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(American Journal of Pathology. 2004;165:63-69.)
© 2004 American Society for Investigative Pathology

TRIO Amplification and Abundant mRNA Expression Is Associated with Invasive Tumor Growth and Rapid Tumor Cell Proliferation in Urinary Bladder Cancer

Min Zheng*, Ronald Simon*, Martina Mirlacher*, Robert Maurer{dagger}, Thomas Gasser{ddagger}, Thomas Forster{ddagger}, Pierre Andre Diener§, Michael J. Mihatsch*, Guido Sauter* and Peter Schraml*

From the Institute of Pathology,* University of Basel, Basel; the Institute of Pathology,{dagger} City Hospital Triemli, Zürich; the Urologic Clinics,{ddagger} Cantonal Hospital Liestal, Liestal; and the Institute of Pathology,§ Cantonal Hospital St. Gallen, St. Gallen, Switzerland


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Studies by comparative genome hybridization have suggested that 5p amplification is related to tumor progression in urinary bladder cancer. In this study seven genes (TAS2R, ADCY2, DNAH5, CTNND2, TRIO, ANKH, and MYO10) located to 5p15.31-5p15.1 were analyzed by fluorescence in situ hybridization using a tissue microarray containing samples from tumors and cell lines with known 5p amplification by comparative genome hybridization. Amplification frequency was highest for TRIO, which maps to 5p15.2 and encodes a protein with a putative role in cell-cycle regulation. To further investigate the role of TRIO amplification in bladder cancer, a tissue microarray containing samples from 2317 bladder tumors was used for fluorescence in situ hybridization analysis. TRIO amplification was strongly associated with invasive tumor phenotype, high tumor grade, and rapid tumor cell proliferation (Ki67 LI) (P < 0.0001 each). Only 7 of 456 pTaG1/G2 tumors (1.5%) but 62 of 485 pT1-4 carcinomas (12.8%) had TRIO amplification. TRIO amplification was not associated with poor prognosis. Using a frozen bladder tumor tissue microarray RNA in situ hybridization confirmed that TRIO is up-regulated in amplified tumors. It is concluded that TRIO up-regulation through amplification has a potential role in bladder cancer progression.

Studies by comparative genomic hybridization (CGH) demonstrated gains and amplifications at many different chromosomal loci mainly in invasive bladder cancer.1 Gene copy number increase is an important mechanism leading to abnormal expression of known oncogenes such as MYC at 8q24, CCND1 at 11q13, or HER2 at 17q21.2-4 However, most of the chromosomal regions found amplified in bladder cancer1 do not contain known oncogenes, suggesting that many amplification target genes remain to be identified.

Amplifications of the short arm of chromosome 5 are of particular interest. In previous CGH studies 5p amplification was found to be one of the few alterations occurring more frequently in muscle invasive tumors (stage pT2 and higher) than in early invasive cancers (stage pT1).5 Our hypothesis of a role of 5p amplification for bladder cancer progression was further corroborated by the results of a second CGH study finding a significant association between 5p amplifications and an increased risk for tumor progression in a series of pT1 carcinomas.6 Because amplifications often cover large areas of 5p in bladder cancer, it is difficult to narrow down the region of amplification using positional cloning approaches. Based on our CGH studies, one of the most common sites of amplification is in the 5p15-p14 region.5 In an attempt to find oncogene candidates among known genes allocated in this chromosomal area, we became interested in TRIO located at 5p15.2.7 The TRIO protein contains a serine/threonine kinase domain and two guanine nucleotide exchange factor domains for the family of Rho-like GTPases, specific for Rac1 and RhoA.8 These functional domains suggest that this enzyme may play a role in signaling pathways controlling cell proliferation.

