1 is strongly up-regulated in colorectal tumors. Semiquantitative reverse transcriptase polymerase chain reaction con®rmed this ®nding. Thus, a well-known nervous system-associated human gene transcript may also be an important colorectal tumor marker and potential therapeutic target. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Gene expression; Oligophrenin-1; Microarray; Gene discovery array

1. Introduction Colorectal cancer (CRC) is one of the most common cancers in the western world [1]. It is the most common gastrointestinal malignancy in the United States, and is the fourth most common cause of cancer mortality in men and the ®fth in women in Brazil [2,3]. Considering the signi®cant health threat that CRC poses, efforts to prevent or detect it at a potentially curable stage would seem to be the best way to decrease morbidity and mortality. The progression of normal colon mucosa to adenomatous tissue and then to carcinoma occurs over a * Corresponding author. Tel.: 155-11-270-4922; fax: 155-11270-7001. E-mail address: [email protected] (A.J.G. Simpson).

period of years and in a stepwise fashion. A molecular model describing the events involved in this progression has been proposed [4,5]. The further characterization of colorectal tumor cells at the molecular level and the correlation of phenotypic changes and tumor progression with speci®c gene expression will shed light on mechanisms of tumor development and provide useful genetic markers for diagnosis and prognosis. In this regard, the rapid progress in the identi®cation of human genes and the development of high throughput parallel gene expression pro®ling technologies have initiated a major change in the strategy of gene expression analysis. Thus, the simultaneous study of numerous genes without prior knowledge of their function or expression is now possible, although changes in post-tran-

0304-3835/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(01)00625-5

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scriptional level of proteins cannot be identi®ed with this method. In a large-scale study involving the cDNA arrays, with 18 376 individual cDNA clones, we found an apparent and consistent strong up-regulation of the gene encoding oligophrenin in colorectal tumors. This was subsequently con®rmed by reverse transcription polymerase chain reaction (RT-PCR) in several individual colorectal tumors. Signi®cantly enhanced individual gene expression in tumors is relatively infrequent, as compared with down-regulation, and all such genes identi®ed are of signi®cant and potential importance, either as diagnostic markers or as drug targets or both. 2. Materials and methods 2.1. Samples Colorectal adenocarcinomas were collected from patients during surgery at the Hospital do Cancer A.C. Camargo and rapidly frozen in liquid nitrogen. From all patients, paired tumor and normal tissues were obtained. The latter were isolated from a region at least 5 cm from the tumor margin. The colon and rectum adenocarcinoma specimens were of clinical stages II±IV. A pool of nine and a separate pool of three tumors, and respective normal colorectal tissue pools were used in the studies described (Table 1). From the frozen tumor tissue, 5-mm cryostat sections were cut and stained with toluidine to permit histopathological identi®cation of normal and tumor

regions. Tumor tissues were microdissected free from other tissue components and cells by the same pathologist in all cases (F.S.), using ®ne surgical scalpels. Each tumor sample was estimated to be more than 85% `homogeneous' as determined by microscopic visualization following microdissection. Routine histopathological analysis was used for diagnosis. 2.2. Gene discovery array (GDA) (Incyte Genomics) cDNA microarray ®lters consisting of a 22 £ 22 cm nylon membrane containing 18 376 human cDNAs in duplicate were obtained from Incyte-Genomics, Inc. (http://reagents.incyte.com/GDA/). Two versions of the array were used. Both membranes contain over 18 000 non-redundant DNA clones from the Image Consortium Clone set. In version 1.2, some of the clones on the ®lter were housed in DH10B or SOLR cells, while in version 1.3, all the clones were housed in DH10B and have been streaked to single colonies. Previous analysis of the reproducibility of the hybridization data generated with membranes suggests that spots with hybridization intensity differing more than ®ve-fold between samples are statistically differentially expressed. 2.3. RNA preparation High-quality total RNA was isolated from the frozen tissues disrupted using a Polytron homogenizer. Extraction of total RNA was performed with Trizol w Reagent total RNA isolation reagent (Gibco BRL, Life Technologies, Gaitherburg, MD, USA). All

Table 1 Tissue samples and pools used in experiments using the microarray membranes Tissue

Topography

Morphology

Clinical stage

Number of samples

Experiment code name/ membranes utilized

Nine tissues

Colon, NOS a

II

1

NIC1/(GDA 1.2)

Colon, NOS a Colon, ascending Colon, descending Recto-sigmoid

Adenocarcinoma, moderately differentiated Tubular, adenocarcinoma Mucinous, adenocarcinoma Adenocarcinoma Adenocarcinoma

III IV IV IV

2 1 3 2

Colon, NOS a Colon, NOS a Recto-sigmoid

Adenocarcinoma Adenocarcinoma Adenocarcinoma

III IV IV

1 1 1

Three tissues

a

NOS, not otherwise speci®ed.