In this study we used a mini tissue microarray (TMA) containing 5p-amplified bladder tumors for gene prescreening and a recently manufactured tissue microarray (TMA)9,10 composed of 2317 bladder cancers to examine TRIO copy number changes and its association with tumor phenotype, cell proliferation, and patient prognosis. The correlation of TRIO mRNA expression and amplification was analyzed in 80 arrayed frozen bladder tumors.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
CGH

A review of the CGH profiles of 278 primary bladder carcinomas and 20 cell lines previously examined in our laboratory revealed 16 primary tumors and 5 cell lines with 5p amplification. Examples of CGH profiles showing circumscribed 5p amplifications in the 5p15.31-5p15.1 area are shown in Figure 1 .



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  		Figure 1. CGH results in tumors with 5p amplification. CGH profile examples of two primary bladder cancers showing distinct peaks in the 5p15.31-p15.1 region suggesting amplification. The number of observations is shown on the bottom of each profile. The vertical lines indicate the base line ratio (1.0), and the threshold selected to define relative chromosomal gains (1.2, right) and losses (0.8, left).

 
Bladder Cancer Tissue Microarrays (TMAs)

Three different TMAs were used in this study. A mini TMA containing 10 5p-amplified bladder tumors and four cell lines (5-HTB, RT11-D21, RT112, and CRL-7930) served as a prescreening tool to determine the amplification frequency and copy numbers of seven 5p15 genes in these samples. The second bladder cancer prognosis TMA was previously described.9,10 One pathologist (G.S.) reviewed all slides of all tumors. Tumor stage and grade were defined according to International Union Against Cancer and World Health Organization classification.11,12 Time to recurrence and time to progression (to stage pT2 or higher) were selected as clinical endpoints for pTa and pT1 tumors, if regular follow-up cystoscopies had been performed at least at 3, 9, and 15 months, then annually until the endpoint of this study (recurrence, last control). To include a patient for analyses of time to progression longer intervals between controls were accepted if the last follow up control ruled out progression.

An additional TMA composed of 80 histologically not further characterized frozen bladder carcinomas was manufactured as described.13 A home-made semiautomated tissue-arraying device equipped with a 0.6-mm drill for recipient hole making was used. An H&E-stained section of this frozen TMA is shown in Figure 3A .



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  		Figure 3. TRIO expression and amplification in bladder cancer. A: H&E-stained section of a TMA containing 80 bladder tumors from fresh frozen tissues. B: RISH phosphor image of the TMA hybridized with TRIO-specific oligomers after 48 hours of exposure time. C: Autoradiography of a bladder tumor array element showing tumor cells with high TRIO mRNA expression. D: Schematic illustration of the RISH result. E: Schematic illustration of the FISH result obtained from a consecutive TMA section. F: Northern blot analysis of three bladder tumor cell lines. Abundant TRIO mRNA expression (9.4 kb) in 5p amplified 5-HTB and low expression in the not amplified cell lines 3-HTB and 4-HTB. The Northern blot was rehybridized with GAPDH probe as a control.

 
Fluorescence in Situ Hybridization (FISH)

The tissue microarray sections were treated according to the Paraffin Pretreatment Reagent kit protocol (Vysis, Inc., Downers Grove, IL) before hybridization. FISH was performed with digoxigenated BAC probes specific for seven genes located to 5p15.31-5p15.1 (Table 1) (Invitrogen, Carlsbad, CA) and a Spectrum Red-labeled chromosome 6 centromeric probe as a reference (Vysis). Hybridization and posthybridization washes were according to the Locus Specific Identifier (LSI) procedure (Vysis). Probe visualization using fluorescein isothiocyanate (FITC)-conjugated sheep anti-digoxigenin (Roche Diagnostics, Rotkreuz, Switzerland) was as described.14 Slides were counterstained with 125 ng/ml of 4',6-diamino-2-phenylindole in an anti-fade solution. Amplification was defined as presence (in ≥5% of tumor cells) of either more than 10 gene signals or more than three times as many gene than centromere 6 signals.


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  		Table 1. Amplification Frequency and Copy Numbers of 5p15 Genes in 5p-Amplified Bladder Cancers

 
RNA in Situ Hybridization (RISH)

Four oligonucleotides of 40 bases specific for TRIO were designed using the Vector NTI program (www.vectornti.co.kr). Each of the oligonucleotides was labeled separately in a reaction volume of 4.5 µl using [{alpha}-33P]-dATP (Amersham Pharmacia Biotech, Buckinghamshire, UK) and terminal transferase (Catalys, Wallisellen, Switzerland). Unincorporated nucleotides were removed according to the manufacturer’s instructions (Qiagen, Basel, Switzerland). Denhardt’s hybridization solution (120 µl) containing 50% formamide, 4x standard saline citrate, 0.02 mol/L NaPO4 (pH 7), 7% dextran sulfate, 5% sarcosyl, Cot1 DNA (40 µg/ml), 0.15 mol/L dithiothreitol, and radiolabeled oligos (5,000,000 cpm/ml hybridization solution) were mixed and added to the microarray and hybridized overnight at 42°C in a moist chamber. Slides were washed for 4 x 15 minutes in 1x standard saline citrate at 55°C, for 1 hour with 1x standard saline citrate at room temperature, dehydrated in distilled water, 60% ethanol, and 95% ethanol (30 seconds for each step) and air-dried for 10 minutes. Hybridization signals were visualized after 48 hours of exposure to a high-resolution screen with Cyclone Phosphor Imager (Canberra Packard, Zurich, Switzerland). Specificity of RISH was evaluated by treating the TMA with Hypercoat emulsion LM-1 according to the protocol of Amersham Pharmacia Biotech.

Northern Blot Analysis

Total RNA was extracted from three bladder cancer cell lines (3-HTB, 4-HTB, and 5-HTB) using Trizol (Invitrogen AG, Basel, Switzerland). Two µg of poly(A)+ mRNA of each cell line was selected with the DynaBead-kit (Dynal, Oslo, Norway), separated on a 1% agarose-formaldehyde gel and transferred to a nylon membrane (Amersham Pharmacia Biotech). The membrane was hybridized with [{alpha}-32P]-dATP (Amersham Pharmacia Biotech)-labeled TRIO-specific oligonucleotides at 42°C for 5 hours in ExpressHyb solution (Clontech, Heidelberg, Germany). Northern analysis was performed with Cyclone phosphor imager (Canberra Packard).

Statistics

Only the first biopsy was used for statistical analyses in patients having more than one tumor on the TMA. Chi-square tests were applied to study the relationship between histological tumor type, grade, stage, and TRIO amplification. Student’s t-tests were used to examine the associations of the Ki67 LI with TRIO amplification, tumor stage, and grade. Survival curves were plotted according to the Kaplan-Meier method and analyzed for statistical differences using a log-rank test. Patients with pTa/pT1 tumors were censored at the time of their last clinical control showing no evidence of disease or at the date when cystectomy was performed. Patients with pT2-4 carcinomas were censored at the time of their last clinical control or at the time of death if they died from causes not related to their tumor.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
FISH Prescreening

Prescreening and establishing optimal hybridization conditions was completed on a mini-TMA containing four cell lines and 10 primary tumors that had shown 5p amplification by CGH. FISH analysis of seven 5p15 genes on the mini-TMA revealed highest frequency of amplification for TRIO according to our definition. TRIO was amplified in all four cell lines and eight of nine interpretable primary tumors (Table 1) . The average TRIO copy numbers in four of these tumors were 14, 16, 18, and more than 20 clustered signals. Lower amplification rates and copy numbers were detected for the remaining genes located in close vicinity to TRIO. Based on these results the role of TRIO in bladder cancer was further examined using a prognosis TMA.

TRIO Gene Amplification

Large-scale TMA analysis showed TRIO amplification in a total number of 111 of 1636 interpretable tumors. FISH related problems (weak hybridization, background, tissue damage) were responsible for approximately two-thirds, TMA-linked problems (too few or absence of tumor cells on the TMA spot) were causing approximately one third of the noninformative cases. Examples of amplified and nonamplified tumors are shown in Figure 2, A and B . The associations with tumor phenotype are summarized in Table 2 . TRIO amplification was not only frequent in muscle-invasive transitional cell carcinomas (TCC) (15.9%) but also in small cell (35.7%), sarcomatoid (two of eight amplified), and squamous cell cancers (5.7%) as well as in adenocarcinomas (one of four amplified). Within TCC, which is by far the most common bladder cancer subtype, TRIO amplification was strongly associated with tumor grade and stage (P < 0.0001 each). Most strikingly, TRIO amplification was rare in pTaG1/G2 tumors (7 of 456; 1.5%) whereas 12.8% (62 of 485) of the invasively growing TCC (pT1-4) were amplified.