NIC2/(GDA 1.2) NIC3/(GDA 1.3)

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the RNAs used in this study were exhaustively treated with RNAse-free DNAse I to remove residual DNA. The ef®cacy of this treatment was evaluated ®rstly by RT-PCR using primers that amplify 379 bp from mitochondria D-loop (5 0 -AATAACAATTGAATGTCTGCAC-3 0 and 5 0 -TTGAGGAGGTAAGCTACATA-3 0 ) as well as a reaction undertaken using primers ¯anking p53 exons 5 and 6 that amplify 268 bp (5 0 -TCACTTGTGCCCTGACTT-3 0 and 5 0 -AACCAGCCCTGTCGTCTC-3 0 ). The fragments ampli®ed with these p53 primers are of different sizes if genomic DNA or c-DNA is present. Both ampli®cations were followed by Southern blotting. The RNA preparations in which positive ampli®cation with Dloop primers was detected were submitted to another round of DNAse treatment. Only RNA preparations shown to be free of DNA as judged by the RT-PCR experiments were used in this study. Poly(A) 1 RNA was isolated using the mRNA isolation kit ± for total RNA (Miltenyi Biotec, Inc.) with MACS Oligo (dt) MicroBeads. From 300 mg of total RNA, 2 mg of poly(A) 1 RNA was isolated. 2.4. cDNA synthesis and labeling [a 32P]dCTP (3000 Ci/mmol, 10 Ci/ml) labeled cDNA was prepared from 2 mg of mRNA using Superscript II reverse transcriptase (LTI Inc.) and oligo-dT according to Incyte-Genomics protocols. Thus, 2.0 mg of mRNA, and 50 mm 5 0 -oligodT18MN were incubated at 708C for 10 min, then 5 £ ®rst-strand buffer, 0.1 M dithiothreitol, 10 mM dA/dG/dT mix, [a 32P]dCTP (3000 Ci/mmol, 10 mCi/ml) and Superscript II reverse transcriptase (200 U/ml) were added and the mixture was incubated for 1 h at 428C. The labeled cDNA was puri®ed using a G-50 spin column. 2.5. Hybridization and scanning of GDA membranes Pairs of GDA membranes were separately hybridized using pools of three or nine colorectal tumors or corresponding normal tissues. The ®rst hybridization was with the pool of nine colorectal tumors and normal tissues using GDA 1.2 membranes. The second hybridization was with the pool of three colorectal tumors and normal tissues. This hybridization used both GDA 1.2 and 1.3 membranes (Table 1). Pre-hybridization was performed at 658C for 30 min with 20 ml of pre-hybridization solution and

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hybridization was performed at 658C overnight [6]. After a high-stringency wash, the hybridization pattern was analyzed using both a Storm 840 PhosphorImager and the GSI images service (Incyte-Genomics, Inc.). 2.6. Semi-quantitative RT-PCR The semi-quantitative analysis of Oligophrenin-1 and GAPDH mRNA levels was carried out by RTPCR where the number of ampli®cation cycles used was not suf®cient to reach the plateau of product accumulation. For this RT-PCR, six microdissected individual colorectal tumors with respective normal tissue were used. One hundred nanograms of poly(A) 1 mRNA isolated from tumor and normal colorectal tissue was reverse transcribed using a Thermoscript RTPCR System (Gibco BRL). For PCR, 5 ng of template was included in a 25 ml PCR mix that contained 4 pmol of forward and reverse primers that amplify 273 bp of the oligophrenin-1 gene (5 0 -GCACTGGGAGATATACCTAATGCTA-3 0 ; 5 0 -TTCTAAGGTAGCTGCAGATAAGTGG-3 0 ), or 195 bp of GAPDH gene (5 0 -CTGCACCACCAACTGCTTA-3 0 ; 5 0 CATGACGGCAGGTCAGGTC-3 0 ), 5 U of recombinant Taq DNA polymerase (Gibco BRL), 125 mM of each dNTP, 1.5 mM MgCl2, 10 mM Tris±HCl (pH 8.0) and 50 mM KCl (Gibco BRL). The reaction was performed with cycling times of 3 min at 948C, 1 and 30 s at 728C, followed by two times (30 s at 948C, 35 s at 628C and 50 s at 728C) and by two times (30 s at 948C, 35 s at 588C and 50 s at 728C) and followed by 28 times (30 s at 948C, 35 s at 548C, and 50 s at 728C). The exponential phase of ampli®cation reaction was established for Oligophrenin at 28 cycles and the GAPDH at 23 cycles. 3. Results 3.1. Detection of differential gene expression pattern by GDA analysis A set of 18 376 human cDNAs coding for proteins of different functional classes arrayed on nylon membranes (GDA 1.2 and GDA 1.3) were used for hybridization. The labeled cDNA probes were synthesized from poly(A) 1 mRNA derived from colorectal