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  		Figure 2. TRIO analysis on a bladder cancer tissue microarray. A: TRIO amplification: tumor cell showing clusters of green TRIO signals and two red centromere 6 signals. B: Nonamplified tumor cell with two copies each of TRIO (green) and centromere 6 (red).

 

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  		Table 2. TRIO Amplification and Tumor Phenotype

 
TRIO mRNA Expression in Bladder Cancer

To determine whether TRIO amplification leads to elevated TRIO mRNA expression, RISH and FISH was performed on two consecutive sections of a microarray containing 80 fresh frozen bladder tumors. TRIO mRNA expression and DNA copy number analyses was evaluated in 59 tumors. There was a high correlation between overexpression and TRIO amplification. Fifteen of 17 tumors (88%) with strong TRIO mRNA expression had more than six TRIO gene copies. Five of 28 tumors (18%) with moderate and only 1 of 14 tumors (7%) with low or no detectable TRIO mRNA expression were amplified (Figure 3; B to E) . Northern blot hybridization of mRNA extracted from three bladder tumor cell lines confirmed the binding specificity of the oligo probes to TRIO mRNA (Figure 3F) .

TRIO Amplification and Tumor Cell Proliferation [Ki67 Labeling Index (LI)]

TRIO amplification was significantly associated with rapid tumor cell proliferation (P < 0.0001). The separate analyses of tumors of identical grades and stages lead to significant differences in the proliferation between TRIO nonamplified and amplified tumors (Table 3) .


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  		Table 3. TRIO Amplification in Relation to Ki-67 LI

 
TRIO Amplification and Prognosis

TRIO amplification was not associated with poor tumor-specific survival neither if all patients were included in the analysis nor within the subgroup of pT2-4 TCCs. TRIO amplification provided no prognostic significance among pTa/pT1 tumors, neither for recurrence nor for progression.


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
In this study, TMA technology was used to screen oncogene candidates located within the 5p15.31-5p15.1 region and to examine gene amplification and mRNA expression of TRIO on chromosome 5p15.2 in urinary bladder cancer. Besides bladder cancer, overrepresentations of chromosome 5p have been found in various other human tumor types including breast,15 colon,16 head and neck,17 lung cancer,18 and osteosarcoma.19 Although frequently altered in these tumor types, for example 70% of non-small-cell lung tumors showed 5p gains,18 potential oncogene candidates residing in this chromosomal area have hardly been described in the literature. The limited resolution of the CGH method (>10 Mb), and the large size of most CGH peaks on 5p make it difficult to exactly define the chromosomal location of interest. However, previous CGH analysis at almost 300 bladder carcinomas allowed us to identify several cases with more distinct peaks around 5p15.31-5p15.1.

No known oncogene has previously been assigned to chromosome 5p15.31-p15.1. A rapid search on the publicly available genome databases (http://www.ensembl.org/) revealed that TRIO, a large gene composed of 58 exons distributed throughout more than 250 kb, has been mapped to 5p15.2. The gene codes for a protein, which contains two guanine nucleotide exchange factor (GEF) domains for the family of Rho-like GTPases and a serine/threonine kinase domain.8 These functional domains suggest that the TRIO protein may play a key role in several pathways that control cell cycle, migration, and cell-cell interactions. It is conceivable that up-regulation of GEFs by TRIO amplification may lead to an increased activation of Rho-like GTPases, thus driving cell motility and invasion.20 Increased amounts of Rho-like GTPases were described in head and neck,21 breast, colon, lung,22 pancreas,23 and testicular germ cell tumors,24 as well as T-lymphoma25 and melanoma.26 It is also well known that dysregulation of serine/threonine kinases may lead to uncontrolled cell proliferation and hence to cancer. For example, amplification of serine/threonine kinase genes such as CDK2 at 12q13, CDK4 at 12q14, PS6K at 17q23, and STK15 at 20q13 were observed in tumors of the ovary,27 breast,28,29 colon,30 brain,31,32 and cervix.33 The strong association of TRIO amplification with high grade, advanced stage, and tumor cell proliferation suggests TRIO as appropriate oncogene candidate when amplified and overexpressed in bladder tumors.