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tumors and normal colorectal tissues. The ®rst experiment was performed with a pool of nine colorectal tumors and corresponding normal colorectal tissue using a pair of GDA 1.2 version membranes from Incyte-Genomics (Experiment code name NIC1). In the second experiment, pools of three colorectal tumors or normal colorectal tissues were used against the same membrane GDA 1.2 after a wash and strip (Experiment NIC2). A third experiment was performed with the same pool of three tumors using the GDA 1.3 version (Experiment NIC3) (Table 1). Many cDNAs were found to be consistently upregulated in colorectal tumors in all experiments. These include genes encoding transcription factors, immunoglobulin kappa constant region, excision repair cross-complementing rodent repair de®ciency, complementation group 2 (xeroderma pigmentosum D), Spi-B transcription factor (Spi-1/PU.1 related), tripeptidyl peptidase II, inhibitor of DNA binding 2, dominant negative helix±loop±helix protein as well as several anonymous expressed sequence tags (ESTs). The most striking and consistent result, however, was obtained with a cDNA encoding oligophrenin-1 (GenBank accession R81942 and Unigene Hs.128824). Between 85 and 90% of the up-regulated genes were identi®ed in all three analyses. Oligophrenin was found to be the most highly over-expressed clone in NIC3 (ratio ˆ 99.9999) and was also found to be over-expressed in NIC2 (ratio ˆ 11.100) and NIC1 (ratio ˆ 14.155) (Table 2). Typical scanned phosphorimages of one of the experiments, NIC3, are shown in Fig. 1. The scanned image shows alterations in the expression of oligophrenin cDNA. To seek in silico corroborating evidence for our unexpected ®nding of signi®cant up-regulation of oligophrenin, we interrogated serial analysis of gene expression (SAGE) map, a component of the Cancer Genome Anatomy Project that provides a central locaTable 2 Results of GDA analysis for Oligophrenin-1 gene GBAcc a Unigene GDA analysis Score Ratio a

GenBank access.

R81942 Hs.128824 NIC1 184.59 11.100

NIC2 564.32 14.155

NIC3 14271.09 99.9999

Fig. 1. Part of the scanned phosphorimages of cDNA microarrays (experiment NIC3). The images were aligned to show visually apparent changes in gene expression, in (A) membrane probed with the pool of three normal colorectal tissues and (B) membrane probed with the pool of three colorectal tumors. Circles indicate the increased expression the oligophrenin gene in tumors.

tion for depositing, retrieving, and analyzing human gene expression data (http://www.ncbi.nlm.nih.gov/ SAGE). The website provides public access to NCI's Cancer Genetic Anatomy Project (CGAP) SAGE data from human colon and brain tissues, and a statistical test for differential analysis between data sets (or libraries). By accessing the SAGEmap the user can compare transcript populations between any of the posted libraries. A virtual Northern tool, vNorthern, has been designed to accept sequence as input. When we used the complete sequence of oligophrenin as input in virtual northern tools we found high-level expression in other malignant library as SAGE CPDR LNCaP-T (prostate carcinoma cell line). 3.2. Oligophrenin mRNA expression in colorectal tumors by semi-quantitative RT-PCR To experimentally con®rm the over-expression of

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Fig. 2. Veri®cation of Oligophrenin-1 expression levels by semi-quantitative RT-PCR analysis. (A) Samples 1±6 correspond, respectively, to rectal adenocarcinoma (stage II), rectosigmoid adenocarcinoma (stage IV), rectal adenocarcinoma (stage III), colon epidermoid adenocarcinoma (stage IV), descending colon adenocarcinoma (stage IV), and ascending colon adenocarcinoma (stage III). Genomic DNA was used as a positive control (DNA). NO represents the negative control in which no DNA was added. (B) GAPDH levels analyzed by semiquantitative RT-PCR.

oligophrenin in colorectal tumors, we performed a semi-quantitative RT-PCR with six individual tumors. The cDNA was produced by RT-PCR from 1.0 mg of RNA from human colorectal tumors. The constitutive gene used as control was glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In the samples from six patients investigated (paired samples of mRNA from normal colonic mucosa and tumor tissue) Oligophrenin-1 was detectable by RT-PCR in six tumor colorectal tissues but barely or not at all detectable in the correspondingly normal colorectal tissue samples (Fig. 2). These data thus con®rmed the over-expression of Oligophrenin-1 in colorectal tumors. The PCR products were analyzed on polyacrylamide gels, since this method provides the highest sensitivity, thus permitting the very strong up-regulation of the gene to be illustrated, i.e. it is really not detectably expressed in the majority of normal colon samples. Nevertheless, the speci®city of the separation on nondenaturing polyacrylamide gels is determined not only

by the size and charge of the PCR product, but also by the presence of secondary structures [7]. In Fig. 2A, the speci®c PCR product appears as two bands in some of the lanes (2, 4, and 5). The same products run on agarose gels show only one speci®c band. To ensure that only one product was present, we cloned the PCR products shown in Fig. 2A. Following the cloning and sequencing of ten PCR products we found in all cases that the clones had 100% identity with the oligophrenin sequence (GenBank accession no. NM_002547). No other sequence was observed. The presence of two bands can therefore be interpreted as being due to conformational artifacts.

4. Discussion Neoplastic transformation arises from multiple defects in cell growth and differentiation. Gene

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expression changes and/or genomic DNA mutations are central to cancer formation and in its progression. Several methods are available for monitoring gene expression or detecting differentially expressed genes. Techniques for studying mRNA by comparative and subtractive hybridization in gels have been used for a number of years; however, they are generally both cumbersome and relatively insensitive [8,9]. Sequencing of cDNA libraries is a more direct approach, but requires a great deal of effort and is not sensitive to the presence of less abundant messages. The SAGE method is an ef®cient variation of the cDNA sequencing approach [10]. This method, however, involves fairly complicated procedures for sample preparation, requires a large amount of sequencing, and is not highly sensitive. Since the initial development of cDNA microarray hybridization [11], there has been considerable interest in this rapidly emerging technology. The ability to compare the relative level of thousands of mRNA transcripts simultaneously in a single hybridization experiment has the potential to contribute signi®cantly to our understanding of tumorigenesis. In this study, we compared the expression of 18 376 cDNA clones in colorectal tumors. We used pools of tissues in our experiment because relatively large amounts of microdissected tumor are required for each cDNA probe synthesis for use in hybridization against the GDA membranes. Pools also favor the detection of consistently up-regulated genes that may be of more long-term diagnostic and therapeutic relevance. Oligophrenin-1 was found to be up-regulated in colorectal tumors in three individual microarray analyses with GDA membranes and this expression was con®rmed by RT-PCR in other colorectal tumor samples. Moreover, we found high expression level of this gene in other data banks such as SAGEmap in a prostate carcinoma cell line. There are no records in the literature regarding the expression of oligophrenin-1 in colorectal tumors. Interestingly, Ljubimova et al. (2001) examined the differential expression of genes by GDA, semiquantitative RT-PCR, and Northern blot between histologically normal adjacent tissue and the brain tumor tissue. By GDA analysis, Oligophrenin-1 gene gave the highest ratio compared to the other genes in brain tissues adjacent to the glioblastoma multiforme (GBM) vs. GBM. By RT-PCR, Oligophrenin-