Preferential amplification with high copy numbers in all four cell lines and eight of nine interpretable tumors with 5p amplification by CGH provided evidence for an involvement of TRIO in bladder cancer and prompted us to further analyze its role in urinary bladder tumors. Using a TMA containing 2317 bladder carcinomas, TRIO amplification was found in 9.6% of minimally invasive (pT1) tumors and in 15.9% of muscle-invasive (pT2-4) tumors. This result is consistent with a previous CGH study in which 5p aberrations occurred significantly more frequent in pT2-4 tumors than in pT1 tumors.5 The rate of muscle-invasive tumors with TRIO amplifications was somewhat higher than that of pT2-4 tumors analyzed with CGH. It is likely that in some cases regional amplifications were interpreted as gain rather than amplification because of technical or DNA quality reasons. Additional RISH and FISH analyses on 80 arrayed frozen bladder tumors confirmed high correlation of TRIO amplification and strong mRNA expression.

In this study a very conservative cutoff was used to define amplification. Therefore, the group of nonamplified tumors contained a fraction of cancers with TRIO gains that were cautionary not considered amplified for statistical calculations. However, the portion of pT2-4 tumors with TRIO copy numbers ≥3 was 30%, which is similar to that observed by CGH analysis.5,34 It is of note that 5p gains detected by CGH were associated with high risk of progression in early invasive (pT1) bladder cancer in one study.6 This finding was interpreted with caution because only 5 of 54 pT1 tumors analyzed demonstrated 5p gains. Although the rate of TRIO-amplified pT1 tumors with clinical follow-up information (9.6%) was comparable to the results obtained by CGH, there was no significant correlation with tumor progression. This holds also true if tumors with average TRIO copy numbers of less than seven were included in the statistical analysis.

Oncogenes such as CCND1, ERBB2, and MYC are commonly highly amplified in different human tumors and appear as tight clustered signals representing up to a few hundred gene copies.35 In contrast, 91% of TRIO-amplified bladder tumors showed signals ranging between 7 and 14 gene copies, which were more or less equally distributed within tumor cell nuclei. This observation suggests a mechanism of gene amplification that differs to that seen for most of the known oncogenes. Cytogenetic analysis demonstrated that 5p might be involved in translocations and/or formation of isochromosomes in a substantial number of bladder tumors.36 An accumulation of 5p isochromosomes because of increasing genomic instability described in invasively growing tumors36 may account for the TRIO amplification pattern seen in most amplified bladder cancers.

In summary, we show that TRIO amplification is strongly linked to high-grade, advanced stage, bladder tumors and rapid tumor cell proliferation in urinary cancer. Preferential amplification within 5p15.31-5p15.1 and the strong association between amplification and mRNA abundance suggests TRIO as an oncogene candidate within this amplicon.


   Acknowledgements
 
We thank Ursula Dürmüller, Yvonne Knecht, Anne Kunzelmann, Hedvika Novotny, Sandra Schneider, and Barbara Stalder for their excellent technical support.


   Footnotes
 
Address reprint requests to Peter Schraml, Ph.D., Institute of Pathology, University of Basel, Schoenbeinstrasse 40, CH-4031 Basel, Switzerland. E-mail: peter.schraml{at}unibas.ch

Supported by the Swiss National Science Foundation (grant 31-059254.99).

Present address of M.Z.: Department of Gynecology, Tumor Center of Sun Yat-Sen University, Guang Zhou, China.

Accepted for publication March 9, 2004.


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