1 was found expressed in both tumors and tumor-adjacent tissues, whereas meningioma and corpus callosum were negative [12]. Thus, the results shown here are novel and, given the normal nervous system restriction of the expression of this gene, unexpected. Oligophrenin-1 possesses 25 exons, which span at least 500 kb. The gene is mapped to chromosome Xq12 and is related to the X-linked mental retardation [13]. The open reading frame (ORF) of oligophrenin1 is 2406 bp long. It contains a domain typical of a Rho-GTPase-activating protein (rhoGAP), and its amino-terminal domain is similar to a highly conserved protein of unknown function identi®ed in C. elegans, mouse, and human. Inactivation of oligophrenin-1 might affect the activity of interacting proteins or cause constitutive activation of potential rho-GTPase targets. Such constitutive activation of Rho family members affects cell migration, axon outgrowth, and morphogenesis in vivo [14,15]. Oligophrenin-1 is expressed at high levels in all parts of the developing neuroepithelium of the neural tube. Billuart et al. (1998) used in situ hybridization analysis to examine the expression of the homologous mouse gene during development. In later stages of differentiation and in mature brain, a signi®cant level of expression is visible in different brain structures. In a multiple tissue expression array (Clontech, Inc.) we observed high expression levels in normal human brain tissues, similar to the literature for oligophrenin-1 (data not shown). In summary, we have consistently observed an over-expression of Oligophrenin in colorectal tumors when compared to their normal counterparts. The implication of this phenomenon on the etiology of colorectal tumors needs to be further explored. Acknowledgements We would like to thank Waleska Kerlen Martins, Alex Fiorini de Carvalho, and Patricia MendoncËa Mattos for technical support. This work was supported by grants from the FAPESP and Ludwig Institute for Cancer Research. N.A.P. was the recipient of a fellowship from FundacËaÄo de Amparo a Pesquisa do Estado de SaÄo Paulo ± FAPESP. This work was supported in part by FAPESP/CEPID grant number 98/14335-2.

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References [1] S.J. Winawar, Screening for colorectal cancer, N. Engl. J. Med. 329 (18) (1993) 1354. [2] D.M. Parkin, N.E. Day, Evaluating and planning screening programs, IARC Sci. Publ. 66 (1985) 45±63. [3] Boletim do INCA ± MinisteÂrio da SauÂde-Registro Nacional de Patologia Tumoral-DiagnoÂsticos de CaÃncer no Brasil, Rio de Janeiro, Instituto Nacional do CaÃncer, MinisteÂrio da SauÂde, 2000. [4] B. Vogelstein, E.R. Fearon, S.R. Hamilton, S.E. Kern, A.C. Preisinger, M. Leppert, Y. Nakamura, R. White, A.M. Smits, J.L. Bos, Genetic alterations during colorectal-tumor development, N. Engl. J. Med. 319 (9) (1988) 525±532. [5] E.R. Fearon, B.A. Vogelstein, Genetic model for colorectal tumorigenesis, Cell 61 (1990) 759±767. [6] G.M. Church, W. Gilbert, Genomic sequencing, Proc. Natl Acad. Sci. USA 81 (1984) 1991±1995. [7] A. Rolfs, I. Schuller, U. Finckh, I. Weber-Rolfs, Methods of identi®cation of ampli®ed PCR products, PCR: Clinical Diagnostics and Research, Springer, Berlin, 1992 pp. 112±135. [8] S.W. Lee, C. Tomasseto, R. Sager, Positive selection of candidate tumor-suppressor genes by subtractive hybridization, Proc. Natl Acad. Sci. USA 88 (1991) 2825±2829. [9] P. Liang, A.B. Pardee, Differential display of eukaryotic

[10]

[11] [12]

[13]

[14]

[15]

73

messenger RNA by means of the polymerase chain reaction, Science 257 (1992) 967±971. V.E. Velculescu, L. Zhang, B. Vogelstein, K.W. Kinzler, Serial analysis of gene expression, Science 270 (1995) 484± 487. M. Schena, D. Shalon, R.W. Davis, P.O. Brown, Quantitative monitoring of gene expression patterns with a complementary DNA microarray, Science 270 (1995) 467±470. J.Y. Ljubimova, N.M. Khazenzon, Z. Chen, Y.I. Neyman, L. Turner, M.S. Riedinger, K.L. Black, Gene expression abnormalities in human glial tumors identi®ed by gene array, Int. J. Oncol. 18 (2001) 287±295. P. Billuart, T. Bienvenu, N. Ronce, V. des Portes, M.C. Vinet, R. Zemni, H.R. Crollius, A. Carrie, F. Fauchereau, M. Cherry, S. Briault, B. Hamel, J.-P. Fryns, C. Beldjord, A. Kahn, C. Moraine, J. Chelly, Oligophrenin-1 encodes a rhoGAP protein involved in X-linked mental retardation, Nature 392 (1998) 823±826. L. Luo, T.K. Hensch, L. Ackerman, S. Barbel, L.Y. Jan, Y.N. Jan, Differential effects of the Ras GTPase on Purkinje cell axons and dendritic trunks and spines, Nature 379 (1996) 837± 840. I.D. Zipkin, R.M. Kindt, C.J. Kenyon, Role of a new Rho family member in cell migration and axon guidance in C. elegans, Cell 90 (1997) 883±894.